Caldicellulosiruptor is a genus of thermophilic, anaerobic, Gram-positive, non-spore-forming cellulolytic bacteria and is capable of fermenting a wide spectrum of carbohydrates. Both Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor kristjanssonii were found to be able to use the raw cellulosic material???switchgrass???for producing H2. Therefore, experiments were designed and conducted to select the optimal substrate and investigate the parameters involved in the fermentation process. C. saccharolyticus and C. kristjanssonii were cultivated on different sugars including glucose, xylose, cellobiose, xylan, avicel (PH105), and switchgrass. The highest H2 production was obtained on glucose by C. saccharolyticus with 2.9 mol (H2)/mol (glucose), whereas the yield by C. kristjanssonii was only 1.7 mol (H2)/mol (glucose). Moreover, the sample of C. saccharolyticus on xylose gained higher cell density, compared with glucose. During the cell growth, there was a small decrease of the H2/acetate ratio from 20 hour???s data to 40 hour???s along with the existence or significant increase of lactate. This suggested that a possible direction shift of metabolism happened in between the two time points, perhaps due to the H2 end product inhibition. A scanning electronic microscope (SEM) and confocal microscope were used to detect the adhesion between cells of these two microorganisms and cellulose and hemicellulose substrates in real time. A series of microscope pictures revealed non-specific and specific attachments between the cells and cellulosic materials, such as avicel and switchgrass, as well as the attachment behavior corresponding to the amount of H2 production. Proteomic analysis of C. saccharolyticus showed the existence of proteins related to mobility of the microbes such as flagella and chemotaxis. Other proteins found??? that contribute to the attachment between the cell and substrates, are the family 3 cellulose-binding module (CBM 3), the fibronectin-binding-A domain-containing proteins, the s-layer proteins, and a lysine motif protein. Groups of proteins like glycoside hydrolases (GHs), alcohol dehydrogenase (ADH) and hydrogenase that are responsible for the breakdown of cellulose and hemicellulose substrates, and the production of ethanol and H2, were also found in the proteome.

The objective was to create a feasibility framework for assessing the feasibility of a cellulosic ethanol refinery. In addition, the research aimed to create a base case scenario based on data from literature and conduct sensitivity analysis to determine significant parameters of a cellulosic ethanol refinery. The base case was found to be not feasible in the financial and economic analysis given the assumptions used.

The objective was to create a feasibility framework for assessing the feasibility of a cellulosic ethanol refinery. In addition, the research aimed to create a base case scenario based on data from literature and conduct sensitivity analysis to determine significant parameters of a cellulosic ethanol refinery. The base case was found to be not feasible in the financial and economic analysis given the assumptions used.

This thesis describes a way to achieve higher conversion rates of sugars from lignocellulosic biomass that can then be used for cellulosic ethanol production. Using oat hulls as the biomass, several chemical and physical pretreatment techniques were explored to overcome the recalcitrance and allow access to cellulose and hemicellulose. Experimentation with enzyme cocktails and dosing was done to obtain the highest conversions of cellulose and xylan to produce sugars. High solids-loading of the substrate, 14-16%, enabled higher conversion rates and would amount to lower cost of production in a commercial facility; however, end-product inhibition by the accumulation of inhibitors is also realized. To remove inhibition, a solid-liquid separation step was implemented which allowed enzymes to operate at a higher efficiency. The best combination of pretreatment and enzymatic hydrolysis led to a glucose of 89% and xylose yield of 84%, for trials conducted in a 20L bioreactor.

Thermotoga petrophila is a hyperthermophilic anaerobic bacterium that grows optimally at 80?? C. It can utilize plant biomass to produce biofuels, including ethanol and hydrogen, which are alternative and renewable sources of energy. Xylan, microcrystalline avicel PH105, switchgrass, corn husks and wheat straw were used as growth substrates to determine T. petrophila???s capability to use different types of plant biomass for the production of ethanol and hydrogen. The metabolism of cellulosic substrates was analyzed by integrating proteomics analysis, gene identification, cellulase and xylanase activities, growth, metabolic products and cell adhesion. T. petrophila showed best growth on xylan, followed by corn husks, switchgrass, avicel PH105 and wheat straw. The optimal pH for higher biofuel yield was within the range of 8.0 to 8.5. The metabolic end products were H2, CO2, acetate, lactate, formate and succinate when T. petrophila grew on all the tested cellulosic materials. The highest yield of hydrogen (9.6 mM) and the highest yield of ethanol (0.95 mM) were both detected when T. petrophila was grown on xylan. No growth was observed on xylose, which was not expected because T. petrophila grew very well on xylan, a ??-1, 4-xylopryranose polymer from which xylose can be produced upon hydrolysis. The possible reason for this phenomenon may be that T. petrophila has no specific sugar transporters for xylose, although it contains all the genes encoding xylose metabolizing enzymes. The majority of exoglucanases and endoglucanases presented in T. petrophila were extracellular enzymes. The highest specific activities of exoglucanase (1361.3 mU/mg) and endoglucanase (1032.1 mU/mg) in T. petrophila were found in the supernatants of the growth culture with xylan as the sole substrate, indicating that xylan, not cellulose, is the best inducer to increase the expression of extracellular cellulases. Compared to the huge discrepancy of extracellular cellulases activities among different substrates (from 0 to 1361.3 mU/mg), intracellular cellulase (endoglucanase) activity was relatively steady (around 150 mU/mg). Xylanase activity was also detected in both the supernatant and the cell free extract; thus T. petrophila contains both extracellular and intracellular xylanase. The highest xylanase activity was detected in the cell free extract when T. petrophila was grown on cellobiose and xylan (3732.4 mU/mg and 3152.8 mU/mg, respectively), indicating that the majority of xylanase is an intracellular enzyme, and xylan and cellobiose are the best inducers to increase the expression of xylanase. Adhesion of T. petrophila cells to xylan, with many filaments connecting all the cells, was observed using scanning electron microscope and fluorescent staining microscope, whereas there was no attachment between cells and cellulose. This difference may explain why T. petrophila grew much better on xylan than on cellulose because cell adhesion increases enzyme concentration near the substrate to improve the efficiency of cellulosic material utilization. Furthermore, proteomics analysis was used to quantify all the expressed proteins in different growth media with various substrates. The proteomics data revealed that the most important enzymes for cellulose and hemicellulose utilization were ATP binding cassette (ABC) transporters, S-layer proteins and membrane binding proteins, which were up-regulated when T. petrophila was grown on cellulosic materials. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) also indicated the up-regulated proteins from the media with cellulosic substrates were probably ABC transporters and S-layer proteins based on the size of proteins. Based on the gene identification, end product determination and proteomics analysis, the tentative cellulosic material metabolic pathways in T. petrophila were completely profiled. Overall, our results suggest that the ability of T. petrophila to convert cellulosic materials into hydrogen and ethanol exceeds T. maritima, which is a model strain for studying hyperthermophiles. T. petrophila has great potential in applications of producing highly thermostable cellulases and biofuels from cellulosic materials. However, the mechanism of cell adhesion between T. petrophila and xylan and the regulation of the entire cellulosic materials metabolic pathway need further investigation.

This thesis describes a way to achieve higher conversion rates of sugars from lignocellulosic biomass that can then be used for cellulosic ethanol production. Using oat hulls as the biomass, several chemical and physical pretreatment techniques were explored to overcome the recalcitrance and allow access to cellulose and hemicellulose. Experimentation with enzyme cocktails and dosing was done to obtain the highest conversions of cellulose and xylan to produce sugars. High solids-loading of the substrate, 14-16%, enabled higher conversion rates and would amount to lower cost of production in a commercial facility; however, end-product inhibition by the accumulation of inhibitors is also realized. To remove inhibition, a solid-liquid separation step was implemented which allowed enzymes to operate at a higher efficiency. The best combination of pretreatment and enzymatic hydrolysis led to a glucose of 89% and xylose yield of 84%, for trials conducted in a 20L bioreactor.

The objective of this research was to study the catalytic graphitization of cellulose fibers coated with iron oxide nanoparticles. Bleached cellulose fibers and iron oxide nanoparticles coated cellulose fibers were pyrolyzed at five elevated temperatures. The crystallographic structures of carbon-encapsulated iron oxide nanoparticles were then investigated by the following techniques: Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD), Raman Spectroscopy, Transmission Electron Microscopy (TEM), and Selected-Area Electron Diffraction (SAED). The graphitization of cellulosic fibers was enhanced by the presence of iron oxide nanoparticles. Moreover, iron oxide nanoparticles deposited on cellulosic fiber samples pyrolyzed above 800°C produced graphitic structures. TEM and XRD were performed to identify and characterize the phase transitions of carbon-encapsulated iron oxide nanoparticles after pyrolysis treatment at four temperatures: 500°C, 800°C, 1000°C, and 1600°C. TEM of samples pyrolyzed at or above 800°C showed resulting units were core-shell structures consisting of dark grains and a light matrix with graphitic structure.

Many recent studies on the relative costs and benefits of biofuels have raised the need for a detailed and rigorous analysis of the operations of a biorefinery that is focused on optimization. The current thesis concentrates on the design and optimization of plants for producing biodiesel and ethanol from cellulosic biomass. We have performed numerical simulations combined with systematic parametric analyses to investigate the effect of various parameters on the overall material and energy balances of each biorefinery. The efficiency of the simulated processes was investigated by introducing and/or estimating various metrics in order to select the more beneficial directions for process improvements. Particular emphasis has been paid on heat integration and the design of highly efficient combined heat and power (CHP) units that generate the steam and electricity needed for the purification of biofuels and their co-products. The first part of the thesis is focused on biodiesel production via transesterification of soybean oil with methanol, under alkali-catalyzed conditions. We have analyzed the performance of several reactor configurations in order to improve the conversion of the reversible transesterification reactions. The effect of the oil to alcohol ratio has also been extensively explored. Furthermore, the energy requirements of the simulated process have been rigorously calculated. Since biodiesel facilities can be used either for small-scale, distributed applications or for large-scale production, we have explored whether it is more energy efficient to burn the glycerol-rich stream in a combined heat and power (CHP) plant, or purify the glycerol and use it a feedstock for producing higher-value chemicals with further biotechnological processes. The second part of the thesis focuses on the production of cellulosic ethanol. Having developed the process model, a detailed parametric analysis was carried out to determine how the energy balances and overall efficiency of the biorefinery were influenced by changes in (a) the composition of the biomass feedstock, and (b) the conversion levels of the hydrolysis and fermentation stages. Furthermore, the requirements of the utility section of the ethanol plant were calculated. The utility section included a combined heat and power unit where by-product streams of the production process were utilized for energy generation. The parametric analysis indicated that these streams were in most cases an insufficient fuel source for meeting the energy requirements of the plant and thus, additional fuel was required (biomass, coal, or natural gas). The calculations of this section indicated a significant trade-off between ethanol production and external energy inputs, thus casting some doubt on the ultimate effectiveness of efforts to develop genetically modified energy crops (with high carbohydrate content) in order to maximize fuel production.

Farmers will supply the raw ingredients for the emerging cellulosic ethanol industry. The long-term relationship between a farmer and a processing firm is expected to be contractual. A processing firm has an incentive to sign long-term contracts to ensure a cost-efficient level of raw ingredient supply. However, farmers generally prefer to operate with either no contract or a short-term contract in order to maintain options for adjustments in future acreage allocations due to changes in relative prices. Of interest in this research is to understand the incentives of farmers and calculating the efficient level of the “inflexibility premium”, which a processing firm must provide to a farmer when a long term contract is signed. A stochastic dynamic programming model is solved and with the help of Microsoft Excel numerically evaluated to illustrate the marginal inflexibility premium is increasing with contract length and the level of price variability, and is decreasing with the size of acreage adjustment costs.

Farmers will supply the raw ingredients for the emerging cellulosic ethanol industry. The long-term relationship between a farmer and a processing firm is expected to be contractual. A processing firm has an incentive to sign long-term contracts to ensure a cost-efficient level of raw ingredient supply. However, farmers generally prefer to operate with either no contract or a short-term contract in order to maintain options for adjustments in future acreage allocations due to changes in relative prices. Of interest in this research is to understand the incentives of farmers and calculating the efficient level of the “inflexibility premium”, which a processing firm must provide to a farmer when a long term contract is signed. A stochastic dynamic programming model is solved and with the help of Microsoft Excel numerically evaluated to illustrate the marginal inflexibility premium is increasing with contract length and the level of price variability, and is decreasing with the size of acreage adjustment costs.