Progress 10/01/06 to 09/30/12
Outputs
OUTPUTS: Two new invention disclosures were submitted on a new method to make single activity enzymes. These enzymes could be used for a variety of purposes. The enzyme we produced was one used for hydrolysis of xylans in switchgrass and other grasses and has direct application for biorefineries. A new trickle bed reactor for enzyme production was constructed. PARTICIPANTS: Collaborators: Raymond L. Huhnke, OSU, Hasan Atiyeh, OSU, and Rolf Prade, OSU Training and Professional Development: Michael Mueller and Jennine Terrill completed his PhD dissertation with data from this project. TARGET AUDIENCES: The target audiences are ethanol producers, government officials involved in bioenergy policy, enzyme producers, farmers and ranchers that may be involved in biomass production, undergraduate and graduate students interested in biofuels, and other biofuels researchers. Educational efforts to inform target audiences of the outputs of this project included a presentation at the 2012 American Society of Agricultural and Biological Engineers Meeting and the 2012 Oklahoma EPSCoR meeting. These presentations did not disclose details that we are seeking to protect through patents. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A trickle bed reactor was successfully used to produce xylanase enzyme using a genetically modified fungus. Additional techniques were also developed to increase production of enzyme; however, these techniques are the subject of a potential patent application and cannot be disclosed in this report. Oklahoma State University is seeking potential licensees of this technology.
Publications
- Mueller, M.M. 2012. Utilization and production of single activity enzyme from Aspergillus nidulans. Ph.D. Dissertation. Stillwater, OK Oklahoma State University. 155 p.
- Terrill, J.B. 2011. Evaluation of best parameters in simultaneous saccharification and fermentation process for maximum ethanol yield by K. marxianus IMB3. Ph.D. Dissertation. Stillwater, OK: Oklahoma State University. 151 p.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: The cost of media used in laboratory fermentations to produce ethanol from cellulosic materials is cost prohibitive for industrial applications. To address this, a series of experiments were conducted to identify key components of yeast fermentation media to determine which components could be reduced or eliminated and which components could be increased. Also, the media developed in these experiments were tested in simultaneous saccharification and fermentation experiments with switchgrass to determine if they were suitable for this method of producing ethanol. PARTICIPANTS: Collaborators: Raymond L. Huhnke, OSU, Hasan Atiyeh, OSU, Andrew Mort, OSU, Rolf Prade, OSU and Ibrahim M. Banat, University of Ulster, United Kingdom. Training and Professional Development: Michael Mueller is now studying to complete a Ph.D. degree and will continue to work on the project. Jennine Terrill is completing her PhD dissertation with data from this project.. TARGET AUDIENCES: The target audiences are ethanol producers, government officials involved in bioenergy policy, enzyme producers, farmers and ranchers that may be involved in biomass production, undergraduate and graduate students interested in biofuels, and other biofuels researchers. Educational efforts to inform target audiences of the outputs of this project included a presentation at the 2011 American Society of Agricultural and Biological Engineers Meeting and the 2011 Oklahoma EPSCoR meeting. In addition results from this project were reported at the S-1041 multistate research committee annual meeting in Stillwater, OK last August. This meeting included several government officials and university researchers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
It was determined that increasing manganese in the media was detrimental to ethanol production, but adding calcium to a level of 3 mM was beneficial. We also developed a completely defined medium that did not contain any yeast extract or other complex media components. This medium allows us to test individual components of the media to determine their effect on ethanol production.
Publications
- No publications reported this period
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The effect of when switchgrass is harvested on its cell wall composition and its subsequent conversion to ethanol is being investigated. Switchgrass was harvested on the 22nd day of July, August, September, October, and November (November was a post-freeze sample). Samples were analyzed for structural carbohydrates and lignin by standard National Renewable Energy Laboratory (NREL) protocols. A portion of each sample was then pretreated using a liquid, hot water treatement at 200 degrees C for 10 min. This pretreatment was to dissolve hemicellulose, disrupt lignin, and open up the cell wall for cellulose hydrolysis to glucose by enzymes. After pretreatment, the sample is filtered to separate the solids from liquid and dissolved solids. The liquid, known as prehydrolyzate, is analyzed by NREL protocols to determine the concentration monomer and oligomer sugars dissolved and the concentration of acids and furfurals that could later inhibit fermentation organisms. The solids are washed with warm water to remover soluble solids and the washed solids are analyzed for lignin and structural carbohydrates. Washed solids containing 4 g of cellulose are added to a 250 ml culture flask and diluted with water, fermentation media, and cellulase enzyme at (15 FPU/g cellulose) for a total volume of 100 ml. Then 20 mg of S. cerevisiae D5A yeast are added to each flask and the flasks are incubated at 37 degrees C for 7 days while being shaken at 150 rpm. Samples are taken each day and analyzed for ethanol, glucose, xylose, acetic acid, glycerol, xylitol, succinic acid and lactic acid by HPLC using a NREL protocol. Results were presented at the 2010 ASABE Annual Conference. PARTICIPANTS: Collaborators: Raymond L. Huhnke, OSU, Hasan Atiyeh, OSU, Andrew Mort, OSU, Rolf Prade, OSU and Ibrahim M. Banat, University of Ulster, United Kingdom. Training and Professional Development: Brian Faga and Michael Mueller have worked on this project and received their MS degrees in May 2009. Michael Mueller is now studying to complete a Ph.D. degree and will continue to work on the project. Michael Matousek and Jennine Terrill are also graduate students working on the project. TARGET AUDIENCES: The target audiences are ethanol producers, government officials involved in bioenergy policy, enzyme producers, farmers and ranchers that may be involved in biomass production, undergraduate and graduate students interested in biofuels, and other biofuels researchers. Educational efforts to inform target audiences of the outputs of this project included presentation at the 2010 American Society of Agricultural and Biological Engineers Meeting and the 2010 Oklahoma EPSCoR meeting. In addition results from this project were reported at the S-1041 multistate research committee annual meeting in Wyndmoor, PA last July. This meeting included several government officials and university researchers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The harvest effect study showed that lignin increased over the harvest season while hemicellulose decreased, which was expected. Cellulose did not change appreciably. Initial SSF tests indicate that ethanol yields are related to the lignin concentration in the original switchgrass, not the lignin concentration in the pretreated grass. Thus, greater amounts of lignin in the harvested switchgrass resulted in lower yields of ethanol from SSF. Dry matter yields from grass harvesting in these plots are not known, so a calculation of change in ethanol produced per acre was not possible.
Publications
- Faga, B.A., M.R. Wilkins, I.M. Banat. 2010. Ethanol production through simultaneous saccharification and fermentation of switchgrass using Saccharomyces cerevisiae D5A and thermotolerant Kluyveromyces marxianus IMB strains. Bioresource Technol. 101:2273-2279.
- Kundiyana, D., D.D. Bellmer, R.L. Huhnke, M.R. Wilkins, P.L. Claypool. 2010. Influence of temperature, pH and yeast on the in-field ethanol production from sweet sorghum. Biomass Bioenergy 34:1481-1486.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Five strains of Kluyveromyces marxianus known as the IMB strains, a yeast known for its ability to grow using xylose at temperatures up to 45 degrees C, were tested for their ability to produce xylitol and ethanol from xylose under microaerobic conditions. The effects of temperature, yeast strain, and sugar used to produce the inoculum were tested. Preliminary results were presented through a poster at the 31st Symposium on Biotechnology for Fuels and Chemicals and more results will be presented through a poster at the Bioenergy Engineering 2009 Conference. Additionally, the effect of when switchgrass is harvested on its cell wall composition and its subsequent conversion to ethanol is being investigated. Switchgrass was harvested on the 22nd day of July, August, September, October, and November (November was a post-freeze sample). Samples were analyzed for structural carbohydrates and lignin by standard National Renewable Energy Laboratory (NREL) protocols. A portion of each sample was then pretreated using a liquid, hot water treatement at 200 degrees C for 10 min. This pretreatment was to dissolve hemicellulose, disrupt lignin, and open up the cell wall for cellulose hydrolysis to glucose by enzymes. After pretreatment, the sample is filtered to separate the solids from liquid and dissolved solids. The liquid, known as prehydrolyzate, is analyzed by NREL protocols to determine the concentration monomer and oligomer sugars dissolved and the concentration of acids and furfurals that could later inhibit fermentation organisms. The solids are washed with warm water to remover soluble solids and the washed solids are analyzed for lignin and structural carbohydrates. Washed solids containing 4 g of cellulose are added to a 250 ml culture flask and diluted with water, fermentation media, and cellulase enzyme at (15 FPU/g cellulose) for a total volume of 100 ml. Then 20 mg of S. cerevisiae D5A yeast are added to each flask and the flasks are incubated at 37 degrees C for 7 days while being shaken at 150 rpm. Samples are taken each day and analyzed for ethanol, glucose, xylose, acetic acid, glycerol, xylitol, succinic acid and lactic acid by HPLC using a NREL protocol. Preliminary results were presented at the 2009 ASABE Annual Conference. PARTICIPANTS: Collaborators: Raymond L. Huhnke, OSU, Andrew Mort, OSU, Rolf Prade, OSU and Ibrahim M. Banat, University of Ulster, United Kingdom. Training and Professional Development: Brian Faga and Michael Mueller have worked on this project and received their MS degrees in May 2009. Michael Mueller is now studying to complete a Ph.D. degree and will continue to work on the project. Michael Matousek and Jennine Terrill are also graduate students working on the project. TARGET AUDIENCES: The target audiences are ethanol producers, government officials involved in bioenergy policy, enzyme producers, farmers and ranchers that may be involved in biomass production, undergraduate and graduate students interested in biofuels, and other biofuels researchers. Educational efforts to inform target audiences of the outputs of this project included 2 posters at the 31st Symposium on Biotechnology for Fuels and Chemicals and a presentation at the 2008 American Society of Agricultural and Biological Engineers Meeting. In addition results from this project were reported at the S-1041 multistate research committee annual meeting in Richland, WA last September. This meeting included several government officials and university researchers. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The Kluyveromyces marxianus IMB yeasts produced little ethanol from xylose. The IMB1 and IMB2 strains produced the highest yields of xylitol. Fermentation at 40 and 45 degrees C resulted in more xylitol production than fermentation at 25, 30 or 35 degrees C. Using xylose to produce inoculum for xylose fermentation instead of glucose, which is typically used, resulted in greater xylitol production. Xylitol yields were from 0.62 to 0.67 g xylitol/g xylose for xylose-grown cells while yields were less than 0.57 g/g for glucose-grown cells. Growth rates of up to 0.20 h-1 were observed during xylose fermentation. The harvest effect study is ongoing, but preliminary results were that lignin increase over the harvest season while hemicellulose dereases. Cellulose did not change appreciably. Initial SSF tests indicate that ethanol yields are related to the lignin concentration in the original switchgrass, not the lignin concentration in the pretreated grass. Thus, greater amounts of lignin in the harvested switchgrass resulted in lower yields of ethanol from SSF.
Publications
- Faga, B. 2009. Simultaneous Saccharification and Fermentation of Pretreated Switchgrass by Thermotolerant IMB strains of Kluyveromyces marxianus. Thesis. Stillwater, OK: Oklahoma State University. 123 p.
- Kundiyana, D., M.R. Wilkins, R.L. Huhnke, I.M. Banat. 2009. Effect of furfural addition on xylose utilization by Kluyveromyces marxianus IMB4 under anaerobic and microaerobic conditions. Biological Eng. (in press).
- Mueller, M. 2009. Fermentation of Xylose and Xylans by Kluyveromyces marxianus IMB Strains. Thesis. Stillwater, OK: Oklahoma State University. 155 p.
- Suryawati, L., M.R. Wilkins, D.D. Bellmer, R.L. Huhnke, N.O. Maness, I.M. Banat. 2009. Effect of hydrothermolysis process conditions on pretreated switchgrass composition and SSF ethanol yield using Kluyveromyces marxianus IMB4. Process Biochem. 44:540-545.
- Widmer, W., J. Narciso, K. Grohmann, M. Wilkins. 2009. Simultaneous saccharification and fermentation of orange processing waste to ethanol using Kluyveromyces marxianus. Biological Eng. (in press).
- Wilkins, M.R. 2009. Effect of orange peel oil on ethanol production by Zymomonas mobilis. Biomass and Bioenergy 33:538-541.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Five strains of Kluyveromyces marxianus, a yeast known for its ability to ferment glucose to ethanol at temperatures up to 50 degrees C, were tested in a simultaneous saccharification and fermentation (SSF) process. The feedstock was switchgrass pretreated using a liquid, hot water process at 200 degrees C for 10 min. This pretreatment was chosen as a result of previous work reported in last year's report. The SSF temperature was 45 degrees C. All experiments were done in 250 ml flasks using a 50mM sodium citrate buffer at pH 5.5. An amount of cellulase preparation (Rapidase, Iogen, Ottawa, Ontario, Canada) with an activity of 15 filter paper units (FPU)/ml was used to hydrolyze the switchgrass. The amount of switchgrass used resulted in a concentration of approximately 40 g glucan/L. SSFs were carried out for 168h. In addition a control SSF was done using Saccharomyces cerevisiae D5A at 37 degrees C. Also, an additional SSF using S. cerevisiae D5A with a 50mM sodium citrate buffer at pH 4.8 was done, which was consistent with the National Renewable Energy Laboratory SSF procedure. After these tests were complete, the effect of enzyme loading on ethanol production by K. marxianus IMB3 and S. cerevisiae D5A during SSF of switchgrass was assessed. For K. marxianus IMB3, the SSF temperature was 45 degrees C and for S. cerevisiae, the SSF temperature was 37 degrees C. All experiments were done in 250 ml flasks using a 50mM sodium citrate buffer at pH 5.5. Cellulase loadings of 5, 10 or 15 FPU/ml were used. Results were presented through posters at the 2008 American Society of Agricultural and Biological Engineers Meeting and at the 30th Symposium on Biotechnology for Fuels and Chemicals. PARTICIPANTS: Collaborators: Danielle D. Bellmer, OSU, Raymond L. Huhnke, OSU, Andrew Mort, OSU, Niels O. Maness, OSU and Ibrahim M. Banat, University of Ulster, United Kingdom. Training and Professional Development: Lilis Suryawati was an M.S. student who worked on this project and received her degree in December 2007. Brian Faga is currently working on a M.S. degree while working on the project, and is expected to graduate in May 2007. Michael Matousek and Jennine Terrill are new graduate students working on the project. In addition, an Oklahoma-Louis Stokes Alliance for Minority Participation Scholar, Manny Cortez, has started to work on research related to inhibition of K. marxianus IMB3 ethanol production at high temperature. TARGET AUDIENCES: The target audiences are ethanol producers, government officials involved in bioenergy policy, enzyme producers, farmers and ranchers that may be involved in biomass production, undergraduate and graduate students interested in biofuels, and other biofuels researchers. Educational efforts to inform target audiences of the outputs of this project included posters at the 2008 American Society of Agricultural and Biological Engineers Meeting and at the 30th Symposium on Biotechnology for Fuels and Chemicals. In addition results from this project were reported at the SDC-325 multistate research committee annual meeting in Washington, DC last September. This meeting included several government officials and university researchers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
K. marxianus IMB3 was the best strain in terms of ethanol production of the five K. marxianus strains tested. The maximum concentration of ethanol produced by IMB3 was 18.6 g/L after 96h, which equals a theoretical yield of 80 percent. All of the other strains tested achieved less than a 75% theoretical yield. K. marxianus IMB4 ceased fermentation after 96h while the other four strains ceased fermentation after 72h. S. cerevisiae D5A achieved a maximum concentration of 21.9 g/L, or a 90 percent theoretical yield, after 168h. When the buffer pH was changed to 4.8, S. cerevisiae D5A's maximum ethanol concentration produced was only 17.6 g/L, or 78 percent theoretical, after 168 h. This is comparable to a previous study done under this project last year, except that fermentation was slower in last year's study. This may be due to a finer particle size being used this year as opposed to last year. Reducing enzyme loading had a negative impact on ethanol production by both K. marxianus IMB3 and S. cerevisiae, but moreso on IMB3. Maximum ethanol concentrations produced by IMB3 were 10 g/L, 5 FPU; 15 g/L, 10 FPU; and 18.7 g/L, 15 FPU. Maximum ethanol concentrations produced by S. cerevisiae D5A were 13 g/L, 5 FPU; 19.4 g/L, 10 FPU; and 20.9 g/L, 15 FPU. Our findings indicate that though the K. marxianus strains can ferment glucose to ethanol at 45 degrees C, they are less efficient at doing so than is S. cerevisiae D5A at 37 degrees C. It was also noted that the cellulase used did not hydrolyze cellulose faster at 45 degrees C than at 37 degrees C. Typically, cellulases have more than 50 percent more activity at 45 degrees C than at 37 degrees C. Due to this observation, we are testing newer cellulases specifically designed for biomass hydrolysis that claim to have a greater activity at higher temperatures. Using a cellulase in SSF with a much greater activity at higher temperature could give K. marxianus IMB3 at 45 degrees C an advantage over S. cerevisiae D5A. Also, these findings have directed us to study which factors may be inhibiting ethanol production by K. marxianus IMB3 at 45 degrees C.
Publications
- Suryawati, L. 2007. Simultaneous saccharification and fermentation of hydrothermolysis pretreated switchgrass for ethanol production. M.S. Thesis. Oklahoma State University. Submitted December 2007.
- Suryawati, L., M.R. Wilkins, D.D. Bellmer, R.L. Huhnke, N.O. Maness, I.M. Banat. 2008. Simultaneous saccharification and fermentation of Kanlow switchgrass pretreated by hydrothermolysis using Kluveromyces marxianus IMB4. Biotechnol. Bioeng. DOI 10.1002/bit.21965.
- Faga, B.A., M.R. Wilkins, and I.M. Banat. 2008. Simultaneous saccharification and fermentation of pretreated switchgrass with Kluyveromyces marxianus IMB strains. 2008 ASABE Annual Meeting Paper No. 083578.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Experiments were conducted to evaluate hydrothermolysis as a pretreatment for preparation of switchgrass for enzymatic hydrolysis. Hydrothermolysis involves heating a slurry of biomass and water under pressure to keep the water in liquid form. Three temperatures (190, 200 and 210 degrees C) and three hold times (10, 15 and 20 minutes) were evaluated. After pretreatment, the remaining insoluble solids were recovered by filtration and hydrolyzed by a cellulase preparation to glucose. A thermotolerant yeast (Kluyveromyces marxianus IMB4) was added at the same time to ferment glucose to ethanol in a process known as simultaneous saccharification and fermentation (SSF). The temperature for SSF was 45 degrees C. In a subsequent experiment, K. marxianus IMB4 was evaluated at 37, 41 and 45 degrees C and compared to a control of Saccharomyces cerevisiae D5A at 37 degrees C. Switchgrass was prepared by hydrothermolysis at 200 degrees C and 10 minutes. SSF was initially conducted at pH
4.8. Due to a premature end to fermentation at pH 4.8 and 45 degrees C, an experiment was also done at initial pH 5.5 using K. marxianus IMB4 at 45 degrees C. K. marxianus IMB4 is of great interest because it can ferment sugars at 45 degrees C, which is an ideal temperature for cellulase hydrolysis. Other yeasts cannot survive at this temperature, so lower temperatures must be used during SSF. This results in a slower hydrolysis and fermentation. Results have been disseminated through a conference presentation and paper to the American Association of Agricultural and Biological Engineers Meeting and through a poster at the Oklahoma GROW Conference.
PARTICIPANTS: Collaborators: Danielle D. Bellmer, OSU, Raymond L. Huhnke, OSU, Andrew Mort, OSU, Niels O. Maness, OSU and Ibrahim M. Banat, University of Ulster, United Kingdom.
TARGET AUDIENCES: The target audiences are ethanol producers, government officials involved in bioenergy policy, enzyme producers, farmers and ranchers that may be involved in biomass production, undergraduate and graduate students interested in biofuels, and other biofuels researchers. Educational efforts to inform target audiences of the outputs of this project included a poster at the Oklahoma GROW Conference, a gathering of biofuels industry officials, Oklahoma farmers and ranchers, government officials, and academic researchers interested in biofuels. Also, a new course has been implemented at Oklahoma State University on renewable energy with several lectures focusing on activities and subjects related to this project.
Impacts
Hydrothermolysis was determined to be a suitable pretreatment for hydrolysis of cellulose in switchgrass to glucose by cellulase. As temperature and hold time increased, more glucose was released through hydrolysis and more ethanol was produced. The conditions of 200 degrees C and 10 minutes were determined to be the best conditions for ethanol production. Also, K. marxianus IMB4 was found to be a promising candidate for use in SSF of biomass. K. marxianus IMB4 at 41 and 45 degrees C produced more ethanol than the control at 72 hours, but they ceased fermentation after 96 and 72 hours, respectively, while the control continued fermentation for 168 hours. Increasing pH to 5.5 resulted in greater ethanol productivity and fermentation continued for 96 h as opposed to 72 h at pH 4.8. SSF using K. marxianus IMB4 at 45 degrees C resulted in faster cellulose hydrolysis and faster ethanol production as compared to the most commonly used ethanol-producing yeast, S. cerevisiae
D5A. The implementation of numerous standard procedures developed for analyzing biomass composition, enzymatic hydrolyses, and fermentations have been implemented as a result of this project. Three graduate students and the primary investigator have been trained in these methods, which has greatly expanded the capability of the biofuels program at Oklahoma State University (OSU). These procedures are commonly used in the biofuels industry and will make students trained during this project more marketable to employers. Also, more experienced graduate students have passed on their knowledge regarding enzymatic hydrolysis techniques and analysis procedures to new students, ensuring continuity of procedures and the ability to compare results over the course of the project.
Publications
- Suryawati, L., M.R. Wilkins, D.D. Bellmer, R.L. Huhnke, N.O. Maness, and I.M. Banat. 2007. Effect of hydrothermolysis on ethanol yield from Alamo switchgrass using a thermotolerant yeast. Paper No. 077071. 2007 ASABE Annual Meeting. June 17-20, 2007.
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