BIOHYDROGEN PRODUCTION by <I>Thermotoga

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5-2013 BIOHYDROGEN PRODUCTION BY Thermotoga neapolitana IN AGRICULTURAL MEDIA David Morris Clemson University, [email protected]

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BIOHYDROGEN PRODUCTION BY Thermotoga neapolitana IN AGRICULTURAL MEDIA

A Thesis Presented to the Graduate School of Clemson University

In Partial Fulfillment of the Requirements for the Degree Master of Science Biosystems Engineering

by David Stephen Morris May 2013

Accepted by: Dr. Caye Drapcho, Committee Chair Dr. Terry Walker Dr. Kevin Finneran

ABSTRACT

In this study the production of hydrogen gas by eubacteria Thermotoga neapolitana was investigated. The organism was grown using varying media of cull peach solids and soybean meal. Hydrogen, co-products, and sugar concentrations were observed and compared.

T. neapolitana grew in medium containing peaches and soybean meal as carbon and nitrogen sources, respectively, and produced hydrogen gas. A standard medium containing glucose and yeast extract produced the most hydrogen at 31.5 mmol H2/L- medium. Withholding the vitamins and minerals from the standard the media had a significant effect on hydrogen production reducing the amount of gas produced to 25.8 mmol H2/L-medium. There was no significant difference in hydrogen production among

T. neapolitana cultures grown in peach and soybean media with or without vitamins and/or trace elements. Approximately 23 mmol H2/L-medium was produced from these cultures.

Acetate and lactate were produced by T. neapolitana grown in all media. The amount of acetate produced was half that of hydrogen produced. When cultured in the standard media without vitamins and trace elements T. neapolitana produced the most lactate, 5.99 mmol/L. In standard media it produced 3.73 mmol/L and in peach media it produced approximately 0.1 - 0.2 mmol/L lactate.

ii DEDICATION

This is dedicated to my best friend, Karen.

iii ACKNOWLEDGMENTS

I would like to thank Dr. Caye Drapcho for her willingness to let me work on this project in her lab. Her direction and support were vital in completing this work. I would also like to think Dr. Terry Walker and Dr. Kevin Finneran for taking the time to help with this project.

Thanks to Louis Hill and Preston Epley that helped day to day with feedback and ideas that were key to completing this experiment. Thanks to Jovan Popovic who sacrificed too much time to help me test late night samples.

Thanks to all my family and friends who were supportive and made life enjoyable while here at Clemson University.

iv TABLE OF CONTENTS

Page

TITLE PAGE...... i

ABSTRACT...... ii

DEDICATION...... iii

ACKNOWLEDGMENTS ...... iv

LIST OF TABLES...... vii

LIST OF FIGURES ...... iix

CHAPTER

I. Introduction to Biohydrogen...... 1

1.1.1.Introduction...... 1 1.1.2.Biologically derived Hydrogen...... 3 1.1.3.Background Thermotoga Genus ...... 6 1.1.4.Thermotoga Metabolism...... 7 1.1.5.Thermotoga Fermentation...... 8 1.1.6.Conclusion ...... 11 1.2.Goals of the study ...... 12 1.3.Chapter Two...... 13 1.4.Chapter Three...... 13 References...... 13

II. Simplification of Peach and Soybean media for Biohydrogen Production with Thermotoga neapolitana ...... 19

2.1.Introduction...... 19 2.2.Methods and Materials...... 24 2.2.1.Media ...... 24 2.2.2.Culture...... 25 2.2.3.Growth ...... 25 2.2.4.Gas Analysis ...... 26 2.2.5.Chemical Oxygen Demand...... 27

v Table of Contents (Continued)

Page

2.2.6.Sugar and Organic Acid Analysis...... 28 2.2.7.Statistical Analysis...... 28 2.3.Results and Discussion ...... 28 2.3.1.Gas Analysis ...... 28 2.3.1.1.Hydrogen...... 28 2.3.1.2.Carbon Dioxide...... 31 2.3.2.Organic Acids Analysis ...... 32 2.3.3.Sugars Analysis...... 35 2.3.4.Chemical Oxygen Demand...... 39 2.4.Conclusions...... 41 References...... 41

III. Conclusions and Ideas for Further Research ...... 45

APPENDICES ...... 48

A: Gas Analysis Data...... 49 B: Organic Acid and Standard Media Glucose Data...... 57 C: Peach Sugar Data ...... 65 D: COD Data...... 71

vi LIST OF TABLES

Table Page

2.1 Batch Treatments ...... 25

2.2 Hydrogen Production Values resulting from Different Treatments...... 29

2.3 Carbon Dioxide Production Values resulting from Different Treatments...... 31

2.4 Acetate and Lactate produced during Fermentation ...... 32

2.5 Summary of Acetate, Hydrogen, and Carbon Dioxide...... 34

2.6 Glucose Consumption...... 36

A-1 Hydrogen Chromatography Data...... 50

A-2 Carbon Dioxide Chromatography Data ...... 52

A-3 Gauge Pressure, pH, Hydrogen, and Carbon Dioxide Concentrations ...... 53

B-1 Acetate Concentration Data ...... 58

B-2 Lactate Concentration Data...... 60

B-3 Glucose Concentrations in Standard Media...... 61

C-1 Glucose and Sucrose Concentration Data ...... 65

D-1 Total COD Data ...... 72

D-2 Soluble and Particulate COD Data ...... 73

vii LIST OF FIGURES

Figure Page

1.1 Emben-Meyerhoff Glucose Catabolism of T. maritima ...... 8

2.1 Sucrose and Glucose consumed for media containing Peach Solids...... 38

2.2 Change in COD...... 40

A-1 Percent Hydrogen Standard Curve...... 49

A-2 Percent Carbon Dioxide Standard Curve...... 51

A-3 Standard Medium Example Gas Analysis Chromatograph. Hydrogen peak is at 1.37 min; Nitrogen peak is at 1.65 min; and Carbon Dioxide peak is at 6.6 min ...... 54

A-4 Standard Medium Example Gas Chromatograph. This shows a close up of the Carbon Dioxide peak...... 54

A-5 Peach and Soybean Medium Example Gas Chromatograph. Hydrogen peak is at 1.29 min; Nitrogen peak is at 1.65 min; and Carbon Dioxide peak is at 6.6 min ...... 55

A-6 Peach and Soybean Medium Example Gas Chromatograph This shows a close up of the Carbon Dioxide peak ...... 55 A-7 Soybean Medium Example Gas Chromatograph. Hydrogen peak is at 1.24 min; Nitrogen peak is at 1.51 min; and Carbon Dioxide peak is at 6.6 min ...... 56

A-8 Soybean Medium Example Gas Chromatograph. This shows a close up of the Carbon Dioxide peak ...... 56

B-1 Acetate Standard Curve ...... 57

B-2 Lactate Standard Curve...... 59

viii List of Figures (Continued)

Figure Page

B-3 Glucose Standard Curve ...... 61

B-4 Example Standard Medium Pre-fermentation Chromatograph of Glucose (RT-9.3 min), Lactic Acid (RT-12 min), and Acetate (RT-14.9 min) ...... 62

B-5 Example Standard Medium Post-fermentation Chromatograph of Glucose (RT-9.3 min), Lactic Acid (RT-12 min), and Acetate (RT-14.9 min) ...... 62

B-6 Example Peach and Soybean Medium Pre-fermentation Chromatograph of Glucose (RT-9.3 min), Lactic Acid (RT-12 min), and Acetate (RT-14.9 min) ...... 63

B-7 Example Peach and Soybean Medium Post-fermentation Chromatograph of Glucose (RT-9.3 min), Lactic Acid (RT-12 min), and Acetate (RT-14.9 min) ...... 63

B-8 Example Soybean Medium Pre-fermentation Chromatograph of Glucose (RT-9.3 min), Lactic Acid (RT-12 min), and Acetate (RT-14.9 min) ...... 64

B-9 Example Soybean Medium Post-fermentation Chromatograph of Glucose (RT-9.3 min), Lactic Acid (RT-12 min), and Acetate (RT-14.9 min) ...... 64

C-1 Glucose Standard Curve ...... 65

C-2 Sucrose Standard Curve...... 65

C-3 Peach and Soybean with Vitamin Medium Pre-Fermentation Chromatograph. Sucrose has a retention time of 10.30 min and glucose has a retention time of 12.35 min...... 67

C-4 Peach and Soybean with Vitamin Medium Post-Fermentation Chromatograph. Sucrose has a retention time of 10.30 min and glucose has a retention time of 12.35 min...... 68

ix List of Figures (Continued)

Figure Page

C-5 Peach and Soybean with Vitamin and Trace Element Medium Pre-Fermentation Chromatograph Sucrose has a retention time of 10.30 min and glucose has a retention time of 12.35 min...... 69

C-6 Peach and Soybean with Vitamin and Trace Element Medium Post-Fermentation Chromatograph. Sucrose has a retention time of 10.30 min and glucose has a retention time of 12.35 min...... 70

D-1 Chemical Oxygen Demand (COD) Standard Curve ...... 71

x CHAPTER ONE

INTRODUCTION TO BIOHYDROGEN

1.1.Introduction

As of today most of the United State’s energy comes from fossil fuels, 81.8%, or nuclear energy, 8.5% (EIA, 2012). Both of these sources of energy are finite. Concerns of the increasing atmospheric carbon dioxide and its link to global warming also contribute to the impracticality of burning fossil fuels (Solomon, 2009). Given this petroleum fuels will become too expensive to use as transportation fuel. In looking to replace today’s gasoline with alternatives, hydrogen gas stands as a potentially carbon- free energy source (Benemann, 1996, Momirlan and Veziroglu, 2002)

Hydrogen has the highest energy density of all known fuels containing 143 KJ/g

(Brown, 2003). Comparatively gasoline has a density of 44 KJ/g and methane, 54 KJ/g.

This high amount of stored energy means that less fuel is needed for similar amounts of work. This energy can be easily and efficiently utilized via a fuel cell, which chemically converts the hydrogen into water and electrical current (Winter, 2005). The current can be used to drive a motor if used in transportation or can be utilized as electrical power for back-up power systems. The hydrogen is oxidized to produce water and energy. Unlike carbon based fuels hydrogen combusts cleanly with no green house gas emissions.

Given the huge potential H2 has for providing clean energy in the new century much more of it should be produced both as a fuel and for a number of industrial chemical processes. In 2011 9,303,000 kg of hydrogen were produced in the US (DOE

1 2012). A large portion of hydrogen in today’s markets is used for making ammonia.

Ammonia itself plays a major part in industrial chemistry and is a critical component in fertilizer. In 2010 the US produced 8.29 million metric tons and consumed 13.8 million metric tons of ammonia. Of this amount 86% was used for fertilization (USGS 2012).

Modern agriculture relies so exclusively on chemical fertilizer for a crop’s nitrogen source that a drop in fertilizer production or even sharp price increases could be disastrous to food production across the world.

Today the most common way of producing hydrogen is from reforming of fossil fuels (Levin et al, 2004). The reforming process extracts the hydrogen bound in the organic compounds found in petroleum and natural gas. Steam-methane reforming is the most common technique in which hydrogen is produced in the US accounting for nearly

95% of all the hydrogen produced (USDOE, 2012). The process involves reacting methane and steam at very high temperature (700-1000 °C) and pressure (3-25 bar) into carbon monoxide and hydrogen. The CO is further reacted in a gas-shift reaction with water to produce carbon dioxide and hydrogen. Other reforming processes capture the hydrogen that is released in the rearranging of larger organic molecules found in petroleum reforming.

To create hydrogen through electrolysis an electrical current (DC) is applied to water. The result is the reduction of hydrogen in the water molecules to hydrogen gas at the anode. The energy recovered in the gas however can be much less than put into the process. Thus, it is counter intuitive to use fossil fuel produced electricity to produce hydrogen for energy. Using renewable sources such as wind energy or hydroelectric to

2 drive the process could make this technique a part of America’s hydrogen production portfolio, but only for certain sections of the country (Christopher and Demitrios, 2012).

Thus a new way of producing hydrogen that is carbon neutral, sustainable, and economical must be developed.

1.1.2.Biologically Derived Hydrogen

One promising method being developed for generating hydrogen utilizes biological means (Hallenbeck and Benemann, 2002, Claassen et al. 2010). A number of organisms have been shown to naturally produce hydrogen gas through either fermentation or photobiological processes.

Photobiologically produced hydrogen implies the use of light to create hydrogen.

A number of organisms have been identified that can produce hydrogen using light

(Dasgupta et al. 2010). Three techniques have been identified: direct biophotolysis, indirect biophotolysis, and photofermentation.

Direct biophotolysis utilizes the electrons provided after the cleaving of water by photosystem II (PS II). Normally, these electrons are shuttled through PS II then into PS

I where they are used to reduce ferredoxin. The reduced ferredoxin is then used to convert NADP+ to NADPH, which then shuttles electrons to the Calvin cycle. In direct biophotolysis, the electrons from ferredoxin are shuttled instead to hydrogen ions to produce hydrogen gas (Eq. 1).

3 + − − + 2H2O + hν → O2↑ + 4H + Fd (red) (4e ) → Fd (red) (4e ) + 4H → Fd (ox) + 2H2

(Dasqupta et al. 2010)

This method has been observed in many species of green algae including

Chlamydomonas reinhardtii and Scenedesmus obliquus (Laurinavinchene et al. 2006,

Vignais et al. 2001).

Similarly indirect biophotolysis utilizes the energy from light and the electrons from water. However, it produces hydrogen separately from the light reactions. First the organism produces carbohydrates from CO2, H2O and light. Afterward the organism is exposed to oxygen-depleted conditions and light. Utilizing the light energy, algae oxidize the starch and cyanobacteria glycogen then the electrons are transferred to protons to produce H2 and CO2. As seen in the following equation:

6CO2 + 6H20 + hv → C6H12O6 + 6O2

C6H12O6 + 6H20→ 6CO2 +12H2

Spirulina, Anabeana, and Oscillatoria are cyanobacteria that have shown indirect photobiolysis. (Aoyama et al. 1997, Berberoglu et al. 2008, Phlips and Matsui 1983).

The photosynthizing purple non-sulfur bacteria like Rhodobacter sphearoides produce hydrogen through photofermentation (Yedis et al. 2000). During this process outside substrate can be consumed under anaerobic conditions to produce H2. This process is catalyzed by nitrogenase compared to hydrogenase in the case of the direct and

4 indirect photobiolysis. Electrons from organic substrates are shuttled via ferrodoxin to the nitrogenase, which works to fix nitrogen in the air. In the N-fixing process H2 gas is produced. This process is energy inefficient due to the necessary input of 2 electrons and 4 ATP to produce one mole of hydrogen but does provide the capacity of using and treating waste to produce hydrogen.

Species utilizing fermentation include but are not limited to Clostridium pastueurianum, Clostridium thermolacticum, Thermoanaerobacterium thermosaccharylyticum, and Caldicellulosiruptor saccharolyticus (van Niel et al. 2002;

Talabardon et al. 2000). These organisms produce a variety of coproducts along with hydrogen gas depending on the growth conditions and the organism. Butyrate and acetate are coproducts produced by C. pastueurianum. Under high partial pressures of

H2 gas (1 atm) 1 mol of glucose is used to create 2.6 mol H2, 0.6 mol acetate, 2 mol CO2, and 0.7 mol butyrate and 3.3 mol ATP. In comparison with low pressures; 4 mol H2, 2 mol acetate, 2 mol CO2, and 4 mol ATP are produced per mol of glucose (Shroder et al,

1994). If butyrate is the primary fermentative product half the amount of hydrogen gas is produced.

- - + C6H12O6 + H2O → CH3CH2CH2COO + 2HCO3 + 3H + 2H2 (Zinder 1984)

Other products generated through fermentation include ethanol, propionate, butanol, and lactate (Hawkes et al. 2002, George et al. 1983, Hwang, M. H., et al. 2004).

5 When ethanol is the product of fermentation only 2 mol of H2 are created (Hwang, M.H.. et al. 2004).

Mixed cultures of anaerobic sludge have also been investigated in their ability to create hydrogen gas from a variety of agricultural products (Han and Shin, 2004). These systems have been grown on potato starch, molasses, sugar beets and lactate (Logan et al.

2002, Hussy et al 2005). Pretreated cornstalks have also been used as feedstock for anaerobic sludge hydrogen generation (Wang et al. 2012, Abreu et al. 2012).

The genera Thermotoga has also shown great potential for producing hydrogen through fermentation. Much research has been put into understanding this group of organisms and utilizing their unique ability to turn common sugars into hydrogen.

1.1.3.Background Thermotoga Genus

T. maritima was the first of the genus to be discovered in 1986 in Vulcano, Italy

(Belkin et al. 1986). This organism was found in geothermally heated marine sediments.

During the same time period another species T. neapolitana was discovered on hydrothermal vents in the Bay of Naples (Jannasch 1988). Since then numerous species have been discovered around the world. T. maritima and T. neapolitana have also been isolated in other parts of the world since their original discovery.

Thermotoga, as named, lives in very hot environments. Naturally it thrives in the extreme heat of hot springs, geothermal vents, and other thermally warmed environments.

Living in temperatures up to 90° C and having optimum temperatures at or above 65° C

6 they are hyperthermophilic bacteria (Huber et al, 1986). They are also strictly anaerobic and are killed in the presence of oxygen.

1.1.4.Thermotoga Metabolism

The fermentation process of Thermotoga is quite unique. It uses the Embden-

Meyerhoff pathway like other fermentative processes but uses a unique ferrodoxin- coupled reaction to oxidize NADH and replenish NAD+ (Schroder et al. 1994). It also derives approximately 15% of its energy needs from the Entner-Doudoroff pathway as well with lactate being produced (Selig et al., 1997. d’Ippolito, 2010). In fact research suggests that the mechanism behind the proton reduction is a unique enzyme that simultaneously oxidizes reduced ferrodoxin and NADH to reduce the hydrogen ion (Shut and Adams, 2009). This type of enzyme has never before been documented and is a good example of the distinctive characteristics of this organism (Atomi et al. 2011). The process is shown in Figure 1.

The overall reaction of the EMP pathway fermentation is as follows (Shroder et al. 1994):

- + C6H12O6 + 2H2O + 4ADP  2CH3COO + 2H + 2CO2 + 4H2 + 4ATP

Through this breakdown of glucose 4 mol of ATP are formed per mole of glucose. This is higher than the normal 2 mol of ATP produced in other fermentation processes. The stoichiometry also shows a maximum yield of 4 mol of hydrogen per mol of glucose.

7 This has not been reproduced in laboratory experiments however and the largest yield being produced by T. neapolitana is 3.8 mol of H2 per mol of glucose (Munro et al.

2009).

Figure 1.1 – Embden-Meyerhoff Glucose catabolism of T. maritima. Adapted from

Schroder et al. 1994

1.1.5.Thermotoga Fermentations

T. neapolitana has mostly been cultured in batch reactors with the hydrogen being collected in the head space. In these experiments strict anaerobic conditions are

8 maintained. T. neapolitana Monod kinetic parameters have been estimated at 77° C with

-1 µmax = 0.94 h and Ks = 0.57 g glucose/liter (Yu and Drapcho 2011). The product yield was found to be 0.0286 g H2/g glucose and the biomass yield was found to be 0.248 g

Xb/g glucose (Yu and Drapcho 2011).

Though pure glucose has given the best yield of hydrogen per mol of carbon source other feedstocks have shown promise as well. Substrates containing hexose and pentose sugars like rice flour, cellobiose, starch, xylose, and sucrose have all been used to culture T. neapolitana (Van Ooteghem 2002;Yu and Drapcho 2011). Yu reports no significant difference in mmol H2/L-medium between using sucrose, xylan, or glucose as the carbon source at approximately 31 mmol H2/L-medium. Culled peaches that contain high levels of sucrose have also been used as a substrate (Hill, unpublished). Rice flour, cellobiose, corn starch, and xylose all produced between 23-26 mmol H2/L-medium (Yu and Drapcho, 2011). Another agricultural byproduct being researched is rice straw. A process that uses rice straw has been patented to researchers in Korea (Sun and Jun,

2010). This process uses ammonia and sulfuric acid in pretreatments to remove lignin and breakdown hemicellulose (Nguyen et al. 2010). These products are then used as substrates for fermentation. Another study showed pretreatment of miscanthus provided usable substrate for T. elfii (de Vrije. 2002). Instead of the ammonia and acid treatment this study used sodium hydroxide to remove lignin then followed by enzymatic hydrolysis. Recently, cheese whey and molasses has also shown potential for hydrogen production for four species of the genera: T. neapolitana, T. maritima, T. naphtophila, and T. petrophila. Of the four species T. neapolitana produced the highest amount of

9 hydrogen/liter medium (Cappelletti, 2012). The ability of Thermotoga to utilize these waste products is invaluable in a new generation of biofuels from biowastes (Demirbas et al. 2011)

T. neapolitana has been reported to lack the mechanisms to use more reduced substrates such as alcohols and volatile acids (Jannasch et al. 1988). However, recently new research has shown that both glycerol and untreated glycerol waste from biodiesel production has been used to produce hydrogen (Ngo et al. 2011), though not at as high a yield per mol as other carbon sources.

Multiple nitrogen sources have been investigated. Yeast extract, soybean meal, canola meal, and linseed meal have successfully been used to culture T. neapolitana as the nitrogen source (Yu and Drapcho, 2011). No significant difference was found between medium using glucose or sucrose as the carbon source and soybean meal or yeast extract as the nitrogen source.

T. neapolitana has also been grown in a fed batch reactor as well (Tien et al.

2011). In these experiments the pH was controlled during the fermentation and product yield when glucose was used as the carbon source was 3.2 mol H2 per mol glucose. This yield was 1.6 times higher than when the fed batch was not pH controlled. T. neapolitana has repeatedly been shown to have a pH optimum of 7 and has a range from

5-9 (Van Ooteghem 2002, Munro et al. 2009) and any practical reactor would need to be pH controlled. No continuous system has been published for Thermotoga, however a continuous system of mixed thermophilic bacteria has been conducted (Qiu et al 2011,

10 Abreu et al. 2012, Hussy 2005). The substrate for this system was ethanol distillery wastewater. They had a yield of 196 ml hydrogen/ g volatile solids.

The process of making hydrogen gas by any member of Thermogales has been patented (Van Ooteghem. 2005). The patent protects any fermentation process containing these organisms at any temperature above 45°C and with a pH between 4-9.

The patent also covers almost every known substrate requiring any commercialization of this process to go through this patent.

1.1.6.Conclusions

For Thermotoga to play some part in hydrogen production more progress must be made. Much research has been done on different feedstocks for Thermotoga fermentations which have shown that the organisms can produce hydrogen from many types of sugars and larger molecules. The ability of the Thermotoga to utilize so many types of substrates is one strong argument for this process. Especially the ability to utilize xylose which is a huge component of biomass and cannot be fermented easily into ethanol.

Also favorable growth conditions have been determined such as temperature and pH. The high temperature of the system could be a large energy cost in future applications, but efficient insulation and recycling of waste heat in a large biorefinery could cut these cost.

The next step seems to be how to make more gas faster as well as cheaper. These conditions can now be applied to scale ups and continuous reactors. Scaling up and

11 running the system continuously can produce more hydrogen, which could then make the system economically competitive. Also these experiments could zero in on growth parameters that could allow optimization of the process in general. Simplification of media will bring the cost of production down. By using waste agricultural products such as culled peaches, feedstock costs can be practically eliminated. The generation of hydrogen gas could become much more sustainable with this new process and with a sustainable way to produce hydrogen we could then make our society more sustainable.

1.2.Goals of this Study

The overall goal of this study is to determine the impact of withholding vitamins and trace elements from a standard medium containing glucose and yeast extract and an agricultural-based medium containing blended peaches and soybean meal as the carbon and nitrogen sources, respectively. The specific objectives are as follows:

1. To determine hydrogen gas production by Thermotoga neapolitana grown in

standard medium and agricultural medium with and without vitamins and trace

elements

2. To determine the organic acid production and sugars consumption by T.

neapolitana grown in standard medium and agricultural medium with and without

vitamins and trace elements

12 1.3.Chapter Two

This chapter presents the study of using peach and soybean media as carbon and nitrogen sources for fermentation of T. neapolitana. The vitamins and trace elements were withheld from a standard medium and from a media using peaches and soybeans.

The hydrogen and carbon dioxide produced by T. neapolitana from all media is presented in this chapter. The production of acetate and lactate is also documented. Lastly the sugar consumption used in the growth of this organism was analyzed through HPLC and

COD and the results are provided.

1.4.Chapter Three

Chapter three presents the conclusions of this study. It also provides a direction for further investigation of T. neapolitana fermentations. The impact of this study on commercial production of biohydrogen through agricultural byproduct is also discussed.

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18 CHAPTER TWO

SIMPLIFICATION OF PEACH AND SOYBEAN MEDIA FOR

BIOHYDROGEN PRODUCTION WITH Thermotoga neapolitana

2.1.Introduction

Both energy and synthetic nitrogen fertilizers play major roles in modern society.

As of today both are cheap and abundant. However, both rely on inexpensive sources of fuel. This cheap fuel is and has been traditionally found in the form of fossil fuels.

These fuels may not remain cheap though as their price is increasing and will continue to rise as their availability diminishes. Fertilizers are created using natural gas and syngas from coal and petroleum refining. Through their combustion and refining fossil fuels like coal, natural gas, and petroleum introduce high levels of greenhouse gases into the atmosphere. These greenhouse gases are directly linked to global warming (Solomon,

2009). Hydrogen gas however does not produce greenhouse gas when combusted.

Hydrogen is also one of the main ingredients in ammonia production, a valuable fertilizer. Therefore, many predict it will become a significant source of energy in the future (Benemann, 1996, Momirlan and Vesiroglu, 2002).

Hydrogen has a number of properties that make it an attractive alternative. First, hydrogen contains 143 KJ/g which is the highest energy density of all known fuels that undergo combustion (Brown, 2003). To compare, gasoline has 44 KJ/g and methane has

54 KJ/g. When combusted the hydrogen gas is combined with oxygen to produce only

19 water. This oxidation can take place in a fuel cell or in an engine. These characteristics of hydrogen are driving many to discover how to adopt it more effectively.

Modern agriculture relies very heavily on hydrogen via ammonia. The production of ammonia uses nitrogen and hydrogen. This ammonia is then used as a nitrogen fertilizer. The US produced 8.29 million metric tons of ammonia and consumed 13.8 million metric tons in 2010 (USGS 2012). Of the ammonia consumed 86% was used as fertilizer. The farms of America are strongly connected to the hydrogen market.

As of today by far the most common way to produce hydrogen is through reforming of fossil fuels (Levin et al, 2004). Steam-methane reforming produced 95% of the 9,303 metric tons of hydrogen manufactured in the US in 2011 (USDOE, 2012). This practice breaks the methane into carbon dioxide and hydrogen. Electrolysis is also used to produce hydrogen by applying an electrical current to water. The water is split into oxygen and hydrogen. This process is very inefficient and is not in widespread use. Both steam-methane reforming and electrolysis are non-sustainable.

However, a developing method for generating hydrogen is through biological means (Hallenbeck and Benemann, 2002, Claassen et al., 2010, Yu and Drapcho, 2011).

This biohydrogen can effectively be produced sustainably for it uses natural systems.

Many organisms have been shown to produce hydrogen through fermentation.

These include Thermoanaerobacterium thermosaccharylyticum, Clostridium pastueurianum, Clostridium thermolacticum, and Caldicellulosiruptor saccharolyticus

(van Niel et al. 2002, Talabardon et al. 2000). These organisms have been shown to produce hydrogen gas along with a number of other fermentation products such as

20 ethanol, propionate, butyrate, lactate, and acetate (Hawkes et al. 2002, George et al. 1983,

Hwang et al. 2004). The amount of hydrogen produced is dependent on the coproduct with more produced when acetate is a coproduct, 4 mol H2, than when butyrate is formed,

2.6 mol H2, per mole of glucose (Shroder et al, 1994).

The genera Thermotoga shows great potential for the production of hydrogen. T. maritima was first discovered off the coast of Italy in 1986 (Belkin et al. 1986).

Thermotoga neapolitana was discovered shortly after in the Bay of Naples (Jannasch et al.1988). As the name implies these organism are hyperthermophilic living in temperatures up to 90° C and at or above 65°C.

T. neapolitana derives most of its energy via the Embden-Meyerhof pathway

(EM) similar to other fermentative processes. It utilizes a ferrodoxin-coupled reaction to oxidize NADH to replenish NAD+ (Schroder et al. 1994). This reaction is driven by the simultaneous oxidation of reduced ferrodoxin and NADH to reduce the hydrogen ion and produce hydrogen gas (Shut and Adams, 2009). It also takes advantage of another pathway as well, the Entner-Doudoroff pathway (ED). This pathway produces approximately 15% of the organisms energy needs with lactate being the final product

(Selig et al., 1997, d’Ippolito, 2010). The EM pathway converts 1 mol glucose to produce 2 mol acetic acid, 2 mol carbon dioxide, 4 mol ATP, and 4 mol hydrogen gas.

The ED pathway produces 1 mol ATP and 2 mol lactate per mol of glucose. Given this the theoretical maximum yield is 4 mol hydrogen when all glucose is shuttled through the

EM pathway. The highest observed yield has been 3.8 mol H2 per mol glucose (Munro et al. 2009).

21 Monod kinetic parameters for T. neapolitana have been estimated. At 77° C the

-1 µmax was determined to be 0.94 h and Ks was equal to 0.57 g glucose/liter (Yu and

Drapcho). The product yield was 0.0286 g H2/g glucose. The biomass yield was 0.248 g

Xb/g glucose. (Yu and Drapcho, 2011).

` Many substrates have been used to culture T. neapolitana. Glucose has consistently provided the best yields however Yu reports no significant difference in hydrogen production between using sucrose, xylan, or glucose as the carbon source at approximately 31 mmol H2/L-medium (Yu and Drapcho, 2011). Other feedstocks for the fermentation that have successfully produced hydrogen include rice flour, starch, xylose, and cellobiose which all produced between 23-26 mmol H2/L-medium (Yu and Drapcho,

2011, Van Ooteghem 2002). Agricultural byproducts such as molasses and cheese whey act as substrate for growth (Cappelletti 2012). Peaches contain 8.74 % sugars wet weight.

These sugars consist mostly of sucrose, glucose, and fructose (Li et al, 2002). The sugars in cull peaches have also been used to culture T. neapolitana (Hill unpublished). T. neapolitana cannot utilize cellulose (Yu and Drapcho, 2011)

Another source of carbohydrate for fermentation has come from pretreated cellulosic feedstocks. Rice straw was pretreated with ammonia and sulfuric acid to remove lignin and degrade hemicellulose then used as a substrate for successful culture of T. neapolitana. Pretreated miscanthus was also used to produce hydrogen through the growth of Thermotoga Elfii. In this study the pretreatment used was sodium hydroxide for lignin removal and enzymatic hydrolysis broke down the cellulose (de Vrije et al.

2002).

22 Multiple nitrogen sources have been investigated. Yeast extract, soybean meal, canola meal, and linseed meal have successfully been used to culture T. neapolitana as the nitrogen source (Yu and Drapcho, 2011). No significant difference was found between medium using glucose or sucrose as the carbon source and soybean meal or yeast extract as the nitrogen source (Yu and Drapcho, 2011).

In this study the ability of T. neapolitana to grow and produce hydrogen from low-cost agricultural byproducts is further investigated. The standard media produces shigh levels of hydrogen (Yu and Drapcho, 2011). This medium identified as the

Standard Medium was shown to produce hydrogen at a 96.25% efficiency (d’Ippolito,

2010). This media however requires meticulous measurements of dozens of chemicals.

These chemicals are expensive and would make any production outside of the laboratory unachievable.

T. neapolitana, as already mentioned, has been shown to grow on the sugars found in culled peaches. In the same study the organism was also shown to grow with soybean meal substituted as a nitrogen source (Yu and Drapcho, 2011). Both these complex substrates contain much more than carbohydrate and nitrogen however. They have the metals and coenzymes necessary for common life processes. In the standard media a solution of vitamins (DSM Media 141) and a solution of trace elements (DSM

Media 141) must be added to supply these resources. The objective of this study is to examine the effects of withholding the vitamins and trace element solutions from the standard medium containing glucose and yeast extract and from an agricultural-based medium containing peach flesh and soybean meal on hydrogen production.

2.2.Materials and Methods

2.2.1. Media

The experiment consisted of 7 treatments. Each treatment was repeated with four separate 565 ml serum bottle filled with 200 ml of designated media leaving 365 ml of headspace. The following medium identified here as the Standard Medium contains as follows: 5g of glucose, 2.0g of yeast extract, 2.0g of Trypticase, 10.0g of NaCl, 0.121 g of THAM, 1.114 g of cysteine HCl monohydrate, 10.0ml of vitamin solution (DSM media 141), 10.0 ml of trace element solution (DSM media 141), a salt solution, and 1.0

L of H2O.

Glucose was substituted with blended peaches as a carbon source for four of the treatments. The peaches were obtained from Musser Farms, Clemson, SC are the Red

Prince varietal. The pits were removed and all peaches were blended with skins together for 5 minutes. The peach slurry was allocated into 0.5 liter bags and stored at -40° C.

For all peach treatments 5 g of peach solids were added per liter of medium. To determine solids content peach slurry was dried in an oven at 105°C. Percent solids weight was determined to be 13.09%. Thus to obtain 5 g/l of solids, 38.19 g of peach slurry was added to each liter of peach media.

Soybean meal was substituted as a nitrogen source for 5 of the treatments.

Southern States manufactured the soybean meal. The meal was screened to contain particles ≤1 mm. The screened meal was then dried at 60° C prior to addition to media.

24 The experiment consisted of the following treatments found in table 2.1.

Table 2.1 – Batch Treatments

2.2.2.Culture

The culture of Thermotoga neapolitana was obtained from DSMZ, strain 4359

(German Collection of Microorganisms and Cell Cultures).

2.2.3.Growth Procedure

The medium preparation/inoculation procedure was as follows: the initial pH of the medium was adjusted to 8.0 with 5 N NaOH and 1 ml of 1% resazurin was added as an oxygen indicator. 200 ml of the medium was transferred into 4 serum bottles. The headspace of the bottles was then sparged for 1 minute with nitrogen to remove oxygen.

The serum bottles with media were capped with septa and allowed to rest for 90 minutes to allow the Cysteine HCl to scavenge oxygen from the system. The medium was then

25 inoculated with 5ml of inoculum with a sterile syringe. The culture was incubated in an orbital shaker bed at 150 RPM at 77° C for 40 hours.

2.2.4.Gas Analysis

After incubation the bottles were then placed in a water bath for 30 minutes to cool the temperature to 25° C. Afterwards the pressure was measured through the septum with a manometer to detect pressure.

The concentration of hydrogen and carbon dioxide in the headspace was measured using a Gow-Mac Series 400 - gas partitioner with a thermal conductivity detector. The carrier gas was argon set at a pressure of 22 psi. The column used was a

10’ X 1/8” and packed with Molecular Sieve 5A, alkali alumino silicate. The pressure was set at 22 kPa. A 0.5 ml of headspace was sampled with a 0.5 ml syringe and then manually injected into the GP. Using a standard curve the percentage of hydrogen and carbon dioxide was obtained for each serum bottle’s headspace.

The percentage of hydrogen and carbon dioxide and the pressure within the headspace was then used to determine the moles of gas by utilizing the ideal gas law (Yu and Drapcho, 2011). The partial pressure of each gas was determined using the percentage of gas and the absolute pressure. By substituting the partial pressure of hydrogen or CO2 and the headspace volume, the number of moles in the headspace could be found. The concentration of gas was found by dividing the moles of gas by the volume of media and multiplying by 1000 mmol/mol.

26 To further take account of dissolved species of carbon dioxide Henry’s law was

used.

CCO 2, aq = kh * pCO 2

In this equation, kh represents Henry’s constant in M/atm. At 25 °C, kh is equal to

€ 0.036 M/atm (Sanders, 1999). CCO2, aq is the concentration of carbon dioxide in mol/L -4 and pCO2 is the partial pressure of CO2. For calculating dissolved H2 a kh of 6.08 x 10

M/atm was used (Sanders, 1999).

2.2.5.Chemical Oxygen Demand

The total and soluble chemical oxygen demand, COD, of the media before and

after fermentation was also measured. Prior to testing COD each serum bottle was

uncapped and its contents poured into a 400 ml beaker. The pH was then measured with

a Thermo Scientific pH probe. Then the media , both post and pre-fermentation, was

blended with a handheld emersion blender for 2 minutes to homogenize the particulates.

The blended media was then allowed to sit for 5 minutes to allow gas bubbles to rise. The

beaker was then stirred as the particulate COD sample was taken. Each COD sample

volume was 0.2 ml of media. Afterward the soluble COD was obtained by filtering the

media through a 0.45 µm filter.

27 2.2.6.Sugar and Organic Acid Analysis

For analysis of glucose, acetate, butyrate, and lactate; 0.45 µm filtered samples of the pre- and post-fermented media was analyzed using HPLC with a refractive index detector. The mobile phase was 0.01 N H2SO4 with a flow rate of 0.6 ml/min. The column was a Bio-Rad Aminex HPX-87H. Standards were made in a 10 g/l NaCl solution.

To measure sucrose and glucose in peach media filtered samples were also tested on HPLC using a Bio-Rad Aminex HPX-87P column. The mobile phase was distilled water with a flow rate of 0.6 ml/min.

2.2.7.Statistical Analysis

The software JMP 10 was used to test significant difference of the hydrogen and carbon dioxide produced for each treatment. A Tukey’s Studentized range test was used with a p-value of 0.05.

2.3.Results and Discussion

2.3.1.Gas Analysis

2.3.1.1.Hydrogen

The mean hydrogen concentrations determined for each treatment are found in table 2.2.

Table 2.2. Hydrogen Concentration Values resulting from Different Treatments[a]

[a] Means followed by different letters are significantly different using Tukey’s

Studentized range test (p < 0.05)

[b] n = 4

The standard medium, treatment 1, had the highest pressure with a mean pressure of 57.3 kPa. The standard without the added vitamins and trace elements, treatment 2, had a lower pressure of 46.7 kPa. All peach and soybean media, treatments 3,4,5, and 6, had similar pressures to one another while the soybean meal without peach medium, treatment 7, had a pressure of 18.4 kPa.

After the pressures were recorded the percent hydrogen was analzyed using a

Gow-Mac gas partitioner. Treatment 1 significantly had the highest percentage of H2 in the headspace gas having a percentage of 26.76 %. Treatment 2 had 23.48 % hydrogen.

29 Treatments 3,4,5, and 6 again showed similar percents hydrogen, 21-22 %, following similar though small trends as the headspace pressure. Treatment 7 contained 11.07 % hydrogen.

Treatment 1, the standard media culture, produced 31.5 mmol H2/L. Treatment 2 did not have as much gas with a concentration of 25.8 mmol H2/L. The peach media treatments again performed similarly to each other. The dissolved concentration was calculated for each treatment and was found to be 0.25, 0.21, 0.19, 0.18, 0.19, 0.19, and

0.08 mmol H2/L respectively for treatments 1-7.

The results of the Tukey test showed a significant difference between treatment 1 and all other treatments in hydrogen production. This is consistent with previous tests

(Yu and Drapcho, 2011). The vitamins and trace metals in the standard medium allowed the fermentation to produce more hydrogen. However, there was no significant difference between hydrogen concentration for treatments 2 – 6; the standard medium without the added vitamins and trace metals and the peach/soybean media with and without vitamins and metals. These results suggest that the media containing peach flesh and soybean meal contained sufficient vitamins and trace metals to meet the needs of the organism.

Treatment 7 had significantly less than all other treatments. This is not surprising given that the only carbon source available was the starch in the soybean meal.

The pH of all the cultures dropped after incubation, Table 2.2. Treatment 1 ended with the lowest pH and treatment 7 ended with the highest. The more hydrogen detected for each treatment the larger the drop in pH.

2.3.1.2.Carbon Dioxide

Table 2.3 presents the concentration of CO2 for each treatment.

Table 2.3. Carbon Dioxide Production Values resulting from Different Treatments[a]

[a]Means followed by different letters are significantly different using Tukey’s

Studentized range test (p < 0.05)

[b] n = 4

The percent carbon dioxide varied between the treatments. Because CO2 is fairly soluble in water, the concentration of dissolved CO2 was also calculated. The trend is similar to that seen in the hydrogen tests with treatment 1 showing the highest percentage of CO2. Treatment 7 as well displayed the lowest fraction of CO2. Treatments 2-6 did not

31 exhibit a statistically significant different amount of CO2. Treatment 7’s level of CO2 was significantly lower. The similar trends between headspace pressures, mmol CO2/L, and mmol H2/L can be attributed to the build up of gas in the headspace. Gas was produced as fermentation proceeded in each reactor. The more hydrogen and carbon dioxide created, the higher the pressure in the serum bottle since no air was exchange with the outside environment.

2.3.2. Organic Acids

The concentrations of acetate and lactate can be seen in table 2.4.

Table 2.4 – Acetate and Lactate Produced during Fermentation

[a] n = 4

32 The acetate levels follow the same trend as hydrogen and carbon dioxide concentrations. Treatment 1 had the highest concentration, treatment 7 had the lowest concentration, and the concentrations of all peach treatments were very similar. The explanation for these similar trends can be found in the overall metabolic equation T. neapolitana utilizes to produce ATP.

- + C6H12O6 + 2H2O + 4ADP  2CH3COO + 2H + 2CO2 + 4H2 + 4ATP

As can be seen for every 4 moles of hydrogen produced, 2 moles of carbon dioxide are created. One mole of glucose is consumed and 2 moles of acetate are produced. Thus all products of this fermentation pathway should show a ratio of 2 mol

H2: 1 mol CO2: 1 mol acetate. The concentrations of these compounds have been summarized and are presented in the following table 2.5.

Table 2.5 – Summary of Acetate, Hydrogen, and Carbon Dioxide

[a] n = 4

The ratios of the products of all treatments support the expected ratio. In treatment

4, for example, given the 22.87 mmol/L concentration of hydrogen the expect amount of acetate created would be expected to be 11.435 mmol/L. The actual level detected was

11.36 mmol/L, for a percent difference of only 0.7%.

For treatment 7 the amount of carbon dioxide detected is much higher than expected. This is most likely due to the chromatography used. The HPLC tests of the acetate and the GP analysis of hydrogen provided more consistent results (Appendix

A,B). The chromatograph response peaks for these compounds had good resolution. The

34 thermal conductivity detector of the GP did not provide a strong response for carbon dioxide (Appendix A). This poor response may have misrepresented the level of carbon dioxide.

Inspecting the levels of acetate, carbon dioxide, and hydrogen revealed a similar pattern due to the fact all three products were derived via the same pathway. But the production of lactate however does not follow the same pattern (Table 2.4). Instead the culture in treatment 2 produced significantly more lactate than all other treatments (5.99 mmol lactate/L). Treatment 1 resulted in the prodution of 3.73 mmol/L. Treatments 3 – 7 had concentrations of only 0.07 – 0.22 mmol lactic acid/L.

The production of the lactate is derived via the Entner-Duodoroff (ED) pathway and not the acetate producing Embden-Meyerhoff (EM) pathway. Each pathway is controlled with different enzymes. The reason why treatment 1 had more acetate and hydrogen than treatment 2 which instead had more lactate may have to do with these two pathways. The vitamins and trace elements could have an effect on these pathways. The organism may rely more upon lactate production as a means of restoring NAD+ and producing ATP when the vitamins and metals are not available.

For all treatments no butyrate was detected.

2.3.3.Sugar Analysis

The concentration of glucose both before and after fermentation from treatments

1, 2, and 7 are in Table 2.6 and were found using the Aminex HPX-87H.

35 Table 2.6 – Glucose Consumption

[a] n = 2

[b] n = 4

Incomplete utilization of glucose was observed for both treatments 1 and 2, likely due to inhibition caused by decline in pH and accumulation of hydrogen gas. Both treatments 1 and 2 contained the same concentration of glucose; however the data suggest that the presence of the vitamins and trace metals in treatment 1 medium allowed for more complete utilization of glucose by T. neapolitana. Treatment 7 also showed a decrease in the concentration of glucose but much smaller than that of the other two treatments.

T. neapolitana’s theoretical yield for hydrogen is 4 mol hydrogen gas per mol of glucose consumed. The standard medium culture produced 72.5% the theoretical yield.

A yield of 69.8% was measured in treatment 2.

Lactate was detected in all fermentations as a coproduct. The theoretical yield for lactate is 2 mmol lactate per mmol glucose consumed. Treatment 1 produced 3.73 mmol lactate/L which means 1.87 mmol glucose was shuttled via the ED pathway.

36 Summing the glucose that was used to produce acetate (EMP pathway) and the glucose that was used in the ED pathway, 8.875 mmol of glucose was fermented into organic acids for ATP production for the standard media. For the standard media without added vitamins and trace metals a total of 9.065 mmol of glucose was fermented into organic acids.

Sugars in treatments 3-6 were not tested on the HPX-87H column. The fructose in the peach flesh eluded very close to glucose. The poor separation of sugars was seen as a broadly split-peak on the chromatograph (Appendix B). The split peak did not allow for accurate measurements of glucose in these samples.

In an attempt to quantify the amount of sucrose, glucose, and fructose used in the peach medias, samples from these treatments were run on an Aminex HPX-87P column.

The NaCl, however, found in the media eluded at the same retention time as fructose and did not allow for an accurate measurement of fructose. The utilization of these sugars is in the following figure.

Figure 2.1 – Sucrose and Glucose consumed for media containing peach solids (Error

Bars indicate one St. Dev.)

The amount of sucrose and glucose consumed within treatment 3 were substantially higher than other three treatments.

However, in all sucrose readings there was a high variability. The large error found in these readings may be related to the column used and the high salt concentration within the media. The Aminex HPX-87P column is a lead based column. The column is designed to test for mono- and disaccharides. The lead ions form temporary ligands with the sugars and thus separate the sugars via adsorption to the bound lead. High concentrations of salts like NaCl can however form ionic salt complexes with the lead

38 forming a lead salt. This salt will then elute from the column and be read by the detector.

All samples contained 10 g/L NaCl including all standards. The baseline from all samples exhibited a sustained ramping. The ramping was not entirely consistent but started at the beginning of elution for each run. Near the end of the run and when most of the sample had eluded from the column the baseline returned back to zero. The values reported in Figure 2.1 were all derived by using the ramped portion as the new baseline.

2.3.4.Chemical Oxygen Demand

The chemical oxygen demand (COD) of the media pre and post fermentation was investigated. The COD gives a measurement of how much oxygen would be needed to completely oxidize the organic compounds in a sample. The total COD concentration should decline in the medium during fermentation as the sugars are oxidized to carbon dioxide and the reduced product hydrogen gas escapes to the headspace. The results of the COD tests can be seen below in Figure 2.2.

Figure 2.2 – Change in COD (Error Bars represent 1 St. Dev)

The results of the COD tests were not as conclusive as many of the other tests run.

However, for treatment 1 and 2 both significantly reduced the COD of the media. The peach and soybean treatments all reduced the COD but not to a significant level. The soybean meal exhibited no significant increase in COD.

The other treatments exhibited much less reliable data as can be seen in the standard deviations most likely due to sampling error. The sampling volume was 0.2 ml and due to the large size of particulates the variance was quite high. Some samples could contain large pieces of soybean meal while others contained no large pieces.

2.4.Conclusion

T. neapollitana was able to grow and produce hydrogen gas in all media. Final concentrations of hydrogen gas in all media containing peach solids as the carbon source were approximately 75% of the values obtained from the standard medium containing glucose. Withholding vitamins and trace elements showed no significant impact on hydrogen production when peach solids were used as the carbon source and soybean meal was used as the nitrogen source. On the other hand, there was a significant impact on hydrogen production when vitamins and trace elements were withheld from the standard medium. The reduction in hydrogen gas produced may partly be due to the increased utilization of the Entner-Doudoroff pathway. The data suggest that both standard media treatment cultures utilized this pathway with the treatment without vitamins and minerals eliciting twice as much lactate as the standard media. Growth in the peach and soybean medium resulted in approximately ten times less lactate than the standard media without minerals and vitamins even though there was no significant difference in hydrogen produced per liter of media.

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44 CHAPTER THREE

CONCLUSION AND IDEAS FOR FURTHER RESEARCH

In this experiment the thermophilic anaerobe Thermotoga neapolitana was grown in seven different media to quantify the effects of adding vitamins and trace elements on hydrogen production. The media used was a standard media with vitamins and trace elements, a standard media without vitamins and trace element, a peach and soybean media with vitamins and trace elements, a peach and soybean media with vitamins and no trace elements, a peach and soybean media with trace elements and no vitamins, a peach and soybean media without trace elements or vitamins, and a soybean media with vitamins and trace elements.

Withholding trace elements and vitamins from a standard media and from a peach and soybean media produced two different outcomes. Using standard medium showed a significant decrease in the amount of hydrogen produced during fermentation without the vitamins and trace elements, 31.5 mmol H2/L compared to 25.8 mmol H2/L. Also the product yield was lower when the additional nutrients were not added, 2.9 mmol

H2/mmol glucose compared to 2.8 mmol H2/mmol glucose. The research also showed a significant increase in the amount of lactate produced by the organism, 5.99 mmol/L and

3.73 mmol/L. These two results are not independent of one another. Hydrogen production occurs through the utilization of the Embden-Meyerhof (EM) pathway that converts glucose to acetate and carbon dioxide. On the other hand, Lactate production occurs via the Entner-Doudoroff (ED) pathway and the glucose is instead converted to lactate. The EM pathway produces 4 ATP compared to the ED pathway that only

45 produces 1 ATP. T. neapolitana when provided adequate vitamins and trace elements were preferentially able to utilize the EM pathway to provide more ATP with each glucose. The culture that was not given these nutrients was not able to utilize this pathway as effectively and thus had to use the ED pathway to provide itself with ATP.

The organism was still able to produce hydrogen in this medium since the yeast extract most likely contained these nutrients but in smaller quantities.

The growth in peach and soybean media did not show a significant change in hydrogen production or in lactate production when vitamins or trace elements were withheld from the media. These samples produced approximately 23-24 mmol H2/L.

The lactate production was approximately 0.1-0.2 mmol /L. Thus the addition of vitamins and trace elements did not significantly affect the production of hydrogen when peach solids substituted the carbon source of the standard media and soybeans as the nitrogen source.

The organisms in the peach and soybean media did not produce as much hydrogen as the standard media. This may be due to a number of reasons including the presence of less sugars in peach solids as a carbon source. Though most of these solids are sugars a portion is cellulose and other non-utilized matter. Also the amino acids in the yeast extract and Trypticase™ peptone found in the standard medium were probably more easily processed by the organism compared to the proteins in soybean meal. These proteins had to be broken down then utilized by the organism. Of note is the greatly decreased amount of lactate produced in the peach and soybean media. These trials produced a very small amount of lactate compared to the standard media. These trials

46 produced only 0.1-0.2 mmol lactate/L. This difference may be due to a number of reasons and could be the focus of further studies. The peaches or soybean meal may have inhibited the metabolic pathway that produces the lactate. What type of inhibition is yet to be determined however. The inhibition of lactate could be used beneficially by pushing T. neapolitana more towards the EM pathway and hydrogen production instead of the ED pathway, which produces lactate.

This research showed that the trace elements and vitamins were not necessary to produce hydrogen by fermenting T. neapolitana. This discovery could drop the price of commercial hydrogen production when complex substrates are used. To further bring down the cost of media a number of steps could be taken given experimental support.

The recycling of spent media could help reduce costs because a good portion of sugar is not used after growth. Also controlling pH or venting of hydrogen and its inhibitory effects further utilization of the glucose can be attained. Care must be put to reduce acetate concentrations as to prevent potential product inhibition of the EM pathway. Also the recycling of media would allow the salts to be recycled as well. By bringing the cost down of this process commercial applications could be made cost effective. This would bring us closer a cleaner hydrogen fueled society and one more step further from fossil fuels.

APPENDICES

Appendix A: Gas Analysis Data

Figure A-1: Percent Hydrogen Standard Curve

49 Table A-1: Hydrogen Chromatography Data

Figure A-2: Percent Carbon Dioxide Standard Curve

51 Table A-2: Carbon Dioxide Chromatography Data

52 Table A-3: Gauge Pressure, pH, Hydrogen and Carbon Dioxide concentrations

Figure A-3: Standard Medium Example Gas Analysis Chromatograph. Hydrogen peak is at 1.37 min; Nitrogen peak is at 1.65 min; and Carbon Dioxide peak is at 6.6 min.

Figure A-4: Standard Medium Example Gas Chromatograph. This shows a close up of the Carbon Dioxide peak.

Figure A-5: Peach and Soybean Medium Example Gas Chromatograph. Hydrogen peak is at 1.29 min; Nitrogen peak is at 1.65 min; and Carbon Dioxide peak is at 6.6 min.

Figure A-6: Peach and Soybean Medium Example Gas Chromatograph. This shows a close up of the Carbon Dioxide peak.

Figure A-7: Soybean Medium Example Gas Chromatograph. Hydrogen peak is at 1.24 min; Nitrogen peak is at 1.51 min; and Carbon Dioxide peak is at 6.6 min.

Figure A-8: Soybean Medium Example Gas Chromatograph. This shows a close up of the

Carbon Dioxide peak.

56 Appendix B: Organic Acid and Standard Media Glucose Data

Figure B-1: Acetate Standard Curve

57 Table B-1: Acetate Concentration Data

Figure B-2: Lactate Standard Curve

59 Table B-2: Lactate Concentration Data

Figure B-3: Glucose Standard Curve

Table B-3: Glucose Concentrations in Standard Media Data

Figure B-4: Example Standard Medium Pre-Fermentation Chromatograph of Glucose

(RT-9.3 min), Lactate (RT-12.9 min), and Acetate (RT 14.9 min)

Figure B-5: Example Standard Medium Post-Fermentation Chromatograph of Glucose

(RT-9.3 min), Lactate (RT-12.9 min), and Acetate (RT-15 min).

Figure B-6: Example Peach and Soybean Medium Pre-Fermentation Chromatograph of

Glucose (RT-9.3 min), Lactate (RT-12.9 min), and Acetate (RT-15 min).

Figure B-7: Example Peach and Soybean Medium Post-Fermentation Chromatograph of

Glucose (RT-9.3 min), Lactate (RT-12.9 min), and Acetate (RT-15 min).

Figure B-8: Example Soybean Medium Pre-Fermentation Chromatograph of Glucose

(RT-9.3 min), Lactate (RT-12.9 min), and Acetate (RT-15 min).

Figure B-9: Example Soybean Medium Post-Fermentation Chromatograph of Glucose

(RT-9.3 min), Lactate (RT-12.9 min), and Acetate (RT-15 min).

64 Appendix C: Peach Sugars Data

Figure C-1: Glucose Standard Curve

Figure C-2: Sucrose Standard Curve

65 Table C-1: Glucose and Sucrose Concentration Data

Figure C-3: Peach and Soybean with Vitamin Medium Pre-Fermentation Chromatograph.

Sucrose has a retention time of 10.30 min and glucose has a retention time of 12.35 min.

Figure C-4: Peach and Soybean with Vitamin Medium Post-Fermentation

Chromatograph. Sucrose has a retention time of 10.30 min and glucose has a retention time of 12.35 min.

Figure C-5: Peach and Soybean with Vitamin and Trace Element Medium Pre-

Fermentation Chromatograph. Sucrose has a retention time of 10.30 min and glucose has a retention time of 12.35 min.

Figure C-6: Peach and Soybean with Vitamin and Trace Element Medium Post-

Fermentation Chromatograph. Sucrose has a retention time of 10.30 min and glucose has a retention time of 12.35 min.

70 Appendix D: COD Data

Figure D-1: Chemical Oxygen Demand (COD) Standard Curve

71 Table D-1: Total COD Data

72 Table D-2: Soluble and Particulate COD Data