PRODUCTION AND CHARACTERIZATION OF XYLANASES AND THEIR APPLICATIONS IN BLEACHING

Abstract

Annual production of paper, paperboard and newsprint in India is 10.11 million tonnes while consumption of paper, paperboard and newsprint in the country is 11.15 million tonnes/ annum till 2012. Indian pulp and paper industry will require 22.0 million tonnes of paper and paperboard till 2025 with an average growth rate of 7.8% per annum. In, India the major raw materials for paper, paperboard and newsprint production were forest-based (31%), agricultural residues (22%) and secondary fibres (47%) in year 2011. There is an acute shortage of forestbased raw materials globally. Natural forests, planted timberlands and agro-residues are the main sources of lignocellulosic raw materials for paper production. Due to increased demand of pulp products, there will be more harvesting pressure on these resources. To mitigate the gap between demand and supply, various fast-growing and high yielding annual and perennial, nonconventional and cellulosic raw materials have been identified and assessed for their suitability for pulp production. The process of papermaking from virgin fibre requires pulping and bleaching chemicals for delignification of raw material. Due to growing environmental concerns and legislative pressures the pulp and paper industry is forced to modify its current pulping, bleaching and effluent treatment technologies. Today, the pulp and paper industry is adopting the eco-friendly technologies to reduce the pollution and to meet the challenges of globalization. It is evident that about 20 to 25% degradation of carbohydrates occurs during pulping and chlorine-based bleaching technologies exerted an impact to our eco-system. Therefore, efforts should be made to develop such pulping technologies which mitigate the carbohydrate degradation during pulping. The selection of molecular chlorine free bleaching sequences, substitution of molecular chlorine with less hazardous bleaching chemicals or mitigation of total chlorine demand during bleaching by extended delignification should be the aim of future studies. With these goals, ethanol-soda pulping process for E. binata was developed to minimize carbohydrate degradation during pulping and compared with other conventional pulping methods suitable for non-woods or grasses. Similarly, all the three major bleaching processes like conventional, ECF and TCF bleaching sequences were studied and effect of enzymes on pulp yield, optical properties, mechanical strength properties and effluent characteristics were studied. ii Enzymes are the biocatalysts which can offer potential advantages to paper industry like in biopulping, biobleaching, biorefining, biodeinking, and effluent treatment. Enzymes can serve as a promising biotechnological tool being the best alternative to polluting chemical technologies. Xylanases are gaining popularity as the catabolic agent for delignification in the bleaching process. This work depicted the production of xylanase by under solid-state fermentation (SSF) conditions using various agro-waste materials as the carbon sources. Morphological and proximate chemical studies of Eulaliopsis binata were carried out to evaluate its papermaking potential. Pulping conditions were optimized to obtain maximum pulp yield with better optical and physical strength properties. The enzyme produced in crude form was used for prebleaching of ethanol-soda E. binata pulp. In this work, out of 20 isolates obtained after screening for xylanase activity, two isolates namely ARC-11 and ARC-12 were selected for further studies. Xylanase producing fungi were identified as Schizophyllum commune ARC-11 and Aspergillus flavus ARC-12 isolated from wood and soil samples, respectively. The efforts were made to find out the optimum fermentation conditions for enhanced production of xylanase. Recent studies are focused on SSF with special reference to enzyme production using different agro-industrial wastes. Eleven agro-residues were tested as the carbon sources for xylanase production for both the fungal strains. In case of Schizophyllum commune ARC-11, the best carbon source for enzyme production was rice straw (4288.36 IU/gds) while pearl millet stover (1345.44 IU/gds) for Aspergillus flavus ARC-12. Depending on the fungal growth, incubation time range tested for Schizophyllum commune ARC-11 was 1-12 days and 12-96 hours for Aspergillus flavus ARC-12. Xylanase production was found to be the maximum (5199.02 IU/gds) after 8th day of incubation for Schizophyllum commune ARC-11 and 2nd day of incubation for Aspergillus flavus ARC-12 (1424.69 IU/gds). To optimize the incubation temperature, both the fungal strains were tested under different temperature ranging from 26-46°C keeping at an interval of 4°C. An incubation temperature of 30°C resulted maximum xylanase production for Schizophyllum commune ARC-11 (5358.93 IU/gds) and Aspergillus flavus ARC-12 (1431.19 IU/gds). An Initial pH of 7.0 was found to be the optimum for Schizophyllum commune ARC- 11 (6340.71 IU/gds) and 6.0 for Aspergillus flavus ARC-12 (1663.72 IU/gds). Initial moisture iii content of 70% was observed to be the optimum for Schizophyllum commune ARC-11 (6721.96 IU/gds) and 77.5% for Aspergillus flavus ARC-12 (1699.50 IU/gds). The nature and amount of nitrogen sources affects the enzyme production. Ammonium sulphate (0.08% N) for Schizophyllum commune ARC-11 and beef extract (1.2%, w/v) for Aspergillus flavus ARC-12, gave maximum xylanase production of 8591.38 and 2219.85 IU/gds, respectively. Among various surfactants tested for xylanase production Tween-20 (0.10% w/v) produced maximum xylanase activity, 10196.53 IU/gds for Schizophyllum commune ARC-11 and Tween-60 (0.10%) produced maximum xylanase activity (2539.54 IU/gds) for Aspergillus flavus ARC- 12. Characterization of the xylanase of Schizophyllum commune ARC-11 clearly showed the activity in a wide range of pH 4.0-7.0 (optimum 5.0) and temperature range of 35-60°C (optimum at 55 °C). Xylanase from Aspergillus flavus ARC-12 was observed to have a broad pH range from 4.0 to 8.0 (optimum pH 6.0) and temperature range from 35-65°C (optimum 50°C). The fraction of 50-70% ammonium sulphate precipitation gave a yield of 41.86% with 2.75 fold purification for xylanase from Schizophyllum commune ARC-11. With the same fraction of 50-70% ammonium sulphate precipitation xylanase from Aspergillus flavus ARC-12 gave a yield of 45.05% with 2.85 fold purification. The detailed morphological and anatomical features of E. binata were determined. The results of proximate chemical analysis revealed that E. binata contain higher holocellulose (73.1%) and α-cellulose contents (46.0%) respectively, which are directly related to good strength and high pulp yield for papermaking. Optimization studies were performed for alkali charge (active alkali as Na2O), cooking temperature, time and moisture content and its effects on unscreened pulp yield, screened pulp yield, kappa number and rejects were studied. E. binata produced a screened pulp yield of 43.58% with kappa number 17.38, and rejects 0.88% at optimum cooking conditions like active alkali charge 12% (as Na2O)), maximum cooking time 120 min and temperature 130 °C. A comparative study of soda, ethanol-soda and bio-soda pulping was carried out. Addition of ethanol (30%), along with alkali at optimum conditions gave maximum screened pulp yield (47.48%) compared to 42.76 and 43.58% for bio-soda and soda pulping respectively. The kappa number was reduced by 7.19 and 7.24% during ethanolsoda and bio-soda pulping of E. binata compared to soda pulping. Mechanical strength properties were determined for all the three types of pulps at a beating level of 35±1 °SR. iv Improvement in strength properties was observed in case of ethanol-soda and bio-soda pulping processes compared to soda pulping. The addition of 30% ethanol, improved pulp brightness (6.6%), tensile index (32.18%), burst index (35.40%) and double fold numbers (77.31%) compared to soda pulping. Tear index decreased by 9.95% in ethanol-soda pulping of E. binata compared to soda pulping. The main objective of bleaching is to make all the chemical pulps whiter or brighter without compromising the strength of the pulps. The major source of dioxins in the pulp and paper industry is the bleaching process in which Cl2 is used as chemical of choice. Lignin removal is more selective in the chlorination and extraction stages than in the pulping process. The crude xylanases produced by Schizophyllum commune ARC-11 and Aspergillus flavus ARC-12 were successfully used in bio-bleaching of E. binata ethanol-soda pulp. A xylanase dose of 10 IU/g (OD pulp basis), reaction time 120 min and consistency 10% were found optimum for pre-bleaching treatment by xylanases from Schizophyllum commune ARC-11 and Aspergillus flavus ARC-12. Xylanase pretreated pulps showed an improvement in brightness during all the bleaching sequences, compared to untreated pulps. In conventional bleaching, chlorine demand mitigated by 23.50 and 24.50% using xylanases from Aspergillus flavus ARC- 12 and Schizophyllum commune ARC-11 respectively in X1ECEHH and X2ECEHH bleaching sequences compared to CEHH. Due to reduction in chemical demand release of AOX in effluents reduced by 21.49 and 28.50% using xylanase from Aspergillus flavus ARC-12 and Schizophyllum commune ARC-11 respectively compared to control. Brightness and tear index improved in case X1DEPP and X2DEPP compared to DEPP at the same chemical dose. Brightness was improved by 2.8 and 1.4% (ISO) during X1QOPP and X2QOPP bleaching sequences compared to QOPP in TCF bleaching. Finally we it is concluded that the sequence X1DEDP was found most effective in bleaching of pulp of E. binata.