Progress 09/01/14 to 08/31/18
Outputs
Target Audience:Research funded by this grant serves both West Virginia and national objectives to advance bioproducts and bioenergy. The research involved both theoretical and practical aspects of producing carboxylates (short chain fatty acids) with mixed microbial cultures and developing a research/teaching facility for bioprocessing reactor applications. The first target audience is environmental scientists and engineers who are interested in developing microbiomes for the conversion of waste biomass into industrially useful products such as platform chemicals, fatty acids, and green chemistry technologies. Because anaerobic digestion is the best existing technology for developing specialized carboxylate reactors, those industries that already utilize anaerobic digesters could also benefit from this research. For example, CAFO (concentrated animal feeding operations) facilities with large quantities of livestock manures could benefit because the primary waste biomass used in anaerobic digesters in the US is animal wastes. Other industrial and agricultural production facilities that use anaerobic digestion (such as food and paper manufacturing) could also benefit because this research advances new applications of anaerobic digestion technology and the development of new value-added products from organic waste management. The grant will also support graduate and undergraduate research training in environmental biotechnology at WVSU and strengthen the linkages of WVSU with an international university (Universidad Autonoma Chapingo, Mexico) and its program in agroindustrial engineering. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This grant supported the training of two MS degree (biotechnology) students. Both have graduated (theses titles are reported below). Two additional graduate students (MS degree, biotech) also participated part-time by assisting in data collection, particularly running bioreactors and performing gas chromatography analysis. One full time technician has been working on this project. The technician has been assisting the graduate students and is the primary engineer who is assembling the pilot-scale bioprocessing reactor. In addition, two other temporary technicians participated in the project. The grant provided technical training for all of them. Three undergraduate students received training support from the grant. One of them, a chemistry major, also conducted an independent research project on carboxylate production that was presented at the West Virginia NASA Space Grant Consortium, April 2017. Student project title: Measuring biochemical methane potential (BMP) during glycerol-induced stress in a thermophilic methanogenic microbiome. Two visiting international graduate student trainees from Universidad Politécnica de Puebla, San Mateo Cuanalá, Puebla, Mexico received training. All of the students received training in environmental biotechnology, environmental microbiology, environmental chemistry, and microbial genomics. How have the results been disseminated to communities of interest?During the grant period, the results were disseminated through twelve national and international professional conferences: 1) American Ecological Engineering Society, 16th Annual Meeting, June 7-9, 2016, Knoxville, TN. David H. Huber, Teodoro Espinosa-Solares, Emmanuel Chavarria-Palma, Natalia Montenegro-Garcia, Alejandro Ramirez-Garcia, Vadesse Lhilhi Noundou, Ifeoma Rosemary Ugwuanyi. Evaluating Co-Digestion in a Pilot-Scale Thermophilic Digester Stabilized on Poultry Litter Substrate: Performance, Resilience, and Microbial Community Adaptation. 2) 6th International Conference on Engineering for Waste and Biomass Valorisation, May 23-26, 2016, Albi, France. Meneses-Reyes, J.R., T. Espinosa-Solares, G. Hernandez-Eugenio, N. Balagurusamy, David H. Huber. Chlorella vulgaris and glycerol used in co-digestion with chicken litter enhance methane production. 3) USDA Project Director's Conference, September 2016. David H. Huber, Micheal Fultz, Sridhar Malkaram, Marak Krasnansky. Bioengineering the carboxylate platform in thermophilic anaerobic microbiomes: progress report. 4) USDA Association for Research Director's Conference, Atlanta (GA), April 1-5, 2017. Increasing Carboxylate Production in Model Methanogenic Microbiomes. A. Ramirez-Garcia, E. Chavarria, N. Montenegro, V. L. Noundou, A. Adeleye, C. Martin, and D. H. Huber. 5) USDA Association for Research Director's Conference, Atlanta (GA), April 1-5, 2017. Building the Bioenergy and Bioengineering Research Facility at West Virginia State University. D. H. Huber, E. Chavarria, N. Montenegro. 6) American Society for Microbiology, ASM Microbe 2017 Meeting, New Orleans (LA), June 1-5. Crude Glycerol Induced Stress and Slow Resilience in Thermophilic Anaerobic Digestion. D.H. Huber, T. Espinosa-Solares, A. Ramirez-Garcia, J.E. Chavarria-Palma, N. A. Montnegro-Garcia, V.Lhilhi Noundou, C. Martin. 7) Vadesse Lhilhi Noundou, Teodoro Espinosa-Solares, David H. Huber. Evaluation of Stress and Resilience in a Model Fluidic Anaerobic Ecosystem. Ohio River Basin Research and Education Symposium/ Ohio River Basin Alliance Summit, Sept 27-29, 2017; Marshall University. 8) Vadesse Lhilhi Noundou, Teodoro Espinosa-Solares, David H. Huber. Evaluation of Stress and Resilience in a Model Fluidic Anaerobic Ecosystem. Ohio River Basin Research and Education Symposium/ Ohio River Basin Alliance Summit, Sept 27-29, 2017; Marshall University. 9) Vadesse Lhilhi Noundou, Sridhar A. Malkaram, Jesus E. Chavarria-Palma, Natalia A. Montenegro-Garcia, Ifeoma R. Ugwuanyi, Teodoro Espinosa-Solares, David H. Huber. American Society for Microbiology Annual Meeting, Atlanta, GA, June 7-11, 2018. Deciphering Niche Differentiation Among Phylotypes in a Thermophilic Digester. 10) Vadesse Lhilhi Noundou, Jesus E. Chavarria-Palma, Alejandro Ramirez-Garcia, Natalia A. Montenegro-Garcia, Ifeoma R. Ugwuanyi, Sridhar A. Malkaram, Teodoro Espinosa-Solares, David H. Huber. 2018. Universities Council on Water Resources(UCOWR)/The National Institutes for Water Resources (NIWR) Annual Water Resources Conference, June 26-28, Pittsburgh, PA. The Water-Energy-Waste Biomass Nexus: Ecological Engineering of Anaerobic Digestion Improves Bioenergy Recovery and Water Use Efficiency. 11) Huber, David H., Vadesse Lhilhi Noundou, Teodoro Espinosa-Solares, Jesus E. Chavarria-Palma, Sridhar A. Malkaram, Natalia A. Montenegro-Garcia, Ifeoma R. Ugwuanyi. 2018. Experimental tests of methanogenic microbiome resilience following resource-induced stress in model bioreactors. Ecological Society of America Annual Meeting, New Orleans (LA), Aug 5-10. 12) Espinosa-Solares, Teodoro, Maria del Carmen Gonzalez-Rangel, David H. Huber. 2018. Resilience evaluation to overloading feed disturbance in thermophilic anaerobic digestion. 17th International Symposium on Microbial Ecology, August 12-17, Leipzig, Germany. In addition, results from this research have been incorporated into the Project Director's teaching in the following classes at WVSU: Microbial Genetics, Current Concepts in Biotechnology, and Biotechniques II. 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 major objectives of this grant were to build a Biomass Conversion Research Facility at West Virginia State University (WVSU) and to advance the development of a thermophilic microbiome with increased carboxylate production. Carboxylates, or short chain fatty acids, are valuable precursor chemicals that are used in the chemical synthesis industry for many processes. Carboxylates are also the major metabolite intermediates produced in anaerobic digestion. Therefore, we have been working on engineering the microbiome of anaerobic digesters to route carbon flow into increased carboxylate production rather than methane production. This first major objective of the grant was to build a pilot-scale bioprocessing reactor that will have versatile applications for the biotechnology program and STEM field education at WVSU. We have built a thermophilic bioprocessing reactor in an enclosed concrete shelter at the Agricultural and Environmental Research Station on the campus of WVSU. The reactor vessel is 1000 gallons, stainless steel, insulated, and can be heated to about 60C. The facility includes heavy-duty centrifugal pumps for circulating the reactor liquid and pumping feed into the reactor vessel. Additional pumps include one to remove effluent from the reactor and a grinder pump for feed that contains high amounts of solids. The system is controlled with a PLC (programmable logic controller) automation system that controls pumps, and monitors and adjusts temperature and liquid levels. Two additional 1000 gallon plastic tanks are used for feed and effluent storage. The bioprocessing reactor is currently used as a thermophilic anaerobic digester but could have other bioprocessing applications in the future. The grant also funded several experiments that utilized an ecological engineering approach to increase carboxylate production in a previously established thermophilic anaerobic digester microbiome. One experiment evaluated different substrate-based methods for increasing carboxylate production. For this experiment, three replicate one liter reactors were run for more than 400 days. Three methods were tested to see if feedstock (substrate) modifications could be used to bioengineer microbiome metabolism. In this experiment, we evaluated whether carboxylate production could be increased by reducing hydraulic retention time versus using a feedstock with higher COD (chemical oxygen demand) content. We also evaluated whether the addition of two co-substrates (crude glycerol and ethanol) would increase carboxylate production. This experiment showed that increasing COD of the feed did significantly increase carboxylate production, but decreasing hydraulic retention time had minimal effect. We found that acetate, propionate, butyrate and valerate production could be increased several-fold. Based on these results, another experiment was conducted that evaluated whether stable, high-level carboxylate production could be maintained in this microbiome by increasing the organic loading rate. This experiment utilized three 10 liter reactors that were operated for more than 500 days. We found that high levels of acetate and propionate could be stably maintained in these thermophilic reactors. In another long-term experiment that used a different set of three 10 liter reactors, we tested whether the capacity of this microbiome to tolerate crude glycerol could be improved by subjecting it to repeated pulses of this substrate. These reactors were also run for more than 500 days. We found that the microbiome could be adapted to this stressful substrate. Following the pulses, the reactors were able to tolerate a higher percentage of crude glycerol and higher levels of carboxylate intermediates were produced. The microbiome of these digesters was also evaluated using Illumina sequencing of 16S rRNA gene diversity. Nearly 8 million paired-end reads were obtained. The dominant bacterial phyla were Firmicutes, Proteobacteria, Thermotogae and Bacteroidetes. Microbiome structure was measured before and after the crude glycerol pulses. Beta diversity analysis showed that community structure following the pulses was altered and these microbiomes became adapted to processing higher concentrations of crude glycerol.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
1) Meneses-Reyes, J.C., G. Hernandez-Eugenio, D.H. Huber, N. Balagurusamy, T. Espinosa-Solares. 2016. Biochemical methane potential of oil-extracted microalgae and glycerol in co-digestion with chicken litter. Bioresource Technology (dx.doi.org/10.1016/j.biotech.2016.11.012)
2) Meneses-Reyes, Jose Carlos, Guadalupe Hernandez-Eugenio, David H. Huber, Nagamani Balagurusamy, Teodoro Espinosa-Solares. 2018. Oil-extracted Chlorella vulgaris biomass and glycerol bioconversion to methane via continuous anaerobic co-digestion with chicken litter. Renewable Energy 128:223-229. (doi.org/10.1016/j.renene.2018.05.053)
3) A third paper was submitted for review at the time of writing this termination report.
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Progress 09/01/16 to 08/31/17
Outputs
Target Audience:This research advances both theoretical and practical aspects of producing carboxylates and bioenergy using bioreactors. The first target audience is environmental scientists and engineers who are interested in developing microbiomes for the conversion of waste biomass into industrially useful products such as platform chemicals, fatty acids, and green chemistry technologies. However, because anaerobic digesters are the best existing technology for developing specialized carboxylate reactors, those industries that already utilize anaerobic digesters could also benefit from this research. For example, CAFO operations that produce large quantities of manures could benefit because the primary waste biomass used in anaerobic digesters in the US is animal wastes. Other industrial and agricultural production facilities that use anaerobic digestion could also benefit because this research advances new applications of digestion technology and the development of new value-added products from organic waste management. The grant will also support graduate and undergraduate research training in biotechnology at WVSU and strengthen the linkages of WVSU with an international collaborating institution (Universidad Autonoma Chapingo, Mexico). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This grant supported the training of two MS degree (biotechnology) students during 2016-2017. One of the students graduated in May 2017. The second student graduated in December 2017. One full time technician has been working on this project. The technician has been assisting the graduate students and is the primary engineer who is assembling the pilot-scale reactor. One undergraduate student (chemistry) also conducted an independent research project on carboxylate production during 2016-17. All of the students have received training in environmental biotechnology, environmental microbiology, environmental chemistry, and microbial genomics. How have the results been disseminated to communities of interest?Results have been disseminated through four professional conferences during 2017: 1) USDA Association for Research Director's Conference, Atlanta (GA), April 1-5, 2017. Increasing Carboxylate Production in Model Methanogenic Microbiomes. A. Ramirez-Garcia, E. Chavarria, N. Montenegro, V. L. Noundou, A. Adeleye, C. Martin, D. H. Huber; 2) USDA Association for Research Director's Conference, Atlanta (GA), April 1-5, 2017. Building the Bioenergy and Bioengineering Research Facility at West Virginia State University. D. H. Huber, E. Chavarria, N. Montenegro; 3) American Society for Microbiology, ASM Microbe 2017 Meeting, New Orleans (LA), June 1-5. Crude Glycerol Induced Stress and Slow Resilience in Thermophilic Anaerobic Digestion. D.H. Huber, T. Espinosa-Solares, A. Ramirez-Garcia, J.E. Chavarria-Palma, N. A. Montnegro-Garcia, V.Lhilhi Noundou, C. Martin; and 4) Vadesse Lhilhi Noundou, Teodoro Espinosa-Solares, David H. Huber. Evaluation of Stress and Resilience in a Model Fluidic Anaerobic Ecosystem. Ohio River Basin Research and Education Symposium/ Ohio River Basin Alliance Summit, Sept 27-29, 2017; Marshall University. In addition, results from this research have been incorporated into the Project Director's teaching through the following classes: Environmental Microbiology, Current Concepts in Biotechnology, and Biotechniques II. What do you plan to do during the next reporting period to accomplish the goals?During 2018, we will conduct another experiment with our 10 liter, high-carboxylate reactors to test whether we can increase propionate and butyrate levels even further. We will investigate the response of the microbiome to the new reactor operating conditions using time series analysis of 16S rRNA marker gene diversity, and we will continue to measure stability of the reactors which is an important property for industrial applications. In addition, we will complete the set-up of the pilot-scale (1000 gallon) thermophilic bioreactor. This project will continue to be used to train MS degree (biotechnology) and undergraduate science students at WVSU.
Impacts
What was accomplished under these goals?
The major objectives of this grant are to build the biomass conversion research facility at West Virginia State University (WVSU) and to advance the development of a thermophilic microbiome with increased carboxylate production. Carboxylates (short chain fatty acids) are valuable precursor chemicals that are used in industry for many processes. Carboxylates are also the major metabolite intermediates produced in the process called anaerobic digestion. Therefore, we (and others) are working on engineering the microbiome of anaerobic digesters to route carbon flow into increased carboxylate production rather than methanogenesis. During 2017, two major experiments were completed. The first experiment concerns the evaluation of methods for increasing carboxylate production in thermophilic digesters. This experiment utilized three replicate one liter reactors that were run for more than 400 days. Three methods were tested to see if feedstock (substrate) modifications could be used to bioengineer microbiome metabolism. In this experiment, we first compared the reduction of hydraulic retention time to the increase of COD (chemical oxygen demand) in the feedstock as methods for increasing carboxylates. We also evaluated whether the addition of two co-substrates (crude glycerol and ethanol) would increase carboxylate production. This experiment showed that increasing COD of the feed did increase carboxylate production dramatically while decreasing the hydraulic retention time had minimal effect. We found that acetate, propionate, butyrate and valerate production could be increased several-fold. Based on these results, a second experiment was conducted that evaluated whether stable, high-level carboxylate production could be maintained in this microbiome by increasing the organic loading rate. This experiment utilized three 10 liter thermophilic reactors that were operated for more than 500 days. We found that high levels of acetate and propionate could be stably maintained in these thermophilic reactors. A third project funded by this grant is the development of a 1000 gallon, thermophilic, pilot-scale reactor facility. We have continued to make progress in setting up this reactor but installation is not yet complete. The electrical infrastructure has been put in place, but we still need to install pumps and the control system. This grant has received a one year no-cost extension so that the set-up the pilot reactor and the carboxylate experiments can be completed.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2017
Citation:
Alejandro DeJesus Ramirez-Garcia. 2017. Increasing carboxylate production in thermophilic methanogenic microbiomes. MS Thesis (Biotechnology), West Virginia State University.
Vadesse Lhilhi Noundou. 2017. Measuring Stability and Resilience in a Thermophilic Anaerobic Digester Microbiome. MS Thesis (Biotechnology), West Virginia State University.
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Progress 09/01/15 to 08/31/16
Outputs
Target Audience:The target audience is environmental scientists and engineers who are interested in using mixed culture microbiomes for producing platform chemicals, fatty acids, and green chemistry technology. This research advances both theoretical and practical aspects of producing carboxylates and bioenergy in bioreactors. Industrial and agricultural production facilities that utilize anaerobic digestion will also be interested in the research because it advances new applications of digestion technology and the development of value-added products from organic waste management. The grant will also support graduate and undergraduate research training in biotechnology at WVSU. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The grant supported the training of two MS degree (biotechnology) students at WVSU during 2016. One full time technician has been assisting with the set-up of the new thermophilic reactor. The graduate students are operating the laboratory-scale reactors for their MS degree research that involves carboxylate production experiments. They are also assisting in the set-up of the large pilot-scale reactor. One undergraduate (chemistry/mathematics) student is also conducting an independent research project on carboxylate production that is supported by this grant. The students are being trained in environmental biotechnology, environmental microbiology, environmental chemistry, and microbial genomics. How have the results been disseminated to communities of interest?Research has been presented at the American Ecological Engineering Society meeting, Knoxville (TN), June 2016. Research was also presented at the USDA CBG Project Director's Conference, Virginia Beach (VA), September, 2016. One publication in a research journal (Bioresource Technology) was produced. Results are also incorporated into the Project Director's classes and teaching: Environmental Microbiology, Current Concepts in Biotechnology, Biotechniques II. What do you plan to do during the next reporting period to accomplish the goals?The set-up of the new pilot-scale thermophilic reactor facility has progressed well during the last year. During 2017 we will complete the installation which requires set-up of the electrical system, plumbing, reactor vessel pumps, and computer control. We plan to have the set-up completed by summer 2017. Current experiments using bench-scale reactors will be continued as we attempt to further increase the production of intermediate-length carboxylates and to achieve steady state, stable reactor performance. Microbiome analysis of the reactors will be done using metagenomics. This project will continue to be used to train MS degree (biotechnology) students at WVSU.
Impacts
What was accomplished under these goals?
The major objectives of this grant are to build the biomass conversion research facility at West Virginia State University (WVSU) and to advance the development of a thermophilic microbiome with increased carboxylate production. During the previous year, we continued the set-up of a 1000 gallon, thermophilic, discontinuously stirred reactor vessel. The vessel has been placed in a dedicated, high roof, concrete walled building at the WVSU Research Station. Large tanks for feed mixing and effluent storage have been placed into the building and spill containment has been constructed to meet WV environmental regulations. Other tasks underway include insulating the reactor and routing electricity to the building. We anticipate that the new reactor will be operational during summer 2017. A second major objective of the grant is to bioengineer a thermophilic anaerobic digester so that it produces high levels of carboxylates from waste biomass. Carboxylates are important platform chemicals for many industrial syntheses. We have been operating three small (1 liter) and three larger (10 liter) thermophilic reactors to test whether carboxylate production can be increased in a microbiome that was previously designed for maximum methane production. We found that by increasing the organic loading rate, short-chain fatty acids (acetate, propionate, butyrate) can be increased. Presentations of this research were made at the 2016 USDA Project Director's meeting (Virginia Beach).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Meneses-Reyes, J.C., G. Hernandez-Eugenio, D.H. Huber, N. Balagurusamy, T. Espinosa-Solares. 2016. Biochemical methane potential of oil-extracted microalgae and glycerol in co-digestion with chicken litter. Bioresource Technology (dx.doi.org/10.1016/j.biotech.2016.11.012)
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