STUDIES ON PRODUCTION OF XYLANASES FROM WHITE ROT FUNGI FOR BIOBLEACHING OF WHEAT STRAW PULP

Abstract

Indian pulp and paper industry is facing multifarious issues and challenges like, tough competition from imports, obsolete technology, soaring energy and environmental costs, minimum profit level and inadequate supply of low-cost fiber to survive in the global competitive market. The dearth of wood fiber sources has compelled the paper technocrats to search for alternative and hitherto unexploited sources of fast growing plants which may fulfill the demand of pulp and paper industry. Agricultural residues are emerging out as a significant alternative raw material resource for pulp and paper industry. Of these, bagasse and straws are the most important. Although the availability of bagasse is likely to improve due to the expected increase in sugarcane production, yet due to an increase in its demand from sugar mills for producing steam in the boilers, its availability to paper industry would be limited. Thus, looking into the availability and cost, the straws look to be a promising raw material for paper industry. The scarcity of raw materials for paper industry has grown in parallel with environmental pollution, where pulping and bleaching processes cause major pollution problems. The untreated effluent from bleaching section of pulp and paper industry without chemical recovery, considerably pollute the receiving waters since they have high BOD, COD, AOX and suspended solids. It was found that in chlorine-based pulp bleaching the C-stage is generally the first point in which 2,3,7,8-TCDD (tetrachlorodibenzodioxin), 2,3,7,8-TCDF (tetrachlorodibenzofuran) and 1,2,7,8-TCDF congeners are always present. The E-stage filtrate contained the highest concentrations ii of dioxins. The chlorinated aromatic derivatives are toxic and persistent, constituting a potential environmental hazard if untreated before discharge to natural water bodies. Thus, strict legislation and environmental concerns regarding imposition of the effluent discharge norms with reference to AOX in Indian paper industry is forcing the paper mills to adopt alternative techniques to reduce or eliminate the use elemental chlorine in bleaching sequences. Biotechnology has a high potential in pulp bleaching sector since it allows the development of more sustainable and environment friendly products and processes by reducing chemical usage, saving energy and water, and minimizing waste products. Several workers have approached biological bleaching with lignolytic and hemicellulolytic enzymes (mainly xylanases). The most important application of xylanase enzymes is in the prebleaching of pulps. Xylanases are being tested as bleaching agents for different raw materials and bleaching sequences. Xylanases also help to increase pulp fibrillation and reduce beating time for achieving the targeted beating level. This has focused the attention of researchers towards xylanases obtained from newer microbial isolates, for the use in pulp and paper industry. Microorganisms including bacteria, yeast, actinomycetes and filamentous fungi have been reported to produce xylanolytic enzymes. Keeping the huge potential of xylanases in view, the present study aims at conducting laboratory studies on enzymatic bleaching of wheat straw soda-AQ pulp using crude xylanases obtained from indigenously isolated white rot fungal strains. The present study has been focused to produce bleached pulp of good mechanical strength by adopting environment friendly bleaching sequences, through enzymatic treatment. iii The main objectives of the present study are as follows: > to isolate and screen for potential xylanase producers > to produce xylanases from the best of the isolated strains under optimum conditions of solid state fermentation (S.S.F.) using low-cost substrate for subsequent application in biobleaching experiments > to study morphological, anatomical and chemical characterization of wheat straw > to prepare soda-AQ pulp of wheat straw at optimized pulping conditions > to optimize various operating parameters of enzymatic treatment for soda-AQ wheat straw pulp > to study the impact of enzymatic bleaching, using xylanases obtained from the test isolates, on different bleaching sequences with regard to pulp viscosity, optical and mechanical strength properties and pollution load generated during bleaching Studies on xylanase production Isolation and screening of potential white rot xylanase producers was done. Of the seven wood-rotting basidiomycetous isolates that exhibited xylanase activity, Coprinellus disseminatus SH-1 (NTCC 1163) and Coprinellus disseminatus SH-2 (NTCC 1164) were selected for further studies as they exhibited the highest and the second highest xylanase activities, with very low cellulase activities. The effect of various factors (incubation period, temperature, initial pH, complex nitrogen source, lignocelluloses as solid substrates, moisture level, simple sugars i.e. glucose and lactose, and fermentation conditions) on xylanase production by the test strains iv (C. disseminatus SH-1 and SH-2) under S.S.F. were studied. Plate assays for lacasse and amylase were done. The test strains exhibited higher xylanase activities under S.S.F. as compared to L.S.F. SDS-PAGE protein profiling of the crude xylanases and zymogram analysis of xylanases was done. Under optimized conditions, C. disseminatus strains SH-1 and SH-2 showed a xylanase activity of 727.78 and 227.99 IU/mL, cellulase activity of 0.925 and 0.660 IU/mL, laccase activity of 0.640 and 0.742 U/mL and protein concentration of 5.480 and 4.900 mg/mL, respectively. Morphological, anatomical, and chemical characterization of wheat straw In order to assess the suitability of wheat straw for pulp and papermaking, anatomical, morphological and chemical characterization of wheat straw was done. The morphological characteristics included fiber length, fiber diameter, cell wall thickness, lumen diameter and its derived values (slenderness ratio, flexibility coefficient and Runkel ratio). Chemical characterization included water solubility, 1% NaOH solubility, alcohol-benzene solubility, lignin, holocellulose, a-cellulose, pentosan, ash and silica content. Preparation of wheat straw soda-AQ pulp Wheat straw was delignified at different operating conditions (temperature, time and alkali doses). Wheat straw produced 45.05% screened pulp yield of kappa number 18.25 by soda-AQ pulping at optimum cooking temperature 150 °C, maximum cooking time 60 minutes and active alkali dose 12% (as Na2O) and fixed AQ dose (0.1%) at liquor to raw material ratio 4:1. Bauer-McNett fiber classification of soda-AQ pulp produced at optimum cooking conditions was done using screens of 20, 48, 100 and 200 mesh sizes. The pulp was beaten at various beating levels in PFI mill to optimize various mechanical strength properties. Xylanase-aided bleaching of soda-AQ pulp of wheat straw The effect of crude xylanases, respectively obtained from the test strains C. disseminatus SH-1 and SH-2 on the bleachability of soda-AQ wheat straw pulp wa studied using conventional (CEHH), ECF (ODED and ODEP) and TCF (OQPP bleaching sequences. The effect of crude xylanases obtained from both the test strain on pulp viscosity, optical and mechanical strength properties and pollution low generated during non-oxygen-delignified soda-AQ wheat straw pulp bleached b: XECEHH bleaching sequence at a chlorine demand of 4.5 and 2.25%, respectively, wa: studied and compared with CEHH bleaching sequences at total chlorine demand o 4.5%. The effect of crude xylanases obtained from both the test strains was also studiec on pulp viscosity, optical and mechanical strength properties and pollution low generated for soda-AQ wheat straw pulp bleached by two ECF bleaching sequences i.e OXEDED and OXEDEP and compared with ODED and ODEP bleaching sequences respectively. The Soda-AQ pulp of wheat straw was bleached by single TCF bleaching sequence i.e. OQPP. For both the test strains, kappa number of enzyme treated pulps was lower that that of respective control pulps. In all bleaching sequences, an increase in brightnes: and viscosity of enzyme treated pulps was observed compared to respective controls fo crude xylanase preparations obtained from both the test strains. In order to attain a beating level of 40 °SR, enzyme treated pulps (for both the strains SH-1 and SH-2) in CEHH and OQPP bleaching sequences required lesser PF mill revolutions but ODED and ODEP bleaching sequences required slightly highe PFI revolutions, as compared to their respective controls. The tensile and burst indices of enzyme treated pulps (for both the strains SH-and SH-2) in all the bleaching sequences slightly decreased as compared to thei vi respective controls. The double fold of enzyme treated pulps (for both the strains SH-1 and SH-2) in CEHH and ODEP bleaching sequences increased but decreased for those in ODED and OQPP bleaching sequences, as compared to their respective control pulps. The enzyme treated pulps (for both the strains SH-1 and SH-2) in all bleaching sequences showed an increase in tear index as compared to respective controls. The COD and color for enzyme treated pulps increased in all the bleaching sequences, compared to their respective controls (except in case of CEHH bleaching sequence at a total chlorine demand of 2.25%). The AOX of combined bleach effluents generated, was significantly lower for enzyme treated pulps in CEHH, ODED and ODEP bleaching sequences, as compared to their respective controls. Fiber surface morphology of the xylanase treated and untreated pulps was studied by scanning electron microscopy (SEM). Significant changes on the fiber surface of the xylanase treated pulps, due to xylan hydrolysis, were observed, while no such changes were observed in the untreated pulps. In oxygen-delignified pulps, the crude xylanases obtained from the test strains seemingly produced greater fiber fibrillation, compared to non-oxygen-delignified pulps. The observations supported the view that xylanase treatment improved the accessibility of the pulps to bleaching chemicals, decreased the diffusion resistance to outward movement of the degraded lignin fragments and allowed the removal of less degraded lignin fragments from the fiber wall, thereby increasing brightness of the enzyme treated pulps.