Determination of cellulase activities from microbial ecosystems found in the digestive tracts of herbivores

The first objective of this study was to develop an efficient method of extracting cellulase complexes from anaerobic ecosystems found in the digestive tracts of herbivores. Various methods of cellulase extraction from ruminal ingesta were investigated. It was found out that extracts from a combination of sonication and triton treatment produced significantly higher cellulase activity. followed by triton- treated extracts, then by sonicated extracts and the untreated samples having the least activity. In all extraction methods investigated, homogeneous suspensions and lOOxg supernatants showed more activity than the 20,000xg supernatants. These results indicate that cell associated celluloses are firmly attached and a combination of physical and chemical processes of cellulase extraction is needed for liberation of considerable amount of cellulases and that the liberated cellulase may re-attach to the cells of microorganisms or fibres. In the second objective. assay conditions of cellulase extracts were investigated for CM Case activity and FPA. Optimal conditions for cellulase assays were pH 5.8-60 at temperature of 50“C and incubation period of 30 min. for CM Case and pH 6.0 at temperature of 55°C and (fill) incubation period between 60-90 min. for FPA. The optimal pH and temperatures found in this study support the hypothesis that assay conditions of cellulase extracts from the rumen ecosystem approximate those of isolated cellulase complexes. The third objective was to assess the cellulases extracted from the rumen and hindgut ecosystems of herbivores in cellulosic biomass conversion in vitro using model substrates. Cellulase extraction was carried by a combination of sonication and triton and CM Case activity and FPA determined under optimal conditions developed in this study using lOOxg supernatants. CM Case degradation (g l-1d_1) amounted to 443, 404, 326, 326 and FP degradation (g l-1d-1) to 56. 51, 37, 35 for cellulase extracts from ruminal ingesta of cows and goats and hindgut ingesta of rabbits and guinea pigs respectively. When compared with in vivo degradation of organic matter the data of this study support the hypothesis that rumen ecosystems have a high potential for application in biomass conversion on a large scale. These results also indicate that different ecosystems (i.e. rumen and hindgut) differ in biomass conversion rates.