BAMBOO BIOCHAR AND CARBON NANOTUBE SOIL AMENDMENT EFFECTS ON

“MICRO-TOM” TOMATO DEVELOPMENT AND QUALITY

By

RATNA SUTHAR

A THESIS PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2016

© 2016 Ratna Suthar

To my dear parents

4

ACKNOWLEDGMENTS

I would like to take this opportunity to express my gratitude to everyone who supported

my aspirations throughout the course of my degree. Thankful to my close friends, who lift my

spirits in times of need and keep me in check with reality when necessary, even through the

miles of distance. I have to thank my parents, Girish and Rekha Suthar, for their unfaltering love

and supporting my decisions.

Thankful to have an advisor like Dr. Bin Gao for his always optimistic attitude, enduring

patience and kindness. For introducing me to a career of research in academia while I was a

curious undergraduate, I am grateful for Dr. Cecilia Nunes. Thank you for serving my

committee. My deep gratitude to Dr. Ray Bucklin who was pivotal in initiating my graduate

studies here at UF. I’d like to thank Dr. Steve Sargent for his insight as a valuable committee

member. I’d like to thank Dr. Don Huber for his inspiring course on postharvest biology. I am

thankful to Dr. Jeff Brecht for offering me opportunities outside of my research to further my

knowledge and professional development. Also thanks to my co-chair Dr. Jianjun Chen for

providing the materials and greenhouse space at the MREC in Apopka, FL to grow the tomato

plants used in this study. Thankful for Mr. Wang Cun, who diligently collected the plant growth

data used in this study. Thanks to Dr. Andrew Zimmerman for helping with biochar

characterization.

Thankful for Dr. Shensen Wang, Mr. Isaac Duerr, Mr. Daniel Preston, Mr. James Lee and

Ms. Kim Cordasco their technical support and maintaining good humor amidst the countless and

inevitable technical difficulties during lab and field work.

5

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...............................................................................................................4

LIST OF TABLES ...........................................................................................................................7

LIST OF FIGURES .........................................................................................................................8

LIST OF ABBREVIATIONS ........................................................................................................10

ABSTRACT ...................................................................................................................................11

CHAPTER

1 INTRODUCTION ..................................................................................................................13

Importance of Tomato Quality ...............................................................................................13

Overview of Biochar ...............................................................................................................15

Biochar Production ..........................................................................................................15

Importance of Feedstock .................................................................................................15

Pyrolysis Method and Temperature .................................................................................16

Soil Fertility ............................................................................................................................16

Biochar as a Soil Amendment ................................................................................................17

Related Studies ................................................................................................................19

Gap in the Knowledge .....................................................................................................19

Overview of Carbon Nanotubes as Soil Amendment .............................................................20

Carbon Nanotubes ...........................................................................................................20

Related CNT Studies .......................................................................................................20

Gap in the Knowledge .....................................................................................................21

Objectives ...............................................................................................................................22

2 INLFLUENCE OF PYROLYSIS TEMPERATURE OF BIOCHAR SOIL

AMENDMENT EFFECTS ON TOMATO GROWTH AND FRUIT QUALITY ................23

Introduction .............................................................................................................................23

Materials and Methods ...........................................................................................................26

Biochar Production and Characterization ........................................................................26

Plant Growth ....................................................................................................................27

Preparation of growth medium .................................................................................27

Seed germination and growth ...................................................................................27

Leachate collection and analysis ..............................................................................28

Yield .........................................................................................................................29

Fruit Quality ....................................................................................................................29

Physical analysis ......................................................................................................29

Compositional analysis ............................................................................................29

Statistical Analysis ..........................................................................................................31

Results and Discussion ...........................................................................................................31

6

Biochar Properties ...........................................................................................................32

Plant Growth ....................................................................................................................34

Fruit Quality ....................................................................................................................36

Conclusions .............................................................................................................................38

3 EFFECT OF CARBON NANOTUBES AS SOIL AMENDMENT IN SAND

CULTURE ON TOMATO GROWTH AND QUALITY ......................................................52

Introduction .............................................................................................................................52

Materials and Methods ...........................................................................................................54

Carbon Nanotubes ...........................................................................................................54

Plant Growth ....................................................................................................................55

Preparation of growth medium .................................................................................55

Seed germination and growth ...................................................................................55

Leachate collection and analysis ..............................................................................56

Yield .........................................................................................................................57

Fruit Quality ....................................................................................................................57

Physical analysis ......................................................................................................57

Compositional analysis ............................................................................................57

Statistical Analysis ..........................................................................................................59

Results and Discussion ...........................................................................................................59

Plant Growth ....................................................................................................................59

Fruit Quality ....................................................................................................................61

Conclusion ..............................................................................................................................62

4 CONCLUSION AND FUTURE WORK ...............................................................................70

Conclusion: Influence of Biochar Pyrolysis Temperature on Soil Amendment Effects on

Tomato Growth and Fruit Quality.......................................................................................70

Future Work ............................................................................................................................70

Conclusion: Effect of Carbon Nanotubes as Soil Amendment in Sand Culture on

Tomato Growth and Quality ...............................................................................................71

Future Work ............................................................................................................................71

LIST OF REFERENCES ...............................................................................................................73

BIOGRAPHICAL SKETCH .........................................................................................................82

7

LIST OF TABLES

Table page

2-1 CO2 and N2 Surface Area and Pore Volume Comparative Cation and Anion

Exchange Capacities and pH of Bamboo Biochar (BB) Produced at 300°C, 450°C or

600°C .................................................................................................................................51

8

LIST OF FIGURES

Figure page

2-1 Micro-Tom cultivar is known for its small plant height and fast growth cycle. ................40

2-2 Locally sourced bamboo feedstock was dried and cut into 10 inch pieces and

pyrolysed at temperatures of 300, 450 and 600C in a large kiln. ......................................40

2-3 Pots prepared with different concentrations of biochar in sand media. .............................41

2-4 Growth Index (cm2) of Micro-Tom plants at week 3 (above) and week 7 (below) of

all biochar treatments. Bars represent standard error (n = ~15) ........................................42

2-5 Yield of average number of full red fruits obtained per plant from each treatment

from harvests during Cycle 1 (2/5/16) and Cycle 2 Harvest 1 (6/2/16) and Harvest 2

(6/9/16) ...............................................................................................................................43

2-6 Concentrations (mg/L) of Ca, Mg, P and NO3 in plant leachate samples measured at

weeks 5, 8 and 10 during cycle 1 and week 10 in cycle 2. Means assigned same letter

groups are not significantly different. ................................................................................44

2-7 Color coordinates, a* and L* values, of tomatoes cultivated in standard (control) and

biochar amended media. Bars represent standard error (n = ~30). ....................................45

2-8 Hue angle values and firmness of tomatoes cultivated in standard (control) biochar

amended media. Bars represent standard error (n = ~30). .................................................46

2-9 Whole and cut appearance of fruit from Cycle 2 Harvest 1. .............................................47

2-10 Fructose and glucose contents of tomatoes cultivated in standard (control) and

biochar amended media. Bars represent standard error (n = 6) .........................................47

2-11 Soluble solids content (SSC) on the left and titratable acidity (TA) on the right for

tomatoes cultivated in standard (control) and biochar amended media. Bars represent

standard error (n = 3). (There was not adequate tissue of Cycle 1 600 High treated

fruits for TA.) .....................................................................................................................48

2-12 AA value of tomatoes cultivated in standard (control) biochar amended media. Bars

represent standard error (n = 6) ..........................................................................................49

2-13 Sugar to acid ratio of (soluble solids content to titratable acidity) all three growth

cycles of control and CNT treated tomatoes. .....................................................................50

2-14 Statistical analysis of sugar to acid ratio for Cycle 2 Harvest 1 data shown in figure

2-13. ...................................................................................................................................50

3-1 Pots of prepared growth medium control of CNT treatments. ...........................................64

9

3-2 Growth Index (cm2) of CNT treated Micro-Tom plants at week 3 (above) and week 7

(below). ..............................................................................................................................64

3-3 Concentrations (mg/L) of Ca, Mg, P and NO3 in plant leachate samples measured at

week 10 in the growth cycle. Means assigned same letter groups are not significantly

different ..............................................................................................................................65

3-4 Yield of average number of full red fruits obtained per plant from each treatment

from Harvest 1 (6/2/16) and Harvest 2 (6/9/16) ................................................................65

3-5 Color a* and L* and Hue Angle values of control and CNT treated tomatoes. Bars

represent standard error (n = ~30) .....................................................................................66

3-6 Ascorbic acid (AA), titratable acidity (TA) and firmness of control and CNT treated

tomatoes. Bars represent standard error (n = ~30 in Force, n = 3 in TA, n = 6 in AA). ....67

3-7 Soluble solids content (SSC), glucose, fructose and of control and CNT treated

tomatoes. Bars represent standard error (n = 6 in glucose and fructose and n = 3 in

SSC). ..................................................................................................................................68

3-8 Sugar to acid ratio of (soluble solids content to titratable acidity) all three growth

cycles of control and CNT treated fruit. ............................................................................69

3-9 Statistical analysis of sugar to acid ratio for Cycle 2 Harvest 1 data. ................................69

10

LIST OF ABBREVIATIONS

AA

BB

Ascorbic Acid

Bamboo Biochar

CEC Cation Exchange Capacity

CNT

SSC

TA

Carbon Nanotubes

Soluble Solids Content

Titratable Acidity

11

Abstract of Thesis Presented to the Graduate School

of the University of Florida in Partial Fulfillment of the

Requirements for the Degree of Master of Science

BAMBOO BIOCHAR AND CARBON NANOTUBE SOIL AMENDMENT EFFECTS ON

“MICRO-TOM” TOMATO DEVELOPMENT AND QUALITY

By

Ratna Suthar

December 2016

Chair: Bin Gao

Cochair: Jianjun Chen

Major: Agricultural and Biological Engineering

Biochar is a highly stable form of black carbon produced by pyrolysis of natural biomass

materials. As a soil amendment, biochar can increase overall soil quality and promote crop

growth. Few studies have explored variables such as pyrolysis temperature on efficacy of biochar

as soil amendment. Carbon nanotubes are widely used in the industry due to their exceptional

chemical and physical properties; however, they may enter the environment as a potential

pollutant. Studies have shown their positive effect on plant growth but no studies have assessed

resulting crop quality. This study was designed to investigate the soil amendment effects of

different temperature bamboo biochar as well as carbon nanotubes on plant growth and quality of

tomato fruit. Micro-Tom tomato cultivar plants were grown in soil amended with biochar

prepared at high and low concentrations of three temperatures: 300°C, 450°C and 600°C. Carbon

nanotubes were added at concentrations of 0.5%, 1%, and 3% by weight. To assess the quality,

tomato fruit were harvested at the red stage and analyzed for color, texture, soluble solids

content, sugars, and acidity. Overall, 300°C and 450°C biochar treatments increased plant

growth index and yielded fruit with higher sugars and acids. Also, higher concentration of carbon

nanotubes resulted in higher plant growth index as well as fruit with higher individual sugar

12

contents. Results will help optimize biochar use by targeting biochar production conditions that

result in desirable soil amendment and fruit quality effects. Secondly, findings will also help in

evaluating impacts of carbon nanotubes on soil and crop systems.

13

CHAPTER 1

INTRODUCTION

Importance of Tomato Quality

Florida’s warm climate is ideal for cultivating tomatoes. Today, Florida is the nation’s

largest producer of fresh tomatoes, shipping more than 1.1 billion pounds of fresh tomatoes to

the US, Canada and abroad (Floridatomatoes.org). Micro-Tom, a dwarf tomato cultivar

(Solanum lycopersicum L.), is known for its small plant and fruit size, and rapid growth,

constituting a convenient model system for research on tomato development and quality (Sun et

al., 2006, Gomez et al., 2009).

Quality is a measure of how much the end-user will value the product. The quality of

fresh tomato fruit is determined by various attributes such as color, firmness, flavor and

nutritional value. Tomatoes are a rich source of many compounds beneficial to health, such as

vitamin C, vitamin E, carotenoids (lycopene and β-carotene), and phenolic compounds such as

flavonoids. Due to the above compounds’ antioxidant activity, studies suggest protective roles of

tomato consumption in the prevention of chronic diseases such as cancer and cardiovascular

diseases (Arab and Steck, 2000, Sesso et al., 2003).

Because consumers purchase tomatoes largely based on appearance, physical

characteristics such as color and firmness are key. High-quality tomato fruit have a firm, turgid

appearance with red color that is uniform and shiny and show no signs of mechanical injury,

shriveling, or decay (Sargent and Moretti, 2014). Color is one of the most important quality

attributes that affect tomato appearance and is determined by skin and flesh pigmentation

(Brandt et al., 2006). Color measurements can also be used as an estimate of the levels of

specific chemical components (i.e., lycopene content) as indices of quality. Tomato color is

greatly correlated with the lycopene content; the change in color from mature green stage to the

14

red stage is reflective of significant increase in lycopene concentration (Helyes et al., 2006,

Dumas et al., 2003, Brandt et al., 2006). Furthermore, tomato firmness is a major factor in

consumer acceptability because it is associated with good eating quality and shelf life (Nunes,

2009).

In addition to color and firmness, measurements of sugars and acidity predict desirable

sweet and sour flavor attributes (Baldwin et al., 1998). Sugar content can be measured by

quantifying the soluble solids content (SSC) and individual reducing sugars: fructose and

glucose. SSC is particularly important to the processing industry and has probably received more

attention than any other quality trait (Aoun et al., 2013). Acidity can be measured in the form of

titratable acidity (TA) and ascorbic acid (AA) or vitamin C content which tends to increase as the

fruit matures and is usually higher in the vine-ripe tomatoes than green (Nunes, 2009). The major

organic acids in tomato are citric and malic acids, with citric acid predominating (Davies et al.,

1981). As tomatoes mature on the plant, sugar-to-acid ratio increases higher soluble solids and

lower acidity (Nunes, 2009).

Much of the variation encountered in the sensory and physicochemical characteristics of

produce include pre-harvest factors such as weather, soil fertility, moisture content of the soil,

use of growth regulators and other cultural practices (Shewfelt, 1990). It is important to know the

pre-harvest factors that contribute to producing fruits with superior quality at harvest while

appropriate postharvest handling and treatment methods should be used to maintain fruit quality

after harvest (Arah et al., 2016). Few reviews have been published regarding the effects of pre-

harvest factors, specifically soil fertility, on postharvest quality attributes such as vitamin C (Lee

and Kader, 2000), sugars (Beckles, 2012), appearance (Kays, 1999) , and texture (Sams, 1999)

along with overall quality attributes (Weston and Barth, 1997). Arah et al. (2016) concluded that

15

postharvest quality status of tomatoes partly depended on pre-harvest practices such as soil

nutrient factors carried out during production.

Overview of Biochar

Biochar Production

Salvaging carbon-rich biomass such as forest and agricultural residues and converting

them into value-added products such as biochar has attracted great attention because of its

potential to improve soil fertility while concurrently help mitigate climate change (Lehmann,

2007). In creating biochar, biomass is heated with little to no available oxygen and at relatively

low temperature (< 1000 °C). This process, called pyrolysis, results in the thermochemical

decomposition of the organic matter, resulting in a porous, carbon-rich material called biochar.

Bio-charcoal, or biochar, is no different from commercial charcoal; however, its application is

intended for biological and agricultural purposes. Use of biochar particularly as soil amendment

has recently gained focus.

Not all biochars are created equal. Potential applications of biochar are governed by their

physical properties such as surface area, functional groups, and elemental composition (Downie

et al., 2009). Such physical properties are influenced by biochar production factors such as type

of feedstock, pyrolysis method, and pyrolysis temperature (Sun et al., 2014).

Importance of Feedstock

Biochar can and should be made from biomass waste materials such as agricultural and

forest residues. Feedstock types include crop residues (both field residues and processing

residues such as nut shells, fruit pits and peels, bagasse, etc.), as well as yard, food and forestry

wastes, and animal manures (Biochar-International, 2016) . Ideally, making biochar from such

materials should create no competition for land with any other land use option, such as food

production, supporting sustainability. Previous studies have demonstrated that feedstock type can

16

play an important role in controlling the quality and functions of biochar (Sun et al., 2014, Kloss

et al., 2012). In particular, feedstock type may strongly affect biochar’s elemental composition

and thus nutrient content.

Pyrolysis Method and Temperature

Biochar pyrolysis temperature usually ranges from 200-1000˚C, depending on the desired

properties. Generally, increasing biochar production temperature, increases surface area

(Lehmann and Joseph, 2015). Increasing the pyrolysis temperature of biochars increases their

degree of carbonization, which increases their surface area (Chen et al., 2008) but reduces the

abundance of amorphous organic matter. An in-depth comparison of feedstock types and

pyrolysis temperature by Sun et al. (2014) showed that even amongst different feedstocks, chars

made at higher temperature had larger surface areas. Additionally, Uchimiya et al. (2011) found

that pyrolysis temperature can affect the presence of surface functional groups on biochars and

thus control their heavy metal sequestration ability in soils.

Recent studies have also suggested that difference in conversion/production methods

such as dry or wet pyrolysis play an important role in controlling biochar properties (Libra et al.,

2011). Overall, pyrolysis temperature and feedstock types are the most important factors in

determining biochar properties and thus their application.

Soil Fertility

By legal definition, the term fertilizer refers to soil amendments that guarantee the

minimum percentages of nutrients (at least the minimum percentage of nitrogen, phosphate, and

potassium) (Whiting, 2015). However, excessive application of the fertilizer may result in

release of high concentrations of nutrient elements, such as nitrogen and phosphorus, into aquatic

systems (Yao et al., 2012). Leaching of nutrients from soils may deplete soil fertility, increase

fertilizer costs for the farmers, reduce crop yields, but most importantly impose a threat to

17

environmental health (Bhargava and Sheldarkar, 1993, Laird et al., 2010, Özacar, 2003). High

nutrient levels can contaminate surface and/or groundwater, promoting eutrophication from

excessive production of photosynthetic aquatic microorganisms in freshwater and marine

ecosystems (Karaca et al., 2004). It is therefore imperative to develop technologies such as

biochar to prevent nutrient leaching.

Peat moss and vermiculite, common nursery potting amendments, have additional

environmental costs. Vermiculite must be exfoliated at temperatures exceeding 900 °C and is

often shipped great distances (Fulton et al., 2013). Peat mining involves the draining and

destruction of sensitive wetlands and must also be shipped. Replacing such products with

biochars produced onsite from locally available waste biomass, with associated capture of

process heat, would be a beneficial not only to nurseries, but to the environment as well.

Biochar as a Soil Amendment

A key advantage to biochar as soil amendment is its longevity; in contrast to other

organic matter in soil, biochars remain particulate over long periods of time (Lehmann et al.,

2011, Skjemstad et al., 1996), even though particle sizes may decrease on a decadal time scale

(Nguyen et al., 2008). Research indicates that biochar is recalcitrant and it may endure for

hundreds or thousands of years (Seiler and Crutzen, 1980). Use of biochar as a soil amendment

dates back to pre-Columbian civilizations in the Americas incorporating charcoal and fish bones

into their soil, creating rich loam such as the “terra preta” (black earth) soils of the Amazon,

which were found to be nine times more fertile than the surrounding un-amended soils (Sohi et

al., 2010, Schumann, 2012). Today in agriculture, biochar is receiving a new interest as a source

of renewable bioenergy because it can be produced from waste biomass materials.

No legal claims are made about nutrient content or other helpful (or harmful) effects that

soil amendments may have on the soil and plant growth (Whiting, 2015). Biochar’s benefits to

18

soil have been attributed to increasing soil water-holding capacity, cation exchange capacity,

nutrient availability and crop yield (Keith et al., 2011, Quilliam et al., 2012, Liu et al., 2013,

Olmo et al., 2014, Olmo et al., 2016). The highly porous structure of biochar increases the water-

holding capacity of sandy soils, improving the efficiency of water use in agricultural production.

Additionally, biochar’s negative charge contributes to a higher cation exchange capacity (CEC),

allowing higher availability of plant mineral nutrients such as calcium (Ca), potassium (K),

phosphorous (P) and magnesium (Mg) in the soil.

Due to its highly aromatic structure, biochar is chemically and biologically more stable

than the organic matter from which it was made (Spokas, 2010). This results in a slower rate of

degradation to CO2 than most other organic matter. Therefore, one of the advantages of using

biochar as opposed to other soil amendments, is the increase of long-term soil carbon content and

thus sequestration of carbon (Lehmann, 2007, Sohi et al., 2010).

Soils high in organic matter or clay content have naturally high CECs and retain mineral

nutrients well, making those nutrients available to plants as they are needed. Conversely, sandy

soils are considered unproductive because of their low CEC and water holding capacity, limiting

the required water and nutrients for the plant because they are easily leached by rain or irrigation

water (Andry et al., 2009). Therefore, the largest impact from biochar amendment would likely

be seen in sandy soils like those found throughout Florida (Schumann, 2012).

Increased crop growth and yield is a commonly reported benefit of adding biochar to

soils (Atkinson et al., 2010, Major et al., 2010, Olmo et al., 2014). The effects of biochar on crop

productivity are, however, diverse (Spokas et al., 2012, Biederman and Harpole, 2013). The

effect of the biochar amendment depends on the feedstock source, the biochar pyrolysis process,

soil properties and the scheme of biochar application.

19

However, the employment of biochar as a soil amendment in agriculture systems should

result not only in a yield or growth increase but also in a positive impact on the overall quality

and nutritional value of crops (Petruccelli et al., 2015).

Related Studies

Success of biochar application in most published studies has limited their definition as

either increased yield or above-ground biomass. However, very little is known about biochar

effects on nutritional attributes in fruits and vegetables (Schmidt et al., 2014). Studies of

biochar’s effects on fruit quality are limited, particularly on tomatoes. Petruccelli et al. (2015)

found that tomatoes (cv. Rio Grande) grown in substrate amended with biochars made from

straw and olive residues produced at a relatively high temperature (1200°C) had higher

secondary metabolites, phenolic compounds, and lycopene compared to those grown in a wheat

straw biochar amended soil. A mix of rice husk and cotton seed biochar produced through

pyrolysis at 400 °C was also shown to enhance soil water holding capacity under reduced

irrigation and also produced fruit with quality comparable to that of tomatoes grown under full

irrigation (Akhtar et al., 2014).

Gap in the Knowledge

While previous studies have tested various types of biochars (from different feedstock) as

soil amendment to the growth of crops, few studies have focused on how pyrolysis temperatures

affect biochar as soil amendment with respect to development of plants.

To date, there are no previous studies comparing the effect of biochar pyrolysis

temperature and its soil amending effects on fruit quality. The work presented in this thesis can

help elucidate the relationship between biochar pyrolysis temperature and the quality of the fruit

grown using the amended soil.

20

Overview of Carbon Nanotubes as Soil Amendment

Carbon Nanotubes

Since their discovery in 1991 (Iijima, 1991, Iijima and Ichihashi, 1993), carbon

nanotubes (CNTs) have remained one of the most interesting nanomaterials due to their unique

mechanical, electrical, thermal, and chemical properties (Eatemadi et al., 2014). Both single-wall

carbon nanotubes (SWCNTs) and multi-wall carbon nanotubes (MWCNTs) are utilized for a

wide range of applications from medical science, aerospace, electronics, and defense industries

(Lacerda et al., 2006, Liu et al., 2006, Wang et al., 2009, Singh, 2010).

Generally, CNTs have a strong tendency to aggregate in aqueous media, which limits

their application and disposal (Maynard et al., 2004). Fortunately, they can be dispersed by

surface modification or dispersant to offset the disadvantages. Due to their wide use, CNTs are

commonly found in the environment. Therefore, environmental occurrence and fate of CNTs

have become the focus of attention of many governments, and public concern has increased

regarding whether the exposure to them may be a potential health concern (Maynard et al.,

2010).

Most CNT studies have been related to tissues of humans and other animals such as

targeted drug delivery, tissue regeneration, and implants (Eatemadi et al., 2014). Success of

application of nanotechnology to these areas has generated interest in introducing

nanotechnological approaches in agricultural and food systems (Sozer and Kokini, 2009).

However, investigations of CNT effects on plants are limited.

Related CNT Studies

The first evidence of positive effects of multi-walled carbon nanotubes (MWCNTs) on

crop plants was reported by Khodakovskaya et al. (2009). By coating tomato seeds with a range

of CNT concentrations from 10 to 40 mg l−1

, they observed that CNTs could penetrate the plant

21

seeds and increase germination rates, and stimulate growth in young seedlings. In another study,

Khodakovskaya et al. (2013) demonstrated that MWCNTs introduced into the soil through

watering, produced a similar quantity of leaves but two-times more flowers and fruits than plants

grown in the control soil. In a study of the molecular mechanisms by which CNTs may act on

plant physiology of tobacco, a correlation between the up-regulation of genes involved in cell

division/cell wall formation and water transport, such as those controlling synthesis of aquaporin,

was found (Khodakovskaya et al., 2012). Aquaporins are crucial for root water uptake, seed

germination, cell elongation, reproduction and photosynthesis (Maurel, 2007). However, it has

also been demonstrated that MWCNTs enhance water uptake by creating new pores in the cell

wall and plasma membrane allowing greater water transport by developing tomato and wheat

seedlings (Liu et al., 2010, Gao et al., 2011). The effect of CNTs on plants was found to be

dependent on the size, concentration, and solubility of the applied CNTs.

Gap in the Knowledge

While these studies gave some insight to CNTs effects as positive growth regulators, all

studies limited their crop analysis to water uptake, yield, and fresh weight. Few studies have

assessed the quality of the fruit produced from the CNT treatments. Therefore, it is important to

assess the effect of these CNT treatments on the overall postharvest quality of the fruit as well as

consumption safety. Hence, there is a need for further investigation on the effects of crop quality

before the application of CNTs can be considered as a widely-applicable means of increasing

crop growth and productivity.

22

Objectives

The main objectives of this work were as follows:

1. To determine and compare the effects of biochar from different pyrolysis temperature

treatments as amendment in sand culture on tomato growth and fruit quality.

2. To determine the effects of carbon nanotubes as amendment into sand culture on tomato

growth and fruit quality.

23

CHAPTER 2

INLFLUENCE OF PYROLYSIS TEMPERATURE OF BIOCHAR SOIL AMENDMENT

EFFECTS ON TOMATO GROWTH AND FRUIT QUALITY

Introduction

Bio-charcoal, or biochar, is no different from commercial charcoal other than its

application is intended for biological and agricultural purposes. Today in agriculture, biochar is

receiving a new interest as a source of renewable bioenergy because: 1) biochar supports

sustainability because it is made from forest and agricultural residues, ideally creating no

competition for land with any other land use option, such as food production, 2) the highly

aromatic structure of biochar is more stable than the organic matter from which it was made,

increasing the long-term soil carbon content and sequestration of carbon due to slower rate of

degradation to CO2 than most other organic matter (Lehmann, 2007, Sohi et al., 2010, Spokas,

2010).

In creating biochar, biomass is heated with little to no available oxygen and at relatively

low temperature (< 1000 °C). This process, called pyrolysis, results in the thermochemical

decomposition of the organic matter, resulting in a porous, the carbon-rich material. However,

not all biochars are created equal. Potential applications of biochar are governed by their

physical properties such as surface area, functional groups, and elemental composition (Downie

et al., 2009). Such physical properties are influenced by biochar production factors such as type

of feedstock, pyrolysis method, and pyrolysis temperature (Sun et al., 2014).

Generally, increasing biochar production temperature, increases surface area (Lehmann

and Joseph, 2015). An in-depth comparison of feedstock types and pyrolysis temperature by Sun

et al. (2014) showed that even amongst different feedstocks, chars made at higher temperature

had larger surface areas. Overall, pyrolysis temperature and feedstock types are the most

important factors in determining biochar properties and thus their application.

24

A key advantage to biochar as soil amendment is its longevity; research indicates that

biochar is recalcitrant and it may endure for hundreds or thousands of years (Seiler and Crutzen,

1980). Use of biochar as a soil amendment dates back to pre-Columbian civilizations in the

Americas incorporating charcoal and fish bones into their soil, creating rich loam such as the

“terra preta” (black earth) soils of the Amazon, which have been found to be nine times more

fertile than the surrounding un-amended soils (Sohi et al., 2010, Schumann, 2012). Biochar’s

benefits to soil have been attributed to increasing soil water-holding capacity, cation exchange

capacity, and nutrient availability (Keith et al., 2011, Quilliam et al., 2012, Liu et al., 2013, Olmo

et al., 2014, Olmo et al., 2016). The highly porous structure of biochar increases the water-

holding capacity of sandy soils, improving the efficiency of water use in agricultural production.

Biochar’s negative charge contributes to a higher cation exchange capacity (CEC), allowing

higher availability of plant mineral nutrients by preventing them from leaching by rain or

irrigation. The largest impact from biochar amendment would likely be seen in sandy soils like

those found throughout Florida that have low CEC(Schumann, 2012).

Increased crop growth and yield is a commonly reported benefit of adding biochar to

soils (Atkinson et al., 2010, Major et al., 2010, Olmo et al., 2014). The effects of biochar on crop

productivity are, however, diverse (Spokas et al., 2012, Biederman and Harpole, 2013). The

employment of biochar as a soil amendment in agriculture systems should result not only in a

yield or growth increase but also in a positive impact on the overall quality and nutritional value

of crops (Petruccelli et al., 2015).

Micro-Tom, a dwarf tomato cultivar (Solanum lycopersicum L.), is known for its small

plant size (Figure 2-1) and rapid growth cycle constituting a convenient model system for

research on tomato development and quality (Sun et al., 2006, Gomez et al., 2009).The quality of

25

fresh tomato fruit is determined by various attributes such as color, firmness, flavor and

nutritional value. High-quality tomato fruit have a firm, turgid appearance with red color that is

uniform and shiny and show no signs of mechanical injury, shriveling, or decay (Sargent and

Moretti, 2014). Color is one of the most important quality attributes that affect tomato

appearance and is greatly correlated with the lycopene content (Brandt et al., 2006) (Helyes et

al., 2006). Tomato firmness is a major factor in consumer acceptability because it is associated

with good eating quality and shelf life (Nunes, 2009). In addition to color and firmness,

measurements of sugars and acidity predict desirable sweet and sour flavor attributes (Baldwin et

al., 1998).

Variation encountered in the sensory and physicochemical characteristics of produce

include pre-harvest factors such as weather, moisture content of the soil, use of growth regulators

and soil fertility (Shewfelt, 1990). It is important to know which pre-harvest factors contribute to

producing fruits with superior quality at harvest while appropriate postharvest handling and

treatment methods should be used to maintain fruit quality after harvest (Arah et al., 2016). Few

reviews have been published regarding the effects of pre-harvest factors, specifically soil

fertility, on postharvest quality attributes such as vitamin C (Lee and Kader, 2000), sugars

(Beckles, 2012), appearance (Kays, 1999), and texture (Sams, 1999) along with overall quality

attributes (Weston and Barth, 1997). Arah et al. (2016) reviewed soil nutrient factors that affect

tomatoes specifically and concluded that postharvest quality status of tomatoes partly depended

on such pre-harvest practices such as soil amendments carried out during production.

Success of biochar application in most published studies has limited their definition as

either increased yield or above-ground biomass. However, very little is known about biochar

effects on nutritional attributes in fruits and vegetables (Schmidt et al., 2014). Furthermore,

26

studies of biochar’s effects on fruit quality are limited, particularly on tomatoes. Petruccelli et al.

(2015) reported that tomatoes (cv. Rio Grande) grown in substrate amended with biochars made

from straw and olive residues produced at 1200°C had higher secondary metabolites, phenolic

compounds, and lycopene compared to those grown in a wheat straw biochar amended soil. A

mix of rice husk and cotton seed biochar produced through pyrolysis at 400 °C was also shown

to enhance soil water holding capacity under reduced irrigation and also produced fruit with

quality comparable to that of tomatoes grown under full irrigation (Akhtar et al., 2014).While

previous studies have tested various types of biochars (from different feedstock) as soil

amendment to the growth of crops, few studies have focused on how pyrolysis temperatures

affect biochar as soil amendment with respect to development of plants. Furthermore, there are

no studies comparing the effect of soil amendment effects biochar pyrolysis temperature on fruit

quality. The overarching objective of this work thus was to determine and compare the effects of

biochars from different pyrolysis temperatures as soil amendment on tomato growth and quality.

Greenhouse growth studies and laboratory analysis of resulting fruit quality were conducted to

accomplish the objective.

Materials and Methods

Biochar Production and Characterization

Bamboo feedstock sourced locally (Gainesville, FL) was dried and pyrolyzed at

temperatures of 300°C, 450°C, and 600°C using a furnace apparatus (Bartlett 3K-CF, Fort

Madison, IA ) (Figure 2-2). The resulting biochars were used in their pristine form after ground

into fine particles of size smaller than 0.45 mm. The bulk density of the biochars was

approximately 0.29 g/cm3.

Major inorganic elements of the biochars were determined by acid digestion of the

samples followed by inductively-coupled plasma atomic emission spectroscopic (ICP-AES)

27

analysis. The surface areas of the biochars were determined using the Brunauer–Emmett–Teller

(BET) method on Quantachrome Autosorb1 at 77 K in the 0.01–0.3 relative pressure range of the

N2 adsorption isotherm.

Plant Growth

Preparation of growth medium

Coarse white sand was used as a main medium component. Sand was washed with tap

water five times and finally with deionized water and dried at 80 oC for one month. Small pots

(bottom and top diameters of 7 and 9.5 cm, a height of 7.5 cm, and a volume of 300 ml) were

filled with coarse sand (up to a volume of 270 ml; the mass of coarse sand at this volume is 435

g) that was pre-mixed (on a mass basis) with 0% (control), 1% or 3% of each biochar type

(300°C, 450°C and 600°C), respectively (Figure 2-3). Each pot was considered as an

experimental unit, and there were either 15 or 10 replicate pots per treatment in different

replicated experiments.

The resulting treatments will be henceforth referred to as: 300 Low (1% biochar), 300

High (3% biochar), 450 Low, 450 High, 600 Low and 600 High, and control (0% biochar).

Seed germination and growth

Micro-Tom tomato seeds were purchased from Total Tomatoes (Randolph, WI) and

germinated in 72-cell plug trays with a soilless substrate. Two weeks after germination, seedlings

were transplanted singly into the aforementioned pots.

All plants were grown in a greenhouse at the Mid-Florida Research and Education Center

(MREC) in Apopka, FL. The light intensity of the greenhouse was 2000 μm-2

s-1

temperature

ranged from 20-30 ˚C and relative humidity of 50% to 100%. A solution containing 200 mg/L N

was prepared weekly using Peters Professional 20-20-20 General Purpose fertilizer (Scotts,

Maryville, OH). Plants were fertigated (i.e., both fertilization and irrigation occur at the same

28

time) with the solution once or twice a week depending on plant size.Two biochar experiments

were conducted, which are hereafter referred to as Cycles 1 and 2:

Cycle 1 was transplanted on December 7 and treatments 300 Low, 600 Low and 600

High were harvested on February 5, 2016 and the remaining treatments were harvested on

February 12, 2016. Cycle 2 was transplanted on March 29, 2015 and the first harvest of all

treatments was done on June 2, 2016 which will be referred to as Cycle 2 Harvest 1. A second

harvest of Cycle 2 (Cycle 2 Harvest 2) was conducted to achieve a second replicate; however,

the plants underwent a greenhouse temperature and humidity malfunction resulting in exposure

to high temperatures and low humidity. Some fruit harvested showed signs of immaturity and

splitting and these fruits were not used for quality analysis.

To monitor plant growth, canopy height, widest width, and width perpendicular to the

widest width of each plant were recorded, The three measurements were multiplied to give the

canopy volume, commonly called plant growth index (GI) as described by (Chen and Beeson Jr,

2013).

Leachate collection and analysis

Three plants from each treatment were randomly selected for leachate collection using

the pour through method (Yeager et al., 1983). Deionized water (DI) was applied to the plant

until approximately a 10 mL leachate volume was collected.

Elemental Mg, Ca and P in the leachate were quantified by acid digestion of the samples

followed by inductively-coupled plasma atomic emission spectroscopic (ICP-AES)

analysis.NO3-N in the leachate was determined using the AQ2 Discrete Analyzer (Seal

Analytical Inc., Mequon WI).

29

Yield

Approximately after 9 weeks of transplanting, full red tomato fruit were harvested from

each plant and weighed. Yield was assessed by counting the total number of full red fruit

harvested per plant.

Fruit Quality

Physical analysis

Color. A total of two color measurements were taken on the opposite sides of the fruit at

the equatorial region of each tomato that was large enough to cover the 8 mm aperture of the

colorimeter. A hand-held tristimulus reflectance colorimeter (Model CR-400, Minolta Co., Ltd.,

Osaka, Japan) equipped with a glass light-projection tube (CR-A33f, Minolta Co., Ltd., Osaka,

Japan) was used. The color was recorded using the CIE-L*a*b* uniform color space (CIE-Lab),

L* (lightness), a* (redness), and b* (yellowness) values. Numerical values of a* and b* were

converted into hue angle using the Konica Minolta CR-400 Utility software CR-S4w (2002-2010

Konica Minolta Sensing, Inc., Osaka, Japan).

Texture. Firmness was measured using a TA.XT Plus Texture Analyzer fitted with a 35

mm flat compression plate and equipped with a 50-kg load cell. A tomato fruit was placed stem

end down on the flat surface of the texture analyzer, thus applying pressure on the blossom-end

of the fruit. The plate was then driven with a crosshead speed of 1 mm s−1, and the compression

force was recorded at 3 mm deformation. The force required to compress the fruit by 3 mm was

recorded in kgf and then converted to Newton (N = kgf x 9.8).

Compositional analysis

Samples intended for compositional measurements were homogenized on the day of

harvest, and kept frozen at −30 °C in air-tight plastic bags until analysis.

30

Ascorbic acid. Total ascorbic acid (AA) was quantified using the method described in

(Nunes, 2015). Tomato tissue was homogenized and 4 g of homogenate was mixed with a 20 mL

metaphosphoric acid mixture (6% HPO3 containing 2 N acetic acid). Samples were then filtered

(0.22 μm) prior to HPLC analysis. Ascorbic acid analysis was conducted using a Hitachi

LaChromUltra UHPLC system with a diode array detector and a LaChromUltra C18 2 μm

column (2 × 50 mm) (Hitachi, Ltd., Tokyo, Japan). The analysis was performed under isocratic

mode at a flow rate of 0.5 mL/min with a detection of 254 nm. Sample injection volume was 5

μL, each with duplicate HPLC injections. Mobile phase was buffered potassium phosphate

monobasic (KH2PO4, 0.5%, w/v) at pH 2.5 with metaphosphoric acid (HPO3, 0.1%, w/v). The

retention time of the ascorbic acid peak was 2.5 min. Peak was identified after comparison of

retention time with the ascorbic acid standard. Total ascorbic acid content was expressed in

terms of fresh weight (mg AA 100 g−1

).

Individual sugars. Individual sugar analysis was conducted using a Hitachi HPLC

system with a refractive index detector and a 300 mm × 8 mm Shodex SP0810 column (Shodex,

Colorado Springs, CO) with a SP-G guard column (2 mm x 4 mm). Aliquots of 4 grams fruit

puree were mixed thoroughly with 4 mL ultrapure water, vortexed, and centrifuged at 1,600 gn

for 20 min. Supernatant was decanted and filtered through 0.45 µm nylon syringe filter. Sample

injection volume was 5 µL, each with duplicate HPLC injections. Isocratic solvent delivery of

water was set at 1.0 mL∙min–1. Sample injection volume was 5 µL. Standard solutions of

sucrose, glucose, and fructose (Fisher Scientific Company, Pittsburgh, PA) were used to identify

sample peaks. The peaks were identified by comparing retention times with those of the

standards. The amount of total sugars in tomato was quantified using calibration curves obtained

from different concentrations (2, 4, 6, 10, and 20 mg∙mL–1) of sucrose, glucose, and fructose

31

standards. Three samples per treatment (2 g fruit puree) were used, each with duplicate HPLC

injections. Total sugar and individual sugar contents were expressed in g·100 g–1 fresh weight.

Titratable acidity and soluble solids content. The supernatants of thawed and filtered

homogenates were analyzed for titratable acidity (TA), and soluble solids content (SSC). For TA

6.0 g aliquots of the supernatant were diluted with 50 ml distilled water and titrated with 0.1 N

NaOH to an end point of pH 8.2 with an automatic titrimeter. Results were converted to percent

citric acid using the method described in (Nunes and Delgado, 2014). The SSC of the

supernatant was determined with a digital ATAGO PR-101 refractometer with a 0% to 45%

range (Atago, Tokyo, Japan).

Statistical Analysis

Data were analyzed by analysis of variance (ANOVA) and Duncan’s multiple range tests

with the package ‘agricolae’ (de Mendiburu and de Mendiburu, 2015) using R Version 3.1.1.

Significant differences between treatments were determined at the 95% confidence level (p ≤

0.05). All harvests showed significantly different effects for all treatments evaluated, and were

therefore analyzed separately.

All figures show means treatment means ranked from highest to lowest, with means

assigned the same letter having no significant difference.

Results and Discussion

It is well known that fruit quality and composition varies greatly among different origins

and cultivation practices (Kays, 1991). There were many experimental variables that were not

able to be controlled during the plant growth experiments such as pest control, weather and

mechanical difficulties during all both growth cycles. From all experiments, Cycle 2-Harvest 1

had the least complications, most complete data collection and adequate yield for quality

analysis; therefore it will be the focus of the discussion.

32

Biochar Properties

As expected, biochars made at higher temperatures had larger surface areas and pore

volumes (Table 2-1). Biochar made at 600 °C showed highest surface areas while biochar made

at 300 °C had the lowest surface area. When increasing biochar production temperature, the

surface area increased which is in agreement with the findings of previous studies that showed

that temperature can highly affect biochars’ surface area (Joseph and Lehmann, 2009). Surface

area is one of the most important factors that influence biochars functionality, as larger surface

area means more porous structures within biochar (Inyang et al., 2010, Yao et al., 2011). The

CO2 surface area indicates presence of micropores (< 1.5 nm diameter) while the N2 surface area

is indicative of mesopores (< 50 nm diameter) which was not as abundant in the chars. Both

macro- and micropores, inherited from the architecture of the feedstock, can hold air or water,

greatly influencing the water holding capacity and reducing the bulk density of the entire biochar

particle (Downie et al., 2009). Because the density of biochar is lower than that of some

minerals, biochar application can also change soil bulk density (Major et al., 2010); with possible

effects on soil water relations, rooting patterns and soil fauna (Lehmann et al., 2011).

It is also noted that the surfaces of low temperature biochar can be hydrophobic, and this

may limit the capacity to store water in soil (Sohi et al., 2010). The biochar formed at high

temperature is more brittle compared to biochar formed at lower temperature, being prone to

abrade into fine fractions once incorporated into the mineral soil due to its fragile structure

(Claoston et al., 2014). It may be proposed that the surface area over the long term of weathered

biochar is not greatly affected by this parameter (Sohi et al., 2010).

Elemental analysis of the biochars revealed the concentration of key minerals that may

affect plant growth as an amendment (Table-2-1). Also as anticipated, higher temperature

biochars contained higher concentrations of elemental components (Yao et al., 2011, Sun et al.,

33

2014). Statistical analysis (not shown) showed no significant difference between mineral

compositions amongst different temperature chars. However, the 450 °C biochar showed the

highest concentration of potassium (K). It has been shown that an adequate supply of potassium

in tomato production improves fruit color and enhances the titratable acidity of the fruit (Passam

et al., 2007) which in the present study supports the fruit quality findings (discussed below).

The pH of the biochars ranged from 5.2-7.9, which are within the range of values

reported in the literature (Yao et al., 2011, Sun et al., 2014). The BB 450 had the most acidic pH

at 5.2, BB 600 had the most basic at 7.9 and lowest temperature biochar of BB 300 had a pH of

6.7 (Table 2-1).

All the three bamboo biochars had relatively high Cation Exchange Capacity (CEC), but

low anion exchange ability (Table 2-1). The CEC values of previously made bamboo biochars

were determined in a previous study (unpublished data from the environmental nanotechnology

lab at UF) showing higher temperature biochars having higher CEC. However, it is difficult to

accurately measure the CEC of biochar (Mukherjee et al., 2011). While “fresh” biochar does not

have a very high CEC, it is still higher than weathered sandy tropical soils, and the CEC

increases over time in soil (Glaser et al., 2002, Lehmann, 2007). It is important to stress that

CEC of biochar surfaces develops over time and varies greatly according to the soil, but the role

of feedstock and production parameters in determining initial and ultimate activity of surfaces

are key factors (Sohi et al., 2010). High CEC is essential for retaining plant nutrients in the soil

media available for the plant; thereby reducing leaching.

Gaskin et al. (2008) compared 400 and 500 °C chars of three different feedstocks (peanut

hull, pine chip and poultry litter) resulting in the lower temperature biochar having CEC possibly

due to presence of acidic functional groups. Chun et al. (2004) found decreasing acidity with

34

increasing pyrolysis temperature. Furthermore, Guo and Rockstraw (2007) found decreasing

number of acidic functional groups with increasing temperature; with the highest decrease

occurring between 300°C and 400°C, slowing loss of these acidic groups after 400°C. Tsutomu

et al. (2004) indicated that lignin and cellulose undergo thermolysis at 400°C to 500°C, which

creates acidic functional groups such as carboxyls and phenolic hydroxyls. This could be the

case for the bamboo feedstock that was used in this study.

Overall the significance of temperature suggests that biochar created at low temperature

may be suitable for controlling release of fertilizer nutrients (Day et al., 2005), while high

temperatures would lead to a material analogous to activated carbon (Ogawa et al., 2006).

Plant Growth

Growth index (GI) values showed that all biochar-treated plants had significantly larger

GI than the control. A more significant difference in GI was seen at the later stage measurements

at week 7 (Figure 2-4). Particularly, the lower temperature biochar treatments of 450 Low and

300 High had the largest growth index, nearly twice that of the control treatment.

Application of biochar is suggested to increase plant growth by supplying elemental

nutrients to growing plants, and improve water and nutrient holding capacity (Akhtar et al., 2014,

Glaser et al., 2002), and increased CEC ultimately preventing leaching of nutrients. Such

combined characteristics improve soil fertility and plant growth (Lehmann et al., 2003, Graber et

al., 2012, Laird et al., 2010). As previously mentioned, at low pyrolysis temperatures (< 500°C),

feedstock composition has the greatest effect on characteristics of biochar that impact

agricultural productivity such as nutrient content and possibly CEC (Gaskin et al., 2008).

High surface area biochar has been shown to decrease the efficacy of herbicide

application due to sorption (Graber et al., 2012). Specifically, surface area and pore volume may

change upon contact with soil by pores clogging from sorbed organic (Pignatello et al., 2006)

35

and mineral material (Joseph et al., 2010) or, conversely, possibly by mineralization of volatile

matter that may be blocking pores. These properties have shown to change sorption behavior of

mineral (Liang et al., 2006) and organic matter (Kasozi et al., 2010) which in turn may influence

nutrients available for plant uptake.

Yield was recorded for both cycles and results from all three harvests are shown in Figure

2-5. There was no trend amongst the treatments in regards to yield. Similarly, (Vaccari et al.,

2015) reported that biochar stimulates plant growth but not tomato fruit yield of tomato. While a

mean yield increase of 10% has been reported, averaging different crops, soils and climates

through meta-analysis by (Jeffery et al., 2011) it is also well known that all biochars are not

created equal and therefore the site specific and biochar specific effects must also be considered

(Mukherjee and Lal, 2014).

Plant leachate was analyzed for concentrations of elements Mg, Ca, P and NO3 which

showed no trends amongst treatments or growth cycle duration (Figure 2-6). Levels of

phosphorous in leachate showed the most dramatic decrease throughout the duration of the cycle,

and were significantly higher in lower temperature 300˚C biochar treatments during the

beginning of the growth cycle. However, some studies have shown that the variation of

phosphorus supply in soils for growing tomato crops does not influence quality traits such as the

total soluble solids (Senevirathna and Daundasekera, 2010), pH, acidity of the tomato juice, or

the fruit color characteristics (Oke et al., 2005).

Another possible mechanism for variance in plant growth promotion is the possible

production of ethylene from the biochar amendment. Ethylene is part of the remaining non-

aromatic compounds in fresh biochars and, recent studies by (Spokas et al., 2010) showed that

ethylene is in fact generated by fresh biochars. Ethylene’s negative hormonal impact on plant

36

growth and development, and positive impact on fruit abscission and senescence has been well

established (Abeles et al., 1992, Frankenberger Jr and Arshad, 1995, Arshad and Frankenberger

Jr, 2012). A study by (Fulton et al., 2013) suggested that, in order to reduce ethylene release and

its adverse effects on plants, biochar should be stored in an open environment for 90 days prior

to use in nursey plants. Although not measured in this study, the likely presence of ethylene in

the biochar used could have affected plant growth, and as discussed below, some fruit quality

attributes. The evolution of ethylene from biochar and its role on root growth remains an

interesting mechanism to be investigated.

Fruit Quality

To date, information on the effects of biochar on the tomato yield and quality are still

scarce (Vaccari et al., 2015). Color and firmness of tomatoes are important quality attributes

because they are highly correlated with sensory attributes such as taste and aroma (Resurreccion

and Shewfelt, 1985). For simplicity, only the L* (lightness), a* (redness) and hue angle are

reported. Throughout Cycle 2, fruit from higher temperature biochar treatments, specifically 450

and 600, were darker (lower L* values) and more red (higher a* values) in colorthan the fruit

from other treatments (Figure 2-5). Hue angle of full red tomato is generally within the range of

30° to 50°, with lower values indicating deeper red or purple color and higher values indicating

more orange color (Perkins-Veazie et al., 2007). In this study, it can be assumed that fruits from

all treatments were roughly at the same maturity because the hue angle (Figure 2-6) was not

significantly different among treatments. Increased a* values was shown to be directly associated

with lycopene synthesis, whereas a*/b* ratio has been reported to be a good indicator of

lycopene content and therefore could be used to characterize tomato ripeness stage (Arias et al.,

2000, Helyes et al., 2006).

37

Among the biochar treatments, fruit from the 450˚C treatments were firmer (Figure 2-6).

However, none of the biochar treatments showed statistically significant difference from the

control. Tomato firmness is an important quality factor because it is associated with good eating

quality and longer postharvest life (Nunes, 2009). Appearance of the fruit were qualitatively

evaluated for differences size and locular structure and seed population and showed no

significant differences amongst treatments (Figure 2-7). Petruccelli et al. (2015) evaluated

effects of three different types of biochars (wheat straw biochar, poplar biochar and olive

residues biochar) at concentrations of 10% and 20% (w/w) on tomato growth, and also found no

significant differences in fruit size and weight amongst treatments.

Reducing sugars such as glucose and fructose are the major components of the SSC with

the remainder consisting of organic acids, lipids, minerals and pigments. In both Cycle 2

harvests, SSC and sugar levels (fructose and glucose) of biochar-treated fruit were significantly

higher than the control (Figures 2-8 and 2-9). The values are in agreement with literature as SSC

of mature tomato ranges from 4.5 to 8.5% of its fresh weight (Pedro and Ferreira, 2005).

The acids measured in the form of TA and AA (Figures 2-9 and 2-10) showed that fruits

treated with lower temperature biochars were inclined to have higher acidity and AA levels than

the other treatments.

As tomatoes mature on the plant, sugar-to-acid ratio increases, resulting in higher soluble

solids and lower acidity (Nunes, 2009). The sugar to acid ratio did not change significantly with

respect to the treatments indicating that they were roughly all the same stage of maturity (Figures

2-11 and 2-12).

In a previous study, Petruccelli et al. (2015) also showed that the type of feedstock

influenced the biochar soil amendment and found that tomatoes (cv. Rio Grande) grown in

38

substrate amended with biochars made from straw and olive residues produced at 1200°C had

higher secondary metabolites, phenolic compounds, and lycopene compared to those grown in a

poplar biochar amended soil.

Conclusions

Biochar has been described as a possible means to sustainably improve soil fertility and

sequester carbon (C) to mitigate climate change. This is the first study in which temperature of

biochar production and its effect on fruit quality cultivated in amended soil were compared.

Temperature optimization of biochar production is important to produce biochar with desirable

properties efficiently at lowest possible temperature. Biochar can improve the acidity of the soil,

increasing the CEC, and therefore nutrient and water holding capacity. The objective was to

evaluate the effect of different pyrolysis temperatures of bamboo biochar on plant growth and

nutritional quality. Overall, the lower temperature biochars, 450 ˚C and 300 ˚C, had most

significant effect on plant growth and positive effect on fruit quality. These results are not as

anticipated since higher temperature biochars have higher surface area and nutrient content.

Since there are no previous studies comparing effect of biochar pyrolysis temperature on plant

growth and fruit quality, the mechanism is not well understood. According to other studies,

possible reasons for plant growth being positively affected by the lower temperature biochar

could be due to increased CEC and lower surface area for sorption. The lower temperature

biochar treatments produced firmer fruit with darker red color, higher sugars and acids; also

higher nutritional value indicated by higher content of vitamin C. The superior quality of these

fruit can be attributed to the treatments and not to difference in maturity as the sugar to acid

ratios and hue angles amongst the treatments did not vary significantly, indicating that the fruit

were at similar maturity stage. Thus, biochar may have resulted in a modification of maturation,

39

possibly increase in lycopene synthesis, and an effect on ripening physiology (hormonal

regulation) may be considered.

Future work. Further characterization of biochar functional groups can be done through

FTIR to quantify these acidic functional groups that influence the soil fertility. Also, because

feedstock has a strong influence on biochar properties, other types of feedstock such as hard

woods, grasses, or crop residues can be used instead of bamboo. Because this study was

conducted in a greenhouse under relatively controlled growing conditions, the effects of biochar

may vary under commercial field conditions where there is a complex interplay between the soil,

climate, and other factors. Further studies are needed to understand plant and soil responses to

biochar and to develop recommended rates for particular biochars in different soils. Lastly, a

shelf-life study can be done to further assess the influence on the pre-harvest quality of the crop.

40

Figure 2-1. Micro-Tom cultivar is known for its small plant height and fast growth cycle.

(Photos courtesy of author, Ratna Suthar)

Figure 2-2. Locally sourced bamboo feedstock was dried and cut into 10 inch pieces and

pyrolysed at temperatures of 300, 450 and 600C in a large kiln. (Photos courtesy of

author, Ratna Suthar)

41

Figure 2-3. Pots prepared with different concentrations of biochar in sand media. (Photos

courtesy of author, Ratna Suthar)

42

Figure 2-4. Growth Index (cm

2) of Micro-Tom plants at week 3 (above) and week 7 (below) of

all biochar treatments. Bars represent standard error (n = ~15)

43

Figure 2-5. Yield of average number of full red fruits obtained per plant from each treatment

from harvests during Cycle 1 (2/5/16) and Cycle 2 Harvest 1 (6/2/16) and Harvest 2

(6/9/16)

44

Figure 2-6. Concentrations (mg/L) of Ca, Mg, P and NO3 in plant leachate samples measured at

weeks 5, 8 and 10 during cycle 1 and week 10 in cycle 2. Means assigned same letter

groups are not significantly different.

Mg P NO3

Groups, Treatments and means Groups, Treatments and means Groups, Treatments and means

a 3H 53.84 a 3H 219.8 a Control 298.9

a 4H 51.92 ab 6H 202.0 ab 450 High 287.3

ab 6H 43.56 ab 4H 201.7 ab 300 High 263.9

ab 3L 41.08 abc 3L 186.7 ab 600 High 224.7

bc 4L 32.82 bc Control 168.6 ab 300 Low 209.9

bc Control 30.86 c 4L 142.2 ab 450 Low 206.1

c 6L 26.96 c 6L 140.7 b 600 Low 148.7

Mg P NO3

Groups, Treatments and means Groups, Treatments and means Groups, Treatments and means

a Control 39.59 a 3H 160.3 a 450 Low 204

ab 3H 30.39 ab 4H 144.9 ab Control 100.3

ab 4L 29.65 abc 3L 123.9 ab 600 Low 99.26

ab 3L 29.19 abcd 6H 113.5 ab 300 High 94.31

ab 6L 27.74 bcd 6L 88.03 ab 300 Low 92.62

b 4H 15.72 cd 4L 69.85 ab 600 High 42.47

b 6H 14.70 d Control 59.01 b 450 High 21.73

Mg P NO3

Groups, Treatments and means Groups, Treatments and means Groups, Treatments and means

a 3H 50.18 a 4L 91.59 a 300 High 171.3

ab 4H 43.48 a 3L 88.08 ab 300 Low 150

abc 6H 37.98 a 3H 87.99 b 450 High 139.5

bcd 3L 31.20 a 6L 86.21 c 600 High 110.2

cd 6L 23.91 a Control 78.30 cd 600 Low 90.74

d 4L 17.04 ab 6H 34.53 cd 450 Low 86.12

d Control 14.19 b 4H 21.53 d Control 66.4

Mg P NO3

Groups, Treatments and means Groups, Treatments and means Groups, Treatments and means

a 3L 17.98 a 3L 33.40 a 450 Low 17.36

a 4H 17.65 a 6H 32.47 a 450 High 14.8

a Control 17.64 a 3H 29.68 a 300 Low 14.23

a 6H 17.36 a 4H 28.54 a Control 10.89

a 4L 16.11 a 6L 27.55 a 600 High 10.57

a 3H 14.74 a Control 23.31 a 300 High 9.484

a 6L 14.51 a 4L 21.15 a 600 Low 8.683

a 6L 23.37

a 3H 20.51

Groups, Treatments and means

Groups, Treatments and means

Groups, Treatments and means

Groups, Treatments and means

a 3L 27.36

a Control 27.17

a 4L 26.44

a 4H 26.41

a 6H 26.00

a Control 28.60

a 6H 24.60

a 4H 22.70

c Control 22.46

c 4L 27.10

Ca

a 4H 71.21

ab 6H 57.16

ab 3L 54.40

bc 4L 41.68

bc Control 39.70

c 6L 33.09

a 3H 48.54

a 4L 48.14

a 6L 45.18

a 3L 41.81

bc 6L 40.29

bc 3L 40.72

ab 4H 57.06

a 3H 60.98

a 6H 61.43

Cycle 1 week 5

Cycle 1 week 8

Cycle 1 week 10

Cycle 2 week 9

a 3H 72.63

Ca

Ca

Ca

45

Figure 2-7. Color coordinates, a* and L* values, of tomatoes cultivated in standard (control) and

biochar amended media. Bars represent standard error (n = ~30).

46

Figure 2-8. Hue angle values and firmness of tomatoes cultivated in standard (control) biochar

amended media. Bars represent standard error (n = ~30).

47

Figure 2-9. Whole and cut appearance of fruit from Cycle 2 Harvest 1. (Photos courtesy of

author, Ratna Suthar)

Figure 2-10. Fructose and glucose contents of tomatoes cultivated in standard (control) and

biochar amended media. Bars represent standard error (n = 6)

48

Figure 2-11. Soluble solids content (SSC) on the left and titratable acidity (TA) on the right for

tomatoes cultivated in standard (control) and biochar amended media. Bars represent

standard error (n = 3). (There was not adequate tissue of Cycle 1 600 High treated

fruits for TA.)

49

Figure 2-12. AA value of tomatoes cultivated in standard (control) biochar amended media. Bars

represent standard error (n = 6)

50

Figure 2-13. Sugar to acid ratio of (soluble solids content to titratable acidity) all three growth

cycles of control and CNT treated tomatoes.

Figure 2-14. Statistical analysis of sugar to acid ratio for Cycle 2 Harvest 1 data shown in figure

2-13.

51

Table 2-1. CO2 and N2 Surface Area and Pore Volume Comparative Cation and Anion Exchange Capacities and pH of Bamboo

Biochar (BB) Produced at 300°C, 450°C or 600°C

Material

N2

Surface

Area

CO2

Surface

Area

CO2

Pore

volume

CEC AEC pH Elemental Composition (% mass based)

m2 g-1 m2 g-1 mL g−1

mmol

100g−1

mmol

100g−1 Ca Fe K Mg Mn Na P Zn

BB 300 °C ~0 178.8 0.053 30.3 1.13 6.7 0.135 0.033 0.794 0.131 0.037 0.248 0.675 0.026

BB 450 °C 0 330.9 0.092 41.4 1.15 5.2 0.146 0.047 1.067 0.158 0.048 0.48 0.925 0.025

BB 600 °C 247.2 493.4 0.132 73.1 ~0 7.9 0.158 0.057 1.022 0.182 0.047 0.552 0.948 0.033

52

CHAPTER 3

EFFECT OF CARBON NANOTUBES AS SOIL AMENDMENT IN SAND CULTURE ON

TOMATO GROWTH AND QUALITY

Introduction

Since their discovery in 1991, carbon nanotubes (CNTs) have remained one of the most

interesting nanomaterials due to their unique mechanical, electrical, thermal, and chemical

properties (Eatemadi et al., 2014). Both single-wall carbon nanotubes (SWCNTs) and multi-wall

carbon nanotubes (MWCNTs) are utilized for a wide range of applications from medical science,

aerospace, electronics, and defense industries (Lacerda et al., 2006, Liu et al., 2006, Wang et al.,

2009, Singh, 2010).

Generally, CNTs have a strong tendency to aggregate in aqueous media, which limits

their application and disposal (Maynard et al., 2004). Fortunately, they can be dispersed by

surface modification or dispersant to offset the disadvantages. Due to their wide use, CNTs are

commonly found in the environment. Therefore, environmental occurrence and fate of CNTs

have become the focus of attention of many governments, and public concern has increased

regarding whether the exposure to CNTs may be a potential health concern (Maynard et al.,

2010).

Most CNT studies have been related to tissues of humans and other animals such as

targeted drug delivery, tissue regeneration, and implants (Eatemadi et al., 2014). Success of

application of nanotechnology to these areas has generated interest in introducing

nanotechnological approaches in agricultural and food systems (Sozer and Kokini, 2009).

However, investigations of CNT effects on plants are limited.

A convenient model system for research on tomato development and quality is the drawf

tomato cultivar Micro-Tom (Solanum lycopersicum L.), because of its rapid growth cycle and

small plant size (Figure 2-1) (Sun et al., 2006, Gomez et al., 2009). Quality of fresh tomato fruit

53

is determined by various attributes such as color, firmness, flavor and nutritional value. Color of

tomato appearance is a key quality attributes that is greatly correlated with the lycopene content

(Brandt et al., 2006) (Helyes et al., 2006). In addition to firmness, which is associated with good

eating quality and shelf life, measurements of sugars and acidity predict desirable sweet and sour

flavor attributes (Nunes, 2009, Baldwin et al., 1998).

Variation encountered in the sensory and physicochemical characteristics of produce

include pre-harvest factors such as weather, soil fertility, moisture content of the soil, use of

growth regulators and other cultural practices (Shewfelt, 1990). It is important to know the pre-

harvest factors that contribute to producing fruits with superior quality at harvest while

appropriate postharvest handling and treatment methods should be used to maintain fruit quality

after harvest (Arah et al., 2016). Few reviews have been published regarding the effects of pre-

harvest factors, specifically soil fertility, on postharvest quality attributes such as vitamin C (Lee

and Kader, 2000), sugars (Beckles, 2012), appearance (Kays, 1999) , and texture (Sams, 1999)

along with overall quality attributes (Weston and Barth, 1997). Arah et al. (2016) reviewed soil

nutrient factors that affect tomatoes specifically and concluded that postharvest quality of

tomatoes partly depended on pre-harvest practices, such as soil amendment, carried out during

production.

The first evidence of positive effects of multi-walled carbon nanotubes (MWCNTs) on

crop plants was reported by Khodakovskaya et al. (2009). By coating tomato seeds with a range

of CNT concentrations from 10 to 40 mg l−1

, they observed that CNTs could penetrate the plant

seeds and increase germination rates, and stimulate growth in young seedlings. In another study,

Khodakovskaya et al. (2013) demonstrated that MWCNTs introduced into the soil through

watering, produced a similar quantity of leaves but two-times more flowers and fruits than plants

54

grown in the control soil. In a study of the molecular mechanisms by which CNTs may act on

plant physiology of tobacco, a correlation between the up-regulation of genes involved in cell

division/cell wall formation and water transport, such as those controlling synthesis of aquaporin,

was found (Khodakovskaya et al., 2012). Aquaporins are crucial for root water uptake, seed

germination, cell elongation, reproduction and photosynthesis (Maurel, 2007). However, it has

also been demonstrated that MWCNTs enhance water uptake by creating new pores in the cell

wall and plasma membrane allowing greater water transport by developing tomato and wheat

seedlings (Liu et al., 2010, Gao et al., 2011). The effect of CNTs on plants was found to be

dependent on the size, concentration, and solubility of the applied CNTs.

While these studies gave insight to CNTs affects as positive growth regulators, they

limited their analysis to crop yield and fresh weight. No studies have assessed the nutritional

quality of the fruit produced from this treatment. The documented ability of plants to uptake

carbon nanotubes from soil and accumulate these nano-particles in reproductive organs also

raises questions in regard to their effect on the fruit (Cañas et al., 2008, Chen et al., 2015). It is

important to assess the effect of CNT treatments on the postharvest quality of the fruit for safety

and consumption. Hence, there is a need for further investigation on the effects of crop quality

before the application of CNTs can be considered as a widely-applicable means of increasing

crop growth and productivity.

Materials and Methods

Carbon Nanotubes

Multi-wall carbon nanotubes (CNT) were purchased from Dykjchina (dykjchina.com).

The CNTs have an inner and outer diameter of 20 nm and 50 nm, respectively. The length of

CNTs is in the range of 10-30 microns with conductivity greater than 100 s/cm and specific

surface area of 110 m2/g. The surface areas of the CNTs were determined using the Brunauer–

55

Emmett–Teller (BET) method on Quantachrome Autosorb1 at 77 K in the 0.01–0.3 relative

pressure range of the N2 adsorption isotherm.

Plant Growth

Preparation of growth medium

Coarse white sand was used as a main medium component. Sand washed with tap water

five times with deionized water and finally dried at 80 oC for one month. Small pots (bottom and

top diameters of 7 and 9.5 cm, a height of 7.5 cm, and a volume of 300 ml) were filled with

coarse white sand (up to a volume of 270 ml, the mass of coarse sand at this volume is 435 g)

that was pre-mixed (wt/wt) with 0.5%, 1% and 3% CNT (Figure 3-1). Each pot was considered

as an experimental unit, and there were 15 replicates (pots) per treatment for Cycle 2 and 10

replications per treatment for Cycle 3.

The resulting treatments will be henceforth referred to as: 3% CNT, 1% CNT and 0.5%

CNT and Control.

Seed germination and growth

Micro-Tom tomato seeds were purchased from Total Tomatoes (Randolph, WI) and

germinated in 72-cell plug trays with a soilless substrate. Two weeks after germination, seedlings

were transplanted singly into the aforementioned pots.

All plants were grown in a greenhouse at the Mid-Florida Research and Education Center

(MREC) in Apopka, FL. The light intensity of the greenhouse was 2000 1 m-2 and temperature

ranged from 20-30 ˚C and relative humidity of 50% to 100%. A solution containing 200 mg/L N

was prepared weekly using Peters Professional 20-20-20 General Purpose fertilizer (Scotts,

Maryville, OH). Plants were fertigated (both fertilization and irrigation occur at the same time)

with the solution once or twice a week depending on plant size.

56

Two biochar experiments were conducted, which are hereafter referred to as Cycles 1 and

2: Cycle 1 was transplanted on December 7 and treatments 300 Low, 600 Low and 600 High

were harvested on February 5, 2016 and the remaining treatments were harvested on February

12, 2016.

Two harvests were done of the same cycle that was transplanted on March 29, 2015. The

first harvest of all the treatments was done on June 2, 2016. A second harvest of Cycle 2 was

conducted to achieve a second replicate; however, the plants underwent a greenhouse

temperature and humidity malfunction resulting in exposure to high temperatures and low

humidity. Some fruit harvested showed signs of immaturity and splitting and these fruits were

not used for quality analysis.

To monitor plant growth, canopy height, widest width, and width perpendicular to the

widest width of each plant were recorded, The three measurements were multiplied to give the

canopy volume, commonly called plant growth index (GI) as described by (Chen and Beeson Jr,

2013).

Leachate collection and analysis

Three representative plants from each treatment were selected for leachate collection

using the pour through method (Yeager et al., 1983). Deionized water (DI) was applied to the

plant until approximately a 10 mL leachate volume was collected.

Elemental Mg, Ca and P in the leachate were quantified by acid digestion of the samples

followed by inductively-coupled plasma atomic emission spectroscopic (ICP-AES) analysis.

Total N in the leachate was determined using the CNS Auto-Analyzer (VarioMAX, Elemental

Americas, Inc. Mt Laurel, NJ).

57

Yield

Red fruit were harvested from each plant about 9 weeks after transplanting. Yield was

assessed by total full red fruit harvested.

Fruit Quality

Physical analysis

Color: A total of two color measurements were taken on the opposite sides of the fruit at

the equatorial region of each tomato that was large enough to cover the 8 mm aperture of the

colorimeter. A hand-held tristimulus reflectance colorimeter (Model CR-400, Minolta Co., Ltd.,

Osaka, Japan) equipped with a glass light-projection tube (CR-A33f, Minolta Co., Ltd., Osaka,

Japan) was used. The color was recorded using the CIE-L*a*b* uniform color space (CIE-Lab),

L* (lightness), a* (redness), and b* (yellowness) values. Numerical values of a* and b* were

converted into hue angle using the Konica Minolta CR-400 Utility software CR-S4w (2002-2010

Konica Minolta Sensing, Inc., Osaka, Japan).

Texture. Firmness was measured using a TA.XT Plus Texture Analyzer fitted with a 35

mm flat compression plate and equipped with a 50-kg load cell. A tomato fruit was placed stem

end down on the flat surface of the texture analyzer, thus applying pressure on the blossom-end

of the fruit. The plate was then driven with a crosshead speed of 1 mm s−1, and the compression

force was recorded at 3 mm deformation. The force required to compress the fruit by 3 mm was

recorded in kgf and then converted to Newton (N = kgf x 9.8).

Compositional analysis

Samples intended for compositional measurements were homogenized on the day of

harvest, and kept frozen at −30 °C in air-tight plastic bags until analysis.

Ascorbic acid. Total ascorbic acid (AA) was quantified using the method described in

Nunes (2015). Tomato tissue was homogenized and 4 g of homogenate was mixed with a 20 mL

58

metaphosphoric acid mixture (6% HPO3 containing 2 N acetic acid). Samples were then filtered

(0.22 μm) prior to HPLC analysis. Ascorbic acid analysis was conducted using a Hitachi

LaChromUltra UHPLC system with a diode array detector and a LaChromUltra C18 2 μm

column (2 × 50 mm) (Hitachi, Ltd., Tokyo, Japan). The analysis was performed under isocratic

mode at a flow rate of 0.5 mL/min with a detection of 254 nm. Sample injection volume was 5

μL, each with duplicate HPLC injections. Mobile phase was buffered potassium phosphate

monobasic (KH2PO4, 0.5%, w/v) at pH 2.5 with metaphosphoric acid (HPO3, 0.1%, w/v). The

retention time of the ascorbic acid peak was 2.5 min. Peak was identified after comparison of

retention time with the ascorbic acid standard. Total ascorbic acid content was expressed in

terms of fresh weight (mg AA 100 g−1

).

Individual sugars. Individual sugar analysis was conducted using a Hitachi HPLC

system with a refractive index detector and a 300 mm × 8 mm Shodex SP0810 column (Shodex,

Colorado Springs, CO) with a SP-G guard column (2 mm x 4 mm). Aliquots of 4 grams fruit

puree were mixed thoroughly with 4 mL ultrapure water, vortexed, and centrifuged at 1,600 gn

for 20 min. Supernatant was decanted and filtered through 0.45 µm nylon syringe filter. Sample

injection volume was 5 µL, each with duplicate HPLC injections. Isocratic solvent delivery of

water was set at 1.0 mL∙min–1. Sample injection volume was 5 µL. Standard solutions of

glucose, and fructose (Fisher Scientific Company, Pittsburgh, PA) were used to identify sample

peaks. The peaks were identified by comparing retention times with those of the standards. The

amount of total sugars in tomato was quantified using calibration curves obtained from different

concentrations (2, 4, 6, 10, and 20 mg∙mL–1) of glucose, and fructose standards. Three samples

per treatment (2 g fruit puree) were used, each with duplicate HPLC injections. Total sugar and

individual sugar contents were expressed in g·100 g–1 fresh weight.

59

Titratable acidity and soluble solids content. The supernatants of thawed and filtered

homogenates were analyzed for titratable acidity (TA), and soluble solids content (SSC). For TA

6.0 g aliquots of the supernatant were diluted with 50 ml distilled water and titrated with 0.1 N

NaOH to an end point of pH 8.2 with an automatic titrimeter. Results were converted to percent

citric acid using the method described in (Nunes and Delgado, 2014). The SSC of the

supernatant was determined with a digital ATAGO PR-101 refractometer with a 0% to 45%

range (Atago, Tokyo, Japan).

Statistical Analysis

Data were analyzed by analysis of variance (ANOVA) and Duncan’s multiple range tests

with the package ‘agricolae’ (de Mendiburu and de Mendiburu, 2015) using R Version 3.1.1.

Significant differences between treatments were determined at the 95% confidence level (p ≤

0.05).

All figures show means treatment means ranked from highest to lowest, with means

assigned the same letter having no significant difference.

Results and Discussion

There were many experimental variables that were not able to be controlled during the

plant growth experiments such as pest control, weather and mechanical difficulties during all

both growth cycles. From all data collected, Harvest 1 had the least complications, most

complete data collection and adequate yield for quality analysis; therefore it will be the focus of

the discussion.

Plant Growth

Overall, there was not much significant difference in growth index (GI) of CNT treated

plants than the control. However, highest CNT concentration (3%) treated plants displayed

significantly higher growth index throughout the growth cycle compared to the other treatments

60

(Figure 3-2). This was as expected since Khodakovskaya et al. (2009) demonstrated earlier that

when tomato seeds were coated with a range of CNT concentrations (10 to 40 mg l−1

), CNTs

penetrated the plant seeds increasing germination rates, and stimulating growth of young

seedlings. One possible mechanism suggested by Khodakovskaya et al. (2012) was that CNTs

stimulate growth and activate gene and protein expression of aquaporin in tobacco cells.

Aquaporins are crucial for root water uptake, seed germination, cell elongation, reproduction and

photosynthesis (Maurel, 2007). Another possible mechanism is that MWCNTs enhance water

uptake by creating new pores in the cell wall and plasma membrane allowing greater water

transport by developing tomato and wheat seedlings (Liu et al., 2010, Gao et al., 2011).

Plant leachate was analyzed for concentrations of elements Mg, Ca, P and NO3 which

showed no trends amongst treatments (Figure 3-3). Phosphorous leaching was significantly

higher in 3% CNT treatment. However, the variation of phosphorus supply in soils for growing

tomato crops does not significantly influence quality traits such as the total soluble solids

(Senevirathna and Daundasekera, 2010), pH, acidity of the tomato juice, or the fruit color

characteristics (Oke et al., 2005).

Yield of full red fruit per plant was recorded for both harvests and data is shown in

Figure 3-4. All CNT treated plants had higher yield than the control, with 1% CNT having the

highest yield. This is supported by results from similar a growth study in which Khodakovskaya

et al. (2013) demonstrated the influence of irrigation with MWCNTs on the tomato plant (cv.

Micro-Tom) from the germination to the flowering stage. CNT treated plants had significantly

higher plant height, twice as many flowers and fruits than plants grown in the control soil. The

50 μg mL−1

had the same influence as that obtained by 200 μg mL−1

, indicating that the effect

61

may not be concentration dependent. It was inferred from the findings that carbon nanotubes

may influence the reproductive system although the mechanism is yet to be understood.

Fruit Quality

Color and firmness of tomatoes are important because they are highly correlated with

sensory attributes such as taste and aroma (Resurreccion and Shewfelt, 1985). CNT treated fruits,

specifically 1% and 0.5%, had lower L* and higher a* values, indicating a darker and more red

skin color (Figure 3-5). Increasing a* value measured by colorimeter was shown to be directly

associated with lycopene synthesis, whereas a*/b* ratio has been reported to be a good indicator

of lycopene content and therefore could be used to characterize fresh tomato ripeness stage

(Arias et al., 2000, Helyes et al., 2006).

All CNT treated fruit had significantly higher hue angle than the control, particularly the

3% CNT treatment indicating a more reddish orange than red color (Figure 3-5). Hue angle of

full red tomato is generally within the range of 30° to 50°, with lower values indicating deeper

red or purple color and higher values indicating more orange color (Perkins-Veazie et al., 2007).

Tomato firmness is an important quality factor because it is associated with good eating

quality and longer postharvest life (Nunes, 2009). Overall, CNT treated tomato fruits were firmer

than the control. Specifically, firmness of the 3% CNT treated tomatoes was significantly higher

than the control (Figure 3-6).

Appearance of fruit was subjectively analyzed for size, differences in locular structure

and seed population, which were not affected by application of CNTs to tomato plants. These

results were similar to results reported by Khodakovskaya et al. (2013).

Reducing sugars such as glucose and fructose are major components of the SSC with the

remainder consisting of organic acids, lipids, minerals and pigments. Fruit from the 3% CNT

treatment showed significantly higher levels of SSC and individual sugars, glucose and fructose,

62

(Figure 3-8). These results are in agreement with data previously reported in the literature as SSC

of mature tomato ranged from 4.5 to 8.5% of its fresh weight (Pedro and Ferreira, 2005).

The acids measured in the form of TA and AA (Figure 3-7). AA levels were significantly

higher in 3% CNT treatments than in the control, indicating higher Vitamin C. and therefore fruit

of higher nutritional quality. There was not a significant difference in TA of tomato fruit among

CNT treatments; however, 3% CNT had significantly lower TA than the control.

As tomatoes mature on the plant, sugar-to-acid ratio increases, resulting in higher soluble

solids and lower acidity (Nunes, 2009). When comparing the sugar to acid ratios, 3% and 1%

CNT treatments had higher ratios, with 3% having a significantly higher ratio (Figure 3-8 and 3-

9). In combination with the significantly higher hue angle of the 3% CNT fruit, the sugar to acid

ratio can possibly indicate the treatment was more mature.

Presence of CNT in edible part of tomato plant was not measured in this study. Because

effect of carbon nanotubes depends greatly on their size, concentration and solubility,

contradicting reports have been received regarding the influence of carbon nanotubes on various

plants. Chen et al. (2015) observed that the MWCNTs permeated into the roots of intact living

mustard plants (three months old) and were then transported to the edible portion of the crop.

However, Cañas et al. (2008) showed that application of SWCNTs inhibited root elongation in

tomato and additionally no SWCNTs entered the root of the plant.

Conclusion

Increasing application of nanotechnologies leads to growing concern regarding the

environmental effects of CNTs on environmental and human health. Evaluating the effect of

CNTs to plants can provide data in both areas; especially because plants are consumed routinely

by organisms, including humans. Therefore, we evaluated the effect of CNTs on plant growth

and nutritional quality of tomato fruit.

63

This is the first study in which nutritional quality of a CNT treated crop have been

evaluated, particularly that of tomato. Overall, higher concentration CNT had positive effect on

plant growth. Fruits from the CNT treatments had firmer and redder skin, with significantly

higher Vitamin C and sugars. There is a possibility that harvested fruits were at different

maturities, due to sugar to acid ratio and hue angle being significantly different amongst

treatments.

This study is the first to confirm that application of CNTs to tomato plants not only

improves growth but also improves nutritional quality of tomato fruit. This could be of

significant economic importance for agriculture, horticulture, and the energy sector, such as for

production of biofuels. Although in some cases, carbon nanomaterials are known to penetrate

edible portion of plant and their bioavailability must be assessed if they pose risk to man and

animals.

Future work. The higher quality fruit produced from the CNT treatments could indicate

a potential longer shelf-life. Therefore, a shelf-life study can be done to further assess the CNT

influence on quality of the crop. Molecular mechanisms of CNT-induced water uptake inside

plants seeds are not clear and require further investigation. Thus, the plant-CNT interaction

needs to be thoroughly investigated, from the molecular to the cellular and organ levels. Finally,

since CNTs penetrating the roots and into the edible portion of the plant is a concern, tracing

CNTs with radioactive labeling methods could provide insight to safety of practical CNT

application in soils.

64

Figure 3-1. Pots of prepared growth medium control of CNT treatments.

Figure 3-2. Growth Index (cm

2) of CNT treated Micro-Tom plants at week 3 (above) and week 7

(below).

65

Figure 3-3. Concentrations (mg/L) of Ca, Mg, P and NO3 in plant leachate samples measured at

week 10 in the growth cycle. Means assigned same letter groups are not significantly

different

Figure 3-4. Yield of average number of full red fruits obtained per plant from each treatment

from Harvest 1 (6/2/16) and Harvest 2 (6/9/16)

Ca P NO3

Groups, Treatments and means Groups, Treatments and means Groups, Treatments and means Groups, Treatments and means

a CNTHigh 32.44 a CNTHigh 17.88 a CNTHigh 22.55 a 3% 7.481

a CNTLow 30.03 a ControlCNT 15.22 b CNTLowest 12.35 a Control 5.393

a ControlCNT 28.18 a CNTLow 14.95 b CNTLow 10.45 a 0.50% 4.146

a CNTLowest 26.42 a CNTLowest 13.33 b ControlCNT 10.21 a 1% 4.11

Mg

Cycle 1 week 9

Treatment Harvest 1 Harvest 2

Control 4.8 2.2

CNT 0.5 % 6 4

CNT 1 % 7.4 1.8

CNT 3% 5.4 3.1

66

Figure 3-5. Color a* and L* and Hue Angle values of control and CNT treated tomatoes. Bars

represent standard error (n = ~30)

67

Figure 3-6. Ascorbic acid (AA), titratable acidity (TA) and firmness of control and CNT treated

tomatoes. Bars represent standard error (n = ~30 in Force, n = 3 in TA, n = 6 in AA).

68

Figure 3-7. Soluble solids content (SSC), glucose, fructose and of control and CNT treated

tomatoes. Bars represent standard error (n = 6 in glucose and fructose and n = 3 in

SSC).

69

Figure 3-8. Sugar to acid ratio of (soluble solids content to titratable acidity) all three growth

cycles of control and CNT treated fruit.

Figure 3-9. Statistical analysis of sugar to acid ratio for Cycle 2 Harvest 1 data.

70

CHAPTER 4

CONCLUSION AND FUTURE WORK

Based on the findings from experimental studies conducted on bamboo biochar and

carbon nanotubes, the conclusions drawn for this research work were twofold. Both materials

were applied in the same manner, through mixture in sand as part of the growth medium.

Conclusion: Influence of Biochar Pyrolysis Temperature on Soil Amendment Effects on

Tomato Growth and Fruit Quality

Biochar has been described as a possible means to sustainably improve soil fertility and

plant growth. The objective was to assess the effects of biochar production temperature on plant

growth and fruit quality. This is the first study in which plant growth and nutritional quality of

fruit affected by different pyrolysis temperatures were studied. Results indicated:

Plant growth was significantly higher in lower temperature biochar treatments,

specifically 300 °C and 450 °C temperatures. Studies showed that acidic functional

groups in lower temperature biochar can improve the acidity of the soil, increasing the

CEC, and therefore nutrient and water holding capacity.

Higher quality and more nutritious fruit resulted from lower temperature biochar

treatments, this was indicated by fruit with darker red color, firmer skin, higher sugars

and acids, and higher levels of vitamin C. Sugar to acid ratios and hue angles amongst the

treatments did not vary significantly, indicating that the fruit were at similar maturity

stage. Thus, biochar may have resulted in a modification of maturation, possibly increase

in lycopene synthesis, and an effect on ripening physiology (hormonal regulation of

ethylene and auxin) may be considered.

Results suggest that lower temperature bamboo biochars could serve as a sustainable soil

amendment due to their positive effect on plant growth and most importantly, positive

effect on resulting fruit quality.

These results are not as anticipated since higher temperature biochars have higher surface

area and nutrient content. Since there are no previous studies comparing effect of biochar

pyrolysis temperature on plant growth and fruit quality, the mechanism is not well

understood.

Future Work

Further characterization of biochar functional groups can be done through methods such

as Fourier Transform Infrared Spectroscopy (FTIR) to quantify the functional groups that

influence the soil fertility.

71

Different types of feedstock such as hard woods, grasses, or crop residues can be used

instead of bamboo, due to strong influence of feedstock type on biochar properties.

Effects of biochar may vary under commercial field conditions where there is a complex

interplay between the soil, climate, and because this study was conducted in a greenhouse

under relatively controlled growing conditions, further studies are needed to understand

plant and soil responses to biochar.

A shelf-life study of biochar treated fruit can be done to further assess the influence on

the quality of the crop.

Conclusion: Effect of Carbon Nanotubes as Soil Amendment in Sand Culture on Tomato

Growth and Quality

The increasing application of nanotechnologies throughout various industries leads to

growing concern regarding the environmental pollutions of CNTs and their consequences on

environmental and human health. Evaluating the effect of CNTs to plants can provide data in

both areas; because crop producing plants are commonly consumed by humans. Therefore, we

evaluated the effect of CNTs on plant growth and quality of tomato fruit. This is the first study in

which quality of a CNT treated crop, particularly tomato, have been evaluated.

Higher concentration CNT treatments had positive effect on plant growth. Previous

studies have suggested that CNTs stimulate growth by increasing water uptake.

Fruit from the CNT treatments had firmer and redder skin, with significantly higher

Vitamin C and sugars. There is a possibility that fruits were harvested at different

maturities, due to sugar to acid ratio and hue angle showing significant differences

amongst treatments.

This study is the first to confirm that application of CNTs to tomato plants not only

improves growth but also improves nutritional quality of tomato fruit. This could be of

significant economic importance for agriculture, horticulture, and the energy sector, such

as for production of biofuels.

Future Work

The higher quality fruit produced from the CNT treatments could indicate a longer shelf-

life; therefore, a shelf-life study can further assess the CNT influence on quality of the

crop.

72

Plant-CNT interaction needs to be thoroughly investigated, from the molecular to the

cellular and organ levels. This could lead to understanding the molecular mechanisms of

CNT-induced water uptake that have been proposed by other studies.

Penetration of CNTs to roots and into the edible portion of the plant is a concern. Future

studies tracing CNTs with radioactive labeling techniques, which are potentially useful

for assessing nutrient uptake, could provide insight to safety of practical CNT application

in soils.

The research studies presented here have exciting avenues to explore the possibility of

application of biochar to improve tomato plant growth and quality. Also it demonstrates the

consequences of CNT presence in plant growth media on tomato plant growth and fruit quality.

73

LIST OF REFERENCES

ABELES, F., MORGAN, P. & SALTVEIT JR, M. 1992. Jr. Ethylene in plant biology. San

Diego: Academic.

AKHTAR, S. S., LI, G., ANDERSEN, M. N. & LIU, F. 2014. Biochar enhances yield and

quality of tomato under reduced irrigation. Agricultural Water Management, 138, 37-44.

ANDRY, H., YAMAMOTO, T., IRIE, T., MORITANI, S., INOUE, M. & FUJIYAMA, H.

2009. Water retention, hydraulic conductivity of hydrophilic polymers in sandy soil as

affected by temperature and water quality. Journal of Hydrology, 373, 177-183.

AOUN, A. B., LECHIHEB, B., BENYAHYA, L. & FERCHICHI, A. 2013. Evaluation of fruit

quality traits of traditional varieties of tomato (Solanum lycopersicum) grown in Tunisia.

African Journal of Food Science, 7, 350-354.

ARAB, L. & STECK, S. 2000. Lycopene and cardiovascular disease. The American journal of

clinical nutrition, 71, 1691s-1695s.

ARAH, I. K., AHORBO, G. K., ANKU, E. K., KUMAH, E. K. & AMAGLO, H. 2016.

Postharvest Handling Practices and Treatment Methods for Tomato Handlers in

Developing Countries: A Mini Review. Advances in Agriculture, 2016.

ARIAS, R., LEE, T.-C., LOGENDRA, L. & JANES, H. 2000. Correlation of lycopene measured

by HPLC with the L*, a*, b* color readings of a hydroponic tomato and the relationship

of maturity with color and lycopene content. Journal of Agricultural and Food

Chemistry, 48, 1697-1702.

ARSHAD, M. & FRANKENBERGER JR, W. T. 2012. Ethylene: agricultural sources and

applications, Springer Science & Business Media.

ATKINSON, C. J., FITZGERALD, J. D. & HIPPS, N. A. 2010. Potential mechanisms for

achieving agricultural benefits from biochar application to temperate soils: a review.

Plant and soil, 337, 1-18.

BALDWIN, E., SCOTT, J., EINSTEIN, M., MALUNDO, T., CARR, B., SHEWFELT, R. &

TANDON, K. 1998. Relationship between sensory and instrumental analysis for tomato

flavor. Journal of the American Society for Horticultural Science, 123, 906-915.

BECKLES, D. M. 2012. Factors affecting the postharvest soluble solids and sugar content of

tomato (Solanum lycopersicum L.) fruit. Postharvest Biology and Technology, 63, 129-

140.

BHARGAVA, D. & SHELDARKAR, S. 1993. Use of TNSAC in phosphate adsorption studies

and relationships. Literature, experimental methodology, justification and effects of

process variables. Water Research, 27, 303-312.

74

BIEDERMAN, L. A. & HARPOLE, W. S. 2013. Biochar and its effects on plant productivity

and nutrient cycling: a meta‐analysis. GCB bioenergy, 5, 202-214.

BIOCHAR-INTERNATIONAL. 2016. Feedstocks [Online]. Available: http://www.biochar-

international.org/technology/feedstocks.

BRANDT, S., PÉK, Z., BARNA, É., LUGASI, A. & HELYES, L. 2006. Lycopene content and

colour of ripening tomatoes as affected by environmental conditions. Journal of the

Science of Food and Agriculture, 86, 568-572.

CAÑAS, J. E., LONG, M., NATIONS, S., VADAN, R., DAI, L., LUO, M., AMBIKAPATHI,

R., LEE, E. H. & OLSZYK, D. 2008. Effects of functionalized and nonfunctionalized

single‐walled carbon nanotubes on root elongation of select crop species.

Environmental Toxicology and Chemistry, 27, 1922-1931.

CHEN, B., ZHOU, D. & ZHU, L. 2008. Transitional Adsorption and Partition of Nonpolar and

Polar Aromatic Contaminants by Biochars of Pine Needles with Different Pyrolytic

Temperatures. Environmental Science & Technology, 42, 5137-5143.

CHEN, G., QIU, J., LIU, Y., JIANG, R., CAI, S., LIU, Y., ZHU, F., ZENG, F., LUAN, T. &

OUYANG, G. 2015. Carbon Nanotubes Act as Contaminant Carriers and Translocate

within Plants. Scientific Reports, 5, 15682.

CHEN, J. & BEESON JR, R. C. ACTUAL EVAPOTRANSPIRATION OF ASPLENIUM

NIDUS AND CHAMAEDOREA ELEGANS DURING PRODUCTION FROM LINERS

TO MARKETABLE PLANTS. 2013. International Society for Horticultural Science

(ISHS), Leuven, Belgium, 339-344.

CHUN, Y., SHENG, G., CHIOU, C. T. & XING, B. 2004. Compositions and sorptive properties

of crop residue-derived chars. Environmental science & technology, 38, 4649-4655.

CLAOSTON, N., SAMSURI, A., HUSNI, M. A. & AMRAN, M. M. 2014. Effects of pyrolysis

temperature on the physicochemical properties of empty fruit bunch and rice husk

biochars. Waste Management & Research, 32, 331-339.

DAVIES, J. N., HOBSON, G. E. & MCGLASSON, W. 1981. The constituents of tomato fruit—

the influence of environment, nutrition, and genotype. Critical Reviews in Food Science

& Nutrition, 15, 205-280.

DAY, D., EVANS, R. J., LEE, J. W. & REICOSKY, D. 2005. Economical CO 2, SO x, and NO

x capture from fossil-fuel utilization with combined renewable hydrogen production and

large-scale carbon sequestration. Energy, 30, 2558-2579.

DE MENDIBURU, F. & DE MENDIBURU, M. F. 2015. Package ‘agricolae’. Version.

DOWNIE, A., CROSKY, A. & MUNROE, P. 2009. Physical properties of biochar. Biochar for

environmental management: Science and technology, 13-32.

75

DUMAS, Y., DADOMO, M., DI LUCCA, G. & GROLIER, P. 2003. Effects of environmental

factors and agricultural techniques on antioxidantcontent of tomatoes. Journal of the

Science of Food and Agriculture, 83, 369-382.

EATEMADI, A., DARAEE, H., KARIMKHANLOO, H., KOUHI, M., ZARGHAMI, N.,

AKBARZADEH, A., ABASI, M., HANIFEHPOUR, Y. & JOO, S. W. 2014. Carbon

nanotubes: properties, synthesis, purification, and medical applications. Nanoscale Res

Lett, 9, 393.

FLORIDATOMATOES.ORG. Facts [Online]. Available: http://www.floridatomatoes.org/facts.

FRANKENBERGER JR, W. T. & ARSHAD, M. 1995. Phytohormones in soils: microbial

production and function, Marcel Dekker Inc.

FULTON, W., GRAY, M., PRAHL, F. & KLEBER, M. 2013. A simple technique to eliminate

ethylene emissions from biochar amendment in agriculture. Agronomy for sustainable

development, 33, 469-474.

GAO, J., WANG, Y., FOLTA, K. M., KRISHNA, V., BAI, W., INDEGLIA, P., GEORGIEVA,

A., NAKAMURA, H., KOOPMAN, B. & MOUDGIL, B. 2011. Polyhydroxy fullerenes

(fullerols or fullerenols): beneficial effects on growth and lifespan in diverse biological

models. PLoS One, 6, e19976.

GASKIN, J., STEINER, C., HARRIS, K., DAS, K. & BIBENS, B. 2008. Effect of low-

temperature pyrolysis conditions on biochar for agricultural use. Transactions of the

ASABE, 51, 2061-2069.

GLASER, B., LEHMANN, J. & ZECH, W. 2002. Ameliorating physical and chemical properties

of highly weathered soils in the tropics with charcoal–a review. Biology and fertility of

soils, 35, 219-230.

GOMEZ, P., FERRER, M., FERNÁNDEZ‐TRUJILLO, J. P., CALDERÓN, A., ARTÉS, F.,

EGEA‐CORTINES, M. & WEISS, J. 2009. Structural changes, chemical composition

and antioxidant activity of cherry tomato fruits (cv. Micro‐Tom) stored under optimal

and chilling conditions. Journal of the Science of Food and Agriculture, 89, 1543-1551.

GRABER, E., TSECHANSKY, L., GERSTL, Z. & LEW, B. 2012. High surface area biochar

negatively impacts herbicide efficacy. Plant and Soil, 353, 95-106.

GUO, Y. & ROCKSTRAW, D. A. 2007. Activated carbons prepared from rice hull by one-step

phosphoric acid activation. Microporous and Mesoporous Materials, 100, 12-19.

HELYES, L., PÉK, Z. & LUGASI, A. 2006. Tomato fruit quality and content depend on stage of

maturity. HortScience, 41, 1400-1401.

IIJIMA, S. 1991. Helical microtubules of graphitic carbon. Nature, 354, 56-58.

76

IIJIMA, S. & ICHIHASHI, T. 1993. Single-shell carbon nanotubes of 1-nm diameter. Nature,

363, 603-605.

INYANG, M., GAO, B., PULLAMMANAPPALLIL, P., DING, W. & ZIMMERMAN, A. R.

2010. Biochar from anaerobically digested sugarcane bagasse. Bioresource Technology,

101, 8868-8872.

JEFFERY, S., VERHEIJEN, F. G. A., VAN DER VELDE, M. & BASTOS, A. C. 2011. A

quantitative review of the effects of biochar application to soils on crop productivity

using meta-analysis. Agriculture, Ecosystems & Environment, 144, 175-187.

JOSEPH, S., CAMPS-ARBESTAIN, M., LIN, Y., MUNROE, P., CHIA, C., HOOK, J., VAN

ZWIETEN, L., KIMBER, S., COWIE, A. & SINGH, B. 2010. An investigation into the

reactions of biochar in soil. Soil Research, 48, 501-515.

JOSEPH, S. & LEHMANN, J. 2009. Biochar for environmental management: science and

technology, London, GB: Earthscan.

KARACA, S., GÜRSES, A., EJDER, M. & AÇıKYıLDıZ, M. 2004. Kinetic modeling of liquid-

phase adsorption of phosphate on dolomite. Journal of Colloid and Interface Science,

277, 257-263.

KASOZI, G. N., ZIMMERMAN, A. R., NKEDI-KIZZA, P. & GAO, B. 2010. Catechol and

humic acid sorption onto a range of laboratory-produced black carbons (biochars).

Environmental science & technology, 44, 6189-6195.

KAYS, S. J. 1991. Postharvest physiology and handling of perishable plant products, Van

Nostrand Reinhold Inc.

KAYS, S. J. 1999. Preharvest factors affecting appearance. Postharvest Biology and Technology,

15, 233-247.

KEITH, A., SINGH, B. & SINGH, B. P. 2011. Interactive Priming of Biochar and Labile

Organic Matter Mineralization in a Smectite-Rich Soil. Environmental Science &

Technology, 45, 9611-9618.

KHODAKOVSKAYA, M., DERVISHI, E., MAHMOOD, M., XU, Y., LI, Z., WATANABE, F.

& BIRIS, A. S. 2009. Carbon nanotubes are able to penetrate plant seed coat and

dramatically affect seed germination and plant growth. ACS nano, 3, 3221-3227.

KHODAKOVSKAYA, M. V., DE SILVA, K., BIRIS, A. S., DERVISHI, E. &

VILLAGARCIA, H. 2012. Carbon nanotubes induce growth enhancement of tobacco

cells. ACS nano, 6, 2128-2135.

KHODAKOVSKAYA, M. V., KIM, B. S., KIM, J. N., ALIMOHAMMADI, M., DERVISHI,

E., MUSTAFA, T. & CERNIGLA, C. E. 2013. Carbon nanotubes as plant growth

regulators: effects on tomato growth, reproductive system, and soil microbial community.

Small, 9, 115-123.

77

KLOSS, S., ZEHETNER, F., DELLANTONIO, A., HAMID, R., OTTNER, F., LIEDTKE, V.,

SCHWANNINGER, M., GERZABEK, M. H. & SOJA, G. 2012. Characterization of

slow pyrolysis biochars: effects of feedstocks and pyrolysis temperature on biochar

properties. Journal of environmental quality, 41, 990-1000.

LACERDA, L., BIANCO, A., PRATO, M. & KOSTARELOS, K. 2006. Carbon nanotubes as

nanomedicines: from toxicology to pharmacology. Advanced drug delivery reviews, 58,

1460-1470.

LAIRD, D., FLEMING, P., WANG, B., HORTON, R. & KARLEN, D. 2010. Biochar impact on

nutrient leaching from a Midwestern agricultural soil. Geoderma, 158, 436-442.

LEE, S. K. & KADER, A. A. 2000. Preharvest and postharvest factors influencing vitamin C

content of horticultural crops. Postharvest biology and technology, 20, 207-220.

LEHMANN, J. 2007. Bio-energy in the black. Frontiers in Ecology and the Environment, 5,

381-387.

LEHMANN, J., DA SILVA JR, J. P., STEINER, C., NEHLS, T., ZECH, W. & GLASER, B.

2003. Nutrient availability and leaching in an archaeological Anthrosol and a Ferralsol of

the Central Amazon basin: fertilizer, manure and charcoal amendments. Plant and soil,

249, 343-357.

LEHMANN, J. & JOSEPH, S. 2015. Biochar for environmental management: science,

technology and implementation, Routledge.

LEHMANN, J., RILLIG, M. C., THIES, J., MASIELLO, C. A., HOCKADAY, W. C. &

CROWLEY, D. 2011. Biochar effects on soil biota–a review. Soil Biology and

Biochemistry, 43, 1812-1836.

LIANG, B., LEHMANN, J., SOLOMON, D., KINYANGI, J., GROSSMAN, J., O'NEILL, B.,

SKJEMSTAD, J., THIES, J., LUIZAO, F. & PETERSEN, J. 2006. Black carbon

increases cation exchange capacity in soils. Soil Science Society of America Journal, 70,

1719-1730.

LIBRA, J. A., RO, K. S., KAMMANN, C., FUNKE, A., BERGE, N. D., NEUBAUER, Y.,

TITIRICI, M.-M., FÜHNER, C., BENS, O. & KERN, J. 2011. Hydrothermal

carbonization of biomass residuals: a comparative review of the chemistry, processes and

applications of wet and dry pyrolysis. Biofuels, 2, 71-106.

LIU, H. K., WANG, G. X., GUO, Z., WANG, J. & KONSTANTINOV, K. 2006. Nanomaterials

for lithium-ion rechargeable batteries. Journal of nanoscience and nanotechnology, 6, 1-

15.

LIU, Q., ZHAO, Y., WAN, Y., ZHENG, J., ZHANG, X., WANG, C., FANG, X. & LIN, J.

2010. Study of the inhibitory effect of water-soluble fullerenes on plant growth at the

cellular level. ACS nano, 4, 5743-5748.

78

LIU, X., ZHANG, A., JI, C., JOSEPH, S., BIAN, R., LI, L., PAN, G. & PAZ-FERREIRO, J.

2013. Biochar’s effect on crop productivity and the dependence on experimental

conditions—a meta-analysis of literature data. Plant and Soil, 373, 583-594.

MAJOR, J., RONDON, M., MOLINA, D., RIHA, S. J. & LEHMANN, J. 2010. Maize yield and

nutrition during 4 years after biochar application to a Colombian savanna oxisol. Plant

and soil, 333, 117-128.

MAUREL, C. 2007. Plant aquaporins: novel functions and regulation properties. Febs Letters,

581, 2227-2236.

MAYNARD, A. D., BARON, P. A., FOLEY, M., SHVEDOVA, A. A., KISIN, E. R. &

CASTRANOVA, V. 2004. Exposure to carbon nanotube material: aerosol release during

the handling of unrefined single-walled carbon nanotube material. Journal of Toxicology

and Environmental Health, Part A, 67, 87-107.

MAYNARD, A. D., WARHEIT, D. B. & PHILBERT, M. A. 2010. The new toxicology of

sophisticated materials: nanotoxicology and beyond. Toxicological sciences, kfq372.

MUKHERJEE, A. & LAL, R. 2014. The biochar dilemma. Soil Research, 52, 217-230.

MUKHERJEE, A., ZIMMERMAN, A. & HARRIS, W. 2011. Surface chemistry variations

among a series of laboratory-produced biochars. Geoderma, 163, 247-255.

NUNES, C. 2009. Color atlas of postharvest quality of fruits and vegetables, John Wiley &

Sons.

NUNES, M. C. D. N. 2015. Correlations between subjective quality and physicochemical

attributes of fresh fruits and vegetables. Postharvest Biology and Technology, 107, 43-54.

NUNES, M. C. N. & DELGADO, A. Quality of organic compared to conventionally grown

strawberries at the retail level. 2014. International Society for Horticultural Science

(ISHS), Leuven, Belgium, 723-730.

OGAWA, M., OKIMORI, Y. & TAKAHASHI, F. 2006. Carbon sequestration by carbonization

of biomass and forestation: three case studies. Mitigation and adaptation strategies for

global change, 11, 421-436.

OKE, M., AHN, T., SCHOFIELD, A. & PALIYATH, G. 2005. Effects of phosphorus fertilizer

supplementation on processing quality and functional food ingredients in tomato. Journal

of agricultural and food chemistry, 53, 1531-1538.

OLMO, M., ALBURQUERQUE, J. A., BARRÓN, V., DEL CAMPILLO, M. C., GALLARDO,

A., FUENTES, M. & VILLAR, R. 2014. Wheat growth and yield responses to biochar

addition under Mediterranean climate conditions. Biology and Fertility of Soils, 50, 1177-

1187.

79

OLMO, M., VILLAR, R., SALAZAR, P. & ALBURQUERQUE, J. A. 2016. Changes in soil

nutrient availability explain biochar’s impact on wheat root development. Plant and Soil,

399, 333-343.

ÖZACAR, M. 2003. Adsorption of phosphate from aqueous solution onto alunite. Chemosphere,

51, 321-327.

PASSAM, H. C., KARAPANOS, I. C., BEBELI, P. J. & SAVVAS, D. 2007. A review of recent

research on tomato nutrition, breeding and post-harvest technology with reference to fruit

quality. The European Journal of Plant Science and Biotechnology, 1, 1-21.

PEDRO, A. M. & FERREIRA, M. M. 2005. Nondestructive determination of solids and

carotenoids in tomato products by near-infrared spectroscopy and multivariate

calibration. Analytical Chemistry, 77, 2505-2511.

PERKINS-VEAZIE, P., ROBERTS, W. & COLLINS, J. K. 2007. Lycopene content among

organically produced tomatoes. Journal of vegetable science, 12, 93-106.

PETRUCCELLI, R., BONETTI, A., TRAVERSI, M. L., FARALONI, C., VALAGUSSA, M. &

POZZI, A. 2015. Influence of biochar application on nutritional quality of tomato

(Lycopersicon esculentum). Crop and Pasture Science, 66, 747-755.

PIGNATELLO, J. J., KWON, S. & LU, Y. 2006. Effect of natural organic substances on the

surface and adsorptive properties of environmental black carbon (char): attenuation of

surface activity by humic and fulvic acids. Environmental Science & Technology, 40,

7757-7763.

QUILLIAM, R. S., MARSDEN, K. A., GERTLER, C., ROUSK, J., DELUCA, T. H. & JONES,

D. L. 2012. Nutrient dynamics, microbial growth and weed emergence in biochar

amended soil are influenced by time since application and reapplication rate. Agriculture,

Ecosystems & Environment, 158, 192-199.

RESURRECCION, A. & SHEWFELT, R. 1985. Relationships between sensory attributes and

objective measurements of postharvest quality of tomatoes. Journal of Food Science, 50,

1242-1245.

SAMS, C. E. 1999. Preharvest factors affecting postharvest texture. Postharvest Biology and

Technology, 15, 249-254.

SARGENT, S. A. & MORETTI, C. L. 2014. Tomato: Scientific Name and Introduction. In:

LUO, Y. (ed.) Agriculture handbook number 66 (HB-66): the commercial storage of

fruits, vegetables, and florist and nursery stocks. Washington United States Department

of Agriculture (USDA) Agricultural Research Service (ARS) Beltsville Area (BA).

SCHMIDT, H.-P., KAMMANN, C., NIGGLI, C., EVANGELOU, M. W., MACKIE, K. A. &

ABIVEN, S. 2014. Biochar and biochar-compost as soil amendments to a vineyard soil:

Influences on plant growth, nutrient uptake, plant health and grape quality. Agriculture,

Ecosystems & Environment, 191, 117-123.

80

SCHUMANN, A. 2012. Opportunities for biochar production anduse in Florida citrus. IFAS

Extension Trade Journal, April 2012.

SEILER, W. & CRUTZEN, P. J. 1980. Estimates of gross and net fluxes of carbon between the

biosphere and the atmosphere from biomass burning. Climatic change, 2, 207-247.

SENEVIRATHNA, P. & DAUNDASEKERA, W. 2010. Effect of postharvest calcium chloride

vacuum infiltration on the shelf life and quality of tomato (cv.'Thilina'). Ceylon Journal

of science (Biological Sciences), 39.

SESSO, H. D., LIU, S., GAZIANO, J. M. & BURING, J. E. 2003. Dietary lycopene, tomato-

based food products and cardiovascular disease in women. The Journal of nutrition, 133,

2336-2341.

SHEWFELT, R. L. 1990. Sources of variation in the nutrient content of agricultural commodities

from the farm to the consumer. Journal of food quality, 13, 37-54.

SINGH, S. 2010. Nanomedicine–nanoscale drugs and delivery systems. Journal of nanoscience

and nanotechnology, 10, 7906-7918.

SKJEMSTAD, J. O., CLARKE, P., TAYLOR, J., OADES, J. & MCCLURE, S. G. 1996. The

chemistry and nature of protected carbon in soil. Soil Research, 34, 251-271.

SOHI, S. P., KRULL, E., LOPEZ-CAPEL, E. & BOL, R. 2010. Chapter 2 - A Review of

Biochar and Its Use and Function in Soil. Advances in Agronomy. Academic Press.

SOZER, N. & KOKINI, J. L. 2009. Nanotechnology and its applications in the food sector.

Trends in Biotechnology, 27, 82-89.

SPOKAS, K. A. 2010. Review of the stability of biochar in soils: predictability of O: C molar

ratios. Carbon Management, 1, 289-303.

SPOKAS, K. A., BAKER, J. M. & REICOSKY, D. C. 2010. Ethylene: Potential key for biochar

amendment impacts. Plant and Soil, 333, 443-452.

SPOKAS, K. A., CANTRELL, K. B., NOVAK, J. M., ARCHER, D. W., IPPOLITO, J. A.,

COLLINS, H. P., BOATENG, A. A., LIMA, I. M., LAMB, M. C. & MCALOON, A. J.

2012. Biochar: a synthesis of its agronomic impact beyond carbon sequestration. Journal

of environmental quality, 41, 973-989.

SUN, H.-J., UCHII, S., WATANABE, S. & EZURA, H. 2006. A highly efficient transformation

protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant and Cell

Physiology, 47, 426-431.

SUN, Y., GAO, B., YAO, Y., FANG, J., ZHANG, M., ZHOU, Y., CHEN, H. & YANG, L.

2014. Effects of feedstock type, production method, and pyrolysis temperature on biochar

and hydrochar properties. Chemical Engineering Journal, 240, 574-578.

81

TSUTOMU, I., TAKASHI, A., KUNIAKI, K. & KIKUO, O. 2004. Comparison of removal

efficiencies for ammonia and amine gases between woody charcoal and activated carbon.

Journal of health science, 50, 148-153.

UCHIMIYA, M., WARTELLE, L. H., KLASSON, K. T., FORTIER, C. A. & LIMA, I. M.

2011. Influence of Pyrolysis Temperature on Biochar Property and Function as a Heavy

Metal Sorbent in Soil. Journal of Agricultural and Food Chemistry, 59, 2501-2510.

VACCARI, F. P., MAIENZA, A., MIGLIETTA, F., BARONTI, S., DI LONARDO, S.,

GIAGNONI, L., LAGOMARSINO, A., POZZI, A., PUSCEDDU, E., RANIERI, R.,

VALBOA, G. & GENESIO, L. 2015. Biochar stimulates plant growth but not fruit yield

of processing tomato in a fertile soil. Agriculture, Ecosystems & Environment, 207, 163-

170.

WANG, Y., MIRKIN, C. A. & PARK, S.-J. 2009. Nanofabrication beyond Electronics. ACS

Nano, 3, 1049-1056.

WESTON, L. A. & BARTH, M. M. 1997. Preharvest factors affecting postharvest quality of

vegetables. Hortscience, 32, 812-816.

WHITING, D., CARD, A., MORAVEC, C., WILSON, C., REEDER, J., AND GOLDHAMER,

D. 2015. Soil Amendments. Colorado State University Extension, Colorado Master

Gardener Program.

YAO, Y., GAO, B., INYANG, M., ZIMMERMAN, A. R., CAO, X.,

PULLAMMANAPPALLIL, P. & YANG, L. 2011. Biochar derived from anaerobically

digested sugar beet tailings: Characterization and phosphate removal potential.

Bioresource Technology, 102, 6273-6278.

YAO, Y., GAO, B., ZHANG, M., INYANG, M. & ZIMMERMAN, A. R. 2012. Effect of

biochar amendment on sorption and leaching of nitrate, ammonium, and phosphate in a

sandy soil. Chemosphere, 89, 1467-1471.

YEAGER, T., WRIGHT, R. & DONOHUE, S. 1983. Comparison of pour-through and saturated

pine bark extract N, P, K, and pH [nitrogen, phosphorus, potassium and hydrogen ion

concentration] levels [Soil testing, soilless media, nutrient extraction]. Journal American

Society for Horticultural Science.

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BIOGRAPHICAL SKETCH

Ratna Suthar was born in New Jersey. She received her bachelor’s degree in chemistry

from University of South Florida in 2013. Before her graduation, she served as an undergraduate

researcher in the Food Quality Laboratory at University of South Florida with Dr. Cecilia Nunes

where she was inspired to continue research. After graduation, she continued working for a year

as a supplementary instructor at the Polk State College Lakeland TLCC, teaching chemistry and

math before coming to University of Florida to receive her Master of Science Degree in

Agricultural and Biological Engineering Department. At the end of her Master’s program, she

intends to continue working on research by pursuing her doctorate degree in Agricultural and

Biological Engineering at the University of Florida.