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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
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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
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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
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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
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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
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LIST OF ABBREVIATIONS
AA
BB
Ascorbic Acid
Bamboo Biochar
CEC Cation Exchange Capacity
CNT
SSC
TA
Carbon Nanotubes
Soluble Solids Content
Titratable Acidity
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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
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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.
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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
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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
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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
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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
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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
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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.
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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.
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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
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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.
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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.
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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
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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.