Bioprospecting for cellulase enzymes of Soil-Borne Bacteria for possible application into Bioethanol production

MSc (Biology), North-West University, Mafikeng Campus, 2016Rising crude oil prices and strict emission standards have prompted a global demand for alternative fuels that are able to reduce the consumption of fossil resources, improve energy security and mitigate climate change. Bioethanol produced from vastly abundant lignocellulosic biomass has been found to be the ideal fuel for such an endeavor. Cellulases have been found to play an important role in producing fermentable sugars from the degradation of lignocellulose for bioethanol production. Although many industrial cellulases have been extracted from a variety of fungal species, researchers have in recent years begun paying attention to various bacterial species that have the ability to produce cellulases. This is due to their ability to produce multi-enzyme complexes, resistance to extreme conditions and rapid growth. In this study, soil samples within lake ecosystems of the Ngaka Modiri Molema District were investigated for the presence of cellulolytic bacteria with the potential to produce cellulases that could one day be utilized in the production of bioethanol. To determine this, cellulose activity was taken as an important parameter to determine the performance and efficiency of the bacterial cellulases. A total of 8 bacterial isolates identified as LD04 ($Bacillus cereus$), LD08 ($Bacillus thuringiensis$), LD09 ($Brevibacterium halotolerans$), LD11 ($Bacillus cereus$), LD19 (Bacillus anthracis), LD25 ($Bacillus tequilensis$) and LD28 ($Bacillus subtilis$) were found to have favourable cellulolytic potential. A DNS assay with CMC as sole carbon source showed that isolate LD25 and LD28 had the highest enzyme activity at 0.125 UI/ml and 0.167 UI/ml respectively and were selected for further analysis, 0.1UI/ml was selected as the minimum favourable enzyme activity. A substrate-based enzymatic assay was employed to determine the ability of bacterial isolates to produce endoglucanase (CMC), exoglucanase (Avicel) and β-glucosidase (cellobiose). LD25 and LD28 produced high volumes of reducing sugars during the degradation of CMC followed by cellobiose (0.081 UI/ml and 0.14UI/ml respectively), indicating that the isolates had the ability to produce endoglucanase and β-glucosidase. The isolates showed minimal degradation of avicel as a carbon source indicating the possible inability to produce the exoglucanase enzyme. Partial purification of cellulose enzyme showed the highest protein yield and activity at 60% ammonium sulphate saturation. Optimization studies indicated that the highest enzyme activity for isolate LD25 was 0.125 UI/ml and 0.167 UI/ml for LD28, these activities occurred after a 24 hour incubation period, during the log phase of bacterial growth. Enzymatic activity peaked at pH 4 for isolate LD25 with an activity of 0.132 UI/ml whereas the activity for isolate LD28 peaked at pH 5 with an activity of 0.167 UI/ml. The enzymatic activity of LD25 and LD28 was greater when incubated at 50°C and 60°C yielding activities of 0.125 UI/ml and 0.195 UI/ml respectively, indicating that enzymes from both isolates were thermostable. Peptone was found to be the ideal nitrogen source to stimulate high enzyme activity among both isolates.. In- silico cloning showed that the celluloytic enzyme activity of Bacillus subtilis (LD28) was attributed to the presence of the CelDr endoglucanase gene. The Cel Dr gene was successfully cloned into a pGEM-T vector to yield a pGEM-CelDr recombinant. The cellulolytic enzyme activity of Bacillus tequilensis (LD25) was found to be attributed to the presence of the 1,3-1,4-β-glucanase gene. Successful cloning of this gene was accomplished through insersin of the gene fragment into a pGEM-T vector to yiend the recombinant, pGEM-E-b-Glu. $B$. $tequilensis$ (LD 25) and $B$. $subtilis$ (LD28) have been shown to be competitive cellulase producers even though they do not fully exhibit the synergistic tri-enzyme complex required for the complete degradation of lignocellulosic biomass. The study does, however, contribute to a pool of knowledge concerning the possible usage of soil borne cellulolytic bacteria from the Ngaka Modiri Molema District in the production of bioethanol or other emerging enzyme industries.Master