EFFECT OF FRUIT FLAVORS ON ANAEROBIC DIGESTION: …hb.diva-portal.org/smash/get/diva2:877097/FULLTEXT01.pdf · 2015. 12. 4. · about the effect of fruit flavors on anaerobic digesting

Digital version: http://hdl.handle.net/2320/14222ISBN 978-91-87525-27-8 (printed)ISBN 978-91-87525-28-5 (pdf)ISSN 0280-381X, Skrifter från Högskolan i Borås, nr. 54

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EFFECT OF FRUIT FLAVORS ON ANAEROBIC DIGESTION: INHIBITIONS

AND SOLUTIONSRachma Wikandari

Rachm

a Wikandari EFFECT OF FRUIT FLAVORS ON ANAEROBIC DIGESTION

: INHIBITIONS AND SOLUTIONS

This thesis presents work that was done within the Swedish Centre for Resource Recovery (SCRR).

Research and education performed within SCRR identifies new and improved methods to convert

residuals into value-added products. SCRR covers technical, environmental and social aspects of

sustainable resource recovery.

Approximately 402 million tons of fruit wastes are annually generated worldwide, that is a serious environmental challenge. Biogas emerges as an attractive solution to handle this waste, since conversion of fruit waste into biogas combines waste treatment and energy production. In theory, 32 billion m3 biogas could be produced from this fruit wastes, which is equivalent to ca 20 billion liters of gasoline. However, in reality, the biogas production from the fruits is less than

theoretically possible. One of the reasons is the presence of toxic compound in the fruits that inhibit growth of biogas producing microorganisms. D-limonene, a flavor compound in orange is an example of the chemicals that causes failure in biogas production from orange peel waste. What are toxic compounds in other fruit and how to solve the toxicity problem? These questions were investigated in this thesis.

Various flavor compounds available in different fruits were examined. Among the compounds examined, myrcene and octanol were shown to be strong inhibitors for the process, which reduces 50% of biogas production at low concentration (0.005–0.05%). These compounds belong to fruit flavors of oranges, strawberries, grapes, plums, and mangoes. The toxicity of α-pinene, hexanal, E-2-hexenal, nonanal, γ-decalactone, epicatechin, and quercetin were moderate (0.05–0.5%). Meanwhile, the rest flavors including methyl butanoate, ethyl butanoate, ethyl hexanoate, hexyl acetate, furaneol, mesifurane, and γ-hexalactone had a little effect on the digestion process.

In order to overcome inhibition challenges of the flavors, the present study proposed two approaches: fruit flavors removal and protection of the digesting microorganism against fruit flavors. In this work, orange peel waste (OPW) and limonene were used as a model for the fruit waste and the inhibitor, respectively. The first approach used was solid-liquid pretreatment, which resulted in increased biogas production from OPW by more than three times. The second approach performed was using encased digesting cells in membranes, which were impermeable to limonene. This method resulted in increasing the biogas production from OPW by more than six times. More importantly, this method can be applied in single-stage digestion that is industrially popular.

Thesis for the Degree of Doctor of Philosophy

Effects of fruit flavors on anaerobic

digestion: inhibitions and solutions

Rachma Wikandari

ii

Copyright© Rachma Wikandari

Swedish Centre for Resource Recovery University of Borås SE-50190 Borås (Sweden)

Handle-id: http://hdl.handle.net/2320/14222

ISBN 978-91-87525-27-8 (printed)

ISBN 978-91-87525-28-5 (pdf)

ISSN 0280-381X

Skrifter från Högskolan i Borås, nr. 54

Printed in Sweden by Ale Tryckteam

Bohus, 2014

iii

Abstract

Fruits are among the most important commodities in global trading due to its fundamental nutritional values. In 2012, the fruits supply was 115 kg/person/year, however, only 50 % of the fruits reached their consumers and the rest ended up as waste during the long fruit supply chain. The waste from fruits is mostly dumped or burned, creating a serious environmental problem. A more sustainable handling of the waste is therefore highly desirable. One of them is conversion of the fruits wastes into biogas through anaerobic digestion. One challenge with the conversion of fruits wastes into biogas is the presence of antimicrobial compounds in the fruits, which reduce the biogas yield or even cause a total failure of the process. Fruit flavors have been reported to have antimicrobial activity against several microorganisms and being responsible for the defense system in the fruits. However, there is only scarce information about the effect of fruit flavors on anaerobic digesting microbia. The objectives of the present thesis were: 1) to investigate the inhibitory activity of the fruit flavors on anaerobic digestion; 2) to remove the flavor compound by pretreatment; and 3) to protect the cell from the flavor compounds using a membrane bioreactor.

The inhibitory activity of the fruit flavors was examined from different groups of flavors by adding a single flavor compound into the batch anaerobic digesting system, at three different concentrations. Among the flavors added, myrcene and octanol were found to exhibit a strong inhibitory activity, with 50 % reduction of the methane production at low concentrations, ca. 0.005–0.05 %. These flavors can be found in oranges, strawberries, grapes, plums, and mangoes. The other flavors tested showed moderate and low inhibitory activity, which might not affect the anaerobic digestion of the fruits wastes.

In order to overcome the inhibitory effects of the fruit flavor, two approaches were proposed in this thesis, namely, fruit flavor removal by leaching pretreatment and cell protection from fruit flavor using a membrane bioreactor. Orange peel waste and D-limonene were used as a model of fruit waste and inhibitor, respectively. The leaching pretreatment uses solvent to extract the limonene from the orange peel. The methane yield increased by 356 % from 0.061 Nm3/kg VS to 0.217 Nm3/kg VS, by pretreating the peel using hexane with peel and a hexane ratio of 1:12 at room temperature for 10 min. Alternative to limonene removal, the cells were encased in a hydrophilic membrane, which is impermeable to hydrophobic limonene. This method yielded more than six times higher methane yield, compared to the free cell. At the highest organic loading rate, examined in this work, 3 g VS/L/day, the methane yield of the reactor containing the free cell was only 0.05 Nm3/kg VS, corresponding to 10 % of the theoretical yield, whereas 0.33 Nm3/kg VS methane yield was achieved using a membrane bioreactor corresponding to 75 % of the theoretical yield.

ii

Copyright© Rachma Wikandari

Swedish Centre for Resource Recovery University of Borås SE-50190 Borås (Sweden)

Handle-id: http://hdl.handle.net/2320/14222

ISBN 978-91-87525-27-8 (printed)

ISBN 978-91-87525-28-5 (pdf)

ISSN 0280-381X

Skrifter från Högskolan i Borås, nr. 54

Printed in Sweden by Ale Tryckteam

Bohus, 2014

iii

Abstract




Abstract




Keywords: Fruits wastes, Fruit flavors, Anaerobic digestion, Biogas, Leaching pretreatment, Membrane bioreactor.

iv

List of publications included in this thesis

1. Wikandari R, Gudipudi S, Pandiyan I, Millati R, Taherzadeh MJ: Inhibitory effects of

fruits flavors on methane production during anaerobic digestion. Bioresource

Technology 2013, 145:188-192.

2. Heriyanti, Wikandari R, Millati R, Niklasson C, Taherzadeh MJ: Ester compounds on

biogas production: beneficial or detrimental? Energy Science and Engineering 2014,

2 : 22-30

3. Wikandari R, Ahmada QA, Kartikasari N, Millati R, Cahyanto MN, Niklasson C,

Taherzadeh MJ: Effect of Ketone, Lactone, and Phenol on Methane Production and

Metabolic Intermediates during Anaerobic Digestion. Submitted manuscript.

4. Wikandari R, Nguyen H, Millati R, Niklasson C, Taherzadeh MJ: Improvement of

biogas production from orange peel waste by leaching of limonene. Biomed Research

International. In Press.

5. Wikandari R, Youngsukkasem S, Millati R, Niklasson C, Taherzadeh MJ:

Performance of semi-continuous membrane bioreactor in biogas production from

toxic feedstock containing D-limonene. Bioresource Technology 2014, 170:350-355.

6. Wikandari R, Millati R, Cahyanto MN, Taherzadeh MJ: Biogas Production from

citrus waste by membrane bioreactor. Membranes 2014, 4: 596-607

Statement of Contribution

Paper 1: Responsible for the experimental design, part of the experimental work,

conducting data analyses, and manuscript writing

Paper 2: Responsible for the experimental design and active participant in the

preparation and organization of the manuscript

Paper 3: Responsible for the experimental design, conducting data analysis, and

manuscript writing

Paper 4: Responsible for part of the experimental work, conducting data analyses, and

manuscript writing

Paper 5: Responsible for major part of the experimental work and data analyses as well

as manuscript writing



v

Table of Contents

1. Introduction………………………………………………………………………… 1

2. Fruits……………………………………………………………………………...... 3

2.1. Fruits……………………………………………………………….. 3

2.2. Global fruit production……………………………………………... 6

2.3. Fruits wastes………………………………………………………… 8

3. Fruit flavors………………………………………………………………………… 13

3.1. Overview of fruit flavors…………………………………………….. 13

3.2. Biosynthesis of fruit flavors…………………………………………. 16

3.3. Antimicrobial activity of fruit flavors……………………………….. 21

4. Anaerobic digestion………………………………………………………………... 23

4.1. Biogas trends and biogas market……………………………………..23

4.2. Anaerobic digestion: complexities of the process and challenges…... 26

4.2.1. Overview of the biochemical reactions……………….. 26

4.2.2. Different characteristics of the microorganisms……… 28

4.2.3. Structural complexity and recalcitrance of the biomass. 29

4.2.4. Inhibition……………………………………………… 31

4.3. Substrates for anaerobic digestion…………………………………... 33

5. Anaerobic digestion from fruits wastes……………………………………………. 35

5.1. Biogas potential from fruits wastes………………………………… 35

5.1.1. Theoretical approach………………………………….. 35

5.1.2. Experimental approach………………………………... 37

5.2. Inhibition effect of fruit flavors……………………………………. 38

6. Addressing the challenges of the inhibitory effects of fruit flavors……………..... 41

6.1. Pretreatment for the removal of fruit flavors ……………………….. 42

6.2. Microbial protection of fruit flavors by using a membrane bioreactor46

6.2.1. MBR for cell protection………………………………. 46

6.2.2. Outlook and future perspective on anaerobic MBR

for biogas production………………………………... 50

Conclusion…………………….…………………………………………….................53

Future work………………………………………………….……………………..……... 55

Nomenclature………………………………………………………….……….……….. 57

iv

List of publications included in this thesis

1. Wikandari R, Gudipudi S, Pandiyan I, Millati R, Taherzadeh MJ: Inhibitory effects of

fruits flavors on methane production during anaerobic digestion. Bioresource

Technology 2013, 145:188-192.

2. Heriyanti, Wikandari R, Millati R, Niklasson C, Taherzadeh MJ: Ester compounds on

biogas production: beneficial or detrimental? Energy Science and Engineering 2014,

2 : 22-30

3. Wikandari R, Ahmada QA, Kartikasari N, Millati R, Cahyanto MN, Niklasson C,

Taherzadeh MJ: Effect of Ketone, Lactone, and Phenol on Methane Production and

Metabolic Intermediates during Anaerobic Digestion. Submitted manuscript.

4. Wikandari R, Nguyen H, Millati R, Niklasson C, Taherzadeh MJ: Improvement of

biogas production from orange peel waste by leaching of limonene. Biomed Research

International. In Press.

5. Wikandari R, Youngsukkasem S, Millati R, Niklasson C, Taherzadeh MJ:

Performance of semi-continuous membrane bioreactor in biogas production from

toxic feedstock containing D-limonene. Bioresource Technology 2014, 170:350-355.

6. Wikandari R, Millati R, Cahyanto MN, Taherzadeh MJ: Biogas Production from

citrus waste by membrane bioreactor. Membranes 2014, 4: 596-607

Statement of Contribution

Paper 1: Responsible for the experimental design, part of the experimental work,

conducting data analyses, and manuscript writing

Paper 2: Responsible for the experimental design and active participant in the

preparation and organization of the manuscript

Paper 3: Responsible for the experimental design, conducting data analysis, and

manuscript writing

Paper 4: Responsible for part of the experimental work, conducting data analyses, and

manuscript writing





v

Table of Contents

1. Introduction………………………………………………………………………… 1

2. Fruits……………………………………………………………………………...... 3

2.1. Fruits……………………………………………………………….. 3

2.2. Global fruit production……………………………………………... 6

2.3. Fruits wastes………………………………………………………… 8

3. Fruit flavors………………………………………………………………………… 13

3.1. Overview of fruit flavors…………………………………………….. 13

3.2. Biosynthesis of fruit flavors…………………………………………. 16

3.3. Antimicrobial activity of fruit flavors……………………………….. 21

4. Anaerobic digestion………………………………………………………………... 23

4.1. Biogas trends and biogas market……………………………………..23

4.2. Anaerobic digestion: complexities of the process and challenges…... 26

4.2.1. Overview of the biochemical reactions……………….. 26

4.2.2. Different characteristics of the microorganisms……… 28

4.2.3. Structural complexity and recalcitrance of the biomass. 29

4.2.4. Inhibition……………………………………………… 31

4.3. Substrates for anaerobic digestion…………………………………... 33

5. Anaerobic digestion from fruits wastes……………………………………………. 35

5.1. Biogas potential from fruits wastes………………………………… 35

5.1.1. Theoretical approach………………………………….. 35

5.1.2. Experimental approach………………………………... 37

5.2. Inhibition effect of fruit flavors……………………………………. 38

6. Addressing the challenges of the inhibitory effects of fruit flavors……………..... 41

6.1. Pretreatment for the removal of fruit flavors ……………………….. 42

6.2. Microbial protection of fruit flavors by using a membrane bioreactor46

6.2.1. MBR for cell protection………………………………. 46

6.2.2. Outlook and future perspective on anaerobic MBR

for biogas production………………………………... 50

Conclusion…………………….…………………………………………….................53

Future work………………………………………………….……………………..……... 55

Nomenclature………………………………………………………….……….……….. 57

vi

Acknowledgment……………………………………………………………………….. 61

References……………………………………………………………………………….. 63

1

CHAPTER 1

Introduction

Fruits are one of the most important trading commodities in the global market. As the

production of fruits increases every year, so does the generation of fruits wastes. The

accumulation of fruits wastes creates serious environmental problems such as strong odors as

well as attracting flies and rats. At the same time, the inevitable oil depletion and the

reduction of greenhouse emission drives public interest on finding and exploiting renewable

energy such as biogas.

Since fruits wastes have a high organic content, it has a potential as a raw material for biogas

production. The conversion of fruits wastes into biogas not only contributes to solving the

environmental problem but also the energy challenge. However, biogas production from

fruits wastes has been reported to be very low, compared to their theoretical yields. One

possible explanation is the presence of antimicrobial compounds in fruits. D-limonene, a fruit

flavor in oranges, has been proved to cause failure of the biogas production from citrus waste.

However, there is scarce information about the inhibitor compound in other fruits and their

effects on anaerobic digestion.

The present thesis deals with finding the inhibitor compound, with the main focus on fruit

flavors and overcoming the inhibitor problem using two strategies: pretreatment for inhibitor

recovery by leaching method and cell protection by using a membrane bioreactor. In this

thesis, orange peel which contains the inhibitor D-limonene was used as a model of fruit

waste since orange peel waste is abundant, and the most frequent inhibitor compound found

in this work are hydrophobic like D-limonene. The main goal of the work in this thesis was to

increase the biogas production from the fruits wastes. To reach this goal, the work was

divided into three parts:

1. Investigation of the inhibitor compound in fruit flavors for several groups of flavor

compounds (Papers I–III)

2. Pretreatment of orange peel waste by leaching method (Paper IV)

vi

Acknowledgment……………………………………………………………………….. 61

References……………………………………………………………………………….. 63

1

CHAPTER 1

Introduction

Fruits are one of the most important trading commodities in the global market. As the

production of fruits increases every year, so does the generation of fruits wastes. The

accumulation of fruits wastes creates serious environmental problems such as strong odors as

well as attracting flies and rats. At the same time, the inevitable oil depletion and the

reduction of greenhouse emission drives public interest on finding and exploiting renewable

energy such as biogas.

Since fruits wastes have a high organic content, it has a potential as a raw material for biogas

production. The conversion of fruits wastes into biogas not only contributes to solving the

environmental problem but also the energy challenge. However, biogas production from

fruits wastes has been reported to be very low, compared to their theoretical yields. One

possible explanation is the presence of antimicrobial compounds in fruits. D-limonene, a fruit

flavor in oranges, has been proved to cause failure of the biogas production from citrus waste.

However, there is scarce information about the inhibitor compound in other fruits and their

effects on anaerobic digestion.

The present thesis deals with finding the inhibitor compound, with the main focus on fruit

flavors and overcoming the inhibitor problem using two strategies: pretreatment for inhibitor

recovery by leaching method and cell protection by using a membrane bioreactor. In this

thesis, orange peel which contains the inhibitor D-limonene was used as a model of fruit

waste since orange peel waste is abundant, and the most frequent inhibitor compound found

in this work are hydrophobic like D-limonene. The main goal of the work in this thesis was to

increase the biogas production from the fruits wastes. To reach this goal, the work was

divided into three parts:

1. Investigation of the inhibitor compound in fruit flavors for several groups of flavor

compounds (Papers I–III)

2. Pretreatment of orange peel waste by leaching method (Paper IV)

2

3. Biogas production from orange peel waste by using a membrane bioreactor (Papers

V–VI)

The thesis consists of six chapters and six papers, summarized as follows:

Chapter 1 introduces the thesis and the main objectives of the research work.

Chapter 2 begins with a comprehensive overview of fruits, including definition of fruits,

followed by the fruit’s function in plants, fruit classification, fruit composition, and fruit

microbiology. The following section presents the global fruit production, and the last section

discusses how the fruits turn into fruits wastes and how much fruits wastes are generated.

Chapter 3 introduces fruit flavors and presents various pathways for the biosynthesis of

flavors. The following section discusses the antimicrobial activity of fruit flavors on diverse

microorganisms, and the last section provides information on the industrial application and

market for fruit flavors.

Chapter 4 presents the current situation of biogas trends and markets. The following section

discusses the complexities and challenges of anaerobic digestion. This section begins with an

overview of the biochemical reaction of anaerobic digestion, followed by a discussion of

several challenges in anaerobic digestion including different characteristics of the

microorganisms, structural complexity and recalcitrance of the biomass, and inhibition. The

last section provides information on various substrates for anaerobic digestion.

Chapter 5 provides an estimate of the biogas potential from fruits wastes using theoretical

and experimental approaches. The following section discusses the inhibitory activity of fruit

flavors on anaerobic digestion as the challenge of anaerobic digestion of fruits wastes (Papers

I-III).

Chapter 6 discusses possible solutions for the inhibition of fruit flavors using two

approaches, that is, fruit flavor removal and cell protection from the inhibitor by using a

membrane bioreactor. This chapter reviews the different pretreatment methods for the

removal of fruit flavors, introduces a leaching method, and presents the results of the current

study using a leaching method (Paper IV). This chapter also provides the basic concept of cell

protection, introduces a membrane bioreactor and its application, and presents the results of

the present study using a membrane bioreactor (Papers V–VI).

3

CHAPTER 2

Fruits

2.1. Fruit

What is fruit?

There are two types of definitions for the term ‘fruits.’ In a biological context, fruit is defined

as part of a flowering plant that is derived from specific tissues of the flower and by which a

flowering plant disseminates the seeds [1]. Meanwhile, in a culinary context, fruit is defined

as any sweet tasting plant product associated with seeds [1]. Thus, occasionally there is a

conflicting classification of fruit. For instance, some fruits, in a biological sense, such as

cucumbers, eggplants, tomatoes, pumpkins, corn, peas, squash, beans, sweet peppers, and

chilies are treated as a vegetable since they are not sweet. In this thesis, the definition will be

based on a biological context.

Fig. 2.1. Anatomy of Flower [2].

2

3. Biogas production from orange peel waste by using a membrane bioreactor (Papers

V–VI)

The thesis consists of six chapters and six papers, summarized as follows:

Chapter 1 introduces the thesis and the main objectives of the research work.

Chapter 2 begins with a comprehensive overview of fruits, including definition of fruits,

followed by the fruit’s function in plants, fruit classification, fruit composition, and fruit

microbiology. The following section presents the global fruit production, and the last section

discusses how the fruits turn into fruits wastes and how much fruits wastes are generated.

Chapter 3 introduces fruit flavors and presents various pathways for the biosynthesis of

flavors. The following section discusses the antimicrobial activity of fruit flavors on diverse

microorganisms, and the last section provides information on the industrial application and

market for fruit flavors.

Chapter 4 presents the current situation of biogas trends and markets. The following section

discusses the complexities and challenges of anaerobic digestion. This section begins with an

overview of the biochemical reaction of anaerobic digestion, followed by a discussion of

several challenges in anaerobic digestion including different characteristics of the

microorganisms, structural complexity and recalcitrance of the biomass, and inhibition. The

last section provides information on various substrates for anaerobic digestion.

Chapter 5 provides an estimate of the biogas potential from fruits wastes using theoretical

and experimental approaches. The following section discusses the inhibitory activity of fruit

flavors on anaerobic digestion as the challenge of anaerobic digestion of fruits wastes (Papers

I-III).

Chapter 6 discusses possible solutions for the inhibition of fruit flavors using two

approaches, that is, fruit flavor removal and cell protection from the inhibitor by using a

membrane bioreactor. This chapter reviews the different pretreatment methods for the

removal of fruit flavors, introduces a leaching method, and presents the results of the current

study using a leaching method (Paper IV). This chapter also provides the basic concept of cell

protection, introduces a membrane bioreactor and its application, and presents the results of

the present study using a membrane bioreactor (Papers V–VI).

3

CHAPTER 2

Fruits

2.1. Fruit

What is fruit?

There are two types of definitions for the term ‘fruits.’ In a biological context, fruit is defined

as part of a flowering plant that is derived from specific tissues of the flower and by which a

flowering plant disseminates the seeds [1]. Meanwhile, in a culinary context, fruit is defined

as any sweet tasting plant product associated with seeds [1]. Thus, occasionally there is a

conflicting classification of fruit. For instance, some fruits, in a biological sense, such as

cucumbers, eggplants, tomatoes, pumpkins, corn, peas, squash, beans, sweet peppers, and

chilies are treated as a vegetable since they are not sweet. In this thesis, the definition will be

based on a biological context.

Fig. 2.1. Anatomy of Flower [2].

4

The formation of fruits starts with pollination, in which the pollen moves from the stamen to

the stigma of the flowers. The pollen is subsequently transferred via a tube that grows from

the stigma to the ovary. Inside the ovary, the sperm of the pollen unites with the egg cell in

the ovule and the diploid zygote is formed (Fig. 2.1). The ovule develops into seeds, whereas

the ovary wall develops into the pericarp of the fruit. While the seeds are analogized as the

egg development in the ovary of a fowl, the pericarp may be like the uterus [3]. Thus, fruits

are often referred to as a ripened ovary. However, some fruits are derived from the accessory

tissues of the flowers.

What is the function of fruits in plants?

The seeds derived from the ovule contain an embryo, which germinates under favorable

conditions and becomes a new plant. The function of the fruits is twofold: to protect the seeds

during development and to disseminate the mature seeds [1]. In some multi-seeded fruits, the

extent to which the flesh develops is related to the number of fertilized ovules [1]. Since

fruits play an important role in protecting the embryo, they are equipped with a natural

defense against microbial attacks, particularly during the seed development. Most of the fruits

are covered with several protective layers known as skin, which may consist of bloom,

cuticle, epidermis, and hypodermis [4]. The bloom is a powdery deposit of wax that can be

found on the surface of fruits such as plums, grapes, and apples. The cuticle is the thin waxy

layer, which prevents water loss from the fruit and protects the fruit from mechanical injury.

Under the cuticle is the thick walled cells known as the epidermis, which gives the skin most

of its mechanical strength. Underlying the epidermis is the hypodermis, which gives the skin

added mechanical strength. Besides having thicker epidermal tissue, the fresh fruits exhibit a

stronger defense system against microbial attacks with a high concentration of an

antimicrobial organic compound [5]. During the ripening process, the function of the fruit

shifts from seed protection into seed dissemination [1]. Thus, the characters of the fruits

change during the ripening process in order to make fruits become more attractive to vectors,

which help to spread the seeds. Following the maturation, the texture of the flesh becomes

softer, the taste becomes sweeter, the color of some fruits changes into yellowish or reddish,

and pleasant flavors are accumulated.

Fruit classification

According to anatomical attributes, fruits are divided into simple fruits, aggregate fruits, and

compound fruits [3]. Simple fruits are derived from a single ovary containing one or many

5

seeds. Examples of simple fruits are red currants, avocadoes, plums, cherries, peaches,

apricots, and olives. Aggregate fruits, such as strawberries, raspberries, and blackberries, are

formed from a single compound flower, which contains many ovaries. Multiple fruits are

generated from the fused ovaries of many separate but closely clustered flowers. Fruit can

also be classified into ‘true fruits’ and ‘false fruits.’ True fruits are derived from one ovary,

whereas false fruits are derived from more than one ovary and parts of the flower other than

the ovary such as sepals, stamens, and stigma. For instance, an apple is formed from the

flower stalk surrounding the ovary wall, which fuses to the ovary wall. Based on the growing

region, fruits are classified into tropical and subtropical fruits [6].

Fruit microflora

The fruit surface harbors a diverse range of microorganisms including pathogens, non-

pathogens, and opportunist pathogens, which are normal microflora of fruits. The normal

fruit microflora consists of bacteria, yeast, and molds. Fruits are protected by the skin, which

acts as a barrier for most pathogens. The source of the microbes in the fruits comes from the

soil, air, insects such as fruit flies (Drosophila melanogaster), and farmers picking the fruits.

Hence, various microbes remain on the skin surface such as Staphylococcus, Enterobacter,

Shigella, Salmonella, E. coli 0157:H7, Bacillus cereus, Pseudomonas, Erwinia, Enterobacter,

and Lactobacillus sp. [7], Rhizopus, Aspergillus, Penicillium, Eurotium, Wallemia,

Saccharomyces, Zygosaccharomyces, Hanseniaspora, Candida, Debaryomyces, and Pichia

sp. [5]. These microbia remain adhered to the outer skin of the fruits as long as the skin does

not get damaged. Based on their invasion of the fruits, pathogens are divided into true

pathogens and opportunist pathogens. The true pathogens are able to infect the fruits by

having cellulolytic and pectinolytic enzymes break the outer covering of the fruits tissue.

Meanwhile, due to the lack of a degradative enzyme, the opportunist pathogens can only

infect the fruits when the fruits defense system weakens, such as by mechanical injury or

curticular damage. Improper or harsh handling causes damage to the fruit’s skin, increasing

the potential microbial attack [5].

Fruit composition

For nature, fruit is a developmental manifestation of the seed disseminating part in the plant,

which plays an important role in the generation of a new organism. However, for humans,

fruit is part of the human diet since it is one of the most important nutritional sources for our

health, particularly vitamins, minerals, fiber, and other health promoting phytochemicals [8].

4

The formation of fruits starts with pollination, in which the pollen moves from the stamen to

the stigma of the flowers. The pollen is subsequently transferred via a tube that grows from

the stigma to the ovary. Inside the ovary, the sperm of the pollen unites with the egg cell in

the ovule and the diploid zygote is formed (Fig. 2.1). The ovule develops into seeds, whereas

the ovary wall develops into the pericarp of the fruit. While the seeds are analogized as the

egg development in the ovary of a fowl, the pericarp may be like the uterus [3]. Thus, fruits

are often referred to as a ripened ovary. However, some fruits are derived from the accessory

tissues of the flowers.

What is the function of fruits in plants?

The seeds derived from the ovule contain an embryo, which germinates under favorable

conditions and becomes a new plant. The function of the fruits is twofold: to protect the seeds

during development and to disseminate the mature seeds [1]. In some multi-seeded fruits, the

extent to which the flesh develops is related to the number of fertilized ovules [1]. Since

fruits play an important role in protecting the embryo, they are equipped with a natural

defense against microbial attacks, particularly during the seed development. Most of the fruits

are covered with several protective layers known as skin, which may consist of bloom,

cuticle, epidermis, and hypodermis [4]. The bloom is a powdery deposit of wax that can be

found on the surface of fruits such as plums, grapes, and apples. The cuticle is the thin waxy

layer, which prevents water loss from the fruit and protects the fruit from mechanical injury.

Under the cuticle is the thick walled cells known as the epidermis, which gives the skin most

of its mechanical strength. Underlying the epidermis is the hypodermis, which gives the skin

added mechanical strength. Besides having thicker epidermal tissue, the fresh fruits exhibit a

stronger defense system against microbial attacks with a high concentration of an

antimicrobial organic compound [5]. During the ripening process, the function of the fruit

shifts from seed protection into seed dissemination [1]. Thus, the characters of the fruits

change during the ripening process in order to make fruits become more attractive to vectors,

which help to spread the seeds. Following the maturation, the texture of the flesh becomes

softer, the taste becomes sweeter, the color of some fruits changes into yellowish or reddish,

and pleasant flavors are accumulated.

Fruit classification

According to anatomical attributes, fruits are divided into simple fruits, aggregate fruits, and

compound fruits [3]. Simple fruits are derived from a single ovary containing one or many

5

seeds. Examples of simple fruits are red currants, avocadoes, plums, cherries, peaches,

apricots, and olives. Aggregate fruits, such as strawberries, raspberries, and blackberries, are

formed from a single compound flower, which contains many ovaries. Multiple fruits are

generated from the fused ovaries of many separate but closely clustered flowers. Fruit can

also be classified into ‘true fruits’ and ‘false fruits.’ True fruits are derived from one ovary,

whereas false fruits are derived from more than one ovary and parts of the flower other than

the ovary such as sepals, stamens, and stigma. For instance, an apple is formed from the

flower stalk surrounding the ovary wall, which fuses to the ovary wall. Based on the growing

region, fruits are classified into tropical and subtropical fruits [6].

Fruit microflora

The fruit surface harbors a diverse range of microorganisms including pathogens, non-

pathogens, and opportunist pathogens, which are normal microflora of fruits. The normal

fruit microflora consists of bacteria, yeast, and molds. Fruits are protected by the skin, which

acts as a barrier for most pathogens. The source of the microbes in the fruits comes from the

soil, air, insects such as fruit flies (Drosophila melanogaster), and farmers picking the fruits.

Hence, various microbes remain on the skin surface such as Staphylococcus, Enterobacter,

Shigella, Salmonella, E. coli 0157:H7, Bacillus cereus, Pseudomonas, Erwinia, Enterobacter,

and Lactobacillus sp. [7], Rhizopus, Aspergillus, Penicillium, Eurotium, Wallemia,

Saccharomyces, Zygosaccharomyces, Hanseniaspora, Candida, Debaryomyces, and Pichia

sp. [5]. These microbia remain adhered to the outer skin of the fruits as long as the skin does

not get damaged. Based on their invasion of the fruits, pathogens are divided into true

pathogens and opportunist pathogens. The true pathogens are able to infect the fruits by

having cellulolytic and pectinolytic enzymes break the outer covering of the fruits tissue.

Meanwhile, due to the lack of a degradative enzyme, the opportunist pathogens can only

infect the fruits when the fruits defense system weakens, such as by mechanical injury or

curticular damage. Improper or harsh handling causes damage to the fruit’s skin, increasing

the potential microbial attack [5].

Fruit composition

For nature, fruit is a developmental manifestation of the seed disseminating part in the plant,

which plays an important role in the generation of a new organism. However, for humans,

fruit is part of the human diet since it is one of the most important nutritional sources for our

health, particularly vitamins, minerals, fiber, and other health promoting phytochemicals [8].

6

Besides its nutritional value, a low fat content in fruits promotes its consumption [9].

Moreover, a daily consumption of fruits is reported to have a positive impact on reducing the

risk of developing cancer, heart disease, diabetes, stroke, obesity, birth defects, cataracts, and

osteoporosis, [8]. The composition of various fruits is presented in Table 2.1.

Table 2.1. Carbohydrate, protein, and fat contents of various fruits [10].

Fruits Carbohydrate (% dry weight)

Protein (% dry weight)

Fat (% dry weight)

Avocado 65.21 12.33 13.39 Grape 83.48 7.40 2.56 Apple 91.20 4.17 2.66 Star Fruit 83.17 7.84 2.40 Guava 71.24 7.64 14.10 Orange 74.53 7.68 10.80 Longan 83.26 8.83 2.54 Mango 75.89 7.77 13.50 Mangosteen 91.78 5.00 1.46 Melon 77.59 10.47 0.74 Pineapple 68.89 6.00 7.09 Papaya 70.37 8.06 10.53 Rambutan 88.16 8.54 0.58 Snack Fruit 79.74 5.83 7.08 Watermelon 69.80 20.17 0.88

2.2. Global fruit production

Due to its beneficial nutrition, fruits are produced commercially. According to the Food and

Agriculture organization , in the last 30 years, there has been a steady average increase of

2.65 % of global fruit production, and in 2012 a total amount of 804.4 million tons of fruits

was produced globally (Fig. 2.2). This is equal to fruits supply of 115 kg per person in a year

worldwide. The rise in the global fruit production accompanies an increase in the

consumption of fresh fruits, which is driven by the rising population growth, better living

standard, and successful promotion from the government health agencies as well as greater

awareness of a healthy lifestyle from the present generation.

7

Fig. 2.2. Global fruit production [11].

The top ten fruit producing countries in the world in 2012 are presented in Table 2.2. China

was the leading fruit producer in the world in 2012 with a total production of 154.6 million

tons and contributed to 23.1 % of the global production. China was followed by India, Brazil,

and the United States of America. Other top ten global fruit producers were: Turkey, Mexico,

Philippines, Spain, and Italy. For Asian countries, Indonesia was the top producer

contributing with 2.7 % of the global market.

Table 2.2. Top ten fruit producing countries in the world [11].

Countries Fruit production (million tons)

% Total world production

China (mainland) 154.6 23.1

India 72.1 10.8

Brazil 38.9 5.8

United States of America 27.5 4.1

Indonesia 17.9 2.7

Turkey 16.7 2.5

Mexico 16.5 2.5

Philippines 16.4 2.5

Spain 14.9 2.2

Italy 14.4 2.1

0

100

200

300

400

500

600

700

800

900

1982 1985 1988 1991 1994 1997 2000 2003 2006 2009 2012

Glo

bal

Pro

du

ctio

n (m

illio

n t

on

s)

Year

6

Besides its nutritional value, a low fat content in fruits promotes its consumption [9].

Moreover, a daily consumption of fruits is reported to have a positive impact on reducing the

risk of developing cancer, heart disease, diabetes, stroke, obesity, birth defects, cataracts, and

osteoporosis, [8]. The composition of various fruits is presented in Table 2.1.

Table 2.1. Carbohydrate, protein, and fat contents of various fruits [10].

Fruits Carbohydrate (% dry weight)

Protein (% dry weight)

Fat (% dry weight)

Avocado 65.21 12.33 13.39 Grape 83.48 7.40 2.56 Apple 91.20 4.17 2.66 Star Fruit 83.17 7.84 2.40 Guava 71.24 7.64 14.10 Orange 74.53 7.68 10.80 Longan 83.26 8.83 2.54 Mango 75.89 7.77 13.50 Mangosteen 91.78 5.00 1.46 Melon 77.59 10.47 0.74 Pineapple 68.89 6.00 7.09 Papaya 70.37 8.06 10.53 Rambutan 88.16 8.54 0.58 Snack Fruit 79.74 5.83 7.08 Watermelon 69.80 20.17 0.88

2.2. Global fruit production

Due to its beneficial nutrition, fruits are produced commercially. According to the Food and

Agriculture organization , in the last 30 years, there has been a steady average increase of

2.65 % of global fruit production, and in 2012 a total amount of 804.4 million tons of fruits

was produced globally (Fig. 2.2). This is equal to fruits supply of 115 kg per person in a year

worldwide. The rise in the global fruit production accompanies an increase in the

consumption of fresh fruits, which is driven by the rising population growth, better living

standard, and successful promotion from the government health agencies as well as greater

awareness of a healthy lifestyle from the present generation.

7

Fig. 2.2. Global fruit production [11].

The top ten fruit producing countries in the world in 2012 are presented in Table 2.2. China

was the leading fruit producer in the world in 2012 with a total production of 154.6 million

tons and contributed to 23.1 % of the global production. China was followed by India, Brazil,

and the United States of America. Other top ten global fruit producers were: Turkey, Mexico,

Philippines, Spain, and Italy. For Asian countries, Indonesia was the top producer

contributing with 2.7 % of the global market.

Table 2.2. Top ten fruit producing countries in the world [11].

Countries Fruit production (million tons)

% Total world production

China (mainland) 154.6 23.1

India 72.1 10.8

Brazil 38.9 5.8

United States of America 27.5 4.1

Indonesia 17.9 2.7

Turkey 16.7 2.5

Mexico 16.5 2.5

Philippines 16.4 2.5

Spain 14.9 2.2

Italy 14.4 2.1

0

100

200

300

400

500

600

700

800

900

1982 1985 1988 1991 1994 1997 2000 2003 2006 2009 2012

Glo

bal

Pro

du

ctio

n (m

illio

n t

on

s)

Year

8

There were five fruit commodities that dominated the global market in 2012, as shown in Fig.

2.3. Watermelon was the highest produced commodity with a total production of 105.4

million tons, corresponding to 13.1 % of the total fruits produced worldwide. The five top

watermelon producers in the world were: China, Turkey, Iran, Brazil, and Egypt. The second

top commodity was bananas, which were mostly produced in India, China, Philippines,

Ecuador, and Brazil. The world banana production reached 102 million tons, and it was

counted as 12.7 % of the total fruit production.

Fig. 2.3. Major fruit commodities in the global production [11].

The third, fourth, and fifth fruit commodities were apples, oranges, and grapes, which were

counted as 9.5, 8.5, and 8.3 % of the total fruits, respectively. The main apple producers were

China, United States of America, Turkey, Poland, and India. Orange production was

dominated by Brazil, United States of America, China, India, and Mexico whereas the five

top grape producers were China, United States of America, Italy, France, and Spain.

2.3. Fruits wastes

In 2012, the production of fruits reached 804.4 million tons worldwide [11], which equals to

approximately 115 kg/person. However, not all of these amounts of fruits are being

consumed, since fruit losses occur at various stages in the fruit supply chain as shown in Fig.

2.4.

Apple 9.5 %

Banana12.7 %

Grapes8.3 %

Oranges8.5 %

Watermelon13.1 %

Other fruits47.9 %

9

Fig. 2.4. Losses in various stages of supply chain of fruits [12].

The fruits wastes can be generated from various steps during the distribution chain, from the

field to the household level, including injured fruits, rotten fruits, contaminated fruits, non-

edible part of the fruits and fruits that do not meet the standard requirement. In the field,

losses occur due to mechanical damage during the product harvest such as careless picking.

Inadequate facilities, poor management, lack of knowledge, and careless handling can cause

mechanical injury, physiological deterioration, parasitic diseases resulting in fruit losses

during the post-harvest and storage stage [13]. Fruits wastes are also generated during the

fruit processing in juice production, canning, and bread baking such as washing, peeling,

slicing, and boiling or when crops are sorted, if not suitable to process. In the distribution

chain, fruit losses occur in the market system such as wholesale markets, supermarkets,

retailers, and wet market, which is dominated by unsalable fruits. In the final stage of the

distribution chain, the fruit losses occur during consumption at the household level.

Harvesting

Threshing

Drying, transport, and distribution

Storage

Processing

Product evaluation and quality control

Packaging

Marketing, selling, distribution

Post-consumer

End of life disposal of food waste

Edible crops left in the field, ploughed into soil, eaten by pests, timing of harvest not optimal, crop damaged during harvesting

Loss due to poor technique

Quality and quantity loss during drying, poor transport infrastructure, loss owning to spotting/bruising

Pests and disease attacks, spoilage, contamination, natural drying out of food

Process losses, contamination in process causing loss if quality

Product disregarded/out-grade in supply chain

Inappropriate packaging damage produces; grain spillage from sacks, attack by pests

Damage during transport: spoilage, poor handling, losses caused by poor storage

Poor storage/stock management; discarded before serving, poor food preparation, expiration

Food waste discarded may be separately treated, fed to animals, mixed with other wastes/landfilled

8

There were five fruit commodities that dominated the global market in 2012, as shown in Fig.

2.3. Watermelon was the highest produced commodity with a total production of 105.4

million tons, corresponding to 13.1 % of the total fruits produced worldwide. The five top

watermelon producers in the world were: China, Turkey, Iran, Brazil, and Egypt. The second

top commodity was bananas, which were mostly produced in India, China, Philippines,

Ecuador, and Brazil. The world banana production reached 102 million tons, and it was

counted as 12.7 % of the total fruit production.

Fig. 2.3. Major fruit commodities in the global production [11].

The third, fourth, and fifth fruit commodities were apples, oranges, and grapes, which were

counted as 9.5, 8.5, and 8.3 % of the total fruits, respectively. The main apple producers were

China, United States of America, Turkey, Poland, and India. Orange production was

dominated by Brazil, United States of America, China, India, and Mexico whereas the five

top grape producers were China, United States of America, Italy, France, and Spain.

2.3. Fruits wastes

In 2012, the production of fruits reached 804.4 million tons worldwide [11], which equals to

approximately 115 kg/person. However, not all of these amounts of fruits are being

consumed, since fruit losses occur at various stages in the fruit supply chain as shown in Fig.

2.4.

Apple 9.5 %

Banana12.7 %

Grapes8.3 %

Oranges8.5 %

Watermelon13.1 %

Other fruits47.9 %

9

Fig. 2.4. Losses in various stages of supply chain of fruits [12].

The fruits wastes can be generated from various steps during the distribution chain, from the

field to the household level, including injured fruits, rotten fruits, contaminated fruits, non-

edible part of the fruits and fruits that do not meet the standard requirement. In the field,

losses occur due to mechanical damage during the product harvest such as careless picking.

Inadequate facilities, poor management, lack of knowledge, and careless handling can cause

mechanical injury, physiological deterioration, parasitic diseases resulting in fruit losses

during the post-harvest and storage stage [13]. Fruits wastes are also generated during the

fruit processing in juice production, canning, and bread baking such as washing, peeling,

slicing, and boiling or when crops are sorted, if not suitable to process. In the distribution

chain, fruit losses occur in the market system such as wholesale markets, supermarkets,

retailers, and wet market, which is dominated by unsalable fruits. In the final stage of the

distribution chain, the fruit losses occur during consumption at the household level.

Harvesting

Threshing

Drying, transport, and distribution

Storage

Processing

Product evaluation and quality control

Packaging

Marketing, selling, distribution

Post-consumer

End of life disposal of food waste

Edible crops left in the field, ploughed into soil, eaten by pests, timing of harvest not optimal, crop damaged during harvesting

Loss due to poor technique

Quality and quantity loss during drying, poor transport infrastructure, loss owning to spotting/bruising

Pests and disease attacks, spoilage, contamination, natural drying out of food

Process losses, contamination in process causing loss if quality

Product disregarded/out-grade in supply chain

Inappropriate packaging damage produces; grain spillage from sacks, attack by pests

Damage during transport: spoilage, poor handling, losses caused by poor storage

Poor storage/stock management; discarded before serving, poor food preparation, expiration

Food waste discarded may be separately treated, fed to animals, mixed with other wastes/landfilled

10

Fig. 2.5. Global fruits and vegetable losses at different stages and regions during the supply chain [14].

It has been reported that during the fruit supply chain, approximately 36–56 % of the fruits

ends up as waste worldwide (Fig. 2.5). In industrialized countries, fruit loss occurs mostly

during the post-harvest and consumption stage due to the quality standards set by retailers

and by consumers who discard purchased fruits. In developing countries, fruit loss occurs

mainly in post-harvest and distribution due to the deterioration of perishable crops in warm

and humid climates [14].

During the fruit processing, two types of waste are generated including solid wastes (e.g.,

peel, seed, and stones) and liquid (e.g., juice and wastewater). The liquid waste/ effluent

contains high organic loads, cleansing and blanching agent, salt, and suspended solids such as

fiber and soil particles and might also contain pesticide residue. The solid and liquid waste of

various fruits is presented in Table 2.3.

0

10

20

30

40

50

60

Per

cent

age

of L

osse

s (%

)Agriculture postharvest Processing Distribution ConsumptionPost-harvest

11

Table 2.3. Target loads per unit of production in the fruit processing industry [15].

Fruits Waste volume (m3/ U)

BOD (kg/U) Solid waste (kg/t product)

Apricots 29 15.0 Apples 90

All products 3.7 5.0 All except juice 5.4 6.4 Juice 2.9 2.0

Cranberries 5.8 2.8 10 Citrus 10.0 3.2 Sweet cherries 7.8 9.6 Sour cherries 12.0 17.0 Bing cherries 20.0 22.0 Dried Fruits 13.0 12.0 Grapefruits

Canned 72.0 11.0 Pressed 1.6 1.9

Olives 38.0 44.0 20 Peaches 180

Canned 13.0 14.0 Frozen 5.4 12.0 200

Pears 12.0 21.0 Pineapples 13.0 10.0 Plums 5.0 4.1 Raisins 2.8 6.0 Strawberries 13.0 5.3 60

10

Fig. 2.5. Global fruits and vegetable losses at different stages and regions during the supply chain [14].

It has been reported that during the fruit supply chain, approximately 36–56 % of the fruits

ends up as waste worldwide (Fig. 2.5). In industrialized countries, fruit loss occurs mostly

during the post-harvest and consumption stage due to the quality standards set by retailers

and by consumers who discard purchased fruits. In developing countries, fruit loss occurs

mainly in post-harvest and distribution due to the deterioration of perishable crops in warm

and humid climates [14].

During the fruit processing, two types of waste are generated including solid wastes (e.g.,

peel, seed, and stones) and liquid (e.g., juice and wastewater). The liquid waste/ effluent

contains high organic loads, cleansing and blanching agent, salt, and suspended solids such as

fiber and soil particles and might also contain pesticide residue. The solid and liquid waste of

various fruits is presented in Table 2.3.

0

10

20

30

40

50

60

Per

cent

age

of L

osse

s (%

)

Agriculture postharvest Processing Distribution ConsumptionPost-harvest

11

Table 2.3. Target loads per unit of production in the fruit processing industry [15].

Fruits Waste volume (m3/ U)

BOD (kg/U) Solid waste (kg/t product)

Apricots 29 15.0 Apples 90

All products 3.7 5.0 All except juice 5.4 6.4 Juice 2.9 2.0

Cranberries 5.8 2.8 10 Citrus 10.0 3.2 Sweet cherries 7.8 9.6 Sour cherries 12.0 17.0 Bing cherries 20.0 22.0 Dried Fruits 13.0 12.0 Grapefruits

Canned 72.0 11.0 Pressed 1.6 1.9

Olives 38.0 44.0 20 Peaches 180

Canned 13.0 14.0 Frozen 5.4 12.0 200

Pears 12.0 21.0 Pineapples 13.0 10.0 Plums 5.0 4.1 Raisins 2.8 6.0 Strawberries 13.0 5.3 60

12

13

CHAPTER 3

Fruit Flavors

3.1. Overview of fruit flavors

Fruit flavors are one of the most important quality attributes in fruits, which consist of sugar,

acids, salts, and aroma volatiles. Although only present at 0.1–100 ppm of the fresh fruit

weight, the aroma volatile compounds can be detected by human olfaction [3]. The fruit

flavor is formed via many different pathways, mainly classified into volatile compounds

formed from the fatty acids, amino acids, glucosinolates, terpenoid, phenol, and related

compounds [16]. There are several factors that influence the volatile compound formation

such as species, variety, climate, production, as well as pre- and post-harvest handling [9].

The volatile compounds are produced during the secondary metabolism. Once sugar is

produced in the primary metabolism, it is stored as a starch or converted into sucrose and also

flavor compounds. The flavor compound acts as an intermediate metabolite, which becomes a

precursor for other secondary metabolites. Most of the fruits produce the flavor during the

ripening process. Therefore, flavor is a signal of ripeness and acts as an attractant for seed-

dispersing organisms. This is supported by the fact that some substances are specifically

formed when fruits ripen, but are absent in the vegetative tissues and non-ripe fruits.

Fruit flavors are classified either by their chemical structure or biogenesis. According to the

chemical structure, fruit flavor is divided into several groups including alcohol, aldehyde,

ketone, lactone, terpenoid, and ester [6] (Table 3.1). Meanwhile, based on their biogenesis,

fruit flavors are classified into flavor compounds derived from fatty acid, amino acids,

glucosinolates, terpenoid, phenol, and related compounds [16]. The different fruit flavors in

various fruits are presented in Table 3.2.

12

13

CHAPTER 3

Fruit Flavors

3.1. Overview of fruit flavors

Fruit flavors are one of the most important quality attributes in fruits, which consist of sugar,

acids, salts, and aroma volatiles. Although only present at 0.1–100 ppm of the fresh fruit

weight, the aroma volatile compounds can be detected by human olfaction [3]. The fruit

flavor is formed via many different pathways, mainly classified into volatile compounds

formed from the fatty acids, amino acids, glucosinolates, terpenoid, phenol, and related

compounds [16]. There are several factors that influence the volatile compound formation

such as species, variety, climate, production, as well as pre- and post-harvest handling [9].

The volatile compounds are produced during the secondary metabolism. Once sugar is

produced in the primary metabolism, it is stored as a starch or converted into sucrose and also

flavor compounds. The flavor compound acts as an intermediate metabolite, which becomes a

precursor for other secondary metabolites. Most of the fruits produce the flavor during the

ripening process. Therefore, flavor is a signal of ripeness and acts as an attractant for seed-

dispersing organisms. This is supported by the fact that some substances are specifically

formed when fruits ripen, but are absent in the vegetative tissues and non-ripe fruits.

Fruit flavors are classified either by their chemical structure or biogenesis. According to the

chemical structure, fruit flavor is divided into several groups including alcohol, aldehyde,

ketone, lactone, terpenoid, and ester [6] (Table 3.1). Meanwhile, based on their biogenesis,

fruit flavors are classified into flavor compounds derived from fatty acid, amino acids,

glucosinolates, terpenoid, phenol, and related compounds [16]. The different fruit flavors in

various fruits are presented in Table 3.2.

14

Table 3.1. Various groups of fruit flavors [9].

Esters Alcohols Ketones Lactones Terpenoids Aldehydes

Butyl acetate Butyl hexanoate Butyl-2-methyl Butanoate Propanoate Ethyl acetate Ethyl butanoate Ethyl-9-deceonoate Ethyl hexanoate Ethyl 2-methylbutanoate Ethyl 3-methylbutanoate Ethyl 2-methylpropanoate Ethyl nonanoate (E)-2-hexenyl acetate Hexyl acetate Hexyl butanoate Hexyl propanoate Hexyl-2- methyl butanoate Methyl acetate Methyl cinnamate Methyl butanoate 2-Methylbutyl acetate 3-Methylbutyl acetate 2-Methylpropyl acetate (Z)-6-nonenyl acetate (Z, Z)-nonadenyl acetate Pentyl acetate Benzyl acetate Propyl acetate Propyl-2-methyl butanoate

Benzyl alcohol Butan-1 ol (E)- cynnamyl alcohol 1-hexanol (E)-2- hexenol (Z)-3- hexenol 1-Octanol (Z)-6-nonenol (Z,Z)-3.6-nonadienol 1-Phenylethanol 2-Phenylethanol

2,3-Butanedione Eugenol β-Damsenone eucalyptol 2-Heptanone 4-(p-Hydroxyphenyl)-2-butanone 3-hydroxy-2-butanone β-Ionone Linalool 6-Methyl-5-heptene-2-one Nerolidol 1-Octen-3-one 2-Pentanone (Z)-1,5-octadien-3-one 2-penranone (Z)-1,5-octadien-3-one Terpenes

γ-Butyrolactone γ- Decalactone δ-Decalactone γ- Dodecalactone δ- Dodecalactone γ- Jasmolactone γ- Octalactone δ- Octalactone

β-caryophyllene 1,8-cineole Citral β-Damsenone Dihydroedulan Farnesyl acetate Geraniol Hotrienol α-Ionone β -Ionone Limonene Linalool Myrtenol Nerol α-Phellandrene α–Pinene β - Pinene Terpinen-4-ol α-Terpineol Terpinolene α- Farensense

(E,E)-2.4-decadienal (E)-2-hexenal Benzaldehyde (E)-cinnamaldehyde (E)-2-nonenal

Esters are commonly used as flavoring agents contributing to “fruity” aromas and are present

in fairly low concentrations, mostly between 1 and 100 ppm [17]. Esters are widely employed

in diverse fruit-flavored products (i.e., beverages, candies, jellies, and jams), baked goods,

wines, and dairy products (i.e., cultured butter, sour cream, yogurt, and cheese). Acetate

esters, such as ethyl acetate, hexyl acetate, isoamyl acetate, and 2-phenylethyl acetate are

considered as important flavor compounds in wine and other grape-derived alcoholic

beverages. Two pathways have been proposed in ester formation: (1) the alcoholysis of acyl -

CoA compounds and (2) the direct esterification of an organic acid with an alcohol [3].

15

Table 3.2. Fruit flavors in various fruits.

Group Compound Fruits References

Aldehydes Hexanal Apple, grape, Australian mango, orange, strawberry, plum

[18], [19], [20-22]

E-2-hexenal Apple, plum, Australian mango, strawberry, cashew apple, peach

[18, 19, 21-24]

Nonanal Cashew apple, orange, strawberry, plum, peach [20-24]

Acetaldehyde Apple, strawberry [18, 25] Benzaldehyde Strawberry, peach [24, 25] Octanal Strawberry [25]

Alcohols Octanol Plum, orange, strawberry, grape [20-22] Hexanol Apple, strawberry, peach, grape [18, 24, 25] Butanol Apple, strawberry [18, 25] Benzyl alcohol Peach [24]

Terpenoid α-pinene African atemoya, Tommy Atkins and Keitt mango, Cuban

atemoya, Venezuelan mango, plum, orange, Brazilian mango, strawberry

[19-21, 26-29]

Car-3-ene Cashew apple, Venezuelan mango, orange, Australian mango, Tommy Atkins and Keitt mango, Brazilian mango

[19, 20, 23, 27, 30]

Myrcene Orange, mango, Tommy Atkins and Keitt mango, Australian mango

[19, 20, 30, 31]

Esters Ethyl butanoate Apple, strawberry, pear [18, 25] Hexyl acetate Strawberry, peach, grape [24, 25] Ethyl acetate Strawberry, grape, papaya [25] [32] Ethyl hexanoate Strawberry, grape [25]

Ketone DMMF Strawberry, Pineapple, Alphonso mango, tomato, yellow

passion fruit, blackberry [9, 33-37]

DMMF Strawberry, mango, blackberry var. marion [37-39]

Polyphenol Quercetin Cashew apple bog, whortleberry, lingonberry, cranberry, choke berry, sweet rowan, rowanberry, sea buckthorn berry, crowberry, strawberry, apple, red currant, apricot, pear, sweet cherry, plum, black grape, white grape, peach, highblush blueberry, Thornless blackberry

[40-43]

Epicatechin Grape berries, blackberry, strawberry, blueberry, grape, black, grape, white, apple, pear, peach, apricot, plum, cherry, sweet, raspberry, commercial apple, pomache, pear

[44-46]

Lactone γ-Decalactone Plum, pineapple, blackberries, mango, apricot, peach, [37, 47-49] γ-Hexalactone Papaya, pineapple, apricot, blackberries, plum [9, 37, 49, 50]

Alcohols play a modest and often indirect role in fruit flavors. They are essential to flavor

quality in wine and distilled beverages. They also play an indirect role as precursors for the

preparation of other flavors such as in aldehyde formation [51]. Aldehyde contains carbonyl

14

Table 3.1. Various groups of fruit flavors [9].

Esters Alcohols Ketones Lactones Terpenoids Aldehydes

Butyl acetate Butyl hexanoate Butyl-2-methyl Butanoate Propanoate Ethyl acetate Ethyl butanoate Ethyl-9-deceonoate Ethyl hexanoate Ethyl 2-methylbutanoate Ethyl 3-methylbutanoate Ethyl 2-methylpropanoate Ethyl nonanoate (E)-2-hexenyl acetate Hexyl acetate Hexyl butanoate Hexyl propanoate Hexyl-2- methyl butanoate Methyl acetate Methyl cinnamate Methyl butanoate 2-Methylbutyl acetate 3-Methylbutyl acetate 2-Methylpropyl acetate (Z)-6-nonenyl acetate (Z, Z)-nonadenyl acetate Pentyl acetate Benzyl acetate Propyl acetate Propyl-2-methyl butanoate

Benzyl alcohol Butan-1 ol (E)- cynnamyl alcohol 1-hexanol (E)-2- hexenol (Z)-3- hexenol 1-Octanol (Z)-6-nonenol (Z,Z)-3.6-nonadienol 1-Phenylethanol 2-Phenylethanol

2,3-Butanedione Eugenol β-Damsenone eucalyptol 2-Heptanone 4-(p-Hydroxyphenyl)-2-butanone 3-hydroxy-2-butanone β-Ionone Linalool 6-Methyl-5-heptene-2-one Nerolidol 1-Octen-3-one 2-Pentanone (Z)-1,5-octadien-3-one 2-penranone (Z)-1,5-octadien-3-one Terpenes

γ-Butyrolactone γ- Decalactone δ-Decalactone γ- Dodecalactone δ- Dodecalactone γ- Jasmolactone γ- Octalactone δ- Octalactone

β-caryophyllene 1,8-cineole Citral β-Damsenone Dihydroedulan Farnesyl acetate Geraniol Hotrienol α-Ionone β -Ionone Limonene Linalool Myrtenol Nerol α-Phellandrene α–Pinene β - Pinene Terpinen-4-ol α-Terpineol Terpinolene α- Farensense

(E,E)-2.4-decadienal (E)-2-hexenal Benzaldehyde (E)-cinnamaldehyde (E)-2-nonenal

Esters are commonly used as flavoring agents contributing to “fruity” aromas and are present

in fairly low concentrations, mostly between 1 and 100 ppm [17]. Esters are widely employed

in diverse fruit-flavored products (i.e., beverages, candies, jellies, and jams), baked goods,

wines, and dairy products (i.e., cultured butter, sour cream, yogurt, and cheese). Acetate

esters, such as ethyl acetate, hexyl acetate, isoamyl acetate, and 2-phenylethyl acetate are

considered as important flavor compounds in wine and other grape-derived alcoholic

beverages. Two pathways have been proposed in ester formation: (1) the alcoholysis of acyl -

CoA compounds and (2) the direct esterification of an organic acid with an alcohol [3].

15

Table 3.2. Fruit flavors in various fruits.

Group Compound Fruits References

Aldehydes Hexanal Apple, grape, Australian mango, orange, strawberry, plum

[18], [19], [20-22]

E-2-hexenal Apple, plum, Australian mango, strawberry, cashew apple, peach

[18, 19, 21-24]

Nonanal Cashew apple, orange, strawberry, plum, peach [20-24]

Acetaldehyde Apple, strawberry [18, 25] Benzaldehyde Strawberry, peach [24, 25] Octanal Strawberry [25]

Alcohols Octanol Plum, orange, strawberry, grape [20-22] Hexanol Apple, strawberry, peach, grape [18, 24, 25] Butanol Apple, strawberry [18, 25] Benzyl alcohol Peach [24]

Terpenoid α-pinene African atemoya, Tommy Atkins and Keitt mango, Cuban

atemoya, Venezuelan mango, plum, orange, Brazilian mango, strawberry

[19-21, 26-29]

Car-3-ene Cashew apple, Venezuelan mango, orange, Australian mango, Tommy Atkins and Keitt mango, Brazilian mango

[19, 20, 23, 27, 30]

Myrcene Orange, mango, Tommy Atkins and Keitt mango, Australian mango

[19, 20, 30, 31]

Esters Ethyl butanoate Apple, strawberry, pear [18, 25] Hexyl acetate Strawberry, peach, grape [24, 25] Ethyl acetate Strawberry, grape, papaya [25] [32] Ethyl hexanoate Strawberry, grape [25]

Ketone DMMF Strawberry, Pineapple, Alphonso mango, tomato, yellow

passion fruit, blackberry [9, 33-37]

DMMF Strawberry, mango, blackberry var. marion [37-39]

Polyphenol Quercetin Cashew apple bog, whortleberry, lingonberry, cranberry, choke berry, sweet rowan, rowanberry, sea buckthorn berry, crowberry, strawberry, apple, red currant, apricot, pear, sweet cherry, plum, black grape, white grape, peach, highblush blueberry, Thornless blackberry

[40-43]

Epicatechin Grape berries, blackberry, strawberry, blueberry, grape, black, grape, white, apple, pear, peach, apricot, plum, cherry, sweet, raspberry, commercial apple, pomache, pear

[44-46]

Lactone γ-Decalactone Plum, pineapple, blackberries, mango, apricot, peach, [37, 47-49] γ-Hexalactone Papaya, pineapple, apricot, blackberries, plum [9, 37, 49, 50]

Alcohols play a modest and often indirect role in fruit flavors. They are essential to flavor

quality in wine and distilled beverages. They also play an indirect role as precursors for the

preparation of other flavors such as in aldehyde formation [51]. Aldehyde contains carbonyl

16

group (C=O), which contributes to a unique character of flavor. Generally, the C2- C7

aldehydes are volatile and characterized by unpleasant irritating odors, on the contrary the C8-

C13 aldehydes are characterized as floral and attractive aromas [51]. One of the most well-

known compounds is benzaldehyde, which is the second most important flavor molecule in

quantity after vanillin [52].

Ketones contribute primarily to cheese flavors, particular in mold-ripened cheeses [51]. One

important example of ketone is furaneol, which was first identified as a natural aroma

component in pineapple [53]. Due to its low odor threshold and characteristic flavor, furaneol

is one of the most important aroma compounds in strawberries [54].

Lactones are cyclic esters of primarily γ- and δ-hydroxy acids. It is ubiquitously found in

food and contributes to coconut-like, buttery, creamy, sweet, or nutty taste, and flavor.

Lactones are generally synthesized by successive β-oxidations of saturated and unsaturated

hydroxyl acids or their lipid precursors to form a hydroxylated carbon at the C4 or C5 position.

These C4 or C5 hydroxyl groups are then internally esterified (lactonized) with a carboxylic

acid group on the same molecule to form γ- and δ- lactones, respectively [51].

Terpenes are responsible for the characteristic odors of essential oils [17]. These compounds

are made of 5-carbon isoprene units (2-methyl–1,3-butadiene) as their skeletal building

block. The isoprene unit is linked together in groups of two (monoterpenes), three

(sesquiterpenes), four (diterpenes), six (triterpenes), nine (tetraterpenes), or more

(polyterpenes). The structures of these compounds can be open or closed chain, cyclic,

saturated, or unsaturated [51].

3.2. Biosynthesis of fruit flavors

A flavor compound is naturally synthesized in fruits, but it also can be manufactured in the

flavor industry by employing a number of microorganisms such as bacteria, fungi, and yeast.

Flavor compounds have a widespread application in food, feed, cosmetic, chemical, and

pharmaceutical industry. The worldwide flavor production continually increases. According

to the World Flavor and Fragrance Freedonia group, the global demand for flavors and

fragrances has increased 4.4 % per year to US $18.6 billion in 2008, and it is predicted to

reach 21.6 billion in 2016 [55]. The largest markets for flavors and fragrances are in Europe

(36 %), North America (32 %), followed by Asian Pacific (26 %), in which eight major

17

global companies share 60 % of the world market [56]. Among the markets for flavors, the

food and beverage industry is the largest which contributes to 47 % of the total demand in

2003 [57].

There are two types of biotechnological approaches for flavor production, namely, using

microorganisms or enzymes [51]. Based on the process, the flavor production can be divided

into two major groups: (1) De novo synthesis in the course of microbial fermentation, by the

use of metabolizing cells and (2) bioconversion of suitable precursors either by

microorganisms or by enzymes [51, 58]. However, these biotechnological flavor processes

are not the focus here, since this chapter will only present the biosynthesis of various flavor

compounds in plants/ fruits. As mentioned in the previous section, fruit flavors can be derived

from fatty acids, amino acids, glucosinolates, terpenoid, phenol, and related compounds.

Flavor compounds derived from fatty acids

Flavor compounds derived from fatty acids can be produced via several pathways, including

α- and β-oxidation, lipoxygenase pathway, and autoxidation. The intermediates and products

from these pathways can be metabolized to synthesize alcohol, aldehydes, carboxylic acids,

lactones, esters, and ketones. Two fatty acids, linoleic, and linolenic acids, are major

precursors for flavor production from fatty acids.

There are several enzymes that are involved in the flavor synthesis from linolenic acid such

as alcohol acyltransferase (AAT), alcohol dehydrogenase (ADH), allene oxide synthase

(AOS), divinil ether synthase (DES), hydroperoxide lyase (HPL), jasmonate

methyltransferase (JMT), and lipoxygenase (LOX) (Fig. 3.1). The unsaturated linolenic acid

is first converted into a saturated hydroperoxy fatty acid by adding oxygen (oxygenation),

with the help of lipoxygenase. The hydroperoxy fatty acid is metabolized into aldehydes and

further converted into alcohol, by the alcohol dehydrogenase. The flavors derived from the

linolenic acid are: (Z)-3-hexanal, n-hexanal, (Z)-3-hexen-1-ol, (Z)-3-hexen-1-yl acetate, 3

(Z)-nonenal, 2-(E)- nonenal, 3-(Z), 6-(Z)-nonadienal, and 2-(E), 6-(Z)-nonadienal.

16

group (C=O), which contributes to a unique character of flavor. Generally, the C2- C7

aldehydes are volatile and characterized by unpleasant irritating odors, on the contrary the C8-

C13 aldehydes are characterized as floral and attractive aromas [51]. One of the most well-

known compounds is benzaldehyde, which is the second most important flavor molecule in

quantity after vanillin [52].

Ketones contribute primarily to cheese flavors, particular in mold-ripened cheeses [51]. One

important example of ketone is furaneol, which was first identified as a natural aroma

component in pineapple [53]. Due to its low odor threshold and characteristic flavor, furaneol

is one of the most important aroma compounds in strawberries [54].

Lactones are cyclic esters of primarily γ- and δ-hydroxy acids. It is ubiquitously found in

food and contributes to coconut-like, buttery, creamy, sweet, or nutty taste, and flavor.

Lactones are generally synthesized by successive β-oxidations of saturated and unsaturated

hydroxyl acids or their lipid precursors to form a hydroxylated carbon at the C4 or C5 position.

These C4 or C5 hydroxyl groups are then internally esterified (lactonized) with a carboxylic

acid group on the same molecule to form γ- and δ- lactones, respectively [51].

Terpenes are responsible for the characteristic odors of essential oils [17]. These compounds

are made of 5-carbon isoprene units (2-methyl–1,3-butadiene) as their skeletal building

block. The isoprene unit is linked together in groups of two (monoterpenes), three

(sesquiterpenes), four (diterpenes), six (triterpenes), nine (tetraterpenes), or more

(polyterpenes). The structures of these compounds can be open or closed chain, cyclic,

saturated, or unsaturated [51].

3.2. Biosynthesis of fruit flavors

A flavor compound is naturally synthesized in fruits, but it also can be manufactured in the

flavor industry by employing a number of microorganisms such as bacteria, fungi, and yeast.

Flavor compounds have a widespread application in food, feed, cosmetic, chemical, and

pharmaceutical industry. The worldwide flavor production continually increases. According

to the World Flavor and Fragrance Freedonia group, the global demand for flavors and

fragrances has increased 4.4 % per year to US $18.6 billion in 2008, and it is predicted to

reach 21.6 billion in 2016 [55]. The largest markets for flavors and fragrances are in Europe

(36 %), North America (32 %), followed by Asian Pacific (26 %), in which eight major

17

global companies share 60 % of the world market [56]. Among the markets for flavors, the

food and beverage industry is the largest which contributes to 47 % of the total demand in

2003 [57].

There are two types of biotechnological approaches for flavor production, namely, using

microorganisms or enzymes [51]. Based on the process, the flavor production can be divided

into two major groups: (1) De novo synthesis in the course of microbial fermentation, by the

use of metabolizing cells and (2) bioconversion of suitable precursors either by

microorganisms or by enzymes [51, 58]. However, these biotechnological flavor processes

are not the focus here, since this chapter will only present the biosynthesis of various flavor

compounds in plants/ fruits. As mentioned in the previous section, fruit flavors can be derived

from fatty acids, amino acids, glucosinolates, terpenoid, phenol, and related compounds.

Flavor compounds derived from fatty acids

Flavor compounds derived from fatty acids can be produced via several pathways, including

α- and β-oxidation, lipoxygenase pathway, and autoxidation. The intermediates and products

from these pathways can be metabolized to synthesize alcohol, aldehydes, carboxylic acids,

lactones, esters, and ketones. Two fatty acids, linoleic, and linolenic acids, are major

precursors for flavor production from fatty acids.

There are several enzymes that are involved in the flavor synthesis from linolenic acid such

as alcohol acyltransferase (AAT), alcohol dehydrogenase (ADH), allene oxide synthase

(AOS), divinil ether synthase (DES), hydroperoxide lyase (HPL), jasmonate

methyltransferase (JMT), and lipoxygenase (LOX) (Fig. 3.1). The unsaturated linolenic acid

is first converted into a saturated hydroperoxy fatty acid by adding oxygen (oxygenation),

with the help of lipoxygenase. The hydroperoxy fatty acid is metabolized into aldehydes and

further converted into alcohol, by the alcohol dehydrogenase. The flavors derived from the

linolenic acid are: (Z)-3-hexanal, n-hexanal, (Z)-3-hexen-1-ol, (Z)-3-hexen-1-yl acetate, 3

(Z)-nonenal, 2-(E)- nonenal, 3-(Z), 6-(Z)-nonadienal, and 2-(E), 6-(Z)-nonadienal.

18

OHOLinolenic acid

OHO

OOH

9-Hydroperoxide

OHC

OHO

OHC

9-oxo-Nonanonic acid

Nonadienal

HOH2C

Nonadienol

O O

OH

O

HO O

Colnelenic acid

10-OPDA

DES

AOS

9-LOX

HPL

ADH

OHOLinolenic acid

OOH

13-Hydroperoxide

12-oxo-Dodecenoic acid

13-LOX

HPL

ADH

DES

AOSOHO

OHC O

OH

CHO

3-Hexanal

CH2OH

3-Hexanol

AAT

O

O

2-Hexenyl acetate

O

OH

O

Etherolenic acid

O

OHO

Jasmonic acid

JMT

O

CH3

OH3CO

Methyl jasmonate

Fig. 3.1. Biosynthesis of flavor compounds from fatty acids by 9- lipoxygenase (a) and 13- lipoxygenase (b) pathway. AAT, alcohol acyltransferase; ADH, alcohol dehydrogenase; AOS, allene oxide synthase; DES, divinil ether synthase; HPL, hydroperoxide lyase; JMT, jasmonate methyltransferase; LOX, lipoxygenase; 10 - OPDA, 12.oxo – phytodienoic [9].

Besides the lypogenesis pathway, the flavors derived from fatty acids are also generated from

α- and β-oxidation pathways [59]. The flavor compounds derived from these pathways are

a)

b)

19

lactones, alkanolides, aldehyde, and esters. The lactones are important flavors in some fruits

such as pineapples, apricots, and strawberries [60].

Flavor compounds derived from terpenoids

Terpenoids are the largest group of flavor compounds, consisting of more than 40,000

different molecules. Terpenoids are biosynthesized from acetyl-coA via the mevalonate

pathway (MVA). Alternatively, terpenoids can also be produced from pyruvate and

glyceraldehyde 3-phosphate via the methylerythritol-4-phosphate (MEP) pathway [61]. The

terpenoid synthesis can be summarized in three steps as presented in Fig. 3.2. The first step is

the formation of the terpenoid, building C5 isopentenyl diphosphate (IPP) and dimethylallyl

diphosphate (DMAPP). The second step is the sequential head to tail addition of the IPP units

to the DMAPP to form prenyl diphosphates, followed by the conversion of the resulting

prenyl diphosphate to the end products. The end products of the MVA and MEP pathways are

monoterpene, diterpene, and sesquiterpene. One of the most well-known of the monoterpenes

is R-limonene, which is an important flavor in citrus fruits and constitutes over 90 % of the

essential oils in citrus fruits. Monoterpene S-linalool also plays an important role in the

strawberry and tomato flavor. For sesquiterpene and valence, α- and β- sinenesal are

necessary for the orange flavor. Structures of some of the terpenoid compounds are presented

in Fig. 3.3

OPP OPP

IPP (C5) DMAPP (C5)

+DMAPP

OPP

GDP (C10)

+IPP

OPP

+IPP

FDP (C15)

OPP

GGPP (C20)

MEP pathway

Monoterpenes(C10)

Diterpenes(C20)

Sesquiterpene(C15)

Plastid

Fig. 3.2. Biosynthesis of terpenoid flavors. IPP, isopentenyl diphosphate; DMAPP,

dimethylallyl diphosphate; GDP, geranyl diphosphate; FDP, farnesyl diphosphate; GGPP,

geranylgeranyl diphosphate [9].

18

OHOLinolenic acid

OHO

OOH

9-Hydroperoxide

OHC

OHO

OHC

9-oxo-Nonanonic acid

Nonadienal

HOH2C

Nonadienol

O O

OH

O

HO O

Colnelenic acid

10-OPDA

DES

AOS

9-LOX

HPL

ADH

OHOLinolenic acid

OOH

13-Hydroperoxide

12-oxo-Dodecenoic acid

13-LOX

HPL

ADH

DES

AOSOHO

OHC O

OH

CHO

3-Hexanal

CH2OH

3-Hexanol

AAT

O

O

2-Hexenyl acetate

O

OH

O

Etherolenic acid

O

OHO

Jasmonic acid

JMT

O

CH3

OH3CO

Methyl jasmonate

Fig. 3.1. Biosynthesis of flavor compounds from fatty acids by 9- lipoxygenase (a) and 13- lipoxygenase (b) pathway. AAT, alcohol acyltransferase; ADH, alcohol dehydrogenase; AOS, allene oxide synthase; DES, divinil ether synthase; HPL, hydroperoxide lyase; JMT, jasmonate methyltransferase; LOX, lipoxygenase; 10 - OPDA, 12.oxo – phytodienoic [9].

Besides the lypogenesis pathway, the flavors derived from fatty acids are also generated from

α- and β-oxidation pathways [59]. The flavor compounds derived from these pathways are

a)

b)

19

lactones, alkanolides, aldehyde, and esters. The lactones are important flavors in some fruits

such as pineapples, apricots, and strawberries [60].

Flavor compounds derived from terpenoids

Terpenoids are the largest group of flavor compounds, consisting of more than 40,000

different molecules. Terpenoids are biosynthesized from acetyl-coA via the mevalonate

pathway (MVA). Alternatively, terpenoids can also be produced from pyruvate and

glyceraldehyde 3-phosphate via the methylerythritol-4-phosphate (MEP) pathway [61]. The

terpenoid synthesis can be summarized in three steps as presented in Fig. 3.2. The first step is

the formation of the terpenoid, building C5 isopentenyl diphosphate (IPP) and dimethylallyl

diphosphate (DMAPP). The second step is the sequential head to tail addition of the IPP units

to the DMAPP to form prenyl diphosphates, followed by the conversion of the resulting

prenyl diphosphate to the end products. The end products of the MVA and MEP pathways are

monoterpene, diterpene, and sesquiterpene. One of the most well-known of the monoterpenes

is R-limonene, which is an important flavor in citrus fruits and constitutes over 90 % of the

essential oils in citrus fruits. Monoterpene S-linalool also plays an important role in the

strawberry and tomato flavor. For sesquiterpene and valence, α- and β- sinenesal are

necessary for the orange flavor. Structures of some of the terpenoid compounds are presented

in Fig. 3.3

OPP OPP

IPP (C5) DMAPP (C5)

+DMAPP

OPP

GDP (C10)

+IPP

OPP

+IPP

FDP (C15)

OPP

GGPP (C20)

MEP pathway

Monoterpenes(C10)

Diterpenes(C20)

Sesquiterpene(C15)

Plastid

Fig. 3.2. Biosynthesis of terpenoid flavors. IPP, isopentenyl diphosphate; DMAPP,

dimethylallyl diphosphate; GDP, geranyl diphosphate; FDP, farnesyl diphosphate; GGPP,

geranylgeranyl diphosphate [9].

20

OH

S-linalool R-limonene Valencene

O O

Fig. 3.3. Some important flavors derived from terpenoids [9].

Flavor compounds derived from amino acids

OH

O

benzoic acid

O O

O

OHH

O

O

O

benzaldehyde benzyl alcohol

CH3

O

benzyl acetateOH

CH3

methyl salicylate

O

O

CH3

methyl benzoate benzyl benzoate

OH

O

H2N H

OH

O

trans-cinnamic acidphenylalanine

OH

O

HO

OH

O

HOOH

O

HO

OH

H3CO

coumaric acidcaffeic acid

ferulic acid

O OH

2-phenylacetaldehyde 2-phenylethanol

H

O

OCH3

HO

CH2

HO

H3COCH2

HO

OCH3

vanilin eugenol isoeugenolFig. 3.4. Biosythesis of flavor compounds derived from amino acids [9].

There are some flavor compounds that are derived from amino acids. For instance,

phenylalanine is a precursor of various flavors, including benzoic acid, benzaldehyde, benzyl

alcohol, benzyl acetate, methyl salicylate, methyl benzoate, benzyl benzoate, 2-phenyl

acetaldehyde, 2-phenylethanol, vanillin, eugenol, and isoeugenol via a complex series of

branched pathways (Fig. 3.4). Valine is a precursor for 1-N-2–methylpropane, 3-

methylbutylnitrile, 1-N-3-methylbutane, and 2-isobutylthiazole [62]. Isoleucine is a precursor

21

for 2-methylbutanol and 2-methylbutyric acid. In addition, leucine is a precursor for 3-

methylbutanal, 3-methylbutanol, and 3-methylbutanoic acid, which are abundant in various

fruits such as strawberries, tomatoes, and grape varieties [63].

3.3. Antimicrobial activity of fruit flavors

Fruits are equipped with a natural defense either by a physical or chemical barrier against

microbial invasion. Fruit flavors have been reported to be responsible for prolonging the

shelf-life of fruits [64]. Various flavor compounds have been reported to exhibit

antimicrobial activity against diverse microorganisms (Table 3.3). The inhibitory

mechanisms of several compounds have been proposed. For instance, the mechanism action

of various terpenoids was suggested due to membrane disruption by the lipophilic compounds

[65].

The inhibitory mechanism of E-2-hexenal and hexanal could be attributed to the alteration of

membrane fatty acids modulation [66]. For ester, the inhibition mechanism might be due to

the changes in the permeability of the cell membranes, which cause leakage of cellular

components and influence the metabolisms of bacteria [67].

The antimicrobial activity of γ-decalactone is related to its hydrophobic characteristic, which

allows the compound to diffuse to the cell, interact with the lipid membrane, increase the

membrane permeability, and eventually lead to cell death. [68]. Similarly, the inhibitory

mechanism of (-) epicatechin has been suggested with the incorporation of the hydrophobic

side of the compound with the surface of a membrane cell [69], causing disruption to the

membrane cell [70, 71]. Other works [71], have reported that (-) epicatechin inhibits the

transport material between the membrane and the environment.The mechanisms of the

antimicrobial action of quercetin have previously been studied. Quercetin might increase the

permeability of the inner bacterial membrane and dissipate the membrane potential, which

decreases the resistance of the cells to other antibacterial agents, thus, disrupting the proton

motive force of the cells leading to the inhibition of bacterial motility [72]. Another

mechanism previously reported [73] suggests that quercetin binds to the GyrB subunit of the

Escherichia coli’s DNA Gyrase and inhibits the enzyme’s ATPase activity. Quercetin binds

to the nucleic acid bases, which explains the inhibitory action on DNA and RNA synthesis

[74].

20

OH

S-linalool R-limonene Valencene

O O

Fig. 3.3. Some important flavors derived from terpenoids [9].

Flavor compounds derived from amino acids

OH

O

benzoic acid

O O

O

OHH

O

O

O

benzaldehyde benzyl alcohol

CH3

O

benzyl acetateOH

CH3

methyl salicylate

O

O

CH3

methyl benzoate benzyl benzoate

OH

O

H2N H

OH

O

trans-cinnamic acidphenylalanine

OH

O

HO

OH

O

HOOH

O

HO

OH

H3CO

coumaric acidcaffeic acid

ferulic acid

O OH

2-phenylacetaldehyde 2-phenylethanol

H

O

OCH3

HO

CH2

HO

H3COCH2

HO

OCH3

vanilin eugenol isoeugenolFig. 3.4. Biosythesis of flavor compounds derived from amino acids [9].

There are some flavor compounds that are derived from amino acids. For instance,

phenylalanine is a precursor of various flavors, including benzoic acid, benzaldehyde, benzyl

alcohol, benzyl acetate, methyl salicylate, methyl benzoate, benzyl benzoate, 2-phenyl

acetaldehyde, 2-phenylethanol, vanillin, eugenol, and isoeugenol via a complex series of

branched pathways (Fig. 3.4). Valine is a precursor for 1-N-2–methylpropane, 3-

methylbutylnitrile, 1-N-3-methylbutane, and 2-isobutylthiazole [62]. Isoleucine is a precursor

21

for 2-methylbutanol and 2-methylbutyric acid. In addition, leucine is a precursor for 3-

methylbutanal, 3-methylbutanol, and 3-methylbutanoic acid, which are abundant in various

fruits such as strawberries, tomatoes, and grape varieties [63].

3.3. Antimicrobial activity of fruit flavors

Fruits are equipped with a natural defense either by a physical or chemical barrier against

microbial invasion. Fruit flavors have been reported to be responsible for prolonging the

shelf-life of fruits [64]. Various flavor compounds have been reported to exhibit

antimicrobial activity against diverse microorganisms (Table 3.3). The inhibitory

mechanisms of several compounds have been proposed. For instance, the mechanism action

of various terpenoids was suggested due to membrane disruption by the lipophilic compounds

[65].

The inhibitory mechanism of E-2-hexenal and hexanal could be attributed to the alteration of

membrane fatty acids modulation [66]. For ester, the inhibition mechanism might be due to

the changes in the permeability of the cell membranes, which cause leakage of cellular

components and influence the metabolisms of bacteria [67].

The antimicrobial activity of γ-decalactone is related to its hydrophobic characteristic, which

allows the compound to diffuse to the cell, interact with the lipid membrane, increase the

membrane permeability, and eventually lead to cell death. [68]. Similarly, the inhibitory

mechanism of (-) epicatechin has been suggested with the incorporation of the hydrophobic

side of the compound with the surface of a membrane cell [69], causing disruption to the

membrane cell [70, 71]. Other works [71], have reported that (-) epicatechin inhibits the

transport material between the membrane and the environment.The mechanisms of the

antimicrobial action of quercetin have previously been studied. Quercetin might increase the

permeability of the inner bacterial membrane and dissipate the membrane potential, which

decreases the resistance of the cells to other antibacterial agents, thus, disrupting the proton

motive force of the cells leading to the inhibition of bacterial motility [72]. Another

mechanism previously reported [73] suggests that quercetin binds to the GyrB subunit of the

Escherichia coli’s DNA Gyrase and inhibits the enzyme’s ATPase activity. Quercetin binds

to the nucleic acid bases, which explains the inhibitory action on DNA and RNA synthesis

[74].

22

Table 3.3. Antimicrobial activity of fruit flavors.

Group Compound Fruits Microorganism Ref. Terpenoid Limonene Orange Saccharomyces cereviceae,

Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Moraxella catarrhalis, Cryptococcus neoformans, and anaerobic digesting microorganism

[75-78]

Car-3-ene, α-terpinene β-caryophyllene

Cashew apple, Venezuelan mango, orange, Australian mango, Tommy Atkins and Keitt mango, and Brazilian mango

Bacillus subtilis, Brevibacterium ammoniugenes, Staphylococcus aureus, Streptococcus mutans, Propionibacterium acnes, Pseudomom aeruginosa, Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Saccharomyces cerevisiae, Candida utilis, Pityrosporum ovale, Penicillium chrysogenum, and Trichophyton mentagrophytes

[23]

Aldehyde Benzaldehyde, Nonanal

Cashew apple, orange, strawberry, plum, and peach


[23]

Hexanal, 2-(E)-hexenal

Apple, grape, Australian mango, orange, strawberry, plum, and pear

Listeria monocytogenes, Escherichia coli, and Salmonella enteritidis

[79]

Ester Hexyl acetate Strawberry, peach, pear, and grape


[79]

Ketone 2,5 dimethyl-4-hydroxy-2H-furan-3-one (DMHF)

Strawberry, raspberries, pineapple, tomatoes, and mango

Human pathogenic microorganisms, Candida albicans

[80]

Lactone γ-Decalactone Plum, pineapple, blackberries, mango, apricot, and peach

Yarrowia lipolytica, Sporidibolus salmonicolor, Sporidibolus ruinenii

[68]

γ-Hexalactone Papaya, pineapple, apricot, blackberries, and plum

Tetrahymena pyriformis [81]

23

CHAPTER 4

Anaerobic Digestion

4.1. Biogas trends and biogas market

The industrial and commercial growth in today’s society contributes to the ever increasing

global energy demand. Currently, 88 % of the world’s energy demand is supplied by fossil

fuels whereas the rest is covered by renewable energy sources [82]. As a renewable energy

source, biogas appears to be the most efficient product of biomass based on the output and

input energy ratio of 28 MJ/MJ, compared to other products such as ethanol production or

combustion [83]. Biogas produced from renewable sources is considered to significantly

reduce the greenhouse gas emissions, compared to fossil fuels which accounted for 94 % of

the carbon dioxide accumulation in the atmosphere in 2009 [84].

Biogas has several energy applications such as electricity, heat generation, cooking, and

vehicle fuel, with an energy content of 1.0 m3 of purified biogas being equal to 1.1 L of

gasoline, 1.7 L of bioethanol, or 0.97 m3 of natural gas [85]. In 2011, the European Union

energy production from biogas reached the level of 10 million tons of oil equivalents [86]. In

Europe, biogas is typically used for electricity and heat generation, although in some

countries such as Sweden, biogas is mainly utilized as a vehicle fuel in the transportation

sector. Meanwhile, in developing countries, biogas is utilized for cooking, heating, and

lighting [76].

The world biogas market is predicted to increase in the future (Fig. 4.1). The biggest market

for biogas is currently in Europe. The biogas market in Asia Pacific is expected to be more

than three times. Around 10,000 biogas production units/plants are currently operated in

Europe and more than 20 million biogas plants are installed worldwide, including small

homemade biogas reactors [76]. Biogas is produced through anaerobic digestion (AD)

processes where the microorganisms convert complex organic matter into a mixture of

22

Table 3.3. Antimicrobial activity of fruit flavors.

Group Compound Fruits Microorganism Ref. Terpenoid Limonene Orange Saccharomyces cereviceae,

Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Moraxella catarrhalis, Cryptococcus neoformans, and anaerobic digesting microorganism

[75-78]

Car-3-ene, α-terpinene β-caryophyllene

Cashew apple, Venezuelan mango, orange, Australian mango, Tommy Atkins and Keitt mango, and Brazilian mango


[23]

Aldehyde Benzaldehyde, Nonanal

Cashew apple, orange, strawberry, plum, and peach


[23]

Hexanal, 2-(E)-hexenal

Apple, grape, Australian mango, orange, strawberry, plum, and pear


[79]

Ester Hexyl acetate Strawberry, peach, pear, and grape


[79]

Ketone 2,5 dimethyl-4-hydroxy-2H-furan-3-one (DMHF)

Strawberry, raspberries, pineapple, tomatoes, and mango

Human pathogenic microorganisms, Candida albicans

[80]

Lactone γ-Decalactone Plum, pineapple, blackberries, mango, apricot, and peach

Yarrowia lipolytica, Sporidibolus salmonicolor, Sporidibolus ruinenii

[68]

γ-Hexalactone Papaya, pineapple, apricot, blackberries, and plum

Tetrahymena pyriformis [81]

23

CHAPTER 4

Anaerobic Digestion

4.1. Biogas trends and biogas market

The industrial and commercial growth in today’s society contributes to the ever increasing

global energy demand. Currently, 88 % of the world’s energy demand is supplied by fossil

fuels whereas the rest is covered by renewable energy sources [82]. As a renewable energy

source, biogas appears to be the most efficient product of biomass based on the output and

input energy ratio of 28 MJ/MJ, compared to other products such as ethanol production or

combustion [83]. Biogas produced from renewable sources is considered to significantly

reduce the greenhouse gas emissions, compared to fossil fuels which accounted for 94 % of

the carbon dioxide accumulation in the atmosphere in 2009 [84].

Biogas has several energy applications such as electricity, heat generation, cooking, and

vehicle fuel, with an energy content of 1.0 m3 of purified biogas being equal to 1.1 L of

gasoline, 1.7 L of bioethanol, or 0.97 m3 of natural gas [85]. In 2011, the European Union

energy production from biogas reached the level of 10 million tons of oil equivalents [86]. In

Europe, biogas is typically used for electricity and heat generation, although in some

countries such as Sweden, biogas is mainly utilized as a vehicle fuel in the transportation

sector. Meanwhile, in developing countries, biogas is utilized for cooking, heating, and

lighting [76].

The world biogas market is predicted to increase in the future (Fig. 4.1). The biggest market

for biogas is currently in Europe. The biogas market in Asia Pacific is expected to be more

than three times. Around 10,000 biogas production units/plants are currently operated in

Europe and more than 20 million biogas plants are installed worldwide, including small

homemade biogas reactors [76]. Biogas is produced through anaerobic digestion (AD)

processes where the microorganisms convert complex organic matter into a mixture of

24

methane and carbon dioxide. One of the advantages of AD technology is a wide range of

substrates that can be used for biogas production.

Fig. 4.1. World biogas market value by region [87].

In Europe, AD plants already handle 25 % of biological treatment with a total of 13,800

plants in 2012 [88]. In addition, it is estimated that approximately 80 % of the composting

plants in the Netherlands and Belgium will have AD as the primary treatment method by

2015. It is expected that AD will continue to increase on a steady basis (Fig. 4.2) since it is a

combination between waste reduction and energy production. Thus, AD is considered to be

one of the most successful and innovative technologies of waste treatment during the last two

decades. In Europe, the largest capacity of biogas plant is in Germany with about 2 million

tons of annual capacity, which is followed by Spain with a total capacity of 1.6 million tons.

In terms of annual capacity, per million inhabitants, the Netherlands and Switzerland become

the highest with installed annual capacity of 52,400 and 49,000 tons per million people,

respectively.

Anaerobic digestion can be classified into different types based on the operating condition.

According to the operating temperature, AD can be divided into mesophilic and thermophilic.

Mesophilic digesters operate at a temperature between 35 °C and 40 °C, while thermophilic

digesters operate between 50 °C and 55 °C. Based on the separation of the acid forming and

methane forming stages, AD can be operated in a single or two stage process. In single stage,

both the acid and methane forming microorganisms are kept together in a single reactor with

0

5

10

15

20

25

30

35

2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022

Bio

gas

mar

ket (

$ B

illio

n)

Year

Middle East/Africa Latin America Asia PasificEurope North America

Asia Pacific

25

a delicate balance between the two groups, since they differ widely in terms of growth

conditions, whereas in the two stage process, each group of microorganism is grown in two

separate reactors with optimum environmental conditions for each group. Considering the

total solid content in the reactor, AD is divided into wet and dry digestion, in which dry

digestion has more than 15 % of the total solid.

Fig. 4.2. Increase in capacity per million inhabitants per country [89].

The current status of the installed biogas plants in Europe in 2014 is summarized in Table

4.1. Currently, biogas plants in Europe are dominated by mesophilic digestion since they are

considered to be a more stable process and require lower energy for heating. During the last

15 years, co-digestion has rarely been implemented since most of the biogas plants have been

dedicated to one specific waste stream material, often integrated with the pretreatment

operation. However, during the last two years, the trend of co-digestion has increased and

reaches approximately 13 %, which may be due to the improvement of the process or

economics of the plant.

Table 4.1. Current status of the biogas plants installed in Europe [89].

Temperature Complexity Moisture Feedstock

Mesophilic

Thermophilic

One phase

Two phase

Wet Dry Solid waste

Co-digestion

Cumulative installed (%)

67 33 93 7 38 62 89 11

0

10000

20000

30000

40000

50000

60000

1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014

Inst

alle

d ca

paci

ty p

er m

illio

n in

habi

tan

ts

Years

Spain belgium FranceNetherlands Germany Switzerland

Belgium

24

methane and carbon dioxide. One of the advantages of AD technology is a wide range of

substrates that can be used for biogas production.

Fig. 4.1. World biogas market value by region [87].

In Europe, AD plants already handle 25 % of biological treatment with a total of 13,800

plants in 2012 [88]. In addition, it is estimated that approximately 80 % of the composting

plants in the Netherlands and Belgium will have AD as the primary treatment method by

2015. It is expected that AD will continue to increase on a steady basis (Fig. 4.2) since it is a

combination between waste reduction and energy production. Thus, AD is considered to be

one of the most successful and innovative technologies of waste treatment during the last two

decades. In Europe, the largest capacity of biogas plant is in Germany with about 2 million

tons of annual capacity, which is followed by Spain with a total capacity of 1.6 million tons.

In terms of annual capacity, per million inhabitants, the Netherlands and Switzerland become

the highest with installed annual capacity of 52,400 and 49,000 tons per million people,

respectively.

Anaerobic digestion can be classified into different types based on the operating condition.

According to the operating temperature, AD can be divided into mesophilic and thermophilic.

Mesophilic digesters operate at a temperature between 35 °C and 40 °C, while thermophilic

digesters operate between 50 °C and 55 °C. Based on the separation of the acid forming and

methane forming stages, AD can be operated in a single or two stage process. In single stage,

both the acid and methane forming microorganisms are kept together in a single reactor with

0

5

10

15

20

25

30

35

2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022

Bio

gas

mar

ket (

$ B

illio

n)

Year

Middle East/Africa Latin America Asia PasificEurope North America

Asia Pacific

25

a delicate balance between the two groups, since they differ widely in terms of growth

conditions, whereas in the two stage process, each group of microorganism is grown in two

separate reactors with optimum environmental conditions for each group. Considering the

total solid content in the reactor, AD is divided into wet and dry digestion, in which dry

digestion has more than 15 % of the total solid.

Fig. 4.2. Increase in capacity per million inhabitants per country [89].

The current status of the installed biogas plants in Europe in 2014 is summarized in Table

4.1. Currently, biogas plants in Europe are dominated by mesophilic digestion since they are

considered to be a more stable process and require lower energy for heating. During the last

15 years, co-digestion has rarely been implemented since most of the biogas plants have been

dedicated to one specific waste stream material, often integrated with the pretreatment

operation. However, during the last two years, the trend of co-digestion has increased and

reaches approximately 13 %, which may be due to the improvement of the process or

economics of the plant.

Table 4.1. Current status of the biogas plants installed in Europe [89].

Temperature Complexity Moisture Feedstock

Mesophilic

Thermophilic

One phase

Two phase

Wet Dry Solid waste

Co-digestion

Cumulative installed (%)

67 33 93 7 38 62 89 11

0

10000

20000

30000

40000

50000

60000

1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014

Inst

alle

d ca

paci

ty p

er m

illio

n in

habi

tan

ts

Years

Spain belgium FranceNetherlands Germany Switzerland

Belgium

26

Recently, the biogas plants in Europe have mostly been operated using one phase digestion.

The application of a two-stage digestion with a separation process for hydrolysis and

methanogenesis has declined since the beginning of the nineties. This is due to the high

investment and operating costs for running two different processes, at the same time, many

high rates for the one phase system digesters are invented. Dry digestion is till the dominant

process for biogas production in recent years.

4.2. Anaerobic Digestion: Complexities and challenges

4.2.1. Overview of the biochemical reactions

Fig. 4.3. The biochemical reaction series of anaerobic digestion (adapted from [90]).

Anaerobic digestion is a complex degradation process of organic wastes, which involves a

series of biochemical reactions and different metabolic group of microorganisms under

anaoxic conditions. AD requires a syntrophic relation with four metabolic groups, namely

hydrolytic, acidogenic, acetogenic, and methanogenic to produce methane and carbon dioxide

as the final product of the organic waste degradation (Fig. 4.3). A syntrophic relation is a

symbiotic collaboration between two metabolically different types of bacteria with mutual

Complex organic matter Carbohydrates, proteins, lipids

Soluble organic matter Sugars, amino acids, fatty acids

Intermediate products Alcohols and VFA

Acetate Hydrogen Carbon dioxide

Methane Carbon dioxide

Hydrolytic bacteria

Anaerobic oxidizers Fermentative

bacteria

Acetate oxidizing bacteria

Homoacetogenic bacteria

Acetogenic methanogens

Hydrogenotrophic methanogens

27

dependence, one to each other, particularly for the energetic reason for degradation of a

certain substrate that simply cannot be replaced by adding a substrate or any other type of

nutrient.

Hydrolysis

Hydrolysis is the first step in the anaerobic digestion processes. In this step, insoluble

complex organic matters, such as carbohydrates, proteins, and lipids are hydrolyzed into their

monomers such as sugars, amino acids, and fatty acids by the extracellular enzymes, that is,

cellulase, amylase, protease, or lipase [91] secreted from the facultative anaerobes hydrolytic

bacteria. This step is carried out by anaerobic bacteria such as Bacterioides, Clostridium, and

Streptococcus. After the hydrolysis, the monomers become a soluble molecule, which is

available for the cell transport and will be further degraded by the fermentative bacteria.

Hydrolysis can be the rate-limiting step if the substrate contains large molecules (particulates)

with a low surface-to-volume ratio [92]. However, if the substrate is readily degradable, the

rate-limiting step will be acetogenesis and methanogenesis [93].

Acidogenesis

In the acidogenesis step, the soluble organic molecules from the hydrolysis are further

degraded into short chain organic acids, acetic acid, alcohols, hydrogen, and carbon dioxide

by both obligate and facultative anaerobes. In a stable anaerobic digester, the products of this

step is usually 51 % acetate, 19 % H2/CO2, and 30 % reduced products, such as higher VFA,

alcohols, or lactate [90]. This step is often considered as the fastest step among the various

steps involved in AD [92]. The concentration of hydrogen plays an important role on the fate

of the final product. When the concentration of the hydrogen and the formate is high, the

acidogenic bacteria will shift the pathway to produce more reduced metabolites [90].

Acetogenesis

The products from the acidogenesis process cannot be directly used in methanogenesis; thus,

it needs to be further oxidized to acetate and H2 in this step. It is important that the

microorganism involved in this step work in a syntrophic relationship with the next group,

that is, methanogen. This synthropic relation is extremely important for this step since the

acetogenic reaction requires a low H2 to be thermodynamically favorable [94], and

methanogen is able to maintain a low H2 by continuously converting the hydrogen into

methane.

26

Recently, the biogas plants in Europe have mostly been operated using one phase digestion.

The application of a two-stage digestion with a separation process for hydrolysis and

methanogenesis has declined since the beginning of the nineties. This is due to the high

investment and operating costs for running two different processes, at the same time, many

high rates for the one phase system digesters are invented. Dry digestion is till the dominant

process for biogas production in recent years.

4.2. Anaerobic Digestion: Complexities and challenges

4.2.1. Overview of the biochemical reactions

Fig. 4.3. The biochemical reaction series of anaerobic digestion (adapted from [90]).

Anaerobic digestion is a complex degradation process of organic wastes, which involves a

series of biochemical reactions and different metabolic group of microorganisms under

anaoxic conditions. AD requires a syntrophic relation with four metabolic groups, namely

hydrolytic, acidogenic, acetogenic, and methanogenic to produce methane and carbon dioxide

as the final product of the organic waste degradation (Fig. 4.3). A syntrophic relation is a

symbiotic collaboration between two metabolically different types of bacteria with mutual

Complex organic matter Carbohydrates, proteins, lipids

Soluble organic matter Sugars, amino acids, fatty acids

Intermediate products Alcohols and VFA

Acetate Hydrogen Carbon dioxide

Methane Carbon dioxide

Hydrolytic bacteria

Anaerobic oxidizers Fermentative

bacteria

Acetate oxidizing bacteria

Homoacetogenic bacteria

Acetogenic methanogens

Hydrogenotrophic methanogens

27

dependence, one to each other, particularly for the energetic reason for degradation of a

certain substrate that simply cannot be replaced by adding a substrate or any other type of

nutrient.

Hydrolysis

Hydrolysis is the first step in the anaerobic digestion processes. In this step, insoluble

complex organic matters, such as carbohydrates, proteins, and lipids are hydrolyzed into their

monomers such as sugars, amino acids, and fatty acids by the extracellular enzymes, that is,

cellulase, amylase, protease, or lipase [91] secreted from the facultative anaerobes hydrolytic

bacteria. This step is carried out by anaerobic bacteria such as Bacterioides, Clostridium, and

Streptococcus. After the hydrolysis, the monomers become a soluble molecule, which is

available for the cell transport and will be further degraded by the fermentative bacteria.

Hydrolysis can be the rate-limiting step if the substrate contains large molecules (particulates)

with a low surface-to-volume ratio [92]. However, if the substrate is readily degradable, the

rate-limiting step will be acetogenesis and methanogenesis [93].

Acidogenesis

In the acidogenesis step, the soluble organic molecules from the hydrolysis are further

degraded into short chain organic acids, acetic acid, alcohols, hydrogen, and carbon dioxide

by both obligate and facultative anaerobes. In a stable anaerobic digester, the products of this

step is usually 51 % acetate, 19 % H2/CO2, and 30 % reduced products, such as higher VFA,

alcohols, or lactate [90]. This step is often considered as the fastest step among the various

steps involved in AD [92]. The concentration of hydrogen plays an important role on the fate

of the final product. When the concentration of the hydrogen and the formate is high, the

acidogenic bacteria will shift the pathway to produce more reduced metabolites [90].

Acetogenesis

The products from the acidogenesis process cannot be directly used in methanogenesis; thus,

it needs to be further oxidized to acetate and H2 in this step. It is important that the

microorganism involved in this step work in a syntrophic relationship with the next group,

that is, methanogen. This synthropic relation is extremely important for this step since the

acetogenic reaction requires a low H2 to be thermodynamically favorable [94], and

methanogen is able to maintain a low H2 by continuously converting the hydrogen into

methane.

28

Methanogenesis

Methanogenesis is the last step in which the acetate and H2/CO2 are converted into CH4 by

methanogenic archaea. The methanogenic archaea are able to utilize the H2/CO2, acetate, and

other one-carbon compounds, such as formate and methanol [94]. Two genera are known to

use acetate for methanogenesis: Methanosarcina and Methanosaeta. Methanosaeta is a

superior acetate utilizer that is able to use acetate at concentrations as low as 5–20 μM, while

Methanosarcina requires a minimum concentration of about 1 mM. Moreover, the inter-

conversion between hydrogen and acetate, catalyzed by the homoacetogenic bacteria, is also

necessary to direct the methane formation pathway. The homoacetogens can either oxidize or

synthesize the acetate depending on the hydrogen concentration in the system [95]. At high

hydrogen partial pressures, the hydrogenotrophic methanogenesis functions better whereas

the aceticlastic methanogenesis is independent of hydrogen partial pressure. The acetate

oxidation pathway becomes more favorable at higher temperatures [94]. It has been reported

that the methane formation through acetate oxidation can contribute up to 14 % of the total

acetate conversion to methane under thermophilic conditions (60 °C) [96].

4.2.2. Different characteristics of the microorganisms

Table 4.2. Optimal environmental requirements for acid and methane formation [83].

Parameters Acid formation Methane formation

Temperature 25–35 oC Mesophilic: 32–42 oC

Thermophilic: 50–58 oC

pH 5.2–6.3 6.7–7.5

C/N ratio 10– 45 20–30

DM content <40 % <30 %

Redox potential

Required C:N:P:S ratio

Trace elements

+400 to -300 mV

500:15:5:3

No special requirement

<-250 mV

600:15:5:3

Essential: Ni, Co, Mo, Se

Biochemical reaction of anaerobic digestion can be divided into two main steps, namely, acid

and methane formation. The acid forming and the methane forming microorganisms differ

widely in terms of physiology, nutritional needs, growth kinetics, and sensitivity to

environmental conditions [97]. The optimal environmental requirements of the acid and

methane forming microorganism is presented in Table 4.2. The acid forming bacteria has a

faster doubling time (1–1.5 days) than the methane forming archea (5–15 days) [98]. In

addition, the cell membrane structure of methanogens, acidogens, and acetogens is different.

The acid forming bacteria have a hydrophobic lipid bilayer on their membrane cell, whereas

29

the cell membrane of methanogen is made from ether lipid, which lacks L-muramic acid, and

this produces fatty acid-sensitive archea [98].

The differences in these two groups in terms of the environmental requirements and growth

rate create a challenge in anaerobic digestion. The longer doubling time of methanogen

requires a longer retention time to prevent wash out in a continuous reactor; however, at the

same time, a long retention time is not economically favorable for industrial application.

Besides, methanogenesis is considered as the limiting step since it is more vulnerable to

parameters such as temperature, pH, and inhibitory chemical. The environmental condition of

the reactor should maintain the balance between these two groups of microorganisms to avoid

the reactor instability [99]. For instance, the high rate of hydrolysis if it is not followed by a

high activity of methanogen causes the accumulation of intermediate products, which inhibit

methanogen. Therefore, high activity of the methanogens is important for maintaining

efficient anaerobic digestion and avoiding the accumulation of H2 and short chain fatty acids.

Low H2 levels indicate efficient hydrogenotrophic methanogenesis and are usually associated

with stable performance.

4.2.3. Structural complexity and recalcitrance of the biomass

Besides the complexities of the microorganism involved in anaerobic digestion, another

challenge comes from the substrate characteristics. In nature, most plants are equipped with

either physical or chemical defense against degradation. The physical barrier is the inherent

properties of a native cell wall, which is very rigid and complex, making it resistant from

enzymatic attack. The collective resistance of the biomass against microbe and enzyme attack

is known as biomass recalcitrance [100]. It involves a sophisticated and integrated defense

system combining crystalline cellulose in microfibrils, heteropolysaccharides, and lignin.

Biomass recalcitrance causes hydrolysis, which becomes the rate-limiting step since the

substrate is not accessible to the enzyme. This is in the case of a lignocellulosic substrate.

Plant cell walls have several functions such as maintaining the shape of the cell, controlling

the cell expansion, providing protection, and storing food reserves [101]. To properly

function, cell walls must provide the mechanical strength to the plant; thus, they are made of

several layers. Cell walls contain the cellulose, hemicellulose, pectin, and lignin, which are

arranged together. Cellulose is the main component of the cell wall, comprising up to 35–

50 %. There are two types of cellulose, including amorphous and crystalline cellulose.

28

Methanogenesis

Methanogenesis is the last step in which the acetate and H2/CO2 are converted into CH4 by

methanogenic archaea. The methanogenic archaea are able to utilize the H2/CO2, acetate, and

other one-carbon compounds, such as formate and methanol [94]. Two genera are known to

use acetate for methanogenesis: Methanosarcina and Methanosaeta. Methanosaeta is a

superior acetate utilizer that is able to use acetate at concentrations as low as 5–20 μM, while

Methanosarcina requires a minimum concentration of about 1 mM. Moreover, the inter-

conversion between hydrogen and acetate, catalyzed by the homoacetogenic bacteria, is also

necessary to direct the methane formation pathway. The homoacetogens can either oxidize or

synthesize the acetate depending on the hydrogen concentration in the system [95]. At high

hydrogen partial pressures, the hydrogenotrophic methanogenesis functions better whereas

the aceticlastic methanogenesis is independent of hydrogen partial pressure. The acetate

oxidation pathway becomes more favorable at higher temperatures [94]. It has been reported

that the methane formation through acetate oxidation can contribute up to 14 % of the total

acetate conversion to methane under thermophilic conditions (60 °C) [96].

4.2.2. Different characteristics of the microorganisms

Table 4.2. Optimal environmental requirements for acid and methane formation [83].

Parameters Acid formation Methane formation

Temperature 25–35 oC Mesophilic: 32–42 oC

Thermophilic: 50–58 oC

pH 5.2–6.3 6.7–7.5

C/N ratio 10– 45 20–30

DM content <40 % <30 %

Redox potential

Required C:N:P:S ratio

Trace elements

+400 to -300 mV

500:15:5:3

No special requirement

<-250 mV

600:15:5:3

Essential: Ni, Co, Mo, Se

Biochemical reaction of anaerobic digestion can be divided into two main steps, namely, acid

and methane formation. The acid forming and the methane forming microorganisms differ

widely in terms of physiology, nutritional needs, growth kinetics, and sensitivity to

environmental conditions [97]. The optimal environmental requirements of the acid and

methane forming microorganism is presented in Table 4.2. The acid forming bacteria has a

faster doubling time (1–1.5 days) than the methane forming archea (5–15 days) [98]. In

addition, the cell membrane structure of methanogens, acidogens, and acetogens is different.

The acid forming bacteria have a hydrophobic lipid bilayer on their membrane cell, whereas

29

the cell membrane of methanogen is made from ether lipid, which lacks L-muramic acid, and

this produces fatty acid-sensitive archea [98].

The differences in these two groups in terms of the environmental requirements and growth

rate create a challenge in anaerobic digestion. The longer doubling time of methanogen

requires a longer retention time to prevent wash out in a continuous reactor; however, at the

same time, a long retention time is not economically favorable for industrial application.

Besides, methanogenesis is considered as the limiting step since it is more vulnerable to

parameters such as temperature, pH, and inhibitory chemical. The environmental condition of

the reactor should maintain the balance between these two groups of microorganisms to avoid

the reactor instability [99]. For instance, the high rate of hydrolysis if it is not followed by a

high activity of methanogen causes the accumulation of intermediate products, which inhibit

methanogen. Therefore, high activity of the methanogens is important for maintaining

efficient anaerobic digestion and avoiding the accumulation of H2 and short chain fatty acids.

Low H2 levels indicate efficient hydrogenotrophic methanogenesis and are usually associated

with stable performance.

4.2.3. Structural complexity and recalcitrance of the biomass

Besides the complexities of the microorganism involved in anaerobic digestion, another

challenge comes from the substrate characteristics. In nature, most plants are equipped with

either physical or chemical defense against degradation. The physical barrier is the inherent

properties of a native cell wall, which is very rigid and complex, making it resistant from

enzymatic attack. The collective resistance of the biomass against microbe and enzyme attack

is known as biomass recalcitrance [100]. It involves a sophisticated and integrated defense

system combining crystalline cellulose in microfibrils, heteropolysaccharides, and lignin.

Biomass recalcitrance causes hydrolysis, which becomes the rate-limiting step since the

substrate is not accessible to the enzyme. This is in the case of a lignocellulosic substrate.

Plant cell walls have several functions such as maintaining the shape of the cell, controlling

the cell expansion, providing protection, and storing food reserves [101]. To properly

function, cell walls must provide the mechanical strength to the plant; thus, they are made of

several layers. Cell walls contain the cellulose, hemicellulose, pectin, and lignin, which are

arranged together. Cellulose is the main component of the cell wall, comprising up to 35–

50 %. There are two types of cellulose, including amorphous and crystalline cellulose.

30

Crystalline cellulose dominates the cell wall and is less vulnerable to enzymatic degradation,

compared to the amorphous form. In this type, several polymers are linked to each other by

hydrogen bonds to form a polymer chain, which will further join together with other chains to

form cellulose microfibril. In contrast to cellulose, hemicellulose consists of amorphous

polymers, which are easily degradable. Cellulose and hemicellulose are glued with lignin by

a variety of chemical bonds. Lignin is very rigid and considered as a protective shield of the

plant, which makes it resistant to chemical and biological degradation.

The outer layer is the primary cell wall, which is composed of microfibril cellulose with a

random orientation. Following the completion of the primary cell wall synthesis, the

secondary cell wall is formed. The secondary layers consist of three layers known as the outer

(S1), middle (S2), and inner (S3) layers. The orientation of each layer is different, for

instance, almost horizontal for S1, vertical for S2, and again horizontal for S3. The vertical

orientation of S2 provides the cell a mechanical strength for the plant [102]. The cell is

connected to other cells by a thin layer of lignin rich material called the middle lamella.

Besides the complexities in the structure, the recalcitrance is also caused by interaction

between the cell wall polymers. Cellulosic microfibrils and structural protein are connected to

the non-cellulosic heteropolysaccharides, such as pectin and hemicellulose by non-covalent

interaction in the gel matrix [103]. This interaction is important for the cohesiveness of the

cell walls. In addition, there is a covalent interaction between the polysaccharides-

polysaccharides, polysaccharides-lignin, polysaccharides-protein, and lignin-proteins [104].

There are several factors affecting the biomass recalcitrance, including the degree of

polymerization of cellulose, available surface area/porosity, presence of lignin, protection of

cellulose by hemicellulose, and fiber strength. Since cellulose and hemicellulose are sugar

polymers, they are a potential source of sugar for the biofuel production. However, to access

the sugars, an aggressive pretreatment is required. In order to increase the accessible surface

area of cellulose, lignin and hemicellulose are firstly eliminated; thus, cellulose crystallinity

remains as the second challenge. In a native state, the cellulose microfibrils are twisted; thus

making the interruption of the single chain difficult. Pretreatment changes the form of the

cellulose from cellulose I to cellulose II, with lower crystallinity; hence, it become easier to

be hydrolyzed by the cellulose enzyme.

31

4.2.4. Inhibition

Another important reason for the low methane yield and the unstable process of anaerobic

digestion is inhibition by various compounds. Inhibition is indicated by a decrease in the

methane production and accumulation of organic acids [105]. The accumulation of volatile

fatty acids (VFA) and low methane production reflects imbalance rate of VFA production

and consumption which most probably caused by disruption of methanogen. Propionic acid is

suggested to be an excellent indicator of digestion process since its degradation only occurs

in a balanced system [106], hence increasing concentration of propionic acid reflecting

disturbance of the process.

Fig.4.4. Acetic acid and propionic acid of anaerobic digestion with addition of 10 g/L of D-limonene (paper V).

In the present thesis, a major flavor compound in citrus, D-limonene was added to anaerobic

digesting system in thermophilic semi-continuous process. The VFA was observed as

presented in Fig. 4.4. Following addition of D-limonene on day 30, the acetic acid started to

increase, whereas no acetic acid accumulation was observed for control reaction (without

addition of D-limonene) during the digestion process (data not shown). Acid concentration

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70

Ace

tic/

prop

ioni

c ac

id (g

/L)

Digestion time (days)

acetic acid propionic acid

Addition of D-limonene

30

Crystalline cellulose dominates the cell wall and is less vulnerable to enzymatic degradation,

compared to the amorphous form. In this type, several polymers are linked to each other by

hydrogen bonds to form a polymer chain, which will further join together with other chains to

form cellulose microfibril. In contrast to cellulose, hemicellulose consists of amorphous

polymers, which are easily degradable. Cellulose and hemicellulose are glued with lignin by

a variety of chemical bonds. Lignin is very rigid and considered as a protective shield of the

plant, which makes it resistant to chemical and biological degradation.

The outer layer is the primary cell wall, which is composed of microfibril cellulose with a

random orientation. Following the completion of the primary cell wall synthesis, the

secondary cell wall is formed. The secondary layers consist of three layers known as the outer

(S1), middle (S2), and inner (S3) layers. The orientation of each layer is different, for

instance, almost horizontal for S1, vertical for S2, and again horizontal for S3. The vertical

orientation of S2 provides the cell a mechanical strength for the plant [102]. The cell is

connected to other cells by a thin layer of lignin rich material called the middle lamella.

Besides the complexities in the structure, the recalcitrance is also caused by interaction

between the cell wall polymers. Cellulosic microfibrils and structural protein are connected to

the non-cellulosic heteropolysaccharides, such as pectin and hemicellulose by non-covalent

interaction in the gel matrix [103]. This interaction is important for the cohesiveness of the

cell walls. In addition, there is a covalent interaction between the polysaccharides-

polysaccharides, polysaccharides-lignin, polysaccharides-protein, and lignin-proteins [104].

There are several factors affecting the biomass recalcitrance, including the degree of

polymerization of cellulose, available surface area/porosity, presence of lignin, protection of

cellulose by hemicellulose, and fiber strength. Since cellulose and hemicellulose are sugar

polymers, they are a potential source of sugar for the biofuel production. However, to access

the sugars, an aggressive pretreatment is required. In order to increase the accessible surface

area of cellulose, lignin and hemicellulose are firstly eliminated; thus, cellulose crystallinity

remains as the second challenge. In a native state, the cellulose microfibrils are twisted; thus

making the interruption of the single chain difficult. Pretreatment changes the form of the

cellulose from cellulose I to cellulose II, with lower crystallinity; hence, it become easier to

be hydrolyzed by the cellulose enzyme.

31

4.2.4. Inhibition

Another important reason for the low methane yield and the unstable process of anaerobic

digestion is inhibition by various compounds. Inhibition is indicated by a decrease in the

methane production and accumulation of organic acids [105]. The accumulation of volatile

fatty acids (VFA) and low methane production reflects imbalance rate of VFA production

and consumption which most probably caused by disruption of methanogen. Propionic acid is

suggested to be an excellent indicator of digestion process since its degradation only occurs

in a balanced system [106], hence increasing concentration of propionic acid reflecting

disturbance of the process.

Fig.4.4. Acetic acid and propionic acid of anaerobic digestion with addition of 10 g/L of D-limonene (paper V).

In the present thesis, a major flavor compound in citrus, D-limonene was added to anaerobic

digesting system in thermophilic semi-continuous process. The VFA was observed as

presented in Fig. 4.4. Following addition of D-limonene on day 30, the acetic acid started to

increase, whereas no acetic acid accumulation was observed for control reaction (without

addition of D-limonene) during the digestion process (data not shown). Acid concentration

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70

Ace

tic/

prop

ioni

c ac

id (g

/L)


acetic acid propionic acid

Addition of D-limonene

32

reached 3.96 g/L and remained high until the end of digestion. Similarly, propionic acid

concentration increase after the perturbation which indicates the imbalance of the process.

Various substances in substantial amounts have been reported to have inhibitory activity that

causes anaerobic digestion failure. These substances include ammonia, sulfide, light metal

ions, heavy metal, and organic compounds [107].

Ammonia inhibition occurs in the anaerobic digestion of nitrogenous substrates, mostly in

the form of proteins and urea [108]. There are two forms of ammonia in aqueous solution,

including ammonia ion and free ammonia. Free ammonia is permeable to the cell membrane

[105], and the hydrophobic ammonia molecule is able to diffuse to the cell resulting in proton

imbalance and/or potassium deficiency [109]. Additionally, ammonia has been suggested to

change intracellular pH, to increase maintenance energy requirement, and to inhibit a specific

enzyme reaction [110]. Ammonia can be removed using air stripping or chemical

precipitation [107].

Sulfate can be easily found in many industrial wastewaters [111]. Sulfate reducing bacteria

(SRB) present in the anaerobic digester reduces sulfate into sulfide. Sulfate disrupts the

methane production by two mechanisms. The primary mechanism is nutrient competition

between the SRB and microorganism involved in the methane production, whereas the

secondary mechanism is toxicity of sulfide to various groups of bacteria [112, 113]. Several

approaches have been promoted to reduce the inhibition of sulfate, including dilution of the

wastewater, sulfide removal by stripping, coagulation, oxidation, precipitation, and biological

conversions by partial oxidation to elemental sulfur [114, 115].

Both light and heavy metals have been reported to have an impact on anaerobic digestion.

Light metal ions such as potassium, sodium, calcium, and magnesium are present in the

digester, as it is liberated during the breakdown of the organic matter or added to adjust the

pH of the digester [116]. Although they are required for microbial growth at a moderate

concentration, an excessive amount of these ions has an opposite effect and even inhibits the

process at higher concentrations. Heavy metals are commonly present in significant amounts

in municipal sewage and sludge. These heavy metals include chromium, cobalt, copper, zinc,

cadmium, and nickel. The inhibitory mechanism of these metals was reported to be due to

their disruption of the microbial enzyme structure and function [117].

33

Organic chemicals that are poorly soluble in water affect the digester performance, since it

may accumulate to high levels in the digester [107]. Accumulation of these compounds

disrupt the membrane function and eventually results in cell lysis [118]. The toxic organic

compounds include: phenol and alkyl phenol [119], alkyl benzenes [120], halogenated

benzene [121], nitrobenzene [122], alkanes [123], alcohols [124], aldehydes [125], some long

chain fatty acids (LCFA), ketone, and ether [126].

4.3. Substrates for anaerobic digestion

Basically, biogas can be produced from any organic material, although the amount of biogas

production depends on the material. Several factors that affect the methane yield from

particular substrates are digestibility, C/N/S/P ratio, pH, and the presence of an inhibitor

compound. The optimum C/N ratio for biogas production is in the range of 20–30, whereas

the desired pH for anaerobic digestion is in the range from 7 to 8.5. A higher C/N ratio leads

to a decrease in the methane yield since the carbon is not optimally consumed, whereas too

low C/N ratio results in the accumulation of free ammonia, which can inhibit the

microorganism by diffusion to the cell membrane. The C/N ratio of the substrate can be

adjusted either by co-digestion with other substances or addition of a carbon/nitrogen source.

The microbial populations involved in anaerobic digestion require nutrients to grow and

multiply. The methane yield of various substrates is shown in Table 4.3.

Table 4.3. Potential methane production from various substrates ( adapted from [127])

Substrate Methane yields (Nm3/ ton VS)

Food Waste Fruit and vegetable waste

400-800 200-500

Manure from cattle, pigs, or chicken 100-300

Slaughterhouse waste 700

Cereals 300-400

Sugar beets 300-800

Silage grass 350-380

Grass 200-400

Straw 100-320

Municipal sludge 180-350

Distillation waste 300-400

In the beginning of the biogas production, called the first generation of biogas, food crops

such as maize is used due to its simple structure, which makes the process easier. However,

32

reached 3.96 g/L and remained high until the end of digestion. Similarly, propionic acid

concentration increase after the perturbation which indicates the imbalance of the process.

Various substances in substantial amounts have been reported to have inhibitory activity that

causes anaerobic digestion failure. These substances include ammonia, sulfide, light metal

ions, heavy metal, and organic compounds [107].

Ammonia inhibition occurs in the anaerobic digestion of nitrogenous substrates, mostly in

the form of proteins and urea [108]. There are two forms of ammonia in aqueous solution,

including ammonia ion and free ammonia. Free ammonia is permeable to the cell membrane

[105], and the hydrophobic ammonia molecule is able to diffuse to the cell resulting in proton

imbalance and/or potassium deficiency [109]. Additionally, ammonia has been suggested to

change intracellular pH, to increase maintenance energy requirement, and to inhibit a specific

enzyme reaction [110]. Ammonia can be removed using air stripping or chemical

precipitation [107].

Sulfate can be easily found in many industrial wastewaters [111]. Sulfate reducing bacteria

(SRB) present in the anaerobic digester reduces sulfate into sulfide. Sulfate disrupts the

methane production by two mechanisms. The primary mechanism is nutrient competition

between the SRB and microorganism involved in the methane production, whereas the

secondary mechanism is toxicity of sulfide to various groups of bacteria [112, 113]. Several

approaches have been promoted to reduce the inhibition of sulfate, including dilution of the

wastewater, sulfide removal by stripping, coagulation, oxidation, precipitation, and biological

conversions by partial oxidation to elemental sulfur [114, 115].

Both light and heavy metals have been reported to have an impact on anaerobic digestion.

Light metal ions such as potassium, sodium, calcium, and magnesium are present in the

digester, as it is liberated during the breakdown of the organic matter or added to adjust the

pH of the digester [116]. Although they are required for microbial growth at a moderate

concentration, an excessive amount of these ions has an opposite effect and even inhibits the

process at higher concentrations. Heavy metals are commonly present in significant amounts

in municipal sewage and sludge. These heavy metals include chromium, cobalt, copper, zinc,

cadmium, and nickel. The inhibitory mechanism of these metals was reported to be due to

their disruption of the microbial enzyme structure and function [117].

33

Organic chemicals that are poorly soluble in water affect the digester performance, since it

may accumulate to high levels in the digester [107]. Accumulation of these compounds

disrupt the membrane function and eventually results in cell lysis [118]. The toxic organic

compounds include: phenol and alkyl phenol [119], alkyl benzenes [120], halogenated

benzene [121], nitrobenzene [122], alkanes [123], alcohols [124], aldehydes [125], some long

chain fatty acids (LCFA), ketone, and ether [126].

4.3. Substrates for anaerobic digestion

Basically, biogas can be produced from any organic material, although the amount of biogas

production depends on the material. Several factors that affect the methane yield from

particular substrates are digestibility, C/N/S/P ratio, pH, and the presence of an inhibitor

compound. The optimum C/N ratio for biogas production is in the range of 20–30, whereas

the desired pH for anaerobic digestion is in the range from 7 to 8.5. A higher C/N ratio leads

to a decrease in the methane yield since the carbon is not optimally consumed, whereas too

low C/N ratio results in the accumulation of free ammonia, which can inhibit the

microorganism by diffusion to the cell membrane. The C/N ratio of the substrate can be

adjusted either by co-digestion with other substances or addition of a carbon/nitrogen source.

The microbial populations involved in anaerobic digestion require nutrients to grow and

multiply. The methane yield of various substrates is shown in Table 4.3.

Table 4.3. Potential methane production from various substrates ( adapted from [127])

Substrate Methane yields (Nm3/ ton VS)

Food Waste Fruit and vegetable waste

400-800 200-500

Manure from cattle, pigs, or chicken 100-300

Slaughterhouse waste 700

Cereals 300-400

Sugar beets 300-800

Silage grass 350-380

Grass 200-400

Straw 100-320

Municipal sludge 180-350

Distillation waste 300-400

In the beginning of the biogas production, called the first generation of biogas, food crops

such as maize is used due to its simple structure, which makes the process easier. However,

34

in the long-term production, biogas produced from this type of material is still debatable

since it creates a moral dilemma in the utilization of land for food or fuel [128]. The social

impact of biogas production is illustrated by taking food from the people of a developing

country and putting it into the cars of people in a developed country. The United Nations

reported that maize used to produce 50 liters of biofuel is equal with amount of maize to feed

one person for one year. Furthermore, a high demand for food crops for biogas production

increases the food price due to the competition of using natural sources such as nitrogen,

land, and water. In addition, there are growing debates on the justification of positive energy

balance of biogas produced from maize as well as the impact on biodiversity, water

resources, and soil quality [129, 130].

In order to overcome the potential conflict between food and energy, the second generation of

biogas has been developed from lignocellulosic materials with low cost, great abundance, and

sustainable supply [131]. Cellulose as the main constituent of lignocellulose is the most

abundant biopolymer on earth [132]. The lignocellulose materials that can be used for biogas

production varies from agricultural residue, forest residue, industrial processing waste, and

municipal solid waste. Biogas production from the waste streams is beneficial not only to

reduce the environmental problem but also to help alleviate the energy problem.

Lignocellulosic materials can be divided into two groups, including cultivated feedstock and

lignocellulose residuals. The latter group is more beneficial since it does not compete with

the usage of natural resources for food and feed production. Moreover, the utilization of

lignocellulosic waste for biogas production can be simultaneously used for waste treatment

and renewable energy production.

35

CHAPTER 5

Anaerobic Digestion of Fruits wastes

5.1. Biogas potential from fruits wastes

Manure is currently the major raw material for biogas production worldwide. However, it has

a relatively low biogas production rate and yield [133]. The high biogas production rate and

yield can be achieved by utilizing a more biodegradable substrate, crop residues, and

industrial byproducts. Furthermore, the increasing demand for biogas in the future compels

the exploration of any organic raw material for biogas digestion, particularly from the waste

streams [134]. Fruits wastes are an interesting substrate for biogas production since it is

readily available in large quantities, has a low cost, and is less competitive than other food

products. In addition, fruits wastes has a high organic content, which makes it suitable for

biogas production [135].

The accumulation of this waste creates a serious problem, not only for health but also for the

environment. Since it attracts flies and rats, a proper handling of this waste is of importance.

The common practice to handle fruits wastes is landfilling, which is not environmentally

friendly and results in landfill gases and greenhouse gas emissions. Hence, the utilization of

fruits wastes for biogas production is an effort to simultaneously alleviate an environmental

challenge and contribute to the supply of renewable energy.

5.1.1. Theoretical yield calculation

The theoretical methane potential represents the maximum value of methane that can be

produced from a particular organic waste and is often expressed as the biogas volume per unit

of susbtrate. This theoretical methane potential can be predicted from the elemental

composition, component composition, and chemical oxygen demand (COD). If the elemental

composition (C, H, O, N) of the substrate is known, the theoretical yield can be calculated

using the following equation [136]:

CcHhOoNnSs + yH2O → xCH4 + nNH3 + sH2S + (c - x) CO2

34

in the long-term production, biogas produced from this type of material is still debatable

since it creates a moral dilemma in the utilization of land for food or fuel [128]. The social

impact of biogas production is illustrated by taking food from the people of a developing

country and putting it into the cars of people in a developed country. The United Nations

reported that maize used to produce 50 liters of biofuel is equal with amount of maize to feed

one person for one year. Furthermore, a high demand for food crops for biogas production

increases the food price due to the competition of using natural sources such as nitrogen,

land, and water. In addition, there are growing debates on the justification of positive energy

balance of biogas produced from maize as well as the impact on biodiversity, water

resources, and soil quality [129, 130].

In order to overcome the potential conflict between food and energy, the second generation of

biogas has been developed from lignocellulosic materials with low cost, great abundance, and

sustainable supply [131]. Cellulose as the main constituent of lignocellulose is the most

abundant biopolymer on earth [132]. The lignocellulose materials that can be used for biogas

production varies from agricultural residue, forest residue, industrial processing waste, and

municipal solid waste. Biogas production from the waste streams is beneficial not only to

reduce the environmental problem but also to help alleviate the energy problem.

Lignocellulosic materials can be divided into two groups, including cultivated feedstock and

lignocellulose residuals. The latter group is more beneficial since it does not compete with

the usage of natural resources for food and feed production. Moreover, the utilization of

lignocellulosic waste for biogas production can be simultaneously used for waste treatment

and renewable energy production.

35

CHAPTER 5

Anaerobic Digestion of Fruits wastes

5.1. Biogas potential from fruits wastes

Manure is currently the major raw material for biogas production worldwide. However, it has

a relatively low biogas production rate and yield [133]. The high biogas production rate and

yield can be achieved by utilizing a more biodegradable substrate, crop residues, and

industrial byproducts. Furthermore, the increasing demand for biogas in the future compels

the exploration of any organic raw material for biogas digestion, particularly from the waste

streams [134]. Fruits wastes are an interesting substrate for biogas production since it is

readily available in large quantities, has a low cost, and is less competitive than other food

products. In addition, fruits wastes has a high organic content, which makes it suitable for

biogas production [135].

The accumulation of this waste creates a serious problem, not only for health but also for the

environment. Since it attracts flies and rats, a proper handling of this waste is of importance.

The common practice to handle fruits wastes is landfilling, which is not environmentally

friendly and results in landfill gases and greenhouse gas emissions. Hence, the utilization of

fruits wastes for biogas production is an effort to simultaneously alleviate an environmental

challenge and contribute to the supply of renewable energy.

5.1.1. Theoretical yield calculation

The theoretical methane potential represents the maximum value of methane that can be

produced from a particular organic waste and is often expressed as the biogas volume per unit

of susbtrate. This theoretical methane potential can be predicted from the elemental

composition, component composition, and chemical oxygen demand (COD). If the elemental

composition (C, H, O, N) of the substrate is known, the theoretical yield can be calculated

using the following equation [136]:


36

Mole of methane produced (x) = 1/8(4c + h – 2o - 3n – 2s)

If the elemental composition is unknown, the theoretical methane potential can be determined

from the component composition of the substrate such as carbohydrate, fat, and protein

content [137]. Based on the aforementioned equation, the theoretical methane yield of

carbohydrate (C6H10O5), protein (C5H7O2N), and fat (C57H104O6) are 0.42, 0.50, and 1.01

Nm3/kg volatile solid, respectively [138].

The theoretical methane yield can also be calculated from the chemical oxygen demand

(COD) using the following equation:

CH4 + 2O2 CO2 + H2O

Based on the equation, one mole of CH4 needs two moles of oxygen; thus, one kilogram of

COD is equal to 0.35 m3 CH4 at a standard pressure and temperature [139].

The elemental composition of fruits wastes is unknown; however, the component

compositions of several fruits wastes are known and presented in Table 2.1 (Section 2.1).

Hence, the methane potential from the fruits wastes in the present work is predicted

according to the component composition. The average carbohydrate, protein, and fat content

of the fruits wastes are 78.3 %, 8.5 %, and 6 %, respectively.

Table 5.1. Total solid and volatile solid of various fruits [10].

Fruits Total solids (%)

Volatile solids (%)

Avocado 14.9 13.55 Grape 10 8.67 Apple 12 11.76 Star Fruit 7.65 6.95 Guava 9.42 7.53 Orange 9.12 8.95 Longan 16.87 16 Mango 9.01 8.51 Mangosteen 11.99 11.13 Melon 4.87 4.46 Pineapple 10 9.84 Papaya 6.08 5.22 Rambutan 15.92 15.71 Snack Fruit 12 9.41 Watermelon 3.57 2.43

The theoretical methane potential from fruits wastes is further calculated by multiplying the

average percentage of each component with its theoretical methane yield. Accordingly, the

37

methane potential from fruits wastes is 0.43 Nm3/kg volatile solid (VS), which equals to 0.04

Nm3/kg fruits wastes, if the average VS of fruits wastes is 9 % (Table 5.1). The global fruit

production was 804.4 million tons in 2012, of which approximately 50 % ended up as fruits

wastes. Thus, it is estimated that 402.2 million tons of fruits wastes are generated globally,

which can be converted into 16 x 109 Nm3 of methane or 32 x 109 Nm3 of biogas. With an

efficiency of combined heat and power generator for heat and electricity assumed as 50% and

30%, respectively, and 20% loss of energy and each m3 of methane has 11 kWh energy, the

biogas production from fruits wastes is equal to 88 TWh of heat or 52.8 TWh of electricity. If

the produced biogas from fruits wastes is used as fuel, the biogas production from fruits

wastes is equal to 20.8 billion liters of gasoline. If the average gas consumption is assumed to

be 15 kg LPG/month/family, which equals to 34 Nm3biogas/month/family [140], the

generated biogas from the fruits wastes can be used for the cooking needs of 78 million

families in a year.

5.1.2. Experimental approach

The theoretical approach assumes a complete degradation of a substrate into methane. In

practice, however, the theoretical production is difficult to achieve, since the digestibility of

the substrate is commonly in the range of 27–76 % [83]. Moreover, the lack of nutrients, the

presence of an inhibitor compound, and suboptimal conditions during the digestion make the

actual yield lower than that calculated in theory. Therefore, the more accurate method to

obtain the biogas potential of any organic matter is by experiment. The experiment can be

conducted in various scales, from lab to commercial. Basically, biomethane potential (BMP)

assay is conducted by incubating the added waste with the methanogenic inoculum in sealed

vials at a specific temperature. During the digestion, the gas output is monitored regarding the

volume and composition directly using the gas analytical instruments. In this study, the

assessment of the biogas potential was conducted on a lab-scale, both in batch (Papers I–IV)

and semi-continuous method (Papers V–VI).

Batch digestion is a traditional method and mainly used to determine the maximum methane

potential and kinetic measurement of a substrate. In this method, a certain amount of

substrate and inoculum are placed in the reactor, which is then sealed. The reactor is then

incubated under a desired temperature. The batch digestion is widely used for comparison

and evaluation, since many tests can be conducted simultaneously.

36

Mole of methane produced (x) = 1/8(4c + h – 2o - 3n – 2s)

If the elemental composition is unknown, the theoretical methane potential can be determined

from the component composition of the substrate such as carbohydrate, fat, and protein

content [137]. Based on the aforementioned equation, the theoretical methane yield of

carbohydrate (C6H10O5), protein (C5H7O2N), and fat (C57H104O6) are 0.42, 0.50, and 1.01

Nm3/kg volatile solid, respectively [138].

The theoretical methane yield can also be calculated from the chemical oxygen demand

(COD) using the following equation:

CH4 + 2O2 CO2 + H2O

Based on the equation, one mole of CH4 needs two moles of oxygen; thus, one kilogram of

COD is equal to 0.35 m3 CH4 at a standard pressure and temperature [139].

The elemental composition of fruits wastes is unknown; however, the component

compositions of several fruits wastes are known and presented in Table 2.1 (Section 2.1).

Hence, the methane potential from the fruits wastes in the present work is predicted

according to the component composition. The average carbohydrate, protein, and fat content

of the fruits wastes are 78.3 %, 8.5 %, and 6 %, respectively.

Table 5.1. Total solid and volatile solid of various fruits [10].

Fruits Total solids (%)

Volatile solids (%)

Avocado 14.9 13.55 Grape 10 8.67 Apple 12 11.76 Star Fruit 7.65 6.95 Guava 9.42 7.53 Orange 9.12 8.95 Longan 16.87 16 Mango 9.01 8.51 Mangosteen 11.99 11.13 Melon 4.87 4.46 Pineapple 10 9.84 Papaya 6.08 5.22 Rambutan 15.92 15.71 Snack Fruit 12 9.41 Watermelon 3.57 2.43

The theoretical methane potential from fruits wastes is further calculated by multiplying the

average percentage of each component with its theoretical methane yield. Accordingly, the

37

methane potential from fruits wastes is 0.43 Nm3/kg volatile solid (VS), which equals to 0.04

Nm3/kg fruits wastes, if the average VS of fruits wastes is 9 % (Table 5.1). The global fruit

production was 804.4 million tons in 2012, of which approximately 50 % ended up as fruits

wastes. Thus, it is estimated that 402.2 million tons of fruits wastes are generated globally,

which can be converted into 16 x 109 Nm3 of methane or 32 x 109 Nm3 of biogas. With an

efficiency of combined heat and power generator for heat and electricity assumed as 50% and

30%, respectively, and 20% loss of energy and each m3 of methane has 11 kWh energy, the

biogas production from fruits wastes is equal to 88 TWh of heat or 52.8 TWh of electricity. If

the produced biogas from fruits wastes is used as fuel, the biogas production from fruits

wastes is equal to 20.8 billion liters of gasoline. If the average gas consumption is assumed to

be 15 kg LPG/month/family, which equals to 34 Nm3biogas/month/family [140], the

generated biogas from the fruits wastes can be used for the cooking needs of 78 million

families in a year.

5.1.2. Experimental approach

The theoretical approach assumes a complete degradation of a substrate into methane. In

practice, however, the theoretical production is difficult to achieve, since the digestibility of

the substrate is commonly in the range of 27–76 % [83]. Moreover, the lack of nutrients, the

presence of an inhibitor compound, and suboptimal conditions during the digestion make the

actual yield lower than that calculated in theory. Therefore, the more accurate method to

obtain the biogas potential of any organic matter is by experiment. The experiment can be

conducted in various scales, from lab to commercial. Basically, biomethane potential (BMP)

assay is conducted by incubating the added waste with the methanogenic inoculum in sealed

vials at a specific temperature. During the digestion, the gas output is monitored regarding the

volume and composition directly using the gas analytical instruments. In this study, the

assessment of the biogas potential was conducted on a lab-scale, both in batch (Papers I–IV)

and semi-continuous method (Papers V–VI).

Batch digestion is a traditional method and mainly used to determine the maximum methane

potential and kinetic measurement of a substrate. In this method, a certain amount of

substrate and inoculum are placed in the reactor, which is then sealed. The reactor is then

incubated under a desired temperature. The batch digestion is widely used for comparison

and evaluation, since many tests can be conducted simultaneously.

38

Semi-continuous digestion is used to examine the performance and stability of the digester in

the long-term. The advantage of this method over the batch method is the ability to detect

inhibitory compounds at low levels. However, this method is labor intensive and requires

operating experience. The most widely used of the semi-continuous reactor is the

continuously stirred tank reactor (CSTR), since it is available from lab scale to commercial

scale. In the present study, the digestions for Papers V and VI were performed using a CSTR

equipped with an automatic methane potential testing system (AMPTS) to record the biogas

volume continuously.

5.2. Inhibition effect of fruit flavors

Having a high organic content makes fruits an attractive material for biogas production. In

the previous section, the BMP from global fruits wastes was calculated and the result shows

that fruits wastes can produce biogas in considerable amounts. However, in practice, the

actual methane yield from fruits wastes in our preliminary study is far too low, compared to

the BMP (Table 5.1).

Table 5.1. The comparison between actual and theoretical methane yield of several tropical fruits [141].

Fruits Theoretical methane yield (Nm3/kg VS)

Experimental methane yield (Nm3/kg VS)

% from theoretical

Orange peel 0.35 0.09 26 Mangosteen peel 0.21 0 0 Pineapple leaves Rambutan peel

0.35 0.31

0.18 0.12

51 37

These numbers can be explained by the inhibitory activity of the flavor compound on the

anaerobic digesting microorganism, since the fruit flavors have been shown to exhibit

antimicrobial activity against a wide range of microorganisms. Therefore, the effect of

several fruit flavors from each flavor group was investigated in Papers I, II, and III. The

inhibitory activity was determined by the minimum inhibitor concentration (MIC) that

reduces 50 % of the methane production, and the results are presented in Table 5.2.

Among the fruit flavors, myrcene, belonging to the terpenoid group, was found to be the most

toxic compound with a MIC less than 0.005 % (50 ppm). It was followed by octanol with a

MIC between 0.005 to 0.05 %. Since the presence of fruit flavors in the fruits is in the range

of 10–100 ppm, the presence of myrcene and octanol could be one of the reasons for the low

39

methane yield from fruits containing them, such as orange, mango, plum, strawberry, and

grape. This result might answer a question in previous work conducted by Mizuki et al.

[142] about presence of antimicrobial compound in orange peel oil other than D-limonene

which caused faster failure of digestion with addition of orange peel oil than that added with

pure D-limonene since orange peel contains myrcene, car-3-ene, and α-pinene. The toxicity

of, hexanal, E-2- hexenal, and nonanal was moderate, whereas the remaining flavors were

found to be less toxic.

Except for myrcene and octanol, the amount of flavor compounds might not result in an

effect in the anaerobic digestion of fruits wastes. However, since flavor is also produced

synthetically, with a volume of several thousand tons, a considerable amount of fruit flavor

might be present in the wastewater of the flavor industry or other industries that use flavor

such as perfumery, food and beverage, and pharmaceutical. According to our results, the

flavor compounds that have a lower MIC are mostly insoluble in distilled water. This

indicates the relationship between insolubility and the toxicity of flavor compounds.

Therefore, we hypothesized that the inhibitory mechanism of flavor compounds on anaerobic

digesting microorganisms is similar to their inhibitory mechanism toward other

microorganisms, as previously mentioned.

Table 5.2. The minimum inhibitory concentration of various flavor compounds on the anaerobic digesting system.

Flavor group Compound MIC50 (%) Reference Terpenoid α-pinene 0.05–0.5 Paper I Myrcene <0.005 Paper I Car-3-ene 0.05–0.5 Paper I Aldehyde Hexanal 0.05–0.5 Paper I E-2- hexenal 0.05–0.5 Paper I Nonanal 0.05–0.5 Paper I Alcohol Octanol 0.005–0.05 Paper I Esters Methyl butanoate 1–2 Paper II Ethyl butanoate 1–2 Paper II Ethyl hexanoate 1–2 Paper II Hexyl acetate 0.5–1 Paper II Ketone Furaneol >0.5 Paper III Mesifurane >0.5 Paper III Lactone γ-Decalactone 0.05–0.5 Paper III γ-Hexalactone >0.5 Paper III Phenol Epicatechin 0.05–0.5 Paper III Quercetin 0.05–0.5 Paper III

38

Semi-continuous digestion is used to examine the performance and stability of the digester in

the long-term. The advantage of this method over the batch method is the ability to detect

inhibitory compounds at low levels. However, this method is labor intensive and requires

operating experience. The most widely used of the semi-continuous reactor is the

continuously stirred tank reactor (CSTR), since it is available from lab scale to commercial

scale. In the present study, the digestions for Papers V and VI were performed using a CSTR

equipped with an automatic methane potential testing system (AMPTS) to record the biogas

volume continuously.

5.2. Inhibition effect of fruit flavors

Having a high organic content makes fruits an attractive material for biogas production. In

the previous section, the BMP from global fruits wastes was calculated and the result shows

that fruits wastes can produce biogas in considerable amounts. However, in practice, the

actual methane yield from fruits wastes in our preliminary study is far too low, compared to

the BMP (Table 5.1).

Table 5.1. The comparison between actual and theoretical methane yield of several tropical fruits [141].

Fruits Theoretical methane yield (Nm3/kg VS)

Experimental methane yield (Nm3/kg VS)

% from theoretical

Orange peel 0.35 0.09 26 Mangosteen peel 0.21 0 0 Pineapple leaves Rambutan peel

0.35 0.31

0.18 0.12

51 37

These numbers can be explained by the inhibitory activity of the flavor compound on the

anaerobic digesting microorganism, since the fruit flavors have been shown to exhibit

antimicrobial activity against a wide range of microorganisms. Therefore, the effect of

several fruit flavors from each flavor group was investigated in Papers I, II, and III. The

inhibitory activity was determined by the minimum inhibitor concentration (MIC) that

reduces 50 % of the methane production, and the results are presented in Table 5.2.

Among the fruit flavors, myrcene, belonging to the terpenoid group, was found to be the most

toxic compound with a MIC less than 0.005 % (50 ppm). It was followed by octanol with a

MIC between 0.005 to 0.05 %. Since the presence of fruit flavors in the fruits is in the range

of 10–100 ppm, the presence of myrcene and octanol could be one of the reasons for the low

39

methane yield from fruits containing them, such as orange, mango, plum, strawberry, and

grape. This result might answer a question in previous work conducted by Mizuki et al.

[142] about presence of antimicrobial compound in orange peel oil other than D-limonene

which caused faster failure of digestion with addition of orange peel oil than that added with

pure D-limonene since orange peel contains myrcene, car-3-ene, and α-pinene. The toxicity

of, hexanal, E-2- hexenal, and nonanal was moderate, whereas the remaining flavors were

found to be less toxic.

Except for myrcene and octanol, the amount of flavor compounds might not result in an

effect in the anaerobic digestion of fruits wastes. However, since flavor is also produced

synthetically, with a volume of several thousand tons, a considerable amount of fruit flavor

might be present in the wastewater of the flavor industry or other industries that use flavor

such as perfumery, food and beverage, and pharmaceutical. According to our results, the

flavor compounds that have a lower MIC are mostly insoluble in distilled water. This

indicates the relationship between insolubility and the toxicity of flavor compounds.

Therefore, we hypothesized that the inhibitory mechanism of flavor compounds on anaerobic

digesting microorganisms is similar to their inhibitory mechanism toward other

microorganisms, as previously mentioned.

Table 5.2. The minimum inhibitory concentration of various flavor compounds on the anaerobic digesting system.

Flavor group Compound MIC50 (%) Reference Terpenoid α-pinene 0.05–0.5 Paper I Myrcene <0.005 Paper I Car-3-ene 0.05–0.5 Paper I Aldehyde Hexanal 0.05–0.5 Paper I E-2- hexenal 0.05–0.5 Paper I Nonanal 0.05–0.5 Paper I Alcohol Octanol 0.005–0.05 Paper I Esters Methyl butanoate 1–2 Paper II Ethyl butanoate 1–2 Paper II Ethyl hexanoate 1–2 Paper II Hexyl acetate 0.5–1 Paper II Ketone Furaneol >0.5 Paper III Mesifurane >0.5 Paper III Lactone γ-Decalactone 0.05–0.5 Paper III γ-Hexalactone >0.5 Paper III Phenol Epicatechin 0.05–0.5 Paper III Quercetin 0.05–0.5 Paper III

40

41

CHAPTER 6

Addressing the challenges of the inhibitory effects of fruit flavors

In order to improve the biogas production, an effort must be made to solve the challenges

depending on the characteristic of the substrate, such as biomass recalcitrance of the material,

natural presence of an inhibitor compound, or inhibitors produced during the anaerobic

digestion. Biomass recalcitrance mostly related to the inefficient hydrolysis stage due to the

inaccessibility of the microbial enzymes to cleave the monomeric bonds. However, in the

case of readily degradable carbohydrates such as fruits, the issue becomes reverse. The

hydrolysis of fruits wastes occurs rather quickly, resulting in the accumulation of acids and

decreased pH [143]. This problem can be addressed by the co-digestion of nitrogen-rich

materials [144], the addition of a buffering agent [145], and using a two stage process with a

separation acid forming process and a methane forming process [146]. In addition, the

presence of an inhibitor compound is also a major concern in the fruits wastes. Hence, one of

the improvement strategies of biogas production is to deal with the inhibitor compound. In

the present study, two approaches were proposed in order to reduce the inhibition effect: (1)

inhibitor removal by pretreatment of the fruits wastes and (2) developing a robust digesting

system that can protect the microorganism from the inhibitor.

In this work, orange peel waste (OPW) was used as a model, representing fruit waste, since

OPW contains the highest concentration of an inhibitor, namely D-limonene, which

constitutes more than 90 % of the OPW essential oil as 2–3 % component of the dry matter of

OPW. Moreover, OPW is produced in considerable amounts. The generation of OPW is

estimated in the range of 15 to 25 million tons per year [147]. Among these solid wastes,

orange peel contains the highest concentration of D-limonene. The utilization of orange waste

today is limited to pectin, flavonoid, and fiber production, which are mostly applied in food,

functional food, and medicines [148-150]. Furthermore, orange peel waste can be further

processed to animal feed by the drying process. Since orange waste contains a considerable

amount of soluble sugars, it is a potential feedstock for biofuel production such as biogas.

40

41

CHAPTER 6

Addressing the challenges of the inhibitory effects of fruit flavors

In order to improve the biogas production, an effort must be made to solve the challenges

depending on the characteristic of the substrate, such as biomass recalcitrance of the material,

natural presence of an inhibitor compound, or inhibitors produced during the anaerobic

digestion. Biomass recalcitrance mostly related to the inefficient hydrolysis stage due to the

inaccessibility of the microbial enzymes to cleave the monomeric bonds. However, in the

case of readily degradable carbohydrates such as fruits, the issue becomes reverse. The

hydrolysis of fruits wastes occurs rather quickly, resulting in the accumulation of acids and

decreased pH [143]. This problem can be addressed by the co-digestion of nitrogen-rich

materials [144], the addition of a buffering agent [145], and using a two stage process with a

separation acid forming process and a methane forming process [146]. In addition, the

presence of an inhibitor compound is also a major concern in the fruits wastes. Hence, one of

the improvement strategies of biogas production is to deal with the inhibitor compound. In

the present study, two approaches were proposed in order to reduce the inhibition effect: (1)

inhibitor removal by pretreatment of the fruits wastes and (2) developing a robust digesting

system that can protect the microorganism from the inhibitor.

In this work, orange peel waste (OPW) was used as a model, representing fruit waste, since

OPW contains the highest concentration of an inhibitor, namely D-limonene, which

constitutes more than 90 % of the OPW essential oil as 2–3 % component of the dry matter of

OPW. Moreover, OPW is produced in considerable amounts. The generation of OPW is

estimated in the range of 15 to 25 million tons per year [147]. Among these solid wastes,

orange peel contains the highest concentration of D-limonene. The utilization of orange waste

today is limited to pectin, flavonoid, and fiber production, which are mostly applied in food,

functional food, and medicines [148-150]. Furthermore, orange peel waste can be further

processed to animal feed by the drying process. Since orange waste contains a considerable

amount of soluble sugars, it is a potential feedstock for biofuel production such as biogas.

42

6.1. Pretreatment for the removal of fruit flavors

A number of studies have attempted to solve the D-limonene problem in OPW, which can be

classified into three categories: (1) D-limonene removal from OPW; (2) D-limonene recovery

from OPW; (3) D-limonene conversion into other less toxic compounds. Among these

options, D-limonene recovery seems to be the best alternative, since D-limonene is a valuable

product for other industries such as perfumery, chemicals, cosmetics, medical, and food

flavor. In the chemical industry, it is used as a solvent for resins, pigments, inks, and rubber.

The D-limonene recovery technique could adopt the extraction techniques used for essential

oils since that has been established for over a hundred years. Essential oils are comprised of

90 % monoterpenes such as limonene, and the rest is made up of aldehyde, ketone, ester,

ether, acids, and phenol [151]. Essential oils mainly contain lipophilic compounds; thus, there

are several methods that are commonly used to extract the essential oil from the natural

products, including stripping using water vapor, cold pressing, supercritical CO2 extraction,

and leaching extraction.

Stripping using water vapor takes advantage of the higher vapor pressure of the essential

oil than water. This technique can be employed at various scales. On a small scale, this

method can be conducted using a traditional apparatus used in hydrodistillation. After the

stripping, the oil is separated from the water and dried using anhydrous sodium. On a large

scale, this process can be performed both in batch and continuous operation, using water

vapor under vacuum conditions and an elevated temperature, to remove the volatile fatty

acids from the extracted essential oil. In this case, the main product is not in the vapor phase.

The drawback of this method is the degradation of some compounds due to the application of

the high temperature during stripping or water separation, which reduces the quality of the

essential oil.

Cold pressing removes the fluids entrained inside the raw material. The pericarps of the

fruits are pressed and the fluid containing the essential oil is separated. Water is added to

enhance the oil removal from the pericarp, which will be subsequently separated by gravity,

centrifugation, and fractioned distillation. This technique is usually used to extract the

essential oil from the citric fruits.

Supercritical CO2 extraction uses a ‘supercritical state’ of CO2 as a solvent. CO2 is

pressurized at least to 73 bar (critical pressure) at temperature higher than 31 oC (critical

43

temperature). At a supercritical state, the viscosity of CO2 is similar to gas, enabling its

permeation into a solid matrix and its capacity for dissolution is similar to a liquid, thus it can

be used for extraction. After the extraction, the CO2 can be removed by lowering the pressure

to lose its increased ability for dissolution; thereafter, the CO2 can be reused for the next

extraction. The advantages of using this technique are the high efficiency of extraction and

the fact that there is no trace of the solvent in the final product. However, the costs and

technical problems involving high-pressure operation hinder its application in industrial

extraction.

Leaching or solid-liquid extraction was first applied by Robiquet in 1835 to extract the

essential oil from a flower. In this technique, the raw material is dissolved in liquid solvent.

The raw material can be placed in extractors, where the solvent penetrated and dissolves the

flavor. The liquid obtained from the extractors is subsequently pumped into an evaporator to

remove the solvent at the lowest possible temperature. Since essential oils mostly consist of a

lipophilic substance, non-polar solvents should preferably be used. Pentane and hexane are

usually used to attain an adequate selectivity of the extraction. Furthermore, they have low

boiling points, so they can be easily separated from the extract and reused. During the 19th

century, several solvents and patents about the process were registered in several countries of

Europe; gradually, the experiment was conducted independently by industry workers. The

advantage of leaching method is the usage of a lower temperature than that used in

distillation. Therefore, the essential oil obtained by solvent extraction generally has a flavor

that better represents the original flavor in the raw material.

Among the extraction techniques mentioned above, only stripping using water vapor has been

applied as a pretreatment method for citrus waste in biogas and bioethanol production. Table

6.1 presents several pretreatment methods that have been applied in the biogas/bioethanol

production. It can be seen that the most common pretreatment method used for biogas

production is steam explosion. Steam explosion has been found to successfully recover D-

limonene and increase the yield of the biogas. However, this technique is performed under

high pressure and temperature, which requires a high energy consumption. On the other hand,

since the goal of the pretreatment is to improve the biogas as an energy source, the

consumption of energy during the production process could be minimized to obtain a positive

energy balance. Therefore, pretreatments of OPW under an ambient temperature are

favorable. Another method that could be an alternative is biological pretreatment. This

42

6.1. Pretreatment for the removal of fruit flavors

A number of studies have attempted to solve the D-limonene problem in OPW, which can be

classified into three categories: (1) D-limonene removal from OPW; (2) D-limonene recovery

from OPW; (3) D-limonene conversion into other less toxic compounds. Among these

options, D-limonene recovery seems to be the best alternative, since D-limonene is a valuable

product for other industries such as perfumery, chemicals, cosmetics, medical, and food

flavor. In the chemical industry, it is used as a solvent for resins, pigments, inks, and rubber.

The D-limonene recovery technique could adopt the extraction techniques used for essential

oils since that has been established for over a hundred years. Essential oils are comprised of

90 % monoterpenes such as limonene, and the rest is made up of aldehyde, ketone, ester,

ether, acids, and phenol [151]. Essential oils mainly contain lipophilic compounds; thus, there

are several methods that are commonly used to extract the essential oil from the natural

products, including stripping using water vapor, cold pressing, supercritical CO2 extraction,

and leaching extraction.

Stripping using water vapor takes advantage of the higher vapor pressure of the essential

oil than water. This technique can be employed at various scales. On a small scale, this

method can be conducted using a traditional apparatus used in hydrodistillation. After the

stripping, the oil is separated from the water and dried using anhydrous sodium. On a large

scale, this process can be performed both in batch and continuous operation, using water

vapor under vacuum conditions and an elevated temperature, to remove the volatile fatty

acids from the extracted essential oil. In this case, the main product is not in the vapor phase.

The drawback of this method is the degradation of some compounds due to the application of

the high temperature during stripping or water separation, which reduces the quality of the

essential oil.

Cold pressing removes the fluids entrained inside the raw material. The pericarps of the

fruits are pressed and the fluid containing the essential oil is separated. Water is added to

enhance the oil removal from the pericarp, which will be subsequently separated by gravity,

centrifugation, and fractioned distillation. This technique is usually used to extract the

essential oil from the citric fruits.

Supercritical CO2 extraction uses a ‘supercritical state’ of CO2 as a solvent. CO2 is

pressurized at least to 73 bar (critical pressure) at temperature higher than 31 oC (critical

43

temperature). At a supercritical state, the viscosity of CO2 is similar to gas, enabling its

permeation into a solid matrix and its capacity for dissolution is similar to a liquid, thus it can

be used for extraction. After the extraction, the CO2 can be removed by lowering the pressure

to lose its increased ability for dissolution; thereafter, the CO2 can be reused for the next

extraction. The advantages of using this technique are the high efficiency of extraction and

the fact that there is no trace of the solvent in the final product. However, the costs and

technical problems involving high-pressure operation hinder its application in industrial

extraction.

Leaching or solid-liquid extraction was first applied by Robiquet in 1835 to extract the

essential oil from a flower. In this technique, the raw material is dissolved in liquid solvent.

The raw material can be placed in extractors, where the solvent penetrated and dissolves the

flavor. The liquid obtained from the extractors is subsequently pumped into an evaporator to

remove the solvent at the lowest possible temperature. Since essential oils mostly consist of a

lipophilic substance, non-polar solvents should preferably be used. Pentane and hexane are

usually used to attain an adequate selectivity of the extraction. Furthermore, they have low

boiling points, so they can be easily separated from the extract and reused. During the 19th

century, several solvents and patents about the process were registered in several countries of

Europe; gradually, the experiment was conducted independently by industry workers. The

advantage of leaching method is the usage of a lower temperature than that used in

distillation. Therefore, the essential oil obtained by solvent extraction generally has a flavor

that better represents the original flavor in the raw material.

Among the extraction techniques mentioned above, only stripping using water vapor has been

applied as a pretreatment method for citrus waste in biogas and bioethanol production. Table

6.1 presents several pretreatment methods that have been applied in the biogas/bioethanol

production. It can be seen that the most common pretreatment method used for biogas

production is steam explosion. Steam explosion has been found to successfully recover D-

limonene and increase the yield of the biogas. However, this technique is performed under

high pressure and temperature, which requires a high energy consumption. On the other hand,

since the goal of the pretreatment is to improve the biogas as an energy source, the

consumption of energy during the production process could be minimized to obtain a positive

energy balance. Therefore, pretreatments of OPW under an ambient temperature are

favorable. Another method that could be an alternative is biological pretreatment. This

44

method has several advantages such as low energy demand, no chemical requirement, and

mild environmental conditions. However, biological pretreatment occurs very slowly, which

makes the application for industries very unattractive.

Table 6.1. Various pretreatments of citrus waste in biofuel production.

Pretreatment

Methods

Substrate Operation condition Product Improvement Ref.

Physical pretreatment

Steam explosion Orange Heating by 60-bar steam until 150 oC

for 20 min, followed by a pressure

drop.

Biogas 526 % increase in

biogas

[76]

Steam explosion Lemon Heating by 6-bar steam until 160 oC

for 5 min, followed by a pressure drop.

Bioethanol 90 % D-limonene

removal

[152]

Steam explosion Mandarin Heating by 6-bar steam until 160 oC



removal

[153]

Steam explosion Kinnow

mandarin

Heating to 121 oC for 15 min at

pressure of 15 psi, followed by a

pressure drop.


removal

[154]

Steam explosion Orange Heating at 160 oC for 8 min and

injecting steam purge at 120 g/min.


removal

[155]

Steam Distillation Orange Heating using 520 W electric heater

for 1h.

Biogas 70 % D-limonene

removal

[156]

Popping Mandarin Heating at 150 oC for 10 min with

pressure of 15 kgf/cm.

Bioethanol 346 % increase in

bioethanol

[157]

Gamma radiation Orange Radiation at dose of 800 kGy. Bioethanol 60 % D-limonene

removal

[158]

Biological pretreatment

Aspergilus sp. Citrus

unshu

Incubating 50 mg of citrus peel with 1

ml of crude fungal enzyme at 30 oC for

48 h.


removal

[159]

Chemical pretreatment

Leaching method Orange

peel waste

Addition of hexane with OPW/hexane

ratio of 1:12 incubated at room

temperature for 10 minutes


biogas

Paper

IV

The present work investigates the potential of leaching method as an alternative pretreatment

method of OPW, which can be performed at mild conditions (Paper IV). In our preliminary

study, various solvents including ether, dichloromethane, acetate, and hexane were selected.

The results of this preliminary study showed that the usage of hexane as a solvent resulted in

a higher methane production than that of other organic solvents. Thus hexane was used as a

solvent in further studies. Four factors of extraction with two levels, including temperature

45

(20 and 40 oC), time (10 and 300 min), solvent ratio (1:2 and 1:12), and the OPW size

(homogenized and chopped) were investigated. The results showed that the best pretreatment

condition based on the methane yield obtained was for chopped OPW treated at 20 °C for 10

min with a ratio of 1:12. This pretreatment increased the methane yield by more than three

times (Fig. 6.1).

Fig. 6.1. Methane yield of unpretreated and pretreated OPW at room temperature for 10 oC with hexane and OPW ratio of 1:12

(Paper IV).

Although the methane yield was improved by more than three times, the yield was relatively

low compared to other pretreatments for biogas production, mentioned in Table 6.1, which

was in the range of 0.2–0.5 Nm3/kg VS. One explanation could be caused by the toxicity of

the hexane residue in the peel. In order to confirm the toxicity of hexane, batch digestion with

the addition of hexane into the medium was conducted. The results showed that the addition

of hexane at a concentration of 12.67 g/L decreased the methane production by 28.6 %,

compared with the control experiment (Fig. 6.2), whereas the hexane residue in the pretreated

OPW was predicted up to 26.34 g/L (Paper IV). To reduce the hexane residue in the peel, the

pretreated peel can be subjected to e.g., evaporation at 50 oC (Paper IV). The result of this

work revealed that the toxicity of the solvent used in the leaching method as a pretreatment

for biogas or other biological process has to be taken into consideration.

0.061

0.217

0

0.05

0.1

0.15

0.2

0.25

untreated OPW treated OPW

Met

hane

Yie

ld (N

m3 /

kg V

S)

Unpretreated OPW Pretreated OPW

44

method has several advantages such as low energy demand, no chemical requirement, and

mild environmental conditions. However, biological pretreatment occurs very slowly, which

makes the application for industries very unattractive.

Table 6.1. Various pretreatments of citrus waste in biofuel production.

Pretreatment

Methods

Substrate Operation condition Product Improvement Ref.

Physical pretreatment

Steam explosion Orange Heating by 60-bar steam until 150 oC

for 20 min, followed by a pressure

drop.


biogas

[76]

Steam explosion Lemon Heating by 6-bar steam until 160 oC



removal

[152]

Steam explosion Mandarin Heating by 6-bar steam until 160 oC



removal

[153]

Steam explosion Kinnow

mandarin

Heating to 121 oC for 15 min at

pressure of 15 psi, followed by a

pressure drop.


removal

[154]

Steam explosion Orange Heating at 160 oC for 8 min and

injecting steam purge at 120 g/min.


removal

[155]

Steam Distillation Orange Heating using 520 W electric heater

for 1h.


removal

[156]

Popping Mandarin Heating at 150 oC for 10 min with

pressure of 15 kgf/cm.

Bioethanol 346 % increase in

bioethanol

[157]

Gamma radiation Orange Radiation at dose of 800 kGy. Bioethanol 60 % D-limonene

removal

[158]

Biological pretreatment

Aspergilus sp. Citrus

unshu

Incubating 50 mg of citrus peel with 1

ml of crude fungal enzyme at 30 oC for

48 h.


removal

[159]

Chemical pretreatment

Leaching method Orange

peel waste

Addition of hexane with OPW/hexane

ratio of 1:12 incubated at room

temperature for 10 minutes


biogas

Paper

IV

The present work investigates the potential of leaching method as an alternative pretreatment

method of OPW, which can be performed at mild conditions (Paper IV). In our preliminary

study, various solvents including ether, dichloromethane, acetate, and hexane were selected.

The results of this preliminary study showed that the usage of hexane as a solvent resulted in

a higher methane production than that of other organic solvents. Thus hexane was used as a

solvent in further studies. Four factors of extraction with two levels, including temperature

45

(20 and 40 oC), time (10 and 300 min), solvent ratio (1:2 and 1:12), and the OPW size

(homogenized and chopped) were investigated. The results showed that the best pretreatment

condition based on the methane yield obtained was for chopped OPW treated at 20 °C for 10

min with a ratio of 1:12. This pretreatment increased the methane yield by more than three

times (Fig. 6.1).

Fig. 6.1. Methane yield of unpretreated and pretreated OPW at room temperature for 10 oC with hexane and OPW ratio of 1:12

(Paper IV).

Although the methane yield was improved by more than three times, the yield was relatively

low compared to other pretreatments for biogas production, mentioned in Table 6.1, which

was in the range of 0.2–0.5 Nm3/kg VS. One explanation could be caused by the toxicity of

the hexane residue in the peel. In order to confirm the toxicity of hexane, batch digestion with

the addition of hexane into the medium was conducted. The results showed that the addition

of hexane at a concentration of 12.67 g/L decreased the methane production by 28.6 %,

compared with the control experiment (Fig. 6.2), whereas the hexane residue in the pretreated

OPW was predicted up to 26.34 g/L (Paper IV). To reduce the hexane residue in the peel, the

pretreated peel can be subjected to e.g., evaporation at 50 oC (Paper IV). The result of this

work revealed that the toxicity of the solvent used in the leaching method as a pretreatment

for biogas or other biological process has to be taken into consideration.

0.061

0.217

0

0.05

0.1

0.15

0.2

0.25

untreated OPW treated OPW

Met

hane

Yie

ld (N

m3 /

kg V

S)

Unpretreated OPW Pretreated OPW

46

Fig. 6.2. Effect of 13 g/L of n-hexane on anaerobic digesting microorganism at thermophilic batch digestion (Paper IV).

6.2. Microbial protection of fruit flavors by using a membrane bioreactor

6.2.1. MBR for cell protection

Another way to overcome the inhibition problem from D-limonene is by cell protection. This

method is used particularly if we do not intend to recover the D-limonene. In this case, the

OPW can be fed into the digester without any pretreatment prior to the digestion process.

Accordingly, the cost for pretreatment can be omitted. One way to separate the D-limonene

from the cell is by taking advantage of the hydrophobicity of D-limonene. The cells could be

protected in a hydrophilic barrier, which enables the water soluble nutrient to diffuse while

retaining the D-limonene outside. The application of a membrane for a biological process is

known as a membrane bioreactor (MBR). Based on this principle, Pourbafrani et al. [160]

encapsulated the Saccharomyces cerevisiae in an alginate membrane, which prevented the

diffusion of D-limonene to the cell. The results showed that an encapsulated cell could grow

in the presence of 1.5 % D-limonene, whereas 0.5 % of D-limonene already caused the

mortality of the free suspended cell. Youngsukkasem et al. [161] adopted this method to

prevent the D-limonene inhibition on anaerobic digestion. However, the anaerobic digesting

bacteria were able to degrade the membrane made from alginate, which caused a leakage of

the membrane capsule and allowed the D-limonene to pass through and reach the cells [161].

A synthetic membrane, called a polyvinylidene difluoride (PVDF) membrane, was then

tested by Youngsukkasem [162]. This membrane was used to encase the cell in a sachet. The

experiment was conducted by adding different concentrations of D-limonene into the

synthetic medium containing the glucose and the volatile fatty acids in batch digestion. The

0

10

20

30

40

50

60

70

80

0 3 6 9 12 15

Met

han

e yi

eld

(mL

/g V

S))

Digesting time (days)

Water Hexane

47

results showed that the membrane had a positive impact on the protection of the cell from D-

limonene, and unlike alginate, the microbia did not degrade the PVDF membrane. However,

this type of MBR could not be directly applied to OPW or other organic polymers since the

substrate is not soluble in water and has a bigger size than the membrane’s pore. This hinders

the substrate from passing through and being consumed by the encased cell. Using this

method, the digestion process needs to be performed in two stages.

In the present work, organic polymer and OPW were used as the substrates, and a novel MBR

configuration was proposed. In this configuration, the digester contained both the encased

cell in a PVDF membrane sachet and the free suspended cell. Firstly, the free suspended cells

degrade the substrate into soluble sugars, which are able to penetrate the membrane and

further convert the sugars into methane by the encased cells. Having this configuration, the

MBR can be directly applied to any complex material in a single stage process.

Fig. 6.3. Daily methane production of synthetic medium with addition of different concentrations of D-Limonene at day 30 in thermophilic semi-continuous digestion (Paper V).

Prior to the application of MBR using OPW, the new configuration of the MBR was first

examined on a synthetic medium containing an organic polymer, in order to evaluate which

reaction step was most affected by the inhibitor and to investigate the mass transfer of the

substrate. The synthetic medium contained cellulose (Avicel) and a basal medium (Paper V).

Three different concentrations of D-limonene, including 1, 5, and 10 g/L, were added to the

medium. The daily methane production is shown in Fig. 6.3. The results of the present study

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0 10 20 30 40 50 60 70 80

Met

hane

pro

duct

ion

(Nm

3 )


0 g/L D-limonene 1 g/L D-limonene 5 g/L D-limonene 10 g/L D-limonene

46

Fig. 6.2. Effect of 13 g/L of n-hexane on anaerobic digesting microorganism at thermophilic batch digestion (Paper IV).

6.2. Microbial protection of fruit flavors by using a membrane bioreactor

6.2.1. MBR for cell protection

Another way to overcome the inhibition problem from D-limonene is by cell protection. This

method is used particularly if we do not intend to recover the D-limonene. In this case, the

OPW can be fed into the digester without any pretreatment prior to the digestion process.

Accordingly, the cost for pretreatment can be omitted. One way to separate the D-limonene

from the cell is by taking advantage of the hydrophobicity of D-limonene. The cells could be

protected in a hydrophilic barrier, which enables the water soluble nutrient to diffuse while

retaining the D-limonene outside. The application of a membrane for a biological process is

known as a membrane bioreactor (MBR). Based on this principle, Pourbafrani et al. [160]

encapsulated the Saccharomyces cerevisiae in an alginate membrane, which prevented the

diffusion of D-limonene to the cell. The results showed that an encapsulated cell could grow

in the presence of 1.5 % D-limonene, whereas 0.5 % of D-limonene already caused the

mortality of the free suspended cell. Youngsukkasem et al. [161] adopted this method to

prevent the D-limonene inhibition on anaerobic digestion. However, the anaerobic digesting

bacteria were able to degrade the membrane made from alginate, which caused a leakage of

the membrane capsule and allowed the D-limonene to pass through and reach the cells [161].

A synthetic membrane, called a polyvinylidene difluoride (PVDF) membrane, was then

tested by Youngsukkasem [162]. This membrane was used to encase the cell in a sachet. The

experiment was conducted by adding different concentrations of D-limonene into the

synthetic medium containing the glucose and the volatile fatty acids in batch digestion. The

0

10

20

30

40

50

60

70

80

0 3 6 9 12 15

Met

han

e yi

eld

(mL

/g V

S))

Digesting time (days)

Water Hexane

47

results showed that the membrane had a positive impact on the protection of the cell from D-

limonene, and unlike alginate, the microbia did not degrade the PVDF membrane. However,

this type of MBR could not be directly applied to OPW or other organic polymers since the

substrate is not soluble in water and has a bigger size than the membrane’s pore. This hinders

the substrate from passing through and being consumed by the encased cell. Using this

method, the digestion process needs to be performed in two stages.

In the present work, organic polymer and OPW were used as the substrates, and a novel MBR

configuration was proposed. In this configuration, the digester contained both the encased

cell in a PVDF membrane sachet and the free suspended cell. Firstly, the free suspended cells

degrade the substrate into soluble sugars, which are able to penetrate the membrane and

further convert the sugars into methane by the encased cells. Having this configuration, the

MBR can be directly applied to any complex material in a single stage process.

Fig. 6.3. Daily methane production of synthetic medium with addition of different concentrations of D-Limonene at day 30 in thermophilic semi-continuous digestion (Paper V).

Prior to the application of MBR using OPW, the new configuration of the MBR was first

examined on a synthetic medium containing an organic polymer, in order to evaluate which

reaction step was most affected by the inhibitor and to investigate the mass transfer of the

substrate. The synthetic medium contained cellulose (Avicel) and a basal medium (Paper V).

Three different concentrations of D-limonene, including 1, 5, and 10 g/L, were added to the

medium. The daily methane production is shown in Fig. 6.3. The results of the present study

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0 10 20 30 40 50 60 70 80

Met

hane

pro

duct

ion

(Nm

3 )


0 g/L D-limonene 1 g/L D-limonene 5 g/L D-limonene 10 g/L D-limonene

48

showed that using a MBR system, the presence of up to 5 g/L of D-limonene did not affect

the biogas production, whereas in the free cell system, the presence of D-limonene at a

concentration less than 1 g/L already caused a failure of the process [76, 142]. However, the

MBR could not handle the presence of 10 g/L of D-limonene as can be observed by the 50 %

decrease in the methane production and the accumulation of volatile fatty acids that reached

up to 6.75 g/L.

The D-limonene mostly affected the methanogenesis process, as can be observed by the low

methane production and the accumulation of acetic acid up to 4 g/L. This can be explained by

the fact that among the microorganisms involved in anaerobic digestion, methanogen is

considered to be the most sensitive group toward an inhibitor compound [163]. Also, the

structure of the cell membrane of methanogen is different from the other groups, since the

lack of muramic acid makes methanogen more sensitive to fatty acids [98]. The mass transfer

of the nutrient into the encapsulated cell was investigated (Paper V). The results of the

present study showed that the membrane was permeable to glucose and fatty acids as

intermediate products in the digestion system. Thus, the addition of a membrane as a barrier

does not affect the mass transfer of the nutrient to the encased cell (Fig. 6.4).

Fig. 6.4. Membrane permeability for glucose and volatile fatty acids at temperature of 55 oC (Paper V).

Following the success of the novel configuration of MBR to overcome the inhibition problem

by D-limonene on an organic polymer, this method was further tested on OPW as a more

complex material, containing various polymers and D-limonene. Digestion with free cell was

used as a control. The feeding was started with 0.3 g VS/L/day and gradually increased to 3 g

VS/L/day. The daily methane production is presented in Fig. 6.5. The methane production of

0.00

0.50

1.00

1.50

2.00

2.50

0 30 60 90 120 150 180

Con

cent

rati

on (g

/L)

Time (min)

glucose

acetic acid

isobutyric acid

butyric acid

isovaleric acid

valeric acid

caproic acid

49

nic loading rate (OLR) of 2 g VS/L/day.

started to decrease when the OLR was

presence of 6.24 g/L of D-limonene. On the

ncreased until the OLR of 3 g VS/L/day

imonene. The methane yield of OPW using

to 80 % from the theoretical yield at

eld is six times higher, compared to the

in thermophilic semi-

(Paper VI).

r VI).

0

0.5

1

1.5

2

2.5

3

3.5

5 50 55 60 65 70 75 80 85

n time (days)

1.5 2 2.5 3

g Rate (g VS/L/day)reactor Free cell bioreactor

48

showed that using a MBR system, the presence of up to 5 g/L of D-limonene did not affect

the biogas production, whereas in the free cell system, the presence of D-limonene at a

concentration less than 1 g/L already caused a failure of the process [76, 142]. However, the

MBR could not handle the presence of 10 g/L of D-limonene as can be observed by the 50 %

decrease in the methane production and the accumulation of volatile fatty acids that reached

up to 6.75 g/L.

The D-limonene mostly affected the methanogenesis process, as can be observed by the low

methane production and the accumulation of acetic acid up to 4 g/L. This can be explained by

the fact that among the microorganisms involved in anaerobic digestion, methanogen is

considered to be the most sensitive group toward an inhibitor compound [163]. Also, the

structure of the cell membrane of methanogen is different from the other groups, since the

lack of muramic acid makes methanogen more sensitive to fatty acids [98]. The mass transfer

of the nutrient into the encapsulated cell was investigated (Paper V). The results of the

present study showed that the membrane was permeable to glucose and fatty acids as

intermediate products in the digestion system. Thus, the addition of a membrane as a barrier

does not affect the mass transfer of the nutrient to the encased cell (Fig. 6.4).

Fig. 6.4. Membrane permeability for glucose and volatile fatty acids at temperature of 55 oC (Paper V).

Following the success of the novel configuration of MBR to overcome the inhibition problem

by D-limonene on an organic polymer, this method was further tested on OPW as a more

complex material, containing various polymers and D-limonene. Digestion with free cell was

used as a control. The feeding was started with 0.3 g VS/L/day and gradually increased to 3 g

VS/L/day. The daily methane production is presented in Fig. 6.5. The methane production of

0.00

0.50

1.00

1.50

2.00

2.50

0 30 60 90 120 150 180

Con

cent

rati

on (g

/L)

Time (min)

glucose

acetic acid

isobutyric acid

butyric acid

isovaleric acid

valeric acid

caproic acid

49

both free cell and MBR was similar until the organic loading rate (OLR) of 2 g VS/L/day.

However, the methane production of free cell started to decrease when the OLR was

increased to 2.5 g VS/L/day corresponding to the presence of 6.24 g/L of D-limonene. On the

contrary, the methane production of the MBR increased until the OLR of 3 g VS/L/day

corresponding to the presence of 10.45 g/L of D-limonene. The methane yield of OPW using

a MBR reached 0.33 Nm3/kg VS, which corresponds to 80 % from the theoretical yield at

OLR of 3 g VS/L/day (Fig. 6.6). This methane yield is six times higher, compared to the

digestion using free cell at OLR of 3 g VS/L/day.

Fig. 6.5. Daily methane production of OPW digestion using MBR and free cells in thermophilic semi-

continuous digestion (Paper VI).

Fig. 6.6. The methane yield of OPW digestion using MBR and free cell under different organic loading

rates (Paper VI).

0

0.5

1

1.5

2

2.5

3

3.5

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85

Met

hane

pro

duct

ion

(10-3

Nm

3 )


MBR

Free cell

OLR

0

0.1

0.2

0.3

0.4

0.5

0.3 0.6 1 1.5 2 2.5 3

Met

han

e yi

eld

(Nm

3 /kg

VS

)

Organic Loading Rate (g VS/L/day)Theoretical yield membrane bioreactor Free cell bioreactor

50

6.2.2. Outlook and future perspectives on anaerobic MBR for biogas production

Recently, the industrial application of a membrane technology for biological processes has

gained worldwide attention. A separation technique facilitated by the membrane in a MBR

contributes to obtaining high cell density in the reactor [164]. Having high cell density in the

reactor provides many benefits to the process such as increased product concentration and

productivity, reduced product inhibition, and easy recovery of the cells. A membrane

bioreactor has been applied at full scale in various treatments such as municipal/domestic

wastewater [165], industrial wastewater [166], and surface/drinking water [167]. The aerobic

process dominates the application of a MBR in the aforementioned processes; however,

growing interest in the anaerobic membrane bioreactor (AnMBR) has been observed recently,

as can be seen by the increased number of reports on the development of an AnMBR on a

lab/ pilot scale. The operation in an anaerobic digestion provides more benefits to the MBR

process such as lower cost for aeration and sludge handling and more importantly, it also

produces biogas (e.g.,[168]) which has various applications.

Table 6.2. Various applications of an anaerobic digestion MBR.

Application Membrane

types

Configurat

ion

Output Ref

Hydrogen production Ultrafiltration Submerged 50 % increase in

hydrogen

[169]

High-concentration food wastewater Flat-sheet PES External 81.3–94.2 % of COD

removal

[170]

Brewery wastewater +surplus yeast Ceramic tubular External 99 % of COD removal [171]

Cheese whey MF External 98.5 % of COD removal [172]

Distillery produces wastewater flat-sheet Submerged 75–92 % of COD removal [173]

Textile wastewater Hollow fiber MF Submerged 90 % of COD removal [174]

Municipal wastewater Flat-sheet MF

PVDF

Submerged 88 % of COD removal [175]

Domestic wastewater UF membrane External 88 % of COD removal [176]

Slaughterhouse Wastewater MF External 98.75% of COD5 removal [177]

Swine manure Tubular PES,

UF, Membrane

External 86 % of COD removal [178]

Landfill leachate UF External 90 % of COD removal [179]

Orange peel waste Flat sheet Submerged 600 % increase in biogas Paper VI

51

Presently, there are two principally different MBR designs. The membrane can be immersed

in the liquid of the reactor (submerged) or placed outside the reactor where the liquid is

pumped to the membrane (external). A MBR is produced in various types of modules i.e.,

hollow fiber, flatsheet, and tubular. The diverse applications of an AnMBR in various

processes are presented in Table 6.2. Among the various applications of the AnMBR, only

distillery wastewaster treatment has been operated on a full scale, whereas the rest are still at

the level of laboratory and pilot scale. AnMBR is not only applied in digestion, but it can also

be used for biogas purification, as reported by Harasimowitch et al. [180]. The application of

MBR on biogas purification resulted in a better quality of biogas. Moreover, the AnMBR

developed by Youngsukassem et al. [157] was capable of overcoming the inhibition problem

caused by D-limonene. In addition, the results from a very recent study substantiate the

advantages of the application of a MBR on biogas production, from a complex and toxic

substrate where the digester can be performed in a “single-stage process.” This is particularly

important since 93 % of the current installed biogas plants are operated in a single-stage

process.

50

6.2.2. Outlook and future perspectives on anaerobic MBR for biogas production

Recently, the industrial application of a membrane technology for biological processes has

gained worldwide attention. A separation technique facilitated by the membrane in a MBR

contributes to obtaining high cell density in the reactor [164]. Having high cell density in the

reactor provides many benefits to the process such as increased product concentration and

productivity, reduced product inhibition, and easy recovery of the cells. A membrane

bioreactor has been applied at full scale in various treatments such as municipal/domestic

wastewater [165], industrial wastewater [166], and surface/drinking water [167]. The aerobic

process dominates the application of a MBR in the aforementioned processes; however,

growing interest in the anaerobic membrane bioreactor (AnMBR) has been observed recently,

as can be seen by the increased number of reports on the development of an AnMBR on a

lab/ pilot scale. The operation in an anaerobic digestion provides more benefits to the MBR

process such as lower cost for aeration and sludge handling and more importantly, it also

produces biogas (e.g.,[168]) which has various applications.

Table 6.2. Various applications of an anaerobic digestion MBR.

Application Membrane

types

Configurat

ion

Output Ref

Hydrogen production Ultrafiltration Submerged 50 % increase in

hydrogen

[169]

High-concentration food wastewater Flat-sheet PES External 81.3–94.2 % of COD

removal

[170]

Brewery wastewater +surplus yeast Ceramic tubular External 99 % of COD removal [171]

Cheese whey MF External 98.5 % of COD removal [172]

Distillery produces wastewater flat-sheet Submerged 75–92 % of COD removal [173]

Textile wastewater Hollow fiber MF Submerged 90 % of COD removal [174]

Municipal wastewater Flat-sheet MF

PVDF

Submerged 88 % of COD removal [175]

Domestic wastewater UF membrane External 88 % of COD removal [176]

Slaughterhouse Wastewater MF External 98.75% of COD5 removal [177]

Swine manure Tubular PES,

UF, Membrane

External 86 % of COD removal [178]

Landfill leachate UF External 90 % of COD removal [179]

Orange peel waste Flat sheet Submerged 600 % increase in biogas Paper VI

51

Presently, there are two principally different MBR designs. The membrane can be immersed

in the liquid of the reactor (submerged) or placed outside the reactor where the liquid is

pumped to the membrane (external). A MBR is produced in various types of modules i.e.,

hollow fiber, flatsheet, and tubular. The diverse applications of an AnMBR in various

processes are presented in Table 6.2. Among the various applications of the AnMBR, only

distillery wastewaster treatment has been operated on a full scale, whereas the rest are still at

the level of laboratory and pilot scale. AnMBR is not only applied in digestion, but it can also

be used for biogas purification, as reported by Harasimowitch et al. [180]. The application of

MBR on biogas purification resulted in a better quality of biogas. Moreover, the AnMBR

developed by Youngsukassem et al. [157] was capable of overcoming the inhibition problem

caused by D-limonene. In addition, the results from a very recent study substantiate the

advantages of the application of a MBR on biogas production, from a complex and toxic

substrate where the digester can be performed in a “single-stage process.” This is particularly

important since 93 % of the current installed biogas plants are operated in a single-stage

process.

52

53

Conclusion

The inevitable oil depletion and the rising environmental concern promote the development

of renewable energy. Among the alternatives for renewable energy, biogas is found to be the

most efficient energy in terms of energy output/input ratio. The increased demand for biogas

in the future compels the exploration of raw materials for biogas digestion, particularly from

the waste stream since it combines energy production and waste reduction. Fruits wastes are

interesting material for biogas production due to its high organic and water content as well as

its ready availability. However, the results of the recent study revealed that some flavor

compounds in the fruits had a negative impact on biogas production. These compounds

include D-limonene, myrcene, and octanol, which can be found in oranges, mangoes,

strawberries, grapes, and plums.

In order to overcome the inhibition problem caused by the fruit flavors, two approaches have

been proposed in this thesis: (1) pretreatment for the inhibitor removal/recovery, (2)

protection of the cells from the inhibitor using a MBR. Leaching method has been shown to

be a potential pretreatment method of OPW and could increase the methane yield three times

higher than that obtained by the untreated OPW. However, the toxicity of the solvent used for

the pretreatment has to be taken into consideration, in order not to inhibit the digesting

microorganisms. In protecting the cell, MBR is an attractive technology. Not only does MBR

offer cell protection from the inhibitor but it also prevents wash out of the microorganism and

allows for easy cell recovery in the downstream process. A novel MBR configuration

proposed in this work used both encased cells in a membrane sachet and free suspended cells.

Having this configuration, the MBR can be applied directly to the complex toxic substrate;

thus, it is possible to conduct the digestion process in a single stage process. The results

showed that this configuration could handle the digestion of a synthetic medium containing 5

g/L of D-limonene. This configuration also improves the methane yield by more than six

times in the digestion of OPW. It can be concluded that the novel configuration of MBR is a

potential technology for the digestion of any complex substrate, containing an inhibitor.

52

53

Conclusion

The inevitable oil depletion and the rising environmental concern promote the development

of renewable energy. Among the alternatives for renewable energy, biogas is found to be the

most efficient energy in terms of energy output/input ratio. The increased demand for biogas

in the future compels the exploration of raw materials for biogas digestion, particularly from

the waste stream since it combines energy production and waste reduction. Fruits wastes are

interesting material for biogas production due to its high organic and water content as well as

its ready availability. However, the results of the recent study revealed that some flavor

compounds in the fruits had a negative impact on biogas production. These compounds

include D-limonene, myrcene, and octanol, which can be found in oranges, mangoes,

strawberries, grapes, and plums.

In order to overcome the inhibition problem caused by the fruit flavors, two approaches have

been proposed in this thesis: (1) pretreatment for the inhibitor removal/recovery, (2)

protection of the cells from the inhibitor using a MBR. Leaching method has been shown to

be a potential pretreatment method of OPW and could increase the methane yield three times

higher than that obtained by the untreated OPW. However, the toxicity of the solvent used for

the pretreatment has to be taken into consideration, in order not to inhibit the digesting

microorganisms. In protecting the cell, MBR is an attractive technology. Not only does MBR

offer cell protection from the inhibitor but it also prevents wash out of the microorganism and

allows for easy cell recovery in the downstream process. A novel MBR configuration

proposed in this work used both encased cells in a membrane sachet and free suspended cells.

Having this configuration, the MBR can be applied directly to the complex toxic substrate;

thus, it is possible to conduct the digestion process in a single stage process. The results

showed that this configuration could handle the digestion of a synthetic medium containing 5

g/L of D-limonene. This configuration also improves the methane yield by more than six

times in the digestion of OPW. It can be concluded that the novel configuration of MBR is a

potential technology for the digestion of any complex substrate, containing an inhibitor.

54

55

Future Works

The current work is part of efforts to find the best method in overcoming the inhibition

problem in the conversion process of fruits wastes into biogas. The following areas of study

are suggested for the future works:

The study of the inhibition mechanism of fruit flavors on anaerobic digestion is of

importance to further develop the methods to overcome inhibition caused by the

flavor compound.

Fruit flavors in real fruits occur in a mixture, thus, it would be interesting to examine

the effect of fruit flavors extracted from the fruits on anaerobic digestion.

Leaching pretreatment has a potential to be used since it has a low energy

requirement; however, it would be interesting to explore the non-toxic solvents to the

anaerobic digestion microorganisms.

In order to examine the shelf life of the novel MBR configuration, application of this

configuration in a longer period would be of high priority.

54

55

Future Works

The current work is part of efforts to find the best method in overcoming the inhibition

problem in the conversion process of fruits wastes into biogas. The following areas of study

are suggested for the future works:

The study of the inhibition mechanism of fruit flavors on anaerobic digestion is of

importance to further develop the methods to overcome inhibition caused by the

flavor compound.

Fruit flavors in real fruits occur in a mixture, thus, it would be interesting to examine

the effect of fruit flavors extracted from the fruits on anaerobic digestion.

Leaching pretreatment has a potential to be used since it has a low energy

requirement; however, it would be interesting to explore the non-toxic solvents to the

anaerobic digestion microorganisms.

In order to examine the shelf life of the novel MBR configuration, application of this

configuration in a longer period would be of high priority.

56

57

Nomenclature

BOD Biological oxygen demand

AD Anaerobic digestion

MBR Membrane bioreactor

COD Chemical oxygen demand

BMP Biomethane potential

MIC Minimum inhibitory concentration

VFA Volatile fatty acid

OPW Orange peel waste

OLR Organic loading rate

VS Volatile solid


PVDF Polyvinylidene difluoride

PES Polyethersulfone

MF Microfiltration

UF Ultrafiltration

56

57

Nomenclature

BOD Biological oxygen demand

AD Anaerobic digestion


COD Chemical oxygen demand

BMP Biomethane potential

MIC Minimum inhibitory concentration

VFA Volatile fatty acid

OPW Orange peel waste

OLR Organic loading rate

VS Volatile solid


PVDF Polyvinylidene difluoride

PES Polyethersulfone

MF Microfiltration

UF Ultrafiltration

58

59

Acknowledgement

Those who have accomplished a Ph.D. would agree that getting a Ph.D. is a long journey, full of emotional ups and downs. Someone once said that theory is when you know everything but nothing works and practice is when everything works but you don’t know why. The onset of this journey was accompanied with those challenges and even worse, it happened in combination ‘Nothing works and I don’t know why.’ When I look back on those tough days, I remember some great people who have supported me in many ways and made this journey a lot easier and nicer. I believe that reaching the end of this incredible journey would never have been possible without their support and help. Although words will never suffice to show my deep gratitude, I take this opportunity to express my sincere thanks to all of them.

First, my profound thanks and gratitude are addressed to my main supervisor, Prof. Mohammad Taherzadeh for his excellent guidance throughout this work. Thank you for always being available at any time of the day and at any place of the world when I send an email with title ‘discussion.’ You are the one who showed me how to think as a scientist and trained me on how to solve problems. Most importantly, you showed me how to change the word ‘impossible’ to become (I’m) possible. I am forever indebted to you for many of my achievements during the years and in the future.

I wish to express my heartfelt appreciation and gratitude to my co-supervisor Dr. Ria Millati, ST, MT who gave me this opportunity, guided, supported, and encouraged me. We came to know each other seven years ago when I took your course and after that, I accomplished my Bachelor and Master thesis under your supervision. Both your scientific and non-scientific advices were a real support to complete this dissertation. I owe my deepest gratitude to you for many of my accomplishments. Thank you so much for everything.

I would also like to express my deep gratitude to my co-supervisor Prof. Claes Niklasson for giving me this opportunity, support, and concern about the status of my research and organization of my Ph.D. study.

I extend my acknowledgment to my examiner Prof. Michael Skrifars for helping me with the writing of this thesis and the organization of my Ph.D. study.

58

59

Acknowledgement

Those who have accomplished a Ph.D. would agree that getting a Ph.D. is a long journey, full of emotional ups and downs. Someone once said that theory is when you know everything but nothing works and practice is when everything works but you don’t know why. The onset of this journey was accompanied with those challenges and even worse, it happened in combination ‘Nothing works and I don’t know why.’ When I look back on those tough days, I remember some great people who have supported me in many ways and made this journey a lot easier and nicer. I believe that reaching the end of this incredible journey would never have been possible without their support and help. Although words will never suffice to show my deep gratitude, I take this opportunity to express my sincere thanks to all of them.

First, my profound thanks and gratitude are addressed to my main supervisor, Prof. Mohammad Taherzadeh for his excellent guidance throughout this work. Thank you for always being available at any time of the day and at any place of the world when I send an email with title ‘discussion.’ You are the one who showed me how to think as a scientist and trained me on how to solve problems. Most importantly, you showed me how to change the word ‘impossible’ to become (I’m) possible. I am forever indebted to you for many of my achievements during the years and in the future.

I wish to express my heartfelt appreciation and gratitude to my co-supervisor Dr. Ria Millati, ST, MT who gave me this opportunity, guided, supported, and encouraged me. We came to know each other seven years ago when I took your course and after that, I accomplished my Bachelor and Master thesis under your supervision. Both your scientific and non-scientific advices were a real support to complete this dissertation. I owe my deepest gratitude to you for many of my accomplishments. Thank you so much for everything.

I would also like to express my deep gratitude to my co-supervisor Prof. Claes Niklasson for giving me this opportunity, support, and concern about the status of my research and organization of my Ph.D. study.

I extend my acknowledgment to my examiner Prof. Michael Skrifars for helping me with the writing of this thesis and the organization of my Ph.D. study.

60

I would like to thank the Department of Food and Agricultural Product Technology, Universitas Gadjah Mada and the head of the department, Dr. Muhammad Nur Cahyanto for giving me the opportunity to become a sandwich Ph.D. under a joint research between your department and the University of Borås. I also appreciate the invaluable advice and the constructive critique for Papers III and VI.

I am grateful to people at the Swedish Centre for Resource Recovery, University of Borås who have helped me in different ways. Thanks to the present and former head of the department, Dr. Peter Axelberg and Dr. Hans Björk, Dr. Peter Therning, and Dr. Thomas Wahnstöm for their openness toward me and for being available to resolve problems. I would also like to thank Susanne Borg and Sari Sarhamo for their practical support in the administration of the study.

I am also sincerely grateful to my fellow lab-mates and colleagues, Jhosane, Maryam, Solmaz, and Karthik for creating a pleasant working environment that made me enjoy and stay sane during the Ph.D. period. Thank you for the support, help, and warmness that made me feel at home, although I am so far away from my family and home country. I also want to thank Dr. Supansa Youngsukkasem as my co-author for Paper V for her valuable advices, fruitful discussions, support, and kindness. I would also like to thank Dr. Päivi, Dr. Patrik, and Jorge for helping me with the analysis instruments. Thank you also to Dr. Johan, Dr. Gergely, Khamdan, Ram, Julius, Mofoluwake, Adib, Anna, Abbas, Farzad who helped me in different ways. I would also like to thank my master’s and bachelor students for their efforts and contributions to part of this thesis: Sailaja, Ish, Heriyanti, Huong, Ayun, and Tika.

I extend my gratitude to Jonas Hansson and Kristina Laurilla for the technical and practical support in the lab.

I would like to express my gratitude to Dr. Ratna Susandarini from Faculty of Biology, Gadjah Mada University for the critical review on biological context of fruits. I extend my gratitude to Luki for helping in setting layout of the thesis, Queen and ‘Ayun for helping review the text.

I would also like to thank the Indonesian Student Association of Sweden and the Indonesian families in Borås and Gothenburg for their support in many different ways and making me feel at home when I am around them. Special thanks to my best friend and roommate, Xin Luo, for making unforgettable memories during my stay in Sweden.

61

I would like to extend a special thanks and gratitude to the Swedish International Development Agency (SIDA) and the Swedish Gas Centre for their financial support.

Last but most importantly, I would like to thank my family to whom I dedicate this thesis. Without the unwavering love and support from my family, none of my achievements would have been possible. I wish to thank my husband, Indra, for his great understanding, support and unconditional love; my parents, Suhanto and Sri Cahyani who always encouraged me to pursue my dream; to my parents-in-law, Suwardi and Dasilah for their prayers and support; to my sisters and brothers, Hani, Jati, Erni, Anton, Nane, Candra, Dian, Nia; my nieces and nephews Alvin, Bella, Darrel, Dina, Aldan, and Fiza for their love and support. Special thanks to Nia for her help in drawing the molecule structures.

60

I would like to thank the Department of Food and Agricultural Product Technology, Universitas Gadjah Mada and the head of the department, Dr. Muhammad Nur Cahyanto for giving me the opportunity to become a sandwich Ph.D. under a joint research between your department and the University of Borås. I also appreciate the invaluable advice and the constructive critique for Papers III and VI.

I am grateful to people at the Swedish Centre for Resource Recovery, University of Borås who have helped me in different ways. Thanks to the present and former head of the department, Dr. Peter Axelberg and Dr. Hans Björk, Dr. Peter Therning, and Dr. Thomas Wahnstöm for their openness toward me and for being available to resolve problems. I would also like to thank Susanne Borg and Sari Sarhamo for their practical support in the administration of the study.

I am also sincerely grateful to my fellow lab-mates and colleagues, Jhosane, Maryam, Solmaz, and Karthik for creating a pleasant working environment that made me enjoy and stay sane during the Ph.D. period. Thank you for the support, help, and warmness that made me feel at home, although I am so far away from my family and home country. I also want to thank Dr. Supansa Youngsukkasem as my co-author for Paper V for her valuable advices, fruitful discussions, support, and kindness. I would also like to thank Dr. Päivi, Dr. Patrik, and Jorge for helping me with the analysis instruments. Thank you also to Dr. Johan, Dr. Gergely, Khamdan, Ram, Julius, Mofoluwake, Adib, Anna, Abbas, Farzad who helped me in different ways. I would also like to thank my master’s and bachelor students for their efforts and contributions to part of this thesis: Sailaja, Ish, Heriyanti, Huong, Ayun, and Tika.

I extend my gratitude to Jonas Hansson and Kristina Laurilla for the technical and practical support in the lab.

I would like to express my gratitude to Dr. Ratna Susandarini from Faculty of Biology, Gadjah Mada University for the critical review on biological context of fruits. I extend my gratitude to Luki for helping in setting layout of the thesis, Queen and ‘Ayun for helping review the text.

I would also like to thank the Indonesian Student Association of Sweden and the Indonesian families in Borås and Gothenburg for their support in many different ways and making me feel at home when I am around them. Special thanks to my best friend and roommate, Xin Luo, for making unforgettable memories during my stay in Sweden.

61

I would like to extend a special thanks and gratitude to the Swedish International Development Agency (SIDA) and the Swedish Gas Centre for their financial support.

Last but most importantly, I would like to thank my family to whom I dedicate this thesis. Without the unwavering love and support from my family, none of my achievements would have been possible. I wish to thank my husband, Indra, for his great understanding, support and unconditional love; my parents, Suhanto and Sri Cahyani who always encouraged me to pursue my dream; to my parents-in-law, Suwardi and Dasilah for their prayers and support; to my sisters and brothers, Hani, Jati, Erni, Anton, Nane, Candra, Dian, Nia; my nieces and nephews Alvin, Bella, Darrel, Dina, Aldan, and Fiza for their love and support. Special thanks to Nia for her help in drawing the molecule structures.

62

63

References

1. Choudhary, D. and A. Mehta, Fruit Crops. 2010, Jaipur, India: Oxford Book Company. 2. University, T.A.M. Rose Breeding and Genetics Program Lesson Series in Rose Hybridization.

[cited 2014 October 11th]. 3. Hui, Y.H., Handbook of fruit and Vegetable Flavors. 2010, Canada: John Wiley & Sons, Inc. 4. Kessler, G.M., Fruits, vegetables, and flowers: Physiology and Structure in Relation to

Economic Use and Market Quality. Minniepolis, Minnesotta: Burgess Publishing Company. 5. Kalia, A. and R. P. Gupta, Fruit Microbiology, in Handbook of Fruits and Fruit Processing, Y.H.

Hui, Editor. 2006, Blackwell Publishing: Iowa, U.S.A. p. 4. 6. Jiang, Y. and J. Song, Fruits and Fruit Flavor: Classification and Biological Characterization, in

Handbook of Fruit and Vegetable Flavors, Y.H. Hui, Editor. 2010, John Wiley & Sons, Inc: Hoboken, New Jersey. p. 3.

7. Pao, S. and P.D. Petracek Shelf life extension of peeled oranges by citric acid treatment. Food Microbiol., 1997. 14: p. 485-491.

8. Hui, Y.H., Handbook of fruits and fruit processing. 2006, USA: Blackwell publishing. 9. Hui., Y.H., Handbook of fruit and Vegetable Flavors. 2010, Canada: John Wiley & Sons, Inc. 10. Oktavia, N., Karakterisasi Sampah Buah Pasar Buah Gamping sebagai Bahan Baku Alternatif

Produksi Biogas, in Teknologi Pengolahan Hasil Pertanian. 2009, Universitas Gajdah Mada. 11. FAO. Food and Agriculture Organization www.faostat.org. 2014. 12. Parfitt, J., M. Barthel, and S. Macnaughton, Food waste within food supply chains:

quantification and potential for change to 2050. Philosophical Transactions of the Royal Society B: Biological Sciences, 2010. 365(1554): p. 3065-3081.

13. Chattopadhyay, S.K., Handling, Transportation and Storage of Fruits and Vegetables. 2007: India

14. Gustavsson, J., et al. Global Food Losses and Food Waste. 2011. 15. Anonymous, Pollution Prevention and Abatement, Handbook World Bank Group. 1998,

Environment Department: Washington, D.C. 16. Berger, R., Flavours and Fragrances - Chemistry, Bioprocessing and Sustainability. 2007,

Berlin: Springer - Verlag. 17. Janssens, L., et al., Production of flavours by microorganisms Process Biochem 1992. 27: p.

195-215. 18. Rizzolo, J., A. Polesello, and G.Y. Teleky-Vamossy, CGC/Sensoryanalysis of volatile

compounds developed from ripening apple fruit. J High Resolut Chrom, 1989. 12: p. 824-827. 19. MacLeod, A.J. and C.H. Snyder, Volatile components of mango preserved by deep freezing. J

Agric Food Chem 1988. 30(1): p. 137-139. 20. Nursten, H.E. and A.A. Williams, Fruit aromas: a survey of compounds identified. Chem Ind,

1967: p. 486-497. 21. Zabetakis, I. and M.A. Holden, Strawberry fl avour: Analysis and biosynthesis. J Sci Food

Agric, 1997. 74: p. 421-434. 22. Gómez, E., C. Ledbetter, and P. Hartsell, Volatile compounds in apricot, plum and their

interspecific hybrids. J Agric Food Chem., 1993. 41(1669-1676). 23. Muroi, H., A. Kubo, and I. Kubo, Antimicrobial activity of cashew apple flavor compounds. J

Agri Food Chem, 1993. 41: p. 1106-1109. 24. Eduardo, I., et al., Identification of key odor volatile compounds in the essential oil of nine

peach accessions. J Sci Food Agric, 2010. 90: p. 1146-1154.

62

63

References

1. Choudhary, D. and A. Mehta, Fruit Crops. 2010, Jaipur, India: Oxford Book Company. 2. University, T.A.M. Rose Breeding and Genetics Program Lesson Series in Rose Hybridization.

[cited 2014 October 11th]. 3. Hui, Y.H., Handbook of fruit and Vegetable Flavors. 2010, Canada: John Wiley & Sons, Inc. 4. Kessler, G.M., Fruits, vegetables, and flowers: Physiology and Structure in Relation to

Economic Use and Market Quality. Minniepolis, Minnesotta: Burgess Publishing Company. 5. Kalia, A. and R. P. Gupta, Fruit Microbiology, in Handbook of Fruits and Fruit Processing, Y.H.

Hui, Editor. 2006, Blackwell Publishing: Iowa, U.S.A. p. 4. 6. Jiang, Y. and J. Song, Fruits and Fruit Flavor: Classification and Biological Characterization, in

Handbook of Fruit and Vegetable Flavors, Y.H. Hui, Editor. 2010, John Wiley & Sons, Inc: Hoboken, New Jersey. p. 3.

7. Pao, S. and P.D. Petracek Shelf life extension of peeled oranges by citric acid treatment. Food Microbiol., 1997. 14: p. 485-491.

8. Hui, Y.H., Handbook of fruits and fruit processing. 2006, USA: Blackwell publishing. 9. Hui., Y.H., Handbook of fruit and Vegetable Flavors. 2010, Canada: John Wiley & Sons, Inc. 10. Oktavia, N., Karakterisasi Sampah Buah Pasar Buah Gamping sebagai Bahan Baku Alternatif

Produksi Biogas, in Teknologi Pengolahan Hasil Pertanian. 2009, Universitas Gajdah Mada. 11. FAO. Food and Agriculture Organization www.faostat.org. 2014. 12. Parfitt, J., M. Barthel, and S. Macnaughton, Food waste within food supply chains:

quantification and potential for change to 2050. Philosophical Transactions of the Royal Society B: Biological Sciences, 2010. 365(1554): p. 3065-3081.

13. Chattopadhyay, S.K., Handling, Transportation and Storage of Fruits and Vegetables. 2007: India

14. Gustavsson, J., et al. Global Food Losses and Food Waste. 2011. 15. Anonymous, Pollution Prevention and Abatement, Handbook World Bank Group. 1998,

Environment Department: Washington, D.C. 16. Berger, R., Flavours and Fragrances - Chemistry, Bioprocessing and Sustainability. 2007,

Berlin: Springer - Verlag. 17. Janssens, L., et al., Production of flavours by microorganisms Process Biochem 1992. 27: p.

195-215. 18. Rizzolo, J., A. Polesello, and G.Y. Teleky-Vamossy, CGC/Sensoryanalysis of volatile

compounds developed from ripening apple fruit. J High Resolut Chrom, 1989. 12: p. 824-827. 19. MacLeod, A.J. and C.H. Snyder, Volatile components of mango preserved by deep freezing. J

Agric Food Chem 1988. 30(1): p. 137-139. 20. Nursten, H.E. and A.A. Williams, Fruit aromas: a survey of compounds identified. Chem Ind,

1967: p. 486-497. 21. Zabetakis, I. and M.A. Holden, Strawberry fl avour: Analysis and biosynthesis. J Sci Food

Agric, 1997. 74: p. 421-434. 22. Gómez, E., C. Ledbetter, and P. Hartsell, Volatile compounds in apricot, plum and their

interspecific hybrids. J Agric Food Chem., 1993. 41(1669-1676). 23. Muroi, H., A. Kubo, and I. Kubo, Antimicrobial activity of cashew apple flavor compounds. J

Agri Food Chem, 1993. 41: p. 1106-1109. 24. Eduardo, I., et al., Identification of key odor volatile compounds in the essential oil of nine

peach accessions. J Sci Food Agric, 2010. 90: p. 1146-1154.

64

25. Hakala, M., A.T. Lapvetela-Inen, and H.P. Kallio, Volatile Compounds of Selected Strawberry Varieties Analyzed by Purge-and-Trap Headspace GCMS. J. Agric. Food Chem, 2002. 50: p. 1133-1142.

26. Wyllie, S.G., et al., Volatile fl avor components of Annona atemoya (custard apple). J Agric Food Chem, 1987. 35: p. 770-774.

27. MacLeod, A.J. and N.G. Gonzalez de Troconis, Volatile flavor components of mango fruit Phytochemistry, 1982. 21: p. 2523-2526.

28. Etievant, P., E. Guichard, and S. Issanchou, The flavour components of Mirabelle plums. Examination of the aroma constituents of fresh fruits: Variation of the headspace composition induced by deep - freezing and thawing. Sci Aliments., 1986. 6: p. 417-432.

29. Franco, M.R.B., A.D. Rodrignez, and F.M. Lancas, Volatile composition of three cultivars of mango ( Mangifera indica L.). Ciencia e Tecnologia de Alimentos, 2004. 24(2): p. 165-169.

30. MacLeod, A.J. and C.H. Snyder, Volatile components of two cultivars of mango from Florida. J Agric Food Chem, 1985. 33: p. 380-384.

31. Hunter, G.L.K., W. Bucek, and T. Radford, Volatile components of canned Alphonso mango. J Food Sci, 1974. 39: p. 900-903.

32. Flath, R.A., et al., Headspace examination of volatile emissions from ripening papaya ( Carica papaya L. Solo variety). J Agric Food Chem, 1990. 38: p. 1060-1063.

33. Larsen, M. and L. Poll, Odor thresholds of some important aroma compounds in strawberries. Lebensm. Unters. Forsch., 1992. 195: p. 120-123.

34. Idstein, H. and P. Schreier, Volatile Constituents Of Alphonso Mango (Mangifera Indica). Phytochemistry, 1985. 24: p. 2313-2316.

35. Chen, Y. and L.M. Sidisky, Quantification of 4-hydroxy-2,5-dimethyl-3- furanone in fruit samples using solid phase microextraction coupled with gas chromatography-mass spectrometry. J Chromatogr A, 2011. 1218(38): p. 6817-6822.

36. Chassagne, D., R. Boulanger, and J. Crouzet, Enzymatic hydrolysis of edible Passiora fruit glycosides. Food Chem, 1999. 66: p. 281-288.

37. Xiaofen, D., E.F. Chad, and C.Q. Michael, Volatile composition and odour-activity value of thornless ‘Black Diamond’ and ‘Marion’ blackberries. Food Chemistry., 2009.

38. Jetti, R.R., et al., Quantification of Selected Aroma-Active Compounds in Strawberries by Headspace Solid-Phase Microextraction Gas. J Food Sci, 2007. 72(7).

39. Jorge, A.P., et al., Volatile Components from Mango (Mangifera indica L.) Cultivars. 2004. 40. Brito, E.S.d., et al., Determination of the flavonoid components of cashew appl (Anacardium

occidentale) by LC-DAD-ESI/MS. Food Chem, 2007. 105: p. 1112-1118. 41. Häkkinen, S.H., et al., Content of the flavonolsquercetin, myricetin, and kaempferol in 25

edible berries. J Agric Food Chem, 1999. 47(6): p. 2274-2279. 42. Hertog, et al., Content of Potentially Anticarcinogenic Flavonoids of 28 Vegetables and 9

Fruits Commonly Consumed in The Netherlands. J. Agric. Food Chem, 1992. 40: p. 2379-2383. 43. Bilyk, A. and G.M. Sapers, Varietal Differences in the Quercetin, Kaempferol, and Myricetin

Contents of Highbush Blueberry, Cranberry, and Thornless Blackberry Fruits. Journal of Agric. And Food Chem, 1986. 34(4).

44. Tsanova-Savova, S., R. Fany, and G. Maria, (+)-Catechin and (-) Epicatechin in Bulgarian Fruits. J Food Comp Anal, 2005. 18: p. 691-698.

45. Anastasiadi, M., et al., Bioactive Non-Coloured Polyphenols Content of Grapes, Wines and Vinification By-Products: Evaluation of The Antioxidant Activities of Their Extracts. Food Res Int, 2010. 43: p. 805-813.

46. Schieber, A., K. Petra, and C. Reinhold, Determination of Phenolic Acids and Flavonoids of Apple and Pear by High-Performance Liquid Chromatography. Journal of Chromatography A, 2000. 910: p. 265-273.

65

47. Tokitomo, Y., et al., Odor Active Constituents in Fresh Pineapple (Ananas comosus [L.] Merr.) by Quantitative and Sensory Evaluation. Biosci. Biotechnol. Biochem, 2005. 69(7): p. 1323-1330.

48. Pino, J.A., et al., Volatile Components from Mango (Mangifera indica L.). Cultivars., 2004. 49. Guichard, E.I. and M. Souty, Comparison of The Relative Quantities of Aroma Compounds

Found in Fresh Apricot from Six Different Varieties (Prunus armeniaca) from Six Different Varieties. 1987, France.

50. Wei, C.-B., et al., Characteristic Aroma Compounds from Different Pineapple Parts. Molecules, 2011. 16: p. 5104-5112.

51. Welsh, F., W. Murray, and R. Williams, Microbiological and enzymatic production of flavor and fragrance chemicals Crit Rev Biotechnol, 1989. 9(2): p. 105-169.

52. Krings, U. and R. Berger, Biotechnological production of fl avours and fragrances. Appl Microbiol Biotechnol, 1998. 49: p. 1-8.

53. Rodin, J., et al., Volatile flavor and aroma components of pineapple. J Food Sci, 1965 30: p. 280-285.

54. Lavid, N., et al., Aroma biosynthesis in strawberry: S - adenosylmethionine : furaneol O - methyltransferase activity in ripening fruits. J Agric Food Chem, 2002. 50(14): p. 4025-4030.

55. World Flavors and Fragrances. 56. Schwab, W., R. Davidovich-Rikanati, and E. Lewinsohn, Harnessing plant biomass for biofuels

and biomaterials. Biosynthesis of plant-derived flavor compounds. The Plant Journal, 2008. 54: p. 712-732.

57. Medeiros, A.B.P., S.C. Rossi, and C.R. SoccoL, Cell Culture for Flavor Production, in Handbook of Fruit and Vegetable Flavors, Y.H. Hui, Editor. 2010, John Wiley & Sons, Inc.: Hobokyen, New Jerse.

58. Engel, K.-H. and I. Roling, Generation of fl avors by microorganisms and enzymes: An overview, in Biotechnology for Improved Foods and Flavors, G.R. Takeoka, et al., Editors. 1996 American Chemical Society: Washington DC. p. 120-123.

59. Osorio, S., c. Muñoz, and v. Valpuesta, Physiology and Biochemistry of Fruit Flavors, in Handbook of Fruit and Vegetable Flavors, Y.H. Hui, Editor. 2010, John Wiley & Sons, Inc.: Hoboken, New Jersey.

60. Basear, K. and F. Demirci, Chemistry of essential oil, in Flavors and Fragrances, B. RG, Editor. 2007, Springer Heidelberg. p. 43.

61. Rodriguez - Concepcion, M. and A. Boronat, Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol 2002 130: p. 1079-1089.

62. Buttery, R. and L. Ling, Volatiles of tomato fruit and plant parts: Relationship and biogenesis, in Bioactive Volatile Compounds from Plants, R. Teranishsi, R. Buttery, and H. Sugisawa, Editors. 1993 Washington, DC : ACS Books p. 11.

63. Aubert, C., S. Baumann, and H. Arguel, Optimization of the analysis of flavor volatile compounds by liquid - liquid microextraction (LLME). Application to the aroma analysis of melons, peaches, grapes, strawberries, and tomatoes. J Agric Food Chem, 2005. 53: p. 8881-8895.

64. Utama, I., et al., In Vitro Efficacy of Plant Volatiles for Inhibiting the Growth of Fruit and Vegetable Decay Microorganisms. J Agri Food Chem, 2002. 50(22): p. 6371-6377.

65. Cowan, M.M., Plant Products as Antimicrobial Agents. Clin. Mcrobiol. Rev., 1999. 12: p. 564-582.

66. Patrignani, F., Lucci,L., Belletti, N., Gardini, F., Guerzoni, M.E., Lanciotti, R., ,Effects of sub-lethal concentrations of hexanal and 2-(E)-hexenal on membrane fatty acid composition and volatile compounds of Listeria monocytogenes, Staphylococcus aureus,Salmonella enteritidis and Escherichia coli. Int J Food Microbiol, 2008. 123: p. 1-8.

64

25. Hakala, M., A.T. Lapvetela-Inen, and H.P. Kallio, Volatile Compounds of Selected Strawberry Varieties Analyzed by Purge-and-Trap Headspace GCMS. J. Agric. Food Chem, 2002. 50: p. 1133-1142.

26. Wyllie, S.G., et al., Volatile fl avor components of Annona atemoya (custard apple). J Agric Food Chem, 1987. 35: p. 770-774.

27. MacLeod, A.J. and N.G. Gonzalez de Troconis, Volatile flavor components of mango fruit Phytochemistry, 1982. 21: p. 2523-2526.

28. Etievant, P., E. Guichard, and S. Issanchou, The flavour components of Mirabelle plums. Examination of the aroma constituents of fresh fruits: Variation of the headspace composition induced by deep - freezing and thawing. Sci Aliments., 1986. 6: p. 417-432.

29. Franco, M.R.B., A.D. Rodrignez, and F.M. Lancas, Volatile composition of three cultivars of mango ( Mangifera indica L.). Ciencia e Tecnologia de Alimentos, 2004. 24(2): p. 165-169.

30. MacLeod, A.J. and C.H. Snyder, Volatile components of two cultivars of mango from Florida. J Agric Food Chem, 1985. 33: p. 380-384.

31. Hunter, G.L.K., W. Bucek, and T. Radford, Volatile components of canned Alphonso mango. J Food Sci, 1974. 39: p. 900-903.

32. Flath, R.A., et al., Headspace examination of volatile emissions from ripening papaya ( Carica papaya L. Solo variety). J Agric Food Chem, 1990. 38: p. 1060-1063.


34. Idstein, H. and P. Schreier, Volatile Constituents Of Alphonso Mango (Mangifera Indica). Phytochemistry, 1985. 24: p. 2313-2316.

35. Chen, Y. and L.M. Sidisky, Quantification of 4-hydroxy-2,5-dimethyl-3- furanone in fruit samples using solid phase microextraction coupled with gas chromatography-mass spectrometry. J Chromatogr A, 2011. 1218(38): p. 6817-6822.

36. Chassagne, D., R. Boulanger, and J. Crouzet, Enzymatic hydrolysis of edible Passiora fruit glycosides. Food Chem, 1999. 66: p. 281-288.

37. Xiaofen, D., E.F. Chad, and C.Q. Michael, Volatile composition and odour-activity value of thornless ‘Black Diamond’ and ‘Marion’ blackberries. Food Chemistry., 2009.

38. Jetti, R.R., et al., Quantification of Selected Aroma-Active Compounds in Strawberries by Headspace Solid-Phase Microextraction Gas. J Food Sci, 2007. 72(7).

39. Jorge, A.P., et al., Volatile Components from Mango (Mangifera indica L.) Cultivars. 2004. 40. Brito, E.S.d., et al., Determination of the flavonoid components of cashew appl (Anacardium

occidentale) by LC-DAD-ESI/MS. Food Chem, 2007. 105: p. 1112-1118. 41. Häkkinen, S.H., et al., Content of the flavonolsquercetin, myricetin, and kaempferol in 25

edible berries. J Agric Food Chem, 1999. 47(6): p. 2274-2279. 42. Hertog, et al., Content of Potentially Anticarcinogenic Flavonoids of 28 Vegetables and 9

Fruits Commonly Consumed in The Netherlands. J. Agric. Food Chem, 1992. 40: p. 2379-2383. 43. Bilyk, A. and G.M. Sapers, Varietal Differences in the Quercetin, Kaempferol, and Myricetin

Contents of Highbush Blueberry, Cranberry, and Thornless Blackberry Fruits. Journal of Agric. And Food Chem, 1986. 34(4).

44. Tsanova-Savova, S., R. Fany, and G. Maria, (+)-Catechin and (-) Epicatechin in Bulgarian Fruits. J Food Comp Anal, 2005. 18: p. 691-698.

45. Anastasiadi, M., et al., Bioactive Non-Coloured Polyphenols Content of Grapes, Wines and Vinification By-Products: Evaluation of The Antioxidant Activities of Their Extracts. Food Res Int, 2010. 43: p. 805-813.

46. Schieber, A., K. Petra, and C. Reinhold, Determination of Phenolic Acids and Flavonoids of Apple and Pear by High-Performance Liquid Chromatography. Journal of Chromatography A, 2000. 910: p. 265-273.

65

47. Tokitomo, Y., et al., Odor Active Constituents in Fresh Pineapple (Ananas comosus [L.] Merr.) by Quantitative and Sensory Evaluation. Biosci. Biotechnol. Biochem, 2005. 69(7): p. 1323-1330.

48. Pino, J.A., et al., Volatile Components from Mango (Mangifera indica L.). Cultivars., 2004. 49. Guichard, E.I. and M. Souty, Comparison of The Relative Quantities of Aroma Compounds

Found in Fresh Apricot from Six Different Varieties (Prunus armeniaca) from Six Different Varieties. 1987, France.

50. Wei, C.-B., et al., Characteristic Aroma Compounds from Different Pineapple Parts. Molecules, 2011. 16: p. 5104-5112.

51. Welsh, F., W. Murray, and R. Williams, Microbiological and enzymatic production of flavor and fragrance chemicals Crit Rev Biotechnol, 1989. 9(2): p. 105-169.

52. Krings, U. and R. Berger, Biotechnological production of fl avours and fragrances. Appl Microbiol Biotechnol, 1998. 49: p. 1-8.

53. Rodin, J., et al., Volatile flavor and aroma components of pineapple. J Food Sci, 1965 30: p. 280-285.

54. Lavid, N., et al., Aroma biosynthesis in strawberry: S - adenosylmethionine : furaneol O - methyltransferase activity in ripening fruits. J Agric Food Chem, 2002. 50(14): p. 4025-4030.

55. World Flavors and Fragrances. 56. Schwab, W., R. Davidovich-Rikanati, and E. Lewinsohn, Harnessing plant biomass for biofuels

and biomaterials. Biosynthesis of plant-derived flavor compounds. The Plant Journal, 2008. 54: p. 712-732.

57. Medeiros, A.B.P., S.C. Rossi, and C.R. SoccoL, Cell Culture for Flavor Production, in Handbook of Fruit and Vegetable Flavors, Y.H. Hui, Editor. 2010, John Wiley & Sons, Inc.: Hobokyen, New Jerse.

58. Engel, K.-H. and I. Roling, Generation of fl avors by microorganisms and enzymes: An overview, in Biotechnology for Improved Foods and Flavors, G.R. Takeoka, et al., Editors. 1996 American Chemical Society: Washington DC. p. 120-123.

59. Osorio, S., c. Muñoz, and v. Valpuesta, Physiology and Biochemistry of Fruit Flavors, in Handbook of Fruit and Vegetable Flavors, Y.H. Hui, Editor. 2010, John Wiley & Sons, Inc.: Hoboken, New Jersey.

60. Basear, K. and F. Demirci, Chemistry of essential oil, in Flavors and Fragrances, B. RG, Editor. 2007, Springer Heidelberg. p. 43.

61. Rodriguez - Concepcion, M. and A. Boronat, Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol 2002 130: p. 1079-1089.

62. Buttery, R. and L. Ling, Volatiles of tomato fruit and plant parts: Relationship and biogenesis, in Bioactive Volatile Compounds from Plants, R. Teranishsi, R. Buttery, and H. Sugisawa, Editors. 1993 Washington, DC : ACS Books p. 11.

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81. Schultz, T.W. and A.D. Deweese, Structure-Toxicity Relationships for Selected Lactones to Tetrahymena pyriformis. Bull. Environ. Contam. Toxicol, 1999. 62: p. 463-468.

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89. Baere, L.D. and B. Mattheeuws, Anaerobic Digestion of the Organic Fractionof Municipal Solid Waste in Europe – Status, Experience and Prospects –. Waste Management, 2013. 3: p. 517-526.

90. Angelidaki, I., et al., Anaerobic processes, in Environmental biotechnology, A. I, Editor. 2002, Institute of Environment and Resources. Technical University of Denmark (DTU). p. 1-114.

91. Parawira, W., et al., Profile of hydrolases and biogas production during two-stage mesophilic anaerobic digestion of solid potato waste. Proc Biochem, 2005. 40(9): p. 2945-2952.

92. Vavilin, V.A., S.V. Rytov, and L.Y. Lokshina, A description of hydrolysis kinetics in anaerobic degradation of particulate organic matter. Bioresource Technology, 1996. 56(2-3): p. 229-237.

93. Björnsson, L., et al., Evaluation of new methods for the monitoring of alkalinity, dissolved hydrogen and the microbial community in anaerobic digestion. Water Research, 2001. 35(12): p. 2833-2840.

94. Schink, B., Energetics of syntrophic cooperation in methanogenic degradation. . Microbiol Mol Biol Rev, 1997. 61(2): p. 262-280.

95. Kotsyurbenko, O.R., Trophic interactions in the methanogenic microbial community of low-temperature terrestrial ecosystems. FEMS Microbiol Ecol, 2005. 53(1): p. 3-13.

96. Petersen, S.P. and B.K. Ahring, Acetate oxidation in thermophilic anaerobic sewage sluge digester: the importance of non-aceticlastic methanogenesis of acetate. . FEMS Microbial Ecology, 1991. 86: p. 149-158.

97. Pohland, F.G. and S. Ghosh, Developments in anaerobic stabilization of organic wastes - the two-phase concept. . Environ. Lett., 1971. 1(255-256).

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99. Demirel, B. and O. Yenigu¨n, Two-phase anaerobic digestion processes: a review. J. Chem. Tech. Biotechnol., 2002. 77: p. 743-755.

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101. Bidlack, J., M. Malone, and R. Benson, Proceeding of Oklahoma Academy of Science, 1992. 72: p. 51.

102. Brett, C.T., Cellulose microfibrils in plants: Biosynthesis, deposition,and integration into the cell wall. Int Rev Cytol. 2000: Academic Press. 161-199.

103. Taherzadeh, M.J. and A. Jeihanipour, Recalcitrance of Lignocellulosic Biomass to Anaerobic Digestion, in Biogas Production: Pretreatment Methods in Anaerobic Digestion, A. Mudhoo, Editor. 2012, Scrivener Publishing, LLC.

104. Harris, P.J. and B.A. Stone, Chemistry and Molecular Organization of Plant Cell Walls, in Biomass Recalcitrance. 2009, Blackwell Publishing LTd. p. 61-93.

105. Kroeker, E.J., et al., Anaerobic treatment process stability. J. Water Pollut. Control Fed, 1979. 51: p. 718-727.

106. Ahring, B.K., M. Sandberg, and I. Andelidaki, Volatile fatty acids as indicators of process imbalance in anaerobic digesters. . Appl Microbiol Biotechnol, 1995. 43: p. 559-565.

107. Chen, Y., J.J. Cheng, and K.S. Creamer, Inhibition of anaerobic digestion process: A review. Bioresour Technol, 2008. 99: p. 4044-4064.

108. Kayhanian, M., Ammonia inhibition in high-solids biogasification: an overview and practical solutions. Environ. Technol., 1999. 20: p. 355-365.

109. Sprott, G.D. and G.B. Patel, Ammonia toxicity in pure cultures of methanogenic bacteria. System. Appl. Microbiol., 1986. 7: p. 358-363.

110. Whittmann, C., A.P. Zeng, and W.D. Deckwer, Growth inhibition by ammonia and use of pH-controlled feeding strategy for the effective cultivation of Mycobacterium chlorophenolicum. Appl. Microbiol. Biotechnol. Lett., 1995. 44: p. 519-525.

66

67. Wenlian, D., et al., Effect of six-carbon aldehydes and alcohols on bacterial proliferation. J. Agric. Food Chem, 1993. 41: p. 506-510.

68. Aguedo, M., et al., Interaction of Odorous Lactones with Phospholipids: Implications in Toxicity Towards Producing Yeast Cells. Biotechnol Lett 2002. 24: p. 1975-1979.







75. Wilkins, M.R., W.W. Widmer, and K. Grohmann, Simultaneous saccharification and fermentation of citrus peel waste by Saccharomyces cerevisiae to produce ethanol. Proc Biochem, 2007. 42: p. 1614-1619.



78. Van Vuuren, S., F and A.M. Viljoen, Antimicrobial activity of limonene enantiomers and 1,8-cineole alone and in combination. Flavour Fragr. J., 2007. 22: p. 540-544.

79. Lanciotti, R., et al., Application of hexanal, (E)-2-hexenal, and hexyl acetate to improve the safety of fresh-sliced apples. J. Agric. Food Chem, 2003. 51: p. 2958 - 2963.

80. Sung, W.S., et al., 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF); antimicrobial compound with cell cycle arrest in nosocomial pathogens. Life Sci, 2007. 80: p. 586-591.

81. Schultz, T.W. and A.D. Deweese, Structure-Toxicity Relationships for Selected Lactones to Tetrahymena pyriformis. Bull. Environ. Contam. Toxicol, 1999. 62: p. 463-468.

82. Weiland, P., Biogas production: current state and perspectives. Appl Microbiol Biotechnol, 2010. 85(4): p. 849-860.


84. Agency., E.E.E. Why did greenhouse gas emissions fall in the EU in 2009. 2010 [cited 2014 October 1th]; Available from: www.eea.europa.eu/publications/european-union-greenhouse-gas-inventory-2011/why-did greenhouse-gas-emissions.


86. EurObserv’ER. Biogas Barometer. 2012 [cited 2014 October 1th]; Available from: http://www.energies-renouvelables.org/observ-er/stat_baro/observ/baro212biogas.pdf.

87. Pike Research. Renewable biogas. Methane Recovery and Utilization in Landfills and Anaerobic Digesters: Municipal Solid Waste, Agricultural, Industrial, and Wastewater Market Report on Analysis and Forecasts. . 2012: Boulder.

88. European biogas association. EBA presents latest biogas production statistics europe growth continuous. 2013 [cited 2014 August 1th]; Available from: http://european-biogas.eu/2013/12/20/eba-presents-latest-biogas-production-statistics-europe-growth-continuous/.

67

89. Baere, L.D. and B. Mattheeuws, Anaerobic Digestion of the Organic Fractionof Municipal Solid Waste in Europe – Status, Experience and Prospects –. Waste Management, 2013. 3: p. 517-526.

90. Angelidaki, I., et al., Anaerobic processes, in Environmental biotechnology, A. I, Editor. 2002, Institute of Environment and Resources. Technical University of Denmark (DTU). p. 1-114.

91. Parawira, W., et al., Profile of hydrolases and biogas production during two-stage mesophilic anaerobic digestion of solid potato waste. Proc Biochem, 2005. 40(9): p. 2945-2952.

92. Vavilin, V.A., S.V. Rytov, and L.Y. Lokshina, A description of hydrolysis kinetics in anaerobic degradation of particulate organic matter. Bioresource Technology, 1996. 56(2-3): p. 229-237.

93. Björnsson, L., et al., Evaluation of new methods for the monitoring of alkalinity, dissolved hydrogen and the microbial community in anaerobic digestion. Water Research, 2001. 35(12): p. 2833-2840.

94. Schink, B., Energetics of syntrophic cooperation in methanogenic degradation. . Microbiol Mol Biol Rev, 1997. 61(2): p. 262-280.

95. Kotsyurbenko, O.R., Trophic interactions in the methanogenic microbial community of low-temperature terrestrial ecosystems. FEMS Microbiol Ecol, 2005. 53(1): p. 3-13.

96. Petersen, S.P. and B.K. Ahring, Acetate oxidation in thermophilic anaerobic sewage sluge digester: the importance of non-aceticlastic methanogenesis of acetate. . FEMS Microbial Ecology, 1991. 86: p. 149-158.

97. Pohland, F.G. and S. Ghosh, Developments in anaerobic stabilization of organic wastes - the two-phase concept. . Environ. Lett., 1971. 1(255-256).


99. Demirel, B. and O. Yenigu¨n, Two-phase anaerobic digestion processes: a review. J. Chem. Tech. Biotechnol., 2002. 77: p. 743-755.

100. Himmel, M.E. and S.K. Picataggio, Our Challenge is To Acquire Deeper Understanding of Biomass Recalcitrance and Conversion, in Biomass Recalcitrance. 2009, Blackwell Publishing,Ltd. p. 1-6.

101. Bidlack, J., M. Malone, and R. Benson, Proceeding of Oklahoma Academy of Science, 1992. 72: p. 51.

102. Brett, C.T., Cellulose microfibrils in plants: Biosynthesis, deposition,and integration into the cell wall. Int Rev Cytol. 2000: Academic Press. 161-199.

103. Taherzadeh, M.J. and A. Jeihanipour, Recalcitrance of Lignocellulosic Biomass to Anaerobic Digestion, in Biogas Production: Pretreatment Methods in Anaerobic Digestion, A. Mudhoo, Editor. 2012, Scrivener Publishing, LLC.

104. Harris, P.J. and B.A. Stone, Chemistry and Molecular Organization of Plant Cell Walls, in Biomass Recalcitrance. 2009, Blackwell Publishing LTd. p. 61-93.

105. Kroeker, E.J., et al., Anaerobic treatment process stability. J. Water Pollut. Control Fed, 1979. 51: p. 718-727.

106. Ahring, B.K., M. Sandberg, and I. Andelidaki, Volatile fatty acids as indicators of process imbalance in anaerobic digesters. . Appl Microbiol Biotechnol, 1995. 43: p. 559-565.

107. Chen, Y., J.J. Cheng, and K.S. Creamer, Inhibition of anaerobic digestion process: A review. Bioresour Technol, 2008. 99: p. 4044-4064.

108. Kayhanian, M., Ammonia inhibition in high-solids biogasification: an overview and practical solutions. Environ. Technol., 1999. 20: p. 355-365.

109. Sprott, G.D. and G.B. Patel, Ammonia toxicity in pure cultures of methanogenic bacteria. System. Appl. Microbiol., 1986. 7: p. 358-363.

110. Whittmann, C., A.P. Zeng, and W.D. Deckwer, Growth inhibition by ammonia and use of pH-controlled feeding strategy for the effective cultivation of Mycobacterium chlorophenolicum. Appl. Microbiol. Biotechnol. Lett., 1995. 44: p. 519-525.

68

111. O’Flaherty, V., et al., Effect of pH on growth kinetics and sulphide toxicity thresholds of a range of methanogenic, syntrophic and sulphate-reducing bacteria. Process Biochem., 1998. 33(5): p. 555-569.

112. Harada, H., S. Uemura, and K. Monomoi, Interactions between sulphate-reducing bacteria and methane-producing bacteria in UASB reactors fed with low strength wastes containing different levels of sulphate. Water Res, 1994: p. 355-367.

113. Anderson, G.K., T. Donnelly, and K.J. Mckeown, Identification and control of inhibition in the anaerobic treatment of industrial wastewater. Process Biochem., 1982. 17: p. 28-32.

114. Oude Elferink, S.J.W.H., et al., Sulphate reduction in methanogenic bioreactors. . FEMS Microbiol. Rev, 1994. 15: p. 119-136.

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117. Vallee, B.L. and D.D. Ulner, Biochemical effects of mercury, cadmium, and lead. . Annu. Rev. Biochem., 1972. 41: p. 91-128.

118. Heipieper, H.J., et al., Mechanisms of resistance of whole cells to toxic organic solvents. . Trends Biotechnol, 1994. 12: p. 409-415.

119. Sierra-Alvarez, R. and G. Lettinga, The effect of aromatic structure on the inhibition of aceticlastic methanogenesis in granular sludge. . Appl. Microbiol. Biotechnol, 1991. 34: p. 544-550.

120. Yang, J. and R.E. Speece, The effects of chloroform toxicity on methane fermentation. Water Res, 1986. 20: p. 1273-1279.

121. van Beelen, P. and P.V. van Vlaardingen, Toxic effects of pollutants on the mineralization of 4-chlorophenol and benzoate in methanogenic river sediment. . Environ. Toxicol. Chem., 1994. 13(7): p. 1051-1060.

122. Bhattacharya, S., M. Qu, and R.L. Madura, Effects of nitrobenzene and zinc on acetate utilizing methanogens. Water Res. , 1996. 30(12): p. 3099-3105.

123. Mormile, M.R. and J.M. Suflita, The toxicity of selected gasoline components to glucose methanogenesis by aquifer microorganisms. Anaerobe, 1996. 2(5): p. 299-303.

124. Dimirer, G.N. and R.E. Speece, biotransformation of four 3-carbon compounds (acrolein, acrylic acid, allyl alcohol and N-propanol) in UASB reactors. . Water Res, 1998. 32(3): p. 747-759.

125. Gonzales-Gil, G., R. Kleerebezem, and G. Lettinga, Conversion and toxicity characteristics of formaldehyde in aceticlastic methanogenic sludge. . Biotechnol. Bioeng, 2002. 79: p. 314-322.

126. Playne, M.J. and B.R. Smith, Toxicity of organic extraction reagents to anaerobic bacteria. Biotechnol. Bioeng, 1983. 25: p. 1251-1265.

127. Schnürer, A. and Å. Jarvis, Microbiological handbook for biogas plants., in Swedish Gas Centre Report 207. 2010, Swedish Waste Managemen.

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111. O’Flaherty, V., et al., Effect of pH on growth kinetics and sulphide toxicity thresholds of a range of methanogenic, syntrophic and sulphate-reducing bacteria. Process Biochem., 1998. 33(5): p. 555-569.

112. Harada, H., S. Uemura, and K. Monomoi, Interactions between sulphate-reducing bacteria and methane-producing bacteria in UASB reactors fed with low strength wastes containing different levels of sulphate. Water Res, 1994: p. 355-367.

113. Anderson, G.K., T. Donnelly, and K.J. Mckeown, Identification and control of inhibition in the anaerobic treatment of industrial wastewater. Process Biochem., 1982. 17: p. 28-32.

114. Oude Elferink, S.J.W.H., et al., Sulphate reduction in methanogenic bioreactors. . FEMS Microbiol. Rev, 1994. 15: p. 119-136.

115. Song, Z., C.J. Williams, and R.G.J. Edyvean, Coagulation and anaerobic digestion of tannery wastewater. Process Saf. Environ. Prot., 2001. 79: p. 23-28.

116. Grady Jr, C.P.L., G.T. Daigger, and H.C. Lim, BiologicalWaste Water Treatment. 1999, New York: Marcel Dekker.

117. Vallee, B.L. and D.D. Ulner, Biochemical effects of mercury, cadmium, and lead. . Annu. Rev. Biochem., 1972. 41: p. 91-128.

118. Heipieper, H.J., et al., Mechanisms of resistance of whole cells to toxic organic solvents. . Trends Biotechnol, 1994. 12: p. 409-415.

119. Sierra-Alvarez, R. and G. Lettinga, The effect of aromatic structure on the inhibition of aceticlastic methanogenesis in granular sludge. . Appl. Microbiol. Biotechnol, 1991. 34: p. 544-550.

120. Yang, J. and R.E. Speece, The effects of chloroform toxicity on methane fermentation. Water Res, 1986. 20: p. 1273-1279.

121. van Beelen, P. and P.V. van Vlaardingen, Toxic effects of pollutants on the mineralization of 4-chlorophenol and benzoate in methanogenic river sediment. . Environ. Toxicol. Chem., 1994. 13(7): p. 1051-1060.

122. Bhattacharya, S., M. Qu, and R.L. Madura, Effects of nitrobenzene and zinc on acetate utilizing methanogens. Water Res. , 1996. 30(12): p. 3099-3105.

123. Mormile, M.R. and J.M. Suflita, The toxicity of selected gasoline components to glucose methanogenesis by aquifer microorganisms. Anaerobe, 1996. 2(5): p. 299-303.

124. Dimirer, G.N. and R.E. Speece, biotransformation of four 3-carbon compounds (acrolein, acrylic acid, allyl alcohol and N-propanol) in UASB reactors. . Water Res, 1998. 32(3): p. 747-759.

125. Gonzales-Gil, G., R. Kleerebezem, and G. Lettinga, Conversion and toxicity characteristics of formaldehyde in aceticlastic methanogenic sludge. . Biotechnol. Bioeng, 2002. 79: p. 314-322.

126. Playne, M.J. and B.R. Smith, Toxicity of organic extraction reagents to anaerobic bacteria. Biotechnol. Bioeng, 1983. 25: p. 1251-1265.

127. Schnürer, A. and Å. Jarvis, Microbiological handbook for biogas plants., in Swedish Gas Centre Report 207. 2010, Swedish Waste Managemen.

128. Brown, L., Exploding U.S. grain demand for automotive fuel threatens world food security and political stability. Earth Policy Institute, 2006.

129. Groom, M.J., E.M. Gray, and P.A. Townsend, Biofuels and biodiversity: principles for creating better policies for biofuel production. Conserv Biol, 2008. 22: p. 602-609.

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174. Baeta, B.E.L., et al., Use of submerged anaerobic membrane bioreactor (SAMBR) containing powdered activated carbon (PAC) for the treatment of textile effluents. Water Sci. Technol. , 2012. 65: p. 1540-1547.

175. Lin, H., et al., Feasibility evaluation of submerged anaerobic membrane bioreactor for municipal secondary wastewater treatment. Desalination, 2011. 280: p. 120–126.

176. Nagata, N., et al., Cross-flow membrane microfiltration of a bacterial fermentation broth, Biotechnol. Bioeng. . 34, 1989: p. 447–466.

177. Saddoud, A. and S. Sayadi, Application of acidogenic fixed-bed reactor prior to anaerobic membrane bioreactor for sustainable slaughterhouse wastewater treatment. J. Hazard. Mater. , 2007. 149 p. 700-706.

178. Padmasiri, S.I., et al., Methanogenic population dynamics and performance of an anaerobic membrane bioreactor (AnMBR) treating swine manure under high shear conditions,. Water Res., 2007. 41 p. 134-144.

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70

157. Choi, I.S., et al., Bioethanol production from mandarin (Citrus unshiu) peel waste using popping pretreatment. Appl Energ, 2013 102: p. 204-210.

158. Bai, H.-W., et al., Degradation of Limonene by Gamma Radiation for Improving Bioethanol Production. J Korean Soc Appl Biol Chem, 2014. 57: p. 1−4.

159. Akao, T., et al., The Methane Fermentation of Citrus unshu Peel Pretreated with Fungus Enzymes. Bioresour Technol, 1992. 41 p. 35-39.

160. Pourbafrani, M., et al., Protective Effect of Encapsulation in Fermentation of Limonene-contained Media and Orange Peel Hydrolyzate. Int. J. Mol. Sci. , 2007. 8: p. 777-787.

161. Youngsukkasem, S., K.S. Rakshit, and M.J. Taherzadeh, Biogas production by encapsulated methane producing bacteria. Bioresources., 2012. 7: p. 56-65.

162. Youngsukkasem, S., et al., Biogas production by encased bacteria in synthetic membranes: protective effects in toxic media and high loading rates. Environ Technol, 2013. 34(13-14): p. 2077-2084.

163. Traversi, D., et al., Application of a real-time qPCR method to measure the methanogen concentration during anaerobic digestion as an indicator of biogas production capacity. J Environ Manage 2012. 111 p. 173-177.

164. Ylitervo, P., J. Akinbomi, and M.J. Taherzadeh, Membrane bioreactors’ potential for ethanol and biogas production: a review. Environmental Technology, 2013. 34(13-14): p. 1711-1723.

165. Melin, T., et al., Membrane bioreactor technology for wastewater treatment and reuse. Desalination, 2006. 187: p. 271-282.

166. Yang, W., N. Cicek, and J. Ilg, State-of-the-art of membrane bioreactors: worldwide research and commercial applications in North America. J. Membr. Sci. , 2006. 270: p. 201-211.

167. McAdam, E.J. and S.J. Judd, A review of membrane bioreactor potential for nitrate removal from drinking water. Desalination, 2006. 196: p. 135-148.

168. Lin, H., et al., A review on anaerobic membrane bioreactors: Applications, membrane fouling and future perspectives. Desalination, 2013. 314 p. 169-188.

169. Shena, L., D.M. Bagleyb, and S.N. Liss, Effect of organic loading rate on fermentative hydrogen production from continuous stirred tank and membrane bioreactors. Int J Hydrogen Energ, 2 0 0 9. 3 4: p. 3 6 8 9 - 3 6 9 6.

170. He, Y., et al., High-concentration food wastewater treatment by an anaerobic membrane bioreactor. Water Res., 2005. 39: p. 4110-4118.

171. Torres, A., et al., Application of two-phase slug-flow regime to control flux reduction on anaerobic membrane bioreactors treating wastewaters with high suspended solids concentration. Sep. Purif. Technol. , 2011. 79: p. 20-25.

172. Saddoud, A., I. Hassairi, and S. Sayadi, Anaerobic membrane reactor with phase separation for the treatment of cheese whey. Bioresour. Technol., 2007. 98: p. 2102-2108.

173. Kanai, M., et al., A novel combination of methane fermentation and MBR - Kubota Submerged Anaerobic Membrane Bioreactor process. Desalination, 2010. 250: p. 964-967.

174. Baeta, B.E.L., et al., Use of submerged anaerobic membrane bioreactor (SAMBR) containing powdered activated carbon (PAC) for the treatment of textile effluents. Water Sci. Technol. , 2012. 65: p. 1540-1547.

175. Lin, H., et al., Feasibility evaluation of submerged anaerobic membrane bioreactor for municipal secondary wastewater treatment. Desalination, 2011. 280: p. 120–126.

176. Nagata, N., et al., Cross-flow membrane microfiltration of a bacterial fermentation broth, Biotechnol. Bioeng. . 34, 1989: p. 447–466.

177. Saddoud, A. and S. Sayadi, Application of acidogenic fixed-bed reactor prior to anaerobic membrane bioreactor for sustainable slaughterhouse wastewater treatment. J. Hazard. Mater. , 2007. 149 p. 700-706.

178. Padmasiri, S.I., et al., Methanogenic population dynamics and performance of an anaerobic membrane bioreactor (AnMBR) treating swine manure under high shear conditions,. Water Res., 2007. 41 p. 134-144.

71

179. Zayen, A., et al., Anaerobic membrane bioreactor for the treatment of leachates from Jebel Chakir discharge in Tunisia,. J. Hazard. Mater. , 2010. 177: p. 918-923.

180. Harasimowicz, M., et al., Application of polyimide membranes for biogas purification and enrichment. J Hazard Mater., 2007;. 144: p. 698-702.

Paper I

Paper I

Inhibitory effects of fruit flavors on methane production duringanaerobic digestion

Rachma Wikandari a,⇑, Sailaja Gudipudi a, Ishwarya Pandiyan a, Ria Millati b, Mohammad J. Taherzadeh a

a School of Engineering, University of Borås, Borås, SwedenbDepartment of Food and Agricultural Product Technology, Gadjah Mada University, Indonesia

h i g h l i g h t s

" Fruit flavors belonging to terpenoid, aldehyde, and alcohol were inhibitors to anaerobic digestion." Terpenoid could reduce methane production 95% at concentration of 0.5%." Aldehydes decreased methane production up to more than 99% at concentration of 0.5%." Alcohol reduced methane production 99% at concentration of 0.5%." Myrcene required lowest concentration to reduce 50% methane production among the tested flavors.

a r t i c l e i n f o

Article history:Available online 4 February 2013

Keywords:BiogasFruit wasteFlavorInhibition

a b s t r a c t

In order to improve biogas production from fruit wastes, the inhibitory effects of fruit flavors on anaer-obic digestion were investigated. Batch anaerobic digestion was performed for 30 days using syntheticmedium and thermophilic sludge. Three groups of flavor compounds i.e. aldehydes (hexanal, nonanal,and E-2-hexenal), terpenes (car-3-ene, a-pinene, and myrcene), and alcohol (octanol) at concentrationof 0.005%, 0.05%, and 0.5% were examined. All the flavor compounds showed inhibitory effect on methaneproduction. The highest methane reduction was obtained at addition of 0.5% of flavor compounds. For ter-penoids, the presence of 0.5% of car-3-ene, myrcene, and a-pinene reduced 95%, 75%, and 77% of methaneproduction, respectively. For aldehydes, addition of 0.5% concentration resulted in more than 99% meth-ane reduction for hexanal and E-2-hexenal, and 84% methane reduction for nonanal. For alcohol, the pres-ence of 0.5% octanol decreased 99% methane production.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Fruit is one of the most important trading commodities in theworld. Food and Agricultural Organization of United Nation (FAOUN) reported 746 million tons of fruit production in the world in2009. However, during the post-harvest chain from the field untilwholesale or retail markets, losses occur both in quantity and qual-ity of the fruits. Inadequate facilities, poor management, lack ofknowledge and careless handling can cause mechanical injury,physiological deterioration, parasitic diseases resulting in post-harvest losses (Chattopadhyay, 2007). The post-harvest losses in-clude injured fruits, rotten fruits, contaminated fruits and fruitsthat do not meet standard requirement. In practice, fruit waste issimply dumped. However, due to its high organic and water con-tents, it creates a serious environmental problem such as heavy

odor, plenty of leachate during the collection, transportation, andlandfill as well as attracting flies and rats (Lin et al., 2011).

Fruits have high moisture and organic content, thus anaerobicdigestion can be considered as a suitable treatment for fruit wasteand biogas production (Garcia-Peña et al., 2011). Biogas can be uti-lized for electricity production, cooking, heating, and vehicle fuel.Among these applications, fuel production for vehicles receivesconsiderable attention driven by an inevitable depletion of fossilfuel and rising environmental concern. In accordance with risingWaste-to-Energy (WTE) concept worldwide, utilization of fruitwaste as raw material for biogas production is an alternative solu-tion for both environmental and energy challenges.

Fruits have a defense system for protection against microbialinvasion, which can be a challenge in digestion process. Volatilecompounds as an important plant flavor formation in fruits havebeen reported responsible for prolonging the shelf-life of fruit(Utama et al., 2002). Flavor substances can be classified into esters,alcohols, aldehydes, ketones, lactones, and terpenoids. Geraniol, lin-alool, a-terpeniol, citronellol, thymol, and carvacrol belonging to

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.01.041

⇑ Corresponding author. Tel.: +46 33 435 40 86; fax: +46 33 435 40 08.E-mail address: [email protected] (R. Wikandari).

Bioresource Technology 145 (2013) 188–192

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terpenes are reported having cytotoxic capacities on standardorganics (Rosato et al., 2007). Alcohol volatile compound such ashexanol and 3-(z)-hexanol involves in protection of wounded areain fruit against decaymicroorganism (Lanciotti et al., 2004). Amajorconstituent in peel oil, limonene which also belongs to terpenoidswas reported having antimicrobial activity even at very low concen-tration i.e. 0.01%w/v (Grohmann et al., 1994;Winniczuk and Parish,1997). Limonene also inhibited anaerobic digestion process andcaused ultimate failure of the process at concentration of 400 lL/Lon mesophilic continuous system (Mizuki et al., 1990). Similarly, italso impedes biogas production at concentration from 450 to900 lL/L on thermophilic system (Gergely, 2012).

Although several studies were carried out on volatiles com-pounds in fruits against various microorganisms, only scarce infor-mation is available on their effect on methanogenic bacteria.Furthermore, for the best of our knowledge, no report about effectof volatile compounds of fruit on methane production has been de-tected. Thus, this study is aimed to investigate the effect of flavorcompound in fruit on methane production.

2. Method

2.1. Microorganism and chemicals

Microorganism (sludge) with 22 g VS/L (volatile solid per liter)was obtained from a thermophilic biogas plant (55 �C) at Borås En-ergy and Environment AB (Borås, Sweden). The sludge was thenstored in incubator at temperature 55 �C for 2–3 days to acclimatethe bacteria with the operation condition. Flavor compounds (pro-vided fromSigma–Aldirch) fromthreegroups i.e.aldehydes (hexanal,E-2-hexenal, and nonanal), terpenoids (myrcene, a-pinene, car-3-ene), and alcohol (octanol) were examined (Fig. 1). Nutrient brothwasobtained fromSigma–Aldirch,whereasyeast extract andglucosewere supplied byMerck.Nutrient broth contained1 g/L D(+)-glucose,15 g/L peptone, 6 g/L sodium chloride, and 3 g/L yeast extract.

2.2. Anaerobic digestion

Medium was prepared according to OECD (2007) with minormodification. Medium solution contained 20 g/L of nutrient broth,20 g/L of yeast extract, and 20 g/L of D-glucose. The medium wassterilized by filtration through a 0.2 lm membrane filter. Flavorsolution was prepared by diluting pure liquid flavor compoundswith distilled water in order to make concentration of 0.005%,0.05% and 0.5% (w/v).

Batch anaerobic digestion experiments were performed (Hansenet al., 2004). The experiments were carried out at thermophilicconditions (55 �C). The reactors used were sealed serum glass bot-tles of 118 mL, containing 50 mL of sludge, 1.0 mL of nutrient med-ium, and 2.5 mL of flavor solution or distilled water as treatmentcontrol. In order to measure methane production only from thesludge, the sludge was incubated without addition of mediumand flavor compounds (later mentioned as inoculum). The reactorswere closed tightly and flashed with gas mixture of 80% N2 and 20%CO2 for 2 min to purge the air and obtain anaerobic conditions. Itwas then incubated at 55 �C for 30 days. The reactors were shakentwice a day using water bath shaker (55 �C) at 150 rpm for 10 min.During incubation, gas sample was taken from headspace of thereactors through the septum using a syringe with pressure lock.Samples were taken at 3, 6, 9, 12, 15, 20, 25, 30 days. Experimentrun in triplicate and the results were presented in average.

2.3. Analytical methods

Methane production from the digestion experiments were mea-sured using a Gas Chromatograph (Auto System, Perkin Elmer,

USA) equipped with a packed column (Perkin Elmer, 60 � 1.800 OD,80/100, Mesh, USA) and a thermal conductivity detector (Perkin El-mer, USA) with inject temperature of 150 �C. The carrier gas wasnitrogen operated with a flow rate of 20 mL/min at 60 �C. The ini-tial methane production rate was measured as the mean of meth-ane production per day during the first nine days. Completefactorial design was used for experimental design. For statisticalanalysis, Analysis of Variance (ANOVA) with significance level of0.05 was performed using Statistical Package for the Social Sci-ences (SPSS). The data presented are the average ± standarddeviation.

3. Results and discussion

Fruit flavor is considered as one of the most important qualitiesin fresh fruit (Reineccius, 2006). The fruit flavor consists of sugars,acids, salts, bitter compounds such as alkaloids or flavonoids, andaroma volatiles (Song and Forney, 2008). The flavors of fruit are di-verse in different fruit species, which is responsible for the uniquecharacteristic of each fruit. Fruit flavors can be classified into 6groups i.e. aldehydes, alcohols, terpenoids, esters, ketones, and lac-tones. Flavor compounds present in fruits are presented in Table 1.

In order to investigate potential inhibitors in fruits, three groupsof flavor compounds were selected, which were terpenoids, alde-hydes, and alcohols. Then, anaerobic digestions at presence ofthese compounds with three different concentrations i.e. 0.005%,0.05%, and 0.5% were performed. The inhibitory effects of the cor-responding compounds were evaluated by comparing the accumu-lated methane production and the initial methane production rateof the mediumwith the presence of the flavor compounds and con-trol. The rate of methane production corresponds to digestibilitylevel of the substrate to anaerobic digesting bacteria.

3.1. Terpenoid

In terpenoid group, the possible inhibition effects of car-3-ene,a-pinene, and myrcene on biogas production were examined.Scarce information was found on inhibitory effect of these flavorson methane production.

The profile of methane production for all the flavor compoundsexamined at three different concentrations is presented in Fig. 1a–c. These terpenoids inhibited the methane production at all theexamined concentrations which was indicated by decreasing inthe methane production. The maximum methane reductions forcar-3-ene, myrcene, and a-pinene were 95 ± 44%, 75 ± 3%, and77 ± 10%, which were resulted from the addition of the corre-sponding flavor at concentration of 0.5%. Methane reduction ex-ceeds 50% from the medium with the presence of car-3-ene,myrcene, and a-pinene was obtained at concentration of 0.05%,0.005%, and 0.5%, respectively. These results indicate that inhibi-tory effect of myrcene is higher compared to car-3-ene and a-pinene. The reason is still questionable and requires further study.

The effects of these terpenoids on initial digestion rate are pre-sented in Fig. 2. In general, addition of the three flavor compoundsshowed lag phase of methane production during the first ninedays. The highest methane production rate was achieved in thepresence of 0.05% a-pinene, whereas the lowest digestion ratewas obtained with addition of 0.5% car-3-ene. Low initial methaneproduction rate, which showed around 50% production rate com-pared to that of control, was observed with just addition of0.005% of all terpenoids.

The results of this work stress that all the tested terpenoids areinhibitor to anaerobic bacteria. This is in accordance with otherprevious studies. Car-3-ene exhibited antimicrobial activity withminimum inhibitory concentration (MIC) between 50 to more than

R. Wikandari et al. / Bioresource Technology 145 (2013) 188–192 189

800 mg/kg against 14 tested bacteria (Muroi et al., 1993). Similarly,a-pinene was reported to inhibit Bacillus sp. and Saccharomycescerevisiae at concentration normally present in the fir needle dietof Douglas Fir Tussock Moth. a-Pinene was found to disrupt cellu-lar integrity and inhibit respiration in yeast. Scarce informationwas found on antimicrobial activity of myrcene. Although, the inhi-bition mechanism of car-3-ene and myrcene is unclear, the mech-anism action of various terpenoids was predicted due tomembrane disruption by the lipophilic compounds (Cowan, 1999).

D-limonene, another terpenoid compound was reported to re-sult in failing mesophilic or thermophilic anaerobic digestion atconcentration of 400 or 450–900 mg/kg (Gergely, 2012; Mizukiet al., 1990). Our results showed that for 50% reduction in methaneproduction, car-3-ene required 0.05–0.5% (500–5000 mg/kg), myr-cene was less than 0.005% (50 mg/kg), and a-pinene was 0.05–0.5%(500–5000 mg/kg). These findings prove the prediction by Mizukiet al. (1990) about the presence of antimicrobial compound otherthan limonene in citrus peel oil. They reported that addition of cit-rus peel oil caused digestion failure in shorter time than addition of

pure limonene, which might be due to the presence of other inhib-itory compound in the peel oil. Besides, another finding reportedthat myrcene, car-3-ene, and a-pinene are responsible as volatilecompound in citrus (Nursten and Williams, 1967).

3.2. Aldehyde

The inhibitory effect of three flavor compounds belonging toaldehyde, i.e. E-2-hexenal, hexanal, and nonanal were examinedin this work. The profile of methane production with addition of0.005%, 0.05% and 0.5% aldehyde compounds is presented inFig. 1e–g. Similar to terpenoids, the methane reduction was ob-served with the presence of all these aldehydes at all the concen-trations tested. Almost no methane was produced at thepresence of 0.5% of hexanal and E-2-hexenal, while in the presenceof nonanal at the same concentration, about 15% biogas relative tothe control experiment was produced. For all aldehydes examined,the presence of 0.5% of flavor compounds resulted in more than50% methane reduction. A comparison between the aldehydes

Fig. 1. Effect of various flavor compounds (a) car-3-ene, (b) a-pinene, (c) myrcene, (d) octanol, (e) E-2-hexenal, (f) hexanal, (g) nonanal, and (i) control on methane productionat different concentrations of (�) 0.005%, (4) 0.05%, (h) 0.5%, in comparison to (}) control (without flavor compounds), and (N) inoculums (without nutrient medium andflavor compounds).

190 R. Wikandari et al. / Bioresource Technology 145 (2013) 188–192

and terpenoids shows that aldehydes required higher concentra-tion than the terpenoids to reduce 50% methane production. Itmight be interpreted that the aldehydes have lower inhibitoryactivity for methane production than the terpenoids.

The effects of E-2-hexenal, hexanal, and nonanal on initialmethane production rate of the bacteria is presented in Fig. 2.These aldehydes reduced the initial methane production rate, ex-cept with their presence in the media at their lowest concentra-tion, i.e. 0.005%. The highest initial methane production rate wasachieved in the presence of 0.005% hexanal, whereas the lowestinitial digestion rate was obtained with addition of 0.5% E-2-hexenal. Low initial methane production rate, which showedaround 50% production rate compared to that of control, was ob-served at the presence of 0.05% of all the aldehydes.

Antimicrobial activity of hexanal and E-2-hexenal was previouslyreported on a few particular organisms. Hexanal and E-2-hexenalshowed bactericidal effect on Listeria monocytogenes and prolongedthe lag phase of E. coli and Salmonella enteritidis in fresh apple slices(Lanciotti et al., 2003). Bactericidal concentrations of E-2-hexenalagainst E. coli, L. monocytogenes, and S. enteritidis were 20, 50, and100mg/kg examined on agar plate (Lanciotti et al., 2003). Meanwhile,hexanal required higher concentration to showbactericide effect up to100, 250, and 200mg/kg for E. coli, L. monocytogenes, and S. enteritidis,respectively. Furthermore, E-2-hexenal exhibited antibacterial activityagainst Bacillus subtilis, Brevibacterium ammoniagenes, Staphylococcusaureus, Streptococcus mutans, Propionibacterium acnes, Pseudomonasaeruginosa, Enterobacter aerogenes, E. coli, Proteus vulgaris, S. cereviseae,Candida utilis, Pityrosporum ovale, Penicillium chrysogenum, Trichophyton

Table 1Effect of flavor compounds on biogas production at different concentrations.

Compounds Concentration (%) Methane production (mL) ± std* Reduction (%)

Control 0 102 ± 27 0Car-3-ene 0.5 7 ± 3 95

0.05 51 ± 34 830.005 92 ± 27 9

Myrcene 0.5 26 ± 1 750.05 33 ± 30 680.005 38 ± 7 63

a-Pinene 0.5 24 ± 3 770.05 87 ± 6 150.005 59 ± 11 42

Hexanal 0.5 0.4 ± 0.14 1000.05 56 ± 31 450.005 80 ± 6 22

E-2-hexenal 0.5 0.2 ± 0.09 1000.05 72 ± 20 290.005 85 ± 7 17

Nonanal 0.5 16 ± 2 840.05 95 ± 22 60.005 96 ± 15 5

Octanol 0.5 0.7 ± 0.1 990.05 16 ± 16 840.005 69 ± 15 32

* Std: standard deviation.

met

hane

pro

duct

ion

rate

(m

l/day

)

0

1

2

3

4

5

6

7

flavlavoror comcompopounundsds

0.50.0

0.500.050.0050

0

0

0

05

0.50

0.05

0.005

0

50

05

005

Fig. 2. Initial methane production rate with addition of flavor compounds.


mentagrophyteswiththeconcentrationrangeof50–400mg/kg,whereashexanal required 800mg/kg to cease the growth of these microorgan-isms (Muroi et al., 1993). InhibitorymechanismofE-2-hexenal andhex-anal could be attributed in alteration of membrane fatty acidsmodulation (Patrignani et al., 2008).Nonanal reported toexhibit antimi-crobial activity against gram-positive and gram-negative bacteria in therange concentration of 100 tomore than800mg/kg (Muroi et al., 1993).

3.3. Alcohol

For alcohol, inhibitory effect of octanol was examined. Additionof octanol at 0.5, 0.05 and even 0.005% reduced themethaneproduc-tion (Fig. 1d). Higher concentration of octanol resulted in lowermethane production. Almost no gas was produced in the presenceof 0.5% octanol. All added concentration of octanol resulted in lowerinitial methane production rate. As can be seen in Fig. 2, addition of0.005% was enough to reduce 50% initial methane production rate.

No previous report was detected in the literature about themechanism action of octanol against microorganism. It was re-ported that the toxicity of alcohols was directly related with thelength of molecular chain (Ingram, 1976). The longer chain of alco-hol caused higher inhibitory activity.

The results of this work are summarized in Table 1. It is shownthat 50% methane production compared to the control experimentwas obtained in the presence of less than 0.005% myrcene,0.05–0.005% octanol, and 0.05–0.5% a-pinene, car-3-ene, hexanal,E-2-hexenal, and nonanal. This result suggests that myrcene wasthe most toxic compounds among the examined fruit flavors foranaerobic digesting bacteria.

4. Conclusions

The tested fruit flavor compounds including hexanal, a- Pinene,Car-3-ene, Nonanal, E-2-hexenal, myrcene, and octanol were foundto be inhibitors formethaneproduction. Thepresence of theseflavorcompounds could reduce methane production by 0–63%, 29–83%,and 75–100% at concentration of 0.005, 0.05, and 0.5%, respectively.

Acknowledgement

We express our gratitude to Swedish International Develop-ment Agency (SIDA) for the financial support of this work.

References

Chattopadhyay, S.K., 2007. Handling, Transportation and Storage of Fruits andVegetables. Global Media Delhi, India.

Cowan, M.M., 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12,564–582.

Garcia-Peña, E.I., Parameswaran, P., Kang, D.W., Canul-Chan, M., Krajmalnik-Brown,R., 2011. Anaerobic digestion and co digestion of vegetable and fruit residues:process and microbial ecology. Bioresour. Technol. 102, 9447–9455.

Gergely, F., 2012. Biogas Production from Citrus Wastes and Chicken Feather:Pretreatment and Co-Digestion. Chalmers University of Technology, Göteborg,Sweden.

Grohmann, K., Baldwin, E., Buslig, B., 1994. Production of ethanol fromenzymatically hydrolyzed orange peel by the yeast Saccharomyces cerevisiae.Appl. Biochem. Biotechnol. 45–46 (1), 315–327.

Hansen, T.L. et al., 2004. Method for determination of methane potentials of solidorganic waste. Waste Manage. (Oxf.) 24, 393–400.

Ingram, L.O., 1976. Adaptation of membrane lipids to alcohols. J. Bacteriol. 125,670–678.

Lanciotti, R. et al., 2003. Application of hexanal, (E)-2-hexenal, and hexyl acetate toimprove the safety of fresh-sliced apples. J. Agric. Food Chem. 51 (10), 2958–2963.

Lanciotti, R., Gianotti, A., Patrignani, F., Belletti, N., Guerzoni, M.E., Gardini, F., 2004.Use of natural aroma compounds to improve shelf-life and safety of minimallyprocessed fruits. Trends Food Sci. Technol. 15 (3–4), 201–208.

Lin, J. et al., 2011. Effects of mixture ratio on anaerobic co-digestion withfruit and vegetable waste and food waste of China. J. Environ. Sci. 23 (8),1403–1408.

Mizuki, E., Akao, T., Saruwatari, T., 1990. Inhibitory effect of Citrus unshu peel onanaerobic digestion. Biol. Wastes 33 (3), 161–168.

Muroi, H., Kubo, A., Kubo, I., 1993. Antimicrobial activity of cashew apple flavorcompounds. J. Agric. Food Chem. 41 (7), 1106–1109.

Nursten, H.E., Williams, A.A., 1967. Fruit aromas: a survey of compounds identified.Chem. Ind. 2, 486–497.

Organisation for Economic Co-operation and Development (OECD), 2007. OECDGuidelines for the Testing of Chemicals. Test No. 224: Determination of theInhibition of the Activity of Anaerobic Bacteria – Reduction of Gas Productionfrom Anaerobically Digesting (Sewage) Sludge.

Patrignani, F., Lucci, L., Belletti, N., Gardini, F., Guerzoni, M.E., Lanciotti, R., 2008.Effects of sub-lethal concentrations of hexanal and 2-(E)-hexenal on membranefatty acid composition and volatile compounds of Listeria monocytogenes,Staphylococcus aureus, Salmonella enteritidis and Escherichia coli. Int. J. FoodMicrobiol. 123, 1–8.

Reineccius, G., 2006. Flavor Chemistry and Technology. CRC Press, Boca Raton, FL.Rosato, A., Vitali, C., De Laurentis, N., Armenise, D., Antonietta Milillo, M., 2007.

Antibacterial effect of some essential oils administered alone or in combinationwith Norfloxacin. Phytomedicine 14 (11), 727–732.

Song, J., Forney, C.F., 2008. Flavour volatile production and regulation in fruit. Can. J.Plant Sci. 88, 537–550.

Utama, I., Made, S., Wills, R.B.H., Ben-yehoshua, S., Kuek, C., 2002. In vitro efficacy ofplant volatiles for inhibiting the growth of fruit and vegetable decaymicroorganisms. J. Agric. Food Chem. 50 (22), 6371–6377.

Winniczuk, P.P., Parish, M.E., 1997. Minimum inhibitory concentrations ofantimicrobials against micro-organisms related to citrus juice. FoodMicrobiol. 14 (4), 373–381.


Paper II

Paper II

RESEARCH ARTICLE

Effect of ester compounds on biogas production: beneficialor detrimental?Heri Yanti1,2, Rachma Wikandari3, Ria Millati4, Claes Niklasson1 & Mohammad J. Taherzadeh3

1Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden2Department of Chemical Engineering, Universitas Gadjah Mada, Yogyakarta, Indonesia3Swedish Centre of Resource Recovery, University of Bor�as, Bor�as, Sweden4Department of Food and Agricultural Product Technology, Universitas Gadjah Mada, Yogyakarta, Indonesia

Keywords

Ester, inhibition, methane, minimum

inhibitory concentration

Correspondence

Rachma Wikandari, School of Engineering,

University of Boras, Boras, Sweden. Tel: +

46-33-435-44-87; Fax: +46-33-435-40-08;

E-mail: [email protected]

Funding Information

We express our gratitude to Directorate

General of Higher Education, Ministry of

National Education of Republic of Indonesia

through Competitive Research Grant of

International joint Research for International

Publication (grant no LPPM-UGM/696/LIT/

2013), the Swedish International

Development Agency (SIDA), the Swedish

Gas Centre for financial support of this study

Received: 16 September 2013; Revised: 9

January 2014; Accepted: 13 January 2014

Energy Science and Engineering 2014;

2(1): 22–30

doi: 10.1002/ese3.29

Abstract

Esters are major flavor compounds in fruits, which are produced in high vol-

ume. The widespread availability of these compounds in nature attracts interest

on their behavior in anaerobic digestion in waste and wastewater treatments.

The aim of this work was to study the effects of various esters at different con-

centrations in anaerobic digestion followed by determination of their minimum

inhibitory concentration (MIC), and to study the effect of chain length of func-

tional group and alkyl chain of ester on methane production. Addition of

methyl butanoate, ethyl butanoate, ethyl hexanoate, and hexyl acetate at con-

centration up to 5 g L�1 increased methane production, while their higher con-

centrations inhibited the digestion process. The MIC values for these esters

were between 5 and 20 g L�1. Except hexyl acetate, the esters at concentration

5 g L�1 could act as sole carbon source during digestion. For ethyl esters,

increasing number of carbon in functional group decreased methane produc-

tion. For acetate esters, alkyl chain longer than butyl inhibited methane produc-

tion. Effect of ester on methane production is concentration-dependent.

Introduction

Exploring and exploiting renewable and green energy is

necessary in today’s energy life style, not only because of

the inevitable depletion of conventional sources of fossil

energy but also due to the ecological-environmental

effects caused by the conventional energy consumption.

Biogas is a clean and renewable form of energy, which

has a wide range of applications, such as vehicle fuel,

cooking, heating, lighting, and electricity production. Bio-

gas is produced by anaerobic degradation of organic sub-

strates. Anaerobic digestion is one of the oldest processes

and the most efficient treatment technologies, widely used

for treating industrial wastes, municipal waste, and stabil-

ization of wastewater sludge [1]. However, the fermenting

organisms in biogas processes are sensitive to the process

conditions and the substrate used. A wide variety of

chemical substances have been reported to be inhibitory

to the anaerobic digestion processes, resulting in decreas-

ing or stopping the biogas production [2].

One of the chemical substances that could impact bio-

gas production is ester. Ester is one of the most impor-

tant classes of chemicals produced in high volume [3]. It

is widely used in various industries such as solvents,

22 ª 2014 The Authors. Energy Science & Engineering published by the Society of Chemical Industry and John Wiley & Sons Ltd.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution

and reproduction in any medium, provided the original work is properly cited.

RESEARCH ARTICLE

Effect of ester compounds on biogas production: beneficialor detrimental?Heri Yanti1,2, Rachma Wikandari3, Ria Millati4, Claes Niklasson1 & Mohammad J. Taherzadeh3

1Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden2Department of Chemical Engineering, Universitas Gadjah Mada, Yogyakarta, Indonesia3Swedish Centre of Resource Recovery, University of Bor�as, Bor�as, Sweden4Department of Food and Agricultural Product Technology, Universitas Gadjah Mada, Yogyakarta, Indonesia

Keywords

Ester, inhibition, methane, minimum

inhibitory concentration

Correspondence

Rachma Wikandari, School of Engineering,

University of Boras, Boras, Sweden. Tel: +

46-33-435-44-87; Fax: +46-33-435-40-08;

E-mail: [email protected]

Funding Information

We express our gratitude to Directorate

General of Higher Education, Ministry of

National Education of Republic of Indonesia

through Competitive Research Grant of

International joint Research for International

Publication (grant no LPPM-UGM/696/LIT/

2013), the Swedish International

Development Agency (SIDA), the Swedish

Gas Centre for financial support of this study

Received: 16 September 2013; Revised: 9

January 2014; Accepted: 13 January 2014

Energy Science and Engineering 2014;

2(1): 22–30

doi: 10.1002/ese3.29

Abstract

Esters are major flavor compounds in fruits, which are produced in high vol-

ume. The widespread availability of these compounds in nature attracts interest

on their behavior in anaerobic digestion in waste and wastewater treatments.

The aim of this work was to study the effects of various esters at different con-

centrations in anaerobic digestion followed by determination of their minimum

inhibitory concentration (MIC), and to study the effect of chain length of func-

tional group and alkyl chain of ester on methane production. Addition of

methyl butanoate, ethyl butanoate, ethyl hexanoate, and hexyl acetate at con-

centration up to 5 g L�1 increased methane production, while their higher con-

centrations inhibited the digestion process. The MIC values for these esters

were between 5 and 20 g L�1. Except hexyl acetate, the esters at concentration

5 g L�1 could act as sole carbon source during digestion. For ethyl esters,

increasing number of carbon in functional group decreased methane produc-

tion. For acetate esters, alkyl chain longer than butyl inhibited methane produc-

tion. Effect of ester on methane production is concentration-dependent.

Introduction

Exploring and exploiting renewable and green energy is

necessary in today’s energy life style, not only because of

the inevitable depletion of conventional sources of fossil

energy but also due to the ecological-environmental

effects caused by the conventional energy consumption.

Biogas is a clean and renewable form of energy, which

has a wide range of applications, such as vehicle fuel,

cooking, heating, lighting, and electricity production. Bio-

gas is produced by anaerobic degradation of organic sub-

strates. Anaerobic digestion is one of the oldest processes

and the most efficient treatment technologies, widely used

for treating industrial wastes, municipal waste, and stabil-

ization of wastewater sludge [1]. However, the fermenting

organisms in biogas processes are sensitive to the process

conditions and the substrate used. A wide variety of

chemical substances have been reported to be inhibitory

to the anaerobic digestion processes, resulting in decreas-

ing or stopping the biogas production [2].

One of the chemical substances that could impact bio-

gas production is ester. Ester is one of the most impor-

tant classes of chemicals produced in high volume [3]. It

is widely used in various industries such as solvents,


This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution

and reproduction in any medium, provided the original work is properly cited.

plasticizer for cellulose, nail polish removers, perfumery

product, plastic tubing, floor tiles, furniture, automobile

upholstery, insect repellents, and as paint additive [3, 4].

Ester can be manufactured via chemical reaction by con-

densation of alcohols and carboxylic acids. In addition, it

can be found in nature as the major volatile compounds

in fruits. For instance, esters constitute 78–92%, 25–90%,

and 13% of the total volatile mass of apple, strawberry,

and raspberry, respectively [5]. Therefore, it is used as a

flavor and fragrance compound in food industry. The

widespread production and ability of some esters to

migrate, make esters to be easily found in the environ-

ment [6].

Since anaerobic digestion is mostly used to tread waste-

waters, investigation of effect of ester on anaerobic diges-

tion is important. Esters might be beneficial or detrimental

to the anaerobic digestion. Some esters are reported to be

degraded by Acetobacterium woodii and Eubacterium limo-

sum, which are involved in anaerobic digestion process

[7]. However, some phthalate esters (PAE) could reduce

the biogas production at a concentration higher than

60 mg L�1 [8]. Besides, some esters are reported having

antimicrobial activity against some standard microorgan-

isms. Butyl acetate and hexyl acetate at 4 lL and 8 lL,respectively, were toxic to Conidia germination after incu-

bation for 24 h at 22°C [9]. Hexyl acetate at concentration

150 mg L�1 has significant inhibitory effect against E. coli,

S. enteritidis, and L. monocytogenes that were isolated both

in model system and in fresh-sliced apples [10]. To our

knowledge, scarce information is available on the effect of

esters in methane production.

In this study, some esters that are available in nature as

fruit flavor compounds and mainly used in food, cos-

metic, and solvent industries were examined. The aim of

this study was to investigate the effects of the ester com-

pounds on biogas production, and their minimum inhibi-

tory concentration (MIC). In addition, the effect of chain

length of functional group and alkyl chain of ester on

biogas production was investigated.

Material and Methods

Microorganisms and chemicals

Inoculum (sludge) with 22 g VS L�1 (volatile solid per

liter) was obtained from a thermophilic biogas plant

(55°C) at Bor�as Energy and Environment AB (Bor�as,

Sweden). The inoculum was then stored in an incubator

at temperature 55°C for 3 days. A synthetic medium solu-

tion was prepared from 20 g L�1 of nutrient broth

(Sigma-Aldrich, St. Louis, MO), 20 g L�1 of yeast extract

(Merck KgaA, Darmstadt, Germany), and 20 g L�1 of

glucose (Merck). Nutrient broth contained 10 g L�1 beef

extract, 10 g L�1 peptone, and 5 g L�1 sodium chloride.

The ester compounds (Sigma-Aldrich) used in this work

were methyl butanoate, ethyl butanoate, ethyl hexanoate,

hexyl acetate, methyl acetate, ethyl acetate, and butyl ace-

tate.

The experiments were carried out in two steps. The

first step was to investigate the effect of ester compounds

on biogas production. In this step, the concentrations of

methyl butanoate, ethyl butanoate, ethyl hexanoate, and

hexyl acetate were 0.05, 0.5, 5, 10, and 20 g L�1 for each

compound. The second step was to investigate the effect

of chain length of functional group and alkyl chain of

ester on biogas production. In this step, the concentration

of ethyl hexanoate, ethyl butanoate, methyl acetate, ethyl

acetate, butyl acetate, and hexyl acetate was 5 g L�1.

Anaerobic digestion

The method used for anaerobic digestion was adapted

from the method described by Hansen et al. [11] and the

OECD [12] with minor modifications. The experiments

were carried out in a 120-mL glass bottle containing

50 mL of sludge, 1 mL of medium, and 2.5 mL of ester

solution or distilled water (for a control). In order to mea-

sure methane production from the inoculum, the inocu-

lum was incubated without addition of medium and ester

solution in the glass bottle containing 50 mL of sludge

and 3.5 mL of distilled water. The bottles were closed

tightly and the headspace was filled with gas mixture con-

taining 80% N2 and 20% CO2 to achieve anaerobic condi-

tion. The bottles were then incubated at 55°C for 28 days.

During incubation, the bottles were shaken twice a day

using water bath shaker (55°C) at 150 rpm. Gas samples

were taken from headspace of the reactors through the

septum using a syringe with pressure lock (VICI; Precision

Sampling Inc., Baton Rouge, LA). Samples were taken at

3, 6, 9, 12, 15, 20, 25, and 30 days. The initial methane

production rate was measured as the mean of methane

production per day during the first 10 days.

Analytical methods

Biogas production from the digestion experiments was

measured using Gas Chromatography (Varian 450 GC,

Palo Alto, CA) with a capillary column (J 6 W scientific

GS-Gas Pro, bonded silica based 30 m 9 0.32 mm,

Agilent Technologies, Santa Clara, CA) and a thermal

conductivity detector (Varian, Palo Alto, CA). The condi-

tions for the analysis were injector temperature of 75°C,oven temperature of 100°C, detector temperature of

120°C, and column flow (N2) 2 mL min�1.

All experiments were carried out in triplicate batch

experiment; the result was presented in average � standard

ª 2014 The Authors. Energy Science & Engineering published by the Society of Chemical Industry and John Wiley & Sons Ltd. 23

H. Yanti et al. Effect of Ester Compounds on Biogas Production

deviation. At the end of digestion, the pH of the sludge

was analyzed. For statistical analysis, analysis of variance

(ANOVA) with significance level of 0.05 was performed

using a statistical package for the social sciences (SPSS

version 21, International Business Machine Corporation,

Armonk, NY).

Results and Discussion

The widespread application and high-volume production

of esters have caused high possibility of ester found in

wastewater of many industries. Various esters at different

concentrations and different chain lengths of functional

group and alkyl chain were added into a batch anaerobic

digestion system. The gas production was measured to

indicate the inhibitory or enhancement effect of the esters

to the system.

Are esters beneficial or detrimental forbiogas production?

In order to investigate the effect of the ester on biogas

production, methyl butanoate, ethyl butanoate, ethyl hex-

anoate, and hexyl acetate were added at different concen-

trations. The inoculum was incubated in the presence of

these esters at five different concentrations that are 0.05,

0.5, 5, 10, and 20 g L�1. Table 1 and Figure 1 present a

summary of the effects of esters on methane production.

The results show that after 30 days, addition of ethyl

butanoate, ethyl hexanoate, and hexyl acetate at concentra-

tions up to 5 g L�1 resulted in increased biogas production

(Table 1). Similar result was obtained from methyl but-

anoate at concentrations up to 10 g L�1. The highest

methane productions for ethyl butanoate, ethyl hexanoate,

and hexyl acetate were 117.4 � 6.6, 105.0 � 5.1, and

112.0 � 3.8 mL, respectively, in the presence of ester at a

concentration of 5 g L�1. These results correspond to

63.5%, 46.2%, and 56% increase in methane production.

The highest methane production for methyl butanoate was

obtained by adding 10 g L�1 was 96.79 � 0.94 mL, corre-

sponding to 34.8% increase in methane yield. The effect of

the esters on the initial digestion rate is presented in Fig-

ure 2. With the exception of hexyl acetate, adding other

ester compounds up to 10 g L�1 is still beneficial for the

digestion in the early process. This higher methane yield in

the presence of esters is most probably due to the esters

consumption by the anaerobic digesting microorganisms.

However, increasing concentration of the esters to more

than 5 g L�1 had an inhibitory effect. It was even more

obvious when the concentration of the esters added was

Table 1. The effects of esters added during methane production.

Ester

Concentration

(g L�1)

Cumulative

methane (mL)

Methane yield

(L g�1 VS) Enhancement1(%) pH

Inoculum – 46.7 � 11.5 0.3 � 0.19 0 7.74

Control (medium + inoculum) – 71.8 � 11.3 1.2 � 0.19 0 7.74

Methyl butanoate 0.05 65.1 � 2.4 1.08 � 0.04 �9.42 7.94

0.5 82.3 � 3.7 1.33 � 0.06 15.4 7.86

5 89 � 10 1.48 � 0.17 24 7.89

10 96.8 � 0.9 1.61 = 0.02 34.8 7.84

20 3.3 � 0.6 0.05 � 0.01 �95.5 5.42

Ethyl butanoate 0.05 68.9 � 9 1.15 � 0.15 �4.12 7.84

0.5 85.7 � 2.6 1.43 � 0.04 19.4 7.76

5 117.4 � 6.6 1.96 � 0.11 63.6 7.77

10 60.8 � 7 1.01 � 0.12 �15.3 5.8

20 9.3 � 5.4 0.15 � 0.09 �87.1 5.31

Ethyl hexanoate 0.05 78.2 � 2.8 1.3 � 0.05 8.9 7.84

0.5 95.1 � 5.6 1.53 = 0.09 32.4 7.78

5 105 � 5.1 1.75 � 0.08 46.2 7.77

10 74.9 � 6.8 1.19 = 0.01 �12 6.65

20 20.2 � 2.9 0.34 � 0.05 �71.9 6.61

Hexyl acetate 0.05 69.8 � 12 1.16 � 0.19 �2.82 7.81

0.5 92.3 � 0.5 1.54 � 0.01 28.5 7.83

5 112 � 3.3 1.37 � 0.06 56 7.84

10 4.2 � 0.7 0.7 � 0.01 �94.2 7

20 3.9 � 1.5 0.06 � 0.02 �94.6 6.91

1Enhancement ¼ CH4 produced by sample containing ester� CH4 produced by controlCH4 produced by control

� 100%

Negative value shows inhibition.2Not significantly different from the control.


Effect of Ester Compounds on Biogas Production H. Yanti et al.

plasticizer for cellulose, nail polish removers, perfumery

product, plastic tubing, floor tiles, furniture, automobile

upholstery, insect repellents, and as paint additive [3, 4].

Ester can be manufactured via chemical reaction by con-

densation of alcohols and carboxylic acids. In addition, it

can be found in nature as the major volatile compounds

in fruits. For instance, esters constitute 78–92%, 25–90%,

and 13% of the total volatile mass of apple, strawberry,

and raspberry, respectively [5]. Therefore, it is used as a

flavor and fragrance compound in food industry. The

widespread production and ability of some esters to

migrate, make esters to be easily found in the environ-

ment [6].

Since anaerobic digestion is mostly used to tread waste-

waters, investigation of effect of ester on anaerobic diges-

tion is important. Esters might be beneficial or detrimental

to the anaerobic digestion. Some esters are reported to be

degraded by Acetobacterium woodii and Eubacterium limo-

sum, which are involved in anaerobic digestion process

[7]. However, some phthalate esters (PAE) could reduce

the biogas production at a concentration higher than

60 mg L�1 [8]. Besides, some esters are reported having

antimicrobial activity against some standard microorgan-

isms. Butyl acetate and hexyl acetate at 4 lL and 8 lL,respectively, were toxic to Conidia germination after incu-

bation for 24 h at 22°C [9]. Hexyl acetate at concentration

150 mg L�1 has significant inhibitory effect against E. coli,

S. enteritidis, and L. monocytogenes that were isolated both

in model system and in fresh-sliced apples [10]. To our

knowledge, scarce information is available on the effect of

esters in methane production.

In this study, some esters that are available in nature as

fruit flavor compounds and mainly used in food, cos-

metic, and solvent industries were examined. The aim of

this study was to investigate the effects of the ester com-

pounds on biogas production, and their minimum inhibi-

tory concentration (MIC). In addition, the effect of chain

length of functional group and alkyl chain of ester on

biogas production was investigated.

Material and Methods

Microorganisms and chemicals

Inoculum (sludge) with 22 g VS L�1 (volatile solid per

liter) was obtained from a thermophilic biogas plant

(55°C) at Bor�as Energy and Environment AB (Bor�as,

Sweden). The inoculum was then stored in an incubator

at temperature 55°C for 3 days. A synthetic medium solu-

tion was prepared from 20 g L�1 of nutrient broth

(Sigma-Aldrich, St. Louis, MO), 20 g L�1 of yeast extract

(Merck KgaA, Darmstadt, Germany), and 20 g L�1 of

glucose (Merck). Nutrient broth contained 10 g L�1 beef

extract, 10 g L�1 peptone, and 5 g L�1 sodium chloride.

The ester compounds (Sigma-Aldrich) used in this work

were methyl butanoate, ethyl butanoate, ethyl hexanoate,

hexyl acetate, methyl acetate, ethyl acetate, and butyl ace-

tate.

The experiments were carried out in two steps. The

first step was to investigate the effect of ester compounds

on biogas production. In this step, the concentrations of

methyl butanoate, ethyl butanoate, ethyl hexanoate, and

hexyl acetate were 0.05, 0.5, 5, 10, and 20 g L�1 for each

compound. The second step was to investigate the effect

of chain length of functional group and alkyl chain of

ester on biogas production. In this step, the concentration

of ethyl hexanoate, ethyl butanoate, methyl acetate, ethyl

acetate, butyl acetate, and hexyl acetate was 5 g L�1.

Anaerobic digestion

The method used for anaerobic digestion was adapted

from the method described by Hansen et al. [11] and the

OECD [12] with minor modifications. The experiments

were carried out in a 120-mL glass bottle containing

50 mL of sludge, 1 mL of medium, and 2.5 mL of ester

solution or distilled water (for a control). In order to mea-

sure methane production from the inoculum, the inocu-

lum was incubated without addition of medium and ester

solution in the glass bottle containing 50 mL of sludge

and 3.5 mL of distilled water. The bottles were closed

tightly and the headspace was filled with gas mixture con-

taining 80% N2 and 20% CO2 to achieve anaerobic condi-

tion. The bottles were then incubated at 55°C for 28 days.

During incubation, the bottles were shaken twice a day

using water bath shaker (55°C) at 150 rpm. Gas samples

were taken from headspace of the reactors through the

septum using a syringe with pressure lock (VICI; Precision

Sampling Inc., Baton Rouge, LA). Samples were taken at

3, 6, 9, 12, 15, 20, 25, and 30 days. The initial methane

production rate was measured as the mean of methane

production per day during the first 10 days.

Analytical methods

Biogas production from the digestion experiments was

measured using Gas Chromatography (Varian 450 GC,

Palo Alto, CA) with a capillary column (J 6 W scientific

GS-Gas Pro, bonded silica based 30 m 9 0.32 mm,

Agilent Technologies, Santa Clara, CA) and a thermal

conductivity detector (Varian, Palo Alto, CA). The condi-

tions for the analysis were injector temperature of 75°C,oven temperature of 100°C, detector temperature of

120°C, and column flow (N2) 2 mL min�1.

All experiments were carried out in triplicate batch

experiment; the result was presented in average � standard



deviation. At the end of digestion, the pH of the sludge

was analyzed. For statistical analysis, analysis of variance

(ANOVA) with significance level of 0.05 was performed

using a statistical package for the social sciences (SPSS

version 21, International Business Machine Corporation,

Armonk, NY).

Results and Discussion

The widespread application and high-volume production

of esters have caused high possibility of ester found in

wastewater of many industries. Various esters at different

concentrations and different chain lengths of functional

group and alkyl chain were added into a batch anaerobic

digestion system. The gas production was measured to

indicate the inhibitory or enhancement effect of the esters

to the system.

Are esters beneficial or detrimental forbiogas production?

In order to investigate the effect of the ester on biogas

production, methyl butanoate, ethyl butanoate, ethyl hex-

anoate, and hexyl acetate were added at different concen-

trations. The inoculum was incubated in the presence of

these esters at five different concentrations that are 0.05,

0.5, 5, 10, and 20 g L�1. Table 1 and Figure 1 present a

summary of the effects of esters on methane production.

The results show that after 30 days, addition of ethyl

butanoate, ethyl hexanoate, and hexyl acetate at concentra-

tions up to 5 g L�1 resulted in increased biogas production

(Table 1). Similar result was obtained from methyl but-

anoate at concentrations up to 10 g L�1. The highest

methane productions for ethyl butanoate, ethyl hexanoate,

and hexyl acetate were 117.4 � 6.6, 105.0 � 5.1, and

112.0 � 3.8 mL, respectively, in the presence of ester at a

concentration of 5 g L�1. These results correspond to

63.5%, 46.2%, and 56% increase in methane production.

The highest methane production for methyl butanoate was

obtained by adding 10 g L�1 was 96.79 � 0.94 mL, corre-

sponding to 34.8% increase in methane yield. The effect of

the esters on the initial digestion rate is presented in Fig-

ure 2. With the exception of hexyl acetate, adding other

ester compounds up to 10 g L�1 is still beneficial for the

digestion in the early process. This higher methane yield in

the presence of esters is most probably due to the esters

consumption by the anaerobic digesting microorganisms.

However, increasing concentration of the esters to more

than 5 g L�1 had an inhibitory effect. It was even more

obvious when the concentration of the esters added was

Table 1. The effects of esters added during methane production.

Ester

Concentration

(g L�1)

Cumulative

methane (mL)

Methane yield

(L g�1 VS) Enhancement1(%) pH

Inoculum – 46.7 � 11.5 0.3 � 0.19 0 7.74

Control (medium + inoculum) – 71.8 � 11.3 1.2 � 0.19 0 7.74

Methyl butanoate 0.05 65.1 � 2.4 1.08 � 0.04 �9.42 7.94

0.5 82.3 � 3.7 1.33 � 0.06 15.4 7.86

5 89 � 10 1.48 � 0.17 24 7.89

10 96.8 � 0.9 1.61 = 0.02 34.8 7.84

20 3.3 � 0.6 0.05 � 0.01 �95.5 5.42

Ethyl butanoate 0.05 68.9 � 9 1.15 � 0.15 �4.12 7.84

0.5 85.7 � 2.6 1.43 � 0.04 19.4 7.76

5 117.4 � 6.6 1.96 � 0.11 63.6 7.77

10 60.8 � 7 1.01 � 0.12 �15.3 5.8

20 9.3 � 5.4 0.15 � 0.09 �87.1 5.31

Ethyl hexanoate 0.05 78.2 � 2.8 1.3 � 0.05 8.9 7.84

0.5 95.1 � 5.6 1.53 = 0.09 32.4 7.78

5 105 � 5.1 1.75 � 0.08 46.2 7.77

10 74.9 � 6.8 1.19 = 0.01 �12 6.65

20 20.2 � 2.9 0.34 � 0.05 �71.9 6.61

Hexyl acetate 0.05 69.8 � 12 1.16 � 0.19 �2.82 7.81

0.5 92.3 � 0.5 1.54 � 0.01 28.5 7.83

5 112 � 3.3 1.37 � 0.06 56 7.84

10 4.2 � 0.7 0.7 � 0.01 �94.2 7

20 3.9 � 1.5 0.06 � 0.02 �94.6 6.91

1Enhancement ¼ CH4 produced by sample containing ester� CH4 produced by controlCH4 produced by control

� 100%

Negative value shows inhibition.2Not significantly different from the control.



20 g L�1. At the end of digestion when 20 g L�1 was added,

the methane yield decreased by 71.9–95.5% (Table 1). Addi-

tion of 20 g L�1 of ester reduced methane production by

47–90% and showed inhibitory effect since the early stage of

digestion (Fig. 2). These results indicate that the effect of

esters on anaerobic digestion is concentration-dependent.

At concentrations up to 5 g L�1, esters are beneficial,

whereas at higher concentrations than 5 g L�1, the esters

gave negative effect on biogas production.

In order to evaluate the effect of esters on biogas

composition, a methane content ratio was determined

with the assumption that only methane and carbon dioxide

produced during digestion. The results are presented in

Figure 3. The biogas composition of the control experiment

was 63.6% of methane and 36.4% of carbon dioxide. Once

esters were added up to 5 g L�1, the methane content ratio

of the reactors did not significantly vary compared with that

of the control experiment without the esters. However, the

methane content ratio declined with the presence of

10 g L�1 esters, and the biogas only contained 3–15% of

methane in the presence of 20 g L�1 of esters. In the case of

methyl butanoate, the methane production increased up to

34.8% at a concentration of 10 g L�1, but the methane con-

tent ratio reduced from 63.6% to 48.9%.

A MIC is defined as the lowest concentration of ester

that will inhibit the activity of microorganisms during

anaerobic digestion. Accordingly, the MIC of methyl

butanoate was between 10 and 20 g L�1, and for ethyl

butanoate, ethyl hexanoate, and hexyl acetate it was 5–10 g L�1. The precise action of antimicrobial activity of

ester is not yet clear. One explanation could be due to

the changes in permeability of the cell membranes, which

cause leakage of cellular components and influence the

metabolism of bacteria [13].

At the end of anaerobic digestion, the pH was mea-

sured which showed that the pH had decreased from 7.5–8 to 5–6 when ester at concentrations higher than 5 g L�1

(for ethyl butanoate and ethyl hexanoate) and

10 g L�1(for methyl butanoate) were added (Table 1).

The decrease in pH is caused by accumulation of volatile

fatty acids as intermediate products that are produced

during acetogenesis reaction. Inhibition could occur when

accumulation of volatile fatty acids could no longer be

handled by the buffering system of anaerobic digestion

[14, 15]. Infantes et al. [16] reported that high energy is

0

20

40

60

80

100

0 5 10 15 20 25 30 0 5 10 15 20 25 30

0

20

40

60

80

100

120

0 5 10 15 20 25 30 0 5 10 15 20 25 30

(A) (B)

(C) (D)

Digestion time (day)

CH

4 pro

duct

ion(

ml)

Figure 1. Cumulative methane production

with addition of ester compound at different

concentrations (+)20 g L�1, (○) 10 g L�1, (*)

5 g L�1, (x) 0.5 g L�1, (D) 0.05 g L�1, and (□)control.

Hexyl acetateNethyl butanoate

Ethyl butanoateEthyl hexanoate

0

1

2

3

4

5

6

7

8

9

10

Met

hane

pro

duct

ion

rate

(m

l/day

)

Flavor concentrations (g L–1) Flav

or co

mpo

unds

Hexyl acetate Methyl butanoate Ethyl butanoate Ethyl hexanoate

Figure 2. Initial methane production rate with addition of esters at

various concentrations.



consumed to maintain the intercellular pH at low pH

which inhibits the cell metabolism.

Ester consumption

In order to confirm the assumption that esters can act

as a carbon source, experiments using 5 g L�1 of esters

as the sole carbon source were conducted. As control,

the anaerobic glass digester was filled with inoculum

without an added medium. The results are presented in

Figure 4 and show that methyl butanoate, ethyl butano-

ate, ethyl hexanoate, and hexyl acetate gave higher

methane production than did the control. This result is

in accordance with a previous study reporting that

methyl esters could be degraded to carboxylic acid and

alcohol and would be further converted into methane

by A. woodii and E. limosum [7]. Those microorgan-

isms are found in all four stages of anaerobic biochem-

ical reactions which have ability to degrade methyl

ester of acetate, propionate, butanoate, and isobutanoate

into carboxylic acid and methanol under anaerobic con-

dition [7, 14]. According to the mechanisms of methyl

ester degradation, methyl butanoate will be degraded

into methanol and acetic acid. Ethyl butanoate and

ethyl hexanoate probably also follow this mechanism.

0102030405060708090

100

Met

hane

con

tent

(%)

0102030405060708090

100

Met

hane

con

tent

(%)

Concentration (g L–1) Concentration (g L–1)

Control 0.05 0.5 5 10 20

(C) (D)

(A) (B)

Figure 3. Methane content ratio (■) CH4, (□)CO2 with addition of various ester compounds

at different concentrations.

0

20

40

60

80

100

120

140

0 5 10 15 20 25 30

CH

4 pr

oduc

tion

(ml)


Figure 4. Methane production with addition of various ester

compounds (D) methyl butanoate, (x) ethyl butanoate, (*) ethyl

hexanoate, (○) hexyl acetate, and (□) control at 5 g L�1

0102030405060708090

100

ino MB EB EH HA

Met

hane

con

tent

(%)

Compounds

CO2 CH4

Figure 5. Methane content ratio of inoculum (ino), methyl butanoate

(MB), ethyl butanoate (EB), ethyl hexanoate (EH), and hexyl acetate

(HA) as sole carbon source compounds at 5 g L�1.



20 g L�1. At the end of digestion when 20 g L�1 was added,

the methane yield decreased by 71.9–95.5% (Table 1). Addi-

tion of 20 g L�1 of ester reduced methane production by

47–90% and showed inhibitory effect since the early stage of

digestion (Fig. 2). These results indicate that the effect of

esters on anaerobic digestion is concentration-dependent.

At concentrations up to 5 g L�1, esters are beneficial,

whereas at higher concentrations than 5 g L�1, the esters

gave negative effect on biogas production.

In order to evaluate the effect of esters on biogas

composition, a methane content ratio was determined

with the assumption that only methane and carbon dioxide

produced during digestion. The results are presented in

Figure 3. The biogas composition of the control experiment

was 63.6% of methane and 36.4% of carbon dioxide. Once

esters were added up to 5 g L�1, the methane content ratio

of the reactors did not significantly vary compared with that

of the control experiment without the esters. However, the

methane content ratio declined with the presence of

10 g L�1 esters, and the biogas only contained 3–15% of

methane in the presence of 20 g L�1 of esters. In the case of

methyl butanoate, the methane production increased up to

34.8% at a concentration of 10 g L�1, but the methane con-

tent ratio reduced from 63.6% to 48.9%.

A MIC is defined as the lowest concentration of ester

that will inhibit the activity of microorganisms during

anaerobic digestion. Accordingly, the MIC of methyl

butanoate was between 10 and 20 g L�1, and for ethyl

butanoate, ethyl hexanoate, and hexyl acetate it was 5–10 g L�1. The precise action of antimicrobial activity of

ester is not yet clear. One explanation could be due to

the changes in permeability of the cell membranes, which

cause leakage of cellular components and influence the

metabolism of bacteria [13].

At the end of anaerobic digestion, the pH was mea-

sured which showed that the pH had decreased from 7.5–8 to 5–6 when ester at concentrations higher than 5 g L�1

(for ethyl butanoate and ethyl hexanoate) and

10 g L�1(for methyl butanoate) were added (Table 1).

The decrease in pH is caused by accumulation of volatile

fatty acids as intermediate products that are produced

during acetogenesis reaction. Inhibition could occur when

accumulation of volatile fatty acids could no longer be

handled by the buffering system of anaerobic digestion

[14, 15]. Infantes et al. [16] reported that high energy is

0

20

40

60

80

100

0 5 10 15 20 25 30 0 5 10 15 20 25 30

0

20

40

60

80

100

120

0 5 10 15 20 25 30 0 5 10 15 20 25 30

(A) (B)

(C) (D)


CH

4 pro

duct

ion(

ml)

Figure 1. Cumulative methane production

with addition of ester compound at different

concentrations (+)20 g L�1, (○) 10 g L�1, (*)

5 g L�1, (x) 0.5 g L�1, (D) 0.05 g L�1, and (□)control.

Hexyl acetateNethyl butanoate

Ethyl butanoateEthyl hexanoate

0

1

2

3

4

5

6

7

8

9

10

Met

hane

pro

duct

ion

rate

(m

l/day

)

Flavor concentrations (g L–1) Flav

or co

mpo

unds

Hexyl acetate Methyl butanoate Ethyl butanoate Ethyl hexanoate

Figure 2. Initial methane production rate with addition of esters at

various concentrations.



consumed to maintain the intercellular pH at low pH

which inhibits the cell metabolism.

Ester consumption

In order to confirm the assumption that esters can act

as a carbon source, experiments using 5 g L�1 of esters

as the sole carbon source were conducted. As control,

the anaerobic glass digester was filled with inoculum

without an added medium. The results are presented in

Figure 4 and show that methyl butanoate, ethyl butano-

ate, ethyl hexanoate, and hexyl acetate gave higher

methane production than did the control. This result is

in accordance with a previous study reporting that

methyl esters could be degraded to carboxylic acid and

alcohol and would be further converted into methane

by A. woodii and E. limosum [7]. Those microorgan-

isms are found in all four stages of anaerobic biochem-

ical reactions which have ability to degrade methyl

ester of acetate, propionate, butanoate, and isobutanoate

into carboxylic acid and methanol under anaerobic con-

dition [7, 14]. According to the mechanisms of methyl

ester degradation, methyl butanoate will be degraded

into methanol and acetic acid. Ethyl butanoate and

ethyl hexanoate probably also follow this mechanism.

0102030405060708090

100

Met

hane

con

tent

(%)

0102030405060708090

100

Met

hane

con

tent

(%)

Concentration (g L–1) Concentration (g L–1)

Control 0.05 0.5 5 10 20

(C) (D)

(A) (B)

Figure 3. Methane content ratio (■) CH4, (□)CO2 with addition of various ester compounds

at different concentrations.

0

20

40

60

80

100

120

140

0 5 10 15 20 25 30

CH

4 pr

oduc

tion

(ml)


Figure 4. Methane production with addition of various ester

compounds (D) methyl butanoate, (x) ethyl butanoate, (*) ethyl

hexanoate, (○) hexyl acetate, and (□) control at 5 g L�1

0102030405060708090

100

ino MB EB EH HA

Met

hane

con

tent

(%)

Compounds

CO2 CH4

Figure 5. Methane content ratio of inoculum (ino), methyl butanoate

(MB), ethyl butanoate (EB), ethyl hexanoate (EH), and hexyl acetate

(HA) as sole carbon source compounds at 5 g L�1.



Hydrolytic bacteria degrade ethyl butanoate and ethyl

hexanoate into ethanol and butanoic and hexanoic acid,

respectively [15]. The carboxylic acid and alcohol will

be further degraded through acetogenesis stage into

acetic acid and hydrogen, which will finally be con-

verted into methane [14].

In order to evaluate the biogas composition produced

from the esters, the methane content ratio was deter-

mined (Fig. 5). The ratios produced from the methyl

butanoate, ethyl butanoate, and ethyl hexanoate were

varied from 64.7, 55.7%, and 54.1%. These values were

slightly higher compared with those of the control

Table 2. Theoretical methane production from ester and ester consumption.

Sample

Ester conc.

(g L�1)

Cumulative

methane (mL)

Methane produced

from ester (mL)1

Theoretical methane

production from ester

(mL)2Ester consumption

(%)3

Control (medium + inoculum) 0 71.8 � 11.3 –4 – –

Medium+ 0.05 65.1 � 2.4 – 1.9 –

Methyl butanoate 0.50 82.8 � 3.7 11.0 19.1 57.8

5.00 89 � 10 17.2 190.9 9.0

10.0 96.8 � 0.9 25.0 381.8 6.5

20.0 3.3 � 0.6 – 763.7 –

Medium + ethyl butanoate 0.05 68.9 � 9 – 2.1 –

0.50 85.7 � 2.6 13.9 20.7 67.5

5.00 117.4 � 6.6 45.6 206.6 22.1

10.0 60.8 � 7 – 413.2 –

20.0 9.3 � 5.4 – 826.5 –

Medium + ethyl hexanoate 0.05 78.2 � 2.8 6.4 2.3 280.5

0.50 95.1 � 5.6 23.3 22.9 101.7

5.00 105 � 5.1 33.2 228.9 14.5

10.0 74.9 � 6.8 – 457.7 –

20.0 20.2 � 2.9 – 915.4 –

Medium + hesylacetate 0.05 69.8 � 12 – 23 –

0.50 92.3 � 0.5 20.5 22.9 89.4

5.00 112 � 3.8 40.2 228.9 17.6

10.0 4.2 � 0.7 – 457.7 –

20.0 3.9 � 1.5 – 915.4 –

Inoculum 0 46.7 � 11.5 – – –

Inoculum + methyl butanoate 5.00 126.2 � 10.28 80.7 190.09 42.5

Inoculum + ethyl butanoate 5.00 95.88 � 18.8 40.8 228.86 19.8


Inoculum + hexylacetate 5.00 3.29 � 0.2 – 228.86 –

1Methane produced from ester = methane produced by sample methane produced by control.2Theoretical methane production from ester was calculated from following equation:CcHhOoNnSs ¼ yH2O ! xCH4 þ nNH3 þ sH2Sþ ðc� xÞCO2

3Ester consumption ¼ Methane produced from esterTheoretical methane production from ester

� 100%

4(–)No production/no consumption.

Table 3. Cumulative methane production with addition of acetate ester and ethyl ester.

Ester

Number of carbonCumulative

methane (mL)

Methane yield

(L g�1 VS)Alkyl group Functional group

Inoculum – – 46.7 � 11.5 0.8 � 0.19

Ethyl hexanoate 2 6 89.4 � 14.5 1.4 � 0.2

Ethyl butanoate 2 4 96.0 � 8.3 1.6 � 0.1

Methyl acetate 1 2 111.5 � 9.8 1.9 � 0.2

Ethyl acetate 2 2 126.1 � 10.3 2.1 � 0.2

Butyl acetate 4 2 111.7 � 15.7 1.9 � 0.3

Hexyl acetate 6 2 3.3 � 0.1 0.1 � 0



(50.2%). However, no methane was produced from hexyl

acetate.

Esters are mostly organic compounds. As an organic

compound, the chemical reaction of degradation of

organic matters is given in equation (1).

CcHhOoNnSs ¼ yH2O ! xCH4 þ nNH3 þ sH2S

þ ðc� xÞCO2:(1)

Thus, theoretically, the potential methane production

from esters can be calculated. According to the methane

obtained from ester, ester consumption can be deter-

mined. The effect of concentrations and the presence of

medium were investigated and the results are presented in

Table 2. The results show that in the presence of a med-

ium, the highest ester consumption was obtained at their

concentration of 0.5 g L�1, which was up to 57.8–100%.

However, almost no esters were consumed at concentra-

tion higher than 10 g L�1 for all esters examined. In the

absence of medium, ester consumption is in the range of

17.8–42.5% at concentration of 5 g L�1. For ethyl but-

anoate and ethyl hexanoate, the ester consumption was

similar either in the presence or absence of medium,

whereas ester consumption of methyl butanoate was

higher in the absence of medium. Interestingly, hexyl ace-

tate exhibited a different behavior in terms of medium. It

was consumed in the presence of medium while it acted

as an inhibitor in the absence of medium, as shown by

the significantly lower methane production than that of

the inoculum. This might indicate a synergy effect

between medium and hexyl acetate, enabling hexyl acetate

to be consumed at this concentration.

Effect of chain length of the functionalgroup (alkyl carboxylic chain) and AlkylChain of ester

Esters consist of a functional group and alkyl chain. In

this study, various carbon lengths of functional group

and alkyl chains of esters were added as sole carbon

source at concentration of 5 g L�1. The results are pre-

sented in Table 3, and Figures 6 and 7. The functional

groups (R1COO-) of the ester were acetate (C2), butano-

ate (C4), and hexanoate (C6). The alkyls (-R2) of the

ester were methyl (C1), ethyl (C2), butyl (C4), and hexyl

(C6).

For the experiments with ethyl ester, an increasing

number of carbons in the functional group required

longer time to achieve maximum methane production

and it decreased the methane production (Fig. 6A). For

the experiments with acetate esters, the cumulative meth-

ane production was much higher compared to that of

the control. When the alkyl was butyl (C4), it exhibited

a longer lag phase compared to those of methyl (C1)

0

20

40

60

80

100

120

140

0 5 10 15 20 25 30

CH

4 pr

oduc

tin

0 5 10 15 20 25 30Digestion time (day)

(a) (b)

Figure 6. Methane production with addition

of various ester compounds (x) Methyl acetate,

(*) Ethyl acetate, (○) Butyl acetate, (D) Hexylacetate, (+) Ethyl butanoate, (□) Ethylhexanoate, and (◊) control at 5 g L�1.

0102030405060708090

100

Ethyl acetate Ethyl Butanoate Ethyl Hexanoate

Met

hane

con

tent

(%)

compounds

0

10

20

30

40

50

60

70

80

90

100

Methyl Acetate Ethyl Acetate Butyl Acetate Hexyl Acetate

Met

hane

con

tent

(%)

Compounds

CO2

CH4

(A)

(B)

Figure 7. Methane content ratio with addition of ethyl esters (A) and

acetate esters (B) at concentration of 5 g L�1.



Hydrolytic bacteria degrade ethyl butanoate and ethyl

hexanoate into ethanol and butanoic and hexanoic acid,

respectively [15]. The carboxylic acid and alcohol will

be further degraded through acetogenesis stage into

acetic acid and hydrogen, which will finally be con-

verted into methane [14].

In order to evaluate the biogas composition produced

from the esters, the methane content ratio was deter-

mined (Fig. 5). The ratios produced from the methyl

butanoate, ethyl butanoate, and ethyl hexanoate were

varied from 64.7, 55.7%, and 54.1%. These values were

slightly higher compared with those of the control

Table 2. Theoretical methane production from ester and ester consumption.

Sample

Ester conc.

(g L�1)

Cumulative

methane (mL)

Methane produced

from ester (mL)1

Theoretical methane

production from ester

(mL)2Ester consumption

(%)3

Control (medium + inoculum) 0 71.8 � 11.3 –4 – –

Medium+ 0.05 65.1 � 2.4 – 1.9 –

Methyl butanoate 0.50 82.8 � 3.7 11.0 19.1 57.8

5.00 89 � 10 17.2 190.9 9.0

10.0 96.8 � 0.9 25.0 381.8 6.5

20.0 3.3 � 0.6 – 763.7 –

Medium + ethyl butanoate 0.05 68.9 � 9 – 2.1 –

0.50 85.7 � 2.6 13.9 20.7 67.5

5.00 117.4 � 6.6 45.6 206.6 22.1

10.0 60.8 � 7 – 413.2 –

20.0 9.3 � 5.4 – 826.5 –

Medium + ethyl hexanoate 0.05 78.2 � 2.8 6.4 2.3 280.5

0.50 95.1 � 5.6 23.3 22.9 101.7

5.00 105 � 5.1 33.2 228.9 14.5

10.0 74.9 � 6.8 – 457.7 –

20.0 20.2 � 2.9 – 915.4 –

Medium + hesylacetate 0.05 69.8 � 12 – 23 –

0.50 92.3 � 0.5 20.5 22.9 89.4

5.00 112 � 3.8 40.2 228.9 17.6

10.0 4.2 � 0.7 – 457.7 –

20.0 3.9 � 1.5 – 915.4 –

Inoculum 0 46.7 � 11.5 – – –

Inoculum + methyl butanoate 5.00 126.2 � 10.28 80.7 190.09 42.5



Inoculum + hexylacetate 5.00 3.29 � 0.2 – 228.86 –

1Methane produced from ester = methane produced by sample methane produced by control.2Theoretical methane production from ester was calculated from following equation:CcHhOoNnSs ¼ yH2O ! xCH4 þ nNH3 þ sH2Sþ ðc� xÞCO2

3Ester consumption ¼ Methane produced from esterTheoretical methane production from ester

� 100%

4(–)No production/no consumption.

Table 3. Cumulative methane production with addition of acetate ester and ethyl ester.

Ester

Number of carbonCumulative

methane (mL)

Methane yield

(L g�1 VS)Alkyl group Functional group

Inoculum – – 46.7 � 11.5 0.8 � 0.19

Ethyl hexanoate 2 6 89.4 � 14.5 1.4 � 0.2

Ethyl butanoate 2 4 96.0 � 8.3 1.6 � 0.1

Methyl acetate 1 2 111.5 � 9.8 1.9 � 0.2

Ethyl acetate 2 2 126.1 � 10.3 2.1 � 0.2

Butyl acetate 4 2 111.7 � 15.7 1.9 � 0.3

Hexyl acetate 6 2 3.3 � 0.1 0.1 � 0



(50.2%). However, no methane was produced from hexyl

acetate.

Esters are mostly organic compounds. As an organic

compound, the chemical reaction of degradation of

organic matters is given in equation (1).

CcHhOoNnSs ¼ yH2O ! xCH4 þ nNH3 þ sH2S

þ ðc� xÞCO2:(1)

Thus, theoretically, the potential methane production

from esters can be calculated. According to the methane

obtained from ester, ester consumption can be deter-

mined. The effect of concentrations and the presence of

medium were investigated and the results are presented in

Table 2. The results show that in the presence of a med-

ium, the highest ester consumption was obtained at their

concentration of 0.5 g L�1, which was up to 57.8–100%.

However, almost no esters were consumed at concentra-

tion higher than 10 g L�1 for all esters examined. In the

absence of medium, ester consumption is in the range of

17.8–42.5% at concentration of 5 g L�1. For ethyl but-

anoate and ethyl hexanoate, the ester consumption was

similar either in the presence or absence of medium,

whereas ester consumption of methyl butanoate was

higher in the absence of medium. Interestingly, hexyl ace-

tate exhibited a different behavior in terms of medium. It

was consumed in the presence of medium while it acted

as an inhibitor in the absence of medium, as shown by

the significantly lower methane production than that of

the inoculum. This might indicate a synergy effect

between medium and hexyl acetate, enabling hexyl acetate

to be consumed at this concentration.

Effect of chain length of the functionalgroup (alkyl carboxylic chain) and AlkylChain of ester

Esters consist of a functional group and alkyl chain. In

this study, various carbon lengths of functional group

and alkyl chains of esters were added as sole carbon

source at concentration of 5 g L�1. The results are pre-

sented in Table 3, and Figures 6 and 7. The functional

groups (R1COO-) of the ester were acetate (C2), butano-

ate (C4), and hexanoate (C6). The alkyls (-R2) of the

ester were methyl (C1), ethyl (C2), butyl (C4), and hexyl

(C6).

For the experiments with ethyl ester, an increasing

number of carbons in the functional group required

longer time to achieve maximum methane production

and it decreased the methane production (Fig. 6A). For

the experiments with acetate esters, the cumulative meth-

ane production was much higher compared to that of

the control. When the alkyl was butyl (C4), it exhibited

a longer lag phase compared to those of methyl (C1)

0

20

40

60

80

100

120

140

0 5 10 15 20 25 30

CH

4 pr

oduc

tin

0 5 10 15 20 25 30Digestion time (day)

(a) (b)

Figure 6. Methane production with addition

of various ester compounds (x) Methyl acetate,

(*) Ethyl acetate, (○) Butyl acetate, (D) Hexylacetate, (+) Ethyl butanoate, (□) Ethylhexanoate, and (◊) control at 5 g L�1.

0102030405060708090

100

Ethyl acetate Ethyl Butanoate Ethyl Hexanoate

Met

hane

con

tent

(%)

compounds

0

10

20

30

40

50

60

70

80

90

100

Methyl Acetate Ethyl Acetate Butyl Acetate Hexyl Acetate

Met

hane

con

tent

(%)

Compounds

CO2

CH4

(A)

(B)

Figure 7. Methane content ratio with addition of ethyl esters (A) and

acetate esters (B) at concentration of 5 g L�1.



and ethyl (C2). It was observed that strong inhibition

occurred when the alkyl was hexyl (C6) and no meth-

ane was produced (Fig. 6B). Moreover, the methane

content ratio decreased with an increasing number of

carbons both in the functional group and alkyl chain

(Fig. 7). The methane content ratio decreased from

59.8% to 54.1% with increasing number of carbon in

functional group and it decreased from 59.8% to zero

with increasing number of carbons in alkyl chain of

esters. A correlation between increasing length of the

ester side-chain and decreasing biodegradability as well as

increasing the toxicity has been confirmed by Gavala et al.

[8] Similarly, Merkl et al. [17] found that the derivative of

phenolic acids, butyl ester is approximately three times

more toxic than methyl ester. The hydrophobicity of ester

increases with increasing chain length of ester. Esters have

both a hydrophilic head and hydrophobic tail, which

resemble to bipolar membrane of the bacterial cell wall.

This similarity might be the reason the hydrophobic tail of

ester targets the microorganisms cell membranes by pene-

trating and disrupting normal function of cellular mem-

branes, thus resulting in the leakage of cells or even killing

the cells [18].

Conclusion

Addition of methyl butanoate, ethyl butanoate, ethyl

hexanoate, and hexyl acetate at concentrations up to

5 g L�1 increased methane production. Addition of

esters to anaerobic digestion at concentrations higher

than 5 g L�1 decreased methane production. The MIC

for methyl butanoate was between 10 and 20 g L�1,

while for ethyl butanoate, ethyl hexanoate, and hexyl

acetate the MIC was between 5 and 10 g L�1. Methyl

butanoate, ethyl butanoate, and ethyl hexanoate at a

concentration of 5 g L�1 could function as sole carbon

source for the anaerobic digesting bacteria. For ethyl

ester, the methane production decreased with increase

in the number of carbons in the functional group. For

acetate esters, with an alkyl chain longer than butyl,

the number of carbons in the functional group started

to give an inhibitory effect for methane production.

Acknowledgment

We express our gratitude to Directorate General of Higher

Education, Ministry of National Education of Republic of

Indonesia through Competitive Research Grant of Interna-

tional joint Research for International Publication (grant

no LPPM-UGM/696/LIT/2013), the Swedish International

Development Agency (SIDA), the Swedish Gas Centre for

financial support of this study, and to Bor�as Energy and

Environment AB (Bor�as, Sweden) for the inoculum.

Conflict of Interest

None declared.

References

1. Yadvika Santosh, T. R. Sreekrishnan, S. Kohli, and V.

Rana. 2004. Enhancement of biogas production from solid

substrates using different techniques – a review. Bioresour.

Technol. 95:1–10.

2. Kroeker, E. J., D. D. Schulte, A. B. Sparling, and H. M.

Lapp. 1979. Anaerobic treatment process stability. J. Water

Pollut. Control Fed. 51:718–727.

3. Papa, E., F. Battaini, and P. Gramatica. 2005. Ranking of

aquatic toxicity of esters modelled by QSAR. Chemosphere

58:559–570.

4. Kirk, R. E., and D. F. Othmer. 1988. Encyclopedia of

chemical technology, 4th ed. Vol. 21. John Wiley and Sons

Inc., New York, NY.

5. Berger, R. G. 2007. Flavours and fragrances – chemistry,

bioprocessing and sustainability. Springer, Germany.

6. Parkerton, T. F., and W. J. Konkel. 2000. Application of

quantitative structure-activity relationships for assessing

the aquatic toxicity of phthalate esters. Ecotoxicol.

Environ. Saf. 45:61–78.

7. Liu, S., and J. M. Suflita. 1994. Anaerobic biodegradation

of methyl esters by Acetobacterium woodii and Eubacterium

limosum. J. Ind. Microbiol. 13:321–327.

8. Gavala, H. N., F. Alatriste-Mondragon, R. Iranpour, and B.

K. Ahring. 2003. Biodegradation of phthalate esters during

the mesophilic anaerobic digestion of sludge. Chemosphere

52:673–682.

9. Filonow, A. B. 1999. Yeasts reduce the stimulatory

effect of acetate esters from apple on the germination

of Botrytis cinerea Conidia. J. Chem. Ecol. 25:1555–

1565.

10. Lanciotti, R., N. Belletti, F. Patrignani, A. Gianotti, F.

Gardini and M. E. Guerzoni. 2003. Application of hexanal,

(E)-2-hexenal, and hexyl acetate to improve the safety of

fresh-sliced apples. J. Agric. Food Chem. 51:2958–2963.

11. Hansen, T. L., J. E. Schmidt, I. Angelidaki, E. Marca,

J. I. C. Jansen, H. Mosbaek, et al. 2004. Method for

determination of methane potentials of solid organic

waste. Waste Manage. 24:393–400.

12. Organisation for Economic Cooperation and Development

(OECD). 2007. Determination of the inhibition of the

activity of anaerobic bacteria - reduction of gas production

from anaerobically digesting sewage sludge, O.f.E.C.-o.a.D.

in Test No. 224. OECD Publishing, Paris, France. ISBN:

9264067337, 9789264067332.

13. Deng, W. L., T. R. Hamilton-Kemp, M. T. Nielsen, R. A.

Andersen, G. B. Collins and D. F. Hildebrand.1993. Effect

of six-carbon aldehydes and alcohols on bacterial

proliferation. J. Agric. Food Chem. 41:506–510.



14. Deublein, D., and A. Steinhauser. 2008. Biogas from waste

and renewable resources. Wiley-VCH, Germany.

15. Gerardi, M. H. 2003. The microbiology of anaerobic

digesters. John Wiley & Sons Inc, Canada.

16. Infantes, D., A. Gonz�alez Del Campo, J. Villase~nor, and

J. Fernandez. 2011. Influence of pH, temperature and

volatile fatty acids on hydrogen production by

acidogenic fermentation. Int. J. Hydrogen Energy

36:15595–15601.

17. Merkl, R., I. Hr�adkov�a, V. Filip, and J. �Smi drkal2010.

Antimicrobial and antioxidant properties of phenolic acids

alkyl esters. Czech J. Food Sci. 28:275–279.

18. Huang, C. B., B. George, and J. L. Ebersole. 2010.

Antimicrobial activity of n-6, n-7 and n-9 fatty acids and

their esters for oral microorganisms. Arch. Oral Biol.

55:555–560.



and ethyl (C2). It was observed that strong inhibition

occurred when the alkyl was hexyl (C6) and no meth-

ane was produced (Fig. 6B). Moreover, the methane

content ratio decreased with an increasing number of

carbons both in the functional group and alkyl chain

(Fig. 7). The methane content ratio decreased from

59.8% to 54.1% with increasing number of carbon in

functional group and it decreased from 59.8% to zero

with increasing number of carbons in alkyl chain of

esters. A correlation between increasing length of the

ester side-chain and decreasing biodegradability as well as

increasing the toxicity has been confirmed by Gavala et al.

[8] Similarly, Merkl et al. [17] found that the derivative of

phenolic acids, butyl ester is approximately three times

more toxic than methyl ester. The hydrophobicity of ester

increases with increasing chain length of ester. Esters have

both a hydrophilic head and hydrophobic tail, which

resemble to bipolar membrane of the bacterial cell wall.

This similarity might be the reason the hydrophobic tail of

ester targets the microorganisms cell membranes by pene-

trating and disrupting normal function of cellular mem-

branes, thus resulting in the leakage of cells or even killing

the cells [18].

Conclusion

Addition of methyl butanoate, ethyl butanoate, ethyl

hexanoate, and hexyl acetate at concentrations up to

5 g L�1 increased methane production. Addition of

esters to anaerobic digestion at concentrations higher

than 5 g L�1 decreased methane production. The MIC

for methyl butanoate was between 10 and 20 g L�1,

while for ethyl butanoate, ethyl hexanoate, and hexyl

acetate the MIC was between 5 and 10 g L�1. Methyl

butanoate, ethyl butanoate, and ethyl hexanoate at a

concentration of 5 g L�1 could function as sole carbon

source for the anaerobic digesting bacteria. For ethyl

ester, the methane production decreased with increase

in the number of carbons in the functional group. For

acetate esters, with an alkyl chain longer than butyl,

the number of carbons in the functional group started

to give an inhibitory effect for methane production.

Acknowledgment

We express our gratitude to Directorate General of Higher

Education, Ministry of National Education of Republic of

Indonesia through Competitive Research Grant of Interna-

tional joint Research for International Publication (grant

no LPPM-UGM/696/LIT/2013), the Swedish International

Development Agency (SIDA), the Swedish Gas Centre for

financial support of this study, and to Bor�as Energy and

Environment AB (Bor�as, Sweden) for the inoculum.

Conflict of Interest

None declared.

References

1. Yadvika Santosh, T. R. Sreekrishnan, S. Kohli, and V.

Rana. 2004. Enhancement of biogas production from solid

substrates using different techniques – a review. Bioresour.

Technol. 95:1–10.

2. Kroeker, E. J., D. D. Schulte, A. B. Sparling, and H. M.

Lapp. 1979. Anaerobic treatment process stability. J. Water

Pollut. Control Fed. 51:718–727.

3. Papa, E., F. Battaini, and P. Gramatica. 2005. Ranking of

aquatic toxicity of esters modelled by QSAR. Chemosphere

58:559–570.

4. Kirk, R. E., and D. F. Othmer. 1988. Encyclopedia of

chemical technology, 4th ed. Vol. 21. John Wiley and Sons

Inc., New York, NY.

5. Berger, R. G. 2007. Flavours and fragrances – chemistry,

bioprocessing and sustainability. Springer, Germany.

6. Parkerton, T. F., and W. J. Konkel. 2000. Application of

quantitative structure-activity relationships for assessing

the aquatic toxicity of phthalate esters. Ecotoxicol.

Environ. Saf. 45:61–78.

7. Liu, S., and J. M. Suflita. 1994. Anaerobic biodegradation

of methyl esters by Acetobacterium woodii and Eubacterium

limosum. J. Ind. Microbiol. 13:321–327.

8. Gavala, H. N., F. Alatriste-Mondragon, R. Iranpour, and B.

K. Ahring. 2003. Biodegradation of phthalate esters during

the mesophilic anaerobic digestion of sludge. Chemosphere

52:673–682.

9. Filonow, A. B. 1999. Yeasts reduce the stimulatory

effect of acetate esters from apple on the germination

of Botrytis cinerea Conidia. J. Chem. Ecol. 25:1555–

1565.

10. Lanciotti, R., N. Belletti, F. Patrignani, A. Gianotti, F.

Gardini and M. E. Guerzoni. 2003. Application of hexanal,

(E)-2-hexenal, and hexyl acetate to improve the safety of

fresh-sliced apples. J. Agric. Food Chem. 51:2958–2963.

11. Hansen, T. L., J. E. Schmidt, I. Angelidaki, E. Marca,

J. I. C. Jansen, H. Mosbaek, et al. 2004. Method for

determination of methane potentials of solid organic

waste. Waste Manage. 24:393–400.

12. Organisation for Economic Cooperation and Development

(OECD). 2007. Determination of the inhibition of the

activity of anaerobic bacteria - reduction of gas production

from anaerobically digesting sewage sludge, O.f.E.C.-o.a.D.

in Test No. 224. OECD Publishing, Paris, France. ISBN:

9264067337, 9789264067332.

13. Deng, W. L., T. R. Hamilton-Kemp, M. T. Nielsen, R. A.

Andersen, G. B. Collins and D. F. Hildebrand.1993. Effect

of six-carbon aldehydes and alcohols on bacterial

proliferation. J. Agric. Food Chem. 41:506–510.



14. Deublein, D., and A. Steinhauser. 2008. Biogas from waste

and renewable resources. Wiley-VCH, Germany.

15. Gerardi, M. H. 2003. The microbiology of anaerobic

digesters. John Wiley & Sons Inc, Canada.

16. Infantes, D., A. Gonz�alez Del Campo, J. Villase~nor, and

J. Fernandez. 2011. Influence of pH, temperature and

volatile fatty acids on hydrogen production by

acidogenic fermentation. Int. J. Hydrogen Energy

36:15595–15601.

17. Merkl, R., I. Hr�adkov�a, V. Filip, and J. �Smi drkal2010.

Antimicrobial and antioxidant properties of phenolic acids

alkyl esters. Czech J. Food Sci. 28:275–279.

18. Huang, C. B., B. George, and J. L. Ebersole. 2010.

Antimicrobial activity of n-6, n-7 and n-9 fatty acids and

their esters for oral microorganisms. Arch. Oral Biol.

55:555–560.



Paper III

Paper III

Effects of lactone, ketone, and phenol on methane production and

metabolic intermediates during anaerobic digestion

Rachma Wikandaria, Noor Kartika Sarib, Qurrotul A’yunb, Ria Millatib, Muhammad Nur

Cahyantob, Claes Niklassonc, and Mohammad J. Taherzadeha

aSwedish Centre for Resource Recovery, University of Borås, Borås, Sweden

bDepartment of Food and Agricultural Product Technology, Universitas Gadjah Mada,

Yogyakarta, Indonesia

cDepartment of Chemical and Biological Engineering, Chalmers University of Technology,

Gothenburg, Sweden

*Corresponding Author:

Email: [email protected]

Tel: + 46 33 435 44 87

Fax: +46 33 435 40 08

1

Effects of lactone, ketone, and phenol on methane production and

metabolic intermediates during anaerobic digestion

Rachma Wikandaria, Noor Kartika Sarib, Qurrotul A’yunb, Ria Millatib, Muhammad Nur

Cahyantob, Claes Niklassonc, and Mohammad J. Taherzadeha

aSwedish Centre for Resource Recovery, University of Borås, Borås, Sweden

bDepartment of Food and Agricultural Product Technology, Universitas Gadjah Mada,

Yogyakarta, Indonesia

cDepartment of Chemical and Biological Engineering, Chalmers University of Technology,

Gothenburg, Sweden

*Corresponding Author:

Email: [email protected]

Tel: + 46 33 435 44 87

Fax: +46 33 435 40 08

1

Abstract

Fruit waste is a potential feedstock for biogas production. However, the presence of fruit

flavors that have antimicrobial activity is a challenge for biogas production. Lactones,

ketones, and phenolic compounds are among the several groups of fruit flavors that are

present in many fruits. This work aimed to investigate the effects of two lactones, i.e., γ-

hexalactone and γ-decalactone; two ketones, i.e., furaneol and mesifuran; and two phenolic

compounds, i.e., quercetin and epicatechin on anaerobic digestion with a focus on methane

production, biogas composition, and metabolic intermediates. Anaerobic digestion was

performed in a batch glass digester incubated at 55oC for 30 days. The flavor compounds

were added at concentrations of 0.05, 0.5, and 5 g/L. The results show that the addition of γ-

decalactone, quercetin, and epicathechin in the range of 0.5–5g/L reduced the methane

production by 50% (MIC50). Methane content was reduced by 90% with the addition of 5 g/L

of γ-decalactone, quercetin, and epicathechin. Accumulation of acetic acid, together with an

increase in carbon dioxide production, was observed. On the contrary, γ-hexalactone,

furaneol, and mesifurane increased the methane production by 83–132% at a concentration of

5 g/L.

Keywords: anaerobic digestion, fruit flavors, ketone, lactone, phenolic compounds

2

1. Introduction

Industrial and commercial growth in today’s societies contributes to the ever increasing

global energy demand. Currently, 88% of the world’s energy demand is supplied by fossil

fuels and the rest is fulfilled by renewable energy sources [1]. As a renewable energy source,

biogas appears to be the most efficient product based on the input and output energy ratio of

the cycle of biomass-to-energy conversion [2]. Biogas is an environmentally friendly energy

source if a close cycle of production and the usage of biogas as fuel is considered. According

to this concept, no net greenhouse gases would be emitted to the atmosphere. Biogas has

several applications such as electricity, heat generation, cooking, and vehicle fuel with an

energy content of 1.0 m3 of purified biogas being equal to 1.1 L of gasoline, 1.7 L of

bioethanol, or 0.97 m3 of natural gas [3]. In 2007, the European energy production from

biogas reached 6 million tons of oil equivalents [4].

Manure is one of the major raw materials for biogas production in the world. Although this

feedstock is an easily available resource from farms, the production rate and yield of biogas is

relatively low (Gerin et al., 2008). Increased biogas production can be achieved by using

energy-rich substrates such as energy crops, crop residues, industrial byproducts, and other

biodegradable wastes in anaerobic digesters (Seppälä et al., 2009). Fruit waste has an

advantage due to its low cost, availability in large quantities, and being less competitive than

other food products. Fruit waste has a high organic content, which makes it suitable for

biogas production [5]. In 2012, the production of fruit reached 668 million tons worldwide

(FAO, 2014). In the supply chain of fruit, 36–56% of the fruit ends up as waste [6]. In

industrialized countries, fruit loss occurs mostly during the post-harvest and consumption

stage due to the quality standards set by retailers and by consumers who discard purchased

fruit. In developing countries, fruit loss occurs mainly in post-harvest and distribution due to

3

Abstract

Fruit waste is a potential feedstock for biogas production. However, the presence of fruit

flavors that have antimicrobial activity is a challenge for biogas production. Lactones,

ketones, and phenolic compounds are among the several groups of fruit flavors that are

present in many fruits. This work aimed to investigate the effects of two lactones, i.e., γ-

hexalactone and γ-decalactone; two ketones, i.e., furaneol and mesifuran; and two phenolic

compounds, i.e., quercetin and epicatechin on anaerobic digestion with a focus on methane

production, biogas composition, and metabolic intermediates. Anaerobic digestion was

performed in a batch glass digester incubated at 55oC for 30 days. The flavor compounds

were added at concentrations of 0.05, 0.5, and 5 g/L. The results show that the addition of γ-

decalactone, quercetin, and epicathechin in the range of 0.5–5g/L reduced the methane

production by 50% (MIC50). Methane content was reduced by 90% with the addition of 5 g/L

of γ-decalactone, quercetin, and epicathechin. Accumulation of acetic acid, together with an

increase in carbon dioxide production, was observed. On the contrary, γ-hexalactone,

furaneol, and mesifurane increased the methane production by 83–132% at a concentration of

5 g/L.

Keywords: anaerobic digestion, fruit flavors, ketone, lactone, phenolic compounds

2

1. Introduction

Industrial and commercial growth in today’s societies contributes to the ever increasing

global energy demand. Currently, 88% of the world’s energy demand is supplied by fossil

fuels and the rest is fulfilled by renewable energy sources [1]. As a renewable energy source,

biogas appears to be the most efficient product based on the input and output energy ratio of

the cycle of biomass-to-energy conversion [2]. Biogas is an environmentally friendly energy

source if a close cycle of production and the usage of biogas as fuel is considered. According

to this concept, no net greenhouse gases would be emitted to the atmosphere. Biogas has

several applications such as electricity, heat generation, cooking, and vehicle fuel with an

energy content of 1.0 m3 of purified biogas being equal to 1.1 L of gasoline, 1.7 L of

bioethanol, or 0.97 m3 of natural gas [3]. In 2007, the European energy production from

biogas reached 6 million tons of oil equivalents [4].

Manure is one of the major raw materials for biogas production in the world. Although this

feedstock is an easily available resource from farms, the production rate and yield of biogas is

relatively low (Gerin et al., 2008). Increased biogas production can be achieved by using

energy-rich substrates such as energy crops, crop residues, industrial byproducts, and other

biodegradable wastes in anaerobic digesters (Seppälä et al., 2009). Fruit waste has an

advantage due to its low cost, availability in large quantities, and being less competitive than

other food products. Fruit waste has a high organic content, which makes it suitable for

biogas production [5]. In 2012, the production of fruit reached 668 million tons worldwide

(FAO, 2014). In the supply chain of fruit, 36–56% of the fruit ends up as waste [6]. In

industrialized countries, fruit loss occurs mostly during the post-harvest and consumption

stage due to the quality standards set by retailers and by consumers who discard purchased

fruit. In developing countries, fruit loss occurs mainly in post-harvest and distribution due to

3

the deterioration of perishable crops in warm and humid climates [6]. The common practice

to handle fruit waste is landfilling, which is not environmentally friendly and results in

landfill gases and greenhouse gas emissions. Hence, the utilization of fruit waste for biogas

production is an effort to simultaneously alleviate an environmental challenge and contribute

to the supply of renewable energy.

There are several challenges encountered in biogas production from fruit waste. For example,

the acidification process, which dominates the biochemical anaerobic reactions, results in the

decrease of the pH, thus, causing failure of the process [7]. However, this problem can be

solved by adding an alkali solution to adjust the pH. Another limitation of the fruit digestion

is the high C/N ratio as opposed to the optimum range for digestion. This can be addressed by

the co-digestion of fruit with other materials having a high nitrogen content such as fish

waste and slaughterhouse wastewater [8]. The flavor compound, which naturally occurs in

the fruit, is also another challenge for anaerobic digestion of fruit waste since the fruit flavor

compounds tend to exhibit antimicrobial activity. However, the inhibition effect caused by

fruit flavors on anaerobic digestion has not been fully explored.

Fruit flavors act as a natural protection against microbial attack and are used as food

preservatives due to its antibacterial and antifungal activity against several standard

organisms such as Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes,

Saccharomyces cerevisiae var. sake and Aspergillus fumigatus TISTR 3180 [9]. Flavor, itself,

is one of the most important quality parameters in the fruit. It comprises of sugars, acids,

salts, bitter compounds, and aroma volatiles [10, 11]. The term ‘flavor’ is usually associated

with volatile /aroma compounds. These volatile compounds are derived from lipids,

carbohydrates, proteins, and amino acids through many metabolic pathways and are usually

produced during the ripening process [12]. During the ripening process, high molecular

4

weight precursors are converted into smaller compounds that help to obtain viable seeds and

to attract seed – dispersing species. Besides determining the sensory properties of fruit, fruit

flavor compounds also possess several important properties such as antimicrobial, antifungal,

antiviral, antioxidant, somatic fat reducing, blood pressure regulating, and anti-inflammatory

properties [13].

Fruit flavors include terpenoids, aldehydes, esters, alcohols, lactones, ketones, and phenolics.

The effects of terpenoids, aldehydes, alcohols, and esters on anaerobic digestion have been

reported [14-16]. From the terpenoid group, D-Limonene, car-3-ene, α-pinene, and myrcene

have been proved to cause the failure of the anaerobic digestion process at concentrations of

0.45–0.9%[14-16][14-16][14-16][14-16]. Meanwhile, almost no methane was produced in

the presence of 0.5% of aldehyde (Hexanal, e-2-Hexenal, and Nonanal) or 0.5% of

octanol[16][16][16][16]. It has also been suggested that ester compounds such as methyl

butanoate, ethyl butanoate, ethyl hexanoate, and hexyl acetate start to inhibit the digestion

process at concentrations of 10 g/L [17]. In searching previous literature, the effects of flavor

compounds belonging to the group of lactone, ketone, and phenolic compounds on anaerobic

digestion has never been reported. Lactone, ketone, and phenolic compounds are present in

several fruits such as strawberries, mangoes, pineapples, tomatoes, passion fruit, blackberries,

plums, apricots, peaches, cashew apples, pears, apples, and grapes in the range of 0.1 to 158

ppm. Therefore, this paper aimed to investigate the effects of those compounds on anaerobic

digestion with a greater focus on the change of metabolic intermediates.

2. Materials and method

2.1. Microorganisms and chemicals

Inoculums (sludge) with 22 g VS/L (volatile solid per liter) was obtained from a thermophilic

biogas plant (55°C) at Borås Energy and Environment AB (Borås, Sweden). The inoculum

5

the deterioration of perishable crops in warm and humid climates [6]. The common practice

to handle fruit waste is landfilling, which is not environmentally friendly and results in

landfill gases and greenhouse gas emissions. Hence, the utilization of fruit waste for biogas

production is an effort to simultaneously alleviate an environmental challenge and contribute

to the supply of renewable energy.

There are several challenges encountered in biogas production from fruit waste. For example,

the acidification process, which dominates the biochemical anaerobic reactions, results in the

decrease of the pH, thus, causing failure of the process [7]. However, this problem can be

solved by adding an alkali solution to adjust the pH. Another limitation of the fruit digestion

is the high C/N ratio as opposed to the optimum range for digestion. This can be addressed by

the co-digestion of fruit with other materials having a high nitrogen content such as fish

waste and slaughterhouse wastewater [8]. The flavor compound, which naturally occurs in

the fruit, is also another challenge for anaerobic digestion of fruit waste since the fruit flavor

compounds tend to exhibit antimicrobial activity. However, the inhibition effect caused by

fruit flavors on anaerobic digestion has not been fully explored.

Fruit flavors act as a natural protection against microbial attack and are used as food

preservatives due to its antibacterial and antifungal activity against several standard

organisms such as Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes,

Saccharomyces cerevisiae var. sake and Aspergillus fumigatus TISTR 3180 [9]. Flavor, itself,

is one of the most important quality parameters in the fruit. It comprises of sugars, acids,

salts, bitter compounds, and aroma volatiles [10, 11]. The term ‘flavor’ is usually associated

with volatile /aroma compounds. These volatile compounds are derived from lipids,

carbohydrates, proteins, and amino acids through many metabolic pathways and are usually

produced during the ripening process [12]. During the ripening process, high molecular

4

weight precursors are converted into smaller compounds that help to obtain viable seeds and

to attract seed – dispersing species. Besides determining the sensory properties of fruit, fruit

flavor compounds also possess several important properties such as antimicrobial, antifungal,

antiviral, antioxidant, somatic fat reducing, blood pressure regulating, and anti-inflammatory

properties [13].

Fruit flavors include terpenoids, aldehydes, esters, alcohols, lactones, ketones, and phenolics.

The effects of terpenoids, aldehydes, alcohols, and esters on anaerobic digestion have been

reported [14-16]. From the terpenoid group, D-Limonene, car-3-ene, α-pinene, and myrcene

have been proved to cause the failure of the anaerobic digestion process at concentrations of

0.45–0.9%[14-16][14-16][14-16][14-16]. Meanwhile, almost no methane was produced in

the presence of 0.5% of aldehyde (Hexanal, e-2-Hexenal, and Nonanal) or 0.5% of

octanol[16][16][16][16]. It has also been suggested that ester compounds such as methyl

butanoate, ethyl butanoate, ethyl hexanoate, and hexyl acetate start to inhibit the digestion

process at concentrations of 10 g/L [17]. In searching previous literature, the effects of flavor

compounds belonging to the group of lactone, ketone, and phenolic compounds on anaerobic

digestion has never been reported. Lactone, ketone, and phenolic compounds are present in

several fruits such as strawberries, mangoes, pineapples, tomatoes, passion fruit, blackberries,

plums, apricots, peaches, cashew apples, pears, apples, and grapes in the range of 0.1 to 158

ppm. Therefore, this paper aimed to investigate the effects of those compounds on anaerobic

digestion with a greater focus on the change of metabolic intermediates.

2. Materials and method

2.1. Microorganisms and chemicals

Inoculums (sludge) with 22 g VS/L (volatile solid per liter) was obtained from a thermophilic

biogas plant (55°C) at Borås Energy and Environment AB (Borås, Sweden). The inoculum

5

was then stored in an incubator at 55oC for 3 days. A synthetic medium solution was prepared

with 20 g/L of nutrient broth (Sigma-Aldrich), 20 g L-1 of yeast extract (Merck), and 20 g L-1

of glucose (Merck). The nutrient broth contained 10 g/L beef extract, 10 g/L peptone, and 5 g

/L sodium chloride. The flavor compounds including furaneol, mesifurane, γ-decalactone, γ-

hexalactone, epicatechin, and quercetin used in this work were obtained from Sigma-Aldrich.


The method used for the anaerobic digestion was adapted from a previous report [16]. The

experiments were carried out in a 120 mL glass bottle containing 50 mL of sludge, 1 mL of

medium, and 2.5 mL of flavor solution or distilled water (for the control experiment). Flavor

solutions were prepared by dissolving epicatechin, quercetin, and γ-decalactone in methanol

while γ-hexalactone, furaneol, and mesifuran were dissolved in deionized water. In order to

measure the methane production from the inoculums, the inoculums were incubated without

the addition of a medium and flavor solution in the glass bottle containing 50 mL of sludge

and 3.5 mL of distilled water. The bottles were closed tightly and the headspace was filled

with gas mixture containing 80% N2 and 20% CO2 to achieve anaerobic conditions. The

bottles were then incubated at 55oC for 30 days. During incubation, the bottles were shaken

twice a day using a water bath shaker (55oC) at 150 rpm. Gas samples were taken from the

headspace of the reactors through the septum using a syringe with a pressure lock (VICI,

Precision Sampling Inc., U.S.A.).


Biogas production from the digestion was measured using Gas Chromatography (Varian 450

GC, U.S.A.) with a capillary column (J 6 W scientific GS-GasPro, bonded silica based 30 m

x 0.32 mm, Agilent Technologies, U.S.A.) and a thermal conductivity detector (Varian,

6

U.S.A.). The injector temperature, oven temperature, and detector temperature were set at

75oC, 100oC, 120oC, respectively, with the carrier gas flow (N2) at 2 mL/min.

Volatile Fatty Acid (VFA) was analyzed using an HPLC (Waters, U.S.A.), equipped with an

ion-exchange column (Aminex HPX-87H, Biorad, Richmond, CA, U.S.A). It was operated at

50oC, with a UV VIS detector (Waters 486, Millipore, Milford, MA) at 210 nm with 5 mM

sulfuric acid solution as an eluent at a flow rate of 0.6 mL/min. Prior to the analyses, the

samples were first centrifuged with 10,000 rpm for 25 minutes. The supernatant was then

filtered using 0.2 µm microfilter.

γ-hexalactone, furaneol, and mesifurane were analyzed using gas chromatography with a

flame ionized detector (Auto System, Perkin Elmer, U.S.A.), equipped with a packed column

(Perkin Elmer, 6’ x 1.8” OD, 80/100, Mesh, U.S.A.) with H2 as a burning gas and N2 as a

carrier gas. For furaneol and mesifurane analysis, the initial temperature of the oven was set

at 80°C and increased to 100°C with a rate of 2°C/min. For γ-hexalactone analysis, the initial

temperature of the oven was set at 80°C and increased to 145°C with a rate of 2°C/min. Prior

to analysis, the flavors were extracted from the samples using dichloromethane with a ratio

solvent and samples were 3:1. The mixture was homogenized by vortex for 1 min and

incubated at room temperature for 4 hours. N-nonane was added as an internal standard.

All experiments were carried out in triplicate batch experiments and the results were

presented in averages. At the end of the digestion, the pH of the sludge was analyzed. For

statistical analysis, analysis of variance (ANOVA) with a significance level of 0.05 was

performed using a statistical package for the social sciences (SPSS version 21, International

Business Machine Corporation).

7

was then stored in an incubator at 55oC for 3 days. A synthetic medium solution was prepared

with 20 g/L of nutrient broth (Sigma-Aldrich), 20 g L-1 of yeast extract (Merck), and 20 g L-1

of glucose (Merck). The nutrient broth contained 10 g/L beef extract, 10 g/L peptone, and 5 g

/L sodium chloride. The flavor compounds including furaneol, mesifurane, γ-decalactone, γ-

hexalactone, epicatechin, and quercetin used in this work were obtained from Sigma-Aldrich.


The method used for the anaerobic digestion was adapted from a previous report [16]. The

experiments were carried out in a 120 mL glass bottle containing 50 mL of sludge, 1 mL of

medium, and 2.5 mL of flavor solution or distilled water (for the control experiment). Flavor

solutions were prepared by dissolving epicatechin, quercetin, and γ-decalactone in methanol

while γ-hexalactone, furaneol, and mesifuran were dissolved in deionized water. In order to

measure the methane production from the inoculums, the inoculums were incubated without

the addition of a medium and flavor solution in the glass bottle containing 50 mL of sludge

and 3.5 mL of distilled water. The bottles were closed tightly and the headspace was filled

with gas mixture containing 80% N2 and 20% CO2 to achieve anaerobic conditions. The

bottles were then incubated at 55oC for 30 days. During incubation, the bottles were shaken

twice a day using a water bath shaker (55oC) at 150 rpm. Gas samples were taken from the

headspace of the reactors through the septum using a syringe with a pressure lock (VICI,

Precision Sampling Inc., U.S.A.).


Biogas production from the digestion was measured using Gas Chromatography (Varian 450

GC, U.S.A.) with a capillary column (J 6 W scientific GS-GasPro, bonded silica based 30 m

x 0.32 mm, Agilent Technologies, U.S.A.) and a thermal conductivity detector (Varian,

6

U.S.A.). The injector temperature, oven temperature, and detector temperature were set at

75oC, 100oC, 120oC, respectively, with the carrier gas flow (N2) at 2 mL/min.

Volatile Fatty Acid (VFA) was analyzed using an HPLC (Waters, U.S.A.), equipped with an

ion-exchange column (Aminex HPX-87H, Biorad, Richmond, CA, U.S.A). It was operated at

50oC, with a UV VIS detector (Waters 486, Millipore, Milford, MA) at 210 nm with 5 mM

sulfuric acid solution as an eluent at a flow rate of 0.6 mL/min. Prior to the analyses, the

samples were first centrifuged with 10,000 rpm for 25 minutes. The supernatant was then

filtered using 0.2 µm microfilter.

γ-hexalactone, furaneol, and mesifurane were analyzed using gas chromatography with a

flame ionized detector (Auto System, Perkin Elmer, U.S.A.), equipped with a packed column

(Perkin Elmer, 6’ x 1.8” OD, 80/100, Mesh, U.S.A.) with H2 as a burning gas and N2 as a

carrier gas. For furaneol and mesifurane analysis, the initial temperature of the oven was set

at 80°C and increased to 100°C with a rate of 2°C/min. For γ-hexalactone analysis, the initial

temperature of the oven was set at 80°C and increased to 145°C with a rate of 2°C/min. Prior

to analysis, the flavors were extracted from the samples using dichloromethane with a ratio

solvent and samples were 3:1. The mixture was homogenized by vortex for 1 min and

incubated at room temperature for 4 hours. N-nonane was added as an internal standard.

All experiments were carried out in triplicate batch experiments and the results were

presented in averages. At the end of the digestion, the pH of the sludge was analyzed. For

statistical analysis, analysis of variance (ANOVA) with a significance level of 0.05 was

performed using a statistical package for the social sciences (SPSS version 21, International

Business Machine Corporation).

7


Furaneol and mesifurane are the major compounds in the ketone group, which can be found

in strawberries, mangoes, pineapples, tomatoes, passion fruit, and blackberries [12, 18, 19]. γ-

decalactone and γ-hexalactone, belonging to the lactone group, are present in plums,

pineapples, blackberries, mangoes, apricots, and peaches [20-22]. Epicatechin and quercetin

are the main phenolic compounds commonly found in cashew apples, berries, pears, apples,

apricots, plums, grapes, and peaches [23, 24]. In the current work, the inhibitory effects of:

two lactones, i.e., γ-hexalactone and γ-decalactone; two ketones i.e., furaneol and mesifuran;

and two phenolic compounds i.e., quercetin and epicatechin on anaerobic digestion were

studied at concentrations of 0.005%, 0.05%, and 0.5%. The evaluated parameters were biogas

production and its composition, pH, and volatile fatty acids.

The accumulated methane after 30 days of digestion is shown in Table 1. The results show

that the addition of γ-decalactone, epicatechin, and quercetin result in a lower methane yield

compared to the control experiment. Increasing the concentration of γ-decalactone,

epicatechin, and quercetin compounds decreased the methane yield, which indicates the

inhibitory activity of these compounds on methane production. On the contrary, the presence

of furaneol, mesifurane, and γ-hexalactone resulted in a higher methane production than that

of the control. Since the flavor compounds exhibited a different influence on the anaerobic

digestion, the discussion is divided into two parts based on their characteristics in the process.

3.1. Toxicity of γ-decalactone, epicatechin, and quercetin

Figure 1 shows the methane production profile during the digestion process with the addition

of γ-decalactone, epicatechin, and quercetin in the medium. When a concentration of a flavor

compound was added up to 0.5 g/L, the result of the methane production was not statistically

8

different compared to the control, except for quercetin. The addition of 0.5 g/L of quercetin

decreased the methane yield by 29%, whereas, almost no methane was produced when γ-

decalactone, epicatechin, and quercetin were individually added at 5 g/L. The minimum

inhibitory concentration that reduced 50% of the methane yield for γ-decalactone, quercetin,

and epicathechin was in the range of 0.5–5 g/L. Regardless of the concentration of the flavor

compounds added to the medium, the maximum methane yield was reached in 15 days and

thereafter, it remained constant until the end of digestion. The methane yield with the

addition of a flavor compound of 5 g/L was even lower than that of the inoculum. This

suggests that the microorganism might be inactivated already from the beginning of the

process, which is why the remaining substrate in the inoculum was not consumed. The

addition of 5 g/L of a flavor compound produced a different biogas composition than that of

the control. The biogas composition from digestion with the addition of 5 g/L of a flavor

compound was 7–13% methane and 85–93% carbon dioxide, whereas it was 80% methane

and 20% carbon dioxide for the control (Fig. 2). This value corresponded to approximately

90% reduction of the methane content.

Gas production, gas composition, and pH are often too slow to detect a sudden change in the

system [25], and VFA has been suggested for a long time as one of the most important

parameters in describing the anaerobic digestion reactions [26-28]. Therefore, in this work

VFA including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid,

isovaleric acid, and caproic acid were analyzed. In order to evaluate which step was inhibited

by the flavor compounds, the VFA was divided into two groups i.e., acetic acid as the main

product of acetogenesis and other VFAs as the products of acidogenesis (later it is referred to

as fatty acids). The result from the digestion with the addition of 5 g/L of a flavor compound

is presented in Fig. 3.

9


Furaneol and mesifurane are the major compounds in the ketone group, which can be found

in strawberries, mangoes, pineapples, tomatoes, passion fruit, and blackberries [12, 18, 19]. γ-

decalactone and γ-hexalactone, belonging to the lactone group, are present in plums,

pineapples, blackberries, mangoes, apricots, and peaches [20-22]. Epicatechin and quercetin

are the main phenolic compounds commonly found in cashew apples, berries, pears, apples,

apricots, plums, grapes, and peaches [23, 24]. In the current work, the inhibitory effects of:

two lactones, i.e., γ-hexalactone and γ-decalactone; two ketones i.e., furaneol and mesifuran;

and two phenolic compounds i.e., quercetin and epicatechin on anaerobic digestion were

studied at concentrations of 0.005%, 0.05%, and 0.5%. The evaluated parameters were biogas

production and its composition, pH, and volatile fatty acids.

The accumulated methane after 30 days of digestion is shown in Table 1. The results show

that the addition of γ-decalactone, epicatechin, and quercetin result in a lower methane yield

compared to the control experiment. Increasing the concentration of γ-decalactone,

epicatechin, and quercetin compounds decreased the methane yield, which indicates the

inhibitory activity of these compounds on methane production. On the contrary, the presence

of furaneol, mesifurane, and γ-hexalactone resulted in a higher methane production than that

of the control. Since the flavor compounds exhibited a different influence on the anaerobic

digestion, the discussion is divided into two parts based on their characteristics in the process.

3.1. Toxicity of γ-decalactone, epicatechin, and quercetin

Figure 1 shows the methane production profile during the digestion process with the addition

of γ-decalactone, epicatechin, and quercetin in the medium. When a concentration of a flavor

compound was added up to 0.5 g/L, the result of the methane production was not statistically

8

different compared to the control, except for quercetin. The addition of 0.5 g/L of quercetin

decreased the methane yield by 29%, whereas, almost no methane was produced when γ-

decalactone, epicatechin, and quercetin were individually added at 5 g/L. The minimum

inhibitory concentration that reduced 50% of the methane yield for γ-decalactone, quercetin,

and epicathechin was in the range of 0.5–5 g/L. Regardless of the concentration of the flavor

compounds added to the medium, the maximum methane yield was reached in 15 days and

thereafter, it remained constant until the end of digestion. The methane yield with the

addition of a flavor compound of 5 g/L was even lower than that of the inoculum. This

suggests that the microorganism might be inactivated already from the beginning of the

process, which is why the remaining substrate in the inoculum was not consumed. The

addition of 5 g/L of a flavor compound produced a different biogas composition than that of

the control. The biogas composition from digestion with the addition of 5 g/L of a flavor

compound was 7–13% methane and 85–93% carbon dioxide, whereas it was 80% methane

and 20% carbon dioxide for the control (Fig. 2). This value corresponded to approximately

90% reduction of the methane content.

Gas production, gas composition, and pH are often too slow to detect a sudden change in the

system [25], and VFA has been suggested for a long time as one of the most important

parameters in describing the anaerobic digestion reactions [26-28]. Therefore, in this work

VFA including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid,

isovaleric acid, and caproic acid were analyzed. In order to evaluate which step was inhibited

by the flavor compounds, the VFA was divided into two groups i.e., acetic acid as the main

product of acetogenesis and other VFAs as the products of acidogenesis (later it is referred to

as fatty acids). The result from the digestion with the addition of 5 g/L of a flavor compound

is presented in Fig. 3.

9

The VFA profile for quercetin and epicatechin exhibited a similar trend. For a substrate

containing 5 g/L of quercetin or epicatechin, the methane production, fatty acids, and acetic

acid concentration were very low. Carbon dioxide production increased in the beginning of

the digestion, whereas no methane is produced. This fact indicates that the acidogenesis stage

was not inhibited; however, the acetogenenesis and methanogenis were affected. An increase

in the acetic acid occurred on day-10 of the digestion in the medium with the addition of

quercetin and on day-20 in the medium with the addition of epicatechin. The production of

acetic acid indicated a recovery of the acetanogenesis reaction. However, this did not occur

with methanogenesis, as can be observed by the low methane and accumulation of the acetic

acid. One explanation could be that methanogen requires a longer doubling time (5–15 days)

compared to acid forming bacteria (1–1.5 days) [29]. Another explanation could be that

methanogen could not tolerate the pH change from 8 to 5.6 (data not shown), as methanogen

is reported to be highly sensitive to pH changes with a narrow optimum pH range from 6.7 to

7.5 [2]. In addition, the cell membrane structure of methanogen, acedogen, and acetogen is

different. The acid forming bacteria have a hydrophobic lipid bilayer on their membrane cell,

whereas the cell membrane of methanogen is made from ether lipids, which lack L-muramic

acid and this produces fatty acid-sensitive bacteria [29]. Furthermore, the membrane of the

acid forming bacteria, particularly gram negative bacteria, could create a negative charge in

the cell surface which could prevent membrane damage by the inhibitor [30].

The inhibitory mechanism of epicatechin on anaerobic digesting consortia is unknown.

However, it has been reported that the hydrophobic side of the (-) epicatechin is incorporated

with the surface of a membrane cell [31] and causes disruption to the membrane cell [30, 32]

or inhibits the transport material between the membrane and the environment [30]. The

mechanisms of the antimicrobial action of quercetin have been previously studied. Quercetin

might increase the permeability of the inner bacterial membrane and dissipate the membrane

10

potential, which may decrease the resistance of the cells to other antibacterial agents and

disrupt the proton motive force of the cells, which can inhibit bacterial motility [33]. Another

mechanism reported by [34] is that quercetin binds to the GyrB subunit of the E. coli DNA

Gyrase and inhibits the enzyme’s ATPase activity. The B ring of quercetin plays a role in the

intercalation or hydrogen bonding with the stacking of the nucleic acid bases and may explain

the inhibitory action on DNA and RNA synthesis [35].

From digestion with 5 g/L γ-decalactone, the carbon dioxide production increased during the

digestion; however, both fatty acids and acetic acid production were consistently low. This

phenomenon could be attributed to the inactivation of acetanogen in the beginning of the

process; thus, no recovery of acetanogen was observed. This indicated that γ-decalactone was

more toxic than other compounds. The antimicrobial activity of γ-decalactone was mainly

based on its hydrophobic characteristic. γ-decalactone diffused to the cell, interacted with the

lipid membrane, and further increased the membrane permeability, which led to cell death.

[36]. In addition, γ-decalactone at a concentration of 300 mg/L could inhibit the plasma

membrane H+ ATPase of Yarrowialipolytica, as suggested by [36].

VFA is not only an indicator of process imbalance, but it is also a compound that could

inhibit the anaerobic digestion at a high concentration. For instance, inhibition occurs at

concentrations of 1, 0.05, and 0.005 g/L acetic acid, isobutyric/ isovaleric, and propionic

acids, respectively. It has also been found that glucose fermentation is inhibited at VFA

concentrations above 4 g/L [37]. Their inhibition action starts with their penetration into the

membrane cell in the form of an undissociated acid and further denaturates the cell protein.

These threshold concentrations are much higher than the concentrations of the corresponding

acids in this work. This underlines the finding that inhibition was mostly caused by activity

flavor compounds in the anaerobic digestion process.

11

The VFA profile for quercetin and epicatechin exhibited a similar trend. For a substrate

containing 5 g/L of quercetin or epicatechin, the methane production, fatty acids, and acetic

acid concentration were very low. Carbon dioxide production increased in the beginning of

the digestion, whereas no methane is produced. This fact indicates that the acidogenesis stage

was not inhibited; however, the acetogenenesis and methanogenis were affected. An increase

in the acetic acid occurred on day-10 of the digestion in the medium with the addition of

quercetin and on day-20 in the medium with the addition of epicatechin. The production of

acetic acid indicated a recovery of the acetanogenesis reaction. However, this did not occur

with methanogenesis, as can be observed by the low methane and accumulation of the acetic

acid. One explanation could be that methanogen requires a longer doubling time (5–15 days)

compared to acid forming bacteria (1–1.5 days) [29]. Another explanation could be that

methanogen could not tolerate the pH change from 8 to 5.6 (data not shown), as methanogen

is reported to be highly sensitive to pH changes with a narrow optimum pH range from 6.7 to

7.5 [2]. In addition, the cell membrane structure of methanogen, acedogen, and acetogen is

different. The acid forming bacteria have a hydrophobic lipid bilayer on their membrane cell,

whereas the cell membrane of methanogen is made from ether lipids, which lack L-muramic

acid and this produces fatty acid-sensitive bacteria [29]. Furthermore, the membrane of the

acid forming bacteria, particularly gram negative bacteria, could create a negative charge in

the cell surface which could prevent membrane damage by the inhibitor [30].

The inhibitory mechanism of epicatechin on anaerobic digesting consortia is unknown.

However, it has been reported that the hydrophobic side of the (-) epicatechin is incorporated

with the surface of a membrane cell [31] and causes disruption to the membrane cell [30, 32]

or inhibits the transport material between the membrane and the environment [30]. The

mechanisms of the antimicrobial action of quercetin have been previously studied. Quercetin

might increase the permeability of the inner bacterial membrane and dissipate the membrane

10

potential, which may decrease the resistance of the cells to other antibacterial agents and

disrupt the proton motive force of the cells, which can inhibit bacterial motility [33]. Another

mechanism reported by [34] is that quercetin binds to the GyrB subunit of the E. coli DNA

Gyrase and inhibits the enzyme’s ATPase activity. The B ring of quercetin plays a role in the

intercalation or hydrogen bonding with the stacking of the nucleic acid bases and may explain

the inhibitory action on DNA and RNA synthesis [35].

From digestion with 5 g/L γ-decalactone, the carbon dioxide production increased during the

digestion; however, both fatty acids and acetic acid production were consistently low. This

phenomenon could be attributed to the inactivation of acetanogen in the beginning of the

process; thus, no recovery of acetanogen was observed. This indicated that γ-decalactone was

more toxic than other compounds. The antimicrobial activity of γ-decalactone was mainly

based on its hydrophobic characteristic. γ-decalactone diffused to the cell, interacted with the

lipid membrane, and further increased the membrane permeability, which led to cell death.

[36]. In addition, γ-decalactone at a concentration of 300 mg/L could inhibit the plasma

membrane H+ ATPase of Yarrowialipolytica, as suggested by [36].

VFA is not only an indicator of process imbalance, but it is also a compound that could

inhibit the anaerobic digestion at a high concentration. For instance, inhibition occurs at

concentrations of 1, 0.05, and 0.005 g/L acetic acid, isobutyric/ isovaleric, and propionic

acids, respectively. It has also been found that glucose fermentation is inhibited at VFA

concentrations above 4 g/L [37]. Their inhibition action starts with their penetration into the

membrane cell in the form of an undissociated acid and further denaturates the cell protein.

These threshold concentrations are much higher than the concentrations of the corresponding

acids in this work. This underlines the finding that inhibition was mostly caused by activity

flavor compounds in the anaerobic digestion process.

11

3.2. Degradability of furaneol, mesifurane, and γ-hexalactone

In contrast with the aforementioned compounds, which inhibit the digestion process, the

addition of furaneol, mesifurane, and γ-hexalactone gave a positive effect on methane

production. Regardless of the concentration, the methane yield reached its maximum

production on day-15 (Fig 4). The maximum methane yields for furaneol, mesifurane, and γ-

hexalactone at a concentration of 5 g/L were 2.02, 2.35, and 2.56, respectively. The biogas

composition was similar, regardless of the type of flavor compound and its concentration

(data not shown). The methane yields of digestion with the addition of 0.05 g/L of flavor

compounds were similar to that of the control. A higher concentration of a flavor compound

added into the medium resulted in a higher methane yield. This result showed that there was a

conversion of the flavor compound into methane. Flavor compounds belong to lactone,

ketone, and phenol groups, which contain carbon, hydrogen, and oxygen atoms and could be

a material source for methane production. In order to confirm this assumption, the flavor

concentration in the system was analyzed.

Figure 5 shows a decrease in the concentration of γ-hexalactone, mesifuran, and furaneol

during the digestion, which was followed by an increase in the volatile fatty acid, carbon

dioxide, and methane production (Fig 5). Approximately, 90% of the γ-hexalactone,

mesifuran, and furaneol were degraded in 1 day, 10 days, and 20 days, respectively. On the

other hand, digestion of γ-hexalactone produced the highest methane production followed by

mesifuran and furaneol. It could be concluded that there is a correlation between the

degradability rate and methane production. γ-hexalactone was completely degraded in 10

days. At the same time, the VFA concentration was low and a high level of methane was

produced. This implies that the conversion of γ-hexalactone into methane was a rapid

process. A similar trend was observed for mesifurane and furaneol. Following a decrease in

12

the mesifurane and furaneol concentration, the concentration of isovaleric acid increased.

After several days, the concentration of isovaleric decreased, followed by an increase in the

acetic acid concentration, carbon dioxide content, and methane production. It could be that

mesifurane and furaneol were converted into isovaleric. Isovaleric is then converted into

acetic acid and carbon dioxide, which is then further converted into methane. As flavor

compounds contain carbon, hydrogen, and oxygen atoms, the conversion of these compounds

into methane can be calculated theoretically using the following equation:


Mol of methane produced (x) = 1/8(4c + h – 2o - 3n – 2s)

Practically, the conversion of flavor compounds into methane can also be measured by

subtracting the methane with that of the control. The methane value was then compared with

the theoretical value. As can be seen in Table 2, the ratio of the flavor compound

consumption for the flavor compound at a concentration of 5 g/L was less than 50%.

Previously, γ-hexalactone, mesifurane, and furaneol have been suggested as the antimicrobial

compounds toward other microorganisms. Furaneol exhibited antimicrobial activity against

gram positive and negative bacteria as well as human pathogenic microorganisms [38].

However, it showed a contrary behavior toward anaerobic digesting microorganism in this

work. γ-hexalactone, mesifurane, and furaneol did not have any inhibition effect on the

anaerobic digestion and in fact, it was used as a substrate for the methane production. Further

studies are warranted to elucidate the inhibition mechanism of some toxic compounds on the

anaerobic digesting microorganism.

4. Conclusion

13

3.2. Degradability of furaneol, mesifurane, and γ-hexalactone

In contrast with the aforementioned compounds, which inhibit the digestion process, the

addition of furaneol, mesifurane, and γ-hexalactone gave a positive effect on methane

production. Regardless of the concentration, the methane yield reached its maximum

production on day-15 (Fig 4). The maximum methane yields for furaneol, mesifurane, and γ-

hexalactone at a concentration of 5 g/L were 2.02, 2.35, and 2.56, respectively. The biogas

composition was similar, regardless of the type of flavor compound and its concentration

(data not shown). The methane yields of digestion with the addition of 0.05 g/L of flavor

compounds were similar to that of the control. A higher concentration of a flavor compound

added into the medium resulted in a higher methane yield. This result showed that there was a

conversion of the flavor compound into methane. Flavor compounds belong to lactone,

ketone, and phenol groups, which contain carbon, hydrogen, and oxygen atoms and could be

a material source for methane production. In order to confirm this assumption, the flavor

concentration in the system was analyzed.

Figure 5 shows a decrease in the concentration of γ-hexalactone, mesifuran, and furaneol

during the digestion, which was followed by an increase in the volatile fatty acid, carbon

dioxide, and methane production (Fig 5). Approximately, 90% of the γ-hexalactone,

mesifuran, and furaneol were degraded in 1 day, 10 days, and 20 days, respectively. On the

other hand, digestion of γ-hexalactone produced the highest methane production followed by

mesifuran and furaneol. It could be concluded that there is a correlation between the

degradability rate and methane production. γ-hexalactone was completely degraded in 10

days. At the same time, the VFA concentration was low and a high level of methane was

produced. This implies that the conversion of γ-hexalactone into methane was a rapid

process. A similar trend was observed for mesifurane and furaneol. Following a decrease in

12

the mesifurane and furaneol concentration, the concentration of isovaleric acid increased.

After several days, the concentration of isovaleric decreased, followed by an increase in the

acetic acid concentration, carbon dioxide content, and methane production. It could be that

mesifurane and furaneol were converted into isovaleric. Isovaleric is then converted into

acetic acid and carbon dioxide, which is then further converted into methane. As flavor

compounds contain carbon, hydrogen, and oxygen atoms, the conversion of these compounds

into methane can be calculated theoretically using the following equation:


Mol of methane produced (x) = 1/8(4c + h – 2o - 3n – 2s)

Practically, the conversion of flavor compounds into methane can also be measured by

subtracting the methane with that of the control. The methane value was then compared with

the theoretical value. As can be seen in Table 2, the ratio of the flavor compound

consumption for the flavor compound at a concentration of 5 g/L was less than 50%.

Previously, γ-hexalactone, mesifurane, and furaneol have been suggested as the antimicrobial

compounds toward other microorganisms. Furaneol exhibited antimicrobial activity against

gram positive and negative bacteria as well as human pathogenic microorganisms [38].

However, it showed a contrary behavior toward anaerobic digesting microorganism in this

work. γ-hexalactone, mesifurane, and furaneol did not have any inhibition effect on the

anaerobic digestion and in fact, it was used as a substrate for the methane production. Further

studies are warranted to elucidate the inhibition mechanism of some toxic compounds on the

anaerobic digesting microorganism.

4. Conclusion

13

Phenolic groups including quercetin and epicatechin inhibited anaerobic digestion at a

concentration of 5 g/L. Ketones such as furaneol and mesifuran can be consumed and used as

a substrate for methane production. The effect on the lactone group is likely depending on the

hydrophobicity of the compound; γ decalatone caused failure of the process, whereas γ-

hexalactone increased the methane yield. According to the metabolic intermediates, the flavor

compound most likely inhibited acetogenesis and methanogenesis.

Acknowledgments

We express our gratitude to the Directorate General for Higher Education, Ministry of

National Education of the Republic of Indonesia through Competitive Research Grant of

International Joint Research for International Publication (Grant no. LPPM-

UGM/696/LIT/2013), the Swedish International Development Agency (SIDA), and Borås

Energy and Environment AB (Borås, Sweden)

REFERENCES

Applied biochemistry and biotechnology

1. Weiland, P., Biogas production: current state and perspectives. Applied Microbiology and Biotechnology, 2010. 85(4): p. 849-860.



4. EurObserv’er, The state of renewable energies in Europe. 2008. p. 47-51.5. Velmurugan, B. and R.A. Ramanujam, Anaerobic Digestion of Vegetable Wastes for

Biogas Production in a Fed-Batch Reactor. Int. J. Emerg. Sci., 2011. 1(3): p. 478-486.

6. Gustavsson, J., et al. Global Food Losses and Food Waste. 2011.7. Bouallagui, H., et al., Bioreactor performance in anaerobic digestion of fruit and

vegetable wastes. Process Biochemistry 2005. 40: p. 989-995.8. Bouallagui, H., et al., Improvement of fruit and vegetable waste anaerobic digestion

performance and stability with co-substrates addition. Journal of Environmental Management, 2009: p. 1844-1849.

9. Chanthaphon, S., S. Chanthachum, and T. Hongpattarakere, Antimicrobial activities of essential oils and crude extracts from tropical Citrus spp. against food-related

14

microorganisms. Songklanakarin Journal of Science and Technology, 2008. 30: p. 125-131.

10. Salunkhe, D. and J. Do, Biogenesis of aroma constituents of fruits and vegetables.CRC Crit Rev Food Sci Nutr, 1976. 8: p. 161-190.

11. Song, J. and C. Forney, Flavour volatile production and regulation in fruit Can J Plant Sci 2008. 88: p. 537-550.

12. Hui, Y.H., Handbook of fruit and Vegetable Flavors. 2010, Canada: John Wiley & Sons, Inc.

13. Berger, R., Biotechnology of flavours - The next generation (Review). . Biotechnol Lett 2009. 31(11): p. 1651-1659.



16. Wikandari, R., et al., Inhibitory effects of fruit flavors on methane production during anaerobic digestion. Bioresource Technology, 2013. 145: p. 188-192.

17. Yanti, H., et al., Effect of ester compounds on biogas production: beneficial or detrimental? Energy Science and Engineering, 2014. 2(1): p. 22-30.


19. Idstein, H. and P. Schreier, Volatile Constituents Of Alphonso Mango (Mangifera Indica). Phyrochemirtry,, 1985. 24: p. 2313-2316.

20. Wei, C.-B., et al., Characteristic Aroma Compounds from Different Pineapple Parts.Molecules, 2011. 16: p. 5104-5112.

21. Guichard, E.I. and M. Souty, Comparison of The Relative Quantities of Aroma Compounds Found in Fresh Apricot from Six Different Varieties (Prunus armeniaca) from Six Different Varieties. 1987, France.

22. Du, X., C.E. Finn, and M.C. Qian, Volatile Composition and Odour-Activity Value of Thornless ‘Black Diamond’ and ‘Marion’ Blackberries. Food Chemistry, 2009.

23. Tsanova-Savova, S., R. Fany, and G. Maria, (+)-Catechin and (-) Epicatechin in Bulgarian Fruits. Journal of Food Composition and Analysis, 2003. 18: p. 691-698.

24. Anastasiadi, M., et al., Bioactive Non-Coloured Polyphenols Content of Grapes, Wines and Vinification By-Products: Evaluation of The Antioxidant Activities of Their Extracts. Food Research International 2010. 43: p. 805-813.

25. Angelidaki, I. and B.K. Ahring, Anaerobic digestion of manure at different ammonia loads: effect of temperature. Water Res, 1994. 28: p. 727-731.

26. Chynoweth, D. and R. Mah, Anaerobic biological treatment processes. Adv Chem Sci 1971. 105: p. 41-53.

27. Fischer, J.R., E.L. Lannotti, and J.H. Porter, Anaerobic digestion of swine manure at various influent concentrations. Biol Wastes, 1983. 6: p. 147-166.

28. Hill, D.T. and J.P. Bolte, Digester stress as related to iso-butyric and iso-valeric acids. Biol Wastes, 1989. 28: p. 33-37.




15

Phenolic groups including quercetin and epicatechin inhibited anaerobic digestion at a

concentration of 5 g/L. Ketones such as furaneol and mesifuran can be consumed and used as

a substrate for methane production. The effect on the lactone group is likely depending on the

hydrophobicity of the compound; γ decalatone caused failure of the process, whereas γ-

hexalactone increased the methane yield. According to the metabolic intermediates, the flavor

compound most likely inhibited acetogenesis and methanogenesis.

Acknowledgments

We express our gratitude to the Directorate General for Higher Education, Ministry of

National Education of the Republic of Indonesia through Competitive Research Grant of

International Joint Research for International Publication (Grant no. LPPM-

UGM/696/LIT/2013), the Swedish International Development Agency (SIDA), and Borås

Energy and Environment AB (Borås, Sweden)

REFERENCES

Applied biochemistry and biotechnology

1. Weiland, P., Biogas production: current state and perspectives. Applied Microbiology and Biotechnology, 2010. 85(4): p. 849-860.



4. EurObserv’er, The state of renewable energies in Europe. 2008. p. 47-51.5. Velmurugan, B. and R.A. Ramanujam, Anaerobic Digestion of Vegetable Wastes for

Biogas Production in a Fed-Batch Reactor. Int. J. Emerg. Sci., 2011. 1(3): p. 478-486.

6. Gustavsson, J., et al. Global Food Losses and Food Waste. 2011.7. Bouallagui, H., et al., Bioreactor performance in anaerobic digestion of fruit and

vegetable wastes. Process Biochemistry 2005. 40: p. 989-995.8. Bouallagui, H., et al., Improvement of fruit and vegetable waste anaerobic digestion

performance and stability with co-substrates addition. Journal of Environmental Management, 2009: p. 1844-1849.

9. Chanthaphon, S., S. Chanthachum, and T. Hongpattarakere, Antimicrobial activities of essential oils and crude extracts from tropical Citrus spp. against food-related

14

microorganisms. Songklanakarin Journal of Science and Technology, 2008. 30: p. 125-131.

10. Salunkhe, D. and J. Do, Biogenesis of aroma constituents of fruits and vegetables.CRC Crit Rev Food Sci Nutr, 1976. 8: p. 161-190.

11. Song, J. and C. Forney, Flavour volatile production and regulation in fruit Can J Plant Sci 2008. 88: p. 537-550.

12. Hui, Y.H., Handbook of fruit and Vegetable Flavors. 2010, Canada: John Wiley & Sons, Inc.

13. Berger, R., Biotechnology of flavours - The next generation (Review). . Biotechnol Lett 2009. 31(11): p. 1651-1659.



16. Wikandari, R., et al., Inhibitory effects of fruit flavors on methane production during anaerobic digestion. Bioresource Technology, 2013. 145: p. 188-192.

17. Yanti, H., et al., Effect of ester compounds on biogas production: beneficial or detrimental? Energy Science and Engineering, 2014. 2(1): p. 22-30.


19. Idstein, H. and P. Schreier, Volatile Constituents Of Alphonso Mango (Mangifera Indica). Phyrochemirtry,, 1985. 24: p. 2313-2316.

20. Wei, C.-B., et al., Characteristic Aroma Compounds from Different Pineapple Parts.Molecules, 2011. 16: p. 5104-5112.

21. Guichard, E.I. and M. Souty, Comparison of The Relative Quantities of Aroma Compounds Found in Fresh Apricot from Six Different Varieties (Prunus armeniaca) from Six Different Varieties. 1987, France.

22. Du, X., C.E. Finn, and M.C. Qian, Volatile Composition and Odour-Activity Value of Thornless ‘Black Diamond’ and ‘Marion’ Blackberries. Food Chemistry, 2009.

23. Tsanova-Savova, S., R. Fany, and G. Maria, (+)-Catechin and (-) Epicatechin in Bulgarian Fruits. Journal of Food Composition and Analysis, 2003. 18: p. 691-698.

24. Anastasiadi, M., et al., Bioactive Non-Coloured Polyphenols Content of Grapes, Wines and Vinification By-Products: Evaluation of The Antioxidant Activities of Their Extracts. Food Research International 2010. 43: p. 805-813.

25. Angelidaki, I. and B.K. Ahring, Anaerobic digestion of manure at different ammonia loads: effect of temperature. Water Res, 1994. 28: p. 727-731.

26. Chynoweth, D. and R. Mah, Anaerobic biological treatment processes. Adv Chem Sci 1971. 105: p. 41-53.

27. Fischer, J.R., E.L. Lannotti, and J.H. Porter, Anaerobic digestion of swine manure at various influent concentrations. Biol Wastes, 1983. 6: p. 147-166.

28. Hill, D.T. and J.P. Bolte, Digester stress as related to iso-butyric and iso-valeric acids. Biol Wastes, 1989. 28: p. 33-37.




15





36. Aguedo, M., et al., Interaction of Odorous Lactones with Phospholipids: Implications in Toxicity Towards Producing Yeast Cells. Biotechnology Letters, 2002. 24: p. 1975-1979.

37. Chen, Y., J.J. Cheng, and K.S. Creamer, Inhibition of anaerobic digestion process: A review. Bioresource Technology, 2008. 99: p. 4044-4064.

38. Sung, W.S., et al., Antimicrobial Effect of Furaneol Against Human Pathogenic Bacteria and Fungi. Journal of Microbiology and Biotechnology, 2006. 16(3): p. 349-354.

16

Paper IV





36. Aguedo, M., et al., Interaction of Odorous Lactones with Phospholipids: Implications in Toxicity Towards Producing Yeast Cells. Biotechnology Letters, 2002. 24: p. 1975-1979.

37. Chen, Y., J.J. Cheng, and K.S. Creamer, Inhibition of anaerobic digestion process: A review. Bioresource Technology, 2008. 99: p. 4044-4064.

38. Sung, W.S., et al., Antimicrobial Effect of Furaneol Against Human Pathogenic Bacteria and Fungi. Journal of Microbiology and Biotechnology, 2006. 16(3): p. 349-354.

16

Paper IV

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 494182, 6 pageshttp://dx.doi.org/10.1155/2014/494182

Research ArticleImprovement of Biogas Production from Orange Peel Wasteby Leaching of Limonene

Rachma Wikandari,1 Huong Nguyen,2 Ria Millati,3

Claes Niklasson,2 and Mohammad J. Taherzadeh1

1 Swedish Centre for Resource Recovery, University of Boras, Allegatan 1, 50190 Boras, Sweden2Department Chemical and Biological Engineering, Chalmers University of Technology, 412 96 Gothenburg, Sweden3Department of Food and Agricultural Product Technology, Faculty of Agricultural Technology, Universitas Gadjah Mada,Bulaksumur, Yogyakarta 55281, Indonesia

Correspondence should be addressed to RachmaWikandari; [email protected]

Received 6 June 2014; Revised 16 August 2014; Accepted 23 August 2014; Published 12 September 2014

Academic Editor: Ilona Sarvari Horvath

Copyright © 2014 RachmaWikandari et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Limonene is present in orange peel wastes and is known as an antimicrobial agent, which impedes biogas productionwhen digestingthe peels. In this work, pretreatment of the peels to remove limonene under mild condition was proposed by leaching of limoneneusing hexane as solvent. The pretreatments were carried out with homogenized or chopped orange peel at 20–40∘C with orangepeel waste and hexane ratio (w/v) ranging from 1 : 2 to 1 : 12 for 10 to 300min. The pretreated peels were then digested in batchreactors for 33 days. The highest biogas production was achieved by treating chopped orange peel waste and hexane ratio of 12 : 1at 20∘C for 10min corresponding to more than threefold increase of biogas production from 0.061 to 0.217m3 methane/kgVS.Thesolvent recovery was 90% using vacuum filtration and needs further separation using evaporation. The hexane residue in the peelhad a negative impact on biogas production as shown by 28.6% reduction of methane and lower methane production of pretreatedorange peel waste in semicontinuous digestion system compared to that of untreated peel.

1. Introduction

Orange as the main citrus fruit is one of top-five fruitcommodities that dominate the global fruit market. Accord-ing to Food and Agriculture Organization, global orangeproduction reached 68 million tons representing 8.5% of thetotal fruit production [1]. The largest orange producers areBrazil, United States of America, China, India, and Mexicoin 2012 [1]. Approximately, 40–60% of oranges are processedfor juice production, of which 50–60% ends up as wasteincluding seed, peel, and segment membrane [2, 3]. Thegeneration of these solid wastes is estimated to be in therange of 15 to 25 million tons per year [3]. Among thesewastes, citrus peel is the major constituent accounting forapproximately 44% of the weight fruit mass [4].

Citrus waste for different applications such as productionof pectin, flavonoid, fiber, and animal feed production hasbeen proposed by several researchers [5–8]. However, a large

amount of this waste is still dumped every year [9], whichcauses both economic and environmental problems such ashigh transportation cost, lack of dumping site, and accumu-lation of high organic content material [10]. Therefore, moreeffective and sustainable alternatives for using orange peelwastes such as biogas are highly desirable.

Biogas is gaseous material produced during anaerobicdigestion of organic compound. Biogas holds wide appli-cations such as fuel for electricity, car, cooking, lightening,and heating. Among these applications, conversion of orangepeels wastes into fuel is attractive, since it gives benefits interms of both energy recovery and environmental aspects.Orange peel waste contains both soluble and insoluble car-bohydrates that can be digested to biogas [11]. However, themain challenge to produce biogas from orange peel is thepresence of an antimicrobial compound “D-limonene.” Thischemical constitutes 90% of oranges essential oil as 2-3% ofdry matter of the orange [11]. Limonene has been reported

2 BioMed Research International

to be highly toxic to anaerobic digestion [11–13]. It causesultimate failure of the process at concentration of 400𝜇𝜇L/L onmesophilic digestion [11] and in the range of 450 to 900𝜇𝜇L/Lon thermophilic digestion [14].

A number of investigations have been carried out totackle the inhibition challenges by limonene [14, 15]. Thesemethods can be classified into three categories of limoneneremoval, limonene recovery, and conversion of limoneneinto less toxic compound. Among these methods, limonenerecovery seems to be the best alternative since this chemicalis a valuable compound used in several industries such asperfumery, chemicals, cosmetics, medical, and food flavor[16, 17]. There are several methods that have been reportedfor limonene recovery including steam explosion [14], steamdistillation [13], and acid hydrolysis [12]. However, thesemethods are performed under harsh conditions, whichrequire high energy consumption. In addition, using acidfor the pretreatment, a further neutralization is essential andexpensive equipment should be applied to handle the cor-rosive behavior of the material. Furthermore, the acids usedhave a negative impact on the subsequent digestion process.On the other hand, since the goal of the pretreatment is toimprove the biogas as a source of energy, the consumption ofenergy during the production process should be minimized.Hence, pretreatment of the orange wastes under ambienttemperature is favorable.

Leaching or solid-liquid extraction is an alternative pre-treatment performed in room temperature. In this technique,the limonene in the orange peel waste is leached into a solventthat has contact with the peel [18]. This technique is widelyused to extract organic compounds from natural materials,where these compounds are present at low concentration.To the best of our knowledge, this technique has not beenemployed for pretreatment of orange peel wastes. Therefore,the objective of this work was to examine leaching techniquefor orange peel waste pretreatment with focus on biogasproduction.

2. Material and Methods

2.1. Material. Orange peel wastes were collected fromBramhults Juice AB (Boras, Sweden). The wastes were fromorange juice process and contained 21.3% total solid (TS).It was then chopped or homogenized prior to pretreatmentprocess. Inoculum was collected from a thermophilic biogasplant (Boras Energy and Environment AB, Boras, Sweden).The inoculumwas kept at 55∘C for 3 days before the digestionprocess. Chemicals including hexane, diethyl ether, andsodium sulfate were purchased from Sigma-Aldrich.

2.2. Methods. Pretreatment of the wastes by leaching wasconducted in Erlenmeyer flasks. Four different solventsincluding hexane, diethyl ether, dichloromethane, andethyl acetate were used. These chemicals are toxic and/orinflammable and should be treated properly. In the exper-iment to select the solvent, each solvent was added to theorange peel with orange peel waste and hexane ratio of 1 : 4.Themixture of solvent and orange peel was shaken vigorously

for 10 minutes followed by incubation for 20 minute at roomtemperature. In the optimization of pretreatment study,hexane was used as a solvent. Two levels of four parametersincluding temperature (20∘C and 40∘C), time (10min and300min), orange peel wastes and hexane ratio (1 : 2 and 1 : 12),and the wastes size (homogenized or chopped) were selected.Forty grams of thewasteswas dissolvedwith a certain amountof hexane in the flasks, followed by shaking vigorously for adetermined period of time. After the settlement, the extractswere removed from residuals by vacuum filtration. Theresidual, pretreated orange waste was then washed threetimes with water in order to remove remaining hexane.Finally, the pretreated waste was digested to produce biogas.

Digestion processes were performed in batch and semi-continuous reactors. The determination of biogas potentialof the orange peels in batch digestion was carried outaccording to a previous study [19]. In the experiment toselect the solvent, the digestion was conducted with differentconcentration of volatile solids (VS) ranging from 0.5 to 2%.In the optimization study, two percent of VS of the untreatedand pretreated peels were placed in 120mL glass bottle. Thetotal volume of the mixture was 30mL including 20mL ofinoculumand the rest was orange peel andwater.The reactorswere then flushed with amixed gas containing 80% of N2 and20% of CO2 for 2min. The reactors were incubated at 55∘Cfor 33 days. Reactors containing only water and inoculumwere used as a blank. The experiments were performed intriplicate. At the end of the digestions, the pHof the digestateswas measured. For semicontinuous digestions, the pretreatedwastes were chosen based on the results obtained fromthe batch digestion. The semicontinuous digestions wereperformed in 2 L reactors (Automatic Methane Potential TestSystem I, Bioprocess Control, Sweden) with a liquid volumeof 1.8 L. The reactors were placed in a water bath at 55∘C.Theorganic loading rates (OLR) for both untreated and treatedorange peels were set at 1 g VS/L/day during the starting upperiod and gradually increased to 3 gVS/L/day.The hydraulicretention timewas set at 30 days. Gas production, pH, volatilefatty acids, and buffer capacity ratio were monitored duringthe digestion process.

Total solid and volatile solid of the untreated and pre-treated wastes were determined using a gravimetric method.The gas productionwasmeasured using a gas chromatograph(Varian 450GC, USA) equippedwith a packed column (J&WScientific GS-Gas Pro, 30m × 0.320mm) and a thermalconductivity detector (TCD). Gas samples of 100𝜇𝜇L werewithdrawn using a 250 𝜇𝜇L pressure tight syringe (VICI,Precision Sampling Inc., USA). The carrier gas was nitrogenwith the flow rate of 2mL/min.The temperature for injection,oven, and detector was 75, 100, and 120∘C, respectively.

Hexane content of the orange wastes was analyzed bydissolving the wastes into 10mL methanol. The methanolextract was then injected to gas chromatography-flame ion-ized detector (Clarus 400, Perkin Elmer) equipped with ZB-WAX-Plus, 30m × 0.25mm × 0.25 𝜇𝜇m.

For statistical analysis, normal probability method andanalysis of variance (ANOVA)were performed usingDesign-Expert 8 package.

BioMed Research International 3

3. Results and Discussion

Orange waste is a potential feedstock for biogas production.Orange waste contains ca 74.5% carbohydrate, 7.7% protein,and 10.6% fat [20]. Even though the theoretical methaneyield is 0.45Nm3/kgVS, the methane yields of 0.061 and0.131 Nm3/kgVS were obtained in this experiment fromchopped and homogenized peel, respectively. This indicatedthe strong inhibition by the limonene. Therefore, this com-pound should be separated from the orange peel before thedigestion process. In the current study, the limonene wasrecovered from the orange peel by solid-liquid pretreatmentusing solvent to extract the limonene.

3.1. Leaching of Orange Wastes and Subsequent Digestion. Inorder to select a proper solvent for limonene recovery, foursolvents including hexane, diethyl ether, dichloromethane,and ethyl acetate were used to extract the limonene followedby digestion of the pretreated orange peel waste for confir-mation. The pretreated orange peel waste was digested atdifferent concentration of volatile solids ranging from 0.5 to2%.The results show that, for all VS concentration added, theorange peel pretreated with hexane gave the highest methaneyield (Figure 1). Hence, the pretreatment using hexane wasfurther investigated in order to obtain the best pretreatmentmethod.

In the optimization study for pretreatment using hexane,four factors of leaching with two levels including temperature(20 and 40∘C), time (10 and 300min), orange peel wasteand hexane ratio (1 : 2 and 1 : 12), and the citrus waste size(homogenized and chopped) were investigated. The pre-treated orange waste was then digested to select the bestcondition of pretreatment. The results are summarized inTable 1. According to the statistical analysis, waste size wasthe only factor that was significant for the methane yield.

Table 1 showed that, for chopped peel, the pretreatedwastes had higher methane production than the untreatedones. The pretreatment of the wastes increased methane pro-duction to the value of 0.076–0.217m3/kgVS correspondingto 25–350%of improvement.Thebest pretreatment conditionbased on the methane yield obtained was for chopped peeltreated at 20∘C for 10min with orange peel waste and hexaneratio of 1 : 12. This pretreatment increased the methane yieldby more than three times. On the other hand, in the caseof homogenized peel, the pretreatment resulted in lowermethane production.

3.2. Hexane Inhibition in Digestion. The toxic effect of hexaneon anaerobic digesting microorganism might be responsiblefor the low yield obtained from the pretreatedwastes. In orderto confirm this hypothesis, batch digestion with addition ofhexane to the digesting system was conducted. For compar-ison, the toxicity of limonene was also examined using thesame method. The result showed that, at the same concen-tration, hexane was more toxic than limonene to anaerobicdigesting system. Addition of hexane at concentration of13 g/L resulted in 28.6% reduction of methane productioncompared with the control experiment (Figure 2).

0.5% VS1% VS

1.5% VS2% VS

Hexane Diethyl Dichloromethane Ethylacetateeter

0

10

20

30

40

50

60

70

80

90

100

Met

hane

pro

duct

ion

(10−

3 Nm

3 )

Figure 1: Methane production of pretreated orange peel waste bydifferent solvents and digestion at different concentration of volatilesolids.


Met

hane

yie

ld (N

m3 /k

g VS

)

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0 3 6 9 12 15

Figure 2: Effect of hexane (I) and limonene (Δ) on biogas produc-tion compared to control (◻).

00.010.020.030.040.050.060.070.080.09

0 5 10 15 20 25 30 35Digestion time (day)

Met

hane

yie

ld (N

m3 /k

g VS

)

Figure 3: Methane production of untreated (◻) and pretreated (Δ)orange peel wastes in semicontinuous digestion at organic loadingrate of 3 gVS/L/day.


Citrus peel waste

Grinding

Biogas

Make-uphexane

Recycled hexane

Extraction tank

Anaerobicdigestion

Condenser

Evaporation

Vapour of hexane

Filtration

Mixture oflimonene

and hexane

D-Limonene

Trea

ted

peel

Hex

ane-

rem

oved

treat

ed p

eel

Evaporation

Vapo

ur o

f hex

ane

Figure 4: Block flow diagram of biogas production from treated orange peel waste by leaching pretreatment and limonene extraction.

The toxicity of hexane might explain the lower methaneyield of the orange peel pretreated with homogenization.Thesmaller size of homogenized peels enabled greater contactsurface between hexane and the peel resulting in higherhexane residue left in the peel. In addition, proportionsof methane in biogas from pretreated homogenized orangewastes (45% to 68.5%) were lower than that of the pretreatedchopped wastes (62.3% to 78.4%) (data not shown).

In order to further examine the accumulation effect ofhexane in the system, semicontinuous digestion was con-ducted. The pretreated orange peel was compared with theuntreated orange peel at organic loading rate of 3 gVS/L/day.Biogas production of the untreated and pretreated orangewastes is presented in Figure 3. The results show that thebiogas production of the pretreated peel was lower than thatof the untreated peel whichmight be due to the accumulationof hexane in the system.

3.3. Hexane Removal from Pretreated Orange Wastes. It wasshown that hexane has inhibitory effect on anaerobic digest-ing system (Figure 2), and thus hexane residue in the peelmust be removed prior to the digestion. Vacuum filtrationwas able to separate 90% of hexane and caused the hexanecontent of the pretreated orange wastes to be 0.2mL/g oforange peel waste, which corresponds to concentration of26 g/L hexane in the digesting system. This hexane residuewas two times higher than the hexane concentration used intoxicity test (13 g/L). Hence, the hexane residue in the peelshould be minimized or eliminated prior to the digestionprocess for both economic and technical reasons. Sincehexane is a highly volatile hydrocarbon, removal of hexanecan be performed by normal or vacuum evaporation process.In order to find the best condition, the evaporation was

conducted at different temperatures and time. The rangeof temperature was between 30 and 70∘C. Evaporation attemperature beyond 70∘C made the orange peel very dried.In addition, boiling point of hexane is 68∘C which is alreadybelow themaximum evaporation temperature.The low rangeof temperature gave advantages in which destruction ofnutrients can be minimized and the energy consumption canbe kept low. Hexane residue in the peel after evaporation wasthen analyzed. The result shows that 66% of hexane can beremoved by evaporation at 50∘C for 10min corresponding to9 g/L of hexane in the peel (Table 2).

3.4. The Overall Process of Methane Production and LimoneneExtraction. One benefit of leaching pretreatment method isto recover the limonene that is a flavor compound in orangebelonging to terpenoid group. As flavor compound, limoneneholds widespread application in food, feed, cosmetic, chemi-cal, and pharmaceutical industry. In themarket of flavor, foodand beverages is the largest which contributes to 47% of totaldemand in 2003 [21].

In this process (Figure 4), orange peel is fed to grindingunit using a conveyer for size reduction. The chopped peel ismixed with hexane for 10min at 20∘C with peel and solventratio of 1 : 2, where limonene is extracted from the peelsand dissolved in the organic phase of hexane. The peel isthen separated from the hexane by vacuum filtration whichseparates ca 90% of the hexane. Since the remaining hexanein the peel inhibits the digestion, it should be separated andrecycled using normal or vacuum evaporation. The treatedpeel is fed into anaerobic digester to produce methane. Themixture of hexane and limonene out from filtration whichhas about 0.55 L limonene per m3 of hexane is fed into rotaryvacuum evaporator operated at 70∘C in order to evaporate the

BioMed Research International 5

Table 1: Methane yield of pretreated orange peel waste at different temperature, time, and peel/solvent ratio.

Temperature (∘C) Time (min) Peel/solvent ratio Citrus waste size Methane yield (Nm3/kgVS)Untreated Homogenized 0.131 ± 0.008

20 10 1 : 2 Homogenized 0.101 ± 0.01140 10 1 : 2 Homogenized 0.097 ± 0.00920 300 1 : 2 Homogenized 0.040 ± 0.00440 300 1 : 2 Homogenized 0.051 ± 0.01020 10 1 : 12 Homogenized 0.071 ± 0.00640 10 1 : 12 Homogenized 0.074 ± 0.00620 300 1 : 12 Homogenized 0.094 ± 0.01640 300 1 : 12 Homogenized 0.060 ± 0.014

Untreated Chopped 0.061 ± 0.00420 10 1 : 2 Chopped 0.177 ± 0.01140 10 1 : 2 Chopped 0.162 ± 0.01520 300 1 : 2 Chopped 0.134 ± 0.01640 300 1 : 2 Chopped 0.102 ± 0.01720 10 1 : 12 Chopped 0.217 ± 0.00940 10 1 : 12 Chopped 0.076 ± 0.01120 300 1 : 12 Chopped 0.120 ± 0.00540 300 1 : 12 Chopped 0.121 ± 0.016

Table 2: Hexane residue in pretreated orange peel waste after evaporation in different conditions.

Pretreated peel waste Evaporation temp. (∘C) Evaporation Time(min)

Hexane concentration(ml/g orange peel waste) % hexane removal

Unevaporated(control) — — 0.12 ± 0.00 —

Evaporated 30 10 0.12 ± 0.01 0Evaporated 30 30 0.06 ± 0.03 54Evaporated 50 10 0.04 ± 0.00 66Evaporated 50 30 0.07 ± 0.00 45Evaporated 70 10 0.08 ± 0.02 31Evaporated 70 30 0.09 ± 0.01 28

volatile hexane and separate it from the limonene. The vaporof hexane is condensed and recycled back to the pretreatmentvessel for extraction of more limonene from fresh peels.

The VS content of the treated orange peel is 11% andthe methane yield was 0.177Nm3/kgVS. Thus, every ton oforange wastes produced 19.47Nm3 of biogas and 1.4 L oflimonene as by-product. The best pretreatment conditionobtained in this work increased the methane yield by 350%compared to the untreated peel. This improvement is lowerthan that obtained from steam-explosion pretreatment whichcould increase the methane yield by 426% [14]. However, inthis current work the pretreatment was conducted at roomtemperature for 10 minutes, whereas the steam explosionpretreatment was carried out at 150∘C for 20 minutes [14].Thus, this pretreatment can be considered as low energydemanding. However, selection of the solvent is a criticalpoint to avoid inhibition problem from the solvent onanaerobic digesting system.

4. Conclusion

Leaching of limonene fromorangewastes can be a low energydemanding method for removing the inhibition effects of thewastes in the digestion process. The highest methane yield

was obtained by pretreatment of the substrate at 20∘C for10min with orange peel waste and hexane ratio of 1 : 12 whichresults in three times higher methane yield compared to theuntreated wastes.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

Acknowledgment

The authors express their gratitude to Swedish InternationalDevelopment Agency (SIDA) for the financial support of thiswork.

References

[1] FAO, Food and Agriculture Organization, 2014,http://www.faostat.org/.

[2] K. Grohmann and E. A. Baldwin, “Hydrolysis of orange peelwith pectinase and cellulase enzymes,” Biotechnology Letters,vol. 14, no. 12, pp. 1169–1174, 1992.


[3] F. R.Marın, C. Soler-Rivas, O. Benavente-Garcıa, J. Castillo, andJ. A. Perez-Alvarez, “By-products fromdifferent citrus processesas a source of customized functional fibres,” Food Chemistry,vol. 100, no. 2, pp. 736–741, 2007.

[4] W. Widmer, W. Zhou, and K. Grohmann, “Pretreatment effectson orange processingwaste formaking ethanol by simultaneoussaccharification and fermentation,” Bioresource Technology, vol.101, no. 14, pp. 5242–5249, 2010.

[5] T. Inoue, S. Tsubaki, K. Ogawa, K. Onishi, and J.-I. Azuma,“Isolation of hesperidin from peels of thinned Citrus unshiufruits by microwave-assisted extraction,” Food Chemistry, vol.123, no. 2, pp. 542–547, 2010.

[6] Y. Jiang, Y. Du, X. Zhu, H. Xiong, M. W. Woo, and J.Hu, “Physicochemical and comparative properties of pectinsextracted from Akebia trifoliata var. Australis peel,” Carbohy-drate Polymers, vol. 87, no. 2, pp. 1663–1669, 2012.

[7] W. C. Kim, D. Y. Lee, C. H. Lee, and C. W. Kim, “Optimizationof narirutin extraction during washing step of the pectinproduction from citrus peels,” Journal of Food Engineering, vol.63, no. 2, pp. 191–197, 2004.

[8] D. Mamma, E. Kourtoglou, and P. Christakopoulos, “Fungalmultienzyme production on industrial by-products of thecitrus-processing industry,” Bioresource Technology, vol. 99, no.7, pp. 2373–2383, 2008.

[9] M. Pourbafrani, G. Forgacs, I. S. Horvath, C. Niklasson, andM. J. Taherzadeh, “Production of biofuels, limonene and pectinfrom citrus wastes,” Bioresource Technology, vol. 101, no. 11, pp.4246–4250, 2010.

[10] M. M. Tripodo, F. Lanuzza, G. Micali, R. Coppolino, and F.Nucita, “Citrus waste recovery: a new environmentally friendlyprocedure to obtain animal feed,” Bioresource Technology, vol.91, no. 2, pp. 111–115, 2004.

[11] E. Mizuki, T. Akao, and T. Saruwatari, “Inhibitory effect ofCitrus unshu peel on anaerobic digestion,” Biological Wastes,vol. 33, no. 3, pp. 161–168, 1990.

[12] M. Lohrasbi, M. Pourbafrani, C. Niklasson, and M. J.Taherzadeh, “Process design and economic analysis of a citruswaste biorefinery with biofuels and limonene as products,”Bioresource Technology, vol. 101, no. 19, pp. 7382–7388, 2010.

[13] M. A. Martın, J. A. Siles, A. F. Chica, and A. Martın, “Biometh-anization of orange peel waste,” Bioresource Technology, vol. 101,no. 23, pp. 8993–8999, 2010.

[14] G. Forgacs, M. Pourbafrani, C. Niklasson, M. J. Taherzadeh,and I. S. Hovath, “Methane production from citrus wastes:process development and cost estimation,” Journal of ChemicalTechnology and Biotechnology, vol. 87, no. 2, pp. 250–255, 2012.

[15] E. Mizuki, T. Akao, and T. Saruwatari, “Inhibitory effect ofCitrus unshu peel on anaerobic digestion,”BiologicalWastes, vol.33, no. 3, pp. 161–168, 1990.

[16] S. Raeissi, S. Diaz, S. Espinosa, C. J. Peters, and E. A. Brignole,“Ethane as an alternative solvent for supercritical extraction oforange peel oils,”The Journal of Supercritical Fluids, vol. 45, no.3, pp. 306–313, 2008.

[17] G. Forgacs, Biogas Production from Citrus Wastes and ChickenFeather: Pretreatment and Co-Digestion, Chalmers University ofTechnology, Goteborg, Sweden, 2012.

[18] J. D. Seader and E. J. Henley, Separation Process Principles, JohnWiley & Sons, New York, NY, USA, 2nd edition, 2006.

[19] T. L. Hansen, J. E. Schmidt, I. Angelidaki et al., “Method fordetermination of methane potentials of solid organic waste,”Waste Management, vol. 24, no. 4, pp. 393–400, 2004.

[20] N. Oktavia, “Karakterisasi Sampah Buah Pasar Buah Gamp-ing sebagai Bahan Baku Alternatif Produksi Biogas,” inTeknologi Pengolahan Hasil Pertanian, Universitas GajdahMada, Yogyakarta, Indonesia, 2009.

[21] A. B. P.Medeiros, S. C. Rossi, and C. R. SoccoL, “Cell culture forflavor production,” in Handbook of Fruit and Vegetable Flavors,Y. H. Hui, Ed., John Wiley & Sons, Hoboken, NJ, USA, 2010.

Paper V

Paper V

Performance of semi-continuous membrane bioreactor in biogasproduction from toxic feedstock containing D-Limonene

Rachma Wikandari a,⇑, Supansa Youngsukkasem a, Ria Millati b, Mohammad J. Taherzadeh a

a Swedish Centre for Resource Recovery, University of Borås, Borås, SwedenbDepartment of Food and Agricultural Product Technology, Gadjah Mada University, Yogyakarta, Indonesia

h i g h l i g h t s

� Addition of 1 g/L limonene did notaffect methane production in MBR.

� A lag phase was observed in MBRwith addition of 5 g/L of limonene.

� Addition of 10 g/L reduced methaneproduction by 50% and no recoverywas observed.

� The MBR worked well with additionof limonene between 5 and 10 g/L inthe medium.

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Addition of D-Limonene

a r t i c l e i n f o

Article history:Received 21 May 2014Received in revised form 25 July 2014Accepted 27 July 2014Available online 7 August 2014

Keywords:Semi continuousMembrane bioreactorEncapsulationBiogasD-Limonene

a b s t r a c t

A novel membrane bioreactor configuration containing both free and encased cells in a single reactor wasproposed in this work. The reactor consisted of 120 g/L of free cells and 120 g/L of encased cells in a poly-vinylidene fluoride membrane. Microcrystalline cellulose (Avicel) and D-Limonene were used as the mod-els of substrate and inhibitor for biogas production, respectively. Different concentrations of D-Limonenei.e., 1, 5, and 10 g/L were tested, and an experiment without the addition of D-Limonene was prepared ascontrol. The digestion was performed in a semi-continuous thermophilic reactor for 75 days. The resultshowed that daily methane production in the reactor with the addition of 1 g/L D-Limonene was similarto that of control. A lag phase was observed in the presence of 5 g/L D-Limonene; however, after 10 days,the methane production increased and reached a similar production to that of the control after 15 days.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

With the energy demands in today’s lifestyles, both energy cri-sis and climate change are key issues throughout the world. Thissituation drives the need for exploration and exploitation of newsources of energy, which are renewable as well as eco-friendly.Biogas is one of the most efficient and effective options amongthe various other alternative sources of renewable energy cur-rently available. Under optimal conditions, the energy output/input can reach 28 MJ/MJ, resulting in a very efficient use of the

biomass (Deublin and Steinhauser, 2008). Biogas is producedthrough anaerobic digestion processes where the microorganismsconvert complex organic matter into a mixture of methane andcarbon dioxide. Biogas is a versatile renewable energy source,which can be used for the replacement of fossil fuels in powerand heat production, and it can also be used as a gaseous vehiclefuel. In Europe, biogas is typically used for generating heat andelectricity although in some countries such as Sweden, biogas ismainly utilized as a vehicle fuel in the transportation sector, whilein developing countries, biogas is utilized for cooking, heating, andlighting (Forgács, 2012).

Around 10,000 biogas plants are currently in operation inEurope, and more than 20 million biogas plants have been installed

http://dx.doi.org/10.1016/j.biortech.2014.07.1020960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +46 33 435 44 87; fax: +46 33 435 40 08.E-mail address: [email protected] (R. Wikandari).

Bioresource Technology 170 (2014) 350–355

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

worldwide, including small homemade biogas reactors (Forgács,2012). Biogas production on an industrial scale is mostly operatedin a continuous process. In a continuous operation, washout of thecells is a serious issue since biogas production involves severalgroups of microorganisms with different doubling times. Amongthe four steps of biogas production including hydrolysis, acidogen-esis, acetogenesis, and methanogenesis, the doubling time of meth-anogens (5–15 days) is 10 times longer than that of acidogens andacetogens (1–1.5 days) (Gerardi, 2003). Consequently, methanogenrequires a longer retention time to grow. Therefore, a high dilutionrate and a short retention time cause a great reduction of the meth-anogen population (Youngsukkasem et al., 2013). The situation isworse when the substrate contains inhibitory substances, whichaffect the growth of the methanogen.

Fruit waste is a potential feedstock for biogas production due toits low cost, availability in large quantities, and high organic mattercontent. A total of 36–56% of fruit and vegetable waste is generatedboth in industrialized and developing countries in various stages ofthe supply chain such as agriculture, post-harvest, processing, dis-tribution, and consumption (Gustavsson et al., 2011). Anotherstudy reported that during the processing of fruit into finished con-sumable products, approximately 10–65% of the raw materialended up as organic waste (Ros et al., 2013). Besides, fruit wastehas a high moisture content of 80–89% and three-fourths of thetotal solids are volatile solids (Das and Mondal, 2013).

Given the very large biodegradable organic content of fruitwaste, one of the major limitations of anaerobic digestion of thesewastes is the presence of antimicrobial compounds in the fruit. Ofthe many antimicrobial compounds naturally present in the fruit,D-Limonene is the most well-known inhibitor compound in anaer-obic digestion, which causes failure of the process even at low con-centrations. It has been reported that D-Limonene ceased theanaerobic digestion at a concentration of 400 lL/L under meso-philic conditions (Mizuki et al., 1990) and between 450 and900 lL/L under thermophilic conditions (Forgács, 2012). D-Limo-nene is a hydrophobic compound that belongs to the terpenoidgroup, which is biosynthetically produced in more than 300 plants(Braddock and Cadwallader, 1995). Since methanogen is very sen-sitive to toxic and chemical compounds, it probably has the mosteffect on the methanogen population. This problem could be over-come by applying membranes that are made from a hydrophilicmaterial to encapsulate the cell and separate the cell from D-Limo-nene, which is theoretically impermeable to the membrane. A pro-cess, which uses both a biological process and a membranemodule, is referred to as a membrane bioreactor (MBR).

Besides protection from the inhibitor, the cell encapsulation inthe membrane offers other benefits i.e., prevention from cell wash-out and easy recovery of the cells in the downstream process (Parkand Chang, 2000). Furthermore, encapsulation protects the micro-organism from harsh conditions such as changes in the pH andtemperature, and accumulation of short chain organic acid, whichnegatively affect the stability of the whole process(Youngsukkasem et al., 2013). Since methanogenesis is the laststep in the production of biogas, enhancing the methanogenesisprocess will significantly improve the biogas productivity.

One challenge in applying the membrane-encapsulated cells tothe fruit waste as an organic polymer is the penetration of the sub-strate through the membrane due to the bigger particle of the sub-strate than the pores of the membrane. To overcome this problem,the waste should first be degraded into smaller particles that candissolve in the liquid and then permeate into the membrane. Thiscan be achieved by adding the microorganism outside the mem-brane to work in parallel with the encapsulated cells. In this work,a novel configuration of membrane bioreactor (MBR), in which freecells and encapsulated cells work simultaneously in a single reac-tor, was tested against D-Limonene in a semi continuous operation.

The performance of this configuration in a long-term process wasevaluated. Avicel was used as a synthetic substrate in order to eval-uate the feasibility of the process. Besides, the mass transfer of thenutrient throughout the membrane was also investigated.

2. Methods

2.1. Inoculum

The inoculum was a digesting sludge obtained from a 3000 m3

thermophilic biogas plant operated at 55 �C (Borås Energy andEnvironment AB, Borås, Sweden). The sludge was acclimated inan incubator at a temperature of 55 �C for 3 days. To obtain ahomogenous inoculum, the sludge was stirred and any remaininglarge particles were removed by passing the sludge through a sievewith a pore size of 1 mm. The sludge was then centrifuged at10,000�g for 10 min and the supernatant was discarded. Thesludge pellet was further used as inoculums.

2.2. Synthetic medium

The substrate for the digestion was a synthetic medium. Avicel(Fluka, Sigma Aldrich, Sweden) was used as a model organic poly-mer. Basal medium was used to supply the necessary nutrients forthe microorganism according to a method previously described(Isci and Demirer, 2007). The compositions of the basal mediumwith minor modifications were as follows in mg/L: NH4Cl (1200);MgSO4�7H2O (400): KCl (400); CaCl2�2H2O (50); (NH4)2HPO4 (80);FeCl2�4H2O (40); CoCl2�2H2O (10); KI (10); MnCl2�4H2O (0.5);CuCl2�2H2O (0.5); ZnCl2 (0.5); AlCl3�6H2O; Na2MoO4�2H2O (0.5);H3BO3 (0.5); NiCl2�6H2O (0.5); Na2WO4�2H2O (0.5); Na2SeO3

(0.5); cysteine (10). The pH of the basal medium was adjusted to7 ± 0.5 using 2 M NaOH.

2.3. Cell encasement preparation

The microbial cell encapsulation was prepared as previouslydescribed (Youngsukkasem et al., 2012). The cells were encapsu-lated using a hydrophilic polyvinylidene fluoride membrane (Dura-pore�, Thermo Fisher Scientific Inc., Sweden). The membranepocket was prepared by making the membrane into the size of3 � 6 cm. Two sides were sealed using a sealer (HPL 450 AS, Hawo,Germany) with a heating and cooling time of 4.5 s each, and oneside was left open for the cell insertion. Three grams of digestingsludge pallet were placed into the pocket. After the insertion ofthe microbial cells, the sachet was immediately sealed. Theencased cells were immediately used for the experiment.

2.4. Membrane bioreactor set up

Semi-continuous anaerobic digestion was carried out in ther-mophilic conditions using 500 mL glass bottle as a reactor. A waterbath was used to control the temperature of 55 �C for 75 days.Total pelleted inoculum of 72 g was used for each reactor in theform of free cell and encased cell. The free cells system was pre-pared by adding 36 g of pelleted sludge into 300 ml of syntheticmedium. Thereafter, the pelleted sludge and the liquid mediumwere mixed for 2 min in order to obtain a homogenous inoculum.Twelve sachets with a total amount of 36 g of pelleted sludge wereloaded into a basket and thereafter immersed in the free cell sys-tem in the reactor. The organic loading rate was set at 1 g VS/L dayfor the starting up period. After 14 days, it was increased to 2 g VS/L day and maintained at this level until the end of the experiment.The hydraulic retention time was 30 days. The reactors were sealedwith rubber stoppers and plastic caps. The reactor included an inlet


transition for feeding and sampling and an outlet transition for col-lecting and measuring the gas. Different concentrations of D-Limo-nene (Fluka, Sweden) i.e., 1, 5, and 10 g/L were investigated in thisexperiment. A reactor without addition of d-Limonene was used asa control. The D-Limonene was added into the bioreactor on day-30of the incubation period. In order to maintain a constant concen-tration of the D-Limonene during the experiment, D-Limonenewas added every day in the same amount as D-Limonene wasremoved from the reactor. All reactors were shaken twice a dayfor better mixing between the microbial, substrate, and inhibitorcompounds. Samples were withdrawn every day for analysis, andthe pH was measured from the effluents immediately.

2.5. Analytical method

The biogas volume from the reactors was automatically moni-tored using a data acquisition system (AMPTS II, Bioprocess con-trol, Sweden AB, Sweden). The gas composition was analyzedusing a gas chromatograph (Clarus 500, Perkin-Elmer, USA)equipped with a packed column (Perkin-Elmer, 60 � 1.800 OD, 80/100, Mesh, USA) and a thermal conductivity detector (Perkin-Elmer, USA) with an inject temperature of 150 �C. The carrier gaswas nitrogen, operated with a flow rate of 20 mL/min at 60 �C.The gas sampling was conducted using a 0.25 mL syringe (VICI,USA). Volatile fatty acids were quantified using Gas Chromatogra-phy (Clarus 500, Perkin-Elmer, USA) on a Flame Ionized Detectorequipped with a capillary column (Zebron ZB-WAX plus, Polyethyl-ene glycol (PEG), 30 m � 0.25 mm � 0.25 lm, USA). The tempera-tures of the injector and the detector were 250 and 300 �C,respectively. Nitrogen, at 2 mL/min and a pressure of 20 �C, wasused as a carrier gas. The oven temperature was initially 50 �Cand gradually increased to 200 �C at the rate of 40 �C/min andmaintained at this temperature for 3.5 min. Autosampler (Clarus500, Perkin-Elmer, USA) was employed for sampling, with an injec-tion size of 1.5 lL and the split was set at 15:1.

The permeability of the membrane for glucose and fatty acids asmetabolic intermediates was tested in order to evaluate the masstransfer of the nutrients to the encased cells. The test was con-ducted by passing a solution containing glucose, acetic acid, propi-onic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid,and caproic through the membrane filter; thereafter, the liquidthat passed the layer of membrane was analyzed.

The analysis of glucose was conducted by HPLC (Waters, Mil-ford, MA, USA) equipped with an ion exchange column (AminexHPX-87H, Biorad, Hercules, CA, USA) working at 60 �C using5 mM H2SO4 with a flow rate of 0.6 mL/min.


3.1. Methane yield and biogas composition

In this work, MBR containing both encapsulated cells and freecells was projected to overcome the inhibition problem by usingD-Limonene with Avicel as a substrate. Having this configuration,the anaerobic digestion of a polymer material can still be carriedout in a single bioreactor. If the bioreactor only has encapsulatedcells without any free cells in the medium, the process would needto be performed in two reactors for the hydrolysis and methano-genesis process. In order to detect the imbalanced system of thedisruption caused by the addition of D-Limonene, several parame-ters including gas production, gas composition, pH, volatile fattyacid profile, and volatile solid degradation have been monitoredas stress indicators (Ahring et al., 1995).

The daily methane yield is presented in Fig. 1. Regardless of theconcentration of the D-Limonene, the methane yield dramatically

decreased on the first day after the addition of D-Limonene. Themethane yield from the MBR added with 1, 5, and 10 g/L of D-Lim-onene were 0.12, 0.09, and 0.09 Nm3/kg VS, respectively. This cor-responded to 65%, 74%, and 75% reduction compared to that of themethane yield before the addition of D-Limonene. The methaneyield obtained from the control experiment was constant at0.37 Nm3/kg VS. However, a fast recovery was observed for thereactor with 1 g/L of D-Limonene, as evidenced by a noticeableincrease in the methane yield up to 0.31 Nm3/kg VS on the secondday and continuous increase to 0.38 Nm3/kg VS on the third dayafter the disturbance. The accumulation of the methane yield with1 g/L of D-Limonene until the end of the digestion period (75 days)was similar to that of the control. This indicated that the encasedcells were able to cope with D-Limonene at a concentration of1 g/L. It was previously stated that the presence of 0.4 g/L of D-Lim-onene in the mesophilic continuous anaerobic digestion of orangepeels with free cells had already caused a total failure of the pro-cess (Mizuki et al., 1990). Similarly, D-Limonene at a concentrationof 0.45–0.9 g/L has been reported to cease the thermophilic batchdigestion of orange peels (Forgács, 2012). The result emphasizesthe benefit of encased cells in the MBR over the free cells system.The cell was encapsulated in hydrophilic membrane, thus theoret-ically prevents the diffusion of hydrophobic D-Limonene. Besides,encapsulation may enhances the cell tolerance due to the high celldensity against an inhibition effect.

A lag phase was observed in the reactor that had the addition of5 g/L of D-Limonene. The methane yield was consistently low fromthe first day until 12 days after the perturbation. However, themethane yield started to rise and reached the same level as thecontrol at 0.38 Nm3/kg VS on the 17th day after the disturbance.It remained constant until the end of digestion. At the end of diges-tion, the accumulation of methane production reached 89% com-pared to what was produced by the control. In anotherexperiment, the addition of 5 g/L of D-Limonene to the free cellsresulted in a total failure of the process after 5 days of digestionand no recovery was observed (data not shown). For the reactorthat had the addition of 10 g/L of D-Limonene, the lag phase wasobserved for 30 days. The methane yield then started to increaseand reached to 0.16 Nm3/kg VS, which corresponded with 50%reduction compared to that of the control. Based on this result, itcan be concluded that the novel configuration of MBR could toler-ate D-Limonene in the range of 5–10 g/L. In general, citrus fruit con-tains 8 g/L of D-Limonene; thus, the novel MBR configuration couldpotentially overcome the inhibition problem of D-Limonene in theanaerobic digestion of fruit wastes.

Generally, the addition of D-Limonene regardless of itsconcentration did not affect the biogas composition. The biogascompositions from all the reactors consisted of 60–74% of methaneand 18–30% of carbon dioxide. Initially, the methane content was

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Fig. 1. Profile of daily methane yield of semi-continuous MBR with addition ofdifferent concentration of D-Limonene in the synthetic medium containing Avicel ascarbon source.


30–40%. It gradually increased up to 60–70% during 7 days andremained constant at 60–75% until the end of the digestion(Fig. 2). Addition of D-Limonene at concentration of 5 and 10 g/Lwas followed by a significant decrease in the methane contentfor several days; thereafter, the methane content increased andbecame normal.

3.2. Permeability of substrates in the PVDF membrane

The methane yield of the control and the MBR with the additionof 1 and 5 g/L of D-Limonene reached 90–93% from the theoreticalyield. This suggests that the operating conditions in the reactorwere optimum for methane production. It also indicates that therewas probably no problem with the mass transfer of the nutrientfrom the liquid medium into the encased cells since the Avicelwas successfully converted into methane. In order to investigatethe effect of the additional barrier (membrane filter) on the diffu-sion of the substrates into the encased cells, a solution with a vol-ume of 5 mL, containing 2 g/L of hydrolysis products (glucose andvolatile fatty acids) was passed through the membrane filter, andthe result is presented in Fig. 3. The diffusion rate of the solutionwas 1 mL/min. Glucose could diffuse completely within 5 min,whereas VFAs including acetic acid, propionic acid, butyric acid,isobutyric acid, valeric acid, isovaleric acid, and caproic acidrequired 30 min to completely permeate. Since the doubling timeof the anaerobic digesting microorganism is between 1 and15 days, a 30 min delay of the nutrient transfer would not be sucha problem. It should be noted that theoretically the D-Limonene isimpermeable to the membrane since D-Limonene is hydrophobicwhile PVDF membrane is hydrophilic. Thus, it is suggested thatencapsulation in a hydrophilic membrane could protect the cellsfrom a hydrophobic inhibitor such as D-Limonene (Pourbafraniet al., 2007).

3.3. Volatile fatty acids profile

Gas production, gas composition, and pH are parameters of theanaerobic digestion process but considered as being too slow todetect a sudden change in the system (Angelidaki and Ahring,1994) compared to the volatile fatty acids. It should be noted that

the pH was adjusted for all the reactors close to the neutral pH byadding 2 M NaOH to the feeding solution during the incubation.Therefore, the effects of the pH to the digestion would be avoided.The profiles of the volatile fatty acids are presented in Fig. 4. Theaccumulation of the volatile fatty acids was observed for all thereactors on the first days during the starting up period. This couldexplain the low methane production in the beginning of the diges-tion. In the steady state period, the methane production increasedas the volatile fatty acids decreased with negligible VFA accumula-tion during this period. The profile of the VFA changed after theperturbation, except for the control. The D-Limonene at a concen-tration of 1 g/L did not alter the VFA profile as can be seen by a sim-ilar VFA profile in the control experiment. However, a slightincrease in the acetic acid occurred only on the first day after thedisturbance. The pH profile was also similar to that of the control.The result suggests that using this configuration, the metabolicstate of the anaerobic digestion did not change regardless of thepresence of 1 g/L D-Limonene.

A significant increase in the VFA was observed in the reactorwith 5 and 10 g/L of D-Limonene. For the reactor with 5 g/L ofD-Limonene, the acetic acid dramatically increased from 0.14 to4.54 g/L in 5 days after the perturbation and thereafter fluctuatedover the next 10 days. It has been reported that an acetic acidconcentration higher than 3.12 g/L indicated an imbalance of the

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Fig. 2. Biogas composition with different concentration of D-Limonene in the synthetic medium. The symbols represent methane (d) and carbon dioxide (s).

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Fig. 3. Permeability of glucose and volatile fatty acids on the PVDF membrane with2 g/L of initial concentration of each compound.


process (Hill et al., 1987). The accumulation of the acetic acid wasfollowed by a decrease in the pH from 7.6 to below 6 and a reduc-tion of the methane yield from 0.35 to 0.11 Nm3/kg VS. Thedecrease in the methane yield was not due to the toxicity of theacetic acid to the microorganism since the concentration of theacetic acid was still below the inhibitory concentration (12 g/L)(Ahring et al., 1995). However, it may be due to the dissociationof the acetic acid at near neutral pH, which increased the acidityof the reactor, thus affecting the methanogen (Capris and Marais,1975). Acid-forming bacteria are more tolerant of the changes inthe pH compared to the methanogen; therefore, they could stillproduce VFA (Stafford, 1982) when the methanogen stopped toconvert the VFA into methane. Then, there was an accumulationof the acetic acid reflecting a stressful situation (Ahring et al.,1995). However, 10 days after the perturbation, the acetic acidstarted to decrease and together with a pH adjustment, the pHincreased. It was followed by an increase in the methane yield,which reached a similar yield as before the perturbation. The con-centration of butyric and isobutyric acid also increased from 0 to0.32 and 1.35, respectively, indicating the disturbance of the pro-cess (data not shown). Isobutyric and butyric acid were suggestedto be good indicators for process monitoring since the concentra-tion increased 1 or 2 days after the perturbation (Ahring et al.,1995).

For the reactor with 10 g/L of D-Limonene, the acetic acidincreased 3 days after the perturbation with the maximum concen-tration of 3.96 g/L and remained high until the end of the digestion.This indicated the imbalance between the acid producers and con-sumers, which may be due to the reduction of the methanogenpopulation. Since there was no decrease in the pH, the methanogenreduction might be due to the toxicity of D-Limonene at very highconcentrations (10 g/L), which the microorganism could not toler-ate and fully recover from, as can be seen from the low methaneyield. Besides, the concentration of the isobutyric and butyric acidalso increased and reached the maximum concentration of 2 and0.38 g/L (data not shown). Their concentration remained high untilthe end of the digestion, which reflects an inhibition effect. Sincethe concentration of the acetic acid, butyric, and isobutyric acidsare still below their inhibitory concentration to the anaerobicdigestion, the low methane yield was caused by the toxicity ofD-Limonene. The inhibitory mechanism of Limonene on the anaer-obic digestion microorganism, particularly methanogen is stillunknown. However, the toxicity of D-Limonene on Saccharomycescerevisiae has been attributed to its disruption on the cellularmembrane, resulting in the leakage of the cell components and dis-turbance of H+ and K+ transport energized by glycolysis (Wilkinset al., 2007). Based on the results in the current work, it can besummarized that a concentration of 1 g/L D-Limonene did not

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affect the system. However, a concentration of 5 g/L affected thesystem by reducing the methanogen; however, adjusting the pHcan solve this problem. Furthermore, a D-Limonene concentrationof 10 g/L was too high and its disturbance to the methanogen couldnot be recovered.

4. Conclusion

A novel membrane bioreactor configuration, containing bothfree and encapsulated cells that work simultaneously, has provedto have a good ability to hydrolyze microcrystalline cellulose andsequentially convert it into methane while maintaining its capabil-ity to overcome an inhibition problem with the addition of up to5 g/L of D-Limonene. This result revealed that this novel configura-tion could have a potential on real solid waste as a complexmaterial.

Acknowledgements

This work was financially supported by the Swedish Interna-tional Development Agency (SIDA). We express our gratitude tothe Directorate General of Higher Education, Ministry of NationalEducation of the Republic of Indonesia through CompetitiveResearch Grant of International Joint Research for InternationalPublication (Grant no. LPPM-UGM/696/LIT/2013) and Borås Energyand Environment AB (Borås, Sweden) for the inoculum.

References

Ahring, B.K., Sandberg, M., Angelidaki, I., 1995. Volatile fatty acids as indicators ofprocess imbalance in anaerobic digestors. Appl. Microbiol. Biotechnol. 43, 559–565.

Angelidaki, I., Ahring, B.K., 1994. Anaerobic digestion of manure at differentammonia loads: effect of temperature. Water Res. 28, 727–731.

Braddock, R.J., Cadwallader, K.R., 1995. Bioconversion of citrus D-limonene. In:Rouseff, R.L., Leahy, M.M. (Eds.), Fruit Flavors: Biogenesis, Characterization andAuthentication, ACS Symposium series N596. Washington, DC, pp. 142–148.

Capris, M.G., Marais, G.V.R., 1975. PH adjustments in anaerobic digestion. WaterRes. 9, 307–313.

Das, A., Mondal, C., 2013. Studies on the utilization of fruit and vegetable waste forgeneration of biogas. Int. J. Eng. Sci. 3 (9), 24–32.

Deublin, D., Steinhauser, A., 2008. Biogas from Waste and Renewable Resources: AnIntroduction. Wiley-VCH, Weinheim.

Forgács, G. 2012. Biogas Production from Citrus Wastes and Chicken Feather:Pretreatment and Co-Digestion (Vol. Ph.D. thesis). Chalmers University ofTechnology, Göteborg, Sweden.

Gerardi, M.H., 2003. The Microbiology of Anaerobic Digesters. John Wiley & SonsInc., Hoboken, New Jersey.

Gustavsson, J., Cederberg, C., Sonesson, U., Otterdijk, R.V., Meybeck, A., 2011. GlobalFood Losses and Food Waste. Food and Agricultural Organization of the UnitedNations.

Hill, D.T., Cobb, S.A., Bolte, J.P., 1987. Using volatile fatty acid relationships to predictanaerobic digester failure. Trans. ASAE 30, 496–501.

Isci, A., Demirer, G., 2007. Biogas production potential from cotton wastes.Renewable Energy 32, 750–757.

Mizuki, E., Akao, T., Saruwatari, T., 1990. Inhibitory effect of Citrus unshu peel onanaerobic digestion. Biol. Wastes 33 (3), 161–168.

Park, J.K., Chang, H.N., 2000. Microencapsulation of microbial cells. Biotechnol. Adv.18, 303–319.

Pourbafrani, M., Talebnia, F., Niklasson, C., Taherzadeh, M.J., 2007. Protective effectof encapsulation in fermentation of limonene-contained media and orange peelhydrolyzate. Int. J. Mol. Sci. 8, 777–787.

Ros, M., Franke-Whittle, I.H., Morales, A.B., Insam, H., Ayuso, M., Pascual, J.A., 2013.Archaeal community dynamics and abiotic characteristics in a mesophilicanaerobic co-digestion process treating fruit and vegetable processing wastesludge with chopped fresh artichoke waste. Bioresour. Technol. 136, 1–7.

Stafford, D.A., 1982. The effects of mixing and volatile fatty acid concentrations onanaerobic digester performance. Biomass 2, 43–55.

Wilkins, M.R., Widmer, W.W., Grohmann, K., 2007. Simultaneous saccharificationand fermentation of citrus peel waste by Saccharomyces cerevisiae to produceethanol. Process Biochem. 42, 1614–1619.

Youngsukkasem, S., Rakshit, K.S., Taherzadeh, M.J., 2012. Biogas production byencapsulated methane producing bacteria. Bioresources 7, 56–65.

Youngsukkasem, S., Akinbomi, J., Rakshit, K.S., Taherzadeh, M.J., 2013. Biogasproduction by encased bacteria in synthetic membranes: protective effects intoxic media and high loading rates. Environ. Technol. 34 (13-14), 2077–2084.


Paper VI

Paper VI

Membranes 2014, 4, 596-607; doi:10.3390/membranes4030596

membranesISSN 2077-0375

www.mdpi.com/journal/membranes Article

Biogas Production from Citrus Waste by Membrane Bioreactor

Rachma Wikandari 1,*, Ria Millati 2, Muhammad Nur Cahyanto 2 and Mohammad J. Taherzadeh 1

1 Swedish Centre for Resource Recovery, University of Borås, Allégatan 1, Borås 50190, Sweden; E-Mail: [email protected]

2 Department of Food and Agricultural Product Technology, Faculty of Agricultural Technology, Universitas Gadjah Mada, Bulaksumur, Yogyakarta 55281, Indonesia; E-Mails: [email protected] (R.M.); [email protected] (M.N.C.)

* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +46-33-435-4487; Fax: +46-33-435-4008.

Received: 29 June 2014; in revised form: 28 July 2014 / Accepted: 19 August 2014 / Published: 27 August 2014

Abstract: Rapid acidification and inhibition by D-limonene are major challenges of biogas production from citrus waste. As limonene is a hydrophobic chemical, this challenge was encountered using hydrophilic polyvinylidine difluoride (PVDF) membranes in a biogas reactor. The more sensitive methane-producing archaea were encapsulated in the membranes, while freely suspended digesting bacteria were present in the culture as well. In this membrane bioreactor (MBR), the free digesting bacteria digested the citrus wastes and produced soluble compounds, which could pass through the membrane and converted to biogas by the encapsulated cell. As a control experiment, similar digestions were carried out in bioreactors containing the identical amount of just free cells. The experiments were carried out in thermophilic conditions at 55 °C, and hydraulic retention time of 30 days. The organic loading rate (OLR) was started with 0.3 kg VS/m3/day and gradually increased to 3 kg VS/m3/day. The results show that at the highest OLR, MBR was successful to produce methane at 0.33 Nm3/kg VS, while the traditional free cell reactor reduced its methane production to 0.05 Nm3/kg VS. Approximately 73% of the theoretical methane yield was achieved using the membrane bioreactor.

Keywords: MBR; encapsulation; anaerobic digestion; D-limonene; citrus waste

OPEN ACCESS

Membranes 2014, 4 597

1. Introduction

According to Food and Agriculture Organization, orange, as the main citrus fruit, is one of top five fruit commodities in the global fruit market [1]. Global orange production reached 69 million tons in 2012 representing 8.5% of the total fruit production. Approximately 40%–60% of citrus production is processed for juice production, of which 50%–60% ends up as waste [2,3]. The global citrus waste production was 15–25 million tons a year [3]. Having high biodegradability, accumulation of citrus waste creates a serious problem to the environment, such as heavy odor, plenty of leachate, as well as attracting flies and rats [4], thus, a sustainable handling of citrus waste is highly desirable. The most promising alternative to incinerating and composting is converting this waste into biogas via anaerobic digestion [5,6]. Biogas holds several applications such as fuel for vehicles, heating, cooking, and electricity production. In addition, the residue of anaerobic digestion can be used as an excellent soil conditioner after minor treatments [7]. Furthermore, conversion of citrus waste into biogas is a combination of pollutant reduction and energy production.

The biogas production from citrus waste process can be classified into two main steps, i.e., acid formation and methane production. The acid forming and methane forming microorganisms differ widely with regards to physiology, nutritional requirements, growth kinetics, and sensitivity to environmental conditions [8]. Failure to maintain the balance between these two groups leads to instability of the process [9]. Moreover, the doubling time of methanogens (5–15 days) is in order of ten time longer than acid forming bacteria (1–1.5 days) [10]. Consequently, high dilution rate and short retention time result in wash out of methanogens [11]. Encapsulation is an attractive solution to prevent wash out of the methanogen by retaining the cells inside the bioreactor.

Other challenges of anaerobic digestion from citrus waste come from the characteristic of the substrate, i.e., rapid acidification and inhibition caused by limonene. Fruit waste in general is very rapidly acidified into volatile fatty acid (VFA) resulting in low pH and tends to inhibit methane production process [12,13]. Furthermore, a number of studies have reported that presence of limonene in the citrus oil hinders the biogas production of citrus waste [14–16]. D-limonene is antimicrobial compound that constitutes 90% of citrus essential peel oil [15]. It causes ultimate failure of the process at concentration of 400 μL/L on mesophilic continuous digestion [15] and in the range of 450 to 900 μL/L on thermophilic batch digestion [14].

Rapid acidification can be overcome by employing two-stage process in two sequential bioreactors for hydrolysis/acidification and methanogenesis. Two-stage system was shown to improve the substrate degradation yield and biogas productivity [13,17,18]. However, two-stage process is less attractive for industry since it requires more complicated design and higher cost for installation and maintenance of the digester [18]. In addition, approximately, 90% of digesters for treatment of organic fraction of municipal solid waste and bio-waste currently in use in Europe are operated in one stage system [19]. Therefore, performing two-stage process in one reactor would be industrially attractive.

Several attempts have been proposed to overcome inhibition problem by limonene, such as pretreatment of the citrus waste to remove the limonene or using the cell protection method [16,20,21]. Citrus waste can be pretreated by several methods including steam explosion [20], steam distillation [16], and acid hydrolysis [22]. However, these methods are performed under harsh conditions, which requires high energy consumption. Hence, cell protection is more favorable in term of energy


consumption. Cell protection can be conducted by employing a selective membrane to prevent diffusion of limonene to the cells while still allowing nutrient to pass. D-limonene is a hydrophobic compound, which belongs to terpenoid group. Thus, it is theoretically not permeable to a hydrophilic membrane.

In our previous work [23], encapsulated cell in Polyvinylidene Difluoride (PVDF) membrane was successful to protect the cell from limonene and improve the biogas production using synthetic medium containing cellulose and limonene. A number of microorganisms was added outside the encapsulated cell to digest the cellulose into a soluble compound, which is able to permeate to the membrane and further converted into methane by the encapsulated cell. Having this configuration, the two-stage process can be conducted in one reactor. However, the application of this configuration of membrane bioreactor (MBR) on real fruit waste as solid material has not yet been examined. Furthermore, there is a scarce report in literature for application of MBR on solid waste. MBR is mostly applied in various dilute wastewater treatments such as municipal/domestic wastewater [24], industrial wastewater [25,26], and surface/drinking water [27]. The aim of this work was to evaluate the performance of MBR for improvement of biogas production from citrus waste.

2. Results and Discussion

Citrus waste contains 74.53% of carbohydrate, 7.68% of protein, and 10.80% of fat [28]. The limonene content of the citrus waste is 3.78% of the dry weight [29]. Based on the composition, the theoretical methane yield that can be obtained from citrus waste calculated by Equation 1 is 0.45 Nm3/kg VS.

CcHhOoNnSs + yH2O → xCH4 + nNH3 + sH2S + (c − x) CO2 (1)

In reality, however, the methane yields of the citrus waste have been reported to be 0.06–0.1 Nm3/kg VS [14] which can be explained by the presence of limonene which inhibit the anaerobic digestion process [15]. In this work, MBR was applied to retain the cell in the reactor and to protect the cell from limonene. The configuration of the MBR used in this work is presented in Figure 1. For comparison, a reactor, which contains only free cell, was used as control. The experiment was carried out in semi-continuous reactor and incubated for 83 days. In order to detect the imbalance due to inhibition by limonene, several parameters have been monitored that include gas production, gas composition, pH, and volatile fatty acid profile as stress indicator [30].

2.1. Methane Yield and Biogas Composition

The citrus waste sludge was fed to the bioreactor at the loads of 0.3 kg VS/m3/day during the startup period. After the methane production was stable, the organic loading rate was gradually increased and reached 3 kg VS/m3/day at the end of the digestion. The change of methane production during the digestion process is presented in Figure 2. For MBR, increasing of the organic loading rate (OLR) was followed by increasing of methane production and the maximum methane production was achieved at OLR of 3 kg VS/m3/day. The methane production for the control increased up to OLR of 2 kg VS/m3/day, which equals to limonene loading of 60 g/m3/day. However when the OLR was increased to 2.5 and 3 kg VS/m3/day, the methane production started to decrease on day 67. This may be explained by the high loading of limonene, which corresponds to limonene loading of 76 and 91 g/m3/day, for OLR of 2.5 and 3 kg VS/m3/day, respectively. The limit of limonene oil loading


obtained using free cell in this study (60 g/m3/day) was slightly higher than that obtained from previous study (55 g/m3/day) [15]. This could be due to the higher cell concentration used in this work (240 g/L) compare to that (137.5 g/L) reported by Mizuki et al. [15]. In addition, final concentration of limonene at 3.293 kg/m3 did not affect the performance of MBR in this study, whereas final concentration of limonene at 378–756 g/m3 has been reported to lead to process failure [14]. The inhibitory mechanism of limonene on anaerobic digestion is unclear. However, the toxicity of limonene on Saccharomyces cerevisiae have been suggested due to membrane cellular disruption resulting in leak out of the cells components and disturbance of H+ and K+ transport energized by glycolysis [31].

Figure 1. Schematic diagram of semi-continuous digestion using MBR. The numbers indicates (1) feeding container containing; (2) MBR containing (a) encapsulated cells in hydrophilic PVDF membranes and (b) free cells; (3) gas measuring system; (4) controller.

The methane yield for control started to decrease from 0.3 Nm3/kg VS at OLR of 1 kg VS/m3/day to 0.21 Nm3/kg VS at OLR of 1.5 kg VS/m3/day. The methane yield was only 0.05 Nm3/kg VS at the highest OLR at 3 kg VS/m3/day corresponding to 11% from theoretical methane yield (Table 1). On the contrary, the methane yield of MBR remained high on high OLR. The methane yield of MBR in various OLR was 0.25–0.38 Nm3/kg VS which corresponded to 55%–84% from theoretical yield. The methane yield at the highest OLR, 3 kg VS/m3/day, was 0.33 Nm3/kg VS corresponding to 73% from the theoretical yield. The results showed that MBR could improve the methane yield by more than six times from 0.05 to 0.33 Nm3/kg VS at the highest OLR and this emphasizes the benefit of MBR over the conventional free cell. Besides improvement of the methane yield, the encapsulation offers other advantage such as easier cell recovery from the bioreactor in downstream process [32]. Moreover, this technique offers other advantage by protection of methanogen from harsh environmental condition such as change of pH, temperature, and accumulation of short chain organic acid which negatively affect the stability of the whole process [33]. In addition, no energy is required for pretreatment. The

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Figure 2. Methane production of anaerobic digestion from citrus waste using MBR and control at different organic loading rate.

Figure 3. Methane content of biogas from citrus waste using MBR (♦) and control (◊) during digestion.

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2.5 0.1 0.32 22 71 3 0.05 0.33 11 73

* Theoretical methane yield of citrus waste is 0.45 Nm3/kg VS.

2.1.1. Volatile Fatty Acids Composition

Anaerobic digestion involved four different groups of microorganism namely hydrolytic bacteria, acidogens, acetogens, and methanogens with mutual dependence one to each other, particularly for energetic reason. The hydrolytic bacteria play a role in the first step by degrading complex organic matters into their monomers such as sugars, amino acids and fatty acids. The soluble monomers were then converted into short chain organic acid, acetic acid, alcohols, hydrogen and carbondioxide by acidogen. The acidogenesis products were then subsequently converted into acetic acid by acetogens and finally converted into methane by methanogen. VFA has been suggested for a long time as one of the most important parameters in depicting the anaerobic digestion process [34–36]. Therefore, in this work VFA including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid were analyzed.

The VFA profiles and pH changes of citrus waste digestion using MBR and free cell are presented in Figure 4. No accumulation of VFA was observed for MBR during the digestion. The concentration of valeric, butyric, and caproic acid did not change during the digestion for both MBR and control (data not shown). For control, no accumulation of VFA was observed during the first 55. However, as the OLR was increased from 1.5 to 2 kg VS/m3/day, the acetic acid sharply increased by almost ten times from 0.64 to 5.75 g/L on day 60 and remained high until the end of digestion. Similarly, the isobutyric acid started to accumulate and reached 1.2 g/L. The accumulation of VFA indicates the inhibition occurred for the methanogen, since the VFA produced by acidogens and acetogens was not converted into methane.

VFA is not only an indicator of process imbalance, but it also can inhibit the microorganism at certain levels. For instance, glucose fermentation is inhibited at VFA concentrations above 4 g/L [37]. Since the VFA concentration for the control reactor was above the inhibitory concentration of VFA, the inhibition of the process was not only caused by limonene but also the VFA. Based on the membrane structure, cell membrane of methanogen is made from ether lipids, which lack L-muramic acid and this produces fatty acid-sensitive cell [10]. The methane production and pH remained stable during the accumulation of VFA. This is in accordance with previous study reported that gas production, gas composition, and pH are often too slow to detect a sudden change in the system [38].


The MBR, on the contrary, no accumulation was observed during the digestion process which underlines no inhibition occurred in this system.

Figure 4. Acetic acid (∆), propionic acid (□), isobutyric acid (○), and pH (---) of anaerobic digestion from citrus waste using (a) MBR and (b) control.

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limiting step for biogas production from cellulose-poor substrate, such as fruit waste [39]. Hence, protection of the cells particularly for methanogen is a major route to improve the performance of anaerobic digesters [40]. The configuration of MBR in this work enabled to separate the acid forming process outside the membrane and methane forming which occurred inside the membrane. Hence, the MBR behaved as a two-stage system although it was carried out in a single reactor. In large scale, the membrane layer can be inserted in the middle of the reactor to separate acid forming and methane forming process.

3. Experimental Section

3.1. Inoculum

Inoculum was a sludge obtained from a 3000 m3 thermophilic biogas plant (55 °C) at Borås Energy and Environment AB (Borås, Sweden). The sludge was stored in incubator at temperature 55 °C for 2–3 days to acclimate the bacteria with the operation condition. The sludge was stirred in order to obtain homogenous inoculum and the any remaining large particle was removed by passing the sludge through a sieve with a pore size of 1 mm. The sludge was then centrifuged at 14,000 rpm for 10 min and the supernatant was discarded. The pellet was further used as inoculums.

3.2. Citrus Waste

The citrus waste used in this work was a residue of orange obtained from Brämhult juice factory (Borås, Sweden) and stored at −20 °C until use. The citrus waste was thawed and grounded to obtain citrus waste slurry. A basal medium was added to the slurry. The composition of basal medium was according to a method previously described [41] with minor modification containing (mg/L): NH4Cl (1200); MgSO4·7H2O (400): KCl (400); CaCl2·2H2O (50); (NH4)2HPO4 (80); FeCl2·4H2O (40); CoCl2·2H2O (10); KI (10); MnCl2·4H2O (0.5); CuCl2·2H2O (0.5); ZnCl2 (0.5); AlCl3·6H2O; Na2MoO4·2H2O (0.5); H3BO3 (0.5); NiCl2·6H2O (0.5); Na2WO4·2H2O (0.5); Na2SeO3 (0.5); cysteine (10). The pH of the basal medium was adjusted to 7 ± 0.5 by adding 2 M NaOH.

3.3. Preparation of Membrane Encapsulated Cells

The encapsulated cell was prepared by inserting three grams of the pellet into a sachet made from PVDF (Durapore®, Thermo Fisher Scientific, Inc., Göteborg, Sweden) as previously reported [11,41]. The properties of PVDF used in this work were: hydrophilic with water flow rate of 2.5 mL/min × cm2; air flow rate of 0.15 mL/min × cm2; pore size of 0.1 µm; thickness of 125 µm; porosity of 70%; and maximum operating temperature of 85 °C. The membrane sachet was prepared by cutting the membrane into size of 3 cm × 6 cm, and sealed on two sides of the membrane using a sealer (HPL 450 AS, Hawo, Mosbach, Germany) with heating and cooling time of 4.5 s of each. After the insertion of the cells, the sachet was sealed to close. The membrane-encapsulated cells were immediately used for the experiment.


3.4. Semi-Continuous Digestion

The anaerobic digestion was carried out in semi-continuous 500 mL glass bottle reactors. The reactor was incubated in waterbath for 75 days at 55 °C. For membrane bioreactor, a total of 72 g of inoculums was used in the form of free cells and membrane encapsulated cells with ratio of 1:1. The free cells were prepared by dissolving 36 g of pellets into 300 mL of distilled water and placed to the reactor. Twelve sachets of encapsulated cell each containing 3 g of pellet were placed in a basket and immersed in the reactor. For control reactor, 72 g of pellet was dissolved in 300 mL of distilled water. The organic loading rate was set at 0.3 kg VS/m3/day during start-up period. The OLR was then gradually increased to 3 kg VS/m3/day. The hydraulic retention time was 30 days. The reactors were sealed with rubber stoppers with two holes, one for feeding and sampling (inlet) and the other one for collecting and measuring the gas (outlet). All reactors were shaken twice a day. Samples were withdrawn every day for analysis and the pH was measured from the samples immediately.

3.5. Analytical Method

Gas production from the outlet of the reactor was automatically monitored using a data acquisition system (AMPTS II, Bioprocess control, Sweden AB, Lund, Sweden). Gas composition was analysed using a gas chromatograph (Clarus 500, Perkin-Elmer, Waltham, MA, USA) equipped with a packed column (Perkin Elmer, 6ʹ × 1.8ʺ OD, 80/100, Mesh, USA) and a thermal conductivity detector (Perkin Elmer, Waltham, MA, USA) with inject temperature of 150 °C. The carrier gas was nitrogen operated with a flow rate of 20 mL/min at 60 °C. Gas sampling was conducted using a 0.25 µL (VICI, Baton Rouge, LA, USA).

Volatile fatty acids were determined using GC (Clarus 500, Perkin-Elmer, Waltham, MA, USA) on a Flame Ionized Detector (FID) equipped with Capillary column (Zebron ZB-WAXplus, Phenomenex, Torrance, CA, USA). The temperatures of the injector and detector were 250 °C and 300 °C, respectively. Nitrogen at 2 mL/min and pressure of 20 °C was used as a carrier gas. The oven temperature was initially 50 °C and gradually increased to 200 °C at the rate of 40 °C/min and maintained at this temperature for 3.5 min. Autosampler (Clarus 500, Perkin-Elmer, Waltham, MA, USA) was employed for sampling with injection size of 1.5 µL and split was set at 15:1.

4. Conclusions

Application of membrane bioreactor on citrus waste successfully improved the biogas production by more than six times compared to the conventional digestion system and reached 73% of theoretical methane yield. This underlines the potential application of MBR for citrus waste and other solid wastes containing hydrophobic inhibitors.

Acknowledgments

We express our gratitude to the Swedish International Development Agency (SIDA) for the financial support and Borås Energy and Environment AB (Borås, Sweden) for the inoculum.


Author Contributions

Rachma Wikandari is the first author and responsible for performing the experiment, writing the paper, and corresponding author. Ria Millati, Muhammad Nur Cahyanto, and Mohammad J. Taherzadeh are the second, third, and fourth authors, respectively, who were responsible for designing the experiment.

Conflicts of Interest

The authors declare no conflict of interest.

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© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Digital version: http://hdl.handle.net/2320/14222ISBN 978-91-87525-27-8 (printed)ISBN 978-91-87525-28-5 (pdf)ISSN 0280-381X, Skrifter från Högskolan i Borås, nr. 54

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EFFECT OF FRUIT FLAVORS ON ANAEROBIC DIGESTION: INHIBITIONS

AND SOLUTIONSRachma Wikandari

Rachm

a Wikandari EFFECT OF FRUIT FLAVORS ON ANAEROBIC DIGESTION

: INHIBITIONS AND SOLUTIONS

This thesis presents work that was done within the Swedish Centre for Resource Recovery (SCRR).

Research and education performed within SCRR identifies new and improved methods to convert

residuals into value-added products. SCRR covers technical, environmental and social aspects of

sustainable resource recovery.

Approximately 402 million tons of fruit wastes are annually generated worldwide, that is a serious environmental challenge. Biogas emerges as an attractive solution to handle this waste, since conversion of fruit waste into biogas combines waste treatment and energy production. In theory, 32 billion m3 biogas could be produced from this fruit wastes, which is equivalent to ca 20 billion liters of gasoline. However, in reality, the biogas production from the fruits is less than

theoretically possible. One of the reasons is the presence of toxic compound in the fruits that inhibit growth of biogas producing microorganisms. D-limonene, a flavor compound in orange is an example of the chemicals that causes failure in biogas production from orange peel waste. What are toxic compounds in other fruit and how to solve the toxicity problem? These questions were investigated in this thesis.

Various flavor compounds available in different fruits were examined. Among the compounds examined, myrcene and octanol were shown to be strong inhibitors for the process, which reduces 50% of biogas production at low concentration (0.005–0.05%). These compounds belong to fruit flavors of oranges, strawberries, grapes, plums, and mangoes. The toxicity of α-pinene, hexanal, E-2-hexenal, nonanal, γ-decalactone, epicatechin, and quercetin were moderate (0.05–0.5%). Meanwhile, the rest flavors including methyl butanoate, ethyl butanoate, ethyl hexanoate, hexyl acetate, furaneol, mesifurane, and γ-hexalactone had a little effect on the digestion process.

In order to overcome inhibition challenges of the flavors, the present study proposed two approaches: fruit flavors removal and protection of the digesting microorganism against fruit flavors. In this work, orange peel waste (OPW) and limonene were used as a model for the fruit waste and the inhibitor, respectively. The first approach used was solid-liquid pretreatment, which resulted in increased biogas production from OPW by more than three times. The second approach performed was using encased digesting cells in membranes, which were impermeable to limonene. This method resulted in increasing the biogas production from OPW by more than six times. More importantly, this method can be applied in single-stage digestion that is industrially popular.

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EFFECT OF FRUIT FLAVORS ON ANAEROBIC DIGESTION: …hb.diva-portal.org/smash/get/diva2:877097/FULLTEXT01.pdf · 2015. 12. 4. · about the effect of fruit flavors on anaerobic digesting