Bioprocesses carried out for the production of fuels and other value-added co-products require effective process control strategies. The objective of this research is to apply Titrimetric Off-Gas Analysis (TOGA) for the on-line estimation of fermentation end products using Clostridium thermocellum. The hydrogen ion production, gaseous H2 and CO2, soluble H2 and CO2, as well as ethanol in the liquid phase and vapour phase, were monitored. All parameters, except the dissolved gases, showed good correlation with concurrent off-line analysis. The resulting mass and electron balances were close to theoretical values, and not significantly different from those determined using off-line analysis. Liquid-to-gas mass transfer limitations caused supersaturation of H2(aq) for a wide-range of operating conditions, and on average, ranged between 8-14 times the expected value at thermodynamic equilibrium. The supersaturation of CO2(aq) was conditional, and could be alleviated by increased sparging at agitation such that no significant mass transfer limitation was present. Simultaneous data on ethanol, CO2, and H2 could be obtained with the MIMS probe placed adjacent to the liquid surface in the reactor headspace. From this data, a metabolic model was proposed for the on-line estimation of formate and acetate using a mass balance and an electron balance. The model estimated formate concentrations with reasonable accuracy. Acetate predictions agreed with the qualitative trends, but the concentrations were inaccurate in comparison with off-line analysis. It was demonstrated that the sensor could provide on-line information on all major end-products synthesized by C. thermocellum. In conclusion, TOGA is a valuable instrument for the on-line monitoring and study of fermentation processes for cellulosic biofuels production

Despite the long-term economic and environmental benefits of cellulosic biofuel production, low rates of cellulose utilization and products syntheses are major techno-economical barriers to the commercialization. Optimized medium composition and low-cost nutrient source could greatly enhance the feasibility of large-scale biofuels synthesis by direct cellulose fermentation using a consolidated bioprocessing (CBP) approach. This study developed an improved growth medium for Clostridium thermocellum, an excellent cadidate for CBP that utilizes cellulose to produce ethanol, hydrogen, and other value-added biochemicals. An experimental design to determine the importance of nutrient components and concentrations on H2 and ethanol production from cellulose by C. thermocellum initially considered seven growth nutrients. Three most significant components - α-cellulose, yeast extract, and magnesium chloride were investigated in detail for their influence on rates and yields of H2 and ethanol production during cellulose fermentation by C. thermocellum. To explore individual and interactive effects of these nutrients on ethanol and hydrogen (H2) production, a central composite face-centered design and the response surface methodology was applied to predict optimum nutrient compositions for H2 and ethanol production. Experimental verification of predicted optima produced about 3-fold and 4-fold more H2 and ethanol respectively compared with the reference medium. These small-scale results were successfully verified in large-volume (7L), atmospheric cultures. Irrespective of culture conditions, relative improvement in rates and productivities of H2 and ethanol in optimized medium compared with reference medium were consistent with small-volume cultures. Various ethanol distillery co-products were tested for their potentials to replace expensive medium ingredients. Medium prepared with these co-products show excellent ability to suppport cell-growth and production of ethanol and H2 at concentrations equivalent to those generated from the reagent grade medium. Utilization of these low-cost nutrient sources to replace expensive reagent ingredients may potentially contribute to the viability of both grain-based ethanol and cellulosic biofuels. With medium optimization, scale-up and use of low-cost nutrient sources, this study represents one of the very few systematic research approaches to improve direct bioconversion of cellulosic biomass into biofuels.

Consolidated bioprocessing, a method that involves cellulase production, substrate hydrolysis, and fermentation all in one step, requires lower energy input and aims at achieving reduced biofuel production costs than traditional processes. It is an economically appealing strategy for the efficient production of biofuels such as ethanol or H2. At present, the yields of fermentative hydrogen and ethanol production are less than the theoretical maximum and vary between anaerobic Clostridia due to the presence of highly branched metabolic pathways. With the recent advancements in ‘Omic technologies, the selected cellulolytic species, in this case, C. termitidis, was extensively studied to identify the key enzymes that are involved in hydrogen and ethanol synthesis pathways in both the genome and proteome under different culture conditions. Metabolic characterization involving growth and end-product synthesis patterns were performed on 2 g L-1 cellobiose and α-cellulose under batch conditions to determine its metabolic potential for hydrogen and/or ethanol production. Initial characterization has shown the ability of C. termitidis to produce hydrogen, ethanol, and various other end-products on the two susbtrates. Continous N2 sparging in the pH-controlled bioreactors with cellobiose and α-cellulose showed a consistent increase in the H2 synthesis and lowered ethanol production compared to batch studies, with the H2 yields of 1.03 and 1.34 mol product per mol hexose equivalent added, respectively. Shotgun 2-D proteome analyses were performed to compare cellulose versus cellobiose grown cultures across exponential and stationary phases of growth. Most of the glycolytic proteins were detected in the proteome with some exceptions and no significant change was observed across both growth conditions. Hydrogen synthesis was regulatd via PFOR and ferredoxin-dependent hydrogenase, where as ethanol synthesis was regulated primarily via bifunctional AdhE activity. Proteomic analyses of C. termitidis cultured on hexose sugars in the absence of xylose suggested possible sequential utilization of xylose and cellobiose for the first time. Putative proteins consistent with xylose fermentation were observed at high levels. The hypothesis that C. termitidis can sequentially utilize xylose and cellobiose was further validated using batch fermentations tests on pure (xylose, cellobiose, xylan) and mixed substrates (xylose + cellobiose).

Bioprocesses carried out for the production of fuels and other value-added co-products require effective process control strategies. The objective of this research is to apply Titrimetric Off-Gas Analysis (TOGA) for the on-line estimation of fermentation end products using Clostridium thermocellum. The hydrogen ion production, gaseous H2 and CO2, soluble H2 and CO2, as well as ethanol in the liquid phase and vapour phase, were monitored. All parameters, except the dissolved gases, showed good correlation with concurrent off-line analysis. The resulting mass and electron balances were close to theoretical values, and not significantly different from those determined using off-line analysis. Liquid-to-gas mass transfer limitations caused supersaturation of H2(aq) for a wide-range of operating conditions, and on average, ranged between 8-14 times the expected value at thermodynamic equilibrium. The supersaturation of CO2(aq) was conditional, and could be alleviated by increased sparging at agitation such that no significant mass transfer limitation was present. Simultaneous data on ethanol, CO2, and H2 could be obtained with the MIMS probe placed adjacent to the liquid surface in the reactor headspace. From this data, a metabolic model was proposed for the on-line estimation of formate and acetate using a mass balance and an electron balance. The model estimated formate concentrations with reasonable accuracy. Acetate predictions agreed with the qualitative trends, but the concentrations were inaccurate in comparison with off-line analysis. It was demonstrated that the sensor could provide on-line information on all major end-products synthesized by C. thermocellum. In conclusion, TOGA is a valuable instrument for the on-line monitoring and study of fermentation processes for cellulosic biofuels production

Despite the long-term economic and environmental benefits of cellulosic biofuel production, low rates of cellulose utilization and products syntheses are major techno-economical barriers to the commercialization. Optimized medium composition and low-cost nutrient source could greatly enhance the feasibility of large-scale biofuels synthesis by direct cellulose fermentation using a consolidated bioprocessing (CBP) approach. This study developed an improved growth medium for Clostridium thermocellum, an excellent cadidate for CBP that utilizes cellulose to produce ethanol, hydrogen, and other value-added biochemicals. An experimental design to determine the importance of nutrient components and concentrations on H2 and ethanol production from cellulose by C. thermocellum initially considered seven growth nutrients. Three most significant components - α-cellulose, yeast extract, and magnesium chloride were investigated in detail for their influence on rates and yields of H2 and ethanol production during cellulose fermentation by C. thermocellum. To explore individual and interactive effects of these nutrients on ethanol and hydrogen (H2) production, a central composite face-centered design and the response surface methodology was applied to predict optimum nutrient compositions for H2 and ethanol production. Experimental verification of predicted optima produced about 3-fold and 4-fold more H2 and ethanol respectively compared with the reference medium. These small-scale results were successfully verified in large-volume (7L), atmospheric cultures. Irrespective of culture conditions, relative improvement in rates and productivities of H2 and ethanol in optimized medium compared with reference medium were consistent with small-volume cultures. Various ethanol distillery co-products were tested for their potentials to replace expensive medium ingredients. Medium prepared with these co-products show excellent ability to suppport cell-growth and production of ethanol and H2 at concentrations equivalent to those generated from the reagent grade medium. Utilization of these low-cost nutrient sources to replace expensive reagent ingredients may potentially contribute to the viability of both grain-based ethanol and cellulosic biofuels. With medium optimization, scale-up and use of low-cost nutrient sources, this study represents one of the very few systematic research approaches to improve direct bioconversion of cellulosic biomass into biofuels.

Consolidated bioprocessing, a method that involves cellulase production, substrate hydrolysis, and fermentation all in one step, requires lower energy input and aims at achieving reduced biofuel production costs than traditional processes. It is an economically appealing strategy for the efficient production of biofuels such as ethanol or H2. At present, the yields of fermentative hydrogen and ethanol production are less than the theoretical maximum and vary between anaerobic Clostridia due to the presence of highly branched metabolic pathways. With the recent advancements in ‘Omic technologies, the selected cellulolytic species, in this case, C. termitidis, was extensively studied to identify the key enzymes that are involved in hydrogen and ethanol synthesis pathways in both the genome and proteome under different culture conditions. Metabolic characterization involving growth and end-product synthesis patterns were performed on 2 g L-1 cellobiose and α-cellulose under batch conditions to determine its metabolic potential for hydrogen and/or ethanol production. Initial characterization has shown the ability of C. termitidis to produce hydrogen, ethanol, and various other end-products on the two susbtrates. Continous N2 sparging in the pH-controlled bioreactors with cellobiose and α-cellulose showed a consistent increase in the H2 synthesis and lowered ethanol production compared to batch studies, with the H2 yields of 1.03 and 1.34 mol product per mol hexose equivalent added, respectively. Shotgun 2-D proteome analyses were performed to compare cellulose versus cellobiose grown cultures across exponential and stationary phases of growth. Most of the glycolytic proteins were detected in the proteome with some exceptions and no significant change was observed across both growth conditions. Hydrogen synthesis was regulatd via PFOR and ferredoxin-dependent hydrogenase, where as ethanol synthesis was regulated primarily via bifunctional AdhE activity. Proteomic analyses of C. termitidis cultured on hexose sugars in the absence of xylose suggested possible sequential utilization of xylose and cellobiose for the first time. Putative proteins consistent with xylose fermentation were observed at high levels. The hypothesis that C. termitidis can sequentially utilize xylose and cellobiose was further validated using batch fermentations tests on pure (xylose, cellobiose, xylan) and mixed substrates (xylose + cellobiose).

Clostridium thermocellum is a fermentative, Gram-positive, thermophile capable of cellulosome-mediated breakdown of hemicellulose and simultaneous biofuels (ethanol and H2) production, and is thus an excellent candidate for consolidated bioprocessing. However, ethanol and/or H2 production yields are below theoretical maxima due to branched product pathways. Biofuel yields may be improved by manipulation of fermentation conditions or implementation of rational metabolic engineering strategies. However, the latter relies on a thorough understanding of gene content, gene product expression, enzyme activity, and intracellular metabolite levels, which can all influence carbon and electron flux. The thesis work represents the first large-scale attempt in combining bioinformatic, enzymatic, proteomic, and culture perturbation approaches to systematically understand these interactions. C. thermocellum was used to investigate how these parameters affect end-product yields. Enzyme activities involved in conversion of pyruvate to end-products were consistent with end-product profiles and draft genome annotation. NADH and NADPH-dependent alcohol dehydrogenase (ADH) activities were comparable, whereas NADPH-dependent hydrogenase activities were higher than NADH and ferredoxin-dependent hydrogenase activities. While product yields changed in response to exogenous end-product additions, most core fermentative enzyme activities did not, suggesting that these changes may be governed by thermodynamics. The lack of major changes (>2-fold) in expression in response to growth and gas sparging was further confirmed by proteomics and RT-qPCR, respectively, although the latter revealed that ADH expression changes in response to gas sparging. Improved genome curation allowed refinement of metabolic pathways. A genomic and end-product meta-analysis of ethanol and/or H2 producing fermentative bacteria revealed that presence/absence of genes encoding hydrogenases and aldehyde dehydrogenases/ADHs had the greatest impacts on biofuel yields. However, genome content alone did not necessarily explain end-product yields. Given that genomic analysis of C. thermocellum revealed the presence of redundant genes encoding enzymes with analogous functions, shotgun and multiple reaction monitoring proteomics was used to refine which proteins are expressed. Absence/low expression of aldehyde dehydrogenase, ferredoxin-dependent hydrogenase and NADH:ferredoxin oxidoreductase suggest that these enzymes may not play a significant role in metabolism. An alternative electron flow pathway is proposed to explain end-product synthesis patterns in response to pyruvate addition or presence of protein inhibitors (CO, hypophosphite).

The Prairie Pothole Region (PPR) of Western Canada is characterized by productive cropland, grasslands, and millions of ‘potholes’ caused by receding glaciers. These potholes fill up with water and form wetlands habitat that is a rich and valuable ecosystem, and is one of the most productive waterfowl habitats in the world. However, the social benefits from wetland ecosystems are not paid to farmers, whose lands support wetlands, leading farmers in the PPR of Canada to drain wetlands. Wetlands habitat in the PPR is also threatened by climate change, due to potentially drier conditions, as well as biofuel policies that are aimed at mitigating climate change (which increase the value of grains relative to wetlands). This research is comprised of four empirical papers that study the optimal level of wetlands retention, as well as the effect of potential future climate change on wetlands. The methods employed include bioeconomic modeling, which maximizes an economic objective (utility of cropping, harvesting ducks) subject to biological constraints (wetlands and waterfowl retention), as well as positive mathematical programming to develop a land use model. In the first paper, a previous bioeconomic model of optimal duck harvest and wetland retention is updated and extended to include the nonmarket value of waterfowl and the ecosystem service and other amenity values of wetlands. Results indicate that wetlands and duck harvests need to be increased relative to historical levels. In the second paper, regression analysis is used to determine the casual effect of climate change on wetlands in the PPR. The model developed in the first paper is then adapted to solve the socially optimal levels of duck harvests and wetlands retention under current climate conditions and various climate change scenarios. Results indicate that the optimal number of wetlands to retain could decrease by as much as 38 percent from the baseline climate. In the third paper, the earlier bioeconomic model is extended to include cropping decisions. Further, the model is solved for disaggregated regions of the PPR. By including cropping decisions, this model can estimate the direct climate effects on wetlands and waterfowl management, as well as land use change due to biofuel policies. The model predicts that climate change will reduce wetlands by 35-56 percent from historic levels, with the majority of this change due to land use change. Wetlands loss is geographically heterogeneous, with losses being the largest in Saskatchewan. Finally, the fourth paper develops a multi-region Positive Mathematical Programming model that calibrates land use in the area to observed acreage in 2006. Policy simulations for both climate effects as well as the effects of biofuel policies determine how climate change will affect land use and wetlands. This model has the advantage of modeling the trade off between all major land uses in the area and is also solved on a region basis. Results indicate that climate change could decrease wetlands in this area by as much as 34 percent; the results are spatially heterogeneous. / Graduate

Naturally occurring microbial communities from high-salt and/or high-temperature environments were collected from sites across the United States and Puerto Rico and screened for their efficacy in the MixAlco biofuel production platform. The MixAlco process, based on the carboxylate platform, is a sustainable and economically viable platform for converting lignocellulosic biomass to biofuels. Using a mixed culture of anaerobic organisms, lignocellulosic biomass is fermented into carboxylic acids, which are neutralized to their corresponding carboxylate salts. These salts can then be converted into a wide variety of chemical products and fuels (alcohols, gasoline, diesel, jet fuel). The central hypothesis is that microbial communities from relatively extreme environments, having evolved to withstand selection pressures similar to the conditions in the carboxylate platform, will exhibit high rates of biomass conversion. A total of 559 soil communities was screened as inocula in established laboratory-scale fermentations. We used pyrotag sequencing of 16S rRNA genes to characterize the bacterial components of the best-performing microbial communities. The best performing communities converted up to 3 times more biomass to acids than a standard marine community inoculum. The community analyses have allowed us to determine the extent to which the same functional types are favored during fermentation, at both laboratory and demonstration plant scales. In all cases, we observed a shift from the more diverse sediment community to post-fermentation communities with relatively low diversity dominated by organisms in the phylum Firmicutes, specifically Bacilli and Clostridia classes. Despite the fact that the inoculum sources were both geographically and ecologically diverse, all of the post-fermentation communities were more similar to each other in community structure than to the corresponding original inoculum community. In addition, studies of the sediments used as inocula revealed that environmental parameters, such as pH and water content, were significantly correlated with bacterial community composition. The wealth of data provided by current sequencing technologies allowed us to question whether communities with high process performances tend to achieve that performance with similar community structures.

Lic.-avh. (sammanfattning) Umeå : Sveriges lantbruksuniversitet, 2009. / Härtill 2 uppsatser.