PRODUCTION OF CELLULASES AND XYLANASES AND THEIR APPLICATIONS IN INDUSTRIAL PROCESSES

Abstract

The world production of paper and paperboard in 2012 was 399 million tonnes. Currently, the production of paper, paperboard and newsprint in India is 10.11 million tonnes. The annual consumption of paper, paperboard and newsprint of country is 11.15 million tonnes and it is expected to reach up to 24 million tonnes by 2025. It is estimated that Indian paper industry will require over 16 million tonnes of waste paper to fulfil this demand by 2025. The Indian paper industry is primarily dependent on three major raw materials viz. forest, agricultural residues and secondary fibres. Pulp and paper industry used 31% of forest based fibre, 22% agro-residues and 47% recycled fibre in 2011. Therefore, use of recovered paper as the source of fibre for paper production is the most promising source. Every metric tonne of recycled fibre saves an average of 17 tree, at least 30,000 litres of water, 3000-4000 kWh of electricity and 95% of air pollution. Most of the conventional chemical deinking techniques require large amount of chemical agents, resulting in a costly wastewater treatment to meet the environmental regulations. Alternatively, use of enzymes such as cellulases and xylanases, for detaching the toner particles from fibres has been reported as efficient process and less polluting solution to overcome this disposal problem. Improved strength and optical properties were obtained along with higher drainage of pulp after enzymatic treatment during deinking of waste paper. Present study aimed at screening and selection of potential microorganisms which were able to synthesize biomass degrading enzymes for deinking of mixed office waste and saccharification process. All the cultural operating parameters were optimized for maximum production of cellulases and xylanases, and partial characterization of cellulases was carried out. Enzymes from selected fungal isolates were evaluated for their potential in deinking of mixed office waste and saccharification of pearl millet stover. A total of 134 fungal isolates were selected during primary screening based on their growth on CMC-agar media. In secondary screening, fungal strains showing zone ratio of 3.0 or more were selected for application based tertiary screening. Two fungal isolates AKB-24 and AKB-25 were selected based on their applications in deinking of mixed office waste (MOW) and saccharification of pearl millet stover after tertiary screening. Fungal isolates AKB-25 and AKB- 24 were identified as Aspergillus nidulans and Penicillium sp. based on morphological features as well as r-DNA sequences. Ten different agricultural residues were tested for the production of enzymes by Aspergillus nidulans AKB-25, Penicillium sp. AKB-24 under solid-state fermentation II conditions. Among different cheap by-products of agricultural residues, black gram residue as the substrate produced maximal endoglucanase (61.68 IU/gds), FPase (1.50 FPU/gds), and β- glucosidase (31.15 IU/gds) activities from Aspergillus nidulans AKB-25 under solid-state fermentation before optimization of cultural parameters. After optimization, Aspergillus nidulans AKB-25 produced maximal endoglucanase (152.14 IU/gds), FPase (3.42 FPU/gds) and xylanase (2441.03 IU/gds) activities at incubation time 4 days, temperature 30 ºC, initial pH 8.0, initial moisture content 77.5%, (NH4)2SO4 (as available N) 0.12% and Triton-X-100 (a surfactant, w/v) 0.05%. The maximum porosity i.e. water retention value (174.94 %) and minimum C/N ratio (22.68) made the wheat bran the best carbon source among different carbon sources for maximal enzyme production by Penicillium sp. AKB-24 under SSF conditions. Penicillium sp. AKB-24 produced maximum endoglucanase (133.94IU/gds), FPase (2.96 FPU/gds), glucosidase (5.88 IU/gds) and xylanase (3592.26 IU/gds) activities at an incubation time of 7 days, temperature 30 ºC, initial pH 7 and moisture content 77.5%, yeast extract 1.2% and sodium dodecyl sulphate 0.10 (w/v). During co-culturing of Penicillium sp. AKB-24 and Aspergillus nidulans AKB-25 showed an enhancement in endoglucanase, FPase, and exoglucanase activities by 33.88, 18.12, and 10.77% respectively compared to A. nidulans AKB-25 alone under optimum conditions. Optimum pH for FPase, endoglucanase, and glucosidase activities were 5.0 for both the fungal strains. Cellulases from A. nidulans AKB-25 were found moderately thermo-stable with optimum endoglucanase activity at 65 °C and optimal FPase and β-glucosidase activities at 60 °C. The maximal endoglucanase, FPase and β-glucosidase activities were observed at 55 °C for fungal strain Penicillium sp. AKB-24. Cellulases from both fungal strains were found stable up to 48 h at 50 °C. The prolonged stability of cellulases made them suitable for saccharification of cellulosic biomass for a long time, without any extra requirement of enzymes. The stability of enzyme for a longer time at higher temperature was desirable features for industrial applications. During chemical deinking of MOW, the pulping time was varied from 10 to 25 min while keeping other variables constant like consistency, temperature and pH. A pulping time of 20 min produced maximum ISO brightness (72.7%) and deinking efficiency (62.66%). In second set of experiments, the pulp consistency was varied from 8 to 14% while keeping other variables constant. A pulp consistency of 10% produced maximal optical and strength properties of paper. Keeping other variables constant except surfactant dose, a dose of 0.2% (w/w) Tween-80 produced maximal pulp brightness, and minimal dirt count keeping the pulp shrinkage in view. The pulp III yield was found to decrease at higher dose of surfactant. Therefore, surfactant dose of 0.2% was taken as optimum for further experiments. The maximum pulp brightness was observed at 60 °C but dirt count (1766 mm2/m2) were at 60 °C compared to 50 °C (954 mm2/m2) because softening of stickies in paper surface. The floatation time of 10 min improved the pulp brightness (75.55%) and deinking efficiency (82.13%) and mitigated the dirt counts (733 mm2/m2). Enzymatic deinking was performed with crude enzymes from Aspergillus nidulans AKB-25 and Penicillium sp. AKB-24. The selection of the optimal enzyme concentration was very important since higher enzyme dose may adversely affect strength properties of paper and its quality. Enzyme dose of 3IU/g of oven dry pulp was found optimum for optical as well as strength properties of paper for both of the fungal strains. Crude enzyme from Aspergillus nidulans AKB-25 at enzyme dose of 3 IU/g resulted improvement in pulp brightness by 72.9% deinking efficiency by 71.22% and mitigating the dirt counts to 560 mm2/m2. The reaction time during deinking experiments varied in the range of 40 to 100 min with an interval of 20 min at 3IU/g of oven dry pulp. A maximum brightness of 73.0% was observed at a reaction time of 60 min and further increase in reaction time up to 100 min improved the pulp brightness insignificantly (73.3%) but strength properties started to deteriorate. Therefore a reaction time of 60 min was taken as optimum reaction time for enzyme from Aspergillus nidulans AKB-25. Similarly, maximum pulp brightness of 73.1% was observed at a reaction time of 80 min and further increase in reaction time insignificantly improved the brightness (73.5%) but strength properties affected adversely. Therefore, a reaction time of 80 min was taken as optimum reaction time for enzyme from Penicillium sp. AKB-24. The pretreatment of pearl millet stover at 3% alkali dose was found to yield 65.85% cellulose, 19.35% hemicelluloses and 9.78% lignin with substantial reduction in extractives (92.5%) and proteins (84.1%). WRV was the measurement of total surface area accessible to water molecules. The water retention value of untreated pearl millet stover was 125.11% and enhanced to 203.11% (+78.66%) at an alkali dose of 3%. The treatment of pearl millet stover at 4% alkali dose enhanced the WRV to 211.20% (+86.09%) compared to control. Brunauer-Emmett-Teller (BET) surface area of untreated pearl millet stover was 3.49 m2/g. The BET surface area pearl millet stover increased with increasing alkali dose up to 3% and reached to 14.76 m2 /g compared to control. Thereafter, the BET surface area of pearl millet stover reduced drastically. Crystallinity index of untreated pearl millet stover was 27.40% while crystallinity index increased with alkali treatment and reached to 41.78% and thereafter, the crystallinity index remained almost constant IV i.e. 41.93% at 4% of alkali dose. Solubilization of hemicelluloses and lignin along with less ordered cellulose occurred during alkali pretreatment which was also observed in chemical compositional analysis of the solid residues. Due to removal of these components during pretreatment, cellulose concentration increased in pretreated samples. Release of reducing sugars from pretreated pearl millet stover increased with increasing alkali doses up to 3% and yield of reducing sugars was 53.13% at a hydrolysis time of 72 h. An enzyme dose of 15 FPU/g dry substrate was found to produce maximum reducing sugars (57.77%) after 72 h of enzymatic hydrolysis. Addition of Tween-20 and Tween-80 (0.15 g/g dry substrate) increased saccharification yield up to 62.14 and 64.77% respectively compared to control (57.64%). From co-culture, an enzyme dose of 15 FPU/g dry substrate was found to produce maximum reducing sugars (64.44%) after 72 h of enzymatic hydrolysis. Compared to enzymes obtained from cocultivation of A. nidulans AKB-25 and Penicillium sp. AKB-24, A. nidulans AKB-25 released the same amount of reducing sugars at an enzyme dose of 15 FPU/g and reaction time 72h.