BIOFUEL ETHANOL PRODUCTION BY Saccharomyces bayanus, THE CHAMPAGNE YEAST

Transcription

1 Clemson University TigerPrints All Theses Theses BIOFUEL ETHANOL PRODUCTION BY Saccharomyces bayanus, THE CHAMPAGNE YEAST Kristen Miller Clemson University, Follow this and additional works at: Part of the Microbiology Commons Recommended Citation Miller, Kristen, "BIOFUEL ETHANOL PRODUCTION BY Saccharomyces bayanus, THE CHAMPAGNE YEAST" (21). All Theses. Paper 98. This Thesis is brought to you for free and open access by the Theses at TigerPrints. It has been accepted for inclusion in All Theses by an authorized administrator of TigerPrints. For more information, please contact

2 BIOFUEL ETHANOL PRODUCTION BY Saccharomyces bayanus, THE CHAMPAGNE YEAST A Thesis Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Master of Science Microbiology by Kristen Publicover Miller December 21 Accepted by: J. Michael Henson, Committee Chair Sarah W. Harcum Harry D. Kurtz, Jr.

3 ABSTRACT The importance of biofuel ethanol is growing as the demand for clean, renewable fuels produced from non-food sources increases. The United States relies mainly on corn and the yeast Saccharomyces cerevisiae for the production of ethanol. A shift to cellulosic feedstocks, as the main source of biomass for ethanol production, would alleviate the pressure on farmers to produce corn for both the food industry and the ethanol industry. Example cellulosic feedstocks include switchgrass, sorghum, and canary grass. The cellulosic feedstocks are typically grown on land that cannot support economic food production, and thus lay unused. For cellulosic feedstocks to be used for ethanol production, the cellulose must be converted to sugars via a process known as hydrolysis. Hydrolyzed switchgrass is an ideal substrate for ethanol production in South Carolina and many southern states because of the humid subtropical climate. Switchgrass hydrolysate contains both five- and sixcarbon sugars, where the six-carbon sugars (mainly glucose) are easily used by yeast to produce ethanol. The five-carbon sugars (mainly xylose) are not directly usable by most yeast to produce ethanol. However, yeast can metabolize xylulose, which can be obtained from xylose by an enzymatic conversion using the enzyme xylose isomerase. In order to economically produce ethanol from cellulosic feedstocks, such as hydrolyzed switchgrass, both the five- and six-carbon sugars need to be metabolized. The long term goal of this research is to produce ethanol cost effectively from hydrolyzed switchgrass using the yeast species Saccharomyces bayanus. ii

4 The specific hypothesis of this study is that S. bayanus, in conjunction with the enzyme xylose isomerase, will metabolize both glucose and the isomer of xylose, xylulose, to produce ethanol. This hypothesis is based on the following observations: 1) S. bayanus can ferment glucose into ethanol; 2) S. bayanus can metabolize xylulose; 3) xylulose can be enzymatically obtained from xylose; 4) S. bayanus can grow in a medium with xylose as the sole carbon source, if xylose isomerase is present. Based on these observations, the experimental focus of this research is to develop and assess methods that allow S. bayanus to convert hydrolyzed switchgrass into ethanol. The experiments are designed to assess the capability and economics of ethanol production using S. bayanus and xylose isomerase. iii

5 ACKNOWLEDGMENTS I would like to acknowledge Dr. Sarah Harcum and Dr. Mike Henson for their valuable advice and guidance, and Tom Caldwell for his technical support in the laboratory. I would like to thank Dr. Charles Turick and Charles Milliken of Savannah River National Laboratory for the analysis of many samples using their high pressure liquid chromatography system. iv

6 TABLE OF CONTENTS TITLE PAGE... i ABSTRACT... ii ACKNOWLEDGMENTS... iv LIST OF TABLES... vii LIST OF FIGURES... viii CHAPTER I. BACKGROUND AND SIGNIFICANCE Significance Background Cellulosic Feedstocks Ethanol Production Yeast Xylose Isomerase... 6 II. EXPERIMENTAL DESIGN Materials Saccharomyces bayanus Hydrolyzed Switchgrass Xylose Isomerase Culture Media Measurement of Cell Growth of S. bayanus High Pressure Liquid Chromatography Gas Chromatography Methods Very High Gravity Fermentations Yeast Growth on Hydrolyzed Switchgrass Page v

7 Table of Contents (Continued) Page Impact of Sodium Azide on Yeast Growth Yeast Growth on Glucose and Xylose with Xylose Isomerase Yeast Growth on Fructose with Xylose Isomerase Statistical Analysis of Growth and Ethanol Production... III. RESULTS AND DISCUSSION Very High Gravity Fermentations Yeast Growth on Hydrolyzed Switchgrass Impact of Sodium Azide on Yeast Growth Yeast Growth on Glucose and Xylose with Xylose Isomerase Yeast Growth on Fructose with Xylose Isomerase IV. CONCLUSION APPENDICES... 4 A. Media B. Experimental Design C. Experimental Results REFERENCES... 6 vi

8 LIST OF TABLES Table Page A-1 Composition of rich medium A-2 Composition of standard minimal medium B-1 Experimental design to determine the effect of xylose isomerase on ethanol production from glucose and xylose by S. bayanus on standard minimal medium C-1 Experimental results for standard minimal medium experiments with statistics... C-2 Experimental results for fructose experiments with statistics... 1 vii

9 LIST OF FIGURES Figure Page 1 Cell density and ethanol concentration profiles for S. bayanus without ph control in 1% rich media during a very high gravity fermentation A comparison of the total ethanol produced and total glucose consumed for S. bayanus fermentations at various ph values in minimal or rich media Glucose concentration profile for S. bayanus grown on minimal medium and/or hydrolyzed switchgrass Glucose consumption (A) and ethanol production (B) for S. bayanus grown on hydrolyzed switchgrass supplemented with minimal medium lacking batch buffer, iron (III) citrate, trace metals, or magnesium sulfate Growth profiles of S. bayanus in minimal medium and various levels of sodium azide Growth of S. bayanus on standard minimal medium lacking iron (III) citrate, trace metals, or magnesium sulfate Growth (A) and ethanol production (B) for S. bayanus on standard minimal medium containing g/l glucose and either 2 g/l or g/l xylose Growth of S. bayanus on standard minimal medium containing A) g/l glucose, B) 8 g/l glucose, or C) 16 g/l glucose. Xylose concentrations are shown for each glucose concentration Production of ethanol by S. bayanus on standard minimal medium containing A) g/l glucose, B) 8 g/l glucose, or C) 16 g/l glucose. Xylose concentrations are shown for each glucose concentration... 3 viii

10 List of Figures (continued) Figure Page 1 Growth profiles of S. bayanus on standard minimal medium containing 4 g/l xylose and A) g/l glucose, B) 8 g/l glucose, or C) 16 g/l glucose Comparison of A) growth profiles, B) maximum observed cell densities, and C) ethanol concentration for S. bayanus on 8 g/l fructose or 8 g/l glucose with and without g/l xylose isomerase Comparison of A) growth profiles, B) maximum observed cell densities, and C) ethanol concentration for S. bayanus on 16 g/l fructose or 16 g/l glucose with and without g/l xylose isomerase... 4 C-1 Growth profiles for S. bayanus on standard minimal medium with g/l glucose... 2 C-2 Growth profiles for S. bayanus on standard minimal medium with 8 g/l glucose... 3 C-3 Growth profiles for S. bayanus on standard minimal medium with 16 g/l glucose... 4 C-4 Fermentation profiles for S. bayanus on g/l glucose... C- Fermentation profiles for S. bayanus on 8 g/l glucose... 6 C-6 Fermentation profiles for S. bayanus on 16 g/l glucose... 7 C-7 Growth profiles for S. bayanus on standard minimal medium with 8 g/l glucose and 8 g/l fructose... 8 C-8 Growth profiles for S. bayanus on standard minimal medium with 16 g/l glucose and 16 g/l fructose... 9 ix

11 CHAPTER ONE BACKGROUND AND SIGNIFICANCE 1.1 Significance The success of this project will result in a method to produce ethanol from hydrolyzed switchgrass. As the dependence on fossil fuels increases, it is vital that a clean source of renewable fuel be easily attainable. The production of ethanol from nonfood based biomass would help alleviate the growing demand for clean fuels that do not contribute to the buildup of greenhouse gases in the atmosphere. 1.2 Background Cellulosic feedstocks are currently being investigated for the production of nonfood based ethanol. S. cerevisiae is commonly used in this process because of its efficient glucose conversion to ethanol and its high ethanol tolerance. This research project investigated S. bayanus, a yeast similar to S. cerevisiae, but with potentially higher ethanol tolerance and lower temperature requirements. The cellulosic feedstock employed was hydrolyzed switchgrass (8 g/l glucose, 4 g/l xylose) or simulated hydrolyzed switchgrass. A major challenge in the biofuel industry is converting xylose into ethanol, as most yeast are not able to metabolize xylose. Thus, xylose isomerase, an enzyme that converts xylose into xylulose, was used. Xylulose can be metabolized by both S. bayanus and S. cerevisiae. 1

12 1.2.1 Cellulosic Feedstocks Currently, the United States uses over 8 million acres of land for growing corn each year, producing nearly 3 billion bushels of corn. In 29, approximately 12 billion bushels of corn were used directly for human consumption, 8 billion bushels for animal feed, and 1 billion bushels for the production of alcohol for fuels. As the demand for fuel increases, the price of corn has also increased. In order to combat higher corn prices, farmers have increased their acreage devoted to corn by planting fewer competing crops, used fallow fields before the fields were nutritionally ready, and reduced the size of their pastures (US Department of Agriculture). To help prevent the price of corn from rising any higher, alternative feedstock sources for ethanol production are being intensively researched, in particular, cellulosic feedstocks. Switchgrass (Panisum virgatum) is a perennial grass native to North America that is adapted to both wet and dry conditions. Due to its high cellulose content and rapid growth, the US Department of Energy has identified switchgrass as a model herbaceous energy crop. Using conventional farming equipment, switchgrass can be easily integrated into existing fields with poor nutritional quality (DOE). Switchgrass is capable of revitalizing a fallow field and has low water and nutritional requirements (Keshwani 29). This hardy grass grows very quickly and can reach up to 1 feet in height. After pretreatment and hydrolysis, the available sugars in hydrolyzed switchgrass are approximately 6% glucose and 4% xylose. 2

13 1.2.2 Ethanol Production Ethanol has been obtained from sugars using yeast for over a millennium. The ethanol concentrations from simple sugars, such as those obtained from corn, can reach 23% volume/volume (Kelsall 1999). The techniques currently used to produce ethanol from corn for biofuel yield between 2 and 4 liters of ethanol per 3 acres of harvested corn. Similarly, switchgrass and other mature cellulosic crops can yield theoretically 2 to 4 L of ethanol per 3 acres of land, with proposed values reaching 6 L of ethanol if xylose is metabolized (Linehart 26). It is important to develop a method with an efficient organism and an easily utilized biomass source to begin relieving the United States dependence on corn for the production of biofuel ethanol Yeast Yeast, specifically the Saccharomyces strains, are facultative anaerobic organisms capable of producing ethanol. Yeast metabolize most sugars through the glycolytic pathway to produce energy and necessary growth intermediates (Ingeldew 1999). To limit high concentrations of acidic end products synthesized in glycolysis, such as pyruvic acid and acetic acid, yeast convert these acids to ethanol and carbon dioxide. Although yeast are not very tolerant of acidic products, they are very tolerant of ethanol. S. cerevisiae are able to produce up to 1.1% weight ethanol/weight glucose from glucose derived from starch (Ingledew 1999). 3

14 Although switchgrass is an ideal candidate for biofuel ethanol production, its major drawback is its high xylose content. Xylose is a five-carbon sugar that is not metabolized by native strains of the yeast Saccharomyces. Alternative organisms capable of metabolizing xylose are often studied; however, these species rarely produce ethanol as efficiently and at the high concentrations of Saccharomyces. For example, Slininger et al. (26) reported ethanol production from Pichia stipitis reaching only approximately 61 g/l ethanol in 3 hours from 4 g/l xylose. The 39% conversion rate is acceptable progress towards xylose utilization; however, the slow growth and ethanol production rates, coupled with low ethanol tolerance and microaerobic requirements of P. stipitis exclude P. stipitis as a candidate for converting xylose to ethanol economically. In order to capitalize on the abundant xylose in cellulosic feedstocks, researchers are also currently modifying S. cerevisiae strains with xylose conversion genes to improve xylose conversion to ethanol. So far, the addition of the bacterial gene for xylose isomerase from Escherichia coli, Bacillus subtilis, Lactobacillus pentosus, and Clostridium thermosulfurogenes has been investigated (Brat 29). Many of the modified S. cerevisiae could metabolize xylose; however, the growth rates were severely reduced and the ethanol production from xylose was limited. Additionally, Brat et al. (29) inserted the gene for xylose isomerase into S. cerevisiae from the bacterium Clostridium phytofermentans. They observed cell growth on 3 g/l xylose with ethanol concentrations of approximately 8 g/l, a conversion rate of 26%. The growth rate, however, was severely limited compared to native S. cerevisiae. Although genetic engineering, in theory, provides many options for improved xylose metabolism, to date, 4

15 the growth rates and ethanol production rates have not reached levels comparable to natural organisms. Although S. cerevisiae is the most commonly used species for biofuel production, there are other Saccharomyces species with robust growth rates and high ethanol tolerance, specifically S. bayanus. S. bayanus is commonly used for wine and champagne fermentations. The genomes of S. bayanus and S. cerevisiae share 4% similarity (Naumov 2). These species also share common environmental niches and are often found together in wine and beer fermentations (Querol 29). Generally, S. cerevisiae prefers 3 C (Querol 29) while S. bayanus can grow and produce ethanol at temperatures ranging from 1 C to 3 C (Pulvirenti 2; Serra 2; Brown 1982). S. bayanus demonstrates good tolerance to ethanol. According to Belloch et al., a strain of S. bayanus isolated from wine in Spain was able to grow in media containing % ethanol (28). It is likely that S. bayanus will survive in media containing more than % ethanol if that ethanol was produced by the cells. Two reports (Brown 1982; Viegas 198) confirm that ethanol added exogenously to growth medium is more toxic than ethanol produced autogenously by the yeast; Viegas specifically studied S. bayanus. In addition to ethanol inhibiting the growth of yeast, very high levels of sugar can also be inhibitory. Most strains of yeast are theoretically able to tolerate up to 4% (weight by volume) solution of sugar, which equals 4 g/l (Yarrow 1998). Studies have shown that S. bayanus is able to grow normally in media containing 2, 2, or 3 g/l glucose (Belloch 28). The tolerable concentrations of xylose are unknown; however,

16 our preliminary results indicate that xylose concentrations up to 8 g/l do not significantly enhance growth or ethanol production Xylose Isomerase A very important aspect of biofuel ethanol production is the complete consumption of the sugars derived from the biomass, mainly glucose and xylose. Glucose consumption and conversion to ethanol is a well-established field. However, the consumption of xylose and subsequent conversion to ethanol is a major hurdle in the production of biofuels. Since xylose is approximately 4% of the cellulosic hydrolysate sugars, for a process to be economical, the xylose must be converted to ethanol. The enzyme xylose isomerase can convert xylose to xylulose. Many yeast species are unable to metabolize xylose; however, many of these species, including S. bayanus, can metabolize xylulose. It has been shown that S. cerevisiae can convert 6% of xylulose into ethanol during fermentation (Gong 1981). The enzyme xylose isomerase performs best at ph 7 and 7 C; however, yeast grows and produces ethanol optimally at ph and 3 C, making a marriage of these two processes a challenge. Initial studies that attempted to convert xylose to xylulose used a two-step process. The two-step process converted xylose to xylulose with xylose isomerase before the sugar was added to the growth medium. The enzymatic reaction reached equilibrium after 2% conversion, limiting the conversion of xylose to a consumable sugar. An alternative one-step process places the xylose and xylose isomerase directly into the growth medium, allowing the reaction to take place in the 6

17 presence of the yeast. Theoretically, the yeast will consume the xylulose as it is created, preventing the reaction from reaching equilibrium, if xylulose is the sole carbon source. Gong et al. (1981) reported % conversion of xylose to ethanol by S. cerevisiae with the one-step process when xylose was the sole carbon source. The initial cell density of the medium in the one-step process was 4 x 1 8 cells/ml, which is very high. No additional cell growth was observed. The cells were not actively growing, xylulose uptake was slow, and the enzymatic reaction reached equilibrium quickly. After % conversion of xylose to xylulose, the reaction stopped and ethanol production ceased. Additionally, the suboptimal growth conditions (4 C, ph 6) may have significantly restricted the cells from growing with their normal efficiency. It was unclear from this study if conversion of xylose to xylulose was limited by the enzyme or by cell health. Recent studies by Rao et al. (28) reported a method of xylose isomerization that creates two different ph environments in the fermentation vessel. Xylose isomerase and urease were co-immobilized in a urea-based growth medium. A ph gradient was created between the enzyme pellet and the medium. This configuration allowed the xylose isomerase to react at the higher ph, while maintaining a lower ph in the medium. This process was most efficient (86% conversion of xylose to xylulose) when tetrahydroxyborate was added to the medium. The addition of urease and tetrahydroxyborate in this system may limit its commercial feasibility due to added expense. Rao et al. states that this configuration will potentially enhance the simultaneous isomerization and fermentation of xylose; however, they did not conduct cell growth studies. 7

18 Studies have shown that growing yeast produce ethanol at a rate approximately 33 times higher than resting cells (Ingledew 1999). Thus, we will use low initial cell densities in a one-step process that will allow S. bayanus to grow through the exponential phase. This method may prevent the enzymatic reaction from reaching equilibrium as the yeast will be actively growing and consuming the xylulose. Thus, it is expected that the enzymatic conversion of xylose to xylulose will be the rate limiting step. Additionally, we will use conditions more suitable for yeast growth (3 C, ph.8). Based on the current standard, it is expected that the sugars will be converted to ethanol with at least % efficiency (not including the production of biomass) (Ingledew 1999). Under the correct growth conditions and with the proper enzyme, S. bayanus will be able to efficiently and totally consume hydrolyzed switchgrass sugars. Producing ethanol in an economical and environmentally-friendly method will help alleviate the dependence on corn for fuel production. Increasing the amount of ethanol produced each year will also provide cleaner fuels, reduce greenhouse gas emissions, and alleviate the demand for fossil fuels. 8

19 CHAPTER TWO EXPERIMENTAL DESIGN 2.1 Materials Saccharomyces bayanus The organism used in this study was Saccharomyces bayanus. S. bayanus was provided by Dr. J. Michael Henson from the Biological Sciences Department at Clemson University. S. bayanus is commercially sold as Liquor Quik Super Yeast X-Press by Winemakeri, Inc (Beaverbank, NS, Canada) and is marketed as a special distiller s yeast Hydrolyzed Switchgrass The hydrolyzed switchgrass used in this study was provided by Dyadic International, Inc (Jupiter, Florida). Dyadic obtained ammonia exploded switchgrass from Dr. Terry Walker of Clemson University. Dr. Walker obtained the switchgrass from Clemson University s Pee Dee Research and Education Center in Florence, South Carolina. 9

20 2.1.3 Xylose Isomerase The enzyme xylose isomerase was purchased from Sigma (Sigma #G4116 glucose isomerase from Streptomyces murinus). Xylose isomerase was used in this study to reversibly isomerize xylose to xylulose. The enzyme also converts glucose to fructose reversibly. The enzyme is also known as Sweetzyme and is produced by Novozymes (Denmark) Culture Media Both a rich medium and a minimal medium were used in these studies. The rich medium contained 1 g/l yeast extract, 2 g/l peptone, and, unless otherwise indicated, g/l glucose and was modified from Gong (1981). The minimal medium, modified from Korz (199) and Sharma (2), contained a phosphate buffer (KH 2 PO 4, (NH 4 ) 2 HPO 4, and citric acid), magnesium sulfate, trace metals (MnCl 2 4 H2O, Zn (CH 3 COO) 2 2 H 2 O, H 3 BO 3, Na 2 MoO 4 2 H 2 O, CoCl 2 6 H 2 O, CuCl 2 2 H 2 O, and EDTA), and iron (III) citrate. The ph was modified to be approximately.8. The standard growth medium was minimal medium supplemented with 1% rich medium and various carbon sources, as indicated for each experiment. The addition of xylose isomerase to the media is noted for each experiment. The media compositions are listed in detail in Appendix A. 1

21 2.1. Measurement of Cell Growth of S. bayanus Cultures of S. bayanus were grown in a bench top shaker at 3ºC and agitated at 13 rpm. The working volume, medium composition, and amount of xylose isomerase are indicated for each experiment. Cell growth in the minimal medium was measured by absorbance at 6 nm with a Spectronic 2 Genesys (Spectronic Instruments, United Kingdom). Cell growth in hydrolyzed switchgrass was indirectly measured by the glucose concentration with the Shimadzu HPLC as described in section Culture flasks were sealed with rubber stoppers that contained a one-way air valve and a sampling syringe. The one-way air valve allowed the carbon dioxide to escape from the shake flasks while minimizing the introduction of oxygen, thus providing an anaerobic environment for ethanol production High Pressure Liquid Chromatography Two different high pressure liquid chromatography (HPLC) systems were used to analyze glucose, fructose, xylose, xylulose, and ethanol. For the very high gravity fermentations and for the fructose experiments, glucose and fructose concentrations were analyzed with a Shimadzu high pressure liquid chromatography (HPLC) system. Glucose samples were injected onto a Bio-Rad Aminex 87-H column in 2 µl volumes at 8 C. The mobile phase was sterile filtered water and flowed at.6 ml/min. Glucose was measured by a refractive index detector and analyzed with Shimadzu software. For the experiments conducted in the standard minimal medium mimicking hydrolyzed switchgrass, glucose, xylose, xylulose and ethanol concentrations were 11

22 measured with a 12 series Agilent HPLC with a Bio-Rad Aminex 87-H column with a cation guard. The samples were injected in µl volumes and run with.6 ml/min sterile filtered water at 6 C. The samples were measured with a refractive index detector and analyzed on ChemStation software. This analysis was performed Savannah River National Laboratory Gas Chromatography For the very high gravity fermentation experiments and the fructose experiments, gas chromatography was used to measure ethanol concentrations (Agilent gas chromatography system). Samples of 1 µl were injected onto the column. The inlet pressure was psi, the total flow was 2. ml/min, and the injector temperature was 23 C. The gas flow through the column was.6447 ml/min. The initial temperature of the column was 6 C, held for 1 minute. The temperature was then ramped to 12 C over 2 min and then ramped to 16 C over 6.13 min. The final temperature was ramped up to 22 C over 4 min and held for 2 min. The mobile phase consists of hydrogen at 3 ml/min, the air at 4 ml/min, and helium (make up gas) at 2 ml/min. 12

23 2.2 Methods Very High Gravity Fermentations The very high gravity fermentations were conducted in a -L vessel with a 2-L working volume at 3ºC and agitated at 2 rpm in rich medium or in the standard minimal medium supplemented with 1% rich medium. Glucose was fed to the fermenters periodically to maintain non-zero glucose levels in the media Yeast Growth on Hydrolyzed Switchgrass S. bayanus was also grown on hydrolyzed switchgrass supplemented with minimal medium and minimal medium lacking various components (batch buffer, iron (III) citrate, trace metals, or magnesium sulfate). The hydrolyzed switchgrass was the source of carbon in this experiment; therefore, the minimal medium did not contain any additional carbon sources. Cells were grown in 2 ml flasks with an 18 ml working volume and g/l xylose isomerase Impact of Sodium Azide on Yeast Growth S. bayanus was grown in the standard minimal medium with various levels of sodium azide. The standard minimal medium supplemented with 1% rich medium contained g/l glucose in 2 ml flasks with a ml working volume. The weight percent of sodium azide in the individual media was.2%,.16%,.7%,.%,.2%, and %. 13

24 2.2.4 Yeast Growth on Glucose and Xylose with Xylose Isomerase Three separate experiments studying the growth of S. bayanus on the standard minimal medium supplemented with 1% rich medium and glucose and xylose were conducted. The first experiment investigated the ability of the standard minimal medium supplemented with 1% rich medium to support growth. S. bayanus was grown in the standard minimal medium lacking iron (III) citrate, trace metals, iron (III) citrate and trace metals, magnesium sulfate, or containing all components. The yeast were grown in 2 ml flasks with a ml working volume. The second experiment provided baseline growth kinetics and ethanol production rates and yields with pure sugars. S. bayanus was grown in the standard minimal medium supplemented with 1% rich medium and xylose isomerase. The media contained g/l glucose and either 2 g/l xylose, g/l xylose, or none at all. The yeast were grown in 2 ml flasks with a ml working volume and g/l xylose isomerase. The third experiment investigated the role of xylose and xylose isomerase in S. bayanus growth and ethanol production and used the standard minimal medium supplemented with 1% rich medium. The baseline sugar concentrations of the medium (8 g/l glucose and 4 g/l xylose) were intended to mimic hydrolyzed switchgrass. Glucose and xylose concentrations investigated were, 8, or 16 g/l glucose and, 4, or 8 g/l xylose. Each condition was cultured with and without g/l xylose isomerase. In all, 2 conditions were investigated in triplicate. These conditions are listed in detail in Appendix B, Table 1. 14

25 2.2. Yeast Growth on Fructose with Xylose Isomerase Experiments were designed to assess the growth and ethanol production of S. bayanus on fructose and xylose isomerase. S. bayanus was grown on 8 g/l or 16 g/l fructose in the standard minimal medium supplemented with 1% rich medium (without glucose) with and without g/l xylose isomerase. Cultures grown on 8 g/l and 16 g/l glucose with and without g/l xylose isomerase were used as controls. The yeast were grown in 2 ml flasks with a ml working volume. Each condition was examined in quadruplicate Statistical Analysis of Growth and Ethanol Production The maximum observed cell densities and ethanol concentrations were examined using statistical analysis software. The statistical analysis software SAS (SAS Institute Inc, Cary, NC) was used to conduct ANOVA analysis (p.) to determine if the cell densities and ethanol concentrations were different due to the carbon source (glucose, xylose, or fructose), or due to enzyme addition (xylose isomerase).

26 CHAPTER THREE RESULTS AND DISCUSSION 3.1 Very High Gravity Fermentations In order to determine the ethanol tolerance and ethanol production of S. bayanus, very high gravity fermentations were conducted. Very high gravity fermentations are classified as having a feedstock of sugar that exceeds 3% weight sugar by volume liquid. Very high gravity fermentation conditions allow for ethanol concentration to reach levels in excess of 1 g/l. The very high gravity fermentations of S. bayanus were conducted across a range of ph levels to determine the best condition for the production of ethanol by S. bayanus. Figure 1 shows the cell density and ethanol concentration profile for S. bayanus without ph control in 1% rich medium. The effect of media was also investigated, where both the standard minimal medium with 1% rich medium and 1% rich medium were examined. Glucose was the sole carbon source in these preliminary experiments. Figure 2A shows the effect of ph on glucose consumption and ethanol production in rich medium. The yeast performed similarly in rich medium for all ph conditions below ph 7. In the rich medium, ethanol production was observed to be slightly higher at ph 6 with % conversion of glucose to ethanol; the final ethanol concentration was 13.8%. Figure 2B shows the effect of ph on glucose consumption and ethanol production in minimal medium. In the minimal medium, the ethanol production was observed to be the highest at ph with 4% conversion of glucose to ethanol; the final ethanol concentration was 1%. All of the fermentations were consistent with robust cell 16

27 health. The cultures grown in rich medium reached higher final cell densities and produced more ethanol. Based on the rich and minimal medium comparison fermentations, it was determined that S. bayanus is capable of growth and ethanol production at ph values less than or equal to ph 6. The fermentation conducted without ph control demonstrates that the yeast can produce ethanol under acidic conditions. Additionally, these data indicate that S. bayanus is ethanol tolerant at levels comparable to S. cerevisiae. 17

28 Cell Density (OD 6nm ) Ethanol (g/l) Time (h) Figure 1. Cell density ( ) and ethanol concentration ( ) profiles for S. bayanus without ph control in 1% rich medium during a very high gravity fermentation. 18

29 Ethanol Produced (g) / Glucose Consumed (g) 6 A uc B ph Figure 2. A comparison of the total ethanol produced and total glucose consumed for S. bayanus fermentations at various ph values in A) 1% rich medium and B) minimal medium with 1% rich medium. UC represents the culture without ph control. Solid bars represent the total ethanol produced (g) and hashed bars represent the total glucose consumed (g). 19

30 3.2 Yeast Growth on Hydrolyzed Switchgrass The very high gravity fermentations demonstrated that S. bayanus had sufficient ethanol production rates and ethanol tolerance to be a potential fermentative organism in cellulosic biofuels (ethanol) production. The next phase of the investigation focused on hydrolyzed switchgrass. S. bayanus was grown on hydrolyzed switchgrass supplemented with minimal medium in order to determine if supplements would be necessary for ethanol production from hydrolyzed switchgrass. Since the hydrolyzed switchgrass renders the medium opaque, cell growth could not be directly monitored; however, the glucose concentrations could be monitored. Thus, the glucose consumption was used as an indirect measure of growth. Figure 3 shows the glucose concentration profiles for the yeast grown in the supplemented hydrolyzed switchgrass. The yeast consumed the glucose in hydrolyzed switchgrass most efficiently when it was supplemented with 11% minimal medium. The glucose consumption rate decreased as the concentration of minimal medium decreased. The 11% minimal medium did not contain glucose, thus no detectable glucose consumption was possible, although cell growth was observed. Thus, some component of the minimal medium improves glucose consumption. 2

31 Glucose (g/l) Time (h) Figure 3. Glucose concentration profiles for S. bayanus grown on hydrolyzed switchgrass supplemented with 11% minimal medium ( ), hydrolyzed switchgrass supplemented with.% minimal medium ( ), and hydrolyzed switchgrass without supplementation ( ), and 11% minimal medium ( ). 21

32 To investigate which component of the minimal medium was responsible for improved glucose consumption, S. bayanus were grown in hydrolyzed switchgrass medium missing one of the four major components of minimal medium (either batch buffer, iron (III) citrate, trace metals, or magnesium sulfate). Again, the glucose concentration profiles were used as an indicator of cell growth. The glucose concentration profiles are shown in Figure 4. Based on the glucose consumption profile, magnesium sulfate was determined to be necessary for efficient glucose consumption, although the other components also improved glucose consumption. The ethanol concentrations were determined at 1 hours, after complete glucose consumption. The ethanol levels were 41 to 46 g/l for the four conditions, which do not appear different. The calculated conversion of glucose to ethanol was on the order of 7% (41 g/l ethanol from 68 g/l glucose), indicating that carbon sources other than glucose in the hydrolyzed switchgrass were used to produce ethanol. The glucose consumption profiles on the hydrolyzed switchgrass were slow compared to pure sugars (1 hours versus less than 24 hours). After these experiments were conducted, it was learned that in the processing of the hydrolyzed switchgrass, sodium azide (.2%) had been added. This level of sodium azide is known to completely inhibit microbial growth. Thus, it was unclear the extent to which sodium azide affected these results, specifically the slow glucose consumption and ethanol production. 22

33 Ethanol (g/l) Glucose (g/l) 7 6 A Time (h) B No Batch No Iron (III) No Trace Buffer Citrate Metals Figure 4. Glucose consumption and ethanol production for S. bayanus grown on hydrolyzed switchgrass supplemented with minimal medium lacking batch buffer ( ), iron (III) citrate ( ), trace metals ( ), or magnesium sulfate ( ). A) glucose concentrations with time; B) ethanol concentrations in the various media at 1 hours. No MgSO 4 23

34 3.3 Impact of Sodium Azide on Hydrolyzed Switchgrass In order to better understand the effect of sodium azide on the hydrolyzed switchgrass results, sodium azide was added to the standard minimal medium supplemented with 1% rich medium, with glucose as the sole carbon source, to mimic the suspected effective dose of sodium azide. Dyadic, Inc. indicated that.2% sodium azide was added to the hydrolyzed switchgrass; however, this level of sodium azide, under normal conditions, should have completely prevented microbial growth. Thus, it appeared that the hydrolyzed switchgrass reduced the inhibitory effects of sodium azide. Figure shows the growth curves for S. bayanus with various levels of sodium azide in the supplemented minimal medium. Interestingly, even.2% sodium azide significantly inhibited growth, more so than was observed in the hydrolyzed switchgrass. Since the overall purpose of this project is to quantify ethanol production from hydrolyzed switchgrass using S. bayanus and the enzyme xylose isomerase, further experiments were designed to use a supplemented minimal medium that would mimic hydrolyzed switchgrass to 1) avoid the sodium azide inhibition effect, and 2) allow for the direct quantification of cell growth. Eventually, the envisioned process for ethanol production from hydrolyzed switchgrass would not include sodium azide. 24

35 Cell Density (OD 6nm ) Time (h) Figure. Growth profiles for S. bayanus in minimal medium and various levels of sodium azide. The weight percent of sodium azide in the media are.2% ( ),.16% ( ),.7% ( ),.% ( ),.2% ( ), and % ( ). 2

36 3.4 Yeast Growth on Glucose and Xylose with Xylose Isomerase Due to the limited availability of hydrolyzed switchgrass and the growth issues associated with sodium azide, experiments were designed to quantify the effect of xylose isomerase on ethanol production in minimal medium that mimics hydrolyzed switchgrass. The standard minimal medium does not contain the undefined organic matter present in hydrolyzed switchgrass, so the ability of the standard minimal medium supplemented with 1% rich medium to support growth was re-examined. The effects of three major medium components were examined. Specifically, S. bayanus were cultured in the standard minimal medium supplemented with 1% rich medium and lacking either iron (III) citrate, trace metals, or magnesium sulfate. The batch buffer was kept in the medium to maintain the ph near.8. Figure 6 shows the growth curves for the media component experiment. As can be clearly seen, the only component that significantly affected growth was the magnesium sulfate. However, the yeast grew to the highest optical density in the presence of all four components, so the complete supplemented minimal medium at approximately ph.8 was used for all further experiments. 26

37 Cell Density (OD 6nm ) Time (h) Figure 6. Growth of S. bayanus in the standard minimal medium lacking iron (III) citrate ( ), trace metals ( ), iron (III) citrate or trace metals ( ), magnesium sulfate ( ), or containing all components ( ). 27

38 Next, experiments were conducted with glucose and xylose separately to provide baseline growth kinetics and ethanol production rates and yields. Initially, growth on glucose was compared to growth on both glucose and xylose. Figure 7 shows both the growth profiles and ethanol production. As shown in Figure 7A, the initial growth rate of all cultures was the same. The final cell densities were observed to be higher for the cultures with xylose; however, the final cell densities for the cultures with 2 g/l or g/l xylose were not different. In Figure 7B, the ethanol production shows that g/l xylose improved ethanol production, whereas 2 g/l xylose did not. The results of this single experiment require further assessment and statistical validation to determine the extent of ethanol production improvement due to xylose. 28

39 Ethanol (g/l) Cell Density (OD 6nm ) 1 A B Time (h) Figure 7. Growth and ethanol production for S. bayanus in the standard minimal medium with xylose isomerase and either g/l glucose ( ), g/l glucose and 2 g/l xylose ( ), or g/l glucose and g/l xylose ( ). 29

40 Preliminary data indicated that xylose was consumed by S. bayanus and may enhance ethanol production; however, the extent of the improvement could not be statistically validated at this point. Thus, the next set of experiments was designed to statistically quantify the enhancement of ethanol production due to xylose and xylose isomerase. The glucose concentrations investigated were g/l, 8 g/l, and 16 g/l; the xylose concentrations were g/l, 4 g/l, and 8 g/l; and the xylose isomerase additions were g/l and g/l. The glucose and xylose concentrations represent typical values obtained for hydrolyzed switchgrass (8 g/l glucose and 4 g/l xylose) and a concentrated hydrolyzed switchgrass (16 g/l glucose and 8 g/l xylose). A concentrated hydrolyzed switchgrass was examined to provide a process condition that would allow for higher ethanol concentrations. The envisioned process includes a concentration stage such that the final ethanol concentration is greater than 7 g/l in the fermentation media, an ethanol concentration considered to be economically viable (Slininger 28). The cultures lacking one or both carbon sources provide a control and will allow for the detection of interaction effects. Triplicate cultures were investigated for all conditions, and all combinations were examined. A list of all the conditions is provided in Appendix B, Table 1. 3

41 The growth of S. bayanus in the standard minimal medium supplemented with 1% rich medium containing various levels of glucose, xylose, and xylose isomerase are shown in Figure 8. As expected, all cultures provided with significant amounts of glucose or xylose grew well. Cell growth in the cultures without significant amounts of glucose or xylose (Figure 8A) was observed due to the basal level of glucose (. g/l) in the medium due to the 1% rich medium addition and the organic material provided in the rich medium components. In Figure 8B, growth on 8 g/l glucose and the three levels of xylose with and without xylose isomerase are shown. As can be clearly observed, the cultures without xylose isomerase had the lowest growth rates and final cell densities, as indicated by the clear separation of the open symbols (no xylose isomerase) and close symbols (xylose isomerase). In Figure 8C, growth on 16 g/l glucose and the three levels of xylose with and without xylose isomerase are shown. Again, it is clear that the cultures with the xylose isomerase addition have better growth profiles (closed symbols) compared to the cultures without xylose isomerase (open symbols). Statistical analysis of the maximum cell densities showed that glucose and xylose isomerase significantly impacted the growth of S. bayanus (p.); however, xylose did not (p.). 31

42 Cell Density (OD 6nm ) A B C Time (h) Figure 8. Growth of S. bayanus on the standard minimal medium with different glucose, xylose, and xylose isomerase levels. The media contained either A) g/l glucose, B) 8 g/l glucose, or C) 16 g/l glucose. Xylose isomerase was either added to the media at g/l (,, ) or not present (,, ). The media also contained xylose at g/l xylose (, ), 4 g/l xylose (, ), or 8 g/l xylose (, ). Error bars represent the standard error. 32

43 Cell growth is important to ethanol production, as many researchers have noted, but ultimately, the ethanol production is the final criteria for a successful process. Figure 9 shows the maximum ethanol concentrations for the glucose, xylose, and xylose isomerase cultures grouped by the glucose concentration in the medium. In Figure 9A the maximum ethanol concentrations are shown for the cultures with. g/l glucose due the 1% rich media addition. As is clearly shown, only minimal amounts of ethanol were produced without significant amounts of glucose. The cultures with xylose isomerase and xylose (4 and 8 g/l) did result in detectable ethanol levels, although it was not statistically different from the cultures without xylose. In Figure 9B the maximum ethanol concentrations are shown for the cultures with 8 g/l glucose, and the three levels of xylose with and without xylose isomerase. The maximum ethanol concentrations were approximately 36 g/l for these cultures. The observed maximum ethanol levels were not statistically different due to the xylose isomerase or xylose. In Figure 9C the maximum ethanol concentrations for the cultures with 16 g/l glucose and the three levels of xylose with and without xylose isomerase are shown. The maximum ethanol concentrations were over 7 g/l. The statistical analysis of the maximum ethanol production results indicate that ethanol production is strongly increased by glucose, as expected (p.). Additionally, the xylose isomerase also statistically impacted the ethanol production (p.). However, the xylose did not significantly impact the maximum ethanol concentration (p.). Comparisons between the cultures with and without xylose isomerase indicate that xylose isomerase increased the ethanol concentration by 12% for the cultures grown 33

44 on 16 g/l glucose (p.); however, xylose isomerase did not increase the ethanol concentration when only 8 g/l glucose was used (p.). 34

45 Ethanol (g/l) 8 7 A B C 4 8 Xylose (g/l) Figure 9. Production of ethanol by S. bayanus on the standard minimal medium with A) g/l glucose, B) 8 g/l glucose, or C) 16 g/l glucose. Xylose concentrations are shown for each glucose concentration. Solid bars represent the addition of g/l xylose isomerase, as the hashed bars represent cultures without xylose isomerase. Error bars represent the standard error. 3

46 In order to better understand the kinetics in the xylose to xylulose reaction, the xylulose profiles were examined in more detail. The cultures with 4 g/l xylose for the three glucose concentrations with xylose isomerase present are shown in Figure 1. The concentration profiles for the and 8 g/l xylose cultures are included in Appendix C. The concentration profiles demonstrate that equilibrium was reached between xylose and xylulose in the presence of three different glucose concentrations with an equilibrium conversion of approximately 12% xylulose. The xylulose equilibrium concentration was approximately g/l, and was maintained until the glucose was consumed for the 8 g/l and 16 g/l glucose cultures. Once the glucose was consumed, the xylulose concentrations decreased due to consumption by S. bayanus. Due to a limited number of cells in the g/l glucose cultures, the xylulose was consumed very slowly. The 12% equilibrium conversion of xylose to xylulose in the culture media is lower than the 2% equilibrium conversion reported for optimal conversion conditions for xylose isomerase (7 C, ph 7.). These concentration profiles indicate that xylose was converted to xylulose until the system reached equilibrium. Once glucose was consumed, xylulose consumption began. The fermentations were halted prior to significant xylulose consumption, so the full extent of the xylulose conversion to ethanol may not have been observed. 36

47 Cell Density (OD 6nm ) and Xylulose (g/l) Glucose and Xylose (g/l) 1 A B C Time (h) Figure 1. Growth profiles of S. bayanus on the standard minimal medium containing 4 g/l xylose, g/l xylose isomerase, and glucose. The glucose levels were A) g/l glucose, B) 8 g/l glucose, or C) 16 g/l glucose. Glucose ( ), xylose ( ), xylulose ( ), and cell density ( ). Error bars represent the standard error. 37

48 3. Yeast Growth on Fructose with Xylose Isomerase The results of the previous experiment showed that glucose and xylose isomerase significantly impacted the growth of S. bayanus. Unexpectedly, the cultures with glucose and xylose isomerase from the previous experiment had the highest observed cell densities. Stepping back, xylose isomerase is also known as glucose isomerase and can convert glucose to fructose, as well as xylose to xylulose, where both reactions are reversible. Thus, S. bayanus was grown on fructose with and without xylose isomerase in the standard minimal medium supplemented with 1% rich medium to determine if the co-metabolism of glucose and fructose could explain the higher cell densities and improved ethanol production. As control conditions, S. bayanus were cultured in media with glucose and xylose isomerase. Figures 11 and 12 show the growth profiles, maximum cell densities, and ethanol production levels for S. bayanus grown on 8 g/l fructose and 16 g/l fructose, respectively, with and without xylose isomerase. Glucose controls are also shown in Figures 11 and

49 Maximum Ethanol (g/l) Maximum Cell Density (OD 6nm ) Cell Density (OD 6nm ) 2 A Time (h) 2 B 1 1 Fructose Glucose C Fructose Glucose Figure 11. Growth profiles, maximum cell densities, and maximum ethanol concentrations for S. bayanus grown on 8 g/l fructose or glucose with and without xylose isomerase. The xylose isomerase addition was g/l. A) Growth profiles, B) maximum observed cell densities, and C) maximum ethanol concentration. Open or hashed symbols represent cultures without xylose isomerase and closed symbols represent cultures with xylose isomerase. In panel A, fructose cultures are represented by circles (, ) and glucose cultures are represented by squares (, ). Error bars represent standard error. 39

50 Maximum Ethanol (g/l) Maximum Cell Density (OD 6nm ) Cell Density (OD 6nm ) 2 A Time (h) 2 B 1 Fructose Glucose 1 C Fructose Glucose Figure 12. Growth profiles, maximum cell densities, and maximum ethanol concentrations for S. bayanus grown on 16 g/l fructose or glucose with and without xylose isomerase. The xylose isomerase addition was g/l. A) Growth profiles, B) maximum observed cell densities, and C) maximum ethanol concentration. Open or hashed symbols represent cultures without xylose isomerase and closed symbols represent cultures with xylose isomerase. In panel A, fructose cultures are represented by circles (, ) and glucose cultures are represented by squares (, ). Error bars represent standard error. 4

51 The initial growth rates for the fructose and glucose cultures containing xylose isomerase were slightly higher than the cultures without enzyme (Figures 11A and 12A) indicating that xylose isomerase significantly affects the growth rate of S. bayanus regardless of the carbon source (glucose or fructose). Additionally, the cultures grown on fructose with xylose isomerase had the highest observed cell densities compared to culture with 8 or 16 g/l glucose. The statistical analysis of the maximum cell density values for the cultures grown on fructose and glucose showed that fructose, glucose, and xylose isomerase were all significant factors (p.). Further analysis showed that xylose isomerase enhanced the maximum cell density of cultures grown on 16 g/l glucose or 8 g/l fructose by 22% and 3%, respectively. The maximum ethanol concentrations for the fructose and glucose with xylose isomerase are shown in Figures 11C and 12C. As expected, the ethanol concentrations were lower for the 8 g/l cultures compared to the 16 g/l cultures for both glucose and fructose. The statistical analysis of the maximum ethanol concentration for the cultures grown on fructose and glucose showed that fructose, glucose, and xylose isomerase were all significant factors (p.). Further analysis showed that 8 g/l fructose and glucose cultures with and without xylose isomerase were not statistically different from each other. Ethanol levels obtained from the from the 8 g/l sugar cultures were approximately 38 g/l, representing a conversion of carbon to ethanol of roughly 48% (Figure 11C). For the 16 g/l fructose and glucose cultures with and without xylose isomerase, the ethanol concentrations were significantly different due to the addition of 41