Progress 10/01/08 to 09/30/14
Outputs
Target Audience: The target audiences were undergraduate and graduate students, farmers and reanchers, and the bioenergy industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
No new accomplishments were achieved during 2013-14 as the project ended September 30, 2013. Previous accomplishments are noted in the annual progress reports.
Publications
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The target audiences were undergraduate and graduate students, farmers and reanchers, and the bioenergy industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? The results were disseminated through peer reviewed articles and presentations at national meetings. What do you plan to do during the next reporting period to accomplish the goals? The project is over.
Impacts
What was accomplished under these goals?
An acid sulfite pretreatment process for Eastern redcedar was developed. Factorial experiments and response surface methodology was used to optimize chemical loadings, pretreatment temperature and reactor residence time for the pretreatment. The results of these experiments were used to pretreat redcedar for conversion process testing. The effect Eastern redcedar oil on hydrolysis of cellulose by cellulase and fermentation of glucose to ethanol by Saccharomyces cerevisiae was investigated. This test was necessary for developing a process to convert redcedar to products via enzymatic hydrolysis and yeast fermentation. The use of dry solids contents up to 20% (w/w) in enzymatic hydrolysis was explored. The use of mixing aids to increase mixing in these tests was also investigated. In addition, the use of a size reduction process developed by Forest Concepts, LLC to prepare biomass for simultaneous saccharification and fermentation (SSF) was evaluated as were the differences in ethanol yields between using sapwood and heartwood in SSF.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2013
Citation:
Ramachandriya, K.D. Ph.D. Biosystems Engineering, Oklahoma State University. Development and optimization of glucose and ethanol production from Eastern redcedar. Completed July 2013.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Ramachandriya, K.D., M.R. Wilkins, N. Dunford, S. Hiziroglu, and H.K. Atiyeh. 2013. Development of an efficient pretreatment process for enzymatic saccharification of Eastern redcedar. Bioresource Technol. 136:131-139.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2014
Citation:
Ramachandriya, K.D., M.R. Wilkins, K. Patil. 2013. Influence of switchgrass generated producer gas pre-adaptation on growth and product distribution of Clostridium ragsdalei. Biotechnol. Bioprocess Eng. (in press).
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: An invention disclosure was filed for a technique for biofuel production and a patent application is being developed. An acid sulfite pretreatment process was developed for Eastern redcedar. An article on biomass pretreatment was posted on the SunGrant BioWeb. Tests to evaluate the effect of redcedar oil on redcedar wood hydrolysis and yeast fermentation were developed. PARTICIPANTS: Participants Co-PIs: Mark R. Wilkins, OSU; Raymond L. Huhnke, OSU; Michael Buser, OSU; Hasan Atiyeh, OSU; Ajay Kumar, OSU. Collaborators: Danielle Bellmer, OSU, , Andrew Mort, OSU, Rolf Prade, OSU and Ibrahim M. Banat, University of Ulster, United Kingdom,. Training and Professional Development Karthikeyan Ramachandriya is a graduate student 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 a presentation at the 2012 ASABE Meeting, and posters at the 2012 Oklahoma EPSCoR conference, 2012 DOE Bioenergy meeting, 2012 National Sun Grant Conference and the 2012 S-1041 Annual Meeting. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Over 85 percent of cellulose in Eastern redcedar was converted to glucose when using an acid sulfite process developed in our lab. The best conditions were 3.75 g sulfuric acid/100 g dry wood, 20 g Na bisulfite/100 g dry wood, 200 degrees C and a hold time of 10 minutes. These conditions are being used to prepare wood for other experiments that are ongoing. Impacts from work related to the invention disclosure cannot be shared at this time.
Publications
- Liu, K., H. K. Atiyeh, R. S. Tanner, M. R. Wilkins and R. L. Huhnke. 2012. Fermentative production of ethanol from syngas using novel moderately alkaliphilic strains of Alkalibaculum bacchi. Bioresource Technology, 104: 336-341.
- Pasangulapati, V., K. D. Ramachandriya, A. Kumar, M. R. Wilkins, C. L. Jones, and R. L. Huhnke. 2012. Effects of cellulose, hemicellulose and lignin on thermochemical conversion characteristics of the selected biomass. Bioresource Technology. 114: 663-669.
- Terrill, J. B., M. R. Wilkins, M. J. M. DeLorme, H. K. Atiyeh and R. S. Lewis. 2012. Effect of energetic gas composition on hydrogenase activity and ethanol production in syngas fermentation by Clostridium ragsdalei. Biological Engineering Transactions. 5: 87-98.
- Ukpong, M. N., H. K. Atiyeh, M. J. M. De Lorme, K. Liu, S. Zhu, R. S. Tanner, M. R. Wilkins and B. S. Stevenson. 2012. Physiological response of Clostridium carboxidivorans during conversion of synthesis gas to solvents in a gas-fed bioreactor. Biotechnology and Bioengineering. 109:2720-2728.
- Wilkins, M.R. 2011. Lignocellulose pretreatment technologies. BioWeb - Sun Grant Initiative and the University of Tennessee: http://www.bioweb.sungrant.org/.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Work continued in the area of producer gas fermentation. The effect of CO and H2 on hydrogenase activities was tested. Also, links between hydrogenase activity, H2 consumption and ethanol and acetate production were investigated. Kluyveromyces marxianus IMB3 was used to produce ethanol from switchgrass using simultaneous saccharification and fermentation (SSF). The effects of enzyme loading, enzyme loading methods, temperature, solids content were investigated. PARTICIPANTS: Co-PIs: Mark R. Wilkins and Raymond L. Huhnke, OSU, Hasan Atiyeh, OSU. Collaborators: Danielle Bellmer, OSU, , Andrew Mort, OSU, Rolf Prade, OSU and Ibrahim M. Banat, University of Ulster, United Kingdom, Ajay Kumar, OSU. Michael Mueller, Karthikeyan Ramachandriya and Xiaoguang Zhu are 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 several posters at the 2011 ASABE Meeting and the 2011 Oklahoma EPSCoR conference. In addition results from this project were reported at the S-1041 multistate research committee annual meeting in Stillwater, OK last July. This meeting included several government officials and university researchers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
CO has a large impact on hydrogenase activity. Increasing CO content from 20% of syngas to 40% of syngas resulted in a large decrease in hydrogenase activity. The highest ethanol production was observed with a gas mixture of 20% CO and 10% H2. Also, the presence of hydrogenase activity was necessary to produce ethanol, even if the activity was very small. A concentration of 0.7 g Accelerase 1500 cellulase enzyme/g glucan was identified as the best enzyme concentration for production of ethanol from SSF of switchgrass using K. marxianus IMB3. Using 45 degrees C with IMB3 gave a similar ethanol yield to an SSF with the conventional ethanol-producing yeast Saccharomyces cerevisiae D5A at 37 degrees C. Also, solids concentrations of up to 12% could be saccharified and fermented without a reduction in ethanol yield.
Publications
- Pessani, N., H.K. Atiyeh, M.R. Wilkins, D.D. Bellmer, I.M. Banat. 2011. Simultaneous saccharification and fermentation of Kanlow switchgrass by thermotolerant Kluyveromyces marxianus IMB3: the effect of enzyme loading, temperature and higher solid loadings. Bioresource Technology. 102:10618-10624.
- Wilkins, M.R. and H.K. Atiyeh. 2011. Fermentative production of ethanol from carbon monoxide. Current Opinion in Biotechnology. 22:326-330.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Work continued in the area of producer gas fermentation. Heat shocking of inoculum was tested in an effort to increase the solvent-producing capabilities of Clostridium strain P11. Heat shocking involves heating an inoculum to a lethal temperature for a short period of time to stimulate sporulation. Sporulation has often been associated with higher alcohol production in alcohol-producing bacteria from the genus Clostridium. The Kluyveromyces marxianus IMB strains were tested for their ability to produce xylitol from xylose. Also, the activities of the enzyme xylose reductase were tested using either NAD(P)H or NADH to determine why ethanol is not produced by these yeast when they consume xylose. PARTICIPANTS: Co-PIs: Mark R. Wilkins and Raymond L. Huhnke, OSU. Collaborators: Danielle Bellmer, OSU, Hasan Atiyeh, OSU, Andrew Mort, OSU, Rolf Prade, OSU and Ibrahim M. Banat, University of Ulster, United Kingdom. Training and Professional Development: Karthikeyan Ramachandriya worked on this project and received his MS degree in December 2009. Dimple Kundiyana also worked on this project and completed his PhD degree in August 2010. Michael Matousek, Michael Mueller, Jennine Terrill, Karthikeyan Ramachandriya and Xiaoguang Zhu 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 several posters at the 2010 ASABE Meeting and the 2010 Oklahoma EPSCoR conference. 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
It was found that heat shocking Clostridium strain P11 at 92 degrees C for 3 minutes promotes increased ethanol production and sporulation. This is the first report of sporulation in this organism. The results of increased ethanol production are similar to other alcohol-producing Clostridium bacteria. IMB2 was found to be the best xylitol producer among the IMB yeast. Product yields up to 0.90 g/g at 40 degrees C with 1.0 g/l initial cell mass and 50 g/l initial xylose concentration were observed for IMB2. Using xylose to produce inocula as opposed to glucose resulted in more xylitol production and xylose consumption at 30 degrees C for all IMB strains. Xylose reductase had no activity when the cofactor NADH was used, but activity was observed when NAD(P)H was used. This result indicates that xylitol production from xylose is observed due to a cofactor imbalance an accumulation of NADH in the cell. NADH accumulation inhibits xylitol dehydrogenase, which leads to accumulation of xylitol. Increasing the NADH activity of xylose reductase would reduce this cofactor imbalance and lead to more ethanol production and less xylitol production from xylose.
Publications
- Kundiyana, D., R.L. Huhnke, M.R. Wilkins. 2010. Syngas fermentation in a 100 L pilot scale fermentor: design and process considerations. J Biosci Bioeng. 109:492-498.
- Kundiyana, D.K., R.L. Huhnke, P. Maddipati, H. K. Atiyeh, M.R. Wilkins. 2010. Feasibility of incorporating cotton seed extract in Clostridium strain P11 fermentation medium during synthesis gas fermentation. Bioresource Technol. 101:9673-9680.
- Panneerselvam, A., M.R. Wilkins, M.J.M. DeLorme, H.K. Atiyeh, R.L. Huhnke. 2010. Effect of reducing agents on syngas fermentation by Clostridium ragsdalei. Biological Eng. 2:135-144.
- Ramachandrya, K.D. 2009. Effect of biomass generated producer gas, methane and physical parameters in producer gas fermentations by Clostridium strain P11. M.S. Thesis. Stillwater, OK: Oklahoma State University. 204 p.
- Ramachandriya, K., M.J.M. DeLorme, M.R. Wilkins. 2010. Heat shocking of Clostridium strain P11 to promote sporulation and ethanol production. Biological Eng. 2:115-131.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Kluyveromyces marxianus IMB3 was evaluated in an SSF process using switchgrass pretreated at 200 degrees C for 10 min with pressurized liquid hot water. SSF was performed in a bioreactor with pH control to evaluate if better pH control results in higher ethanol production than in flask reactors with minimal pH control. SSF was done in a 2 L bioreactor with pH controlled at 5.0 or 5.5. Results of this work were published in a recent M.S. thesis. Five K. marxianus IMB strains were evaluated for their potential to ferment xylose to both xylose and ethanol. Inocula were prepared for each strain using either glucose or xylose. After fully grown, part of the inocula were transferred to flask containing media with 50 g/L xylose. Results from this work were presented at the ASABE Bioenergy Engineering 2009 Conference as a poster. Research investigating Clostridium strain P11 potential to produce ethanol from syngas continued. It was investigated whether the defined media currently used for the process can be replaced with complex media such as corn steep liquor (CSL) or cotton seed extract (CSE). A factorial experiment was conducted to investigate the effect of eliminating certain components of the defined media as well. The results of this study were presented as a poster at the ASABE Bioenergy Engineering 2009 Conference. A study investigating the effects of calcium pantothenate, vitamin B12 and cobalt chloride on ethanol formation was done since these components are key precursors to metabolic enzymes in P11. The results of this study were presented as a poster at the 2009 Society of Industrial Microbiology Meeting. A 59 day batch fermentation was completed in a 75L bioreactor to investigate scaleup issues for P11 fermentation. A manuscript detailing the results of this study was submitted to Journal of Bioscience and Bioengineering, accepted for publication in FY 2010. Finally, the effect of three reducing agents--benzyl viologen, methyl viologen and neutral red--on ethanol production by P11 was studied. Reducing agents promote ethanol production and reduction of acetic acid. The results of this study are in a recent M.S. thesis and a manuscript has been submitted to Biological Engineering. On-farm conversion of sweet sorghum into ethanol is one of the major biofuels research programs at Oklahoma State University. The objective of the current study is to investigate syngas generation from sweet sorghum bagasse using an exploratory downdraft gasifier system. OSU's downdraft gasifier system has an internal separate combustion section where turbulent, swirling high-temperature combustion flows are generated. High temperatures in the cyclonic section facilitate biomass pyrolysis. Combustion products from the cyclonic section pass through a char gasification chamber where additional tar cracking occurs. A series of gasification tests have been conducted. OSU, together with Kansas State University, the University of Arkansas, and South Dakota State University, were recently awarded a USDA Higher Education Challenge grant to develop a graduate certificate program in Biobased products and Bioenergy. Dr. Wilkins is closely involved with this effort. PARTICIPANTS: Co-PIs: Mark R. Wilkins and Raymond L. Huhnke, OSU. Collaborators: Danielle Bellmer, 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, Michael Mueller, and Anushadevi Panneerselvam 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, Jennine Terrill, Karthikeyan Ramachandriya and Xiaoguang Zhu 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 3 posters at the 32nd Symposium on Biotechnology for Fuels and Chemicals, three posters, two presentations at the 2009 ASABE Meeting, and a poster at the 2009 SIM 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
For K. marxianus IMB3 pH testing, the reactor controlled at pH 5.0 resulted in a maximum theoretical ethanol yield of 84% with the reactor controlled at pH 5.5 reached a yield of 78%. Fermentation continued for a longer period of time at pH 5.5, but ethanol production rate was faster at pH 5.0. For the K. marxianus IMB3 xylose fermentation screening, the flasks containing cells grown using xylose consumed xylose faster and produced more xylitol than did cells grom using glucose. Less than 1.0 g/L ethanol was produced by xylose-grown cells while no ethanol was produced by glucose grown cells. A screening of all 5 IMB strains for their ability to ferment xylose at temperatures from 25 to 45 degrees C is almost complete and will be reported in the next report. It was discovered that the defined media that has been used for years to grow P11 can be replaced completed with a media containing 0.5 g/L CSE. The CSE media resulted in faster ethanol production than did the defined media. It was also discovered that reducing calcium pantothenate and vitamin B12 concentrations can enhance ethanol production. For the 70 L scale up fermentation, the ethanol concentration reached over 25 g/L and isopropanol was also produced. For the reducing agent study, P11 ethanol production was greatly enhanced by the addition of 10 mM methyl viologen to media while 10 mM neutral red had only a minimal effect on ethanol production and 10 mM benzyl viologen killed P11. Downdraft gasification tests on sweet sorghum bagasse (11 to 12% wet basis) show the average oxidation zone temperatures in the range of 915 to 997C. Gas components of greatest interest (volume basis) were CO: 14.9-18.7%, H2: 9.7-12.8%, CO2: 14.6-16.2% and CH4: 2.2-3.1%. Dry product gas yield ranged from 1.3 to 1.7 Nm3/kg dry biomass. Hot gas and cold gas efficiencies varied from 42 to 60% and 38 to 54%, respectively while the mass balance varied from 85 to 91%. Producer gas tar and particulate contents at the gasifier exit were 58 and 1.8 g/Nm3, respectively. Recently, a curriculum has been developed for a bioenergy graduate certificate by faculty at Kansas State, South Dakota State, Arkansas, and OSU. These courses are being developed by individual faculty members at the universities. Dr. Wilkins is serving on the Conversion Course Oversight Committee for this certificate program.
Publications
- Faga, B. 2009. Simultaneous Saccharification and Fermentation of Pretreated Switchgrass by Thermotolerant IMB strains of Kluyveromyces marxianus. M.S. Thesis. Stillwater, OK: Oklahoma State University. 123 p.
- Gappa-Fahlenkamp, H. and M. Wilkins. 2009. Utilizing diversity in a bioprocess engineering course for a group project to design and characterize a bioreactor to convert cellobiose to glucose. Proc. 2009 ASEE Annual Conference.
- 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.
- Panneerselvam, A. 2009. Effect of Glucose and Reducing Agents on Syngas Fermentation by Clostridia Species P11. M.S. Thesis. Stillwater, OK: Oklahoma State University. 82 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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