Abstract
There is a rapidly growing interest in the utilization of cellulosic biomass for the production of biofuels and other chemicals. Many cellulolytic organisms have been screened and studied so far but despite nearly half a century of study there is still much that is not understood about the initial disruption stage in cellulose hydrolysis. Four cellulolytic fungi, Aspergillus niger, Trichoderma reesei, Myrothecium verrucaria and Trichurus spiralis were grown and tested for their ability to degrade cellulose. The fungi were grown on defined media in various pH and temperature conditions. Cellulase activity, and protein concentration in the culture filtrates were studied for each set of growth conditions. Trichoderma reesei was found to have the best ability to degrade cellulose. Vogel’s medium with Avicel as carbon source was found to be the best medium for cellulase production by T. reesei when grown at 30°C. Concentrated culture filtrate of T. reesei was fractionated by gel filtration (size exclusion) chromatography. The fractions were assayed for cellulose (filter paper) disruption. Fractions able to disrupt the filter paper as shown by the filter paper disintegration assay (FPDA) were pooled together while the rest of the fractions were pooled according to the protein profile. All the pools were tested for their ability to disrupt cellulose. The results were monitored through SEM imaging and particle size analysis of the degraded cellulose. The pool of fractions from the gel filtration depicting disruption of cellulose was further fractionated on Ion exchange chromatography, followed by hydrophobic interaction chromatography (HIC). None of the fractions obtained from HIC was positive for the FPDA. The fractions were pooled together according to their protein profile. None of the individual pools could disrupt Avicel to a comparable level to that of the culture filtrate of T. reesei. When the pools were recombined in appropriate proportion to the original sample, the disruption activity was restored. The results were confirmed through particle size analysis. To gain an insight into the mechanism and role of cellulose disruption by T. reesei. Avicel was pretreated with the culture filtrate of T. reesei for various lengths of time up to 24 hours. The particle size decreased with time during the course of the incubation. This was called the pre-treatment. Pre-treated cellulose with different particle sizes was then washed and incubated with the fresh culture filtrate of T. reesei. Pre-treatment increased the liberation of reducing sugars during subsequent incubation of the Avicel with the culture filtrate of T. reesei. It was concluded that disruption of cellulose (Avicel) during the pre-treatment process opened up attack sites for further hydrolysis. This supported the view that T. reesei employs disruption of cellulose as a mean to gain accessibility to deeper levels of the substrate. M. verrucaria was unable to attack the cellulose disrupted by this pretreatment. A new technique Esculin Gel Diffusion Assay (EGDA) was developed to screen large number of samples for B-glucosidase activity. The technique was less laborious and many times cheaper than other established assays for β-glucosidase, and showed the same level of sensitivity as them.