BIOBLEACHING OF ETHANOL-SODA PULP OF EULALIOPSIS BINATA BY XYLANASES FROM ASPERGILLUS FLAVUS ARC-12 AND SCHIZOPHYLLUM COMMUNE ARC-11

Environmental pollution can be minimized by using xylanase pretreatment of pulp before chemical bleaching. A. flavus ARC-12 and S. commune ARC-11 produced 234.26 and 1147.11 IU/ml of xylanase under solid-state fermentation that was used for biobleaching of ethanolsoda pulp of Eulaliopsis binata. The brightness of bleached ethanol-soda pulp of E. binata increased by 3.2 and 1.9% (ISO) with A. flavus ARC-12 and S. commune ARC-11 xylanase respectively compared chemical bleaching at the same chlorine dioxide charge. While the consumption of chlorine dioxide were mitigated by 2.98 and 3.82% with A. flavus ARC-12 and S. commune ARC-11 xylanase pretreatment respectively. Moreover, A. flavus ARC-12 and S. commune ARC-11 xylanase pretreatment reduced AOX generation by 23.80 and 19.04% respectively compared to chemical bleaching.

. The bleaching efficiency of xylanases is dependent upon several factors such as reaction temperature, pH, enzyme dose, treatment time, pulp consistency, the type of raw material, and the type of pulping and bleaching process (Bajpai, 1999; Sharma et al., 2020). Bokhari et al. (2010) studied the production of xylanase by Thermomyces lanuginosus and evaluated its efficacy in ECF bleaching of unbleached wheat straw pulp. During biobleaching of pulp kappa number was decreased by 18.6% while brightness was improved 2.63%. Moreover, xylanase pretreatment resulted in a maximum reduction in chlorine demand by 27.3%. The xylanases produced by A. niger and A. flavus were utilized for the prebleaching of Eucalyptus grandis pulp at consistency of 10%. The xylanase pretreatment was performed at 55 °C for 2 h with enzyme dose of 10 IU/g of pulp. Kappa number was decreased by 25.93% and 36.32% by A. niger and A. flavus xylanase pretreatments respectively (de Alencar Guimaraes et al., 2013).

Microorganism and xylanase production
The bleaching studies of ethanol-soda pulp of E. binata were carried out with crude xylanase from A. flavus ARC-12 and S. commune ARC-11, previously isolated and identified. Both of the fungal strains, A. flavus ARC-12 and S. commune ARC-11 were deposited at the National Fungal Culture Collection of India, Agharkar Research Institute, Pune with accession numbers NFCCI 3028 Environmental pollution can be minimized by using xylanase pretreatment of pulp before chemical bleaching. A. flavus ARC-12 and S. commune ARC-11 produced 234.26 and 1147.11 IU/ml of xylanase under solid-state fermentation that was used for biobleaching of ethanol-soda pulp of Eulaliopsis binata. The brightness of bleached ethanol-soda pulp of E. binata increased by 3.2 and 1.9% (ISO) with A. flavus ARC-12 and S. commune ARC-11 xylanase respectively compared chemical bleaching at the same chlorine dioxide charge. While the consumption of chlorine dioxide were mitigated by 2.98 and 3.82% with A. flavus ARC-12 and S. commune ARC-11 xylanase pretreatment respectively. Moreover, A. flavus ARC-12 and S. commune ARC-11 xylanase pretreatment reduced AOX generation by 23.80 and 19.04% respectively compared to chemical bleaching. and NFCCI 3029 respectively. The fungal isolate was maintained over potato dextrose agar slants at 4 °C. Xylanase production by A. flavus ARC-12 and S. commune ARC-11 was carried out under solid-state fermentation using pearl millet stover and wheat bran as substrate (Gautam et al., & 2018.

Enzyme assays
Xylanase activity was determined by using 1% (w/v) of birch wood xylan (Sigma Chemical Co. St Louis, MO, USA) in 50 mM citrate buffer at pH 5.5 according to Bailey method (Bailey et al. 1992). One unit of xylanase activity is defined as the amount of enzyme that librates 1 µmole of xylose per min per ml under assay conditions. Cellulase activity was determined as described by .

Ethanol-soda pulping of E. binata
Fresh E. binata grass was collected from Behat, Saharanpur district, India at the end of rainy season and chopped into small pieces. The cooking of chopped E. binata was performed in an electronically heated WEVERK rotary digester of 0.02 m 3 capacity. The digester contained four bombs of one-liter capacity each. Maximum pulp yield of 47.47% with kappa number, 16.13 was obtained by optimized ethanol-soda pulping. The brightness and viscosity of unbleached pulp were 43.9±0.2 % ISO and 28.2±0.14 cps respectively (Gautam et al., 2016).

Elemental chlorine-free (ECF) bleaching
Unbleached ethanol-soda pulp of E. binata was bleached by DEDP, X1DEDP, and X2DEDP bleaching sequences where stands 'X1' represented xylanase from A. flavus ARC-12 and X2 represented xylanase from S. commune ARC-11, 'D1' and 'D2' stood for chlorine dioxide 1 st and 2 nd stages respectively, 'E' for alkaline extraction stage, 'P' for hydrogen peroxide stage. The xylanase pre-treatment stage was carried out under optimized conditions. The xylanase treatments (X1 & X2) of ethanol-soda pulp of E. binata were performed at enzyme dosages of 10 IU/g of o.d. pulp for 120 min. During xylanase treatment, the temperature was maintained at 50 and 55 °C for X1and X2 respectively. After E-stage, samples were treated with 2% chlorine dioxide in 'D1' and 'D2' stages (o.d. pulp basis) (1.34% in 'D1' and 0.66% in 'D2' stage) at a consistency of 10% at 70 °C for 180 min and pH 4.2. In E-stage NaOH (as such) was conducted at 10% consistency, 60 °C for 60 min, and pH 11.7. In DEDP bleaching sequence, the final stage i.e. peroxide (P) stage was carried out at 10% consistency, temperature 90 °C, pH 10.3 and reaction time 60 min in polythene bag with 0.5% H2O2, 0.1% MgSO4 (as a carbohydrate stabilizer) and 0.5% EDTA (to mask the activities of d-block elements/transition metals). All the chemicals were added on o.d. pulp basis. The strength of H2O2 was determined by the method of Vogel's (2002).

Analysis of bleach effluent
The effluent generated after each stage of bleaching sequence was collected and mixed in equal amounts and were analyzed for COD (closed reflux titrimetric method using Thermoreactor CR2010) (1985), colour (Test method No-204A) as per standard methods for the examination of water and wastewater, American Public Health Association, 1985 (Greenberg et al. 1992) and AOX by column method (2006) with AOX Analyzer Dextar ECS 1200.

Statistical analysis
All the experiments were carried out in triplicate and experimental results were represented as the mean ± standard deviation of three experimental values.

Xylanase production
Xylanase production was carried out under solid-state fermentation conditions by A. flavus ARC-12 and S. commune ARC-11 using millet stover and wheat bran respectively as the carbon source under SSF conditions. A. flavus ARC-12 and S. commune ARC-11 produced 234.26 and 1147.11 IU/ml of xylanase respectively at optimum cultural conditions. Cellulase activity was not detected in crude xylanase from A. flavus ARC-12 while S. commune ARC-11 produced 1.47 IU/ml of cellulase. However, cellulase activity was very low in crude xylanase from S. commune ARC-11, the ratio between xylanase and cellulase activity was 780 to 1 respectively. Campioni et al. (2019) also used T. reesei QM9414 enzyme having xylanase to cellulase ratio 200 to 1 respectively for the biobleaching of kraft pulp. Table 1 showed the results and bleaching conditions of DEDP, X1DEDP, and X2DEDP sequences of ethanol-soda pulp of E. binata and its effect on brightness, viscosity, and bleached pulp yield. The brightness of ethanol-soda pulp by DEDP, X1DEDP, and X2DEDP bleaching sequences was 82.6, 85.8, and 84.5% respectively. Bleached pulp yield was improved up to 45.24 and 45.57% in bleaching sequences X1DEDP, and X2DEDP as compared to DEDP bleaching sequence (44.30%). The brightness of X1DEDP, and X2DEDP bleached ethanolsoda pulp of E. binata increased by 3.2 and 1.9% (ISO) respectively compared DEDP bleaching sequences at the same chlorine dioxide charge. Xylanase treatment enhances the porosity of pulp fibres and which subsequently improves the accessibility of bleaching chemicals into the pulp compared untreated pulp. It allows the lignin fragments to remove from the pulp. Therefore, higher brightness of pulp can be obtained by xylanase treatment at the same bleaching dosage The viscosity of X1DEDP, and X2DEDP bleached E. binata ethanol-soda pulp increased by 3.40 and 2.27% respectively compared to DEDP bleaching sequence. It is well established that the crude xylanase hydrolyzes xylan only and not cellulose chains in pulp (da Silva et al., 1994;Vidal et al., 1997;Roncero et al., 2003). The copper number decreased by 28.0 and 25.33% for E. binata ethanol-soda pulp after X1DEDP, and X2DEDP bleaching sequences compared to DEDP. Xylanase pre-treatment reduced the degree of damage to cellulose of the ethanol-soda pulp after full bleaching sequences in terms of reduction in copper number. The pulp viscosity of ethanol-soda pulp by DEDP, X1DEDP, and X2DEDP bleaching sequences were 8.8, 9.1, and 9.0% cps respectively. This slight increase indicated that there was no adverse effect on cellulose chain polymer. Tear index improved by 20.29 and 15.53% during X1DEDP and X2DEDP sequences compared to DEDP (Figure 1). While improvement in other mechanical strength properties like burst index and double-fold numbers were insignificant except a slight improvement in tensile index during bleaching sequences X1DEDP and X2DEDP compared to DEDP. Agrawal et al. (2016) performed bleaching of plywood veneer soda anthraquinone pulp with xylanopectinolytic enzyme from Bacillus pumilus AJK and observed 8.5, 13.4, and 10.8% increase in breaking length, burst factor and tear factor respectively. Lin et al. (2013) also reported slight improvements in burst, tear, and tensile index on the treatment of wheat straw soda-AQ pulp with recombinant xylanase from B. halodurans during ECF bleaching (Lin et al. 2013). Pretreatment of pulp with xylanase and its subsequent bleaching with sequence CDED1D2 improved various physical properties of the pulp i.e. viscosity, tensile strength, breaking length, burst factor, and tear factor and by 44%, 32%, 21%, 6%, and, 7% respectively, which greatly improves the quality of the paper (Battan et al., 2007). The consumed ClO2 during X1DEDP and X2DEDP bleaching sequences were mitigated by 2.98 and 3.82% respectively. Xylanase pretreatment reduced AOX generation by 23.80 and 19.04% after X1DEDP, and X2DEDP bleaching sequences respectively compared to DEDP ( Table 2). It is well proved that 4-Omethylglucuronic acid side chain of hemicelluloses is converted into hexenuronic acid (HexA) during pulp cooking. Several researchers indicated that the formation of AOX during bleaching has close relationship with HexA content of pulp (Björklund et al., 2002, 2004; Nie et al., 2015; Dai et al., 2016). Various studies proposed that HexA consumes chlorine dioxide during bleaching. It is primarily the in-situ generated hypochlorous acid that reacts with HexA to form AOX (Torngren & Ragnar, 2002; Ventorim et al., 2008). Therefore, when hemicelluloses and HexA were removed from the fibre due to xylanase action, the lignin could easily react with ClO2 and AOX generation decreased at the same dose of ClO2 (Nie et al., 2015). Nie et al. (2015) studied the xylanase-aided chlorine dioxide bleaching of bagasse pulp and concluded that lignin and HexA were the main sources of AOX generation and xylanase pretreatment removed HexA, mitigated AOX formation by 21.4-26.6% to achieve same level of brightness. Dai et al. (2016) also studied the correlation between HexA content of pulp and AOX formation and found that AOX could be reduced up to 29.8% by xylanases treatment (XD0) compared to chlorine dioxide bleaching stage (D0). Chlorophenol compounds were completely removed after xylanase treatment.  On contrary to this, the COD showed an increase of 9.87 and 7.96% respectively in combined bleached effluent obtained from X1DEDP, and X2DEDP bleached pulps compared to DEDP (Figure 2). The increase in COD of combined bleach effluent of xylanase prebleaching sequences may be explained due to the dissolution of xylan and lignin fragments with carbohydrates compared to control   chlorine dioxide bleaching. Xylanases from both fungi were found effective for improvement in ISO brightness. During xylanase bleaching, pulp viscosity improved slightly and physical strength properties were maintained except tear index that increased significantly. Xylanase pretreatment reduced both chlorine dioxide consumption and AOX generation. CODs were increased in combined bleached effluent obtained from X1DEDP and X2DEDP bleaching sequences as compared to DEDP bleaching sequence.