Lignocellulosic biomass is one of the most valuable alternative energy sources because it is renewable, widely available, and environmentally friendly. Unfortunately, enzymatic hydrolysis of biomass has been shown to be a limiting factor in the conversion of biomass to chemicals and fuels. This limitation is due to inherent structural features (i.e., acetyl content, lignin content, crystallinity, surface area, particle size, and pore volume) of biomass. These structural features are barriers that prevent complete hydrolysis; therefore, pretreatment techniques are necessary to render biomass highly digestible. The ability to predict the biomass reactivity based solely on its structural features would be of monumental importance. Unfortunately, no study to date can predict with certainty the digestibility of pretreated biomass. A concerted effort with Auburn University and Michigan State University has been undertaken to study hydrolysis mechanisms on a fundamental level. Predicting enzymatic hydrolysis based solely on structural features (lignin content, acetyl content, and crystallinity index) would be a major breakthrough in understanding enzymatic digestibility. It was proposed to develop a fundamental understanding of the structural features that affect the enzymatic reactivity of biomass. The effects of acetyl content, crystallinity index (CrI), and lignin content on the digestibility of biomass (i.e., poplar wood, bagasse, corn stover, and rice straw) were explored. In this fundamental study, 147 poplar wood model samples with a broad spectrum of acetyl content, CrI, and lignin were subjected to enzymatic hydrolysis to determine digestibility. Correlations between acetyl, lignin, and CrI and linear hydrolysis profiles were developed with a neural network model in Matlab®. The average difference between experimentally measured and network-predicted data were ±12%, ±18%, and ±27% for 1-, 6-, and 72-h total sugar conversions, respectively. The neural network models that included cellulose crystallinity as an independent variable performed better compared to networks with biomass crystallinity, thereby indicating that cellulose crystallinity is more effective at predicting enzymatic hydrolysis than biomass crystallinity. Additionally, including glucan slope in the 6-h and 72-h xylan slope networks and glucan intercept in the 6-h and 72-h xylan intercept networks improved their predictive ability, thereby suggesting glucan removal affects later-stage xylan digestibility.

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Currently biomass is considered to be one of the main options to substitute the fossil fuels. Nevertheless, bioenergy is not the only alternative use for organic waste materials. In recent years, the utilisation of cellulolytic waste from industries, such as the paper industry and agriculture (in fields and in processing plants), is considered to be a good source of renewable carbon to produce chemical intermediates, such as glucose, lactic acid, ethanol and acetic acid, which can be returned to the productive chain. However, the principal obstacle in the use of this material for enzymatic degradation lies in the nature of the cellulose polymer. There are still many engineering, technological and chemistry related issues which remain to be resolved. The main objective of this study is to enable the production of glucose from the enzymatic hydrolysis of cellulose waste, arising from the waste of a recycle paper plant (paper crumb) by using Trichoderma reesei strain directly, instead of the commonly used mixture enzymes. This procedure, known as the single-step glucose production process, aims to reduce the costs associated in the use of pure enzymes and pre-treatments that are usually necessary to carry out the enzymatic degradation. The paper crumb is high in cellulose fibres (32%) with an alkaline characteristic, which carries a wide variety of impurities. This study recommends using existing knowledge with regards the enzymatic activity of the fungus and demonstrates its ability to degrade this substrate; regardless of the complex matrix linked to the cellulose polymer. Due to the nature of paper crumb a number of issues had to be solved during the development of the single-step production process. Firstly, the identification of an analytical method to monitor the enzymatic degradation of the paper crumb without interference of the inorganic compounds present in the substrate. The glucose analyser GL6 proved to be most suitable in this study. Secondly; the verification of the fungus’ ability to grow in this substrate by using PDA/Paper crumb plate, which allowed its adaptation gradually and reduced the time to produce enzymes. Finally, the evaluation of the enzymatic activity under acid and alkaline conditions was undertaken. It is demonstrated that the single-step process is feasible under acid conditions. The study also found that the fermentation time was the key parameter (up to 9 h.) to avoid the consumption of the glucose. The results show that the single-step process produces the same amount of glucose as the multi-step process (0.4 g/l), however the lower glucose production making it less economically attractive and less feasible to be expanded into an industrial scale. Nevertheless, the findings of this research contribute to establishing the basics for the optimisation of the glucose production process as an alternative for cellulose waste management. This adds economic value to the organic waste minimisation, which will lead to reduce cost in production processes.

An enrichment procedure led to the isolation, by the cellulose roll tube method, of a number of actively cellulolytic anaerobic thermophilic bacteria. Two isolates were terminally sporing rods and were tentatively identified as Clostridium thermocellulaseum (Enebo, 1951). Strain M-7 (0.6 μm x 4.0 μm) from manure grew optimally at 58°C to 63°C, pH 6.0 to 6.5 and did not require organic nitrogen. Strain C-19 (0.3 μm x 4.5 μm) from compost was similar but grew optimally at 50°C to 68°C, pH 7.5. Both utilized cellobiose and a wide range of other sugars. Strain C-19 did not utilize glucose, raffinose and inositol but did use inulin. The mean generation times in a rich nutrient medium containing cellobiose were 35 min for strain M-7 and 25 min for strain C-19. Strain M-7 had a mean generation time of 2 hr when grown on cellulose. Yeast extract (0.5%) stimulated growth and cellulase production by strain M-7 but was inhibitory at higher concentrations. Other organic nitrogen sources acted similarly. Cellulose at 1.0% gave maximum cellulase production after 72 hr incubation of strain M-7. Higher concentrations of cellulose were not completely degraded in 72 hr. Strain M-7 did not produce cellulase when grown on any carbon source other than cellulose substrates. The addition of cellobiose (0.3%) and glucose (0.4%) prevented cellulose hydrolysis in cellulose medium. This may have been repression of synthesis but cellulase was inhibited by both sugars. Both C₁, cellulase (degrades native cellulose) and Cx cellulase (β-1,4-glucanase) activities in strain M-7 cultures were assayed by measuring the liberation of reducing sugars, using dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67°C. Cx cellulase could conveniently be assayed by a new automated procedure. Strain M-7 was very actively cellulolytic when compared to previously microbial species. The 48 hr culture contained Cx activity (56 μg glucose/min/ml from carboxymethyl cellulose) and C₁ activity (8 μg glucose/min/ml from cotton fibres); the ratio of C₁,:Cx was 1:7. The cellulase(s) from strain M-7 were extra-cellular, produced during exponential growth but were not free in the growth medium until 50% of the cellulose was hydrolyzed. Glucose and cellobiose were the only soluble products liberated by the cellulase from cellulose. ZnCl₂ precipitation appeared to be a good method for the concentration of cellulase activity but subsequent purification was not successful. Isoelectric focusing indicated the presence of four Cx cellulases (pI 4.5, 6.3, 6.8, and 8.7). DEAE-Sephadex chromatography indicated three Cx components. It is concluded that C. thermocellulaseum M-7 produces cellulase(s) capable of rapidly hydrolyzing native cellulose. The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of cellulose is possible at elevated temperature. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The catalytic domain is separated from the cellulose-binding domain in Cellulomonas fimi endoglucanase CenA by a proline-threonine rich sequence called the Pro-Thr box. To study the function of the Pro-Thr box region, a deletion mutant, cenAAPT, was made from cenA by an oligonucleotide directed in vitro mutagenesis. The truncated enzyme, CenAAPT, was purified to homogeneity by affinity chromatography on cellulose and characterized. Comparing CenAAPT to CenA, the following characteristics were observed: 1) the Pro-Thr box affected the migration of CenA on SDS-PAGE; 2) the deletion of the Pro-Thr box altered the high affinity interaction with cellulose; 3) the truncated enzyme showed 40-50% reduction in catalytic activity towards both microcrystalline and amorphous cellulose; 4) the truncated enzyme was as sensitive as CenA to a C.fimi protease, and both enzymes were cleaved at the same site adjacent to the binding domain. The Pro-Thr box is not essential for the catalytic activity of CenA or its binding to cellulose, but it does contribute to both functions. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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Cellulolytic enzymes capable of efficiently degrading crystalline cellulose are a complex mixture of endo- (endoglucanases) and exo-acting (cellobiohydrolases) enzymes. One approach to separating these enzymes is affinity chromatography. A new ligand, p-aminophenyl l-thio-β-D-cellobioside (APTC), is introduced for this purpose. The property of APTC in affinity chromatography is demonstrated using Trichoderma reesei cellulases. The behavior of these enzymes on APTC-affinity column was essentially equivalent to that reported for the same enzymes on p-aminobenzyl 1-thio-β- D-cellobioside (ABTC)-columns; ABTC being the traditional ligand for affinity chromatography of exocellulases. The primary advantage of the APTC ligand is its ease of preparation. The affinity between CBHs and APTC may be considerably affected by nonspecific interactions. In this study, the significance of nonspecific protein/matrix interactions in affinity chromatography of cellulolytic enzymes is evaluated. The role of pH, NaCl, coupling conditions and stationary phase functional groups (N-hydroxysuccinimide ester and cyanogen bromide) on the affinity purification of Trichoderma reesei CBHs has been systematically determined. The results suggest that the apparent discrepancies in existing methods for the affinity purification of CBHs are due to nonspecific interactions, i.e. ionic interactions, between the enzymes and the stationary phase matrix. Exocellulases can be classified into two classes, based on their hydrolytic specificities. Class I enzymes preferentially hydrolyze cellulose from the reducing end, while Class 11 enzymes preferentially hydrolyze cellulose from the nonreducing end. Trichoderma reesei CBH I is a class I enzyme and CBH 11 is a class II enzyme. CBH I and CBH II are both retained on the APTC-affinity column; showing that both CBH classes bind to immobilized APTC. To further understand the differences in the two CBH classes, the behavior of CBH I and CBH II on the APTC-affinity column was compared. The affinity of CBH I for immobilized APTC was found to decrease when glucose was present in the system. In contrast, glucose was found to increase the affinity of CBH 11 for immobilized APTC. An outcome of this difference is that in the presence of glucose CBH I can be selectively eluted from the column. Equilibrium binding studies with each enzyme clearly reflect that CBH II has a higher affinity for immobilized APTC than CBH I. / Graduation date: 1998

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