The rising concerns about the scarcity of fossil fuels, the emission of green house gasses and air pollution by incomplete combustion of fossil fuel have also resulted in an increasing focus on the use of cellulases to perform enzymatic hydrolysis of the lignocellulosic materials for the generation of bioethanol. The aim of this study was to isolate a potential thermo-solvent tolerant cellulase producing bacterium from natural resources, and then applied for purification and characterization. The purified enzyme was to be accessible for the bioethanol production as well as industrial exploitation (discuss in our next study). It is the first instance when thermo-solvent tolerant cellulase producing bacterium was isolated from soil sample. The culture was identified as Bacillus vallismortis RG-07 by 16S rDNA sequence analysis. Bacillus vallismortis RG-07 reported maximum cellulase production from sugarcane baggase (4105 U ml−1) used as agro-waste carbon source. The cellulase enzyme produced by the Bacillus sp. was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography, with overall recovery of 28.8%. The molecular weight of purified cellulase was 80 kDa as revealed by SDS-PAGE and activity gel analysis. The optimum temperature and pH for enzyme activity was determined as 65°C and 7.0 and it retained 95 and 75% of activity even at 95°C, and 9.0 respectively. The enzyme activity was enhanced in the presence of organic solvents (30%) n-dodecane, iso-octane, n-decane, xylene, toluene, n-haxane, n-butanol, and cyclohexane, after prolonged incubation (7 days). The enzyme activity was also stimulated by Ca2+, mercaptoethanol, Tween-60, and Sodium hypochloride whereas strongly inhibited by Hg. Kinetic analysis of purified enzyme showed the Km and Vmax to be 1.923 mg ml−1 and 769.230 μg ml−1 min−1, respectively. The unique property of solvent-thermostable-alkalophilic, nature proves the potential candidature of this isolate for current mainstream biomass conversion into fuel and other industrial process.

Background: Sugarcane distilleries use molasses for ethanol production and generate large volume of effluent containing high biological oxygen demand (BOD) and chemical oxygen demand (COD) along with melanoidin pigment. Melanoidin is a recalcitrant compound that causes several toxic effects on living system, therefore, may be treated before disposal. The aim of this study was to isolate a potential thermotolerant melanoidin decolorizing yeast from natural resources, and optimized different physico-chemical and nutritional parameters. Results: Total 24 yeasts were isolated from the soil samples of near by distillery site, in which isolate Y-9 showed maximum decolorization and identified as Candida tropicalis by Microbial Type Culture Collection (MTCC) Chandigarh, India. The decolorization yield was expressed as the decrease in the absorbance at 475 nm against initial absorbance at the same wavelength. Uninoculated medium served as control. Yeast showed maximum decolorization (75%) at 45°C using 0.2%, glucose; 0.2%, peptone; 0.05%, MgSO4; 0.01%, KH₂PO₄; pH-5.5 within 24 h of incubation under static condition. Decolorizing ability of yeast was also confirmed by high performance liquid chromatography (HPLC) analysis. Conclusion: The yeast strain efficiently decolorized melanoidin pigment of distillery effluent at higher temperature than the other earlier reported strains of yeast, therefore, this strain could also be used at industrial level for melanoidin decolorization as it tolerated a wide range of temperature and pH with very small amount of carbon and nitrogen sources.

The enzymatic saccharification of sugarcane bagasse was significantly increased by alkali pretreatment under mild conditions. The effectiveness of different concentrations of alkali and acid pretreatment of sugarcane bagasse for improving the enzymatic saccharification of lignocellulose has been evaluated. The sugarcane bagasse was characterized to contain 39.52% celluloses, 25.63% hemicelluloses, and 30.36% lignin. After that, sugarcane bagasse was pretreated with 5 and 10% of H₂SO₄ and NaOH at 121°C for 60 min. FTIR, XRD, and SEM analyses also showed significant molecular and surface structure changes of the sugarcane bagasse with 10% NaOH. Maximum saccharification was 489.5 mg/g from 10% NaOH pretreatment followed by 322.75, 301.25, and 276.6 mg/g from 10% H₂SO₄, 5% NaOH, and 5% H₂SO₄, respectively, which were 55.1, 32.0, 27.1, and 20.6 times higher than the that of the control. Cellulase and xylanase produced by Pseudomonas sp. CVB-10 (MK443365) and Bacillus paramycoides T4 (MN370035) were used to hydrolyze the pretreated bagasse, and the optimal condition was determined to be 30 h of the enzymatic reaction with the 3:1 ratio of enzymes under the temperature of 55°C, pH 5.0, and substrate concentration of 3%, leading to celluloses and hemicelluloses conversion in the enzymatic hydrolysis/saccharification that is more proficient.

In the present study, an extracellular alkali-thermo-tolerant xylanase from Bacillus paramycoides was produced in the presence of an organic solvent. The enzyme was purified by ammonium sulphate precipitation, gel filtration, and ion exchange chromatography, with an overall recovery of 25.9%. The purified enzyme hada 70 kDa molecular weight (MW) confirmed by SDS-PAGE gel analysis. The maximum enzyme activity was reported at 55 °C and pH 7.0. Xylanase activity and stability were improved in the presence of 30% (v/v) n-dodecane, iso-octane, n-decane, and cyclohexane (7 days). The enzyme activity was improved by Co²⁺, EDTA, and Triton-X-100 while vigorously repressed by Hg²⁺ and Cu²⁺. The purified enzyme showed 1.473 mg/mL K_m and 654.017 µg/mL/min V_max values. The distinctive assets of the isolate verified the potential application in the field of biomass conversion into fuel and other industrial processes. Organic solvent-tolerant xylanases can be used for concurrent saccharification and bioethanol production, the amplification of intoxicating beverages, and the fermenting industry.