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Currently, one of the hurdles hindering efficient production of cellulosic biofuel is the recalcitrant nature of cellulose to hydrolysis. A wide variety of cellulase enzymes are found natively in microorganisms that can potentially be used to effectively hydrolyze cellulose to fermentable sugars. Thermobifida fusca is a high G-C content, thermophilic, gram-positive soil actinobacterium with high cellulolytic activity. The phenomenological and mechanistic parameters affecting cellulase activity were studied in T. fusca and two mechanisms have been found: 1) transcriptions of cellulase-related genes were not closely associated with measured differences in cellulase activity and 2) cellular energetics (intracellular ATP) correlated more closely to changes in specific cellulase activity. In T. fusca, CelR is thought to act as the primary regulator of cellulase gene expression by binding to a 14-bp inverted repeat: 5’-(T)GGGAGCGCTCCC(A) that is upstream of many known cellulase genes. An efficient procedure for creating precise chromosomal gene replacements has been developed and this procedure was demonstrated by generating a celR deletion strain. Measurements of mRNA transcript levels in both the celR deletion strain and the wild-type strain indicated that the CelR potentially acts as a repressor for some cellulase genes and as an activator for other cellulase genes. Based on the protocol of disrupting celR gene, the direct conversion of untreated cellulosic biomass to 1-propanol in aerobic growth conditions using an engineered strain of T. fusca was demonstrated. Based upon computational predictions, a bifunctional butyraldehyde/alcohol dehydrogenase (encoded by adhE2) was added to T. fusca leading to production of 1-propanol during growth on glucose, cellobiose, cellulose (Avicel), switchgrass, and corn stover. The highest 1-propanol titer (0.48 g/L) was achieved for growth on switchgrass. The adaptive evolution of T. fusca was conducted to find a high cellulase-yield strain. The evolved strains of T. fusca were generated for two different scenarios: continuous exposure to cellobiose (strain muC with specialist phenotype) or alternating exposure to cellobiose and glucose (strain muS with generalist phenotype). Characterization of cellular phenotypes and whole genome re-sequencing were conducted for both the muC and muS strains and 18 and 14 point mutations in the muC and muS strains, respectively were verified. Among these mutations, the site mutation of Tfu_1867 was found to contribute the specialist phenotype and the site mutation of Tfu_0423 was found to contribute the generalist phenotype. The experiment results were used to test genome-scale metabolic model of T. fusca built in this study.

Shaojun Ding. / "October 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, / Includes bibliographical references (p. 173-200). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

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In this research, the interfacial behavior of Trichoderma reesei CBHI and Thermomonospora fusca E��� cellulases were studied at synthetic surfaces. For this purpose, colloidal silica and polystyrene particles were used to prepare cellulase-particle suspensions that were analyzed by several solution-phase techniques. These included circular dichroism spectroscopy, size exclusion chromatography and filtration, and a spectrophotometric assay for cellulase activity. All techniques were performed in the presence and absence of particles. Circular dichroism spectroscopy (CD) and size exclusion chromatography showed, however, that binding did not occur between either cellulase and silica, presumably because silica is hydrophilic and negatively charged. Binding did occur between each cellulase and polystyrene, most likely mediated through hydrophobic associations. Cellulase-polystyrene complexes were not analyzed using CD because of high light absorption by the polystyrene nanoparticles. Upon adsorption to polystyrene, the activity of the E��� dropped about 95% relative to that of the free enzyme. While this substantial loss in activity may have been the result of binding being mediated through the catalytic domain, strong evidence supporting the thought that adsorption occurs through hydrophobic associations, mediated through the binding domain, suggests that structural or steric factors were partly responsible for the loss. / Graduation date: 1999

Renewable energy sources, such as lignocellulosic biomass, are environmentally friendly because they emit less pollution without contributing net carbon dioxide to the atmosphere. Among lignocellulosic biomass, corn stover is a very useful feedstock to economically produce environmentally friendly biofuels. Corn stover was pretreated with an excess of calcium hydroxide (0.5 g Ca(OH)2/g raw biomass) in non-oxidative and oxidative conditions at 25, 35, 45, and 55oC. The optimal condition is 55oC for 4 weeks with aeration, determined by yields of glucan and xylan. The overall yields of glucose (g glucan hydrolyzed/100 g original glucan) and xylose (g xylan hydrolyzed/100 g original xylan) were 91.3 and 51.8 at 15 FPU/g cellulose, respectively. Furthermore, when considering the dissolved fragments of glucan and xylan in the pretreatment liquors, the overall yields of glucose and xylose were 93.2 and 79.5 at 15 FPU/g cellulose, respectively. The pretreatment liquor has no inhibitory effect on ethanol fermentation using Saccharomyces cerevisiae D5A. At the recommended condition, only 0.073 g Ca(OH)2 was consumed per g of raw corn stover. Under extensive delignification conditions, 87.5% of the initial lignin was removed. Extensive delignfication required oxidative treatment and additional lime consumption. Deacetylation quickly reached a plateau within 1 week. Delignification highly depended on temperature and the presence of oxygen. Lignin and hemicellulose were selectively removed, but cellulose was not affected by lime pretreatment in mild temperatures (25 ?? 55oC). The delignification kinetic models of corn stover were empirically determined by three simultaneous first-order reactions. The activation energies for the oxidative delignification were estimated as 50.15 and 54.21 kJ/mol in the bulk and residual phases, respectively. Crystallinity slightly increased with delignification because amorphous components (lignin, hemicellulose) were removed. However, the increased crystallinity did not negatively affect the 3-d sugar yield of enzyme hydrolysis. Oxidative lime pretreatment lowered the acetyl and lignin contents to obtain high digestibility, regardless of crystallinity. The enzymatic digestibility of lime-treated biomass was affected by the change of structural features (acetylation, lignification, and crystallization) resulting from the treatment. The non-linear models for 3-d hydrolysis yields of glucan and xylan were empirically established as a function of the residual lignin fraction for the corn stover pretreated with lime and air.

The efficiency of cellulose hydrolysis under straight saccharification and simultaneous saccharification and fermentation (SSF) conditions was evaluated using three lignocellulosic materials (switchgrass, cornstover, and poplar), which had been pretreated with dilute sulfuric acid under conditions which optimized xylose concentrations in the prehydrolysate liquid. Yields of glucose, cellobiose and ethanol obtained from the pretreated feedstocks were measured over 168 hrs. The final theoretical conversions of cellulose from pretreated switchgrass, cornstover, and poplar in straight saccharification were 85-100% (average 94%), 84-100% (average 96%), and 75-100% (average 87%), respectively, while in SSF the conversions were 84-90% (average 87%), 91-96% (average 90%), 72%-82% (average 76%), respectively. The conversion rates of poplar in straight saccharification and SSF were significantly lower than those of switchgrass and cornstover. The effects of reaction parameters such as enzyme activity, cellulose availability, and yeast cell viability on the extent of hydrolysis in straight saccharification and SSF were also studied. Results indicate that the lower glucose or ethanol yields associated with some of the poplar were due to the recalcitrant nature of its cellulose. To compare accurately the efficiencies between straight saccharification and SSF, a direct method for determining the cellulose content of the feedstocks residues resulting from SSF experiments has been developed and evaluated. The method improves on classical cellulose assays by incorporating a yeast lysing enzyme to remove yeast glucans from the feedstocks residue prior to acid hydrolysis and subsequent quantification of cellulose derived glucose. A freeze-drying step was identified as necessary to render the SSF yeast cells susceptible to enzyme lysis. The method was applied to the analysis of the cellulose and yeast-glucan content of SSF residues from the three pretreated feedstocks. Cellulose assays employing the lysing enzyme preparation demonstrated relative errors up to 7.2% when yeast-associated glucan were not removed prior to analysis of SSF residues. Enzymatic lysis of SSF yeast cells may be viewed as a general preparatory procedure to be used prior to the subsequent chemical and physical analysis of SSF residues. / Graduation date: 1996

Modified cellulase enzyme assay methods were developed to determine the potential of using enzyme activities to evaluate the decomposition of organic matter in carbon amended mine tailings systems. Cellulase assays are commonly performed in soil science applications, industrial production and bio-energy research to determine organic matter response to physical, chemical or temporal variation but, they have not been applied in a mine waste environment. Heavy metal content is considered to be a potential inhibitor to cellulase enzyme activity. Using samples collected from Greens Creek Mine in Juneau, Alaska, USA, the modified assay was evaluated to develop a method that provided reproducible results. It was determined that a sample mass of three grams is sufficient to provide consistent enzyme measurements. Each sample location was characterized by four replicates to ensure statistically representative data. Matrix interferences were determined to be inconsequential in this system. Despite the low organic content amended to the tailings, heavy metal content and potentially low enzyme activity, the modified enzyme assay method provided reproducible enzyme measurements. Following the development of the cellulase assays, spatial and temporal variations in cellulase enzymes were investigated from carbon amended tailings samples collected at the Greens Creek Mine. Six test cells, containing a mixture of tailings and a combination of peat, spent brewery grain and/or municipal biosolids were sampled in the fall of 2005, 2007 and 2009. Exo-(1,4)-β-D-glucanase (EC 3.2.1.91), Endo-(1,4)-β-D-glucanase (EC 3.2.1.4) and β-glucosidase (EC 3.2.1.21) enzymes were assayed from core sections at five different depths. Enzyme activities were compared to sulfate reducing and acid producing bacterial enumerations, sulfide trends and carbon content. General trends were consistent between enzyme activity and SRB enumerations. The range of total carbon values fell between 3 and 5 wt % in each test cell while the average inorganic carbon content was 3.5 wt %. The range of organic carbon content was between 0.2 and 1.2 wt %. Total, inorganic and organic carbon values were more characteristic of test cell carbon distribution. Cellulase enzyme assays provide valuable information regarding the degradation of cellulose and hemi-cellulose. This study demonstrates that enzymes can be monitored in a tailings environment and that enzyme assays conducted for monitoring purposes may be a useful practice to indicate the sustained or declining performance of organic matter in a carbon amended remedial system.

Cellulose is the most abundant organic compound on the planet, and is found in nearly every ecosystem. Cellulose is also the most abundant waste product produced by human activity. These enormous stores of natural cellulose and cellulose-containing wastes are a potential renewable energy source. The hydrolysis of cellulose is referred to as cellulolysis and is carried out by cellulase enzymes, which are members of certain glycoside hydrolase families. For most of its history, the microbiology of organisms like those that hydrolyze cellulose was based solely on the testing of physiological and biochemical behaviour of isolated organisms in pure cultures. Despite having gained an important foundation of knowledge in the characterization of microorganisms, cultivation-based techniques introduce major bias into understanding the role that specific microorganisms play because the majority of microorganisms are not readily cultured. Two of the most powerful culture-independent approaches for accessing microbial communities are DNA stable-isotope probing (DNA-SIP) and metagenomics. Though each methodology has been used on its own, it is a combination of these two approaches that has enormous potential to generate results for industrial applications and to help characterize biogeochemical cycling. This thesis presents the first research combining DNA-SIP and metagenomics using cellulose, and the first to target glycoside hydrolase genes from Arctic tundra. For this research, two-month DNA-SIP incubations were carried out with 200 mg of 13C-labelled cellulose as a substrate. Denaturing gradient gel electrophoresis (DGGE) provided evidence indicating the successful enrichment of microorganisms able to metabolize cellulose. Multiple displacement amplification (MDA) was applied to both the bulk-soil samples and DNA-SIP samples. Following MDA, all DNA samples were subjected to Illumina sequencing, including DNA from a cellulose-degrading enrichment. Functional annotation for each Illumina library was done using the SwissProt database within MG-RAST. The DNA-SIP enrichment resulted in a ~3 fold increase in the relative abundance of glycoside hydrolases and cellulase enzymes in relation to bulk soil samples. A cellulose degrading enrichment contained the highest relative abundance of glycoside hydrolases and cellulase enzymes, with a five fold increase relative to the DNA-SIP enrichment. The enrichment culture had a much lower relative diversity, which was measured using the Shannon Index. An unrooted neighbor-joining tree constructed using Bray-Curtis similarity coefficients for each sample demonstrated that as a result of a considerably higher proportion of cellulase gene sequences and a lower diversity the enrichment culture was the most distinct library, with the DNA-SIP library most closely related to it. DGGE provided initial evidence that MDA introduced bias into the amplification of DNA from the DNA-SIP sample. This was confirmed following sequencing and annotation as the proportion of glycoside hydrolase enzymes sequences decreased 67% following MDA of DNA-SIP enriched DNA and the mean G+ C content of libraries decreased. This research provides evidence indicating that DNA-SIP enrichment prior to the construction of metagenomic libraries increases the abundance of targeted gene sequences, which should enable greater access to functional genes of active microorganisms for potential industrial applications.

Renewable energy sources, such as lignocellulosic biomass, are environmentally friendly because they emit less pollution without contributing net carbon dioxide to the atmosphere. Among lignocellulosic biomass, corn stover is a very useful feedstock to economically produce environmentally friendly biofuels. Corn stover was pretreated with an excess of calcium hydroxide (0.5 g Ca(OH)2/g raw biomass) in non-oxidative and oxidative conditions at 25, 35, 45, and 55oC. The optimal condition is 55oC for 4 weeks with aeration, determined by yields of glucan and xylan. The overall yields of glucose (g glucan hydrolyzed/100 g original glucan) and xylose (g xylan hydrolyzed/100 g original xylan) were 91.3 and 51.8 at 15 FPU/g cellulose, respectively. Furthermore, when considering the dissolved fragments of glucan and xylan in the pretreatment liquors, the overall yields of glucose and xylose were 93.2 and 79.5 at 15 FPU/g cellulose, respectively. The pretreatment liquor has no inhibitory effect on ethanol fermentation using Saccharomyces cerevisiae D5A. At the recommended condition, only 0.073 g Ca(OH)2 was consumed per g of raw corn stover. Under extensive delignification conditions, 87.5% of the initial lignin was removed. Extensive delignfication required oxidative treatment and additional lime consumption. Deacetylation quickly reached a plateau within 1 week. Delignification highly depended on temperature and the presence of oxygen. Lignin and hemicellulose were selectively removed, but cellulose was not affected by lime pretreatment in mild temperatures (25 ?? 55oC). The delignification kinetic models of corn stover were empirically determined by three simultaneous first-order reactions. The activation energies for the oxidative delignification were estimated as 50.15 and 54.21 kJ/mol in the bulk and residual phases, respectively. Crystallinity slightly increased with delignification because amorphous components (lignin, hemicellulose) were removed. However, the increased crystallinity did not negatively affect the 3-d sugar yield of enzyme hydrolysis. Oxidative lime pretreatment lowered the acetyl and lignin contents to obtain high digestibility, regardless of crystallinity. The enzymatic digestibility of lime-treated biomass was affected by the change of structural features (acetylation, lignification, and crystallization) resulting from the treatment. The non-linear models for 3-d hydrolysis yields of glucan and xylan were empirically established as a function of the residual lignin fraction for the corn stover pretreated with lime and air.