Macromolecules such as proteins and polysaccharides can constitute a significant portion of dissolved organic carbon (DOC) in wastewater, but limited information is available on how these compounds are degraded in biological wastewater treatment systems. Bacteria cannot assimilate intact macromolecules but must first hydrolyze them to monomers or small oligomers. To better understand the mechanism of macromolecule degradation in wastewater treatment systems this study investigates two important questions of macromolecule metabolism. First, does hydrolysis occur in close proximity to the cell or are the hydrolytic enzymes released into bulk solution, and second, if hydrolysis is cell-associated, are hydrolyzed fragments directly assimilated into the cell or are they released back into solution? Fluorescent model substrate analogs were used to determine the location of leucine aminopeptidase and a-glucosidase activity in wastewater inoculated biofilm and suspended cultures and in trickling filter effluent. In biofilm and suspended cultures at least 93% of hydrolytic activity was cell-associated. In trickling filter effluent hydrolysis rates were at least five times higher in contact with cells and sloughed biofilm pieces than in cell-free solution. To determine whether hydrolytic fragments were directly assimilated or released into solution molecular size distributions in bulk solution were monitored using membrane ultrafiltration techniques during the degradation of the model protein, bovine serum albumin, and the model polysaccharides, dextran and dextrin, in batch and continuous suspended cultures, and in fixed-film reactor systems. Measurable amounts of macromolecule hydrolytic fragments accumulated in all reactor configurations, substrates and inocula tested. Relatively larger concentrations of hydrolytic fragments accumulated in pure culture than in wastewater culture inoculated reactors. These results support a generalized model for macromolecule degradation by bacteria that features cellbound hydrolysis of protein and polysaccharides and the subsequent release of hydrolytic fragments back into bulk solution. This hydrolysis and release is repeated until fragments are small enough ( < 1000 amu) to be assimilated by cells. A separate, but related, part of this study adapted pre-column orthophthaldialdehyde derivatization, reverse phase high performance liquid chromatographic separation and fluorometric detection to measure free and combined amino acids in unconcentrated wastewaters and to assess their treatability in wastewater treatment systems.

There are generally four factors recognized as delimiting in the study of lignocelluloses for fuel ethanol production, viz., the source of the cellulase-system and its quality characteristics for cellulose hydrolysis, the substrate and pretreatment method, the process for cellulase production and bioreactor design, and the ability of yeast to ferment mixed hexose and pentose sugars. Wheat straw (WS) and T. reesei mutants were used in the study to evaluate the production of cellulase-systems. Hydrolysis of cellulose revealed the superiority of mild NaOH pretreatment over steam explosion for cellulase production with T. reesei MCG 80 and QMY-1. Response surface models were capable of predicting that NaOH could be used for the pretreatment of WS at 4% (w/w) without urea in the fermentation medium to yield optimum filter paper activity (FPA) of 9.9 IU/mL (247 IU/g WS) and beta-glucosidase activity ($ beta$GA) of 6.4 IU/mL (159 IU/g WS) under solid-state fermentation (SSF) conditions. Multiple regression analysis with multiple coefficients of correlation, R, between 0.957 and 0.99 from the experimental data showed close agreement between the cellulase activities (FPA and $ beta$GA) from the experiments and predicted values. / The superiority of SSF over liquid-state fermentation (LSF) in the production of cellulase-systems was also established, and a prototype pan-bioreactor showed good potential for upgrading cellulase production under SSF conditions. The economics of fuel ethanol production was considered in the optimization model that sought to establish threshold cellulase loadings needed to achieve maximum cellulose hydrolysis for fermentation. High substrate concentrations of up to 7.5% were hydrolyzed with cellulase loadings of 24-30 IU/g and fermented by Pichia stipitis to achieve 90-100% conversion into ethanol. / Crude unextracted cellulase yielded over 90% hydrolysis of delignified wheat straw and proved to be better than extracted cellulase and commercial cellulases for the hydrolysis of pure cellulose and pretreated wheat straw. Studies were also conducted to demonstrate the importance of the ratio of $ beta$GA- to FPA in cellulose hydrolysis which showed that ratios closer to one (1), produced more sugars and lowered the cellobiose content in the hydrolysates. It was also shown that the source of the cellulase is important in eliminating the accumulation of cellobiose during hydrolysis as was demonstrated with cellulase from mixed cultures of T. reesei and Aspergillus phoenicis. Higher $ beta$GA from the latter were implicated since A. phoenicis is a good $ beta$-glucosidase producer. / Delignified wheat straw at 5% concentration when subjected to separate hydrolysis and fermentation and simultaneous hydrolysis and fermentation resulted in similar volumetric productivities (g/L/h) of ethanol.

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.

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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.

Thesis (Ph. D.)--University of Adelaide, 1985. / Includes bibliographical references (leaves 358-406).