Studies on thermophilic cellulytic microorganisms from compost piles and its application in bioethanol, production.

Thesis Report.

ABSTRACT: Cellulose, a major polysaccharide primarily constitutes of agriculture plant residues, industrial and municipal solid wastes. The most ambitious of these has been the conversions of cellulose to fermentable sugars through enzymatic saccharification and bioethanol production. Therefore, aim of this study, screen potent indigenous strains of thermophilic cellulolytic microbes for bioethanol production as an alternative fuel and play an important role in biodegradation and environmental pollution management of substrates. In thirteen different aerated and heated compost piles (≤ 500C) collected, 25 isolates of cellulolytic bacterial including (10 thermophilic and 15 thermotolerant) were isolated. Similarly, 15 cellulolytic isolates of actinomycetes (8 thermophilic and 7 thermotolerant) and 9 isolates of fungi (8 thermophilic and one mesophilic) were isolated. The most potent thermophilic cellulolytic isolates were Bacillus subtilis, Streptomyces spp. and Rhizomucor spp. on the basis of positive clear highest zone of hydrolysis using congo red indicator on 1% cellulose basal agar medium , incubated at 550C for 1-2 weeks. The crude enzyme produced on cellulose basal broth medium by these potent isolates was partially purified by 80-85% ammonium sulphate. Then, the enzyme activity was evaluated by measuring zone of hydrolysis by using carboxymethyl cellulose (CMC) plate diffusion assay. Saccharification substrate into reducing sugar estimated by 3, 5- Dinitrosalicylic acid (DNS) reagent. The enzyme of respective isolates converted 1% CMC substrate into reducing sugar as 18%, 28% and 37% at optimized pH 7.2, 7.4 and 4.8 at 500C. However, 72%, 77% and 90% conversion was found at substrate concentration at 0.2% CMC at similar condition respectively. The enzyme activity was found to be statistically significant (p= 0.001) with tested optimized parameters (Days wise Reducing sugar production, pH, Temperature and substrate concentration). The maximum filter paper assay (Fpase activity) were as 1.83 FPU/mL, 2.31 FPU/mL and 4.05 FPU/mL.Whereas, Carboxymethyl cellulase (CMCase) activity were as 2.2 IU/mL, 3.0 IU/mL and 4.0 IU/mL respectively. The cellulase enzyme of elaborated by Rhizomucor spp. hydrolysed on Cellulose Powder and Carboxymethyl cellulose produced reducing sugar as 8.10% and 7.01% respectively. However, in natural substrates such as in Sugarcane bagasse the reducing sugar as (4.60%), Corn Stover (5.0%), Rice husk (4.40%), Paper waste (4.20%), Household waste (6.50%) was produced.Then saccharified products selected for batch fermentation for bioethanol production using local strains of Saccharomyces cerevisiae. The maximum amount of bioethanol Production was found to be 6.31% per volume of broth on carboxymethyl cellulose substrate. Whereas, in Cellulytic solid waste- sugarcane bagasses (5.99%) followed by corn stover (4.55%), rice husk (4.14%), household waste (4.30%) and Paper waste (3.71%). Therefore, bioethanol production was compared lower by Rhizomucor spp. than Trichoderma viridae (Control) Aspergillus niger (mesophilic) (p= 0.027).Thermophilic cellulytic isolates can be used for production of bioethanol from natural cellulosic wastes. Keywords: Thermophilic cellulolytic microorganism, Cellulase Enzyme, Compost piles, Bioethanol

Eng.