Progress 01/01/09 to 09/30/13
Outputs
Target Audience: Scientists, professionals, graduate students, undergraduate students, extension personnel Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate students, undergraduate research assistants, and a post-doctoral scholar have been trained on this project. How have the results been disseminated to communities of interest? Journal Articles and public presentations What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
A. A baling system that increases bale density has been developed and a patent application submitted. C. I unique bioconversion process to produce solvents and organic acids has been developed a provisional patent issued. D. We continue to teach and develop four courses: Fundamentals of Biorenewable Resources, Biofuel Production and Properties, Thermochemical Processing, and Life Cycle Analysis.
Publications
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Co-culturing of cellulase-producing bacteria (releasing soluble sugars from cellulose) and solvent-producing bacteria (consuming soluble sugars to synthesize solvents) using thermal cycles is a novel aspect of the proposed on-farm bioprocessing approach. Production of solvents and mixed organic acids by thermal cycling of co-cultures on pretreated lignocellulose was achieved in experiments of increasing complexity with respect to substrate (amorphous cellulose versus pretreated lignocellulose) and fermentation conditions (liquid versus solid substrate cultivations and flushed solid substrate cultivations). Bacterial co-cultures of pretreated (by Fenton chemistry) switchgrass, untreated switchgrass and amorphous cellulose demonstrated the conversion of cellulose to solvents and mixed organic acids, with similar production rates across biomass sources in liquid cultivations. Successful long term co-cultivations confirm that soluble sugars are still available to C. beijerinckii after weeks of fermentation, indicating that the C. thermocellum cellulases from the thermophilic cycle continue to liberate sugar from the biomass during the mesophilic cycle. As measured from co-culturing on amorphous cellulose, butanol production dramatically increases with the addition of butyric acid, which is expected to be a generalizable phenomenon for this strain. Tailoring solvent production through the enrichment of a selected product in the flush stream is achievable with a recycle stream, which is a significant benefit of our proposed on-farm biomass processing. High solids reactors with intermittent flushing were developed for co-cultivation experiments at solid state cultivation conditions, envisioned as our bench scale on-farm storage/processing model. Intermittently removing products by flushing improved the yield of the fermentation products, a process which is proposed for product recovery on-farm. Data show Fenton-chemistry treated Miscanthus, switchgrass, and corn stover released more glucose than the untreated control when subjected to cellulase (wheat straw did not show a difference). However acid-insoluble lignin assays showed no change in the amount of acid-insoluble lignin relative to the untreated feedstocks, suggesting that the Fenton chemistry pretreatment is making the cellulose more bioavailable, but not delignifying the material. In contrast, fungal pre-treatment resulted in lignin removal. Data clearly show that fungal pretreatment removed the lignin in corn stover in the majority of the treatments. Similar to other studies our pretreatment effectiveness is dependent on substrate and fermentation method. However the selection of pretreatment process will not be totally dependent on product yields, but rather the LCA of each process/substrate combination. Our preliminary data support the feasibility of this process in several ways. We propose using producer gas from the gasifier (gasifying spent biomass from previous fermentations) to take the bunker anaerobic initially. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Pretreated lignocellulose produced solvents and mixed organic acids in Demonstrating proof of concept for the innovative and high risk aspects of the proposed approach to on-farm biomass processing was the primary focus of 2012. To this end, the feasibility of cycling through pretreatment, in-situ cellulase production for biomass saccharification, and subsequent fermentation for solvent /organic acid production has been demonstrated. This approach to the conversion of biomass in a flushed solid substrate cultivation with recycle exhibits clear advantages at the bench scale, and is envisioned as our on-farm storage/processing model for further scale up. Finally, the framework is prepared for integrating these innovations into the larger goals of the grant, with a focus on harvesting/processing integration, scale up, and analysis of economics and sustainability, with broad participation of all constituents. Broad participation, interest, and feedback from the general public, farmers, manufacturers, equipment dealers, and refiners has been accomplished through outreach activities. Future scientists in this interdisciplinary research area (high school, undergraduate, and graduate students) are being trained. Economic and environmental aspects of this project have been used as case studies in several courses and student group projects. high solids reactors which were thermally cycled between co-cultivation conditions favoring bacterial cellulase production and those favoring fermentation. Intermittent removal of products by flushing improved the yield of the fermentation products. Two approaches to delignification were established, the biomimetic Fenton chemistry and fungal pretreatment. Both approaches resulted in fermentable biomass with increased cellulose accessibility. Preliminary data suggest that the fungal pretreatment delignifies whereas the Fenton chemistry likely modifies the structure but does not remove lignin. The two pretreatments therefore present two very different approaches in terms of the subsequent effluents and the LCA. The collaboration between the University of Kentucky and CNH America has resulted in a patent disclosure related to increasing bale density. Hydraulic conductivities through bales have been quantified and have been shown to be a function of bale density. These values indicate that bale inoculation and product recovery in a flushed bioreactor is feasible, and can be tailored by creating bales of strategic densities using our baling system. Endogenous gas production and the energy from burning spent biomass are sufficient to allow for a self-sustaining co-culturing process. The gas produced is sufficient to take the system anaerobic, and we have demonstrated production even in the presence of small amounts of oxygen. Additionally, roughly 1/10th of the spent biomass will be needed to supply the energy required to keep the insulated bunker at the desired temperatures, leaving spent biomass to provide energy for other on-farm processes.
Publications
- Modenbach, A. A. and S.E. Nokes. 2012. The Use of High-Solids Loadings in Biomass Pretreatment - A Review. Biotechnology and Bioengineering.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Clostridium thermocellum is a cellulolytic anaerobic bacterium that can directly convert cellulosic feedstock into ethanol. However, ethanol yield of this organism is low due to the reallocation of carbon to other fermentation products (lactate, acetate, formate), as well as gases (carbon dioxide, and hydrogen). C. thermocellum at elevated pressure (7.0 MPa, and 13.0 MPa) increased the ethanol: acetate ratio by more than 100-fold compared to that under atmospheric pressure. The observed effect has been attributed to the increased concentration of hydrogen in the culture broth. Hydrogen is hypothesized to inhibit hydrogenase. To separate the increased hydrogen inhibition effect from the additional effect seen at pressure, this project focused on the effect of exogenous hydrogen and other hydrogenase inhibitors on the product formation of C. thermocellum. Objective 2: Incorporate metabolic flux model into whole-cell model that accounts for growth (i. e., dilution) rate and dissolved gas effects on product selectivity. Experimental observations from literature have shown increased ethanol production (the target product) and decreased acetate production (the by-product) under conditions of elevated pressures and/or pH and the presence of increasing dissolved hydrogen gas. These conditions continue to influence the control of NADH/NAD pairs towards target product formation. The model predicted the flux spectrum of metabolic distribution to account for ethanol and acetate yields as functions of dissolved hydrogen gas and pressure alongside the NADH/NAD effect. Acetate and ethanol yields mostly agreed with the corresponding values reported. Product selectivity mainly shifted from acetate to ethanol at elevated pressures. The role of NADH/NAD is significant for controlling product selectivity in fermentation processes. It is expected that more NADH consumption will continuously produce hydrogen in the oxidation-reduction which will in turn inhibit acetate formation and cause acetylcoA to move towards ethanol. This explains the linear relationship observed between NADH consumption flux and hydrogen flux under conditions of elevated pressures which confirms NADH's effect on the metabolic flux distribution. It is also useful to note that the observed linear relationship between NADH consumption and hydrogen flux is analogous to the relationship between NADH/NAD ratio and hydrogen under pressure reported in the literature. For this reason, it was useful to incorporate an NADH flux due to the reversible NADH reaction into the model in order to quantify and interpret the effect of relative changes in NADH/NAD ratio due to hydrogen and pressure on the metabolic flux distribution end-products, especially ethanol. It was determined that ethanol is brought up to an approximate ratio of 1.4:1 of NADH flux due to the reversible NADH reaction when pressure is elevated. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We showed that the ethanol: acetate ratio increased in the presence of exogenous hydrogen and hydrogenase inhibitors, but only under certain conditions. Further experiments and metabolic modeling resulted in the determination that two conditions must be satisfied to have increased ethanol production: hydrogenase must be inhibited such that acetate product is severely impaired, and the substrate uptake mechanism must be impaired such that the cellobiose feed rate into the cell is below a threshold level. The ability to control product selectivity by environmentally manipulating carbon and electron flows offers a novel approach to directing microbial metabolism. The pattern of acetate and ethanol production at different dilutions rates were similar to previous results. The assumed zero flux of glycogen had little or no impact on the predicted flux spectrum and consequently the effect dissolved hydrogen gas and pressure. However, based on model predictions, varying hydrogen flux across dilution rates for each pressure versus keeping hydrogen flux constant for each pressure significantly influenced ethanol yields. These observations provide important information for conditions under which ethanol yields may be highest. It was determined that ethanol yields are at their highest when hydrogen flux is maintained at about 4.81 coupled with pressure at 7 MPa and above.
Publications
- Hsin-Fen Li, Barbara L. Knutson, Sue E. Nokes, Bert C. Lynn, Michael D. Flythe. 2011. Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate. Applied Microbiology and Biotechnology.
- Adotey, B., Nokes, S.E., Knutson, B.L., Lynn, B.C., and M.A. Flythe. 2011. Metabolic Flux and Control Analyses of Wild Type and Ethanol Adapted Clostridium thermocellum . Presented at the 2011 ASABE Annual Meeting, Louisville, KY. August 7-10, 2011.
- Adotey, B., Nokes, S.E., Knutson, B.L., Lynn, B.C., and M.A. Flythe. 2011. Metabolic Flux and Control Analyses of Wild Type and Ethanol Adapted Clostridium thermocellum . Presented at the 2011 Symposium on the Thermochemical Conversion of Biomass to Fuels. Advanced Technology and Research Center, Oklahoma State University, August 2, 2011.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: We have found a promising strain of Scenedesmus, which has a high growth rate at a pH less than 7. Chlorella vulgaris and Scenedesmus have been tested with varying amounts of sulfuric acid. Small amounts (6 ppm) appear to have little effect on the culture growth. Intermediate amounts (60 ppm) inhibit growth, but the algae appear to have the ability to recover. Higher amounts (90 ppm) will kill the culture. The influence of strain selection and media components are also currently under way. There have been three major outputs from this project. The first is pertaining to grant writing. Our involvement in this multistate research project enabled us to compete for a large multiinstitutional grant, which is pending, and also to be awarded a large grant based on the work performed as part of this multistate project. The second output is the dissemination of knowledge through presentations, publications, and course lectures. The third is a set of laboratory techniques and equipment developed specifically for this research which will allow our lab to further explore these questions. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We currently have several potential set-ups for running algae studies. In our environmental chambers we have the ability to run with constant temperature and consistent lighting (16 h days, 8 h nights). This set-up is used for media development, strain selection, and inoculum preparation with cultures up to 400 mL and capacity of 123 flasks. We also have a varying temperature chamber, where the temperature of the cultures is varied using a circulating water bath through the metal base of the chamber. This system has consistent lighting (16 h days, 8 h nights) and uses cultures up to 400 mL with a capacity of up to 27 flasks. We also have a ventilated and constantly stirred system for use with lower flow rates, which also requires stirring. This set-up is also used with simulated flue gas experiments that need to be properly ventilated. This system utilizes cultures up to 400 mL with a capacity of up to 27 flasks. We also have a pilot-scale, continuously harvested system, which is currently under renovation. This system will be able to run a 60 L culture. Clostridium thermocellum is a cellulolytic anaerobic bacterium that can directly convert cellulosic feedstock into ethanol. However, ethanol yield of this organism is low due to the reallocation of carbon to other fermentation products (lactate, acetate, formate), as well as gases (carbon dioxide, and hydrogen). C. thermocellum at elevated pressure (7.0 MPa, and 13.0 MPa) increased the ethanol: acetate ratio by more than 100-fold compared to that under atmospheric pressure. The observed effect has been attributed to the increased concentration of hydrogen in the culture broth. Hydrogen is hypothesized to inhibit hydrogenase. To separate the increased hydrogen inhibition effect from the additional effect seen at pressure, our current work focuses on the effect of exogenous hydrogen and other hydrogenase inhibitors on the product formation of C. thermocellum. Preliminary batch experiments have shown that the ethanol: acetate ratio increased in the presence of exogenous hydrogen and hydrogenase inhibitors. Continuous fermentations will be carried out in a chemostat under treatments of different inhibitors at various pressures. The ability to control product selectivity by environmentally manipulating carbon and electron flows offers a novel approach to directing microbial metabolism.
Publications
- Sharma, B., Nokes, S., Montross, M., and L. Vaillancourt. 2010. A real-time polymerase chain reaction protocol for quantifying growth of Fusarium graminearum during solid substrate cultivation on corn stover. Journal of Biotech Research, 2010. 2:144-155.
- Dhamagadda, V. S., S.E. Nokes, H.J. Strobel, and M.D. Flythe. 2010. Investigation of the metabolic inhibition observed in solid substrate cultivation of Clostridium thermocellum on cellulose. Bioresource Technology. 101(15): 6039-6044.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The annual meeting was held in September, 2009 in Portland, Oregon. Several of the investigators on this project consulted with the Kentucky Energy Cabinet's task force on biomass utilization to develop Kentucky's Energy Plan. Three courses were developed at the University of Kentucky; Fundamentals of Biorenewable Resources; Biofuels; and Thermochemical Conversion of Biomass PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Videos were created of the course lectures. The University of Kentucky collaborated with the University of Idaho and Iowa State University to develop the courses and share lecture material.
Publications
- Coleman, N., C. Crofcheck, S. Nokes and B. Knutson. 2009. Effects of Growth Media pH and Reaction Water Activity on the Conversion of Acetophenone to (S)-1-phenylethanol by Saccharomyces cerevisiae Immobilized on Celite 635 and in Calcium Alginate. Trans of ASABE. 52(2):665-671.
- Timmons, MD, BL Knutson, SE Nokes, H.J. Strobel, BC Lynn. 2009. Analysis of composition and structure of Clostridium thermocellum membranes from wild-type and ethanol-adapted strains. Applied Microbiology and Biotechnology. Vol. 82(5):929-939.
- Chinn, M.S., Nokes, S.E., Strobel, H.J. 2008. Influence of Process Conditions on End Product Formation from Clostridium thermocellum 27405 in Solid Substrate Cultivation on Avicel. Bioresource Technology. Available online 12 July 2007. doi:10.1016 j.biortech.2007.04.052. Vol. 99(7) May 2008: 2664-2671.
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