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Filamentous fungi have been of great importance in the biotechnology industry for many years, being employed in some of the largest commercial bioprocesses, such as citric acid production by Aspergillus niger. Despite their importance, little is still known about the metabolism of these organisms in such processes, and in particular under conditions of stress known to occur within these environments.

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The work presented in this thesis is a study of the growth of Trichoderma koningii in both batch and continuous culture with a special interest in the biosynthesis and regulation of two of the enzymes of the cellulase complex, CM-cellulase and P-glucosidase. CM-cellulose with a degree of substitution of 0.7-0.8 has been shown to be a useful substrate for the synthesis of the two enzymes under study. CM-cellulose was shown to offer 23% of its theoretically available sugar to the organism for utilisation. The production of both CM-cellulase and P-glucosidase is about three-fold greater in continuous culture in fermenters than under similar conditions of batch culture. In batch culture, it has been possible to increase the yield of each enzyme independently of one another, but not simultaneously. Thus, replenishing spent medium in a batch culture improves the density of the culture, but only the yield of P-glucosidase. Aeration of the culture has been shown to be important, particularly for CM-cellulase formation; thus in batch culture, decreasing the aeration rate to + vol/vol from the usual rate of I vol/vol increased the yield of this enzyme threefold and brought it close to the yields found in culture. Under conditions of continuous culture, where autolysis of cells would be minimal, it was found that CM-cellulase was a wholly extracellular (about 99%) enzyme whereas P-glucosidase was almost entirely intracellular (at least 90%). In continuous culture, but not in batch, CM-cellulase has been shown to be an inducible enzyme and can be induced by low concentrations of cellobiose as sole carbon source, but higher concentrations became repressive. In both batch and continuous culture the synthesis of this enzyme was very sensitive to repression by metabolites of cellulose such as glucose and glycerol, for instance, glucose at concentrations as low as up to 30 pg/ml was a very effective repressor of CM-cellulase synthesis when added to the continuous inflow of CM-cellulose. P-glucosidase has also been shown to be induced by cellobiose, but in batch as well as in continuous culture and by high as well as low concentrations of the sugar. The induction is brought about by growing the organism on gradually increasing concentrations of the sugar (in continuous culture) and in absence of other carbon sources such as CM-cellulose. Higher concentrations of cellobiose or a change of substrate to glucose or glycerol caused repression. The use of a continuous inflow of low concentrations of either glucose (30 )ag/ml) or cellobiose (IO fag/ml) has shown the sensitivity of both of these enzymes to these sugars and also made it possible to study their control without the use of large amounts of such sugars; the latter causes excessive increase in cell weight and lowering of the pH, both of which can complicate interpretations of control mechanisms involving these sugars and their effects on enzyme synthesis. Based on the above results a scheme has been proposed for the action of metabolic products of cellulose, such as cellobiose and glucose, in regulating the biosynthesis of CM-cellulase and P-glucosidase and thus the overall breakdown of cellulose itself.

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Components of the cellulase systems produced by a mesophilic fungus, Trichoderma konlngii, and a thermophilic anaerobic bacterium, Clostridium thermocellum, have been purified and their actions studied alone and in mixtures. The cellulase complex to T.koningii was fractionated into eight components by gel filtration on Ultrogel AcA 45 and ion-exchange chromatography on OEAE-Sepharose and Cellex PA6 (p-aminobenzyl) cellulose. The components isolated were a major cellobiohydrolase, a major CM-cellulase, a low molecular weight (1.M.14.) CIA-cellulase, two minor CM-cellulases, p-glucosidase, a minor cellobiohydrolase and a cellobiohydrolase related component with CM-cellulase activity. None of the components alone was effective in solubilising reactive (bacterial) cellulose or cotton (native cellulose) or in forming short-fibres from cotton; but when recombined at their culture filtrate concentrations, the mixture displayed more than 90% of the culture filtrate activity in solubilising both substrates and in forming short-fibres from cotton. Cellobiohydrolases (major and minor) caused little decrease in the viscosity of reactive cellulose and released small amounts of cellobiose; this is connected with their action as exo-glucanases. All the CM-cellulase components rapidly decreased the viscosity of reactive cellulose as expected from the action of endo-glucanases. The major CM-cellulase was most effective in decreasing the viscosity of reactive cellulose and released more sugar (cellobiose) from this substrate than the other components. The L.M.W. Cli-cellulase released an appreciable amount of cellotriose as well as cellobiose with traces of higher oliuosaccharides. The other components also released cellobiose, but with some glucose in the case of the minor CM-cellulases. The major cellobiohydrolase synergised only with the major CM-cellulase and together they showed 70 and 30% of the culture filtrate activity in solubilising reactive cellulose and cotton respectively; addition of the L.M.W. CM-cellulase and/or the minor CM-cellulases markedly improved solubilisation of cotton, but not that of reactive cellulose. Thus these CM-cellulase components appeared to have a different mode of action from the synergistic major CM-cellulase. Effective fragmentation of cotton into short-fibres also required a concerted action of cellulolytic components and cellobiohydrolase (exo-glucanase) as well as the major CM-cellulase was essential. a-Glucosidase made little contribution to cellulolysis. Cell-free culture broth of C.thermocellum contained cellulase activity capable of degrading, in the presence of CaLl and a thiol reducing agent, native cellulose (cotton) and derived forms of cellulose (Avicel and filter paper) at a rate, and to an extent, comparable with that achieved by cellulase from Trichoderma reesei. The cellulase was most active at 70°C and at pH 5.7-6.1. Fractionation of cell-free culture broth of C.thermocellum on Ultrogel AcA 45 provided a low molecular weight CM-cellulase (containing most of the B-glucosidase of the broth) and a high molecular weight CM-cellulase/cellulase fraction. The latter was further resolved into two, CM-cellulase fractions; one of which was free of cellulase and a-glucosidaso activity; The other was active on Avicel (microcrystallinecellulose). This isolated cellulase fraction failed to hydrolyse Avicel extensively and did not synergise with other fractions. Considerable cellulase activity, but not Cli-cellulase activity, was lost during fractionation, particularly on lyophilisation of partially purified preparations. The results provide evidence for the presence of different cellulolytic components in the C.thermocellum cellulase system. One of these may be an exo-glucanase but there is no direct evidence, as yet, for its presence.

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