File S1 - Reduction in Acute Ecotoxicity of Paper Mill Effluent by Sequential Application of Xylanase and Laccase

Supporting equations, figures, and tables. Equation S1, Second order polynomial equation showing pretreatment of agro-residual pulp with B. stearothermophillus xylanase. A, B, C and D are independent variables corresponds to pH, temperature (°C), enzyme dose (U/mg) and retention time (min), respectively. Y1, Y2 and Y3 are final response for permanganate number (PNX), brightness (BX) and yellowness (YX) after pretraetment of agro-residual pulp with B. stearothermophillus xylanase. Equation S2, Second order polynomial equation showing pretreatment of agro-residual pulp with C. subvermispora laccase. where E, F and G are independent variables corresponds to enzyme dose (U/mg), mediator conc. (%) and retention time (min), respectively Y4, Y5 and Y6 are final response for permanganate number (PNL), brightness (BL) and yellowness (YL) after pretreatment of agro-residual pulp with C. subvermispora laccase. Figure S1, Decolorization of Azure-B using natural mediators (syringaldehyde and acetosyringone) and synthetic mediator (1-hydroxybenzatriazole) with C. subvermispora and the commercial laccase. C. subvermispora laccase with HBT (•), SA (▾), AS (▪); the commercial laccase with HBT (○), SA (▵) and AS (□). Figure S2, Effect of different enzymatic treatments on lignin removal. BP: before pulping; AP: after pulping; X: after xylanase treatment; XL: after sequential use of xylanase and laccase; XC: after treatment with commercial xylanase; XC+LC: after sequential treatment with commercial xylanase and laccase. Figure S3, Brightness (%ISO) value of the agro-residual pulp at different bleaching stages for the different strategies. Enz: after enzymatic pretreatment; CD: after chlorination stage; EOP: after alkaline peroxide stage; D1: after first ClO2 stage; D2: after second ClO2 stage; SO2: after final treatment with SO2. Figure S4, Improvement in the different strength properties of hand sheets made from the agro-residual pulp for various enzymatic treatment strategies. BF: burst factor; BI: burst index (kN/g); TF: tear factor; TI: tear index (mNm2/g); DF: double fold (number); TS: tensile strength (Nm/g); BL: breaking length (m); P: porosity (sec/100 ml). Figure S5, X-ray diffraction analysis of (a) control sample and enzymatically pretreated sample after (b) strategy I; (c) strategy II; (d) strategy III and (e) strategy IV. Figure S6, FTIR spectral analysis of (a) control sample and enzymatically pretreated sample after (b) strategy I; (c) strategy II; (d) strategy III and (e) strategy IV. Figure S7, Three dimensional contour plot for the reduction in the permanganate number. *pH and temperature were used as independent variables for xylanase-aided pretreatment. Figure S8, Three dimensional contour plot for the increase in the brightness. *enzyme dose and retention time were considered as independent variables for laccase-mediated pretreatment. Table S1, Study of interaction among the various nutritional and physical parameters on xylanase production through Plackett-Burman design. #xylanase activity in Unit per gram of dry bacterial bran. *Experiments were performed at pH 8.0. a – Beef extract (%); b – Galactose (%); c – KNO3 (%); d – Temperature (°C); e – Incubation time (h); f – Substrate (wheat bran) concentration (%); g – Particle (wheat bran) size (mm); h – Peptone (%); i – Yeast extract (%); j – Dummy variable (blank). Table S2, Different levels of variables affecting the xylanase production through CCD-RSM modeling. Table S3, Study of interaction among different levels of variables and their effect on xylanase production under SSF. *Xylanase activity in Units per gram of dry wheat bran. a – Peptone (%); b – KNO3 (%); c – Temperature (°C); d – Incubation time (h). Table S4, List of values of independent variables used for the pretreatment of the agro-residual pulp. Table S5, Effect of various temperatures on different properties of wheat straw pulp. *Experimentation was carried out with 15 U/mg of laccase from C. subvermispora using 2% of ABTS for 240 min of incubation at pH 5.5. a – temperature in °C; b – permanganate number of pulp; c – Brightness (%ISO) of pulp; d – yellowness (b*) of pulp. Table S6, Effect of various pH values on different properties of wheat straw pulp. *Experimentation was carried out with 15 U/mg of laccase from C. subvermispora using 2% of ABTS for 240 min of incubation at 50°C. a – permanganate number of pulp; b – Brightness (%ISO) of pulp; c – yellowness (b*) of pulp. Table S7, X-ray diffraction analysis of wheat straw pulp after different enzymatic pretreatments. *crystallinity index. XC represents pretreatment with commercial xylanase; XL represent pretreatment with lab-scale xylanase; XC+LC represents sequential pretreatment with commercial xylanase followed by commercial laccase; XL+LL represents sequential pretreatment with lab-scale xylanase followed by lab-scale laccase. Table S8, Characterization of effluents from the entire process for different enzymatic combinations.
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