CHAPTER 1: INTRODUCTION - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/29696/7/07... · 2018-07-02 · to Loihi, a submarine volcano rising from slope of Mauna Loa. Located

ISOLATION AND IDENTIFICATION OF HALOPHILES AND THERMOPHILES AND THEIR

APPLICATION IN THE PROCESS OF BIODIESEL PRODUCTION AND METHANOGENESIS

INTRODUCTION | 4

CHAPTER 1: INTRODUCTION

This chapter includes an introduction to thermophiles, halophiles,

biodiesel, biogas, methods for biodiesel and biogas production,

current scenario of biodiesel and biogas production, enzymes

involved in biodiesel production and methanogenesis.



INTRODUCTION | 5

1.1 INTRODUCTION : HALOPHILES AND THERMOPHILES

Thermophiles are a group of organisms, a type of

extremophiles that thrives at relatively high

temperatures, between 45°C and 120°C.

Thermophiles are found in various geothermally

heated regions of the Earth, such as hot springs,

hydrothermal vents, in decaying plant matter and

sometimes in compost. "Thermophiles" is derived

from the Greek word thermotita-meaning heat,

and philia – meaning love. They are mainly classified into obligate and facultative thermophiles:

Obligate thermophiles (also known as extreme thermophiles) require high temperatures for

growth, whereas facultative thermophiles (also known as moderate thermophiles) can thrive at

moderately high temperatures which below 60°C. Hyperthermophiles are particularly extreme

thermophiles for which the optimal temperatures are above 80°C. But there are also some

extreme thermophiles which require a very high temperature of 80°C to 105°C for their growth.

The cell membranes and proteins of these organisms are unusually stable at high temperatures.

This property makes them very important for many biotechnological processes.

These microorganisms were discovered first in 1977 in deep-sea hydrothermal vents. These

microbes live adjacent to magma heated plumes of fluid which are heated in excess of 400°C.

The waters in which the microbes thrive ranging temperature between 120°C and 150°C. Water

does not boil at these temperatures because the pressure is well above 150 atm. Even more

significant is the fact that these microbes survive in extremely high concentrations of heavy

metals and sulfides. In 1999, scientists in Hawaii conducted an extremophile collecting mission

to Loihi, a submarine volcano rising from slope of Mauna Loa. Located 34 km southeast of the

big island of Hawaii, the summit of Loihi is 1000m below the surface of the ocean. The mission

proved to be a success as many microbial mats, including a never-seen before jelly-like

organisms were found in waters at 160ºC (Ackerman, Anderson, & Anderson, 2008; Alexander

et al., 2002; Delbes, Godon, & Moletta, 1998; Galand, Juottonen, Fritze, & Yrja, 2005).

Currently, scientists are preparing a new mission to Loihi with a submarine capable of collecting

Figure 1.1 Hot water Spring – a source for thermophiles



INTRODUCTION | 6

and bringing these organisms to the surface while keeping them in their natural conditions before

being transferred to onshore bioreactors.

Halophiles are a group of microorganisms again a

class of extremophiles that thrive in environments

with very high concentrations of salt. They are found

in various hypersaline environments of the world

mainly salt lakes and oceans. “Halophile” as a word

is derived from a Greek word which means "salt-

loving". This term is most often applied for Archaea,

but there are also bacterial halophiles and some

eukaryotes, such as alga. Halophiles can be loosely

classified as slightly, moderately or extremely halophilic, depending on their requirement for

NaCl. The extremely halophilic archaea are well adapted to saturating NaCl concentrations

above (15%). Moderate halophiles are able to grow between 3 – 15% of salt concentration.

Microorganisms which can grow below 3% salt concentration are known as slightly halophiles.

Their novel characteristics and capacity for large-scale culturing make halophiles potentially

valuable for biotechnology. Halophiles produce a large variety of stable and unique biomolecules

that may be useful for practical applications. Halophilic microorganisms produce stable enzymes

(including many hydrolytic enzymes such as DNAases, lipases, amylases, gelatinases and

proteases) capable of functioning under conditions that lead to precipitation or denaturation of

most proteins. Halophilic proteins compete effectively with salts for hydration, a property that

may result in resistance to other low-water-activity environments, such as in the presence of

organic solvents (Enache & Kamekura, 2010; Karan, Capes, & Dassarma, 2012). Novel

halophilic biomolecules may also be used for specialized applications, e.g. bacteriorhodopsin for

biocomputing, gas vesicles for bioengineering floating particles, pigments for food colouring,

and compatible solutes as stress protectants.

Figure 1.2 Sea – a source of halophiles



INTRODUCTION | 7

1.2 BIODIESEL AND METHANOGENESIS

Biofuels are alternative fuels produced from biological feedstocks. Demand of alternatives fuels

is increasing day by day to fulfill the present crisis of conventional fuels. They are classified as

first generation, second generation and third generation biofuels (Antoni, Zverlov, & Schwarz,

2007; Askew, 2007). First generation biofuels involve bioethanol produced from sugar and

starch crops, biodiesel from animal fat and oilseed crops. These are produced by simple and

known conversion technologies (Antoni et al., 2007; Askew, 2007; Fukuda, Kondo, &

Tamalampudi, 2009). Second generation biofuels are produced from various feedstocks like

agricultural/forestry residues, algae and other form of wastes which contain high level of organic

matter. Production of these kinds of sources involve highly promising but less proven

technologies (Al-zuhair, 2007; Aresta, Dibenedetto, Carone, Colonna, & Fragale, 2005; Bajpai &

Tyagi, 2006; Canakci & Sanli, 2008; Wang, Ou, Liu, & Zhang, 2007). Third generation biofuels

which have been introduced recently is production of biofuels from microorganisms. Methods

for production varies and depends on the feedstock used (Kalscheuer, Stolting, & Steinbu, 2006;

Li, Du, & Liu, 2008; Ratledge & Cohen, 2008). Among all the biofuels, bioethanol produced

from sugar is the mostly widely produced and utilized fuel (Munack & Krahl, 2007; Pimentel &

Patzek, 2007, 2005).

1.3 GLOBAL SCENARIO OF BIOFUELS

Global markets for biofuels mainly ethanol and biodiesel have shown enormous growth in the

past decade: in the year 2006 they contributed 2.0% of road transport fuels worldwide (over 45

billion liters). Global bioethanol production doubled between the year 2000 and 2005, reaching

over 39 billion liters in 2006, equal to about 3 percent of the 1300 billion liters of gasoline

consumed globally. USA and Brazil produced almost 90 percent of the world’s bioethanol in

2006: the USA produced over 18 billion liters, followed closely by Brazil, with about 17.5

billion liters (Ortiz, 2008). Other countries producing fuel ethanol include Australia, Canada,

China, Colombia, the Dominican Republic, France, Germany, India, Malawi, Poland, South

Africa, Spain, Sweden, Thailand and Zambia. Table 1.1 gives an overview of the 2006 global

biofuel production for the top 15 countries. On the other hand, global biodiesel production



INTRODUCTION | 8

jumped 50 percent in 2006 to over 10 billion liters globally. Half of the world biodiesel

production was done in Germany (Ortiz, 2008). Significant production also increased in Italy and

the USA. In Europe, supported by new policies, biodiesel, produced mostly from rapeseed,

gained broader acceptance and market share.

Table 1.1 An overview of the 2006 global biofuel production for the top 15 countries

Country Fuel ethanol Biodiesel

Billion Liters

USA 18.3 0.85

Brazil 17.5 0.07

Germany 0.5 2.80

China 1.0 0.07

France 0.25 0.63

Italy 0.13 0.57

Spain 0.40 0.14

India 0.30 0.03

Canada 0.20 0.05

Poland 0.12 0.13

Czech Republic 0.02 0.15

Colombia 0.20 0.06

Sweden 0.14 -

Malaysia - 0.14

UK - 0.11

EU total 1.6 4.5

World total 39 6

Aggressive expansion of biodiesel production also occurred in Asia (Malaysia, Indonesia,

Singapore, China), Latin America (Argentina, Brazil), and southeastern Europe (Romania,

Serbia). The following figure 1.3 indicates the proposed production of transport biofuels by

different countries. According to which Brazil, USA and EU will be the leading countries for

biofuel production (Msangi, Sulser, Rosegrant, & Valmonte-Santos, 2007).



INTRODUCTION | 9

It is believed that sugar cane and grains will become the primary feedstock for bioethanol

production by 2019, while secondary feedstocks will involve wheat, molasses, sugar beet etc

(Figure. 1.4) (Ortiz, 2008; Pimentel & Patzek, 2007, 2005). Similarly vegetable oil will be the

most acceptable source for biodiesel sources followed by jatropha and other sources. (Figure.

1.5) (Bajpai & Tyagi, 2006; Canakci & Sanli, 2008; Demirbas, 2008; Pimentel & Patzek, 2005)

B io fu e l (b io e th a n o l+ b io d ie s e l) p ro d u c t io n fo r t r a n s p o r t (M T O E )

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

2 0 0 5 2 0 1 0 2 0 1 5 2 0 2 0 2 0 2 5 2 0 3 0

million tons oil equivalent

C h in a

In d ia

B r a z i l

U S A

E U

Figure 1.3 Proposed productions of transport biofuels by different countries

Figure 1.4 Utilization of different feedstocks for bioethanol production



INTRODUCTION | 10

1.4 INDIAN SCENARIO OF BIOFUELS

India is the fifth largest primary energy consumer and fourth largest petroleum consumer in the

world. India is among one of the fifteen countries which contributes significantly in the production of

biofuels though it is very less compared to other countries like Brazil, Germany and USA (Pathak,

Mandalia, & Rupala, 2012). In 2003, India defined its policy for bioethanol production from

sugarcane molasses, according to which the ministry of petroleum and natural gas made 5% ethanol

blending in petrol mandatory across 9 states and 5 Union Territories (Wright & Aradhey, 2011).

However, it was not implemented completely because of non-availability and insufficient production

of sugarcane. Again in 2006, government mandated 5% ethanol blending in 20 states and 8 union

territories with the collaboration of oil marketing companies (OMC) but they could supply only 540

million liter of ethanol in place of 1.4 million liters expected according to the contract. Again in 2008,

the government mandated 5% blending in the whole country but due to non-availability of sugarcane

it was implemented partially. Now India has targeted 20% blending by 2017 (Wright & Aradhey,

2011).

The biodiesel policy of the country has recognized that Jatropha curcus is one of the most suitable

tree borne oilseed for biodiesel production. The Planning Commission of India had set an ambitious

target covering 11.2 to 13.4 million hectares of land under jatropha cultivation by the end of the 11th

Figure 1.5 Utilization of different feedstocks for biodiesel production



INTRODUCTION | 11

Five Year Plan (2011/12). The central government and several state governments provide fiscal

incentives for supporting planting of Jatropha and other non-edible oilseeds. Several public

institutions, state biofuel boards, state agricultural universities and cooperative sectors are also

supporting the biofuel mission in various capacities (Wright & Aradhey, 2011). The National

Biodiesel Mission, launched by the government of India has initiated development in biodiesel

production in two main phase named demonstration phase (2003-2007) and self sustaining execution

phase (2008-2012). In the first phase, the major focus was on jatropha cultivation, nursery

development, seed procurement and installation of transesterification plants. While the second phase

was focused on large scale cultivation of jatropha as well as production of sufficient biodiesel for

20% blending by the end of the XIth

plan (2008-2012). Both these phase were only partially

successful due to some limitations in jatropha cultivation and availability.

1.5 BIODIESEL

Brief History - The concept of alternative, renewable

energy has been in existence for over a century. Rudolf

Diesel is credited as the inventor of the first diesel

engine which was originally designed to run on fuel

derived from peanut oil. Rudolf Diesel was quoted as

saying; "The diesel engine would help considerably in

the development of agriculture of the countries which

use it." Unfortunately, due to the low cost of mineral

oils at the time, the diesel engine was modified to run

on petroleum oil. Biodiesel technology was overlooked while the demand for crude oil increased

significantly as the automotive and industrial age ensued. Rudolf Diesel was well aware that

renewable fuel would not be of major relevance during his lifetime when he said, "The use of

vegetable oils for engine fuel may seem insignificant today. But such oils may become in course

of time as important as petroleum and the coal tar products of the present time” (Bajpai & Tyagi,

2006).

Figure 1.6 Representative symbol of biodiesel



INTRODUCTION | 12

Biodiesel is derived from triglycerides or free fatty acids by transesterification with short chain

alcohols. Biodiesel can be produced through chemical ways and biological ways (Al-zuhair,

2007; Bajpai & Tyagi, 2006; Canakci & Sanli, 2008; Demirbas, 2008). Chemical catalysts

involve alkali catalysts and acid catalysts. Sodium hydroxide, sodium methoxide, potassium

hydroxide, and potassium methoxide are most widely used alkali catalyst. Sulfuric acid,

hydrochloric acid, phosphoric acid, and organic sulfonic acid are most common acid catalyst.

Acid-catalyzed transesterification is generally suitable for feedstock with high free fatty acid or

water content (Ataya, Dube, & Ternen, 2007; Demirbas, 2008). Recently, enzymatic approaches

for biodiesel production have received much attention since these have many advantages over

chemical methods: moderate reaction conditions, lower alcohol to oil ratio, easier product

recovery, and environmental friendly (Chen, Du, Liu, & Ding, 2008; Du, Xu, Liu, & Zeng, 2004;

Vieira, Silva, & Langone, 2006). Also, free fatty acids contained in waste oils and fats can be

completely converted to alkyl esters (Wang et al., 2007). On the other hand, in general, the

production cost of a lipase catalyst is significantly greater than that of an alkaline one. Currently,

there are extensive reports about enzyme mediated alcoholysis for biodiesel production, and

based on the application forms of biocatalysts, the related research can be classified into

immobilized lipase, whole cell catalyst, and liquid lipase-mediated alcoholysis for biodiesel

production, respectively (Abigor et al., 2000; Al-zuhair, 2005; Hernandez-martin & Otero, 2008;

Modi, Reddy, Rao, & Prasad, 2006; Ognjanoviû, Bezbradica, & Kneževiû, 2008; Shah & Gupta,

2007; Vieira et al., 2006).

Figure 1.7 General reaction of transesterification for biodiesel production



INTRODUCTION | 13

Major sources of biodiesel are based on either animal or plant sources. Rapeseed oil, jatropha oil,

and sunflower oil are very common plant based feedstock, while chicken fat and fish oil are

among the most common animal based sources (Bajpai & Tyagi, 2006; Canakci & Sanli, 2008;

Du, Li, Sun, Chen, & Liu, 2008; Vasudevan & Briggs, 2008). Both these kinds of sources are

facing problem of constant availability for prolong time periods. In case of plant based sources,

the area required for plant cultivation and the time taken for plant growth are major problems.

Animal based sources require sacrificing numbers of animals to fulfill the feedstock demand

which is not acceptable. This has led scientists to find out such sources which could replace all

the other present sources. They found that microorganisms, especially algae and genetically

modified microorganisms could be the best potential candidates (Aresta et al., 2005; Chisti,

2007; Kalscheuer et al., 2006; Sheehan, Dunahay, John, & Rosseler, 1998). Some groups have

also focused on oleaginous microorganisms as they contain higher amount of lipids (Q. Li et al.,

2008; Ratledge & Cohen, 2008; Wackett, 2008).

In general, the procedures for biodiesel production the following steps: 1) lipids extraction from

the feedstock, 2) transesterification of extracted lipids, 3) product collection and purified. For

lipids extraction several physical and/or chemical methods are used (Andrich, Zinnai, Venturi,

Nesti, & Fiorentini, 2006; Bhattacharyya, 1999; White & Frerman, 1967). Selection of method is

depending on the type and quantity of feedstock. Transesterification of lipids is carried out by

either chemical or enzymatic methods (Al-zuhair, 2007; Bajpai & Tyagi, 2006; Canakci & Sanli,

2008). For production purification methods like chromatography, filtration and use of certain

chemical are applied (Du et al., 2008; Pinto, Guarieiro, Rezende, Ribeiro, & Ednildo, 2005).

Once the pure product is obtained it can be used directly or after mixing with petroleum diesel

which is known as blending.

1.6 METHANOGENESIS

Brief History - Since ancient times combustible gas has been known to seep from geological

fissures in certain areas of the world. However, the experiments of Alessandro Volta with

combustible air obtained from sediments and marshy places created widespread interest and laid

the scientific foundation for study of the biological production of methane (Wolfe, 1996). The



INTRODUCTION | 14

results of his experiments are recorded in a series of letters. Carlo Campi, a friend of Volta had

also observed rising gas near a spring and finding that they were capable of catching fire.

Another recorded observation of combustion gas from sediments was made in 1783 by Thomas

Paine and George Washington, who were ignorant of Volta’s discovery. Lavoisier and others had

obtained evidence that Volta’s flammable air was “gas hidrogenium carbonatrum”. Finally the

term methane was proposed in 1865 and the nomenclature was confirmed by the International

Congress on Chemical Nomenclature in 1892. The first definite indication that methane is

formed by a microbiological process was obtained in 1868 by Bechamp, a student of Pasteur. In

his experiments, he had decomposed sugar and starch with simple inorganic media containing

chalk in absence of oxygen.

Methanogenesis or biomethanation is the formation of methane by microbes known as

methanogens. Organisms capable of producing methane have been identified only from the

domain Archaea, a group phylogenetically distinct from both eukaryotes and bacteria, although

many live in close association with anaerobic bacteria (Conrad, Erkel, & Liesack, 2006; Garcia,

Patel, & Ollivier, 2000). The production of methane is an important and widespread form of

microbial metabolism. In most environments, it is the final step in the decomposition of biomass.

Methanogenesis is a continuous process but for the sake of understanding it is divided into three

main steps: degradation, acetogenesis and methanogenesis. Out of these three steps, degradation

could be carried out either aerobically or anaerobically while the later two steps must be carried

out under anaerobic condition. Cowdung is a key initiator as it is a good source of methanogens

and acidophiles. It also acts as a buffering agent to maintain neutral pH during the process.

Along with cowdung certain other feedstocks are also being used, which mainly include plant

fibers, grasses, vegetables etc. Polymers of these sources are first degraded and converted into

monomers which are then converted into lactate or acetate by acidophiles. Ultimately biogas is

produced from lactate and acetate by methanogenesis.



INTRODUCTION | 15

1.7 ENZYMES INVOLVED IN BIODIESEL PRODUCTION AND METHANOGENESIS

LIPASE

Lipase (EC 3.1.1.3) is a subclass of the esterase which

catalyzes the cleavage (hydrolysis) of fats (lipids). Lipases

perform essential roles in the digestion, transport and

processing of dietary lipids (e.g. triglycerides, fats, oils) in

most, if not all, living organisms. Genes encoding lipases are

also present in certain viruses. Most lipases act at a specific

position on the glycerol backbone of lipid substrates and

convert them into diglycerides and monoglycerides.

Although a diverse array of genetically distinct lipase enzymes

are found in nature, and represent several types of protein folds and catalytic mechanisms, most

are built on an alpha/beta hydrolase fold and employ a chymotrypsin-like hydrolysis mechanism

involving a serine nucleophile, an acid residue (usually aspartic acid), and a histidine.

Lipases are involved in diverse biological processes ranging from routine metabolism of dietary

triglycerides to cell signaling and inflammation. Thus, some lipase activities are confined to

specific compartments within cells while others work in extracellular spaces.

• In the instance of lysosomal lipase, the enzyme is confined within an organelle called the

lysosome.

Figure 1.8 General reaction of methanogenesis for biogas production

Figure 1.9 Lipase



• Other lipase enzymes, such as

where they serve to process dietary lipids into more simple forms that can be more easily

absorbed and transported throughout the body.

• Fungi and bacteria may secrete lipases to

medium (or in examples of pathogenic microbes, to promote invasion of a new host).

• Certain wasp and bee venoms contain

payload" of injury and inflammation delivered by a sting.

• As biological membranes

phospholipids, lipases play important roles in

• Malassezia globosa, a fungus that is thought to be the cause of human

lipase to break down

dandruff.

Lipase also has many other appli

medicals, as biosensors, as detergents, in

industries, in environment management

Lipases are generally animal sourced, but can also be sourced

based industries have enabled

purposes.

CELLULASE

Cellulase refers to a group of

fungi, bacteria, and protozoans

are also cellulases produced by a few other types of organisms,

such as some termites and the microbial intestinal symbionts

other termites (Tokuda & Watanabe, 2007; Watanabe, Noda,

Tokuda, & Lo, 1998). Several different kinds of cellulases are



Other lipase enzymes, such as pancreatic lipases, are secreted into


absorbed and transported throughout the body.

Fungi and bacteria may secrete lipases to facilitate nutrient absorption from the external


Certain wasp and bee venoms contain phospholipases that enhance the "biological

payload" of injury and inflammation delivered by a sting.

biological membranes are integral to living cells and are largely composed

, lipases play important roles in cell biology.

, a fungus that is thought to be the cause of human

lipase to break down sebum into oleic acid and increase skin cell production, causing

Lipase also has many other applications in different fields such as in the food industry, in

, as biosensors, as detergents, in the leather industry, in the cosmetic and perfume

industries, in environment management, etc.

Lipases are generally animal sourced, but can also be sourced from microbes

d production of lipase through faster and easier to serve the different

refers to a group of enzymes produced chiefly by

protozoans which catalyze cellulose. There

are also cellulases produced by a few other types of organisms,

and the microbial intestinal symbionts of

(Tokuda & Watanabe, 2007; Watanabe, Noda,

. Several different kinds of cellulases are Figure 1.10



INTRODUCTION | 16

, are secreted into extracellular spaces


facilitate nutrient absorption from the external


that enhance the "biological

are integral to living cells and are largely composed of

, a fungus that is thought to be the cause of human dandruff, uses

and increase skin cell production, causing

food industry, in bio-

cosmetic and perfume

microbes. Biotechnological

faster and easier to serve the different

Figure 1.10 Cellulase



INTRODUCTION | 17

known, which differ structurally and mechanistically. Other endoglucanases are: endo-1,4-beta-

glucanase, carboxymethyl cellulase (CMCase), endo-1,4-beta-D-glucanase, beta-1,4-glucanase,

beta-1,4-endoglucan hydrolase, and celludextrinase.

There are five general types of cellulases based on the type of reaction catalyzed:

• Endocellulase (EC 3.2.1.4) randomly cleaves internal bonds at amorphous sites that

create new chain ends.

• Exocellulase (EC 3.2.1.91) cleaves two to four units from the ends of the exposed chains

produced by endocellulase, resulting in the tetrasaccharides or disaccharides, such as

cellobiose. There are two main types of exocellulases [or cellobiohydrolases (CBH)] -

CBHI works processively from the reducing end, and CBHII works processively from the

nonreducing end of cellulose.

• Cellobiase (EC 3.2.1.21) or beta-glucosidase hydrolyses the exocellulase product into

individual monosaccharides.

• Oxidative cellulases depolymerize cellulose by radical reactions, as for instance

cellobiose dehydrogenase (acceptor).

• Cellulose phosphorylases (EC 2.4.1.20) depolymerize cellulose using phosphates instead

of water.

In the most familiar case of cellulase activity, the enzyme complex breaks down cellulose to

beta-glucose. This type of cellulase is produced mainly by symbiotic bacteria in the ruminating

chambers of herbivores. Aside from ruminants, most animals (including humans) do not produce

cellulase in their bodies and can only partially break down cellulose through fermentation,

limiting their ability to use energy in fibrous plant material. Enzymes that hydrolyze

hemicellulose are usually referred to as hemicellulase and are usually classified under cellulase

in general. Enzymes that cleave lignin are occasionally classified as cellulase, but this is usually

considered erroneous.

Within the above types there are also progressive (also known as processive) and nonprogressive

types. Progressive cellulase will continue to interact with a single polysaccharide strand,



INTRODUCTION | 18

nonprogressive cellulase will interact once then disengage and engage another polysaccharide

strand.

Most fungal cellulases have a two-domain structure, with one catalytic domain and one cellulose

binding domain that are connected by a flexible linker. This structure is adapted for working on

an insoluble substrate, and it allows the enzyme to diffuse two-dimensionally on a surface in a

caterpillar way(Czjzek, Schu, Panine, & Henrissat, 2002; Garsoux, Lamotte, Gerday, & Feller,

2004). However, there are also cellulases (mostly endoglucanases) that lack cellulose binding

domains. These enzymes might have a swelling function.

In many bacteria, cellulases in-vivo are complex enzyme structures organized in supramolecular

complexes, refered to as cellulosomes. They contain roughly five different enzymatic subunits

representing namely endocellulases, exocellulases, cellobiases, oxidative cellulases and cellulose

phosphorylases wherein only endocellulases and cellobiases participate in the actual hydrolysis

of the β(1→ 4) linkage. Recent work on the molecular biology of cellulosomes had led to the

discovery of numerous cellulosome-related “signature” sequences known as dockerins and

cohesins. Depending on their amino acid sequence and tertiary structures, cellulases are divided

into clans and families (Mechaly et al., 2000; Pinheiro, Benedita Andrade Brás, Joana Luís

Armada Najmudin, Shabir Carvalho, Ana Luísa Ferreira, Prates, & Fontes, 2012).

The three types of reactions catalyzed by cellulases:1. Breakage of the noncovalent interactions

present in the amorphous structure of cellulose (endocellulase) 2. Hydrolysis of chain ends to

break the polymer into smaller sugars (exocellulase) 3. Hydrolysis of disaccharides and

tetrasaccharides into glucose (beta-glucosidase).

Cellulases have wide range of applications in many industries which mainly include pulp and

paper industry, textile industry, bioethanol industry, wine and brewery industry, food processing

industry, animal feed industry, agriculture industry, oil extraction industry, pigment extraction

industry, detergent industry, waste management etc.



INTRODUCTION | 19

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AIM AND OBJECTIVES | 25

AIM AND OBJECTIVES

AIM: Isolation and identification of halophiles and thermophiles and their application in the

process of biodiesel production and methanogenesis.

Our primary target was to identify certain halophiles and thermophiles which help in the process

of either biodiesel production or methanogenesis. From the literature, it was found that microbes

having higher lipid contents could provide better feedstock for biodiesel production as it is

produced by transesterification of lipids. It was verified that yeast can accumulate lipids in

higher concentration under certain stress conditions. So certain halophilic yeasts were isolated

and adapted for lipid accumulation. Thermophilic enzymes obtained from thermophiles were

used for production of extracellular enzymes. These enzymes were either used in enzymatic

transesterification for biodiesel production or in the process of methanogenesis. With the help of

these enzymes an attempt was made to increase the rate of methanogenesis.

OBJECTIVES

a) Isolation of halophiles and thermophiles

b) Screening of halophilic microbes capable of lipid accumulation (yeast)

c) Screening of thermophilic microbes producing extracellular enzymes like lipase and

cellulase

d) Identification of microbes by 16s rRNA or 18s rRNA sequencing

e) Developing approaches for biodiesel production (chemical/enzymatic transesterification)

f) Developing approaches to increase the rate of methanogenesis

g) Confirmation of production of biodiesel and biogas by various analyses like high

performance thin layer chromatography, Infrared spectrophotometer, Gas

chromatography etc.

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CHAPTER 1: INTRODUCTION - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/29696/7/07... · 2018-07-02 · to Loihi, a submarine volcano rising from slope of Mauna Loa. Located