Lignocellulose cellulases

A literature survey indicated that very little work has been done to produce an optimal cellulase system as described above. Here, we used solid-state fermentation (SSF) to achieve this objective. SSF processes, such as the "koji" process, have been used extensively for amylase production on wheat bran in Japan its application was extended to cellulase production on wheat bran and Ugnocellulosic materials by Toyama (13), Since then, wheat bran has become an important substrate for producing various products by SSF (14-20), In this study, we tested various lignocellulosic substrates for the production of cellulase and )3-glucosidase from T, reesei QMY-1 by SSF. 

Pretreatment of Substrate. Several different lignocelluloses were pretreated with NaOH. This pretreatment partially solubilizes the hemicelluloses and lignin and swells the cellulose so that the organism can utilize it for its growth and for production of a cellulase system in SSF. The treated lignocelluloses were not washed. The NaOH treatment is done with a minimum amount of water so that, after the addition of nutrient solution and inoculum, the moisture content is less than 80% wt/wt and there is no free water in the medium. More water was added to make suspensions of different lignocellulosic substrates of the desired concentration (1% or 5%) for liquid-state (submerged) fermentation (LSF). 

One of the favored organisms for study of cellulolysis by Trichoderma is T. reesei. Consequently, many mutant strains which hyperproduce cellulase have been obtained by treatment with ultraviolet light, gamma irradiation, the linear accelerator, diethyl sulphate and N-methyl-N -nitro-N-nitroso-guanidine (7). Whereas much of the study of T. reesei has been with cellulose as substrate, it is relevant to consider the other fractions of natural lignocelluloses hemicellulose and holocellulose (the combined cellulose and hemicellulose fraction). 

T. harzianum IMI 275950 isolated from wheat straw grew better on lignocellulose (Table I). Despite these differences, the enzyme yields of the cellulase/xylanase complexes did not correlate with growth rate. These observations from this empirical study indicate that strains isolated or derived for growth on extracted substances may not necessarily be the most useful strains to exploit natural substrates. 

Lignocellulosic biomass is a valuable and plentiful feedstock commodity and its high cellulose and hemicellulose content (about 80% of total) provides considerable potential for inexpensive sugars production. However, enzymatic deconstruction of these polysaccharides remains a costly prospect. Strides in cellulase cost reduction have been made, yet further improvements are needed to reach the goal of 0.10/gal of EtOH expected to enable this new industry. Strategies to reach this goal will combine reduction in the cost to produce the needed enzymes as well as efforts to increase enzyme efficiency (specific activity). As this work proceeds, the more easily attained achievements will be made first, and thus the overall difficulty increases with time. 

Cellulase Retention and Sugar Removal by Membrane Ultrafiltration During Lignocellulosic Biomass Hydrolysis... 

Many reseachers have previously studied the use of ultrafiltration to recycle cellulase during saccharification (5-22). However, much of this previous work involved either purified cellulosic substrates, such as Solka-Floc, which are not representative of feedstocks in large-scale operations, or lignocellulosic substrates at concentrations that would be much too low to be economical in a full-scale process. 

A major problem in the commercialization of this potential is the inherent resistance of lignocellulosic materials toward conversion to fermentable sugars (4). To improve the efficiency of enzymatic hydrolysis, a pretreatment step is necessary to make the cellulose fraction accessible to cellulase enzymes. Delignification, removal of hemicellulose, and decreasing the crystallinity of cellulose produce more accessible surface area for cellulase enzymes to react with cellulose (5). 

Cellulose ethanol has the potential to displace a significant amount of petroleum in the United States, reducing the nation s dependence on foreign imports (1). The biologic processes favored for producing ethanol from lignocellulosic biomass require a pretreatment step before high yields can be realized, and the accessibility of cellulose to cellulase enzymes has... 

Harvest the cellulase-containing transgenic plants at the end of the season (dry), grind this material to release enzyme, and subsequently use it in the enzymatic hydrolysis of lignocellulosic biomass for ethanol production. 

Various authors have shown that non-ionic surfactants have a beneficial effect on the hydrolysis of cellulosic and lignocellulosic substrates, whereas anionic and cationic surfactants alone interfere negatively (Castanon and Wilke, 1981 Helle et al, 1993 Park et al, 1992 Ooshima et al., 1986 Traore and Buschle-Diller, 1999 Ueda el al., 1994 Eriksson el al., 2002). Increases in the amount of reducing soluble sugars and substrate conversion were reported. The effect depends on the substrate and is not observed for soluble substrates, such as carboxymethylcellulose or cellobiose. Nonionic surfactants increased the initial rate of hydrolysis of Sigmacell 100, and when they were added later in the process they were less effective (Helle et al, 1993). They same authors found also that the addition of cellulose increases the critical micelle concentration of the surfactant, which indicates that the surfactant adsorbs to the substrate. Surfactants are more effective at lower enzyme loads and reduce the amount of adsorbed protein (Castanon and Wilke, 1981 Ooshima et al, 1986 Helle et al, 1993 Eriksson et al., 2002) which can be used to increase desorption of cellulase from the cellulosic substrate (Otter et al., 1989). Anyhow, the use of surfactants to enhance desorption of cellulases from textile substrates in order to recover and recycle cellulases was not successful (Azevedo et al., 2002b). 

Baker, J. O., Vinzant, T. B., Ehrman, C. I., Adney, W. S., and Himmel, M. E., Use of a new membrane-reactor saccharification assay to evaluate the performance of cellulases under simulated SSF conditions—Effect on enzyme quality of growing Trichoderma reesei in the presence of taigeted lignocellulosic substrate. Appl. Biochem. Biotechnol. 1997, 63-5, 585-595. 

Wood hydrolysis has been limited to cellulose degradation by cellulase enzymes. These en mes are typically low activity and highly inhibited by the glucose product. As mentioned above, mild acid pretreatment has been found to be an important first step in the biomass utilization process. The pretreatment is used both to break down the hemicellulose to sugars and to disrupt the lignocellulosic structure and tlie crystallinity of the cellulose. 

---