Progress 08/01/08 to 08/31/12
Outputs
OUTPUTS: Undefined mixed consortia of microbes as open cultures (1000s of microbial species) are very successful in engineered systems to generate biogas (methane with anaerobic digesters) from organic wastes. However, they have not played an important role in the production of liquid biofuels. We are changing this with our research program and are promoting the development of an important carboxylate platform within the biorefinery concept. Carboxylates are already used as intermediate molecules for further chemical and fuel production, but we generate these acids from biomass rather than from nonrenewable sources. Here, we are developing a system with undefined mixed consortia (i.e., reactor microbiomes) conversion of lignocellulosic biomass into n-butyrate and subsequent biological conversion into the liquid biofuel n-butanol. Specifically, we are optimizing three subsequent treatment steps in a biorefinery system: 1. chemical/physical pretreatment; 2. undefined-mixed-culture bioprocess to generate n-butyrate; and 3. pure-culture bioprocess to convert n-butyrate into n-butanol (a biological reduction process). We also used ecotoxicology studies to show that the choice for a pretreatment system should be made on very different factors depending on the type of subsequent bioprocess that is desired. For example, certain inhibiting species in the biomass hydrolysate may be a problem when using a yeast-based bioprocess, while this is not a problem for a bioprocess with an undefined mixed consortia. As was outlined in the research objectives of this study, we optimized an undefined-mixed-culture bioprocess to generate n-butryate. During our research it became clear that the biomass to n-butyrate yields reached an unsatisfying maximum, because it is hard to extract n-butyrate from the fermentation broth due to the high maximum solubility concentration for n-butyrate. Fortunately, we discovered during our studies that we could produce a more hydrophobic chemical that is much easier to extract than n-butyrate. Subsequent research under this program found that this bioprocess, indeed, can be optimized after in-line extraction of the new product - n-caproate. As part of this study on n-caproate, we also further developed bioinformatics tools that were helpful for high-throughput DNA sequencing approaches. The reduction of n-butyrate to n-butanol was successful during the course of this study, but we concluded that using sugar as a source of electrons and energy for this metabolic pathway would not be sustainable. Therefore, we concluded that other sources of reducing equivalents (electrons) and energy need to be studied to sustain reduction of carboxylates to alchols. During our lab-scale studies we also educated interested parties in the agronomy sector by providing technological information about our process with the goal to enhance economic opportunities for agricultural producers and to protect and enhance the US natural resources and environmental quality. PARTICIPANTS: Dr. Hanno Richter, Research Associate (Post-doc) to convert butyrate into butanol Dr. Jeffrey Werner, Research Associate (Post-doc) to assist with bioinformatics tools to study the open microbial community Matt Agler, PhD candidate to perform the reactor studies to generate butyrate and caproate Sebastian Heger, MS student at RTWH Aachen who performed ecotoxicological assays Catherine Dispirito, MS student to assist with caproate reactor Joseph Usack, MS student to assist with caproate reactor Ge Shijiang, visiting PhD student to operate the caproate reactor Dr. Bruce Dien, Chemical Engineer at ARS, USDA, Peoria, IL who has managed the pretreatment of corn fiber Loren Iten, ARS, USDA, Peoria, IL who has performed the pretreatment steps Dr. Michael Cotta, ARS, USDA, Peoria, IL who has advised the overall fermentation research Dr. Nasib Qureshi, Chemical Engineer at ARS, USDA, Peoria, IL who has managed the work of Hanno Richter (butanol fermentation) Dr. Henner Hollert, RTWH Aachen, Germany who advised the work of a MS student to perform ecotoxicological assays on our bioreactor influent and effluent samples Dr. Largus Angenent, PD, who has managed the project TARGET AUDIENCES: Bioenergy companies and farmers are target audiences of the outcome of generating biofuels from lignocellulosic materials. One of our conclusions is that pretreatment is essential, and this must be immediately be understood in the field. We also need to educate that other products can be generated from open mixed communities and not only the low-value commodity methane. Caproate is a platform chemical with many uses, so stakeholders need to be educated about the possibilities of generating other products from organic streams that are complex in nature. Finally, target audiences are scientist, who work with undefined mixed cultures for energy generation or to study the microbial ecology in the environment or engineered systems. In the coming years, we will publish research papers with the results to help the field move forward (in addition to attending conferences). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
During the initial phases of the project we showed that pretreatment of our lignocellulosic biomass is necessary to sustain an efficient undefined mixed culture bioprocess. We also found that one type of pretreatment strategy (hot-dilute acid pretreatment) was especially optimum for our application and we obtained maximum n-butyrate yields. This pretreatment technology was known to inhibit yeast because of the production of unwanted side products, such as inhibiting compounds. A collaborative effort between our bioprocessing lab and a lab that works with ecotoxicological assays showed that our undefined mixed microbial consortia degraded the inhibiting components, which explained why the hot-dilute acid pretreatment method was so successful in our study. We found maximum n-butyrate conversion efficiencies with the lignocellulose biomass of our choice (corn fiber) by using the hot-dilute acid pretreatment step and by minimizing as much as possible the inhibiting activity of the final product (n-butyric acid). This was accomplished by diluting the substrate. We hypothesized that continuous in-situ product recovery by liquid/liquid pertraction (extraction) would increase n-butyrate yields further. This was indeed found to be correct, but it was also found that n-butyrate is hard to extract because the maximum solubility concentration is too high. Our breakthrough came when we found that microbial communities catalyzed a biological two-carbon chain-elongation reaction with n-butyrate to form n-caproate, using electrons from externally supplied ethanol that was oxidized to acetate. Importantly, this C6 carboxylate is hydrophobic and relatively easy to separate from water because of its relatively low maximum solubility of ~10 g per liter at 30 degrees C. n-Caproate can only be produced at high levels in our bioreactor when it is continuously removed by our liquid/liquid extraction system because its unionized form n-caproic acid is toxic at the pH values that we operate the reactor (pH of 5.5). Importantly, during the operiod of this program we were able to optimize the n-caproate production and extraction rates to a level that is similar to methane production rates in anaerobic digesters treating solid wastes (5 g n-caproate per liter reactor volume per day). Generating a chemical product with an existing monetary value that is already more than 20 times higher per carbon molecule than methane and twice the value of carbon in ethanol is important for the US renewable industry. For the first time, we can use open mixed communities to produce a product with considerable value. Implementation of mixed community-based engineered systems has a long track record of success; think for example about anaerobic digestion and composting. We were also successful in reducing n-butyrate to n-butanol with a continuously-fed fermentation system with a pure culture of Clostridia by adding sugar to the n-butyrate nutrient stream into the process. n-Butanol was extracted from the fermenter by a stripping and condensation system until phase separation of the n-butanol became apparent.
Publications
- Agler M. T., Werner J. J., Iten L. B., Dekker A., Cotta M. A., Dien B. S. and Angenent L. T. (2012). Shaping reactor microbiomes to produce the fuel precursor n-butyrate from pretreated cellulosic hydrolysates. Environmental Science and Technology, Vol. 46, No. 18, pp. 10229-10238.
- Heger S., Bluhm K., Agler M. T., Maletz S., Schaffer A., Seiler T. B., Angenent L. T. and Hollert H. (2012). Biotests for hazard assessment of biofuel fermentation. Energy and Environmental Science, Vol. 5, No. 12, pp. 9778-9788.
- Agler M. T., Spirito C. M, Usack J. G., Werner J. J. and Angenent L. T. (2012). Chain elongation with reactor microbiomes: upgrading dilute ethanol to medium-chain carboxylates. Energy and Environmental Science, Vol. 5, No. 8, pp. 8189-8192.
- Bluhm K., Heger S., Agler M. T., Maletz S., Schaffer A., Seiler T.-B., Angenent L. T. and Hollert H. (2012). Assessment of the ecotoxicological & environmental effects of biorefineries (Chapter 15, pp. 435-467). In: The role of green chemistry in biomass processing and conversion. Eds.: Xie H. and Gathergood N., John Wiley & Sons, Inc., Hoboken, NJ, USA.
- Angenent L. T.*, Agler M. T., Werner J. J., Heger S., Hollert H., Dien B. S., Iten L. B. and Cotta M. A. (2011). The effect of different lignocellulosic pretreatment methods on diverse microbial consortiums in a bioprocess step to generate fuel precursors. SETAC North America 32nd Annual Meeting, November 13-17, Boston, MA.
- Richter H., Qureshi N., Dien B., Cotta M.A. and Angenent L.T. (2011). Long-term conversion of n-butyrate to n-butanol with Clostridium saccharoperbutylacetonicum using a two-stage continuous culture and in-situ product removal. SIM annual meeting and exhibition, July 24-28, 2011, New Orleans, USA.
- Heger S.*, Bluhm K., Brinkmann M., Winkens K., Schneider A., Wollenweber M., Maletz S., Wolz J., Agler M. T., Angenent L. T., Seiler T. B. and Hollert H. (2011). What's up inside the reactor - Biotests for risk assessment of biofuel fermentation. SETAC North America 32nd Annual Meeting, November 13-17, Boston, MA.
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Progress 08/01/10 to 07/31/11
Outputs
OUTPUTS: Undefined mixed cultures (1000s of microbial species) are very successful in engineered systems to generate biogas (methane) from organic wastes. However, they have not played an important role in the production of liquid biofuels. We are changing this with our research program and are promoting the development of an important carboxylate platform within the biorefinery concept. Carboxylates are already used as intermediate molecules for further chemical and fuel production, but we generate these acids from biomass rather than from nonrenewable sources. Here, we are developing a system with undefined mixed culture (i.e., open microbial community) conversion of lignocellulosic biomass into n-butyrate and subsequent biological conversion into the liquid biofuel n-butanol. Specifically, we are optimizing three subsequent treatment steps in a biorefinery system: 1. chemical/physical pretreatment; 2. undefined-mixed-culture bioprocess to generate n-butyrate; and 3. pure-culture bioprocess to convert n-butyrate into n-butanol. During our lab-scale studies we are education interested parties in the agronomy sector by providing technological information about our process with the goal to enhance economic opportunities for agricultural producers and to protect and enhance the US natural resources and environmental quality. During our studies we also found that we could produce a hydrophobic chemical that is much easier to extract than n-butyrate; and we were requested a no-cost extension to study this further. PARTICIPANTS: Dr. Hanno Richter, Research Associate (Post-doc) to convert butyrate into butanol Dr. Jeffrey Werner, Research Associate (Post-doc) to assist with bioinformatics tools to study the open microbial community Matt Agler, PhD candidate to perform the reactor studies to generate butyrate and caproate Catherine Dispirito, MS student to assist with caproate reactor Joseph Usack, MS student to assist with caproate reactor Ge Shijiang, visiting PhD student to operate the caproate reactor Dr. Bruce Dien, Chemical Engineer at ARS, USDA, Peoria, IL who has managed the pretreatment of corn fiber Loren Iten, ARS, USDA, Peoria, IL who has performed the pretreatment steps Dr. Michael Cotta, ARS, USDA, Peoria, IL who has advised the overall fermentation research Dr. Nasib Qureshi, Chemical Engineer at ARS, USDA, Peoria, IL who has managed the work of Hanno Richter (butanol fermentation) Dr. Largus Angenent, PD, who has managed the project TARGET AUDIENCES: Bioenergy companies and farmers are target audiences of the outcome of generating biofuels from lignocellulosic materials. One of our conclusions is that pretreatment is essential, and this must be immediately be understood in the field. We also need to educate that other products can be generated from open mixed communities and not only the low-value commodity methane. Caproate is a platform chemical with many uses, so stakeholders need to be educated about the possibilities of generating other products from organic streams that are complex in nature. Finally, target audiences are scientist, who work with undefined mixed cultures for energy generation or to study the microbial ecology in the environment or engineered systems. In the coming years, we will publish research papers with the results to help the field move forward (in addition to attending conferences). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We hypothesized that continuous in-situ product recovery by liquid/liquid pertraction would increase n-butyrate yields and result in a more concentrated product. This was indeed found to be correct, but it was also found that n-butyrate is hard to extract because the maximum solubility concentration is too high. Our breakthrough came when we found that microbial communities catalyzed a biological two-carbon chain-elongation reaction with n-butyrate to form n-caproate, using electrons from externally supplied ethanol that was oxidized to acetate. Importantly, this C6 carboxylate is hydrophobic and relatively easy to separate from water because of its relatively low maximum solubility of ~10 g per liter at 30 degrees C. n-Caproare can only be produced at high levels in our bioreactor when it is continuously removed by our liquid/liquid extraction system because its unionized form n-caproic acid is toxic at the pH values that we operate the reactor (pH of 5.5). To generate a valuable fuel, the extracted n-caproate with an energy density of -144.8 kJ per C, should be further upgraded to, for example, n-hexanol with an energy density similar to ethanol and n-butanol (-182.7 and -171.4 kJ per C); or even better alkanes (-178.6 kJ per C), by additional external reactions. Bus this is outside of our current scope. Generating a chemical product with an existing bulk value that is already 10 times higher per carbon molecule than methane and twice the value of carbon in ethanol is important for the US renewable industry. For the first time, we can use open mixed communities to produce a product with considerable value. Implementation of mixed community-based engineered systems has a long track record of success; think for example about anaerobic digestion and composting. We are currently further optimizing this process by using real yeast fermentation beer from the corn to ethanol industry with the goal to circumvent energy-consuming distillation at the plant. We are also educating stakeholders, such as industries and granting agencies about this innovative process.
Publications
- Werner J. J., Zhou D., Caporaso J. G., Knight R. and Angenent L. T. (2012). Comparison of Illumina paired-end and single-direction sequencing for microbial 16S rRNA gene amplicon survey, DOI:10.1038/ismej.2011.186. The ISME Journal.
- Richter H., Qureshi N., Heger S., Dien B., Cotta M. A. and Angenent L. T. (2012). Prolonged conversion of n-butyrate to n-butanol with Clostridium saccharoperbutylacetonicum in a two-stage continuous culture with in-situ product removal. Biotechnology and Bioengineering, DOI:10.1002/bit.24380.
- Agler M. T., Wrenn B. A., Zinder S. H. and Angenent L. T. (2011). Waste to bioproduct conversion with undefined mixed cultures: the carboxylate platform. Trends in Biotechnology, Vol. 29, No. 2, pp. 70-78.
- Werner J. J., Koren O., Hugenholtz P., DeSantis T. Z., Walters W. A., Caporaso J. G., Angenent L. T., Knight R., Ley R. E. (2012). Impact of training sets on classification of high-throughput bacterial 16S rRNA gene surveys. The ISME Journal, Vol. 6, No. 1, pp. 94-103.
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Progress 08/01/09 to 07/31/10
Outputs
OUTPUTS: Undefined mixed cultures (1000s of microbial species) are very successful in engineered systems to generate biogas (methane) from organic wastes. However, they have not played an important role in the production of liquid biofuels. We are changing this with our research program and are promoting the development of an important carboxylate platform within the biorefinery concept. Carboxylates are already used as intermediate molecules for further chemical and fuel production, but we generate these acids from biomass rather than from nonrenewable sources. Here, we are developing a system with undefined mixed culture conversion of lignocellulosic biomass into n-butyrate and subsequent biological conversion into the liquid biofuel n-butanol. Specifically, we are optimizing three subsequent treatment steps in a biorefinery system: 1. chemical/physical pretreatment; 2. undefined-mixed-culture bioprocess to generate n-butyrate; and 3. pure-culture bioprocess to convert n-butyrate into n-butanol. During our lab-scale studies we are education interested parties in the agronomy sector by providing technological information about our process with the goal to enhance economic opportunities for agricultural producers and to protect and enhance the US natural resources and environmental quality. PARTICIPANTS: Dr. Hanno Richter, Research Associate (Post-doc) to convert butyrate into butanol Matt Agler, PhD candidate to perform the reactor studies to generate butyrate Dr. Bruce Dien, Chemical Engineer at ARS, USDA, Peoria, IL who has managed the pretreatment of corn fiber Loren Iten, ARS, USDA, Peoria, IL who has performed the pretreatment steps Dr. Michael Cotta, ARS, USDA, Peoria, IL who has advised the overall fermentation research Dr. Nasib Qureshi, Chemical Engineer at ARS, USDA, Peoria, IL who has managed the work of Hanno Richter (butanol fermentation) Sebastian Heger, MS student at RTWH Aachen University, Germany who has visited our lab Dr. Henner Hollert, Professor at RTWH Aachen University, Germany who is advising Sebastian Heger Dr. Largus Angenent, PD, who has managed the project TARGET AUDIENCES: Bioenergy companies and farmers are target audiences of the outcome of generating biofuels from lignocellulosic materials. One of our conclusions is that pretreatment is essential, and this must be immediately be understood in the field. Finally, target audiences are scientist, who work with undefined mixed cultures for energy generation or to study the microbial ecology in the environment or engineered systems. In the coming years, we will publish research papers with the results to help the field move forward (in addition to attending conferences). PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We hypothesized that continuous in-situ product recovery by liquid/liquid pertraction would increase n-butyrate yields and result in a more concentrated product. Thus, we operated two 5-L anaerobic sequencing batch reactors (ASBR) in semi-batch mode at pH 5.5 and thermophilic temperatures (55 degrees C) for 430 days. In our membrane-based liquid/liquid pertraction system, a hydrophobic solvent selectively extracts the most hydrophobic acidic molecules (longer-chain carboxylic acids), which then are concentrated in an alkaline aqueous stripping solution. After 220 days of normal operation, R1 (control) was contacted with the solvent without stripping, while R2 was subjected to pertraction. At the highest rates, in R2 we extracted about 70 percent of n-butyrate, over 90 percent of n-caproate, and nearly 100 percent of n-caprylate produced. n-Butyrate was concentrated from reactor concentrations of approx. 3.5 g/L to approx. 22 g/L in the stripping solution. Development of ethanol-oxidizing methanogens in the extraction system resulted in accumulation of acetate (from 1.2g/L to 2.7g/L). In R2, the decreased levels of C4-C8 carboxylates, combined with increased levels of acetate were not enough to increase hydrolysis. Next, we hypothesized that supplementing ethanol would drive carboxylate-elongation reactions, thus, increasing extraction rates. Thus, we began adding ethanol to the reactors after 350 days. In R2, rates of n-butyrate, n-caproate, and n-caprylate production increased dramatically relative to the control (48 percent, 33 percent, and 200 percent higher, respectively), although hydrolysis did not increase. We further optimized production parameters: our main challenge was that the Clostridial cultures are not indefinitely stable regarding solvent production. This has been reported previously. We improved the stability of solventogenesis in our system by keeping the n-butyrate concentration and the pH at a level at which solventogenesis is stimulated, similarly to a previous report. Nevertheless, in multiple attempts in which we varied pH and n-butyrate-concentration all fermentations ultimately turned acidogenic - approx. 2 weeks was the longest time we were able to maintain solventogenesis. We then established a method in which we heat-shocked and re-inoculated the smaller 1 st stage fermentor every 5 days with cells grown from spore solution. This way, we provided a constant supply of solventogenic cells and prevented the gradual decline of solventogenic cells in the 2 nd stage fermentor. We are now able to run our 2 nd stage fermentor for months (we stopped the fermentor after a couple of months even though we could have operated it for longer). In addition, we increased the concentration of n-butanol obtained from gas stripping by introducing a 2 nd stage stripping column into our system, which uses part of the dry off-gas from the first gas-stripping loop. We achieved further concentration without having to put a significant amount of additional energy into our system - 80 g n-butanol/L.
Publications
- Richter H., Qureshi N., Cotta M. A. and Angenent L. T. (2010). Producing butanol from syngas with hollow fiber bioreactors. Northeast Sun Grant 2010 Regional Conference. May 24-26 2010, Syracuse, NY.
- Richter H., Qureshi N., Cotta M. A. and Angenent L. T. (2010). Conversion of n-butyrate to n-butanol with continuous fermentation. Institute for Biological Engineering 2010 Annual Conference. March 4-6, 2010, Boston, MA.
- Matthew T. Agler, Loren B. Iten, Michael A. Cotta, Bruce Dien, Largus T. Angenent. (2010). Toward Narrowing Endproduct Distribution in Nondefined Mixed Culture Anaerobic Conversion of Lignocellulosic Corn Fiber to n-Butyrate. 2010 Institute of Biological Engineering Annual Conference, March 4-6, 2010, Cambridge, MA.
- Matthew T. Agler, Loren B. Iten, Michael A. Cotta, Bruce Dien, Largus T. Angenent. (2010). Toward Narrowing Endproduct Distribution in Nondefined Mixed Culture Anaerobic Conversion of Lignocellulosic Corn Fiber to n-Butyrate. 2010 Cornell Engineering Research Conference, March 17, 2010, Ithaca, NY.
- Heger S., Bluhm K., Brinkmann M., Winkens K., Schneider A., Wollenweber M., Maletz S., Wolz J., Agler M. T., Seiler T. B., Angenent L. T. and Hollert H. (2010). Whats up inside the reactor: Biotests for risk assessment of biofuel fermentation. 20th Annual Meeting SETAC Europe, May 23-27, 2010, Seville, Spain.
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Progress 08/01/08 to 07/31/09
Outputs
OUTPUTS: Undefined mixed cultures (1000s of microbial species) are very successful in engineered systems to generate biogas (methane) from organic wastes. However, they have not played an important role in the production of liquid biofuels. We are changing this with our research program and are promoting the development of an important carboxylic acid platform within the biorefinery concept. Carboxylic acids are already used as intermediate molecules for further chemical and fuel production, but we generate these acids from biomass rather than from nonrenewable sources. Here, we are developing a system with undefined mixed culture conversion of lignocellulosic biomass into butyric acid and subsequent biological conversion into the liquid biofuel n-butanol. Specifically, we are optimizing three subsequent treatment steps in a biorefinery system: 1. chemical/physical pretreatment; 2. undefined-mixed-culture bioprocess to generate butyric acid; and 3. pure-culture bioprocess to convert butyric acid into n-butanol. During our lab-scale studies we are education interested parties in the agronomy sector by providing technological information about our process with the goal to enhance economic opportunities for agricultural producers and to protect and enhance the US natural resources and environmental quality. PARTICIPANTS: Dr. Hanno Richter, Research Associate (Post-doc) to convert butyrate into butanol Matt Agler, PhD candidate to perform the reactor studies to generate butyrate Dr. Bruce Dien, Chemical Engineer at ARS, USDA, Peoria, IL who has managed the pretreatment of corn fiber Loren Iten, ARS, USDA, Peoria, IL who has performed the pretreatment steps Dr. Michael Cotta, ARS, USDA, Peoria, IL who has advised the overall fermentation research Dr. Nasib Qureshi, Chemical Engineer at ARS, USDA, Peoria, IL who has managed the work of Hanno Richter (butanol fermentation) Sebastian Heger, MS student at RTWH Aachen University, Germany who has performed the ecotox studies Dr. Henner Hollert, Professor at RTWH Aachen University, Germany who has overseen the ecotox studies Dr. Largus Angenent, PD, who has managed the project TARGET AUDIENCES: Bioenergy companies and farmers are target audiences of the outcome of generating biofuels from lignocellulosic materials. One of our conclusions is that pretreatment is essential, and this must be immediately be understood in the field. Finally, target audiences are scientist who work with undefined mixed cultures for energy generation or to study the microbial ecology in the environment or engineered systems. In the coming years, we will publish research papers with the results to help the field move forward (in addition to attending conferences) PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Pretreatment. We have found with ecotox studies that the biological toxicity for acid-treated corn fiber was as extensive as with corn stover. However, the toxicity for dilute base and hot water was considerably lower. Butyrate fermentation. We operated four 5-L bioreactors (anaerobic sequencing batch reactors, ASBR) in semi-batch mode for the entire year. At a start-up pH of 5.5 and a thermophilic temperature (55 degrees C) all methanogenic activity ceased, which raised the butyric concentration innately. Each reactor (R1, R2, and R3; acid, base, and how water, respectively) utilized one of three pretreatments or nonpretreated (R4) corn fiber to compare their butyric acid yields. R4 (nonpretreated corn fiber) was discontinued after 100-days of operation because slow hydrolysis caused it to fill with undegraded biomass. Reactors R1, R2, and R3 have been operated continuously for 330 days. At a 25-day hydraulic retention time (HRT) we achieved a maximum butyric acid production rate of 0.185 g/L reactor volume/day in R1 (acid pretreatment) at an organic loading rate of 2.33 g chemical oxygen demand (COD)/L reactor volume/day. In terms of yield based on COD, this translates to 14.5%, compared to 8.4% and 9.7% in R2 and R3. We found significant levels of soluble carbohydrates in the effluent (~1 g/L as glucose in R1 and R3, and ~3.5 g/L in R2), which led us to believe that product inhibition was limiting conversion of hydrolyzed substrate. To test the hypothesis, we began diluting products by maintaining our organic loading rate while reducing the HRT to 20 days and then to 15 days (reaching steady state at each HRT). As a result, at the 15-d HRT the butyric acid production rate increased to 0.223 g/L/day in R1, with yields of 17.4%, 10.5%, and 12.8% in R1, R2, and R3, respectively. This proofs that, indeed, product inhibition limits the butyric acid yields. Ecotox studies showed that the mixed culture degraded complex inhibiting compounds that were generated during this pretreatment step. This is, thus, an additional advantage of using undefined mixed cultures compared to pure cultures. Butanol fermentation. We performed a strain comparison of 10 solventogenic Clostridia strains to identify an optimum strain that can convert a mixture of butyric acid and carbohydrates rather than just carbohydrates alone (conventional procedure). The strain with the highest butanol yield was Cl. saccharoperbutylacetonicum N1-4. Next, we determined the optimum conditions for maintenance and growth for strain N1-4 in batch cultures, and found a maximum butanol production rate of up to 1.2 g/L/hour. We then determined the parameters for running continuous culture, including a sufficient butanol-stripping rate. With strain N-14 we were able to operate a two-step fermentation process in a continuous mode. This was accomplished with a pH-auxostat method to keep the pH stable and the butyrate concentration constant by feeding butyric acid at a low pH of 4.8. This culture was able to continuously convert butyrate to butanol for two-week periods (we have not determined the maximum operating period with continuous butanol formation).
Publications
- Heger S., Winkens K., Schneider A., Brinkmann M., Maletz S., Wolz J., Agler M. T., Angenent L. T., Seiler T. B. and Hollert H. (2009). Assessing the ecotoxicological effects of bioenergy extraction processes. 2009 Europe Annual Meeting of the Society of Environmental Toxicology and Chemistry (SETAC), 31 May - 4 June, 2009, Gothenburg, Sweden.
- Agler M. T., Iten L. B., Qureshi N., Cotta M. A., Dien B. and Angenent L. T. (2009). Use of nondefined mixed cultures for anaerobic conversion of lignocellulosic corn fiber to n-butyrate. 2009 Institute of Biological Engineering IBE Annual Conference, March 19-21 2009, Santa Clara, CA.
- Agler M. T., Iten L. B., Qureshi N., Cotta M. A., Dien B. and Angenent L. T. (2008). Mixed-community bioreactors to convert (ligno)cellulosic feedstocks into liquid biofuels. 2008 Northeast Renewable Energy Conference. August 26-28, 2008, State College, PA.
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