Screening and optimization of cellulase- Producing Bacteria Isolated from different Enviromental Samples using Lignocellulosis Biomass and their Utilization in Bioethanol Production

Abstract

This thesis investigates eco-friendly alternatives to fossil fuels in response to the demand for sustainable energy. It focuses on bioethanol production from lignocellulosic biomass, emphasizing the isolation, screening, and optimization of cellulase-producing bacteria. Total 16 cellulose-degrading bacteria were isolated from garden soil, plant residues, rotten wood, and Cow dung of Kirtipur, Chovar and Lainchour sites of Kathmandu, Nepal. Cellulolytic bacteria were screened primarily by plate hydrolysis and further confirmed by well diffusion method along with Congo red staining followed by NaCl treatment. Out of 12 pure bacterial isolates, 10 showed good cellulolytic activity. Notably, the cow dungderived U1C4 isolate displayed the highest cellulolytic potential and was selected for further studies. This isolate, identified as Bacillus cereus spp. strain U1C4 was confirmed through morphological, biochemical, and phylogenetic analyses. Further investigations involved utilizing various lignocellulosic substrates as carbon sources including, Saccharum spontaneum, rice straw, wheat straw, barley straw, and wood for cellulase production after hot water pretreatment of samples. Saccharum spontaneum showed the highest cellulase production with cellulolytic activity 0.488 ± 0.021 IU/mL by U1C4 strain. The crude enzyme was purified using (NH4)2 SO4 precipitation, dialysis, and chromatography, resulting in a 5.4% recovery rate, 6.34-fold purification, and 4.693 U/mg specific activity. The purified enzyme displayed a molecular weight of 50 kDa on SDSPAGE. Upon kinetic analysis, the purified CMCase enzyme showed Km and Vmax values of 5.16 mg/mL and 33.22 µmol/ml/min, respectively. The optimum temperature and pH for maximum cellulase production were obtained as 35OC and 8 respectively including Sacchharum spontaneum (2%) and peptone (1%) as carbon and nitrogen sources for enhancing maximum cellulase production capabilities. Upon scaling up to a 3.5L production medium, the optimized Bacillus sp. (U1C4) showed improved enzyme production in an Electrolab fermenter 360 compared to a 250 mL conical flask. The CMCase activity reached the maximum of 0.595 ± 0.021 U/mL in the fermenter on 4 th day, surpassing the activity of 0.484 ± 0.04 U/mL observed in the conical flask. Ethanolic fermentation was done using a well-optimized CDBT-2 strain of S. cerevisiae after the saccharification of S. spontaneum using U1C4 strain for 7 days and the highest ethanol concentration, 10.73± 0.045 mg/mL was achieved on 3rd day of the fermentation process. Additionally, the efficiency of ethanol production, as measured by the yield in relation to the production of reducing sugars, reached an impressive 82.65%. This yield was obtained by utilizing 17.709 mg/mL of reduced glucose during fermentation. These findings demonstrate that ethanol can be produced from agricultural waste, highlighting the potential of this process to generate wealth from waste. Keywords: Bioethanol, Bacillus sp., Cellulase, Lignocellulose, Saccharum spontaneum, Fermentation, CMCase, Pretreatment.