Nora SzijArto,1 Zsolt Szengyel,1
Gunnar Liden,2 and Kati Reczey*1

1 Department of Agrtcnltnral Chemtcal Technology,
Bndapest Untversity of Technology and Economtcs,
H-1521 Bndapest, Szent Gellbrt ter 4, Hnngary,

E-matl: katt_reczey@mkt. bme. hn;
and 2Department of Chemtcal Engtneertng,

Lnnd Untversity, PO Box 124, SE-221 00 Lnnd, Sweden

Abstract

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily ferment­able low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity prod­ucts with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucose — grown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka- floc (delignified pine pulp), and the response was monitored in terms of CO2 evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO2 evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO2 evolution are analyzed and discussed herein.

Index Entries: Trichoderma reesei; fermentation; cellulase; growth charac­terization; cellulose hydrolysis.

*Author to whom all correspondence and reprint requests should be addressed. Applted Btochemtstry and Btotechnology 115 Vo!. 113-116, 2004

The accelerating accumulation of CO2 and other greenhouse gases in the atmosphere may lead to adverse climate changes that would seriously endanger the sensitive ecologic balance of Earth (1). Energy shortages in the world coupled with environmental considerations have directed applied research toward the development of novel processes to produce renewable fuels with a special emphasis on fuel ethanol production from cellulosic materials (2). Even though CO2 is released during the bioprocess of fuel ethanol production and also during its combustion, the CO2 is reuti­lized to grow new biomass, replacing that harvested for ethanol produc­tion. As a result, the net produced CO2 is small in comparison with that released by the utilization of fossil fuels, thus reducing the hazards of a global climate change (1,3).

The potential for using cellulosic materials to produce fermentable sugars for biotechnological processes—including bioethanol production— is enormous (4,5). Ethanol production from cellulose comprises hydrolysis of cellulosic raw materials to sugars and the subsequent anaerobic fermen­tation of sugar compounds by yeast to produce ethanol. Although enzy­matic hydrolysis is superior, in several aspects, to acid hydrolysis, its economic realization is highly hindered by the presently too high produc­tion cost of cellulose-degrading enzymes.

Cellulases are inducible enzymes, which are synthesized by many microorganisms during their growth on cellulosic materials. Example microorganisms known to produce cellulases include bacterial species of Clostridium and Bacillus and species of filamentous fungi from Penicillium, Aspergillus, and Trichoderma (6). Complete enzymatic degradation of native cellulose requires the synergistic action of three general types of cellu­lolytic enzymes, traditionally classified as endoglucanases, cellobio — hydrolases, and P-glucosidases (7). Endoglucanases preferentially hydro­lyze the amorphous regions of the fibrils by randomly cleaving P-gluco — sidic bonds; cellobiohydrolases are exoglucanases releasing cellobiose, the repeat unit of cellulose from the chain ends; while P-glucosidases complete the degradation process by hydrolyzing cellobiose and other cellodextrins with a low degree of polymerization to glucose units. The high level of synergy among cellulase enzymes results from their different, but comple­mentary, mode of action. This synergy increases the degree of hydrolysis by more than twofold over that achieved with individual enzymes (8).

Because of its ability to produce and secrete the complete set of cellu­lolytic enzymes, thus making it particularly potent in hydrolyzing the cellulose polymer to glucose monomers, the soft-rot fungus Trichoderma, in particular T. reesei has been the focus of cellulase research for decades (8). The preferred substrates used by most researchers for cellulase pro­duction are pure celluloses such as Avicel, Solka-floc, and cotton (9). Cel — lulase production by Trichoderma is controlled by a complex metabolic regulation (10-12). Cellulose acts (indirectly) as an inducer for the produc­tion of cellulases. Expression of cellulases is furthermore subject to repres­sion by the end product of the hydrolysis—glucose. Cellulose-derived inducers, sophorose being the most potent, are likely to provide an effec­tive induction during cultivation on cellulose, but the concentration of the end product, glucose, may negatively affect cellulase production. Glucose concentration is determined by the dynamic balance between the rates of glucose generation (by cellulose hydrolysis) and consumption (by micro­bial uptake). At low concentrations of cellulase and/or cellulose, glucose generation may be too slow to meet the need of active cell growth and function. On the other hand, cellulase synthesis can be halted by glucose repression when glucose generation is faster than its consumption. Glu­cose repression of enzyme expression is an obvious target for strain improvement. Many of the high-producing strains of T. reesei that have been isolated have also been shown to be partly glucose derepressed. This is the case for, e. g., the strain T. reesei Rut-C30 (6), which is used in the present study.

The objective of the current work was to characterize carefully the dynamics of cellulase production and metabolic activity following cellu­lose addition in a batch cultivation of the strain T. reesei Rut-C30. Cells were initially grown on glucose as the carbon source, and after its depletion, cellulose was added. Since it is difficult to follow the growth directly after addition of a solid substrate, on-line measurements of CO2 evolution were used to follow the metabolic activity of the cells. Frequent samples were also taken to measure enzyme activity and sugar concentrations.