Source: OKLAHOMA STATE UNIVERSITY submitted to NRP
BIOFUELS AND BIOPRODUCTS FROM BIOMASS-GENERATED SYNTHESIS GAS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0220041
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2009
Project End Date
Sep 30, 2014
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Biosystems & Ag Engineering
Non Technical Summary
The dominant biofuel in the US is ethanol made via yeast-based fermentation of corn which is a well-developed technology. However, there is a need to produce more biofuels as Congress passed the Energy Independence and Security Act of 2007 that requires the production of 36 billion gallons per year of biofuels by 2022. This total includes cellulosic ethanol and biomass based diesel. The increase in ethanol production is expected to come from feedstocks other than corn grain because of limitations in supply and other uses of corn in food, animal feed and other industrial products. These feedstocks are typically called "biomass" which includes agricultural residues, wood, municipal solid waste and dedicated energy crops such as switchgrass. Oklahoma is well-positioned to take a leading role in the biobased economy and help meeting part of the demand for biofuels from cellulosic biomass. Oklahoma has biomass production and waste biomass generation areas that can be used in biofuels production. Gasification of cellulosic biomass to produce syngas (primarily carbon monoxide, carbon dioxide and hydrogen) used in fermentation for the production of ethanol and other alcohols is a novel technology. Critical bottlenecks that diminish alcohol productivity, lower syngas conversion efficiency, and inhibit the movement of this process to commercial scale include low cell density and low gas to liquid mass transfer in the bioreactor. This project will expand upon previous syngas fermentation studies by exploring and critically evaluating the enhancement of mass transfer and associated alcohol productivity with various types and modifications of reactor designs. The reactor designs will include a continuous stirred tank reactor and a trickle bed reactor. Novel microbial catalysts will be used for the conversion of syngas to ethanol and other useful bioproducts. Successful completion of this work will provide valuable guidance towards designing large scale bioreactors with increased alcohol productivity and syngas utilization. An improved efficiency in alcohol production will increase the cost effectiveness of the syngas fermentation technology, leading towards a more economically viable cellulosic biofuels process to meet a portion of the biofuels production target of 36 billion gallons per year by 2022.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5111629202030%
5114010202070%
Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
1629 - Perennial grasses, other; 4010 - Bacteria;

Field Of Science
2020 - Engineering;
Goals / Objectives
The overall goal of this project is to enhance and critically assess syngas utilization in various reactors to identify reactor designs that increase the alcohol (primarily ethanol) productivity and syngas utilization during the fermentation process. Mathematical models will be developed to describe the kinetics of syngas fermentation and predict the effectiveness of the various reactor designs. Specific objectives are to: (1) Design and construct a trickle bed reactor (TBR) for syngas fermentation and optimize its operating conditions. (2) Explore methods to enhance the gas-liquid mass transfer rate in a continuous stirred tank reactor (CSTR) and determine its optimum operating conditions. (3) Developed mathematical models for alcohol production from syngas in the TBR and CSTR reactors. Data collected from this project will be useful in designing large scale bioreactors and process development.
Project Methods
A TBR reactor will be constructed with the addition of a recirculation loop and pH controller to aid in comparison between the reactors. The effect of liquid flow rate and size of packing materials on syngas conversion efficiency and product formation will be determined. For the CSTR reactor, cell and product formation during syngas fermentation will be assessed at various agitation speeds. The reactors will be operated at 37 C and the inlet gas composition will be the same to aid in the comparison between the two reactors and with previous studies. Mass transfer characteristics will be determined for both reactors. The fermentation medium will have the same composition and the reactors will be inoculated with a similar level of inoculum. Liquid and gas samples will be withdrawn periodically from the reactors to measure cell and product concentrations as well as gas composition. Cell concentration will be determined using a spectrophotometer. Acetic acid, ethanol and butanol concentrations will be measured using a Gas Chromatograph (GC) with a flame ionization detector. The gas analysis will be carried out on a GC with a thermal conductivity detector. Mathematical models that describe syngas fermentation will be developed. The formulation of a fermentation model involves determination of an expression for the growth rate. In many cases this involves the use of the Monod model with additional terms to take into account substrate and product inhibition. Kinetic models for substrate consumption and product formation rates are then formulated using yields coefficients. The simplest types of product formation kinetics arise when there are simple connections between product formation and substrate utilization or cell growth. Kinetic expressions for ethanol formation and acetate production during syngas fermentation will be developed. The effect of various parameters such as syngas composition and pressure on the fermentation process will be included in the models for both reactor designs.

Progress 10/01/09 to 09/30/14

Outputs
Target Audience: The target audiences include biofuels producers, government officials involved in bioenergy policy, farmers interested in biomass production, researchers, and undergraduate and graduate students interested in production of biofuels and biobased products. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two Post Doctorate Research Associates, one PhD student, one MS student and one undergraduate students were trained on syngas fermentation, bioreactor setup and operation and medium design. The undergraduate and graduate students and Post-Doc Fellows had the opportunity to present their work at national and international conferences. How have the results been disseminated to communities of interest? Over the last five years,15 journal articles were published in refereed journal. One book chapter was published and another book chapter is in review. One international patent application was filed and over 15 conference presentations were also delivered at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This research explores and critically evaluates the enhancement of mass transfer and associated alcohol productivity in various types and modifications of reactor designs. The three reactor designs are trickle-bed reactor (TBR), continuous stirred tank reactor (CSTR), and hollow fiber membrane reactor (HFR). We found that the reactor geometry, volume, and operating conditions affect the mass transfer and associated gas utilization and biofuel productivity. These findings are critical in designing reactors for efficient biofuels and chemicals production from syngas. My research team published the results on the comparison of mass transfer characteristics of three reactors in Bioresource Technology in 2013. We also developed a new method to sustain culture activity, gas uptake and improve selectivity for ethanol production during syngas fermentation in the CSTR. The new method resulted in the production of twenty-six times the ethanol concentration compared to the conventional method. This method is described in a US Patent Application, which was filed on August 4, 2014. We have also developed tools and mathematical models to facilitate designing and control of large-scale bioreactors with increased alcohol productivity and selectivity and gas utilization to make the hybrid conversion process more economically viable for the production of biofuels. I extended the work with Alkalibaculum bacchi strain CP15 and examined the effects of hydrogen (H2) to carbon monoxide (CO) ratios and other parameters on alcohol yield and productivity during continuous syngas fermentation. This research will improve the research community’s understanding of the link between the various fermentor operating parameters and alcohol production, and guide efficient operation of bioreactor to convert the syngas to alcohols. We found that the H2 to CO ratios in the syngas affected cells’ uptake rate. High H2 to CO ratios improved ethanol production because more reductants (electron sources) were available to convert acetic acid to ethanol. We also found that opportune contamination at later stages of the fermentation resulted in the production of higher alcohols (n-propanol and n-butanol). We identified the mixed culture using 16S rRNA gene-based survey. The mixed culture was dominated by A. bacchi strain CP15 and Clostridium propionicum. The mixed culture presents a new opportunity for the production of higher alcohols from syngas, which has not been reported in the literature previously. The results from this project were published in two journal papers. My research team and collaborator from other institutions have used and characterized over seven different strains of syngas fermenting microorganisms including Clostridium carboxidivorans, A. bacchi strains CP11, CP13 and CP15 and Clostridium ragsdalei strain P11. We are developing the next generation of syngas fermenting microorganisms for conversion of H2, CO and CO2 to butanol and hexanol. We also designed various syngas fermentation media and investigated addition of reducing agents to enhance ethanol production by C. ragsdalei. We succeeded in developing a completely defined media that provided higher alcohol yield at less than 5% the cost of the current standard syngas fermentation medium. My research team was the first to report the ability of Clostridium ragsdalei to make butanol in addition to ethanol from syngas in yeast extract and corn steep liquor media. My OSU research team over the last five years has published fifteen peer-reviewed publications on the hybrid conversion technology. We have five published conference proceedings and over eighty conference contributions (oral presentations, poster presentations and abstracts). Impact: My research efforts will impact conversion efficiency, cost of production, reactor design and process development of the hybrid conversion technology for implementation in sustainable biorefineries in Oklahoma, the nation, and the world. There are opportunities to apply this hybrid conversion technology in different regions of the country to meet our increasing energy needs. Upon its full development, this hybrid technology can provide 35% more ethanol from the same amount of biomass as compared to the biochemical conversion technology. If biofuel producers adopt this hybrid technology to produce 25% of the mandated 16 billion GPY renewable transportation fuels such as ethanol (i.e., 4 billion GPY), my research suggests a projected annual savings of over $650 million due to the use of 13.1 million tons less biomass with the hybrid technology.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Phillips, J. R., H. K. Atiyeh and R. L. Huhnke. 2014. Method for Design of Production Medium for Fermentation of Synthesis Gas to Ethanol by Acetogenic Bacteria. Biological Engineering Transactions. Accepted 8/26/2014.
  • Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Ramachandriya K. D., M. R. Wilkins, C. L. Goad, N. T. Dunford, H. K. Atiyeh and S. Hiziroglu. 2014. Acid Bisulfite Pretreatment of Eastern Redcedar for Fermentable Glucose Production: Optimization Through Response Surface Methodology. Transactions of the ASABE. 57:881-890.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Ramachandriya K. D., N. T. Dunford, H. K. Atiyeh, S. Hiziroglu and M. R. Wilkins. 2014. Influence of Eastern Redcedar Oil on Enzymatic Hydrolysis of Microcrystalline Cellulose and Saccharomyces cerevisiae Fermentations. Biocatalysis and Agricultural Biotechnology. In press.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Liu, K., H. K. Atiyeh, B. S. Stevenson, R. S. Tanner, M. R. Wilkins and R. L. Huhnke. 2014. Mixed Culture Syngas Fermentation and Conversion of Carboxylic Acids into Alcohols. Bioresource Technology. 152; 337-346.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Liu, K., H. K. Atiyeh, B. S. Stevenson, R. S. Tanner, M. R. Wilkins and R. L. Huhnke. 2014. Continuous Syngas Fermentation for the Production of Ethanol, n-Propanol and n-Butanol. Bioresource Technology, 151; 69-77.
  • Type: Theses/Dissertations Status: Other Year Published: 2014 Citation: Mamatha Devarapalli, Ph.D. Biosystems Engineering, Oklahoma State University, 2009  2014. Dissertation: Analysis of Trickle-Bed Reactor for Ethanol Production from Syngas Using Clostridium ragsdalei. Completed May 2014
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Kan Liu, Ph.D. Biosystems Engineering, Oklahoma State University, 2009  2013. Dissertation: Production of Alcohols via Syngas Fermentation Using Alkalibaculum bacchi Monoculture and a Mixed Culture. Dissertation submitted to the Graduate College on August 13, 2013. Completed December 2013
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2014 Citation: Wilkins, M. R., H. K. Atiyeh and S. K. Khanal, Syngas Fermentation, in Bioenergy: Principles and Applications, Y. Li and S. K. Khanal, Eds, Wiley-Blackwell, IW, USA. In review. Expected publication date is in 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Atiyeh, H. K., K. Liu, B. S. Stevenson, R. S. Tanner, M. R. Wilkins and R. L. Huhnke, Production of Diesel and Jet Fuel Intermediates Using Hybrid Gasification-Syngas Fermentation, BIO Pacific Rim Summit on Industrial Biotechnology and Bioenergy, San Diego, CA, USA, December 8-11, 2013. Poster.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Atiyeh, H.K., K. Liu, B. S. Stevenson, R. S. Tanner, M. R. Wilkins and R. L. Huhnke, Drop-in Biofuels Production Using Mixed Culture Syngas Fermentation, AIChEs 2013 Annual Meeting, San Francisco, CA, November 3-8, 2013. Oral.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Devarapalli, M., H. K. Atiyeh, J. R. Phillips, R. S. Lewis and R. L. Huhnke, Continuous Alcohol Production from Syngas in a Trickle-Bed Reactor, US Department of Energy Biomass 2014 Conference: Growing the Future Bioeconomy, Washington, DC, USA, July 29-30, 2014. Poster.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Atiyeh, H. K., M. Devarapalli, J. R. Phillips, R. S. Lewis and R. L. Huhnke, Ethanol Production in Semi-Continuous and Continuous Syngas Fermentation in Trickle Bed Reactor, 2014 Annual Meeting of the Society for Industrial Microbiology & Biotechnology, St. Louis, MO, July 20-24, 2014. Invited Speaker.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Devarapalli, M., H. K. Atiyeh, J. R. Phillips, R. S. Lewis and R. L. Huhnke, Continuous Syngas Fermentation in Trickle Bed Reactor Using Clostridium ragsdalei, 2014 ASABE and CSBE/SCGAB Annual International Meeting, Montreal, QC, Canada Jul 13-16, 2014. Oral.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Phillips, J. R., H. K. Atiyeh, M. R. Wilkins, R. S. Tanner and R. L. Huhnke, Production of n-butanol and n-hexanol from syngas via syngas fermentation using Clostridium carboxidivorans, 2014 ASABE and CSBE/SCGAB Annual International Meeting, Montreal, QC, Canada Jul 13-16, 2014. Oral.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Liu, K., H. K. Atiyeh, O. P. Planas, K. D. Ramachandriya, M. R. Wilkins, T. Ezeji and R. S. Tanner, Production of Butanol from Eastern Redcear, 2014 ASABE and CSBE/SCGAB Annual International Meeting, Montreal, QC, Canada Jul 13-16, 2014. Oral.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Atiyeh, H.K., M. Devarapalli, R. S. Lewis and R. L. Huhnke, Semi-Continuous Syngas Fermentation in a Trickle Bed Reactor, AIChEs 2013 Annual Meeting, San Francisco, CA, November 3-8, 2013. Oral.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Orgill, J. J., R. S. Lewis and H. K. Atiyeh, Syngas Mass Transfer Analysis in a Hollow Fiber Reactor, AIChEs 2013 Annual Meeting, San Francisco, CA, November 3-8, 2013. Oral.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Atiyeh, H. K., K. Liu, B. S. Stevenson, R. S. Tanner, M. R. Wilkins and R. L. Huhnke, C4 and C6 alcohols production using mixed culture syngas fermentation, US Department of Energy Biomass 2013 Conference: How the Advanced Bioindustry is Reshaping American Energy, Washington, DC, USA, July 31-August 1, 2013. Poster.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Atiyeh, H. K., M. Devarapalli, J. R. Phillips, J.J. Orgill, R. S. Lewis and R. L. Huhnke, Comparison of syngas fermentation in trickle bed, continuously stirred tank and hollow fiber membrane reactors using Clostridium ragsdalei, ASABE 2013 Annual International Meeting, Kansas City, MO, July 21-24, 2013. Oral.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Atiyeh, H. K., J. Gao, J. R. Phillips, M. R. Wilkins and R. L. Huhnke, Development of low cost defined medium for ethanol production from syngas using Clostridium ragsdalei, ASABE 2013 Annual International Meeting, Kansas City, MO, July 21-24, 2013. Oral.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Liu, K., H. K. Atiyeh, B. S. Stevenson, R. S. Tanner, M. R. Wilkins and R. L. Huhnke, Production of drop-in biofuels by mixed culture syngas fermentation, ASABE 2013 Annual International Meeting, Kansas City, MO, July 21-24, 2013. Oral.


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The target audiences include biofuels producers, government officials involved in bioenergy policy, farmers interested in biomass production, researchers, and undergraduate and graduate students interested in production of biofuels and biobased products. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Three PhD students, one MS student and two undergraduate students were trained on syngas fermentation, bioreactor setup and operation and medium design. The graduate students had the opportunity to present their work at national and international conferences. One PhD and one MS students graduated in 2013. How have the results been disseminated to communities of interest? One PhD dissertation and one MS theses resulted from partial funding from this grant. Additionally, two journal articles were published in refereed journals. Two refereed journal articles are under review, one provisional patent application and eight conference papers and presentations resulted from partial funding from this grant. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The hollow fiber membrane reactor (HFR) achieved the highest volumetric mass transfer coefficient for aeration of water, followed by the trickle bed reactor (TBR) and then the continuously stirred tank reactor (CSTR). During syngas fermentation in the three reactors, mass transfer capacities were calculated from uptake of CO in syngas fermentations. The volumetric mass transfer coefficient for CO, kL,COa/VL,in the TBR was dependent on the liquid and gas flow rates. As expected, agitation speed, gas flow and cell concentration affected the kL,COa/VL in the CSTR. Conversion efficiencies of CO and H2 were over 90% and 80% in the TBR and CSTR, respectively. Analysis of mass transfer during syngas fermentation in the HFR is ongoing. Although scale-up issues are different for each reactor, the comparison of the three reactors provides guidance as to which reactor(s) is a better candidate for scale-up, especially if one reactor significantly outperforms the others regarding ethanol yield and productivity, and syngas utilization efficiency. Further, insight gained from the analysis of the three reactors can be extended to other fermentation processes. A novel method that significantly sustains culture activity, gas uptake and improves selectivity for ethanol production during synthesis gas fermentation in continuously stirred tank reactor (CSTR) was developed. The novel method is unique in its ability to sustain biocatalyst activity by over fourfold and resulted in the production of twenty six times the ethanol concentration compared to the conventional method. This invention has applications in many fermentation processes that require gas to liquid mass transfer. This invention was submitted in a provisional patent application. The development of low cost media is critical for the feasibility of the biological conversion of syngas or producer gas to fuels such as ethanol, butanol and hexanol. Various media formulations for Clostridium ragsdalei were designed and compared by reduction, elimination and replacement of expensive nutrients. Cost analysis was performed and the effects of medium components on growth and product formation were investigated. Based on cells’ elemental composition, a minimal mineral concentration medium was formulated, which provided 29% higher ethanol yield from CO at 3% of the cost compared to the standard starting medium. A defined medium was successfully formulated by elimination and reduction in concentrations of several nutrients, which resulted in 36% higher ethanol yield from CO at 5% of the cost compared to the standard medium. The defined medium has the potential for use in large-scale syngas fermentation for ethanol production. The method developed to design a syngas fermentation medium for C. ragsdalei could be extended to other organisms and fermentations. A syngas fermentation medium was also formulated for the moderately alkaliphilic novel strain of Alkalibaculum bacchi CP15 that produced ethanol from syngas. Strain CP15 medium cost was reduced by 27% by removing [N-Tris (hydroxymethyl) methyl]-3-aminopropanesulfonic acid (TAPS buffer) from standard yeast extract (YE) medium and replacing YE, minerals, and vitamins with corn steep liquor (CSL). Over 78% more ethanol was produced with CSL medium compared to YE medium.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Gao J., H. K. Atiyeh, J. R. Phillips, M. R. Wilkins and R. L. Huhnke. 2013. Development of Low Cost Medium for Ethanol Production from Syngas by Clostridium ragsdalei. Bioresource Technology, 147; 508-515.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Orgill, J. J., H. K. Atiyeh, M. Devarapalli, J. R. Phillips, R. S. Lewis and R. L. Huhnke. 2013. A Comparison of Mass Transfer Coefficients Between Syngas Fermentation Reactors. Bioresource Technology, 133; 340-346.
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Jie Gao, M.S. Biosystems Engineering, Oklahoma State University, 2012. Thesis: Development of low cost medium for ethanol production from syngas by Clostridium ragsdalei. Completed December 2012
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Atiyeh, H. K., M. Devarapalli, J. R. Phillips, J.J. Orgill, R. S. Lewis and R. L. Huhnke, Comparison of syngas fermentation in trickle bed, continuously stirred tank and hollow fiber membrane reactors using Clostridium ragsdalei, ASABE 2013 Annual International Meeting, Kansas City, MO, July 21-24, 2013.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Liu, K., H. K. Atiyeh, B. S. Stevenson, R. S. Tanner, M. R. Wilkins and R. L. Huhnke, Production of drop-in biofuels by mixed culture syngas fermentation, ASABE 2013 Annual International Meeting, Kansas City, MO, July 21-24, 2013.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Devarapalli, M., H. K. Atiyeh, R. S. Lewis and R. L. Huhnke, Syngas Fermentation in a Trickle Bed Reactor Using Clostridium ragsdalei, Oklahoma EPSCoR Annual State Conference 2013, Oklahoma State University, Stillwater, OK, April 23, 2013.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Atiyeh, H.K., J. J. Orgill, M. Devarapalli, J. R. Phillips, R. S. Lewis and R. L. Huhnke, Comparison of syngas fermentation reactors for ethanol production, AIChEs 2012 Annual Meeting, Pittsburgh, PA, October 28- November 2, 2012.
  • Type: Theses/Dissertations Status: Other Year Published: 2013 Citation: Randy Phillips, Ph.D. and Research Engineer. Biosystems Engineering, Oklahoma State University, 2010  2013. Dissertation: Fermentation of Synthesis Gas to Ethanol Medium Design, Mass Transfer and Integrated Model. Completed May 2013
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Atiyeh, H. K., K. Liu, B. S. Stevenson, R. S. Tanner, M. R. Wilkins and R. L. Huhnke, C4 and C6 alcohols production using mixed culture syngas fermentation, US Department of Energy Biomass 2013 Conference: How the Advanced Bioindustry is Reshaping American Energy, Washington, DC, USA, July 31-August 1, 2013.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Atiyeh, H. K., J. Gao, J. R. Phillips, M. R. Wilkins and R. L. Huhnke, Development of low cost defined medium for ethanol production from syngas using Clostridium ragsdalei, ASABE 2013 Annual International Meeting, Kansas City, MO, July 21-24, 2013.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Gao J., J. R. Phillips, H. K. Atiyeh, M. R. Wilkins and R. L. Huhnke, Medium Design for Ethanol Production Through Syngas Fermentation, AIChEs 2012 Annual Meeting, Pittsburgh, PA, October 28- November 2, 2012.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Devarapalli, M., H. K. Atiyeh, J. R. Phillips, J. J. Orgill, R. S. Lewis and R. L. Huhnke, Comparison of Syngas Fermentation Reactors for Biological Alcohol Production, The Sun Grant Initiative 2012 National Conference, New Orleans, LA, October 2-5, 2012. Poster. 21- Wilkins, M. R., K. D. Ramachandriya, S. Hiziroglu, N. T. Dunford, and H. K. Atiyeh, Development of an acid sulphite process to pretreat eastern red cedar for cellulose hydrolysis, The Sun Grant Initiative 2012 National Conference, New Orleans, LA, October 2-5, 2012.
  • Type: Journal Articles Status: Under Review Year Published: 2013 Citation: Liu, K., H. K. Atiyeh, B. S. Stevenson, R. S. Tanner, M. R. Wilkins and R. L. Huhnke. 2013. Continuous Syngas Fermentation for the Production of Ethanol, n-Propanol and n-Butanol. Bioresource Technology. in review
  • Type: Journal Articles Status: Under Review Year Published: 2013 Citation: Liu, K., H. K. Atiyeh, B. S. Stevenson, R. S. Tanner, M. R. Wilkins and R. L. Huhnke. 2013. Mixed Culture Syngas Fermentation and Conversion of Carboxylic Acids into Alcohols. Bioresource Technology. In review.


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: The production of "drop-in" biofuels and chemicals using the hybrid gasification-syngas fermentation technology is limited by the low solubility of the gaseous substrates carbon monoxide (CO) and hydrogen (H2) in fermentation medium. Various reactors were proposed to increase the mass transfer of these gases into medium including stirred tank, trickle bed and membrane reactors. A good bioreactor design for syngas fermentation should provide high mass transfer capability that matches the fermentation kinetics to obtain high gas conversion efficiency and productivity. Mass transfer capabilities in trickle bed reactor (TBR), continuous stirred tank reactor (CSTR) and hollow fiber member reactors (HFR) were characterized and compared. Alkalibaculum bacchi CP15 was found to be the most promising among three strains discovered recently in our laboratories for ethanol production. The use of low cost medium is critical to improve the feasibility of syngas fermentation. Syngas fermentations were examined using low cost medium component such as corn steep liquor (CSL) without expensive nutrients such as yeast extract, minerals, vitamins and buffer. Clostridium carboxidivorans P7 is another promising microorganism for the production of advanced and drop-in biofuels through syngas fermentation. Cell growth, gene expression, enzyme activity and product formation by Clostridium carboxidivorans P7 in batch cultures continuously fed syngas was examined. The results from the above outputs have been presented at five conferences. In addition, two peer reviewed journal papers and one conference papers were published. Two more peer reviewed journal papers and one conference papers are in review. PARTICIPANTS: Collaborators: Raymond L. Huhnke and Mark R. Wilkins, Oklahoma State University; Randy Lewis, Brigham Young University. New collaborators: Ralph Tanner and Bradley Stevenson, University of Oklahoma. Kan Liu, Mamatha Devarapalli, Randy Phillips and Jie Gao (graduate students), and Jonathan Overton (undergraduate student) are currently working on this project. TARGET AUDIENCES: The target audiences include biofuels producers, government officials involved in bioenergy policy, farmers interested in biomass production, researchers, and undergraduate and graduate students interested in production of biofuels and biobased products. Educational efforts to inform target audiences of the outputs of this project include two peer reviewed journal papers (published), two peer reviewed journal papers (in review), and two conference papers (one published and one in review). Several presentations were made at national and international meetings as an invited speaker. Several oral and poster presentations were made at the following professional meetings: ASABE 2012 Annual International Meeting (Dallas, TX), 20120ASABE Oklahoma Section Annual Meeting (Stillwater, OK),Oklahoma EPSCoR Annual State Conference 2012 (Stillwater, OK), 2012 S-1041-The Science and Engineering for a Biobased Industry Annual Meeting (Washington, DC), AIChE's 2011 Annual Meeting (Minneapolis, MN). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The volumetric mass transfer coefficient for oxygen in CSTR, TBR and HFR reactors was measured experimentally. The highest volumetric mass transfer coefficient was obtained for the non-porous HFR (1062 h-1) followed by the TBR (421 h-1) and then the CSTR (114 h-1). Reactor configuration, agitation speed, and gas and liquid flow rates greatly affected the mass transfer characteristics in each reactor. Analysis of the three reactors' ability in syngas fermentation is ongoing with regards to gas utilization (including mass transfer assessment), cell growth, and product formation. Although scale-up issues are different for each reactor, the comparison of the three reactors will provide guidance as to which type of reactor(s) should be evaluated for scale-up, especially if one reactor significantly outperforms the other reactors with regards to alcohol productivity and syngas utilization efficiency. Further, insight gained from the analysis of the three reactors can be extended to other important fermentations. To reduce the cost of production medium for syngas fermentation with Alkalibaculum bacchi CP15, yeast extract (YE), minerals and vitamins were replaced with corn steep liquor (CSL). In addition, TAPS buffer previously identified as being important for pH control during growth, was also removed. Syngas fermentations were performed in 250-mL fermentation bottles with 20% CO, 15% CO2, 5% H2 and 60% N2 gas mixture. The results showed that CP15 produced twofold more ethanol using CSL than YE. In addition, over 94% reduction in medium cost was achieved by replacing YE with CSL and elimination of TAPS, minerals and vitamins. These results clearly show the potential for CSL use as a low cost nutrient for syngas fermentation. The maximum concentrations of acetate (41 mM), butyrate (1.4 mM), ethanol (61 mM) and butanol (7.1 mM) were achived by Clostridium carboxidivorans P7 in a 7.5 L reactor. Enzyme activities were highest during cell's growth. However, expression of enzymes for alcohol formation were highest during solvent production stage. These findings show the potential targets for metabolic engineering to enhance ethanol or butanol titers from syngas.

Publications

  • Devarapalli, M., H. K. Atiyeh, J. R. Phillips, J. J. Orgill, R. S. Lewis and R. L. Huhnke, Comparison of Syngas Fermentation Reactors for Biological Alcohol Production, The Sun Grant Initiative 2012 National Conference, New Orleans, LA, October 2-5, 2012 (7 pages), In review.
  • Liu, K., H. K. Atiyeh, R. S. Tanner, M. R. Wilkins and R. L. Huhnke, Low Cost Medium for Ethanol Production Using Novel Moderately Alkaliphilic Alkalibaculum bacchi CP15, ASABE 2012 Annual International Meeting, Dallas, TX, July 29-August 1, 2012 (12 pages), Paper number: ASABE -121337312.
  • 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(11): 2720-2728.
  • 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(2): 87-98.


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Mass transfer and kinetic limitations during syngas (primarily CO, H2 and CO2) fermentation by Clostridium ragsdalei was evaluated in bottle fermentors. CO and H2, sparingly soluble gases are transferred to reactive sites inside the cells to produce fuel and chemicals. The rate of conversion was set by either the transfer of gas into the cell or the kinetics of reaction inside the cell. Gas consumption, cell growth and production of ethanol and acetic acid from syngas were followed to observe the interplay of kinetic and mass transfer limitations in the course of fermentation. The mass transfer analysis for the trickle bed reactor (TBR) with 3 mm and 6 mm glass beads was completed. The overall volumetric mass transfer coefficient (kLa/V) values were measured at various gas and liquid flow rates. The geometry of the CSTR reactor has also been examined in planning mass transfer experiments. A configuration for the CSTR was selected based on analysis of the recommendations reported in the literature. Experiments were run in the 3-L CSTR. The ability of three novel moderately alkaliphilic strains Alkalibaculum bacchi CP11, CP13 and CP15 to produce ethanol and acetate from syngas was investigated. In addition, the ability of Clostridium carboxidivorans and Clostridium ragsdalei to convert acetone into isopropanol in the presence of syngas was examined. The results from the above outputs have been presented at three conferences. In addition, four peer reviewed journal papers and two conference papers were published. PARTICIPANTS: Collaborators: Raymond L. Huhnke, OSU; Mark R. Wilkins, OSU; Randy Lewis, Brigham Young University. Kan Liu, Mamatha Devarapalli, Randy Phillips and Jie Gao are graduate students currently working on this project. TARGET AUDIENCES: The target audiences include biofuels producers, government officials involved in bioenergy policy, farmers interested in biomass production, researchers, and undergraduate and graduate students interested in production of biofuels and biobased products. Educational efforts to inform target audiences of the outputs of this project include four peer reviewed journal papers and two conference papers. In addition, several oral and poster presentations were made at the following professional meetings: ASABE 2011 Annual International Meeting (Louisville, KY), Oklahoma EPSCoR Annual State Conference 2011 (Norman, OK), 2011 S-1041-The Science and Engineering for a Biobased Industry Annual Meeting (Stillwater, OK), AIChE's 2010 Annual Meeting (Salt Lake City, UT). Tours to our bioengineering and bioenergy facilities were organized to explain our biofuels research activities to government officials, visiting scientists and high school, undergraduates and graduate students. In addition, results from this project were incorporated in the graduate course that the PI developed on Bioconversion of Biomass to Alcohol Fuels. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Clostridium ragsdalei consumed CO to greater than 95% converted, and of H2 to 98% converted, in about 50 h per charge of syngas (CO: CO2: H2 = 40: 30: 30% per volume) in bottle fermentors. The consumption of CO and H2 was correlated with mass transfer. Batch fermentation of syngas with a high total pressure of rich syngas did not use the mass transfer capacity of the bottle fermentors efficiently, evidenced by low apparent kLa/V for CO. The fermentation became mass transfer limited as cells grew and CO and H2 were depleted. Under mass transfer limitation, gas was transferred at the maximum capacity of the fermentor, and substrate gas concentrations at the cell were near zero. Kinetic limitation was reestablished when nutrient limitation limited growth and energy demand for the cells. Practioners of fermentation mass transfer generally adhere to the theory that "more is better." However, in an industrial scale, more mass transfer is costly in capital and operating expense of over- designed equipment. We showed that inefficient mass transfer can be detrimental to activity of the fermenting culture and productivity of the fermentation. Application of appropriate mass transfer will optimize productivity in syngas fermentation. Future study will further define "appropriate" mass transfer. The mass transfer analysis for the TBR with 3 mm beads showed no changes in kLa/V at gas flow rates below 80 ml/min. In addition, the increase in liquid flow rate from 50 to 90 ml/min increased the kLa/V at high gas flow rates (117 ml/min to 144 ml/min). However, when the liquid flow rate was above 107 ml/min, the kLa/V values were not affected by the liquid flow rate. The estimated kLa/V values for CO and H2 varied from 1 to 30 h-1 and 1 to 45 h-1, respectively. There was a slight increase in kLa/V values with the decrease in beads size from 6 mm to 3 mm indicating that the kLa/V was more affected by the gas flow rate than surface area of the beads. For the 3-L CSTR, preliminary data showed a marked effect of gas entrained from the headspace that resulted in low kLa/V as agitation speed increased. Significant entrainment of gas from the headspace was observed visually at agitator speeds above 500 rpm. Alkalibaculum bacchi CP11, CP13 and CP15 grew at initial pH between 7.7 and 8.0 and produced ethanol and acetic acid using Syngas I (CO: CO2: H2: N2 = 20: 15: 5: 60) and Syngas II (CO: CO2: H2 = 40: 30: 30) in bottle fermentations. Strain CP15 was found to be the most promising for ethanol production because of its higher growth and ethanol production rates and yield compared to CP11 and CP13. CP15 produced over twofold more ethanol with Syngas I compared to strains CP11 and CP13. In addition, CP15 produced 18% and 71% more ethanol using Syngas II compared to strains CP11 and CP13, respectively. In addition, Clostridium ragsdalei was found to have a secondary alcohol dehydrogenase that enabled it to convert acetone into isopropanol. However, Clostridium carboxidivorans lacks this enzyme.

Publications

  • Liu, K., H. K. Atiyeh, R. S. Tanner, M. R. Wilkins and R. L. Huhnke. 2011. Fermentative production of ethanol from syngas using novel moderately alkaliphilic strains of Alkalibaculum bacchi. Bioresource Technology, doi: 10.1016/j.biortech.2011.10.054.
  • Maddipati, P., H. K. Atiyeh, D. Bellmer and R. L. Huhnke. 2011. Ethanol Production from Syngas by Clostridium Strain P11 Using Corn Steep Liquor as a Nutrient Replacement to Yeast Extract. Bioresource Technology, 102:6494-6501.
  • Wilkins, M. R. and H. K. Atiyeh. 2011. Microbial production of ethanol from carbon monoxide. Current Opinion in Biotechnology, 22:1-5.
  • Ramachandriya, K. D., M. R. Wilkins, M. J.M. Delorme, X. Zhu, D. K. Kundiyana, H. K. Atiyeh and R. L. Huhnke. 2011. Reduction of acetone to isopropanol using producer gas fermenting microbes, Biotechnology and Bioengineering, 108:2330-2338.
  • Phillips, J. R., N. M. Remondet, H. K. Atiyeh, M. R. Wilkins and R. L. Huhnke, Designing Syngas Fermentation Medium for Fuels and Bulk Chemicals Production, ASABE 2011 Annual International Meeting, Louisville, KY, August 7-10, 2011 (12 pages), Paper number: ASABE -1111052.
  • Phillips, J. R., H. K. Atiyeh, R. S. Lewis and R. L. Huhnke, Mass Transfer and Kinetic Limitations During Synthesis Gas Fermentation by Acetogenic Bacteria, ASABE 2011 Annual International Meeting, Louisville, KY, August 7-10, 2011 (12 pages), Paper number: ASABE -1111040.


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: Yeast extract (YE) and corn steep liquor (CSL) were investigated as the primary media constituents in syngas fermentation. Initially, growth and product profiles of Clostridium strain P11 in YE and CSL media were followed during fermentation in 250-mL serum bottles fed with syngas every 24 h. In subsequent runs, the syngas fermentation was performed in a 7.5-L fermentor, in which growth and product profiles were measured during fermentation. Bottled syngas composed of 20% CO, 15% CO2, 5% H2, and 60% N2 (by volume) was used. The preliminary results have been presented at two conferences. The effect of dithiothreitol (DTT) on enhancing ethanol production from syngas using Clostridium strain P11 was investigated in 250-mL serum bottles. Reducing agents help in regeneration of NADH from NAD+. NADH is utilized in the production of alcohol from aldehydes. Strain P11 was fed with syngas every 24 h and samples were collected to measure pH, cell mass and product concentrations. Various concentrations of DTT were examined. The results from this part of the project were presented at two conferences and one paper was published in Biological Engineering. Mass transfer coefficients for a trickle bed reactor (TBR) with 6 mm glass beads at 20C and 37C were determined. Counter current flow of gas and liquid were used. The gas flows from the bottom of the TBR and the liquid flows from the top. Dissolved oxygen (DO) probe was placed inline very close to the TBR liquid outlet to obtain accurate values of DO concentrations. The preliminary results will be presented at the AIChE's 2010 Annual Meeting. PARTICIPANTS: Collaborators: Raymond L. Huhnke, OSU; Mark R. Wilkins, OSU; Randy Lewis, Brigham Young University. Prasanth Maddipati and Balaji Kubandra Babu have worked on some aspect of the project and received their MS degrees in May 2010. Mamatha Devarapalli, Randy Phillips and Don Claus are graduate students currently working on this project. TARGET AUDIENCES: The target audiences include ethanol producers, government officials involved in bioenergy policy, farmers interested in biomass production, researchers, and undergraduate and graduate students interested in production of biofuels and bioproducts. Educational efforts to inform target audiences of the outputs of this project include an invited speaker presentation on Biofuels and Bioproducts from Biomass-Generated Synthesis Gas at the 2010 Oklahoma EPSCoR Annual State Conference (Norman, OK). Several oral and poster presentations were made at the following meetings: 2010 S-1041-The Science and Engineering for a Biobased Industry Annual Meeting (Wyndmoor, PA), ASABE 2010 Annual International Meeting (Pittsburgh, PA), NanoFocus and BioEnergy Oklahoma EPSCoR Annual State Conference 2009 (Oklahoma City, OK), 2009 Bioenergy Engineering Conference (Bellevue, Washington) and The Sixth Annual World Congress on Industrial Biotechnology & Bioprocessing in 2009 (Montreal, QC, Canada). Tours to tour our bioenergy labs were organized to explain our biofuels research activities to government officials, visiting scientists and high school, undergraduates and graduate students. In addition, results from this project were incorporated in a new graduate course the PI developed on Bioconversion of Biomass to Alcohol Fuels. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
After 600 h of syngas fermentation, ethanol concentrations in 250-mL serum bottles with 1 g/L yeast extract (YE) and 10 g/L corn steep liquor (CSL) were 1.3 g/L and 1.5 g/L, respectively. A maximum ethanol concentration of 8.6 g/L was measured after 360 h of syngas fermentation in 7.5-L fermentor with 10 g/L CSL medium. This represents 57% of the theoretical ethanol yield based on CO consumed. About 14% more ethanol was produced in the medium with 10 g/L CSL compared to the medium with 1 g/L YE. The maximum ethanol concentrations after 360 h in the 7.5-L fermentor with 20 g/L CSL media were 9.6 g/L, which represent 60% of the theoretical ethanol yield. These results demonstrate that CSL can replace YE as the primary medium component and significantly enhance ethanol production by Clostridium strain P11. Results of the effect of dithiothreitol (DTT) on syngas fermentations in 250-mL serum bottles showed that more than a 350% increase in ethanol concentration was measured in media that contained at least 7.5 g/L of DTT after 360 h of fermentation compared to the control medium (without DTT) in 1 g/L yeast extract medium. These results suggested that the use of DTT as a reducing agent in the broth enhances ethanol production from syngas. Several mass transfer experiments were conducted in the trickle bed reactor (TBR) with 6 mm glass beads at 20C and 37C in order to calculate overall volumetric mass transfer coefficient (kLa/V) values. Various gas and liquid flow rates were used. Air and nitrogen were used in the gas phase and deionized water was the liquid phase. DO in water was first stripped by nitrogen. Then, air flows in the TBR until DO concentration in water reached saturation. The kLa/V values were then calculated. Preliminary results showed that kLa/V values increased with the increase in gas flow rate. However, less change in kLa/V values was noticed with the increase in liquid flow rates and temperatures used.

Publications

  • Kundiyana, D. K., R. L. Huhnke, P. Maddipati, H. K. Atiyeh and M. R. Wilkins. 2010. Feasibility of Incorporating Cotton Seed Extract in Clostridium strain P11 Fermentation Medium During Synthesis Gas Fermentation. Bioresource Technology 101: 9673-9680.
  • Maddipati, P. 2010. Ethanol Production from Syngas by Clostridium Strain P11 Using Corn Steep Liquor as a Nutrient Replacement. MS Thesis. Stillwater, OK: Oklahoma State University. 164p.
  • Babu, B. K. 2010. Effect of the Reducing Agent Dithiothreitol on Ethanol and Acetic Acid Production by Clostridium Strain P11. MS Thesis. Stillwater, OK: Oklahoma State University. 110p.
  • Babu, B. K., H. K. Atiyeh, M. R. Wilkins and R. L. Huhnke. 2010. Effect of the Reducing Agent Dithiothreitol on Ethanol and Acetic Acid Production by Clostridium Strain P11 Using Simulated Biomass-Based Syngas. Biological Engineering. (In Press).
  • Panneerselvam, A., M. R. Wilkins, M. J. M. DeLorme, H. K. Atiyeh and R. L. Huhnke. 2010. Effects of reducing agents on syngas fermentation by Clostridium ragsdalei. Biological Engineering 2: 135-144.
  • Atiyeh, H. K., B. K. Babu, M. R. Wilkins and R. L. Huhnke. 2009. Effect of the Reducing Agent Dithiothreitol on Ethanol and Acetic Acid Production by Clostridium Strain P11 Using Simulated Biomass-Based Syngas. ASABE BIO-097917. American Society of Agricultural and Biological Engineers, St. Joseph, MI.