Partial Characterization of Halotolerant Microbial Cellulases and Their Application in Bioethanol Production Using Sea Water System

Dash, Indira (2020) Partial Characterization of Halotolerant Microbial Cellulases and Their Application in Bioethanol Production Using Sea Water System. PhD thesis.

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Decreasing fossil fuel reservoir and increasing environment issues has triggered biofuel research. Lignocellulosic biomass is the most abundantly available biomass on earth with immense potential to meet global energy demands in a sustainable manner. Enzymatic hydrolysis of lignocellulosic biomass is advantageous than chemical hydrolysis because of environmental friendly nature and higher glucose yield without production of fermentation inhibitors. Cellulases are critical enzymes in biofuel and food industries. Several studies have been carried out on saccharification ability of bacterial and fungal cellulase. However, all these works have been carried out using fresh water based medium. Recent public threats on fresh water depletion signify the exploration of non-freshwater medium for the production of biofuels. Among the non-freshwater sources, seawater is the best source to be studied as a medium for biomass conversion due to its abundant availability in India. Utilization of halotolerant microorganisms capable of producing salt-tolerant enzymes will be a breakthrough in this field as they can tolerate high salt levels and ionic liquids better than current bacterial and fungal cellulases. Further, there will be advancement in use of sea and brackish water for biomass conversion. The present study focused on isolation and screening of bacterial strains from Gopalpur, Odisha, India capable of producing halotolerant cellulases. All the isolates were screened for their cellulolytic ability, and their enzymatic properties were characterized using soluble cellulose sources in fresh as well as in seawater. Cellulase produced by Bacillus oceanisediminis, Brevibacterium halotolerans, Psychrobacter celer, Pseudomonas aeruginosa, and Bacillus subtilis was characterizedfor different parameters like optimal pH, temperature, and substrate concentration. The optimal pH was found to be at 7.0, and optimal temperature was recorded to be between 50-60°C. The value of Km was 0.5, 0.7, 1.4, 1.3, and 0.3 mg/mL for B. oceanisediminis, B. halotolerans, P. celer, P. aeruginosa, and B. subtilis, respectively. Out the five isolates best three i.e. B. oceanisediminis, B. halotolerans, and P. Celer were selected for further analysis. All the enzymes from the three potent isolates were purified and analyzed for determination of molecular weight by SDS-PAGE and LC-MS studies and were found to be 42, 38, and 53 kDa for B. oceanisediminis, B. halotolerans, and P.celer, respectively. CD spectroscopy and bioinformatics studies were performed for the determination of secondary structure.The purified cellulase samples from B. oceanisediminis, B. halotolerans and P. celer has a characteristics antiparallel β-sheets, α-helix and type I β-turn in the secondary structures respectively. The crude enzyme extract and vii
direct bacterial cultures were independently utilized for saccharification of pretreated rice straw and the treated rice straw was characterizedfor theproduction of reducing sugars using HPLC. The Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy analysis was performed to assess the possible bond breakage and formation during saccharification of cellulose.The pretreated rice straw biomass on treatment with cellulase enzymes by the three halotolerant strains generated strong peaks which were evident at 1033 cm-1 (glucose), 2266 cm-1 (cellulose) and 1465, 1510, and 1600 cm-1 (lignin). The relative fraction and size of crystallites in cellulose was evaluated by X-ray diffraction (XRD) study. The crystallinity of pretreated rice straw was recorded as 59.073%, which decreased to 40.25%, 37.25% and 36.05% on treatment with B. oceanisediminis, B. halotolerans, and P. celer cellulases in freshwater, respectively. However, on treatment with cellulase in the seawater-based system, the crystallinity for B. oceanisediminis, B. halotolerans, and P. celer cellulases was found to be 51.58%, 79.34% and 49.21%, respectively. The saccharified biomass was further utilized for production of bioethanol in fresh water and seawater-based media using Saccharomyces cerevisiae NCIM 3570 and Dekkera naardenensis NCIM 3575. The resulting ethanol concentration in D. Naardenensis was disappointing, contradictory to that S. cerevisiae yielded excellent results with 9.79 g/L and 10.19 g/L in seawater and fresh water, respectively. Performance of S. cerevisiae remains unaffected in seawater and establishes it to one of the promising organisms for fermentation.

Item Type:Thesis (PhD)
Uncontrolled Keywords:Halotolerant; Cellulase; Bioethanol; Psychrobacter celer; Brevibacterium oceanisediminis; Brevibacterium halotolerans
Subjects:Life Science > Biochemistry
Life Science > Microbiology
Divisions: Sciences > Department of Life Science
ID Code:10172
Deposited By:IR Staff BPCL
Deposited On:26 Feb 2021 09:54
Last Modified:26 Feb 2021 09:54
Supervisor(s):Jayabalan, R and Das, Surajit

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