TIRADO-ACEVEDO, OSCAR. Production of Bioethanol from Synthesis Gas Using
Clostridium ljungdahlii as a Microbial Catalyst. (Under the direction of Dr. Amy M. Grunden).
As world energy consumption increases, the main sources of energy, which are fossil fuels, are declining. Biofuels are a promising source of sustainable energy. Nevertheless, feedstocks for biofuels need to be inexpensive and separate from the human food network. Lignocellulosic biomass has been identified as one such feedstock. An innovative way to convert lignocellulosic biomass to biofuels is through a process called gasification- fermentation. There are known microorganisms that can convert synthesis gas components to biofuels. One such organism is Clostridium ljungdahlii which can utilize CO and H 2 and produce ethanol. The purpose of the research described herein was to identify and describe fermentation conditions that support higher ethanol production from synthesis gas by C. ljungdahlii. Earlier reports had shown that certain acetogens could divert electrons and carbon flux away from acetate and towards a number of reductant sinks including ethanol. In the present study, we describe a laboratory-isolated C. ljungdahlii strain (strain OTA1). This strain produces approximately 2-fold more ethanol than the wild-type (WT) strain. Furthermore, we demonstrate that addition of oxygen to the cultures improves ethanol to acetate ratios, as well as total ethanol yields in both C. ljungdahlii WT and OTA1 cultures grown in media with and without reducing agents. In addition, we demonstrate that pre- adapting C. ljungdahlii cultures in a medium containing fructose also improves ethanol formation by this bacterium.
Production of Bioethanol from Synthesis Gas Using Clostridium ljungdahlii as a Microbial Catalyst
by Oscar Tirado-Acevedo
A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy
Microbiology
Raleigh, North Carolina 2010
APPROVED BY:
_______________________________ ______________________________ Dr. Amy M. Grunden Dr. Mari S. Chinn Committee Chair
________________________________ ________________________________ Dr. Jonathan W. Olson Dr. Eric S. Miller ii
DEDICATION To God. To my wife Johanna, for being there for me, loving me unconditionally and standing next to me through these years as a PhD student even if she did not understand what my research was about. To my parents, for giving me a foundation to build my future on. For giving me everything when they did not have much. To my kids, all I do, I will do it for you guys. iii
BIOGRAPHY Oscar Tirado-Acevedo was born in Rincn, Puerto Rico to Oscar Tirado-Hernndez and Blanca I. Acevedo-Lorenzo. He is the oldest of three children and had a happy childhood with a very supportive and united family. Growing up in a coastal town he was interested in marine sciences since the time he was very little. He completed his Bachelors and Masters degrees from the University of Puerto Rico. Somewhere along the way, he got married to Johanna M. Crespo-Torres. Because of a lack of employment opportunities in Puerto Rico and a desire to experience new opportunities abroad, he and his wife moved to the United States. After a year working at a microbial pathogenesis laboratory in Nashville TN, they moved to Raleigh NC, to attend NCSU in the Graduate Program in the Microbiology Department. He received his PhD degree in Microbiology, has a beautiful daughter, is expecting a son and is moving on to keep searching for even better things in the future.
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ACKNOWLEDGEMENTS I would like to thank my thesis committee members, Dr. Mari S. Chinn, Dr. Jonathan Olson, Dr. Eric Miller and especially my thesis advisor Dr. Amy M. Grunden for being a source of ideas, support and knowledge. I would also like to thank my laboratory colleagues, Dr. Casey M. Theriot, Dr. Alice M. Lee, Dr. Jimmy L. Gosse, Dr. Anthony A. Devine, and Rushyannah Killens for ideas, support and laughs, as well as Drs. Julie Du and Mikyoung Ji. Dr. Flickinger for ideas, technical discussions and letting me use the GC. I also appreciate the training Dr. Gosse provided me for the GC operation. I do not know if I would have made it without that instrument. I offer my thanks to the NCSU Microbiology Department faculty who would open their office doors for me any time I had a question, especially Dr. Hassan and Dr. Bruno- Barcena. Also, I would like to thank Cindy Whitehead for keeping me up to date on forms to fill-out, datelines, registration, etc. Thank you, Cindy.
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TABLE OF CONTENTS
LIST OF TABLES ..................................................................................................................vii
LIST OF FIGURES................................................................................................................viii PRODUCTION OF BIOFUELS FROM SYNTHESIS GAS USING MICROBIAL CATALYSTS............................................................................................................................1
Abstract....................................................................................................................2 Introduction..............................................................................................................3 Syngas Chemical Catalytic Conversion.................................................................16 Syngas Biotransformation......................................................................................19 Metabolic Engineering of Syngas Biotransformation Processes...........................33 Acetogens and Oxidative Stress Response............................................................35 Conclusions............................................................................................................37 References Cited....................................................................................................46 METABOLIC RESPONSE OF CLOSTRIDIUM LJUGDAHLII STRAINS TO OXYGEN EXPOSURE.............................................................................................................................64
Materials and Methods...........................................................................................68 Results....................................................................................................................74 Discussion..............................................................................................................82 References Cited..................................................................................................101 INFLUENCE OF CARBON SOURCE PRE-ADAPTATION ON CLOSTRIDIUM LJUGDAHLII GROWTH AND PRODUCT FORMATION.............................................107
Abstract................................................................................................................108 Introduction..........................................................................................................109 vi
Methods................................................................................................................110 Results and Discussion........................................................................................112 Conclusion...........................................................................................................115 References Cited..................................................................................................121 STUDY CONCLUSIONS...............................................................................................123 APPENDIX......................................................................................................................126
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LIST OF TABLES
Table 1-1 Composition of synthesis gas derived from various lignocellulosic biomass sources.................................................................................................................43
Table 1-2 Summary of microbial catalysts capable of producing biofuels from synthesis gas........................................................................................................................44 Table 1-3 Thermochemical reactions that occur during gasification of biomass.................45
Table 2-1 Effect of O 2 on product profiles in C. ljungdahlii strains at maximum production............................................................................................................96
Table 2-2 Oxidative stress enzyme activities in C. ljungdahlii WT and OTA1..................97
Table 2-3 Pentose Phosphate Pathway enzyme activities in C. ljungdahlii WT and OTA1...................................................................................................................98 Table 2-4 Ethanol forming enzyme activities in C. ljungdahlii WT and OTA1..................99 Table 2-5 Intracellular pyridine nucleotide pools of C. ljungdahlii WT and OTA1..........100
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LIST OF FIGURES Figure 1-1. Biofuel production from biomass: (A) hydrolysis-fermentation (B1) gasification-chemical synthesis and (B2) gasification biosynthesis................39
Figure 1-2. Fuel products obtained from synthesis gas transformation..............................40 Figure 1-3. Diagrams of gasification reactors: A) updraft gasifier; B) downdraft gasifier; C) fluid-bed gasifier.........................................................................................41
Figure 1-4. Actetyl-CoA or Wood-Ljungdahl pathway used in autotrophic growth by anaerobes..........................................................................................................42
Figure 2-1. C. ljungdahlii cell growth. A) WT reduced, B) OTA1 reduced, C) WT unreduced, D) OTA1 unreduced.....................................................................87
Figure 2-2. C. ljungdahlii ethanol production. A) WT reduced, B) OTA1 reduced, C) WT unreduced, D) OTA1 unreduced.....................................................................88
Figure 2-3. C. ljungdahlii acetate production. A) WT reduced, B) OTA1 reduced, C) WT unreduced, D) OTA1 unreduced.....................................................................89
Figure 2-4. C. ljungdahlii WT syngas utilization when grown in reduced medium; A) 0% O 2 , B) 8% O 2 , C) 12% O 2 ................................................................................90
Figure 2-5. C. ljungdahlii OTA1 syngas utilization when grown in reduced medium; A) 0% O 2 , B) 8% O 2 , C) 12% O 2 .........................................................................91
Figure 2-6. C. ljungdahlii WT syngas utilization when grown in unreduced medium; A) 0% O 2 , B) 6% O 2 , C) 10% O 2 ........................................................................92
Figure 2-7. C. ljungdahlii OTA1 syngas utilization when grown in unreduced medium; A) 0% O 2 , B) 6% O 2 , C) 8% O 2 .....................................................................93
Figure 2-8. C. ljungdahlii fructose utilization. A) WT reduced, B) OTA1 reduced, C) WT unreduced, D) OTA1 unreduced.....................................................................94
Figure 2-9. Model for the C. ljungdahlii oxidative stress detoxification system. SOD (superoxide dismutase), Cat (catalase), Rr (rubrerythrin), Trx (thioredoxin)..95
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Figure 3-1. Synthesis gas fermentation reactor..................................................................117 Figure 3-2. C. ljungdahlii culture density in the syngas reactor (A) and syngas-fructose reactor (B) inoculated with cells pre-grown on fructose (circles), syngas- fructose (squares), or syngas (triangles)........................................................118
Figure 3-3. C. ljungdahlii production of ethanol (A and B) and acetate (C and D) in the syngas reactor (A and C) and in the syngas-fructose reactor (B and D) inoculated with cells pre-grown on fructose (circles), syngas-fructose (squares), or syngas (triangles).......................................................................119
Figure 3-4. C. ljungdahlii fructose utilization (A) in the syngas-fructose reactor inoculated with cells pre-grown on fructose (circles), syngas-fructose (squares), or syngas (triangles) and syngas utilization (B) in the syngas reactor inoculated with cells pre-grown on syngas.....................................................................120
Figure A-1. C. ljungdahlii-WT metabolism in reduced medium. A) Growth, B) fructose utilization, C) ethanol production, D) acetate production..............................127
Figure A-2. C. ljungdahlii-WT growing in reduced medium headspace, analysis at different O 2 concentrations. A) 0 % O 2 B) 4 % O 2 C) 6 % O 2 D) 8 % O 2 E) 12 % O 2 ....................................................................................................128
Figure A-3. C. ljungdahlii-WT metabolism in unreduced medium. A) Growth, B) fructose utilization, C) ethanol production, D) acetate production.............................131
Figure A-4. C. ljungdahlii-WT growing in unreduced medium headspace analysis at different O 2 concentrations. A) 0 % O 2 B) 4 % O 2 C) 6 % O 2 D) 8 % O 2
E) 10 % O 2 .....................................................................................................132
Figure A-5. C. ljungdahlii-OTA1 metabolism in reduced medium. A) Growth, B) fructose utilization, C) ethanol production, D) acetate production.............................135
Figure A-6. C. ljungdahlii-OTA1 growing in reduced medium headspace analysis at different O 2 concentrations. A) 0 % O 2 B) 4 % O 2 C) 6 % O 2 D) 8 % O 2
E) 12 % O 2 .....................................................................................................136
Figure A-7 C. ljungdahlii-OTA1 metabolism in unreduced medium. A) Growth, B)
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CHAPTER 1
Literature Review
Production of Biofuels from Synthesis Gas Using Microbial Catalysts *
Oscar Tirado-Acevedo 1 , Mari S. Chinn 2 , Amy M. Grunden 1
1 Department of Microbiology, North Carolina State University, 4548 Thomas Hall, Campus Box 7615, Raleigh, NC 27695-7615; 2 Department of Biological and Agricultural Engineering North Carolina State University D S, Weaver Labs 277, Box 7625 Raleigh, NC 27695-7626
* Published in: Advances in Applied Microbiology, 2010 (70) 57-92.
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ABSTRACT
World energy consumption is expected to increase 44% in the next twenty years. Today, the main sources of energy are oil, coal and natural gas, all fossil fuels. These fuels are unsustainable and contribute to environmental pollution. Biofuels are a promising source of sustainable energy. Feedstocks for biofuels used today such as grain starch are expensive and compete with food markets. Lignocellulosic biomass is abundant and readily available from a variety of sources, for example energy crops and agricultural/industrial waste. Conversion of these materials to biofuels by microorganisms through direct hydrolysis and fermentation can be challenging. Alternatively, biomass can be converted to synthesis gas through gasification and transformed to fuels using chemical catalysts. Chemical conversion of synthesis gas components can be expensive and highly susceptible to catalyst poisoning, limiting biofuel yields. However, there are microorganisms that can convert the CO, H 2 and CO 2 in synthesis gas to fuels such as ethanol, butanol and hydrogen. Biomass gasification- biosynthesis processing systems have shown promise as some companies have already been exploiting capable organisms for commercial purposes. The discovery of novel organisms capable of higher product yield, as well as metabolic engineering of existing microbial catalysts, make this technology a viable option for reducing our dependency on fossil fuels.
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1.1 Introduction There are three main sources of non-renewable fuels: oil, natural gas, and coal. The oil market is the largest commodity market in the world (Driesprong et al., 2008). However, in recent years there have been concerns about uncertainty in gasoline prices, oil production peaking, environmental damage caused by oil spills and emissions from oil combustion as well as political instability in many major oil producing nations (Basher and Sadorsky, 2006; Bentley et al., 2007; Sheehan and Himmel, 1999; Wirl, 2008). Coal is an abundant source of fuel. Nevertheless, coal burning has been linked to environmental pollution and human health problems including arsenic poisoning (Liu et al., 2002; Ng et al., 2003; Popovic et al., 2001). Natural gas is the third most used fossil fuel after oil and coal. In addition it is more efficient and less carbon intensive than any other fossil fuel (http://www.eia.doe.gov/oiaf/ieo/world.html, 2009; Lochner and Bothe, 2009). Still, natural gas reserves in the US and Europe are declining rapidly, the gas is difficult to transport, and the liquefied natural gas market is only 9% of the total natural gas market (Dresselhaus and Thomas, 2001; Lochner and Bothe, 2009; Yepes Rodrguez, 2008) For these reasons, countries around the world are exploring different ways to minimize their dependency on fossil fuels (Barnwal and Sharma, 2005; Vicente et al., 2005; Zhao and Melaina, 2006). Biomass has been identified among the renewable energy sources to have the highest potential to minimize some of these problems (Maniatis and Millich, 1998). Biofuels, fuels (liquid or gas) that can be produced from biomass (organic material produced by plants, animals or microbes), can help meet energy demands. Biofuels have the advantage that they
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can be produced from renewable agronomic raw materials using existing farm machinery and grain distribution systems independent of global location (Herrera, 2006). Biofuels have the potential to be sustainable, abundant and environmentally friendly energy sources. With an annual production of approximately 17.3 billion US gallons, ethanol is currently the biofuel produced in the greatest quantity world-wide, with the majority of production in Brazil and the United States (Demirbas and Balat, 2006; http://www.ethanolrfa.org/resource/facts/trade/, 2009). The first automobiles designed by Henry Ford were fueled by ethanol (Al-Hasan, 2003). Ethanol is produced commercially in the US and Brazil from corn and sugar cane, respectively (Herrera, 2006). Some other countries produce it from wheat, and palm oil (Herrera, 2006). This biofuel has been used (10% blend) as a replacement for methyl-tert-butyl ether (MTBE) as a fuel oxygenate in gasoline for the last thirty years (Mackay et al., 2006; Sheehan and Himmel, 1999). In Brazil, all cars run on a 20-26% ethanol blend up to 100% ethanol in flex-fuel vehicles (Goldemberg et al., 2008). In todays US market, ethanol can be used in flexible fuel vehicles as a blend of 85% ethanol and 15% gasoline (E85). Nevertheless, with less than 2000 E85 refueling stations in the whole nation (most states with less than 100 stations total), presently there is insufficient infrastructure to make this feasible (http://www.afdc.energy.gov/afdc/fuels/stations_counts.html, 2009; Sheehan and Himmel, 1999). Butanol is considered a promising biofuel. Biobutanol is part of the widely known acetone-butanol-ethanol (ABE) fermentation and can be produced from corn, whey permeate and molasses (Ezeji et al., 2007; Qureshi and Ezeji, 2008). Its production from fermentation
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was first described by Pasteur in 1869, and it has been produced commercially since the beginning of the 20 th century as part of the ABE fermentation. This process played a crucial part in both World War I and II (Jones and Woods, 1986). Production of butanol by fermentation ended in the 1950s due to lower production cost from petrochemical sources. Today, annual production of butanol is estimated to be around 350 million gallons (Shapovalov and Ashkinazi, 2008). Butanol is used mainly as an industrial intermediate to make chemicals; primarily butyl acrylate, but also butyl acetate, ethylene glycol, and butyl xanthate among others. Butanol has the possibility of being used in car engines up to 100% without need for any modifications (Ramey, 2007). The two major drawbacks of butanol fermentation are the production of side-products (acetone and ethanol) and product inhibition at low concentrations (Maddox, 1980). Ongoing research is focused on identifying solutions to these obstacles (Ezeji et al., 2007; Qureshi and Ezeji, 2008). Hydrogen is viewed as an ideal fuel for future transportation because it can be converted to energy without production of CO 2 ; the only byproduct of its combustion is water (Antoni et al., 2007; Schlapbach and Zuttel, 2001). It can be used in fuel cells as well as in combustion engines (Schlapbach and Zuttel, 2001). To date, hydrogen is produced by chemical processes such as methane steam reforming (MSR), where steam reacts with methane over a nickel catalyst to yield H 2 and CO, and electrolysis of water. These techniques are energy intensive and could be detrimental to the environment (Nath and Das, 2004). Other disadvantages are that it is difficult to transport and store, and would require the development of a new distribution infrastructure (Antoni et al., 2007). Nevertheless, great improvements have been achieved in these areas (Chalk and Miller, 2006). A more
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environmentally friendly way of obtaining hydrogen is by microbial production (biohydrogen generation). However, biohydrogen production is currently still at a laboratory scale, but recent work has shown that it can be produced from a number of biomass feedstocks including sugars, and new microbes with improved hydrogen production capabilities are being isolated and characterized in laboratories around the world (Akhtar and Jones, 2008; Davila-Vazquez et al., 2008; Maeda et al., 2008b). The United States governments Biomass Program adopted a plan to make biofuels production cost competitive by 2012 and to reduce gasoline consumption in the US by 30% by 2030 (http://www1.eere.energy.gov/biomass/biofuels_initiative.html, 2007). The Energy Independence and Security Act of 2007 requests a production of 36 billion gallons of renewable fuels by 2022 (Sastri and Lee, 2008), three times higher than current production (Gura, 2009). To help meet this goal, the US government and federal agencies are planning to spend more than $2 billion to support research and development of advanced biofuel technologies (Akhtar and Jones, 2008; http://www1.eere.energy.gov/biomass/pdfs/nbap.pdf, 2008). To date, most first generation biofuels are produced from starch. Nevertheless, research and industry efforts are moving toward production of biofuels from lignocellulosic biomass. Starch is the most abundant storage carbohydrate in plants. Starch contained in grains or plants is a mixture of amylose (10-30%), and amylopectin (70-90%) (Peters, 2006), both containing alpha-1,4-linked glucose polymers. These polymers differ in that amylose is a linear glucose chain and amylopectin contains alpha-1,6-side chains. Grain starch conversion to biofuels via fermentation is well established and is also the most mature
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technology today (http://www.nrel.gov/biomass/pdfs/39436.pdf, 2006). In the case of corn, ethanol is produced by either one of two processes, dry grind or wet mill. Most of the ethanol is produced by dry grind (Bothast and Schlicher, 2005). In this process, grain is mixed with water, a thermostable alpha-amylase, ammonia, and lime, and the mixture is heated in a reactor at temperatures at approximately 88C. This is where the liquefaction (starch gelatinization and hydrolysis) step occurs. The mixture is then transferred to the saccharification tank where sulfuric acid is added to lower the pH of the slurry. Subsequently, glucoamylase is added and the temperature held at 61C. This process releases glucose, which in turn can be converted to biofuels such as ethanol and butanol through fermentation. The entire process is described in detail in (Kwiatkowski et al., 2006). The feedstock for this process is the most significant cost input (Bothast and Schlicher, 2005); therefore, biofuel production cost is proportionally affected by the feedstock market price (Kwiatkowski et al., 2006). For example, corn price average ranges from $1.94 to $3.24 per bushel (McAloon et al., 2002), but have more than doubled in 2008 (Pimentel et al., 2009). In turn, corn ethanol has doubled as well (O'brien and Wolverton, 2009). Feedstocks most used in commercial production of biofuels (mostly ethanol) are sugar cane, corn, and wheat. This process still relies on economic subsidies as high as $8.7 billion per year (Datar et al., 2004; Kowplow, 2006), and there is evidence that ethanol production from grains gives a negative net energy balance (Patzek et al., 2005; Pimentel et al., 2007; Pimentel and Patzek, 2005), which results in a non-ideal process. The use of crop products for biofuels production has also raised some concerns. For example, the use of corn for ethanol production has increased the prices of US beef, chicken, pork, eggs, breads,
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cereals, and milk by 10% to 20% (Pimentel et al., 2009) and has been linked with recent food shortages and riots around the world (Solomon and Johnson, 2009). Scientists have been investigating the use of more sustainable, accessible and economic feedstocks, namely lignocellulosic biomass. Sources for these feedstocks can be of agricultural, forestry, industrial or municipal residue origin as well as dedicated energy crops such as switchgrass, miscanthus and poplar among others (Clifton-Brown et al., 2004; Demirbas and Balat, 2006; Kim et al., 2009; Schmer et al., 2008). Lignocellulosic (cellulosic) biomass is composed of cellulose (14-70%), hemicellulose (9-22%), and lignin (8-30%). Cellulose, the most abundant biopolymer on Earth (O'Sullivan, 1997), is a fibrous, hard, impermeable homopolysaccharide composed of glucose units. Hemicellulose, the second most common biopolymer in nature (Saha, 2003), is a mixture of pentoses (xylan and arabinan) and hexoses (mannose, glucose and galactose). Hemicellulose is essential for cell wall integrity (Saha, 2003). Lignin, a biopolymer that is considered to be the most recalcitrant of known biopolymers to degradation, (Steffen et al., 2007) forms a matrix with hemicelluloses around the cellulose forming a rigid polymeric network (Kirk and Farrell, 1987). Lignin is made up of the precursor alcohols coumaryl, coniferyl and sinapyl. The application of lignocellulosic biomass for biofuels production though hydrolysis- fermentation is very attractive because of its abundance and higher sugar yield compared to corn starch (Hamelinck et al., 2005). Nevertheless, this technology is not currently commercially available since this process is still not cost effective (Himmel et al., 2007; Mosier et al., 2005). A diagram illustrating the basic hydrolysis-fermentation procedure is shown in Fig. 1-1A. In this process, feedstocks require a chemical pretreatment in which the
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carbohydrates-lignin network is broken. This pretreatment hydrolyzes hemicelluloses and also makes cellulose more accessible for enzymatic hydrolysis (Hamelinck et al., 2005). Briefly, biomass is generally treated at elevated temperatures with chemicals such as dilute sulfuric acid, sulfur dioxide, ammonia, and lime (Yang and Wyman, 2008). Cellulose is then converted to glucose monomers by further hydrolyzing with acids or cellulases. The resulting liquor needs to be treated to remove unwanted components such as acids and degradation products of C5 and C6 sugars, such as furfural and hydroxymethyl furfural. The liquor is separated into solid and liquid fractions and the liquid fraction pH is neutralized. Then the resulting liquid portion is collected and fermented with either yeast or bacteria. Furthermore, the product is purified by distillation, and dehydration. This topic has been extensively reviewed previously (Hamelinck et al., 2005; McMillan, 1992; Mosier et al., 2005; Sun and Cheng, 2002; Yang and Wyman, 2008). Although extensive work has been done to develop the hydrolysis-fermentation process for conversion of biomass to biofuels, there are still significant challenges that need to be addressed before this process can be commercially viable. These challenges include slow kinetics of breaking down cellulose to glucose, low yields of individual sugars from hemicelluloses, and removal of lignin (Himmel et al., 2007). To provide adequate rates of production and total sugar yields, high enzyme concentrations are required for cellulose hydrolysis. Furthermore, enzyme recovery and recycling, which is necessary to mitigate the cost of the hydrolysis-fermentation process, is a complicating factor since the enzymes used for cellulose degradation have a tendency to bind to the residual lignocellulose, and can, therefore, be lost during the solid-liquid separation (Eriksson et al., 2002). In addition, the
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lignin released during biomass hydrolysis can be biocidal and its presence often causes bioreactor failure (Maness and Weaver, 2002), and since this lignin cannot be broken down into fermentable sugars, 8-30% of the original biomass is not utilized for product formation. Also, there is the formation of waste streams such as acid pretreatment materials and toxic compounds found in acidic hydrolysates of biomass (Datar et al., 2004). Moreover, the biochemical composition and structure of the biomass (cellulose, hemicellulose, and lignin) dictates the process performance since this influences the final ethanol yield (Hamelinck et al., 2005). Unlike the sugar-intermediate biosynthesis technology, in synthesis gas- intermediate biosynthesis, feedstock biochemical composition does not materially affect the outcome of the process. Synthesis gas (syngas) is a product of the gasification of biomass. Gasification is a well established technology where carbonaceous material (usually coal, wood and charcoal) is cracked at extreme temperatures (700-1000C). If pure oxygen is used as the oxidant in the gasifier, then the resulting synthesis gas is rich in CO and H 2 . If air is used, then the resulting gas (producer gas) is a mixture of CO, CO 2 , H 2 , CH 4 , N 2 , some light hydrocarbons such as C 2 H 2 and C 2 H 4 as well as heavy hydrocarbons known as tars (Do et al., 2007). The partial oxidation reactions with oxygen that take place within a gasifier are exothermic. Steam can also be used as an oxidant in indirect gasification. The result of these thermochemical reactions is an endothermic and often heat transfer limited, but thermodynamically efficient process (Sipma et al., 2006). The ratio of the components of synthesis gas varies depending on the biomass source and the gasification conditions employed (see Table 1-1).
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Gasification of biomass to provide vehicles with fuel has been in use since the early 1930s (McKendry, 2002). Due to a petroleum products shortage during World War II, this technology flourished in some European countries providing fuel for both civilians and militaries (Dasappa et al., 2004). More recently, in the 1980s and 1990s, synthesis gas was successfully used in the US and Europe for heat and electricity (Faaij, 2006; http://fossil.energy.gov/programs/powersystems/gasification/gasificationpioneer.html, 2008 ). In 2007, the US Department of Defense released plans to use synthesis gas in its military bases and vehicles (http://newsblaze.com/story/2006020806383900003.mwir/topstory.html, 2006). Synthesis gas can also be transformed into a number of different products such as methanol, ethanol, hydrogen, dimethylether and others via chemical catalysts (Fig. 1-2). This idea is not new and has been developed in the past few decades (Wilhelm et al., 2001). Nevertheless, these are expensive processes subjected to high pressures and temperatures (Quinn et al., 2004; Takeguchi et al., 2000). Furthermore, syngas coming out of the gasifier has many contaminants leading to catalyst poisoning (Leibold et al., 2008). Some synthesis gas transformations to biofuels such as ethanol, butanol and hydrogen can be performed using chemical as well as biological catalysts (Fig. 1-1B.1 and 1-1B.2). Biological processes, while slower than chemical reactions, have a number of advantages such as higher yields (even similar to direct fermentation of biomass (Spath and Dayton, 2003)), specificity, and generally greater potential for decreased catalyst poisoning (Younesi et al., 2008). Some key biological transformations related to end product formation are irreversible, these also occur at ambient temperatures and pressures, therefore requiring minimum energy and lower cost
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(Klasson et al., 1992a; Klasson et al., 1992b). Pioneering work by Dr. Clausens group focused on production of biofuels from synthesis gas using microbial catalysts, and they proposed methane production from synthesis gas using a mixed culture of methanogens and hydogenotrophic bacteria (Barik et al., 1988b; Klasson et al., 1990). They also suggested the possibility of biological production of ethanol from synthesis gas when there was no known organism that could perform that reaction (Barik et al., 1988a). The number of investigations on methane production from syngas has declined during the last decade. However, a number of new organisms capable of producing biofuels from synthesis gas components have been isolated (Table 1-2). In addition, microbial ethanol production from synthesis gas has flourished to the point that Coskata, Inc. has a developed a commercial demonstration unit this year (http://www.cleantech.com/news/4995/coskata-leaks-word-demo-plant-and-r, 2009) and has plans to have a full commercial plant operating by 2011. INEOS Bio has a pilot syngas to ethanol production plant in operation (http://www.ineosbio.com/57- Welcome_to_INEOS_Bio.htm, 2009) and Syngas Biofuels Energy, Inc. is making efforts for commercialization of biological conversion of syngas to butanol (http://www.syngasbiofuelsenergy.com/, 2009). Plans and research to make microbial biohydrogen production from biomass cost competitive by 2020 are also in progress. Hyvolution, a multi-country effort funded by the European Union is working on the development of a bioprocess for cost-effective production of hydrogen from biomass (http://www.biohydrogen.nl/hyvolution, 2009). Also, Sapporo Breweries, Ltd. in collaboration with the Brazilian government announced plans to operate a plant for production of hydrogen from industry waste
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(http://www.tradingmarkets.com/.site/news/Stock%20News/2239395/, 2009). None of these biohydrogen projects are planning to utilize synthesis gas from biomass as their feedstock; however, recent published research described briefly in this work (see Section IV. C) appear very promising in regards to the development of industrial biohydrogen production from synthesis gas.
1.2 Biomass gasification Carbonaceous material can be used as feedstocks for gasification to produce syngas streams. Researchers have experimented (Table 1-1) with feedstocks as diverse as wood, sawdust, grass straw, nut and cacao shells, olive husks, even meat and bone meal (a by- product of rendering industries) (Soni et al., 2009). This is important since synthesis gas can then be produced from local biomass resources providing energy independence to both developed and developing countries. The gasification of solid waste and biomass is generally more complex than coal gasification due to the diversity of the carbon based materials. Biomass gasification also needs to occur at lower temperatures since biomass is more reactive than coal (Huber et al., 2006). Synthesis gas produced from biomass contains contaminants such as alkali compounds, H 2 S, HCl, NH 3 , HCN, potassium, sodium, and tars that are detrimental to the gasifier equipment as well as catalysts for Fischer-Tropsch (F-T) reactions (Tijmensen et al., 2002). Gasification occurs through a combination of complex reactions that include drying, pyrolysis, combustion and finally reduction. Pyrolysis occurs in the absence of oxygen and releases volatiles and gases from the biomass and produces char (solid carbonaceous
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material). Combustion occurs when the oxidant reacts with carbon and water from the biomass, producing CO 2 and H 2 O. In the reduction reaction, oxidant as well as CO 2 and H 2 O produced in the combustion step react with the carbon in char producing CO as well as H 2 . Two other important reactions occurring in the gasifier are the water-gas shift reaction in which water reacts with CO to form H 2 and CO 2 and methanation, where CO reacts with H 2
to form CH 4 and H 2 O. If the combustion to provide heat to the process is generated in a separate reactor, the process is called indirect gasification. Table 1-3 shows some of the reactions that take place in gasifiers.
1.2.1. Gasifiers
Although there are many different gasifier designs in use, the three main types are the updraft, the downdraft and fluidized-bed gasifiers. In the updraft gasifier (Fig. 1-3A), the feedstock enters from the top of the chamber where it is dried by the syngas leaving the chamber. Simultaneously, the gas leaving the chamber is cooled by the feedstock. The dried feedstock travels down the vessel where pyrolysis to char occurs. The char that is generated continues moving down the gasifier vessel where it is reduced and also interacts with the oxidant for combustion. Ashes fall through a grate to the bottom of the gasifier. Some of the advantages of updraft gasifiers are their simple construction, low cost, ability to handle high moisture and inorganic content, and their high energy efficiency due to the low temperature of the gas leaving the chamber. Some of the disadvantages are that the resulting syngas can contain high levels of tars and hydrocarbons. Therefore if this gas will be used for F-T
15
conversion, extensive cleanup is required. Furthermore, updraft gasifiers have a feed size limit, and slagging (molten residual ash that can be recycled for many applications) potential. Downdraft gasifiers are very similar to updraft gasifiers (Fig. 1-3B), except that the feedstock and oxidizer in downdraft gasifiers both enter from the top of the gasifier. The gas passes though the hot zone combusting the tars and leaving the reactor from the bottom. Some of the advantages of this design are that it has a fairly simple design and is low cost, and it produces a relatively cleaner gas with very low tar formation. Some of the disadvantages are that the system requires low moisture and ash feedstock, can only use feedstocks within a limited particle size range (between 1-30 cm), and it has low efficiency because the product gas leaves the gasifier at higher temperatures, which requires an additional cooling system as compared to an updraft gasifier. The third type of gasifier is the fluidized bed gasifier (Fig. 1-3C) This is a more recent configuration, but it is also the most popular type (Bricka, 2007). In this design, the feedstock size is reduced to a powder, and is mixed with the fluidizing material, which is usually silica sand, ceramic or alumina. The oxidizer and mixed feedstock enter the reactor from the bottom, where they form a bed of hot fluid, where most of the conversion to synthesis gas occurs. One of the biggest advantages of this type of gasifier is the uniform temperature distribution in the fluid bed. Also, carbon conversion could be up to 100%, and it is a good design for large scale applications. Some of the problems with this type of gasifier are that it produces a gas with high suspended particulates that need to be pre-heated prior to introduction to the fluidized bed and there can be a loss of bed fluidization due to feedstocks ash contents.
16
Gasification technology is significant to chemical or microbial catalytic syngas conversion process development as quality of the gaseous feedstock influences application, product formation and system efficiency. Optimization of the gasification unit operation in combination with downstream catalytic conversion will improve the application of this thermochemcial approach to bioenergy production.
1.3 Syngas chemical catalytic conversion
Fischer-Tropsch (F-T) is a surface catalyzed polymerization process that uses CH x
monomers formed by hydrogenation of adsorbed CO in order to obtain hydrocarbons with a broad chain length and functionality (Iglesia, 1997). This process utilizes metal catalysts such as Co, Ru, Rh and Fe catalysts. F-T plants have been operated around the world since 1938 (Dry, 2002). Syngas can be chemically converted to ethanol and butanol by F-T chemistry and to hydrogen by the water-gas-shift reaction (WGS) (see Fig. 1-2). A successful chemical transformation of syngas requires a purified syngas and a fixed CO/H 2 ratio for maximum conversion. Therefore, removal of H 2 S and organic sulfur compounds as well as excess CO 2
is necessary. This cleanup process typically accounts for 60-70% of capital costs for plant operation (Dry, 2002).
1.3.1. Ethanol Synthesis In this process synthesis gas is heated to 210-350C and compressed up to 1-7 MPa. The gas is converted to alcohol across a fixed bed catalyst as described by Eq. (1). The
17
reaction rate is high, and completion takes seconds to minutes, with up to 60% CO conversion to ethanol (Wei et al., 2009). Next the gas is cooled, this allows the alcohols to condense and separate from the unconverted syngas. The liquid alcohols are then further refined by alcohol separation and purification steps. Some of the catalysts that have been historically used contain rhodium (Rh), cobalt, molybdenum and others including multi- component catalysts (Takeuchi et al., 1985). Rh seems to be the best catalytic metal for synthesis gas to ethanol conversion for its ability to perform all of the four specific functions that a catalyst should perform. These include: dissociation of the adsorbed CO to carbon and oxygen, hydrogenation of the adsorbed carbon to methyl species, insertion of non-dissociated CO into the methyl species to form an adsorbed acyl species, and hydrogenation of the acyl species to form the ethanol product (Haider et al., 2009). Some of the problems with these chemical catalysts are their non-selectivity (i.e. a mix of alcohols is produced) and low conversion yields (Subramani and Gangwal, 2008). Also the high cost of Rh may affect commercialization of this technique. The reader is referred to references (He and Zhang, 2008; Spivey and Egbebi, 2007) for a full review of syngas to ethanol chemical catalysts. 2CO + 4H 2
catalyst CH 3 CH 2 OH + H 2 O (1)
1.3.2 Hydrogen Synthesis Synthesis gas produced from biomass can also be enriched thermochemically for hydrogen production. Although hydrogen production yield from biomass is relatively low (12-14% based on biomass weight), this technology has been viewed as very promising (Asadullah et al., 2002; Demirbas, 2005). Subjection of synthesis gas to the water-gas shift
18
(WGS) reaction is the most widely used process (Nath and Das, 2003). This reaction produces H 2 and CO. In this process, syngas coming from the gasifier is transferred to a WGS reactor where the H 2 concentration is increased. In this reactor, steam and CO react to produce H 2 and CO 2 (Eq. 2). Usually, the operating temperatures for the WGS reaction range from 330 to 530C (Tonkovich et al., 1999). Hydrogen from syngas can also be produced by steam methane reforming (SMR). Similar to natural gas SMR, in synthesis gas SMR, CH 4 and other light hydrocarbons in the gas are reacted with steam over a catalyst, usually nickel (Eq. 3). This reaction is carried out at temperatures ranging from 700 to 1100 C and pressures up to 2.5 MPa (Demirbas, 2007). Out of all chemical production to hydrogen, production from synthesis gas SMR gives the highest conversion efficiency and concentration of hydrogen (Ciambelli et al., 2009). However, again, tars, dust, sulfur alkali compounds and other impurities can cause catalyst block or poisoning in this process (Ciambelli et al., 2009). Still the cost of hydrogen production from biomass gasification is three times higher than the current price of hydrogen from steam methane or natural gas reforming (Balat et al., 2009; Demirbas, 2005).
CO+ H 2 O catalyst H 2 + CO 2 (2) CH 4 + H 2 O catalyst 3H 2 + CO
(3)
1.3.3 Butanol Synthesis Over the last several decades, chemical catalytic systems for synthesis of higher weight alcohols such as butanol, through CO hydrogenation have been developed (the overall
19
reaction is described in Eq. 4). Catalysis for higher alcohol synthesis can be achieved in two ways. One is to produce methanol and branched alcohols, using modified methanol synthesis with Cu/ZnO based catalysts (Christensen et al., 2009). These reactions generally take place at temperatures around 400C and pressures of 20.0 MPa (Smith and Anderson, 1983). The other process to produce higher weight alcohols is to form straight-chain alcohols, using MoS 2 and Co/Cu-based catalysts (Christensen et al., 2009; Surisetty et al., 2009). These catalysts usually operate at temperatures of 275325C, and pressures of 7.5 10MPa (Herman, 2000). The biggest disadvantage of these methods is, like with the ethanol process, low selectivity. This in turn adds cost to the process due to the need for alcohol separation. A maximum butanol selectivity of 4% of total alcohol was achieved when a Cu/ZnO catalyst promoted with 0.5% K 2 CO 3 was used (Smith and Anderson, 1983). Sun and colleagues (Xu et al., 2004) showed that use of a FeCuMnZrO 2 catalyst resulted in butanol formation selectivity as CO conversion increased, but the butanol yield was only 7.5% alcohol by weight.
4CO + 8H 2
catalyst C 4 H 9 OH
+ 3H 2 O
(4)
1.4 Syngas Biotransformation 1.4.1 Acetyl-CoA Pathway and Carbon Monoxide Dehydrogenase Scientists have suggested that before an O 2 atmosphere
appeared,
the first autotrophs on Earth, organisms able to use CO or CO 2 as their sole source of carbon, utilized the acetyl-
20
CoA pathway (Pereto et al., 1999; Ragsdale and Wood, 1991b; Russell and Martin, 2004). Apparently, this pathway is still exclusively used by anaerobes (Henstra et al., 2007). The acetyl-CoA or Wood-Ljungdahl pathway (see Fig. 1-4) was first described in the acetogen Moorella thermoacetica (formerly known as Clostridium thermoaceticum), a heterotroph capable of producing 3 moles of acetate from one mole of glucose. The pathway, which is not cyclic like the Calvin cycle or the reverse TCA cycle, is formed by two branches. These branches have been called the Eastern and Western branches and were the research focus of Lars Ljungdahl and Harland Wood, respectively (Ragsdale, 1997). The Eastern branch produces the methyl group of acetyl-CoA and the Western produces the carbonyl group. The methyl group is formed from the reduction of CO 2 to formate which is converted to formyltetrahydrofolate and is reduced to methyltetrahydrofolate. These steps involve formate dehydrogenase, and a series of tetrahydrofolate dependent enzymes (Drake, 1994; Ragsdale and Wood, 1991b). Even though the pathway was described in a heterotroph, it has been a model for autotrophic growth from CO 2 or CO. Briefly, the organism can synthesize acetyl-CoA by reducing CO 2 with electrons produced by a hydrogenase from H 2 (Eq. 5). If CO is used, it acts as both a source of carbon and electrons (Eq. 6) (Drake, 1994). These reactions are exergonic and therefore allow ATP formation and energy conservation through both substrate-level phosphorylation and the electron transport chain (Ragsdale and Wood, 1991b). Carbon monoxide dehydrogenase (CODH) is the central enzyme in this pathway (Wood et al., 1986), and it has also been characterized in a diverse group of organisms. In acetogens, this enzyme is responsible for reducing CO 2 to CO yielding the carbonyl group of
21
acetyl-CoA and it also catalyzes the final step in synthesizing acetyl-CoA from CH 3 , CO and S-CoA. Acetogens convert acetyl-CoA to acetate gaining an ATP. Some of these organisms can also reduce acetyl-CoA to acetaldehyde and ethanol using electron donors such as NAD(H) and NADP(H). This results in a net ATP consumption (Klasson et al., 1992b). In chemolithoautotrophs, this enzyme also enables the utilization of CO as the sole carbon and electron source by catalyzing the oxidation of CO to CO 2 (Eq. 7). In these organisms, energy is conserved through an electron transport chain. In hydrogenogenic carboxydotrophs, the CODH reaction is the same as Eq. 7. However, the electrons are transferred to a membrane associated hydrogenase that combines the generation of hydrogen with the translocation of protons. This generates the proton gradient needed for ATP formation by ATP synthase. In aceticlastic archaea, the CODH works in a reverse manner (Eq. 8) in which acetyl-CoA is formed from acetate. Acetyl-CoA is cleaved and the methyl group is reduced to CH 4 and CO is oxidized to CO 2 . Energy in this system is also generated through an electron transport chain. In methanogens able to grow on CO 2 /H 2 or CO, CODH drives the formation of acetyl-CoA from methyltetrahydrosarcinapterin and CO as well as the oxidation of CO to CO 2 . In Methanosarcina barkeri a hydrogenase couples generation of hydrogen with the translocation of protons much like the process that occurs in hydrogenogenic carboxydotrophs. In sulfate reducing bacteria, CODH functions very similarly to the reverse acetyl-CoA pathway in methanogens. More recently, there has been evidence of a CODH enzyme in hyperthermophilic archaea able to grow on CO. These CODHs are very similar to counterparts in methanogens, such as Methanosarcina acetivorans C2A and Methanosarcina
22
mazei G1(Lee et al., 2008a). There has even been found a hyperthermophilic bacterium, Carboxydothermus hydrogenoformans, containing five different forms of this enzyme (Wu et al., 2005). For a complete review on CODH the reader is referred to (Ferry, 2003). As is shown in sections IV. A, IV. B and IV. C, microbial production of ethanol, hydrogen and/or butanol from synthesis gas components depends on CODH, the acetyl-coA pathway or both. Therefore, we can expect the acetyl-CoA pathway and Carbon Monoxide dehydrogenase to be critical not only for microorganisms surviving in a CO or CO 2
atmosphere, but to the whole biofuels production from synthesis gas process.
2CO 2 + 4H 2 CH 3 COOH + 2H 2 O (5) 4CO + 2H 2 O CH 3 COOH + 2CO 2 (6) CO + H 2 O CO 2 + 2H + + 2e - (7) CH 3 COOH CH 4 + CO 2 (8)
1.4.2 Ethanol from Syngas Ethanol, butanol, hydrogen, biodiesel and methanol are all currently being evaluated as next generation biofuels. Of these, ethanol is by far the biofuel produced in the greatest quantities world-wide (Demirbas and Balat, 2006). Brazil is the number one ethanol producer in the world with 41% of the total production, followed very closely by the United States (Herrera, 2006). Most of this ethanol is produced by microbial fermentation of sugars from either sugar-cane or corn starch. To make ethanol a commercial fuel contender, the feedstock has to be switched to lignocellulosic biomass. Hydrolysis-fermentation technology
23
has proven to be expensive and labor intensive. Up to 40% of the carbon present in the biomass is lost in the form of lignin and most microorganisms used in this process are unable to utilize 5-carbon sugars in the hydrolysates. Plus, ethanol produced this way has not been able to compete with fossil fuel derivatives like gasoline and diesel. Biomass gasification can yield up to 100% carbon conversion to gas components and fermentation of syngas to ethanol has been shown to be commercially feasible (http://www.cleantech.com/news/4995/coskata-leaks-word-demo-plant-and-r, 2009). The first microorganism shown to catalyze conversion of synthesis gas components to ethanol (Eq. 9 and 10) was the acetogen Clostridium ljungdahlii (Barik et al., 1988a) named after Dr. Lars Ljungdahl to honor his work on clostridia and acetogens. Even though ethanol production from synthesis gas was detected, the main product was acetate. Initially, a molar ratio of ethanol to acetate of 1:9 and an ethanol concentration of less than 1 g/L was obtained in batch cultures (Vega et al., 1989). It was quickly learned that yeast extract had an influence on the product ratio and that the ethanol production was non-growth related. Therefore, yeast extract concentration was greatly reduced or eliminated completely and replaced by cellobiose. This increased both ethanol and cell concentrations. Adding reducing agents to the media seemed to alter electron flow to NADH formation and in turn, increasing ethanol production. These first experiments where done in batch cultures. By applying the culture performance information acquired through experimentation and operating two continuously stirred tank reactors (CSTR) in series (the first to promote growth and the second one for increased ethanol production), they were able to improve ethanol production by 30 fold (Klasson et al., 1991b). A cell recycle apparatus was added to the
24
CSTR and pH was held at 4.5, agitation was set at 450 rpm, gas flow rate was 30 ml/min and liquid flow rate ranged from 3.5 to 12 ml/h. These modifications increased the cell concentration from 800 mg/L to 4000 mg/L, and increased ethanol production to 50 g/L with an ethanol to acetate molar ratio of 21:1 and CO and H 2 conversions of 90% and 70% respectively (Klasson et al., 1993; Phillips et al., 1993a). Investigations also showed that C. ljungdahlii is quite tolerant of sulfur gases. It is able to grow and uptake CO and H 2 in the presence of up to 2.7% H 2 S or 5% carbonyl sulfide (Klasson et al., 1993; Smith et al., 1991). This is relevant since syngas contains a considerable amount of these gases. Work by Ghasem Najafpour and colleagues has investigated the effect of gas pressures in ethanol and acetate production by C. ljungdahlii in batch cultures (Najafpour and Younesi, 2006; Younesi et al., 2005). CO consumption and CO 2 production was the highest when syngas was applied at 1.6 and 1.8 atm, showing that CO uptake is not inhibited by synthesis gas at high pressures. H 2 and CO 2 consumption occurred after 72 h of incubation time in cultures with 1.6 and 1.8 atm of syngas, by which time CO had already been exhausted. This shows that CO was the preferred substrate for cell growth. This was expected since free energy is higher when CO is used as a substrate as compared to CO 2 (Barik et al., 1988a). Growth and acetate formation were not affected by the higher syngas pressures. On the contrary, ethanol production was enhanced at syngas pressures of 1.6 and 1.8 atm by four fold. For their system, ethanol yield was only 0.6 g/L and the ethanol to acetate molar ratio was 0.54. More recently, different synthesis gas compositions as well as different agitation speeds and gas flow rates were compared for their effect on ethanol production by C.
25
ljungdahlii (Younesi et al., 2006). Using synthesis gas with a composition of (volume %) 55% CO, 20% H 2 , 10% CO 2 and 15% Ar, a bioreactor agitation speed of 300 rpm and a gas flow rate of 10 ml/min, CO utilization was only 14% (by volume). When agitation was increased to 400 rpm, CO utilization only increased to 18%. When pure CO or a CO rich gas mix (70% CO, 15% H 2 and 15% Ar) was used, cell concentration increased by 28%, but CO utilization remained low. Throughout the experiment a maximum of 6g/L ethanol and 7g/L acetate was produced. Since ethanol production by C. ljungdahlii is assumed to be non-growth related, the effects of nitrogen limitation and low pH on ethanol and acetate production under a nitrogen atmosphere were investigated (Cotter et al., 2009a). Results showed that C. ljungdahlii requires vitamins and trace elements to maintain high cell viability in media lacking a nitrogen source. Also, ethanol and acetate production were significantly lower in resting cells as compared to growing cultures. Cultures were stable when initial pH of the non-growth media was lowered to 4.5. Nevertheless, cell viability dropped from 100% in the control pH (6.8) to 11.1%. Ethanol production was also significantly lower in cultures at low pH as compared to the control cultures. The effects of pH and gas flow on growing batch cultures have also been studied, under a synthesis gas atmosphere (Cotter et al., 2009b). The gas flow rates tested were 5, 7.5 and 10 ml/min and culture pHs were 6.8 and 5.5. It was reported that initial lower pH conditions resulted in lower cell densities and end product formation. The greatest ethanol formation occurred during cell growth, which is not in agreement with the theory that ethanol production is non-growth associated. Higher ethanol yields were obtained at gas flows of 7.5
26
ml/min with an ethanol to acetate ratio of 1:8. CO 2 concentration coming out of the reactor increased in all treatments tested. CO and H 2 were consumed with CO 2 consumption occurring at the later stages of the culture growth. A few years after C. ljungdahlii was described, Clostridium autoethanogenum, another acetogen able to produce ethanol from CO was isolated from rabbit feces (Abrini et al., 1994). Synthesis gas fermentation studies with C. autoethanogenum are limited. However, ethanol production from syngas components has been shown to be only a fraction of the molar yield of C. ljungdahlii, and the ethanol to acetate molar ratio was around 1:1or less (Abrini et al., 1994; Cotter et al., 2009a; Cotter et al., 2009b). In their non-growing experiments Chinn and colleagues, (Cotter et al., 2009a) observed that C. autoethanogenum culture densities decreased substantially, but were 100% viable. The C. autoethanogenum cells also consumed xylose, meaning that cells were metabolically active even under nitrogen limitation conditions. Nevertheless, ethanol and acetate production were lower than when cells were grown in the presence of a nitrogen source. In batch cultures with constant synthesis gas flow, higher ethanol yields were achieved at a gas flow rate of 10 ml/min with an ethanol to acetate molar ratio of 1:16. CO and H 2 were consumed and CO 2 was produced in similar quantities across all flow rates (Cotter et al., 2009b). The only published research showing growth and ethanol production from actual biomass producer gas has been done with the acetogen Clostridium carboxidivorans P7 (Datar et al., 2004; Liou et al., 2005). This organism (originally named bacterium P7) was isolated from an agricultural settling lagoon and was extensively studied because of its ability to produce six times more ethanol than acetate (Rajagopalan et al., 2002). In these studies,
27
the researchers operated the reactor by switching from bottled gas to producer gas. In the first stages, the culture cells were grown in batch liquid with continuous bottled synthesis gas flow at 180 ml/min and pH was controlled at 5.9. When cell growth plateaued, continuous liquid flow at 1.5 ml/min was initiated. Acid production was shown to be growth associated and ethanol production was minimal at this stage. Both H 2 and CO were consumed during cell growth. When the reactor was operated with producer gas, cell growth and H 2 uptake stopped, CO consumption decreased, and cells began to wash out. Nevertheless, ethanol production increased and acid production decreased, indicating that ethanol production was non-growth associated. When the reactor was returned to bottled syngas and liquid batch mode, cells began to grow again indicating that the cells had not lost viability. CO uptake started again, but not H 2 consumption. The group reported that this H 2 uptake inhibition could be caused by nitric oxide (NO) and acetylene present in the producer gas used (Ahmed et al., 2006; Ahmed and Lewis, 2007). NO is a known hydrogenase inhibitor, and in this organism, it affects H 2 uptake at concentrations above 40 ppm (Ahmed and Lewis, 2007). Therefore, the producer gas used may need to be processed to remove NO to concentrations below 40 ppm. Nevertheless, their results seem promising for the development of commercial processes. Yet another bacterium has been isolated for its ethanol production from synthesis gas. In this case a thermophile, Moorella sp. HUC22-1 (Sakai et al., 2004). Even though lowering pH and cell recycle have improved ethanol production from 1mM to 15 mM, the ethanol to acetate molar ratio obtained was 1:45 (Sakai et al., 2005). An acetaldehyde dehydrogenase (Aldh) and three alcohol dehydrogenases (AdhA, AdhB, and AdhC) have
28
been described from this organism (Inokuma et al., 2007; Sakai et al., 2004). Aldh was shown to catalyze the thioester cleavage of acetyl-CoA, as well as the thioester condensation from CoASH and acetaldehyde. It also was shown to have activity towards both NADP(H) and NAD(H), but activity towards NAD(H) was determined to be eight fold higher. AdhA was observed to catalyze the NADP(H)-dependent reduction of acetaldehyde as well as the oxidation of ethanol. This enzyme can also use NAD(H) as a cofactor but with decreased activity. AdhB was active only when NADP(H) was used as a cofactor. AdhC showed no activity with any of the cofactors used. Both AdhA and AdhB were active towards reduction of a variety of aldehydes. Surprisingly, the highest activities were toward n-butylaldehyde and isobutylaldehyde, even though this organism has not been shown to produce butanol. Finally the study reported higher aldh gene expression when cells were grown on H 2 /CO 2 , but lower adhABC expression in cells grown on H 2 /CO 2 than cells grown on fructose. Another possible use for the discussed autotrophic microorganisms is the conversion of CO 2 emitted from industrial operations such as fossil-fueled power plants. This CO 2 can be converted to ethanol with a suitable electron donor. Shu and Wiesner (Shu and Wiesner, 2008) ran a simulation using either C. ljungdahlii or Moorella sp. HUC22-1 for conversion of CO 2 from a power plant to ethanol. They determined that H 2 was the most favorable electron donor when compared to acetate or methane since the reaction occurs at near ambient temperatures. Hydrogen can be produced by microorganisms (Section IV. C), or with current technologies like steam methane reforming. They propose to use an aerated membrane reactor (AMR) as the bioconversion vessel. As described by Shu and Weiner (2008), inside the AMR there is a membrane bundle that liquid media and gases pass
29
through. Bacteria are attached to the membranes as well as suspended in the medium. This reactor does not require agitation like the others, thus reducing operational costs. In their simulation, the ethanol yield was significantly lower than yields obtained by the Clausen group (Klasson et al., 1993). Nevertheless, when C. ljungdahlii conversion was modeled, ethanol production costs by this method appeared to be less than the current ethanol market price. These findings appear very promising by themselves; imagine if even higher ethanol yields were reached.
2CO 2 + 6H 2
bacteria CH 3 CH 2 OH +3H 2 O (9) 6CO + 3H 2 O bacteria CH 3 CH 2 OH + 4CO 2 (10) 1.4.3 Hydrogen from Syngas Hydrogen is a versatile fuel as it can be used for power generation as well as a transportation fuel (in association with fuel cells). It is considered to be a clean fuel since water is the only byproduct when it is burned. It has been proposed that hydrogen will likely be the most common alternative fuel used for automotive consumption in the next 25 years (Demirbas, 2007). Natural gas and coal SMR are the least expensive known technologies for H 2 production. This process results in a gas mixture of mainly CO and H 2 . Subsequently, CO is chemically converted to CO 2 by the water-gas shift (WGS) reaction producing additional H 2 . Syngas may also be converted to hydrogen via the WGS reaction. At a production cost of up to $50/GJH 2 (www.iea.org/Textbase/techno/essentials.htm, 2007), these processes do not make hydrogen a viable replacement for fossil fuels at present (Ismail et al., 2008).
30
Hydrogen can also be produced by biological systems. Biological hydrogen production is environmentally friendly and requires less energy input compared to chemical processes (Ismail et al., 2008). Rhodospirillum rubrum, Rhodopseudomonas palustris P4, Citrobacter sp. and Rubrivivax gelatinosus CBS are photoautotrophic and chemoheterotrophic microorganisms capable of performing the gas-water shift (GWS) reaction (Ismail et al., 2008; Jung et al., 1999; Klasson et al., 1992b; Maness and Weaver, 2002; Markov and Weaver, 2008; Najafpour et al., 2003; Najafpour et al., 2004). Two enzymes that mainly contribute to the GWS reaction in these organisms are CODH and hydrogenase. The former catalyses the oxidation of CO, and hydrogenase mediates the reduction of protons to H 2 (Maness et al., 2005; Najafpour et al., 2004). Biological GWS is thermodynamically favorable at room temperature, (CO + H 2 O H 2 + CO 2 G= - 20 kJ/mol) and atmospheric pressure, compared to the chemical catalysis where a two-stage process is required as well as high temperature (>200C) (Benemann, 1999). Therefore, minimum energy requirements and low process cost are expected (Ismail et al., 2008). R. rubrum grows quickly and reaches high cell concentrations that uptake CO more rapidly than other similar organisms capable of performing the GWS reaction (Klasson et al., 1992b; Najafpour et al., 2003; Najafpour et al., 2004). It also tolerates small amounts of O 2
and sulfur often present in syngas (Klasson et al., 1992b). As a result, this strain is the favorite organism for studies investigating biohydrogen production from syngas. R. rubrum requires a light source for growth; however, H 2 production is independent of light intensity (Najafpour et al., 2004). An organic carbon source is needed for this organism to efficiently consume CO. The highest CO consumption (90-97 %) has been determined to occur when
31
R. rubrum is provided with acetate as the organic carbon substrate (1-2 g/l), resulting in a 98% hydrogen production yield (Najafpour and Younesi, 2007; Najafpour et al., 2004). Investigations involving continuously stirred tank reactors (CSTR) of R. rubrum and with continuous CO flow resulted in hydrogen yields and CO conversions of 87% and 95% theoretical values, respectively (Younesi et al., 2008). This type of bioreactor was stable for continuous operation for 27 days (Ismail et al., 2008). Most hydrogen production from syngas research has been done using artificial syngas. A mixture of pure gases is normally present in synthesis gas at a fixed concentration (e.g. 56.0% N 2 , 17.2% CO, 16.3% CO 2 , and 8.8% H 2 ). In a study by DiSpirito and colleagues, R. rubrum growing on artificial syngas and on real producer syngas was compared. It was determined that producer syngas had no negative effect on growth rates, biomass production, hydrogen production or carbon monoxide consumption (Do et al., 2007). R. gelatinosus CBS is another promising strain for its use in syngas-to-hydrogen conversion. In the presence of CO, this organism carries out the WGS reaction in both light and dark conditions (Maness and Weaver, 2002). These cells are capable of converting 100% of CO in the gas phase to H 2 in the dark (Markov and Weaver, 2008). Its tolerance for oxygen (Maness and Weaver, 2002) and its capacity to use CO as the sole carbon and energy source (Maness et al., 2005; Markov and Weaver, 2008) make it an attractive biocatalyst.
32
1.4.4 Butanol from Syngas
Butanol, like ethanol, can be produced from fermentable sugars, synthesis gas and glycerol. Butanol has a number of notable qualities that make it a suitable alternative fuel. Its energy content is 30% more than ethanol (Qureshi and Ezeji, 2008). It can be mixed with gasoline in any proportion or be used as the sole fuel component (100% butanol) in unmodified car engines (Ramey, 2007). It carries less water; and therefore, it can be transported through existing gasoline pipelines (Drre, 2007). Reports of biological butanol formation date back to Louis Pasteur. He reported an alcohol product from a clostridial culture (Drre, 2007). The acetone-butanol-ethanol (ABE) fermentation was essential during World War I. Acetone was needed to prepare munitions, and it was in great shortage at the time. Production of acetone by fermentation meant a constant supply of acetone to Britain and its allies (Drre, 2007). C. acetobutylicum has been the model organism for research in ABE fermentation from sugars, but other species have also been extensively investigated. Some of the most studied are Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, and Clostridium saccharobutylicum. Butyribacterium methylotrophicum is an anaerobe capable of using 1-carbon compounds such as CO 2 (in the presence of H 2 ), CH 4 , and formate as carbon sources in addition to fermentable substrates like glucose, sucrose and glycerol (Zeikus et al., 1980). It also possesses the advantageous ability to produce butanol from synthesis gas (Grethlein et al., 1990; Lynd et al., 1982; Zeikus et al., 1980). It is one of the most versatile CO utilizing bacteria (Grethlein et al., 1991). Other fermentation products are ethanol, acetate and butyrate. The first attempts at
33
investigating this strain for butanol production from CO yielded concentrations of 1.4 g/L (Worden et al., 1991). After some changes in fermentation set up, such as operation at pH 5.5 and continuous cell cycle, butanol production from CO was improved by more than 200% (Grethlein et al., 1991). Nevertheless, with classic ABE strains producing butanol at more than 400 g/l (Lee et al., 2008b), B. methylotrophicum is not yet a contender for commercial biobutanol production. Another interesting, but less studied strain for butanol production, is C. carboxidivorans P7. Being able to produce up to 4 times more ethanol than butanol from CO or producer gas, this strain has mostly been studied for its ethanol production capabilities (Section IV. B) (Datar et al., 2004; Liou et al., 2005; Rajagopalan et al., 2002).
1.5 Metabolic Engineering of Syngas Biotransformation Processes
Escherichia coli strain KO11 is the classic example of metabolic engineering for improved biofuels production in a microorganism. In this organism, Ingram and colleagues successfully integrated the pyruvate decarboxylase and alcohol dehydrogenase II genes from Zymomonas mobilis (Ohta et al., 1991). This strain has been the foundation of a significant number of studies (Underwood et al., 2002; Yomano et al., 2008; Yomano et al., 1998). After genetic tools were developed and its genome sequenced, Z. mobilis has become an important part of recent bioethanol production research. Since this organism can only utilize simple C6 sugars, research has been focused on providing it pathways for xylose and arabinose utilization (Rogers et al., 2007).
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Metabolic engineering in E. coli has also been applied for enhanced hydrogen production. Over-expression of the native formate hydrogen lyase (FHL) in E. coli resulted in about three times more hydrogen production than the wild type strain (Yoshida et al., 2005). Through a series of deletions and mutations, Wood and colleagues successfully un- regulated FHL, inhibited hydrogen uptake and redirected glucose metabolism towards formate resulting in around a five-fold hydrogen production increase (Maeda et al., 2007). They also randomly bioengineered the HycE hydrogenase (produces hydrogen from formate) to obtain a 23-fold increase in hydrogen production from E. coli (Maeda et al., 2008a). It was recently shown that similar to E. coli, Citrobacter amalonaticus Y19 possess a FHL complex (Kim et al., 2008). This finding potentially provides many opportunities for engineering this bacterium. Some work has also been done in an effort to redirect metabolism towards H 2 production in R. palustris (Rey et al., 2007). To date, metabolic engineering studies focused on butanol producing bacteria have targeted enhancing butanol production, increasing tolerance to solvents and selecting for butanol over other products. Findings from these studies have provided insight into how to meet some of the challenges of using metabolic engineering strategies to improve biotransformation of synthesis gas. For example, regular plasmid vectors are degraded in ABE Clostridia due to DNA restriction patterns in these bacteria. Also genetic tools for these strains are not as developed as with E. coli or Bacillus subtilis. Some of these hurdles have been overcome (Heap et al., 2007; Mermelstein et al., 1992) and to date, some of the aims have been met by inactivating acetate and butyrate kinases, the solvent formation repressor
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gene solR, and over-expression of heat shock proteins among other approaches (Green et al., 1996; Harris et al., 2001; Tomas et al., 2003). Just recently, attention has been given to modifying or introducing butanol (or its isomers) production pathways in E. coli. Yukawa and colleagues (Inui et al., 2008) successfully cloned and expressed butanol production genes from C. acetobutylicum ATCC 824. Similar approaches have been taken with yeast (Steen et al., 2008). Also, higher isobutanol production has been observed in E. coli cells over-expressing Alcohol Dehydrogenase A from Lactococcus lactis (Atsumi et al., In press).Throughout the literature, many microorganisms have been genetically manipulated for enhance biofuel production. However, synthesis gas has not been the source of energy or growth for organisms in these experiments. This is probably due to the lack of genetic information and tools for syngas utilizing organisms. The work of Nishio and colleagues (Inokuma et al., 2007) is a good start on identifying what enzymes are more active under syngas fermentation and which ones are not, helping to identify good candidate genes for genetic modifications. Metabolic engineering of these microorganisms may further facilitate biomass conversion to biofuels as well as lowering the cost of these processes.
1.6. Acetogens and oxidative stress response The acetyl-CoA pathway used by acetogens relies on enzymes that are highly susceptible to O 2 (Ljungdhal, 1986; Ragsdale and Wood, 1991b). Some of these enzymes are: formate dehydrogenase, methylene-H 4 folate reductase, disulfide reductase, and carbon monoxide dehydrogenase (Ljungdhal, 1986). Therefore, acetogens have been classified as
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anaerobes, even obligate anaerobes (Drake et al., 2008). Nevertheless, acetogens have been isolated from environments that experience transient O 2 exposure such as leaf litter, forest soil, termite guts, and plant roots (Brioukhanov and Netrusov, 2007; Kusel et al., 2001). Thus, it is not surprising that an array of different Reactive Oxygen Species (ROS) protecting enzymes have also been detected in acetogens and characterized. Moorella thermoacetica (Clostridium thermoaceticum), the model obligately anaerobic homoacetogen, studied for the elucidation of the acetyl-CoA pathway contains a five-gene cluster encoding for rubrerythrin (Rbr), rubredoxin oxidoreductase (Rbo), two rubredoxins (Rub) and a type A flavoprotein suggested to protect this bacterium from ROS (Das et al., 2001). In a study by Drake and colleagues, M. thermoacetica, Sporomusa silvacetica and Clostridium magnum were able to grow and reduce O 2 concentrations up to 1.9 % (v/v) in the gas phase (Karnholz et al., 2002). These three organisms exhibited NADH oxidase as well as peroxidase activities. However, superoxide dismutase and catalase activities were not detected. Sporomusa termitida and Sporomusa sp. strain TmAO3, both isolated from termite guts and Acetobacterium woodii, isolated from estuarine sediments show H 2 -dependent O 2 reduction. An NADH oxidase was recently isolated and characterized from Clostridium aminovalericum. This was the first enzyme of this type characterized from the genus Clostridium (Kawasaki et al., 2005). In this the same study, genes encoding for ROS protecting enzymes such as glutathione peroxidase, thioredoxin, Rbr, peroxidase, various SODs and thioredoxin reductase were found in the genome of both Clostridium aminovalericum and C. acetobutylicum.
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It has been shown that acetogens can divert electrons from acetate formation routes to alternative terminal electron-accepting processes when exposed to O 2 (Drake et al., 2008). For example M. thermoacetica can use nitrate as e - acceptor. Additionally, Clostridium glycolicum RD-1 is capable of diverting e - for the formation of H 2 and lactate. Furthermore, this organism shows a 2-fold increase in ethanol formation concomitant with approximately 2-fold reduction in acetate production. These results are encouraging as researchers might be able to increase ethanol production from synthesis gas at the expense of acetate formation by exposing these acetogens to certain amounts of O 2 . This would be beneficial especially for improving biofuel production from organisms where genetic systems are not available. On the other hand, it could also be used in combination with metabolic engineering of organisms where genetic systems are already developed.
1.7. Conclusions
Current US ethanol production from corn starch is not sustainable. It directly impacts food and feed markets, depends on subsidies, and has questionable energy balances. Biofuels from cellulosic biomass have promise to be a renewable alternative to fossil fuels. Industry can take advantage of feedstocks such as energy crops, agricultural residues as well as industrial and municipal waste. Developing technologies around lignocellulosic biomass feedtsocks can make biofuels cost competitive without negatively altering global food and environmental affairs.
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Fermentation of synthesis gas obtained from biomass has proven to be a viable approach to produce biofuels. When compared to lignocellulose hydrolysis-fermentation, gasification-fermentation has higher product yields and lower energy input (Piccolo and Bezzo, 2009; Wei et al., 2009). Microbial catalysts generally can manage synthesis gas contaminants making them more robust than some chemical catalysts. Nevertheless, quality of the synthesis gas coming out of the gasifier has to be closely monitored since contaminants such as NO, acetylene and O 2 can inhibit activities from these microbes. Seemingly most of these problems have been overcome since a commercial demonstration plant utilizing this process is already in operation, with possibilities of several other companies opening new plants in the near future. Furthermore, microbial ethanol production from CO 2 emitted from power plants seems promising and would support value-added, environmentally-conscious fuel production. Even though a number of microorganisms have been isolated with abilities to convert synthesis gas to biofuels, the list is still small and extensive physiological and metabolic research is limited for most of these isolates. Consequently, metabolic tools and techniques are non-existent for many of these microbes. The fact that some acetogens produce higher ethanol concentration as a response to O 2 exposure opens up new possibilities for improvement of biofuels production. Furthermore, the combination of new and better isolates with advancement in metabolic engineering would improve gasification-fermentation process costs, product yields, and overall performance.
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Figure 1-1. Biofuel production from biomass: (A) hydrolysis-fermentation (B1) gasification-chemical synthesis and (B2) gasification-biosynthesis.
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Figure 1-2. Fuel products obtained from synthesis gas transformation, modified from Spath and Dayton (2003).
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Figure 1-3. Diagrams of gasification reactors: A) updraft gasifier; B) downdraft gasifier; C) fluid-bed gasifier.
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Figure 1-4. Actetyl-CoA or Wood-Ljungdahl pathway used in autotrophic growth by anaerobes.
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Table 1-1. Composition of synthesis gas derived from various lignocellulosic biomass sources.
1- (Datar et al., 2004) 2- (Corella et al., 1998), 3- (van der Drift et al., 2001), 4- (Gordillo and Annamalai, In press, doi:10.1016/j.fuel.2009.07.018 ), 5- (Boateng et al., 2007).
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Table 1-2. Summary of microbial catalysts capable of producing biofuels from synthesis gas
Microorganism Source of isolation Growth temperature Biofuel Reference Acetobacterium woodii Estuary sediment Mesophilic Ethanol (Balch et al. 1977; Buschhorn et al. 1989) Butyribacterium methylotrophicum Sewage digester Mesophilic Ethanol, butanol (Zeikus et al. 1980) Caldanaerobacter subterraneus subsp. pacificus Marine hot vent Thermophilic H 2 (Sokolova et al. 2001) Carboxydocella themoautotrophica Hot spring Thermophilic H 2 (Sokolova et al. 2002) Carboxydothermus hydrogenoformans Hot swamp Thermophilic H 2 (Gerhardt et al. 1991) Citrobacter sp. Y9 Wastewater sludge digester Mesophilic H 2 (Oh et al. 2003) Clostridium autoethanogenum Rabbit feces Mesophilic Ethanol (Abrini et al. 1994) Clostridium glycolicum RD-1 Sea grass roots Mesophilic Ethanol (Kusel et al. 2001) Clostridium ljungdahlii Chicken waste Mesophilic Ethanol (Barik et al. 1988; Tanner et al. 1993) Clostridium carboxidivorans Lagoon sediment Mesophilic Ethanol, butanol (Liou et al. 2005) Moorella sp. HUC22-1 Mud Thermophilic Ethanol (Sakai et al. 2004) Rhodopseudomonas palustris P4 Wastewater sludge digester Mesophilic H 2 (Jung et al. 1999) Rhodospirillum rubrum Dead mouse residue Mesophilic H 2 (Zurrer and Bachofen 1979; Gest 1995) Rubrivivax gelatinosus Lake sediment Mesophilic H 2 (Uffen 1976) Thermincola carboxydiphila Hot spring Thermophilic H 2 (Sokolova et al. 2005) Thermococcus strain AM4 Hydrothermal vent Thermophilic H 2 (Sokolova et al. 2004b) Thermolithobacter carboxidivorans Hot spring Thermophilic H 2 (Sokolova et al. 2007) Thermosinus carboxidivorans Hot spring Thermophilic H 2 (Sokolova et al. 2004a)
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Table 1-3. Thermochemcial reactions that occur during gasification of biomass
Reaction Chemistry Enthalpy change Partial Oxidation C + 1/2O 2 CO -268 MJ/kg mole Complete Oxidation C + O 2 CO 2 -406 MJ/kg mole Water Gas Reaction C + H 2 O CO + H 2 +118 MJ/kg mole Water Gas Shift Reaction CO + H 2 O CO 2 + H 2 -42 MJ/kg mole Methane Formation CO + 3H 2 CH 4 + H 2 O -88 MJ/kg mole
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CHAPTER 2
Metabolic Response of Clostridium ljungdahlii Strains to Oxygen Exposure
Oscar Tirado-Acevedo 1 , Jimmy L. Gosse 2 , Mari S. Chinn 2 , and Amy M. Grunden 1
1 Department of Microbiology, North Carolina State University, 4548 Thomas Hall, Campus Box 7615, Raleigh, NC 27695-7615; 2 Department of Biological and Agricultural Engineering North Carolina State University D S, Weaver Labs 277, Box 7625 Raleigh, NC 27695-7626
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ABSTRACT Research in the biofuels area has been moving away from feedstocks that compete with food supplies. Lignocellulosic biomass has been identified among the renewable energy sources to have the highest potential to minimize dependency on fossil fuels. Any carbonaceous material can be converted to syngas (a mixture of CO, CO 2 , N 2 , and H 2 ) by means of gasification. Clostridium ljungdahlii ferments syngas to ethanol resulting in the conversion of the CO and H 2 present in the syngas to ethanol and acetic acid. ATP synthesis resulting from acetate production skews the final liquid products to higher acetate production compared to ethanol. There have been previous studies reporting that certain acetogens can produce higher amounts of ethanol compared to acetate when oxygen is added to the cultures. For this project, a range of oxygen concentrations was added to the culture headspace of both C. ljungdahlii wild type and the laboratory derived OTA1 strains. In the present study, we demonstrate that these acetogens can grow in the presence of oxygen and that oxygen is consumed in the course of the syngas fermentations. Furthermore, we demonstrate that addition of oxygen to the cultures improves ethanol to acetate ratios, as well as total ethanol yields in cultures grown in media with and without reducing agents. In addition, cell extracts generated from the syngas fermentation cultures exposed to the varying amounts of oxygen were assayed for oxidative stress enzyme activities, and NADH oxidase activity was observed; however, catalase activity was not detected. NAD(P)H concentrations decrease after cultures reach mid-log growth phase suggesting these adenine nucleotides were utilized in response to oxygen exposure and for acetyl-CoA reduction to ethanol.
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2.1. Introduction Acetogenic bacteria (acetogens) form acetate as a final product of fermentation. This group of organisms is very significant. They are ubiquitous in the environment and reside at an important step of the anaerobic food chain. Acetogens are indirectly involved in green-house gas formation. Acetate is considered to be the primary precursor for methane (CH 4 ) formation (Chauhan and Ogram, 2006). Methane is the second most important greenhouse gas after CO 2 (Frankenberg et al., 2005; Thauer et al., 2008). Furthermore, approximately 70% of the CH 4 produced on earth is produced by methanogens from acetate (Chauhan and Ogram, 2006). Acetogens can metabolize a wide range of organic substrates such as sugars, formate, methanol and amino acids (Morton et al., 1993). They are an important part of the carbon cycle on Earth because of their ability to utilize CO and CO 2
through the acetyl-CoA pathway as energy and carbon sources. Acetogens have also been shown to participate in the anaerobic metabolism of methyl-tert-butyl-ether (MTBE) (Mackay et al., 2007) as well as the degradation of halogenated compounds (Zinder, 2010). These compounds are particularly problematic due to their persistence in the environment, bioaccumulation in various organisms, and cytotoxic effects on wildlife and humans (Luo et al., 2008; Teuten et al., 2005). Acetogens have shown potential economical uses as well. Recently, it has been demonstrated that some acetogens are able to degrade cellulose, converting it directly to acetate (Karita et al., 2003; Wolin et al., 2003). Also, acetogens have the ability to convert sugars and synthesis gas (a mixture of mainly CO 2 , CO and H 2 ) to acetic acid (a valuable industrial chemical), and/or ethanol, butanol and hydrogen (important biofuels) among other
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products (Allen et al., 2009; Cotter et al., 2009a; Cotter et al., 2009b; Heiskanen et al., 2007; Kundiyana et al., 2010; Munasinghe and Khanal; Tirado-Acevedo et al., 2010). Significant advances have been made by companies for commercialization of syngas fermentation technologies (Gnansounou and Dauriat, 2010; Munasinghe and Khanal). Clostridium ljungdahlii (ATCC 49587T) was the first acetogen isolated for its ability to produce ethanol and acetate from CO (Barik et al., 1988a; Tanner et al., 1993a). Ethanol to acetate production ratios reported have been as low as 1:9 and as high as 21:1 (Vega et al., 1989). Besides CO, this bacterium also utilizes a number of other carbon sources including pyruvate, fructose, and xylose among others. Although a number of studies have been performed to evaluate the ability of C. ljungdahlii to ferment syngas components and to improve ethanol production by this organism, few studies have been done from a functional genomics perspective. However, recently the C. ljungdahlii genome sequence was published, and a genetic system developed for heterologous butanol production in C. ljungdahlii was presented (Kpke et al., 2010). It has been found that in addition to the acetyl-CoA and ethanol fermentation pathways, the C. ljungdahlii genome has genes encoding reactive oxygen species detoxification enzymes, Pentose Phosphate Pathway (PPP) enzymes and aldehyde oxidoreductase (AOR). Acetogens have traditionally been classified as strict anaerobes (Fuchs, 1986), nevertheless, they have been isolated from different aerobic or microaerobic environments (Drake et al., 2008). It has been demonstrated these bacteria are equipped with an assortment of oxidative stress enzymes and that certain acetogens can even reduce oxygen by different means (Boga and Brune, 2003; Das et al., 2005; Karnholz et al., 2002; Ksel et al., 2001). In
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addition, it has been suggested that exposing acetogens to microaerobic conditions trigger a shift in electron flow towards ethanol, lactate, H 2 , and/or NH 4 + production instead of acetate formation (Drake and Daniel, 2004; Drake et al., 2008; Ksel et al., 2001). Clostridium ljungdahlii was isolated from a microaerobic environment (Tanner et al., 1993a). In addition, preliminary data suggests that this bacterium can tolerate relatively high concentrations of O 2 in the gas phase. Therefore, the objectives of this study were to determine the tolerance, metabolic, and physiological response of C. ljungdahlii toward O 2 .
2.2 Materials and Methods 2.2.1 Organisms and inoculum preparation Clostridium ljungdahlii (ATCC 55383), an acetogen isolated from chicken waste, and Clostridium ljungdahlii-OTA1, a laboratory derived strain, were grown on a modified Reinforced Clostridial Medium (mRCMfs). This medium contained (per liter, pH 6.8): proteose peptone (10 g), beef extract (10 g), yeast extract (3 g), fructose (5 g), 5 mg/mL resazurin solution (0.1%, w/v final), 50 ml PETC salt solution [(per L): NH 4 Cl (20 g), KCl (2 g), MgSO 4 .7H 2 O (4 g), NaCl (16 g), KH 2 PO 4 (2 g), and CaCl 2 (0.4 g)], 10 ml PETC trace element solution [(per L): nitrilotriacetic acid (2 g), MnCl 2 .4H 2 O (1.3 g), FeSO 4 .7H 2 O (0.4 g), CoCl 2 .6H 2 O (0.2 g), ZnSO 4 .7H 2 O (0.2 g), CuCl 2 .2H 2 O (0.02 g), NiCl 2 .6H 2 O (0.02 g), Na 2 MoO 4 .2H 2 O (0.02 g), Na 2 SeO 3 (0.02 g), and Na 2 WO 4 .2H 2 O (0.025 g)] and 10 ml modified Wolfes vitamin solution [(per L): biotin (2 mg), folic acid (2 mg), pyridoxine (10 mg), thiamine HCl (5 mg), riboflavin (5 mg), nicotinic acid (5 mg), calcium D (+) pantothenate (5 mg), vitamin B12 (5 mg), p-aminobenzoic acid (5 mg), and lipoic acid
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(thioctic acid) (5 mg)]. The medium was prepared and dispensed in 160 ml serum bottles (50 ml medium/bottle). Bottles were sealed with butyl rubber stoppers and aluminum seals, connected to a vacuum manifold using a needle and a 0.22 m filter and made anaerobic by cycling three times (30 s cycles) between vacuum headspace evacuation and sparging with artificial synthesis gas (a pure bottled mixture containing 20% CO, 20% CO 2 , 10% H 2 with N 2 balance). The bottles were then autoclaved for 30 min (121 C, 15 psig). Prior to inoculation, when needed, cysteineHCl (1 ml 2.5% w/v, 0.5 g final) and sodium sulfide (1 ml 2.5% w/v, 0.5 g final), were added to the bottles. The culture bottles were then inoculated with 5 ml freezer stock cells and incubated with shaking at 100 rpm at 37C. After 24 h, 2.5 ml of culture were transferred to fresh medium and incubated as above. After 12 h, 2.5 ml of actively growing cells were transferred to fresh medium and incubated as above. After inoculation, any excess pressure was released from the bottles by inserting a needle connected to a 0.22 m filter and a hose with its end immersed in a water trap. After 12 h of incubation, O 2 was injected by syringe at the indicated concentration (headspace volume). Liquid and gas samples were taken at 0, 12, 24, 48, and 72 h. Results are reported as an average of six replicates.
2.2.2 Gas and liquid product analysis Headspace gas samples were collected using a sample lock, 5 ml gas-tight syringe (SGE, Ringwood, Australia). The samples were analyzed by gas chromatography using an Agilent 7890A gas chromatograph containing a 13823 molsieve 5 zeolite molecular sieve packed stainless steel column (6 ft x 1/8 in. i.d. Supelco) and a thermal conductivity detector.
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Argon was used as the carrier gas at a flow rate of 30 ml/min. The injector and detector temperatures were 150 C and 250 C respectively, and the oven temperature was 160 C. Liquid samples (500 l) were acidified with 125 l 25% m-phosphoric acid. The acidified samples were centrifuged for 10 min at 14,000 x g at room temperature. The sample supernatants were analyzed for ethanol and acetate concentrations by gas chromatography using an Agilent 7890A gas chromatograph containing a 0.25 m J & W DB-FFAP column (30 m x 0.32 mm i.d.) and a flame ionization detector. Argon was used as the carrier gas at a flow rate of 30 ml/min. The injector and detector temperatures were 250 C and the oven temperature 160 C.
2.2.3 Intracellular enzyme assays Cell extracts were prepared as follows, 694 ml of sterile, modified un-reduced RCMfs in 2 L stoppered bottles were inoculated with 5% (total volume) mid-log C. ljungdahlii culture. These cultures were grown as above for 12 h to an O.D. 600 of 0.3 0.4. Experimental bottles were treated with 6 % O 2 (headspace volume) and returned to the incubator. After 2 h (early response) or 24 h incubation (later response), cultures were transferred to an anaerobic chamber and dispensed into centrifuge bottles. The bottles were sealed and centrifuged (25 min at 11,000 x g, 4 C). The cell pellets were washed with 40 ml anaerobic potassium phosphate buffer (50 mM, pH7.0) and centrifuged as described above. Cell pellets were resuspended in 3 ml anaerobic potassium phosphate buffer (50 mM, pH7.0) per gram of wet cell pellet. Five hundred microliters of the cell suspension were transferred
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to 2 ml screw cap tubes. The cell suspensions were either processed immediately for assays or stored at -80 C. Five hundred microliters of 0.1 mm disruption glass beads (RPI, Illinois, USA) were added to the tubes. Samples were vortexed horizontally (10 min, maximum speed) using a MoBio vortex adapter 13000-V1 for Vortex-Genie 2
(MoBio, California, USA), and
centrifuged (10 min, 21,000 x g, 4 C). Supernatants were collected and kept on ice for assays. All assays were carried out in 3 ml total volume at 25 C in anaerobic cuvettes except for the superoxide dismutase assay that was performed aerobically in 96-well plates. All enzyme assays are reported as an average of six replicates, whereas the nucleotide pool measurements represent three replicates. NADH or NADPH oxidase was assayed as described by Stanton and Jensen, 1993. Reactions were initiated by adding cell extract (10-100 g protein). Catalase was assayed by measuring the disappearance of H 2 O 2 at 340 nm (Hassan and Fridovich, 1978). The assay contained, 2 ml 50 mM potassium phosphate buffer pH 7.0 and 1 ml 59 mM H 2 O 2 solution (150 L 30% H 2 O 2 in 25 mL 50 mM, pH 7.0 potassium phosphate buffer). This mix should give an Abs 240 of 0.858 0.02. The catalase reactions were initiated by adding cell extract (10-100 g protein). Peroxidase was assayed as described by Poole and Ellis, 1996, with the following modification, 200 M NADH or NADPH (final concentration) was used. SOD activity was determined using a commercially available WST SOD assay kit (Dojindo Laboratories, Kumamoto, Japan) (Peskin and Winterbourn, 2000). This kit uses the water- soluble tetrazolium salt WST-1 which produces a formazan dye upon reduction with a superoxide anion. This reduction rate is linearly related to xanthine oxidase activity, which is
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inhibited by SOD. Therefore, the 50% inhibition activity of SOD can be determined by the colorimetric method. Sample volumes were adjusted so that a 10 g protein amount was loaded per well. Enzyme activity, defined as percent inhibition of WST-1 reduction, was calculated according to the manufacturers protocol using the formula SOD activity (inhibition rate %) = {[(A blank 1 - A blank 3) - (A sample - A blank 2)]/ (A blank 1 - A blank 3)} x 100, where blank 1 lacks sample solution, blank 2 lacks xanthine oxidase solution and blank 3 lacks both sample and xanthine oxidase solutions. Alcohol dehydrogenase assays contained 1.5 ml 100 mM Tris-HCl pH 8.5, 0.5 ml 2M ethanol, and 1 ml of 0.025 M NAD + or NADP + . Reactions were initiated by adding cell extract (10-100 g protein) and the appearance of NADH or NADPH was measured at 340 nm. Acetaldehyde dehydrogenase assays were performed as described in Clark and Cronan, 1980, in which the appearance of NADH or NADPH was measured at 340 nm. Glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase were assayed as described by Sugimoto and Shiio, 1987a and Sugimoto and Shiio, 1987b by measuring the increase in absorbance at 340 nm of NADPH with the following modification, the reaction mixture contained 50 mM Tris-HCl buffer, pH 7.5. One unit of the enzyme activity was defined as the amount catalyzing the formation of 1 nmol of NADPH per min.
2.2.4 Quantitative analysis of intracellular pyridine nucleotide pools Pyridine nucleotides were extracted as in (Wimpenny and Firth, 1972). In brief, NAD + and NADP + were extracted with HCl, while NADH and NADPH were extracted with KOH from cells after 2 h (early response) or 24 h (later response) of 6 % headspace v/v
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oxygen exposure. Cells were grown on un-reduced mRCMfs as above. The concentrations of pyridine nucleotides in extracts were measured by spectrophotometric enzyme assays as in (Klingenberg, 1985).
2.2.5 Additional analytical methods Growth was measured as optical density at 600 nm. Protein quantification in cell extracts was performed using Bio-Rads Protein Assay Dye Reagent (Bio-Rad, Hercules, CA, USA), according to manufacturers instructions. The amount of fructose present in the culture media was determined by high-performance liquid chromatography as follows. Liquid samples (600 l) were centrifuged (10 min, 14,000 x g, room temperature). Supernatant was filtered though a 0.22 m syringe filter and analyzed using a Shimadzu LC20 liquid chromatograph containing a HPX-87H column maintained at 65 C. Sulfuric acid (5 mM) was used as the eluent at a rate of 0.6 ml/min. To determine cell density 11 ml of culture were sampled from growing cells. One ml was used for O.D. 600 reading while 10 ml of culture were filtered through a disposable pre- weighed and pre-dried 22 m syringe filter. Flow through was discarded. Filter and cells were washed by passing 10 ml 50 mM phosphate buffer pH 7.0. Filter with cells was then incubated at 80C. These were weighed every 24 h until constant weight. The optical densities were plotted against their corresponding dry cell weights yielding a linear relationship between measured O.D. 600 nm and culture densities (mg/L). The relationship between optical density and dry cell weight was found to be 312 and 353 mg dry cells/L per O.D. 600 unit for C. ljungdahlii-WT and C. ljungdahlii-OTA1 respectively.
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2.3. Results 2.3.1 Effects of oxygen on C. ljungdahlii growth In all of our experiments, cultures reached stationary phase by 48 h. C. ljungdahlii strains were unexpectedly resistant to various concentrations of oxygen in the gas phase (Fig. 2-1 and Appendix Figs. A-1, A-3, A-5 and A-7). C. ljungdahlii-WT grew in reduced medium containing up to 8% O 2 with no apparent differences in cell density when compared to control cultures (Fig. 2-1A). C. ljungdahlii-OTA1 also grew in reduced medium containing up to 8% O 2 in the gas phase. Nevertheless, growth was significantly reduced (p<0.05) in these cells after 12 h of O 2 exposure when compared to control cultures (Fig. 2- 1B). Cell growth was inhibited when either WT or OTA1 cultures were exposed to 12% O 2
in reduced medium. Reducing agents are regularly used to remove O 2 traces from the media as well as to lower the redox potential in the medium (Chalmers and Taylor-Robinson, 1979). Because of that the oxygen exposure growth studies performed for cells grown with reducing agents in their media cannot necessarily be relied upon to provide an accurate assessment of the Clostridium cells resistance to oxygen exposure. Therefore, we removed both cysteine and ammonium sulfide from the medium (un-reduced medium) and proceeded to expose C. ljungdahlii to different O 2 concentrations. Absence of reducing agents in the medium did not have an effect in growth for either of the C. ljungdahlii species, since, as in control experiments, cell numbers were similar in both reduced and unreduced media (Fig 2-1C and D). C. ljungdahlii species were also capable of growing in unreduced media containing relatively high amounts of O 2 in the gas phase (Fig. 2-1 C, D, and Appendix Figs. A-3 and A-
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7). As in reduced media, C. ljungdahlii-WT showed no differences in growth when the culture was exposed to O 2 compared to control cultures (Fig. 2-1C). As expected, cultures grown in un-reduced media showed less resistance to O 2 than cells grown in reduced media. Unlike C. ljungdahlii-WT, C. ljungdahlii-OTA1 growth was significantly reduced (p<0.05) when cells were exposed to 6% O 2 when compared to control cultures (Fig. 2-1 D). C. ljungdahlii-WT and OTA1 cultures failed to continue growth when exposed to 10% O 2 and 8% O 2 respectively, indicating that the OTA1 strain was more sensitive to O 2 exposure than the WT strain.
2.3.2 Effects of oxygen on ethanol production Both in control as well as O 2 exposed conditions, C. ljungdahlii OTA1 produced significantly higher (p < 0.05) concentrations of ethanol than C. ljungdahlii-WT (Fig. 2-2). Ethanol production in C. ljungdahlii-WT reached its peak at 683 mg/L after 48 h of growth in reduced medium (Fig. 2-2A). When these cells were exposed to 8% O 2 in the gas phase, the amount of ethanol produced was significantly higher (p < 0.05). Ethanol production continued beyond 48 h with a production of 1.4 g/L at 72 h. This shows approximately 100% increase in ethanol production when compared to control cells. C. ljungdahlii-OTA1 strain growing in reduced control medium also reached maximum ethanol production at 48 h with 1.2 g/L produced (Fig. 2-2B). It is important to note that in control experiments, OTA1 produced more than 75 % more ethanol than the WT strain (p < 0.05). As with WT, when C. ljungdahlii-OTA1 cells were exposed to 8% O 2 , the amount of ethanol produced was significantly higher (p < 0.05). Ethanol accumulation continued past 48 h with a production
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of 2.0 g/L at 72 h; a 66 % increase compared to the 0% oxygen control experiments and 46% higher when compared to C. ljungdahlii-WT exposed to 8% O 2 (p < 0.05). Neither strain had significant ethanol production when the headspace was supplemented with 12% O 2 because of poor growth (Fig 2-2A and B). Generally, ethanol production by both C. ljungdahlii-WT and OTA1 strains ceased after 72 h (data not shown). For this reason experiments were ended after 72 h of growth. When grown in un-reduced medium, C. ljungdahlii strains produced less ethanol when compared to cells grown in reduced media (Fig. 2-2C, D). These results were expected as reducing agents have previously been shown to enhance ethanol production from syngas (Atiyeh et al., 2009; Klasson et al., 1992c). Ethanol production in C. ljungdahlii-WT strain reached 247 mg/L at 24 h and showed minimal increases in ethanol accumulation at later time points (Fig. 2-2C). When these cells were exposed to 8% O 2 in the gas phase, ethanol production was maintained at increasing levels until 48 h. These cultures also had significantly higher (p < 0.05) ethanol production (741 mg/L) than the 0% O 2 control experiment. A 200 % increase in ethanol production in C. ljungdahlii-WT control versus O 2 treated cultures in un-reduced medium was observed. The C. ljungdahlii-WT strain was unable to produce ethanol when the headspace was supplemented with 10% O 2 . In control experiments, C. ljungdahlii-OTA1 strain continued ethanol production until reaching a maximum (509 mg/L) after 48 h (Fig. 2-2D). When the OTA1 cultures were exposed to 6% O 2 , ethanol production was increased by approximately 120% (1109 mg/L). When we compare both strains exposed to 6% O 2 ,
C. ljungdahlii OTA1 produced 50 % more ethanol
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than the wild type strain. When exposed to 8% O 2 ethanol production was negligible for the OTA1 strain.
2.3.3 Effects of oxygen on acetate production C. ljungdahlii, as do most acetogens, gains an ATP molecule when acetate is made from acetyl-CoA (Russell and Martin, 2004). Therefore, because of this energetic benefit, it produces considerably more acetate than ethanol. We see this in our experiments as C. ljungdahlii strains produce as much as 22 times more acetate than ethanol (Fig. 2-2, 2-3). When growing in reduced medium, C. ljungdahlii-WT strain, accumulated acetate until 48 h of growth in the absence of O 2 in the gas phase (Fig. 2-3A). When these cells were exposed to 8% O 2 the rate of acetate production decreased; however, the acetate concentration reached similar amounts as in control experiments (Fig. 2-3A). C. ljungdahlii-OTA1 strain growing in reduced medium also stopped acetate production by 48 h in control experiments (Fig. 2- 3B). As in the wild type strain, acetate formation rate decreased when C. ljungdahlii-OTA1 cells were exposed to 8% O 2 . Nevertheless, the final acetate concentration was comparable to control experiments (Fig. 2-3B). Under both control and O 2 exposed conditions, C. ljungdahlii-WT produced significantly (p < 0.05) more acetate than C. ljungdahlii-OTA1, 33% and 45% higher amounts, respectively. Both strains cease acetate production when 12% O 2 was added in the gas phase because of poor culture growth. In unreduced medium with no O 2 in the headspace, C. ljungdahlii-WT produced the highest observed acetate concentration of 7.3 g/L. Acetate production continued until 72 h (Fig. 2-3C). Again, cells exposed to O 2 showed a decrease in acetate production rate;
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however, acetate concentration reached similar levels compared to control cells. C. ljungdahlii -OTA1 growing in unreduced medium continued to produce acetate until 48 h. When these cells were exposed to 6% O 2 , acetate continued to accumulate until 72 h of growth. However, acetate in O 2 exposed cells was 35% lower (p < 0.05) than in control cells (Fig. 2-3D). In unreduced medium C. ljungdahlii-WT also produced significantly (p < 0.05) more acetate than C. ljungdahlii-OTA1, 33% and 53% higher amounts in control and O 2
exposure experiments, respectively. We observed that under all our conditions, regardless of the medium, C .ljungdahlii-OTA1 produced significantly less acetate (p < 0.05) than the wild type strain (Fig. 2-3). Acetate production in both wild type and OTA1 strains did not occur when cells were exposed to 10% and 8% O 2 respectively. Table 2-1 summarizes the effects of O 2 exposure on liquid products accumulation for the C. ljungdahlii-WT and OTA1 strains.
2.3.4 Effects of oxygen on syngas utilization Both C. ljungdahlii-WT and OTA1 strains growing in reduced medium showed continuous H 2 and CO consumption until cultures reached stationary phase (Fig. 2-4A, 2- 5A). Consumption rate for these gases was 0.004 mmol/h and 0.015 mmol/h, respectively. CO 2 showed a net production throughout the length of the experiment at a rate of 0.025 mmol/h and 0.045 mmol/h for the WT and OTA1 strain, respectively. When cultures were exposed to 8 % O 2 , O 2 was completely reduced after 36 h of exposure (Fig. 2-4B, 2-5B). Consumption rates for H 2 and CO were not affected by O 2 exposure. Nevertheless, the CO 2 production rate increased (0.069 mmol/h and .061 mmol/h for C. ljungdahlii-WT and
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OTA1, respectively) when cultures were exposed to 8% O 2 (Fig. 2-4B, 2-5B) . Neither consumption of CO and H 2 nor production of CO 2 were observed for cultures supplemented with 12% O 2 , (Fig. 2-4C, 2-5C). C. ljungdahlii-WT and OTA1 growing in unreduced medium also showed continuous H 2 and CO consumption until cultures reached stationary phase (Fig. 2-6A, 2- 7A). Consumption rates for H 2 and CO were very similar for both strains (0.0072 and 0.0075 mmol/h for H 2 and 0.018 and 0.019 mmol/h for CO for WT and OTA1, respectively. Again CO 2 was produced for the duration of the experiment and was accumulated at a rate of 0.018 mmol/h and 0.025 mmol/h in C. ljungdahlii-WT and OTA1cultures, respectively. When cultures were exposed to 6% O 2 , O 2 was completely reduced after 36 h of exposure (Fig. 2- 6B, 2-7B). Again, consumption rates for H 2 and CO were not affected by O 2 exposure; also the CO 2 production rate increased (0.031 mmol/h and .043 mmol/h for C. ljungdahlii-WT and OTA1 respectively) when cultures were exposed to 6% O 2 (Fig. 2-6B, 2-7B) . Neither consumption nor production of gases was observed for cultures supplemented with 10 % O 2 , (Fig. 2-6C, 2-7C).
2.3.5 Effects of oxygen on fructose utilization The medium used for growth was supplemented with 5 g/L fructose as this carbon source improves C. ljungdahlii growth, ethanol production and syngas utilization (see chapter 3). Fructose utilization by C. ljungdahlii strains in control cultures was completed by 48 h of growth (Fig. 2-8). A reduction of the fructose utilization rate was observed when the cells were exposed to 8% and 6% O 2 in reduced and unreduced medium, respectively.
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Nevertheless, fructose was always completely utilized by 72 h. When C. ljungdahlii strains growing in reduced media were exposed to 12% O 2 , fructose utilization did not appear to occur. The same results were obtained when C. ljungdahlii-WT and OTA1 strains growing in unreduced media were exposed to 10% and 8% O 2 , respectively.
2.3.6 Enzyme activities associated with oxygen tolerance and ethanol production and determination of cellular pyridine nucleotide pools
Crude cell extracts from both control and O 2 exposed C. ljungdahlii strains had superoxide dismutase, peroxidase, and oxidase activities. Catalase activity, which detoxifies hydrogen peroxide, was not detected in any of the cell extracts (Table 2-2). C. ljungdahlii WT strain had higher SOD activities than the OTA1 strain (24 and 9.5 U/mg for WT and OTA1 respectively). SOD activity was not detected at early logarithmic phase in control samples (14 h). However, activity was apparent at mid-logarithmic phase (36 h). SOD was induced by O 2 exposure as activity was detected in exposed cells at early logarithmic phase. C. ljungdahlii strain crude extracts generally showed higher oxidase and peroxidase activities (1.5 2.4 fold) when NADPH was used as electron donor (Table 2-2). Nevertheless, this activity was not observed after 36 h of culture growth. Both oxidase and peroxidase activities were detected early in the growth phase. Activities decreased after mid- logarithmic growth phase and did not show induction after cells were exposed to O 2 . In general, oxidase activity was higher in crude extracts from C. ljungdahlii strain OTA1 and peroxidase higher in crude extracts from the WT strain.
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The pentose phosphate pathway plays a crucial role for cells combating oxidative stress. Specially one of its key enzymes, glucose-6-phosphate-dehydrogenase (G6PDH), provides reducing equivalents in the form of NADPH for oxygen species reduction (Lundberg et al., 1999; Pomposiello et al., 2001). Therefore, both NADPH producing enzymes in the pathway, G6PDH and 6-phospho-gluconate dehydrogenase (6PGDH) were assayed. Both enzymes were detected in crude extracts from both C. ljungdahlii species exposed to 6 % O 2 (Table 2-3). Activity was detected in extracts after 2 h (14 h of growth) of O 2 exposure but not after 24 h (36 h of growth) of O 2 exposure. Importantly, activity was not detected in cell extracts of control samples that had not been exposed to oxygen. The C. ljungdahlii-WT strain showed higher G6PDH and 6PGDH activities than the OTA1 strain. To try to understand why ethanol production was higher in O 2 exposed cells, ethanol production enzymes and pyridine nucleotide pools were measured. NAD + dependent aldehyde dehydrogenase (ALDH) activity was found in crude extracts from all samples (Table 2-4). On the other hand, no activity was detected when NADP + was provided as the e -
acceptor. ALDH activity was higher in crude extracts from control cells during early logarithmic phase. However, activity was higher in crude extracts from O 2 exposed cells during mid-logarithmic phase. NAD + dependent alcohol dehydrogenase (ADH) activity was also observed in crude extracts from all samples assayed (Table 2-4). When NADP + was used as e - acceptor, activity was only detected in crude extracts from C. ljungdahlii strains during early logarithmic phase. In addition, it was barely detected in samples from strain OTA1 during mid-logarithmic growth phase. ADH activity was higher in C. ljungdahlii- OTA1 crude extracts when compared to the WT strain. Once more, activity was higher in
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extracts made from cells in early logarithmic phase. However, in O 2 exposed cells, ADH activity remained at steady levels after 24 h of O 2 exposure, while it dropped more than half in control cells. The total pool size of NADH and NAD + was approximately 2.5 fold higher in C. ljungdahlii-WT strain control samples during early logarithmic growth phase compared to the OTA1 strain (Table 2-5). Also, the total pool size of NADPH and NADP + was higher in the WT strain control samples at early logarithmic growth phase when compared to the OTA1 strain (approximately 3 fold higher). In general, the total pool size of NAD(P)H and NAD(P) + for both strains decreased as cell numbers increased. In both strains, the NADPH/NADP + ratio increases upon exposure to O 2 whereas NADH/NAD + generally decrease. This is more apparent in the OTA1 strain compared to WT (Table 2-5).
2.4. Discussion
We have shown that C. ljungdahlii strains are capable of tolerating and enzymatically reducing O 2 at concentrations up to 8% in the gas phase (Fig. 2-1). We also demonstrate that exposure to O 2 increases ethanol production as well as decreases acetate formation (Fig. 2-2, 2-3 and Table 2-1). These results are not surprising as acetate producing bacteria have been shown to tolerate oxygen in previous studies (Hardy and Hamilton, 1981). Also, Drake and colleagues have described an acetogen, Clostridium glycolicum RD-1, capable of tolerating up to 4% O 2 in the gas phase in agitated cultures and showing increased ethanol production at the expense of acetate in glucose-supplemented medium (Kusel et al.,
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2001). However, we have reported that C. ljungdahlii can tolerate and reduce O 2
concentrations of up to 8% in the headspace in agitated unreduced medium (see appendix) and produces up to 10 fold more ethanol than was reported for C. glycolicum RD-1. Furthermore, this study demonstrates that C. ljungdahlii is producing ethanol and acetate while still consuming synthesis gas components (H 2 and CO) (Fig. 2-4 and 2-5), presumably by using the acetyl-CoA pathway, which is known to have enzymes that are readily inactivated by O 2 (Ragsdale and Wood, 1991b). Our results, therefore, suggest that C. ljungdahlii species have effective enzymes to protect its metabolism from limited oxidative stress. Numerous anaerobes posses an array of oxidative stress protection enzymes. These range from the classic enzymes such as SOD, catalase and NADH oxidase, to more recently described, unusual enzymes such as SOR, thioredoxin reductase, and rubrerythrin among others (Brioukhanov et al., 2002; Das et al., 2001; Jenney et al., 1999). In fact, the C. ljungdahlii genome contains genes coding for at least six oxidative stress protection enzymes (Kpke et al., 2010). These include SOD, catalase, NADPH flavin oxidoreductase, NADH oxidase, rubredoxin and rubrerythrin. Cell extracts of C. ljungdahlii showed activities for SOD, oxidase and peroxidase (using either NADH or NADPH as an e - donor), but not catalase (Table 2-2). The C. ljungdahlii genome does not contain genes coding for NADPH oxidase nor peroxidase; nevertheless, NADPH flavin oxidoreductase can show activity towards O 2 (reducing it to H 2 O 2 ) (Bruchhaus et al., 1998). Also rubrerythrin has peroxidase activity using NADPH as an e - donor (Coulter et al., 1999). These enzymes (with the
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exception of SOD), appear to be constitutively expressed as we could detect relatively high activities in control samples of both C. ljungdahlii strains. Classic oxidative stress regulator systems such as SoxRS or PerR are not evident in the genome of this organism. Nevertheless, we saw an increase in NADPH/NADP + ratio in C. ljungdahlii-WT strain as a response to O 2 exposure (Table 2-5). In addition, oxidase and peroxidase activities on both WT and OTA1 strains are higher when NADPH is used as e -
donor. NADPH plays a role in biosynthetic anabolism reactions and is required for many detoxification reactions in bacteria; furthermore, it is produced mainly by the pentose phosphate pathway (PPP) in response to oxidative stress (Briolat and Reysset, 2002; Kabir and Shimizu, 2006). The C. ljungdahlii genome has genes that encode for the main enzymes involved in the PPP. We show that both glucose-6-phosphate dehydrogenase and 6- phosphogluconate dehydrogenase (responsible for producing NADPH) activities in crude extracts are up-regulated after exposing the cells to O 2 (Table 2-3). These results show evidence for some level of regulation of oxidative stress response. On the other hand, we do not observe an increase in NADPH/NADP + ratio in strain OTA1. This suggests that we may have missed this increase (happened between O 2 exposure and 2 h sampling time) or that NADPH consumption increased due to O 2 exposure. There is evidence that demonstrates NADPH turnover rate (consumption) can increase as a response to oxidative stress (Emerson et al., 2003). Higher SOD activity in crude extracts from C. ljungdahlii-WT compared to the OTA1 strain might explain the higher tolerance to O 2 shown by the former versus the latter. A proposed model for C. ljungdahlii oxidative stress detoxification system is shown in figure 2.9. It has been shown that addition of reducing agents to the culture medium alters e -
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flow to increase the formation of NAD(P)H which in turn directs carbon and acid to alcohol production (Drre et al., 1995; Klasson et al., 1991a). We confirm this theory in our experiments as ethanol production in C. ljungdahlii-WT and OTA1 species is approximately 2.5 fold higher when cultures are grown in reduced medium (Table 2-1). Exposure of C. ljungdahlii to O 2 appears to have similar effects on ethanol formation as with addition of reducing agents. O 2 exposed cultures produced ethanol concentrations comparable to cultures in reduced medium and more than 2-fold higher than the control unreduced cultures (Table 2-1). In addition, ethanol forming enzymes remain at relatively higher levels after 24 h of O 2 exposure compared to control cultures (Table 2-4). Furthermore, it appears that the oxygen and reducing agents effect is additive as cells growing in reduced media and exposed to O 2 produced the most ethanol. NAD(H) and NADP(H) levels are strongly regulated in cells by the NAD kinase (encoded in C. ljungdahlii genome) and these levels will fluctuate depending on the growth condition of the cell (Grose et al., 2006). For example, NADH is toxic to the cell during oxidative stress as it contributes electrons for iron reduction, thereby facilitating the hydroxyl radical generating Fenton reaction (Imlay and Linn, 1988; Woodmansee and Imlay, 2002). This triggers NAD + destruction/consumption followed by de novo synthesis. However, NADP(H) has not been shown to enhance the Fenton reaction (Grose et al., 2006; Hashida et al., 2010). Upon restoration of anaerobic conditions, the cell could have excess NADPH and reducing power that were accumulated in response to the oxidative stress conditions, (Kuec et al., 2009; Wimpenny and Firth, 1972) which then must be balanced. We see this as NAD(P)H concentrations decline at 24 h after O 2 exposure (Table 2-5). Under conditions in
86
which C. ljungdahlii has high levels of NAD(P)H, it might reduce acetate to aldehyde and then ethanol by the action of aldehyde oxidoreductase (AOR) enzymes encoded in the genome. This could also assist the cell in energy conservation by forming ATP by acetate production then acetaldehyde and ethanol (Kpke et al., 2010). This might explain an increase of ethanol formation in cultures exposed to oxidative stress. We have isolated a mutant strain (OTA1) that produces approximately 2-fold more ethanol than the WT strain, most likely due a higher ethanol dehydrogenase activity (Table 2- 4). We have also shown that is possible to increase ethanol production along with decreasing acetate formation by exposing C. ljungdahlii to O 2 . The fact that C. ljungdahlii can tolerate high O 2 concentrations and simultaneously produce higher ethanol concentration, is significant for its use in the biofuels industry. It is likely that in a syngas fermentation facility these cells will encounter O 2 contamination in the gas stream, either directly from the syngas or O 2 intrusion along the syngas collection/stream process (Datar et al., 2004; Zainal et al., 2002). Not only would these cells survive this O 2 intrusion, the process would benefit from it, lowering the cost by eliminating the use of reducing agents and stringent anaerobic environment.
87
0 12 24 36 48 60 72 0 500 1000 1500 0% O 2 8% O 2 12% O 2 C u l t u r e
d e n s i t y
( m g / L ) Time (h) 0 20 40 60 80 0 500 1000 1500 0% O 2 8% O 2 12% O 2 Time (h) C u l t u r e
D e n s i t y
( m g / L ) 0 20 40 60 80 0 500 1000 1500 0% O 2 6% O 2 10% O 2 Time (h) C u l t u r e
D e n s i t y
( m g / L ) 0 20 40 60 80 0 500 1000 1500 0% O 2 6% O 2 8% O 2 Time (h) C u l t u r e
D e n s i t y
( m g / L ) A D C B
Figure 2-1. C. ljungdahlii cell growth. A) WT reduced, B) OTA1 reduced, C) WT unreduced, D) OTA1 unreduced.
88 0 12 24 36 48 60 72 0 500 1000 1500 2000 2500 0% O 2 8% O 2 12% O 2 Time (h) E t h a n o l
( m g / L ) 0 12 24 36 48 60 72 0 500 1000 1500 2000 2500 0% O 2 8% O 2 12% O 2 Time (h) E t h a n o l
( m g / L ) 0 12 24 36 48 60 72 0 500 1000 1500 2000 2500 0% O 2 6% O 2 10% O 2 Time (h) E t h a n o l
( m g / L ) 0 12 24 36 48 60 72 0 500 1000 1500 2000 2500 0% O 2 6% O 2 8% O 2 Time (h) E t h a n o l
( m g / L ) C D B A
Figure 2-2. C. ljungdahlii ethanol production. A) WT reduced, B) OTA1 reduced, C) WT unreduced, D) OTA1 unreduced.
89 0 12 24 36 48 60 72 0 2000 4000 6000 8000 10000 0% O 2 8% O 2 12% O 2 Time (h) A c e t a t e
( m g / L ) 0 12 24 36 48 60 72 0 2000 4000 6000 8000 10000 0% O 2 8% O 2 12% O 2 Time (h) A c e t a t e
( m g / L ) 0 12 24 36 48 60 72 0 2000 4000 6000 8000 10000 0% O 2 6% O 2 10% O 2 Time (h) A c e t a t e
( m g / L ) 0 12 24 36 48 60 72 0 2000 4000 6000 8000 10000 0% O 2 6% O 2 8% O 2 Time (h) A c e t a t e
( m g / L ) C D B A Figure 2-3. C. ljungdahlii acetate production. A) WT reduced, B) OTA1 reduced, C) WT unreduced, D) OTA1 unreduced.
90 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) A B C
Figure 2-4. C. ljungdahlii WT syngas utilization when grown in reduced medium; A) 0% O 2 , B) 8% O 2 , C) 12% O 2
91 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 20 40 60 80 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) A B C
Figure 2-5. C. ljungdahlii OTA1 syngas utilization when grown in reduced medium; A) 0% O 2 , B) 8% O 2 , C) 12% O 2
92 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) A C B
Figure 2-6. C. ljungdahlii WT syngas utilization when grown in unreduced medium; A) 0% O 2 , B) 6% O 2 , C) 10% O 2
93
0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 20 40 60 80 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) A C B
Figure 2-7. C. ljungdahlii OTA1 syngas utilization when grown in unreduced medium; A) 0% O 2 , B) 6% O 2 , C) 8% O 2
94 0 12 24 36 48 60 72 0 1 2 3 4 5 6 7 0% O 2 8% O 2 12% O 2 Time (h) F r u c t o s e
( g / L ) 0 12 24 36 48 60 72 0 1 2 3 4 5 6 7 0% O 2 8% O 2 12% O 2 Time (h) F r u c t o s e
( g / L ) 0 12 24 36 48 60 72 0 1 2 3 4 5 6 7 0% O 2 6% O 2 10% O 2 Time (h) F r u c t o s e
( g / L ) 0 12 24 36 48 60 72 0 1 2 3 4 5 6 7 0% O 2 6% O 2 8% O 2 Time (h) F r u c t o s e
( g / L ) D B C A
Figure 2-8. C. ljungdahlii fructose utilization. A) WT reduced, B) OTA1 reduced, C) WT unreduced, D) OTA1 unreduced.
95
Figure 2-9. Model for the C. ljungdahlii oxidative stress detoxification system. SOD (superoxide dismutase), Cat (catalase), Rr (rubrerythrin), Trx (thioredoxin).
96
Table 2-1. Effect of O 2 on product profiles in C. ljungdahlii strains at maximum production
Strain Medium Condition (% O 2 ) EtOH (g/L) Ac (g/L) EtOH/Ac a
ratio Total liq. Prod. (g/L) C. ljung.-WT Reduced 0% 0.68 4.9 0.14 5.7 8% 1.4 4.5 0.30 5.9 Unreduced 0% 0.29 7.2 0.05 7.6 6% 0.81 6.7 0.17 7.5 C. ljung.-OTA1 Reduced 0% 1.3 3.3 0.40 4.6 8% 2.0 2.5 0.80 4.5 Unreduced 0% 0.54 4.9 0.11 5.4 6% 1.1 3.2 0.35 4.3 a EtOH is ethanol, Ac is acetate
97
Table 2-2. Oxidative stress enzyme activities in C. ljungdahlii WT and OTA1 a N.D. is not detected
98
Table 2-3. Pentose Phosphate Pathway enzyme activities in C. ljungdahlii WT and OTA1
Strain Condition Time (h) Enzyme Activity (mU/mg of protein) Glucose-6-P DH 6-P-gluconate DH C. ljungdahlii-WT Control 14 N.D. a N.D. 36 N.D. N.D. O 2 exposed 14 0.70 0.14 0.76 0.19 36 N.D. N.D. C. ljungdahlii-OTA1 Control 14 N.D. N.D. 36 N.D. N.D. O 2 exposed 14 0.30 0.25 0.50 0.21 36 N.D. N.D.
a N.D. is not detected.
99
Table 2-4. Ethanol forming enzyme activities in C. ljungdahlii WT and OTA1
Strain Condition Time (h) Enzyme Activities (mU/mg of protein)
Table 2-5. Intracellular pyridine nucleotide pools of C. ljungdahlii WT and OTA1
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107 CHAPTER 3
Influence of carbon source pre-adaptation on Clostridium ljungdahlii growth and product formation *
Oscar Tirado-Acevedo 1 , Jacqueline L. Cotter 2 , Mari S. Chinn 2 and Amy M. Grunden 1
1-Department of Microbiology, North Carolina State University, 4548 Thomas Hall, Campus Box 7615, Raleigh, NC 27606, USA 2-Department of Biological and Agricultural Engineering, North Carolina State University, 277 Weaver Labs, Campus Box 7625, Raleigh, NC 27695-7625, USA.
O.T.A. and J.L.C. contributed equally to this work.
* Submitted for publication to: Bioresource Technology
108 ABSTRACT Clostridium ljungdahlii was grown on different carbon sources including syngas only, syngas-fructose and fructose only to identify ideal pre-adaptation conditions for ethanol and acetate production from subsequent cultures grown in reactors containing syngas only or fructose-syngas substrates. In syngas only reactors, cultures pre-adapted to fructose had faster growth rates and higher ethanol and acetate formation than cells pre-grown on syngas or syngas-fructose. In syngas-fructose reactors, cultures did not show significant growth or acetate production differences under pre-adaptation treatments. Nevertheless, in these reactors, syngas and syngas-fructose pre-adapted cultures showed higher ethanol production than fructose pre-adapted cultures. Among the pre-adaptation carbon sources tested, fructose showed better results in syngas only reactors. However, the presence of syngas in the pre- adaptation cultures proved to be the better method overall for ethanol production.
109 3.1. Introduction Conversion of synthesis gas to liquid fuels by biological catalysts has been suggested as a promising technology to achieve oil independence (Zeikus, 1980). In this method, cellulosic biomass and other carbon sources (coal, industry and/or municipal waste, etc.) are converted to synthesis gas (syngas) by a well known process called gasification (Bridgwater, 1995). This syngas is then fed to a microorganism which can utilize it as a carbon and energy source. The by-product of this fermentation could be ethanol, butanol or other commodity chemicals. There have been a number of microorganisms isolated for use in this process. In addition, some companies are en route to commercializing their methods (Tirado-Acevedo et al., 2010). Extensive research has been focused on optimizing bioreactor designs and media development (Hurst and Lewis, 2010; Phillips et al., 1993b; Worden et al., 1991). Recent bioreactor processes have made use of micro-bubble gas dispersers, cell recycling, and addition or removal of reducing agents and yeast extract, respectively. However, only a few studies have examined the effects that seed cultures pre-adapted to different carbon sources and carbon source in the production culture media have on product formation and cell yield (Klasson et al., 1992c). Clostridium ljungdahlii, which has been studied in regard to synthesis gas fermentation (Phillips et al., 1993b), was isolated from chicken yard waste and was the first microorganism described to be able to ferment syngas components to ethanol via the acetyl- CoA pathway (Tanner et al., 1993b). In addition to utilizing CO and H 2 , C. ljungdahlii can use sugars such as fructose as a carbon source. The purpose of this study was to compare
110 cell growth and product formation from C. ljungdahlii grown on different carbon sources (syngas and sugar combinations) while using cells pre-adapted to sugar, syngas and mixed sugar-syngas substrates in a continuous gas feed, batch liquid system. 3.2. Methods 3.2.1. Organism and medium preparation C. ljungdahlii (ATCC 55353) was obtained from the American Type Culture Collection. The bacterium was grown at 37 C on a modified Reinforced Clostridial Basal medium with additional salts, vitamins, and trace elements adopted from the ATCC 1754 PETC medium (RCM.NA.SVE) in a nitrogen atmosphere unless otherwise indicated (Cotter et al., 2009a). Cultures provided syngas (7.5 ml/min ) were grown in a fermentation reactor (Fig.3.1) (Cotter et al., 2009b). The bottled syngas used was an artificial mix of 50% N2, 20% CO, 20% CO2, and 10% H2. When added to medium in combination with syngas, fructose was used at 2.5 g/L. All culture transfers were done at 5 % inoculum (v/v).
2.2. Inoculum preparation using different carbon sources
C. ljungdahlii was grown on RCM.NA.SVE (which from here on will be referred to simply as medium). Cultures were initiated from a freezer stock (-80C) and incubated at 37C for 72 hours before 3 serial transfers every 24 hours to achieve steady growth in medium containing fructose (5 g/L). Active cells (472 mg dry cells/ml) were transferred to different carbon sources [medium-syngas, medium-syngas-fructose (2.5 g/L) or medium- fructose (5g/L)] to start the seed cultures for the fermentation studies. Fermentation reactors containing medium-syngas and medium-syngas-fructose at a working volume of 250 ml were inoculated with the three different seed cultures (472 mg dry cells/ml; 5% v/v). Each
111 medium seed culture combination was grown in triplicate. Cell growth was monitored and liquid and gas samples were taken for analysis every 6-8 hours. 3.2.3. Substrate and end product analysis Headspace gases were analyzed by gas chromatography on a Carbosieve S-II, 100/120 mesh stainless steel column (10 x 1/8: 3.05 m x 0.3175 cm) using a thermal conductivity detector (Shimadzu GC-17A), Ethanol and acetate concentrations were determined in acidified samples by gas chromatography (Shimadzu GC-17A, Kyoto, Japan). The chromatography column was packed with Supelco SP 1000 (1% H3 PO4, 100/120 mesh) and analytes were quantified using a flame ionization detector (Cotter et al., 2009a; Cotter et al., 2009b). The amount of fructose present in the experimental cultures was determined by high- performance liquid chromatography (Shimadzu LC20) as follows: liquid samples (600 L) were centrifuged (10 min, 18,400 x g, room temperature). Supernatant was filtered though a 0.22 m syringe filter into a crimp vial and analyzed on a HPX-87H column (65C) using a refractive index detector. Sulfuric acid (5 mM) was used as the eluent at a rate of 0.6 ml/min. 3.2.4 Statistical analysis Cell growth and liquid product analysis from the different inoculum sources were evaluated using the General Linear Model (GLM) in SAS
Version 9.1 (SAS Inc., Cary, NC,
USA). Assessment of statistical significance for pre-adaptation on different carbon sources and different production growth substrates was set at P < 0.05.
112 3.3. Results and discussion 3.3.1. Growth of C. ljungdahlii and product formation in syngas reactors C. ljungdahlii cultures were pre-adapted on three different carbon source combinations (syngas, syngas-fructose or fructose). Subsequently, these cells were transferred to fermentation reactors with either syngas or syngas-fructose medium. In syngas only reactors, cells pre-adapted on fructose produced the highest cell densities reaching maximum growth after 24 hours (Fig. 3-2A). This observation is interesting since one would expect a lag-time given that these cells would have to switch from fructose fermentation to syngas fermentation. Syngas and syngas-fructose pre-adapted cells reached maximum densities after 40 and 56 hours, respectively. Cells pre-adapted on syngas-fructose showed the longest lag-time, contributing to the increased time to reach peak growth, where fructose only cultures had a growth rate of 10.4 mg/ml/h, while syngas and syngas-fructose cultures both had growth rates of 5 mg/ml/h. Interestingly, after 56 hours cell density was statistically the same independent of pre-adaptation medium, possibly due to product inhibition or depletion of an essential nutrient. Ethanol production in syngas reactors was significantly higher (P < 0.05) when cells where pre-adapted on fructose, reaching a maximum of 2.2 mM after 64 hours of growth (Fig. 3-3A). Ethanol production in these cells seems to be non-growth associated since these cells reached stationary phase after 24 hours. Cells pre-adapted on syngas also reached maximum ethanol yield after 64 hours, while cells pre-adapted on syngas-fructose reached a maximum after 48 hours. Although acetate is considered to be a growth associated product, it was continuously accumulated during the growth phase as well as after the stationary phase
113 was reached (Fig. 3-3C). Cells pre-adapted on fructose produced significantly higher acetate concentrations (P < 0.05) with a maximum of 34.3 mM. Again, in syngas reactors, cells pre- adapted to syngas-fructose show a lag time in acetate production when compared to fructose or syngas pre-adapted cells but reached comparable concentrations by the end of the experiment. 3.3.2. Growth and product formation in syngas-fructose reactors No significant growth differences were observed among the three inoculum sources in the syngas-fructose reactors (Fig. 3-2B). These cultures did not show a lag-time and reached a maximum cell density after 24 hours. Cell growth rates were approximately 43 mg/L/h for syngas and fructose pre-adapted cultures and 37 mg/L/h for syngas-fructose pre-adapted cultures. Independent of inoculum source, cells growing in syngas-fructose reactors had significantly higher cell yields (P < 0.05) as compared to cells growing in syngas reactors (787 mg/L vs. 332 mg/L). Ethanol concentration in syngas-fructose reactors was statistically lower compared to cells that were pre-adapted on fructose (Fig 3-3B). Pre-adaptation had no effect on acetate production as all cultures produced statistically the same amount of acetate. Independent of pre-adaptation, both ethanol and acetate production seem to be growth associated as production slows down when cells reach stationary phase. It had been previously suggested that ethanol production is associated with non-growth conditions (Klasson et al., 1992c). We observe this in syngas only medium but not when fructose is present (Fig. 3-3A and B). Independent of the inoculum source, cells grown in syngas-fructose reactors produced significantly more ethanol and acetate than cells grown in syngas reactors (Fig. 3-3), with as
114 much as 8- and 1.5-fold more ethanol and acetate, respectively. This trend has also been observed with addition of 5 g/L of fructose to syngas-grown cultures, where increases up to 25% and 35 % for ethanol and acetate concentration respectively were seen compared to syngas alone (unpublished data). This may indicate that an addition of a small concentration of sugar in the medium could improve C. ljungdahlii ethanol yields. A number of low-cost sugar sources have been identified that could act as a carbon supplement for this process without interfering with food supply demands (Jiang et al., 2009; Lawford and Rousseau, 1997). The use of low concentration sugar supplementation may be worth examining as a method to improve commodity chemical production from C. ljungdahlii. 3.3.3. Gas and fructose analysis Headspace gas composition was monitored in both syngas and syngas-fructose fermentations. Figure 3-4A illustrates a representation of headspace gases exhausting from the reactor. CO and H 2 consumption at 8 to 24 hours coincides with a CO 2 increase. This indicates that C. ljungdahlii is most likely using the acetyl-CoA pathway for the metabolism of CO and H 2 , thereby providing the cell with carbon for biomass and energy production as well as reducing equivalents (Ragsdale and Wood, 1991a). Nevertheless, by the end of the culture growth, the head-space gases return to near starting concentrations. This observation may be related to several conditions, including cells that are no longer consuming gases for growth and metabolism or that the gas flow rate exceeds the cells rate of consumption. More complete utilization of the syngas supplied could be achieved by changing the reactor design to include gas recirculation (Nie et al., 2008).
115 Fructose utilization was analyzed throughout the experiment for the syngas-fructose reactors. Fructose was consumed at equal rates independent of inoculum source and was exhausted by 24 hours (Fig. 3-4B). Fructose utilization and liquid product synthesis appear to be tightly associated as disappearance of fructose in the medium correlates with reduction of ethanol and acetate production. Nevertheless, after fructose is completely consumed, ethanol production rate is still higher on syngas-fructose reactors when compared to syngas reactors. This indicates that fructose is most likely providing the cell with an excess of reducing equivalents , which in turn could be utilized to reduce acetate to ethanol (Kopke et al., 2010). 3.4. Conclusions In syngas reactors, inoculum source was found to have an effect on growth rate and final cell concentration as well as ethanol formation. In these reactors, the highest cell concentration and ethanol production was found in reactors with cells pre-adapted with fructose. These reactors produced around 2.5 and 1.5 times more ethanol than reactors inoculated with syngas and syngas-fructose pre-adapted cells, respectively. Fructose pre- adaptation likely provides higher levels of energy equivalents (ATP/GTP) and reducing power (NADH/NADPH) to the cells, which enables the cells to efficiently utilize a carbon source (syngas) that initially requires an energy and reductant input (Kopke et al., 2010). In syngas-fructose reactors, inoculum source did not appear to have an impact on cell density, liquid product yield, nor fructose utilization. This suggests that independent of pre- adaptation, in syngas-fructose reactors, cells have the capacity to readily utilize fructose as carbon and electron source. Overall, syngas-fructose reactors had approximately 2.5 times
116 higher cell densities and 8 and 1.5 times higher ethanol and acetate, respectively than syngas reactors. This indicates that a small amount of fructose may be needed to increase ethanol yields in our system as excess reducing equivalents translate to better ethanol yields. However, fructose is an expensive carbon source and is also part of the human food supply, but there are cheap alternative carbon sources that could be suitable for C. ljungdahlii culture (Gulln et al., 2008; Jiang et al., 2009). Syngas utilization in the reactors could not be conclusively established possibly due to the gas flow rate being greater than an accurately measureable cell gas utilization rate. If quantification of gas consumption in the continuous gas feed system is desired, changes in reactor design and gaseous substrate flow patterns would be needed. Therefore, it was concluded that for syngas-fructose reactors, pre-adaptation is not necessary as the overall fermentation was not affected. Nevertheless, in syngas only reactors a fructose pre- adaptation will be beneficial as it increases cell numbers and liquid products.
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Figure 3-1. Synthesis gas fermentation reactor.
118
Figure 3-2. C. ljungdahlii culture density in the syngas reactor (A) and syngas-fructose reactor (B) inoculated with cells pre-grown on fructose (circles), syngas-fructose (squares), or syngas (triangles).
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Figure 3-3. C. ljungdahlii production of ethanol (A and B) and acetate (C and D) in the syngas reactor (A and C) and in the syngas-fructose reactor (B and D) inoculated with cells pre-grown on fructose (circles), syngas-fructose (squares), or syngas (triangles).
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Figure 3-4. C. ljungdahlii fructose utilization (A) in the syngas-fructose reactor inoculated with cells pre-grown on fructose (circles), syngas-fructose (squares), or syngas (triangles) and syngas utilization (B) in the syngas reactor inoculated with cells pre- grown on syngas.
121 REFERECES
Bridgwater, A. V., 1995. The technical and economic feasibility of biomass gasification for power generation. Fuel. 74, 631-653.
Cotter, J. L., Chinn, M. S., and Grunden, A. M., 2009a. Ethanol and acetate production by Clostridium ljungdahlii and Clostridium autoethanogenum using resting cells. Bioprocess. Biosyst. Eng. 32, 369-380.
Cotter, J. L., Chinn, M. S., and Grunden, A. M., 2009b. Influence of process parameters on growth of Clostridium ljungdahlii and Clostridium autoethanogenum on synthesis gas. Enzyme. Microb. Technol. 44, 281-288.
Gulln, B., Yez, R., Alonso, J. L., and Paraj, J. C., 2008. l-Lactic acid production from apple pomace by sequential hydrolysis and fermentation. Bioresour. Technol. 99, 308-319.
Hurst, K. M., and Lewis, R. S., 2010. Carbon monoxide partial pressure effects on the metabolic process of syngas fermentation. Biochem. Eng. J. 48, 159-165.
Jiang, L., Wang, J., Liang, S., Wang, X., Cen, P., and Xu, Z., 2009. Butyric acid fermentation in a fibrous bed bioreactor with immobilized Clostridium tyrobutyricum from cane molasses. Bioresour. Technol. 100, 3403-3409.
Klasson, K. T., Ackerson, M. D., Clausen, E. C., and Gaddy, J. L., 1992. Bioconversion of synthesis gas into liquid or gaseous fuels. Enzyme. Microb. Technol. 14, 602-608.
Kopke, M., Held, C., Hujer, S., Liesegang, H., Wiezer, A., Wollherr, A., Ehrenreich, A., Liebl, W., Gottschalk, G., and Durre, P., 2010. Clostridium ljungdahlii represents a microbial production platform based on syngas. P.N.A.S. 107, 13087-13092.
Lawford, H., and Rousseau, J., 1997. Corn steep liquor as a cost-effective nutrition adjunct in high-performance Zymomonas ethanol fermentations. Appl. Biochem. Biotechnol. 63-65, 287-304.
Nie, Y., Liu, H., Du, G., and Chen, J., 2008. Acetate yield increased by gas circulation and fed-batch fermentation in a novel syntrophic acetogenesis and homoacetogenesis coupling system. Bioresour. Technol. 99, 2989-2995.
Phillips, J., Klasson, K., Clausen, E., and Gaddy, J., 1993. Biological production of ethanol from coal synthesis gas. Appl. Biochem. Biotechnol. 39-40, 559-571.
122 Ragsdale, S. W., and Wood, H. G., 1991. Enzymology of the Acetyl-CoA Pathway of CO 2
Tanner, R. S., Miller, L. M., and Yang, D., 1993. Clostridium ljungdahlii sp. nov., an Acetogenic Species in Clostridial rRNA Homology Group I. Int. J. Syst. Bacteriol. 43, 232- 236.
Tirado-Acevedo, O., Chinn, M. S., and Grunden, A. M., Production of Biofuels from Synthesis Gas Using Microbial Catalysts. In: I. L. Allen, S. Sima, M. G. Geoffrey, Eds.), Advances in Applied Microbiology, Vol. Volume 70. Academic Press, 2010, pp. 57-92.
Worden, R. M., Grethlein, A. J., Jain, M. K., and Datta, R., 1991. Production of butanol and ethanol from synthesis gas via fermentation. Fuel. 70, 615-619.
Zeikus, J. G., 1980. Chemical and Fuel Production by Anaerobic Bacteria. Annu. Rev. Microbiol. 34, 423-464.
123
CHAPTER 4 STUDY COCLUSIOS
Fossil fuels are harmful to the environment and human health, and the fossil reserves are dwindling at a dramatic rate. In addition, todays biofuels sources such as corn and sugar cane are expensive, depend on government subsidies and can increase food prices as less of these crops are made available for human consumption as a result of their use for fuel production. Biofuels from cellulosic biomass have been identified as one suitable alternative to fossil fuels and first generation biofuel corn ethanol (hydrolysis-fermentation). Synthesis gas has been used as fuel for decades due to being affordable and can be obtained from any type of biomass regardless of its composition. Being a gas, its use as a transportation fuel is not ideal. Converting it to liquid would condense its energy and make it easier to store and to transport. Fermentation of synthesis gas obtained from biomass has proven to be a viable approach to produce biofuels. This technology has higher product yields and lower energy input than lignocellulose hydrolysis-fermentation. Researchers have isolated several microorganisms capable of converting synthesis gas to more suitable, higher energy fuels. These biocatalysts can manage most synthesis gas contaminants making them more robust than some chemical catalysts. Nevertheless, contaminants such as NO, acetylene and O 2 can inhibit activities from these microbes. The industry and the government have been very interested in this process and a commercial demonstration plant is already in operation (Coskata Inc. Madison, Pennsylvania).
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The syngas-fermenting bacterium C. ljungdahlii has been the subject of a number of investigations to improve its ethanol yields. Nevertheless, extensive physiological and metabolic research is limited for this organism. Several acetogens have been shown to produce higher ethanol concentration as a response to O 2 exposure. This opens up new possibilities for improvement of biofuels production. During our research to increase ethanol production by C. ljungdahlii, one of our aims was to demonstrate that C. ljungdahlii could increase ethanol formation from synthesis gas as a response to O 2 exposure. We have also isolated a mutant strain (OTA1). This strain produces approximately 2-fold more ethanol than the WT strain. Both strains increase ethanol production along with decreasing acetate formation when exposed to O 2 . This is significant for C. ljungdahliis use in the biofuels industry as O 2 contamination is likely to occur in the synthesis gas. We also demonstrate that inoculum source for syngas reactors has an effect on growth rate and final cell concentration as well as ethanol formation. Fructose pre-adaptation improves cell concentration and ethanol production, most likely providing higher levels of energy and reducing power to the cells. Fructose is an expensive carbon source; however, an alternative inexpensive sugar may be utilized to enhance ethanol production instead. Given the lack of genetic tools available for C. ljungdahlii, we have had to rely on other methods to increase ethanol production from synthesis gas. We were successful in doing this by exposing the cells to O 2 and pre-adapting the cells to fructose. The fact that this organism increases ethanol formation at the expense of acetate in the presence of O 2 is beneficial in an
125
industrial production setting since it will lower the cost by eliminating the use of reducing agents and the requirements for stringent anaerobic conditions.
126
APPEDIX
This appendix section contains additional figures from C. ljungdaglii oxygen exposure study presented in chapter 2. These include growth, ethanol and acetate formation, headspace analysis and fructose utilization.
127
0 12 24 36 48 60 72 0 500 1000 1500 2000 0% O 2 4% O 2 6% O 2 8% O 2 12% O 2 Time (h) C u l t u r e
D e n s i t y
( m g / L ) 0 12 24 36 48 60 72 0 1 2 3 4 5 6 7 0% O 2 4% O 2 6% O 2 8% O 2 12% O 2 Time (h) F r u c t o s e
( g / L ) 0 12 24 36 48 60 72 0 500 1000 1500 2000 2500 0% O 2 4% O 2 6% O 2 8% O 2 12% O 2 Time (h) E t h a n o l
( m g / L ) 0 12 24 36 48 60 72 0 2000 4000 6000 8000 0% O 2 4% O 2 6% O 2 8% O 2 12% O 2 Time (h) A c e t a t e
( m g / L ) A D C B
Figure A-1. C. ljungdahlii-WT metabolism in reduced medium. A) Growth, B) fructose utilization, C) ethanol production, D) acetate production.
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Figure A-2. C. ljungdahlii-WT growing in reduced medium, headspace analysis at different O 2 concentrations. A) 0 % O 2 B) 4 % O 2 C) 6 % O 2 D) 8 % O 2 E) 12 % O 2
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0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
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( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
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( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) A B C D
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0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
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( m m o l e s ) E
131
0 12 24 36 48 60 72 0 500 1000 1500 0% O 2 4% O 2 6% O 2 8% O 2 10% O 2 Time (h) C u l t u r e
D e n s i t y
( m g / L ) 0 12 24 36 48 60 72 0 1 2 3 4 5 6 7 0% O 2 4% O 2 6% O 2 8% O 2 10% O 2 Time (h) F r u c t o s e
( g / L ) 0 12 24 36 48 60 72 0 500 1000 1500 2000 2500 0% O 2 4% O 2 6% O 2 8% O 2 10% O 2 Time (h) E t h a n o l
( m g / L ) 0 12 24 36 48 60 72 0 2000 4000 6000 8000 0% O 2 4% O 2 6% O 2 8% O 2 10% O 2 Time (h) A c e t a t e
( m g / L ) A D C B
Figure A-3. C. ljungdahlii-WT metabolism in unreduced medium. A) Growth, B) fructose utilization, C) ethanol production, D) acetate production
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Figure A-4. C. ljungdahlii-WT growing in unreduced medium, headspace analysis at different O 2 concentrations. A) 0 % O 2 B) 4 % O 2 C) 6 % O 2 D) 8 % O 2 E) 10 % O 2
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0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
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( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) C D A B
134
0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
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( m m o l e s ) E
135
0 12 24 36 48 60 72 0 2000 4000 6000 8000 0% O 2 4% O 2 6% O 2 8% O 2 12% O 2 Time (h) A c e t a t e
( m g / L ) 0 12 24 36 48 60 72 0 500 1000 1500 2000 0% O 2 4% O 2 6% O 2 8% O 2 12% O 2 Time (h) C u l t u r e
D e n s i t y
( m g / L ) 0 12 24 36 48 60 72 0 500 1000 1500 2000 2500 0% O 2 4% O 2 6% O 2 8% O 2 12% O 2 Time (h) E t h a n o l
( m g / L ) 0 12 24 36 48 60 72 0 2 4 6 8 0% O 2 4% O 2 6% O 2 8% O 2 12% O 2 Time (h) F r u c t o s e
( g / L ) C A D B
Figure A-5. C. ljungdahlii-OTA1 metabolism in reduced medium. A) Growth, B) fructose utilization, C) ethanol production, D) acetate production
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Figure A-6. C. ljungdahlii-OTA1 growing in reduced medium, headspace analysis at different O 2 concentrations. A) 0 % O 2 B) 4 % O 2 C) 6 % O 2 D) 8 % O 2 E) 12 % O 2
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0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
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( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) A D B C
138
0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
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( m m o l e s ) E
139
0 12 24 36 48 60 72 0 500 1000 1500 0% O 2 2% O 2 4% O 2 6% O 2 8% O 2 Time (h) C u l t u r e
D e n s i t y
( m g / L ) 0 12 24 36 48 60 72 0 1 2 3 4 5 6 7 0% O 2 2% O 2 4% O 2 6% O 2 8% O 2 Time (h) F r u c t o s e
( g / L ) 0 12 24 36 48 60 72 0 500 1000 1500 2000 2500 0% O 2 2% O 2 4% O 2 6% O 2 8% O 2 Time (h) E t h a n o l
( m g / L ) 0 12 24 36 48 60 72 0 2000 4000 6000 8000 0% O 2 2% O 2 4% O 2 6% O 2 8% O 2 Time (h) A c e t a t e
( m g / L ) A D C B
Figure A-7. C. ljungdahlii-OTA1 metabolism in unreduced medium. A) Growth, B) fructose utilization, C) ethanol production, D) acetate production
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Figure A-8. C. ljungdahlii-OTA1 growing in unreduced medium, headspace analysis at different O 2 concentrations. A) 0 % O 2 B) 2 % O 2 C) 4 % O 2 D) 6 % O 2 E) 8 % O 2
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0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
c o n c e n t r a t i o n
( m m o l e s ) 0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s
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( m m o l e s ) A D B B
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0 12 24 36 48 60 72 0 1 2 3 4 H 2 O 2 CO CO 2 Time (h) G a s