The objective of this research was to investigate the influence of physicochemical properties of lignocellulosic biomass on the rate and extent of cellulose conversion, i.e. the "hydrolyzability" of the substrate. The initial two parts of this thesis investigated the interactions between various components of pretreated biomass. The relevance of cellulase partitioning between the cellulose and non-cellulose components of pretreated switchgrass was determined. Furthermore, a method was developed to assess the changes in surface area of the non-cellulose fraction during saccharification. The overall conclusion from these studies was that for an optimally pretreated switchgrass sample the cellulose and non-cellulose components are fully disassociated and the non-cellulose fraction does not appear to interfere with cellulose conversion. The third study determined the changes in physicochemical properties of native and pretreated poplar during simultaneous saccharification and fermentation (SSF). While this study gave an unprecedented insight into the dynamics of SSF, it was also apparent that parameters measuring the complete feedstock are inadequate for explaining cellulose reactivity/hydrolyzability during saccharification. As a consequence of these conclusions, the final study focused on properties of model celluloses and initial rates of hydrolysis by the major cellulase - CBHI. The content of insoluble ends was measured by different modified soluble reducing sugar assays and by tritium labeling through NaB³H₄ reduction reactions. The content of insoluble reducing ends partially explained the relative initial rates of hydrolysis. However, comparisons between celluloses in their crystalline and corresponding amorphous state revealed that crystallinity also accounts for some of the observed rate differences. Furthermore, surface characteristics, such as the presence of pores, must be considered to fully explain cellulose reactivity. / Graduation date: 1999

Lignocellulosic biomass represents a vast supply of fermentable carbohydrates and functional aromatic compounds. Conversion of lignocellulosics to ethanol and other useful products would be of widespread economical and environmental benefit. Better understanding of the behavior of different lignicellulosic feedstocks in fermentation protocols as well as catalytic activities involved in lignocellulosic depolymerization will further enhance the commercial viability of biomass-to-ethanol conversion processes. The relative toxicity of the combined non-xylose components in prehydrolysates derived from three different lignocellulosic biomass feedstocks (poplar, corn stover and switchgrass, or Panicum virgatum L.) was determined using a Pichia stipits fermentation assay. The relative toxicity of the prehydrolysates, in decreasing order, was poplar-derived prehydrolysates > switchgrass-derived prehydrolysates > corn stover-derived prehydrolysates. Ethanol yields averaged 74%, 83% and 88% of control values for poplar, switchgrass and corn stover prehydrolysates, respectively. Volumetric ethanol productivities (g ethanol l⁻¹ h⁻¹) averaged 32%, 70% and 102% of control values for poplar, switchgrass and corn stover prehydrolysates, respectively. Ethanol productivities correlated closely with acetate concentrations in the prehydrolysates; however, regression lines correlating acetate concentrations and ethanol productivities were found to be feedstock-dependent. Differences in the relative toxicity of xylose-rich prehydrolysates derived from woody and herbaceous feedstocks are likely due to the relative abundance of a variety of inhibitory compounds, e.g. acetate and aromatic comounds. Fourteen aromatic monomers present in prehydrolysates prepared from corn stover, switchgrass, and poplar were tentatively identified by comparison with published mass spectra. The concentrations of the aromatic monomers totaled 112, 141 and 247 mg(l)⁻¹ for corn stover, switchgrass and poplar prehydrolysates, respectively. The woody and herbaceous feedstocks differed in both amount and type of aromatic monomers. The cellulases of Trichoderma reesei are the most widely studied for use in the depolymerization of lignocellulosics. The Trichoderma cellobiohydrolases CBH1 and CBH2 are traditionally categorized as exo-acting cellulases. A simple individual-based model was created to explore the potential effects of native endo activity on substratevelocity profiles. The model results indicate that an enzyme with a small amount of endo activity will show an apparent substrate inhibition as substrate levels are increased. Actual hydrolysis studies using affinity chromatography-purified CBH2 preparations from three laboratories indicate that CBH2 has native endo activity, while CBH1 does not. / Graduation date: 1999

The biological degradation of lignocellulose into fermentable sugars for the production of liquid transportation fuels is feasible and sustainable, but equires a variety of enzymes working in synergy as lignocellulose is a complex and recalcitrant substrate. The cellulosome is a multi-enzyme complex (MEC) with a variety of cellulolytic and hemicellulolytic enzymes that appears to facilitate an enhanced synergy and efficiency, as compared to free enzymes, for the degradation of recalcitrant substrates such as lignocellulose and plant cell walls. Most of the studies on cellulosomes have focused on a few organisms; C. thermocellum, C. cellulovorans and C. cellulolyticum, and there is only limited knowledge vailable on similar complexes in other organisms. Some MECs have been identified in aerobic bacteria such as Bacillus circulans and Paenibacillus curdlanolyticus, but the nature of these MECs have not been fully elucidated. This study investigated the cellulolytic and emi-cellulolytic system of Bacillus licheniformis SVD1 with specific reference to the presence of a MEC, which has never been reported in the literature for B. licheniformis. A MEC of approximately 2,000 kDa in size, based on size exclusion chromatography using Sepharose 4B, was purified from a culture of B. licheniformis. When investigating the presence of enzyme activity in the total crude fraction as well as the MEC of a birchwood xylan culture, B. licheniformis was found to display a variety of enzyme activities on a range of substrates, although xylanases were by far the predominant enzyme activity present in both the crude and MEC fractions. Based on zymogram analysis there were three CMCases, seven xylanases, three mannanases and two pectinases in the crude fraction, while the MEC had two CMCases, seven xylanases, two mannanases and one pectinase. The pectinases in the crude could be identified as a pectin methyl esterase and a lyase, while the methyl esterase was absent in the MEC. Seventeen protein species could be detected in the MEC but only nine of these displayed activity on the substrates tested. The possible presence of a β-xylosidase in the crude fraction was deduced from thin layer chromatography (TLC) which demonstrated the production of xylose by the crude fraction. It was furthermore established that B. licheniformis SVD1 was able to regulate levels of enzyme expression based on the substrate the organism was cultured on. It was found that complexed xylanase activity had a pH optimum of between pH 6.0 and 7.0 and a temperature optimum of 55oC. Complexed xylanase activity was found to be slightly inhibited by CaCl2 and inhibited to a greater extent by EDTA. Complexed xylanase activity was further shown to be activated in the presence of xylose and xylobiose, both compounds which are products of enzymatic degradation. Ethanol was found to inhibit complexed xylanase activity. The kinetic parameters for complexed xylanase activity were measured and the Km value was calculated as 2.84 mg/ml while the maximal velocity (Vmax) was calculated as 0.146 U (μmol/min/ml). Binding studies, transmission electron microscopy (TEM) and a bioinformatic analysis was conducted to investigate whether the MEC in B. licheniformis SVD1 was a putative cellulosome. The MEC was found to be unable to bind to Avicel, but was able to bind to insoluble birchwood xylan, indicating the absence of a CBM3a domain common to cellulosomal scaffoldin proteins. TEM micrographs revealed the presence of cell surface structures on cells of B. licheniformis SVD1 cultured on cellobiose and birchwood xylan. However, it could not be established whether these cell surface structures could be ascribed to the presence of the MECs on the cell surface. Bioinformatic analysis was conducted on the available genome sequence of a different strain of B. licheniformis, namely DSM 13 and ATCC 14580. No sequence homology was found with cohesin and dockerin sequences from various cellulosomal species, indicating that these strains most likely do not encode for a cellulosome. This study described and characterised a MEC that was a functional enzyme complex and did not appear to be a mere aggregation of proteins. It displayed a variety of hemi-cellulolytic activities and the available evidence suggests that it is not a cellulosome, but should rather be termed a xylanosome. Further investigation should be carried out to determine the structural basis of this MEC.

Lignocellulosic materials (LM) are an ever present renewable and available energy source. The energy stored by photosynthesis in the form of vegetation is about ten times more than world's annual energy consumption (Zsuffa, 1982). This source is the only alternative for chemical production after fossil fuels. Formation of organic acids (mainly acetic acid) from hemicellulose during steam treatment of LM leads to acid hydrolysis of cell wall components. Solubilization of hemicellulose and depolymerization of lignin are the most important changes that occur during the process. During hydrolysis of LM appreciable amounts of sugar degradation products, organic acids and phenolics are produced. Inhibitory effects of the compounds on yeast during alcoholic fermentation have been reported and several methods have been proposed to overcome the problem. Among the new compounds phenolics derived from lignin depolymerization have received most attention. Another problem during enzymic saccharification of cellulose is partial inactivation of cell free enzymes. The above mentioned constraints were investigated in this study.

The research described in this thesis was aimed at understanding how particle size, porosity, and enzyme accessible surface area influence the rate of saccharification of microcrystalline cellulose. Microcrystalline cellulose (MCC) is a commonly used substrate for the study of cellulolytic enzymes. MCC preparations of different particle size are commercially available. In this study, MCC preparations having average particle sizes of 20, 50, and 90 μm were analyzed with respect to their enzyme accessible surface area, chemical and physical properties and rates of enzymatic saccharification. Saccharification studies were done using a commercially available cellulase preparation from Trichoderma reesei. Pore volume distributions were determined from solute exclusion experiments. Internal surface areas were calculated based on the application of the lamellae model to the pore volume distribution data. External surface areas were calculated based on the average particle size of each MCC preparation assuming that the particles could be represented as solid spheres. The different MCC preparations were found to have nearly equivalent enzyme accessible surface areas per unit weight. Greater than 99 % of the total enzyme accessible surface area for each MCC preparations was found to be within the porous structure of the particles. Enzymatic saccharification experiments demonstrated that the smaller particle size MCCs were more readily digested than those of larger particle size. The similarity of the three MCC preparations with respect to chemical and physical properties (other than particle size), pore volume distribution, and total enzyme accessible surface area suggests that a rate limiting factor in the enzymatic digestion of MCC is a resistance attributable to diffusion within the capillary network of these insoluble substrates. / Graduation date: 1996

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1990. / Vita. Abstract. Includes bibliographical references (leaves 163-174). Also available via the Internet.

Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains xii, 173 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 138-142).

Lignocellulose is one of the most abundant carbon sources in nature. This naturally-occuring substance is an underutilized source of bioenergy. A major bottleneck in biofuel processing is the enzymatic hydrolysis of lignocellulose into its ultimate fermentable product, glucose. Cellulomonas fimi is a well-studied soil organism known for its capabilities to efficiently hydrolyze cellulose. Recently sequenced genomes of Cellulomonas fimi and Cellulomonas flavigena have allowed analysis to reveal previously unidentified cellulases from several glycoside hydrolase (GH) families. This study also includes the expression of secreted cellulases from families GH 5, 6, and 9 at the protein level by the native organism after growth in media supplemented with carboxymethylcellulose or soluble xylan. In order to find enzymes with novel qualities, the cloning and expression of these newly identified cellulases from C. fimi and C. flavigena were done. One of these enzymes is Celf_1230 (Cel6C), a putative cellobiohydrolase from the glycoside hydrolase family 6. Using substituted cellulose derivatives as substrates, we have characterized Celf_1230 to be a thermostable enzyme with endoglucanase activity.

No description available.

Lignocellulose is one of the most abundant carbon sources in nature. This naturally-occuring substance is an underutilized source of bioenergy. A major bottleneck in biofuel processing is the enzymatic hydrolysis of lignocellulose into its ultimate fermentable product, glucose. Cellulomonas fimi is a well-studied soil organism known for its capabilities to efficiently hydrolyze cellulose. Recently sequenced genomes of Cellulomonas fimi and Cellulomonas flavigena have allowed analysis to reveal previously unidentified cellulases from several glycoside hydrolase (GH) families. This study also includes the expression of secreted cellulases from families GH 5, 6, and 9 at the protein level by the native organism after growth in media supplemented with carboxymethylcellulose or soluble xylan. In order to find enzymes with novel qualities, the cloning and expression of these newly identified cellulases from C. fimi and C. flavigena were done. One of these enzymes is Celf_1230 (Cel6C), a putative cellobiohydrolase from the glycoside hydrolase family 6. Using substituted cellulose derivatives as substrates, we have characterized Celf_1230 to be a thermostable enzyme with endoglucanase activity.