STUDIES ON CELLULASE PRODUCTION FOR ENZYMEASSISTED REFINING OF PULP

Abstract

The present thesis envisages the biotechnological approach for the reduction in refining energy requirement during papermaking process. Refining, one of the crucial step for development of paper strength properties, consumes a large proportion of total electrical energy required by the paper mills. Therefore, there is a need to decrease refining energy demand. Energy reduction is required globally which is directly related to total production cost and profitability. Thus, in the present research work, a biotechnological solution for refining energy reduction is investigated for energy conservation along with pulp/paper quality improvement. Cellulase is one of the most demanded industrial enzymes. Out of its three components viz. exoglucanase, endoglucanase and β-glucosidase, endoglucanase has been paid more attention with a key role in refining behavior (Chapter 1 and 2). This research work includes the isolation and screening of cellulase producing fungi. Subsequently, optimization for cellulase production was carried out under solid state fermentation using agro-based materials. Thus obtained crude cellulase was characterized for optimum pH and temperature with their stabilities. In the present research work, optimization studies for pre-treatment of bleached mixed hardwood pulp at different enzyme dosages and reaction times were performed keeping in view the industrial process conditions for temperature, pH and consistency. The enzyme treated pulps were subjected to refining in laboratory PFI mill to achieve a fixed value of freeness. After refining, both untreated as well as enzyme treated pulps were analyzed for reducing sugars content, fines content, water retention value and viscosity of pulps. The handsheets were prepared and the impact of different enzyme treatments on paper strength properties was studied. Fibrillation and inter-fiber bonding were observed through scanning electron microscope images of pulp handsheets. Crude fungal enzymes were evaluated for their potential use in reduction of refining energy. Furthermore, commercial enzymes (CA-endoglucanase and CB-cellulase+xylanase) were also tested for comparative evaluation of refining efficiency with laboratory produced fungal enzymes (Chapter 3). Newly isolated cellulase producing fungal strains were identified as Rhizopus microsporus (UPM0810n/RS1-NFCCI 2927) and Penicillium oxalicum (UPS1010B/RS2-NFCCI 2926) isolated from soil and wood samples, respectively. The finding from this work favors that solid state fermentations can be used for cellulase production by using agro-wastes. Among various carbon sources (agro-wastes) tested for cellulase production wheat bran was observed to be the best carbon source for both of the isolated fungal strains. Particle size of substrate was found to have a little ii effect on cellulase production. For R. microsporus mesh size 36 and for P. oxalicum mesh size 44 were observed to give maximum cellulase production. An incubation temperature of 38 C was found to be optimum for R. microsporus and 34 C for P. oxalicum. Endoglucanase production peaked to maximum after 5 days for R. microsporus and 4 days for P. oxalicum. Total cellulase production peaked to maximum after 6 days for R. microsporus and 4 days for P. oxalicum. Optimized inoculum size was 6 discs for R. microsporus and 8 discs for P. oxalicum. An initial pH 6.5 was found to be optimum for R. microsporus and 5.5 for P. oxalicum for endoglucanase production. Moisture content of 1:3.5 (Solid:Nutrient salt solution ratio) was observed to be the optimum for R. microsporus and 1:3 for P. oxalicum. Nitrogen sources were found to affect biomass and cellulase production. For R. microsporus soya peptone-2 g/L (organic nitrogen source), ammonium nitrate-0.03 M (inorganic nitrogen source) and mustard cake+wheat bran in 1:3 ratios were best (agro-waste). However, for better production ammonium nitrate was chosen among three best sources. For P. oxalicum yeast extract-4 g/L (organic nitrogen source), ammonium nitrate-0.03 M (inorganic nitrogen source) and soyabean hulls+wheat bran in 1:3 ratios (agro-waste) were best. Soyabean hull was chosen as best nitrogen source for P. oxalicum. Among various surfactants studied Tween 80 gave the best cellulase production for both R. microsporus and P. oxalicum (Chapter 4a). After optimization, cellulase production was found to be enhanced for both the fungi on agro-wastes by upto two to three fold. Crude enzyme of R. microsporus was found to be active in a wide range of pH 4.0-9.0 (optimum 5.5-6.0) and temperature, 40-60 °C (optimum at 55 °C). Crude enzyme of P. oxalicum has been observed to have broad pH range from 3.0 to 8.0 (optimum pH 4.5-5) and temperature, 40-60 °C (optimum 50-55 °C) (Chapter 4a). Fungal enzymes have shown the potential for refining of bleached mixed hardwood pulp. Crude enzyme can be used directly for treatment of pulp before refining under optimized conditions to get improved pulp/paper quality in addition to energy conservation. An enzyme dosage of 0.08 IU/g OD pulp for reaction time 1 h was found to be favorable for laboratory produced enzyme of R. microsporus (LA). At this optimized condition of pulp pre-treatment, PFI revolutions were decreased by 32% than those for control pulp. Paper properties improved for tensile index by 25%, burst index by 22% and tear index reduced by 9% in comparison to control pulp. For laboratory produced enzyme of P. oxalicum (LB) an enzyme dosage of 0.05 IU/g OD pulp for reaction time 1.5 h was the best. At this optimized range PFI revolutions were decreased by 28% compared to control pulp. iii Paper properties improved for tensile index by 22%, burst index by 20% and tear index reduced by 8% in comparison to control pulp (Chapter 4b). Refining efficiency of commercial enzymes was also tested for comparative evaluation of refining potential between laboratory produced enzyme and commercial enzymes. For commercial enzyme, CA 0.07 IU/g OD pulp and 1.5 h pre-treatment time was found to be optimum. At this optimized range PFI revolutions were decreased by 25% than control pulp. Paper properties improved for tensile index by 21%, burst index by 19% and tear index reduced by 10% compared to control pulp. For commercial enzyme, CB 0.06 IU/g OD pulp and 2 h reaction time was optimal for refining application. At this optimized range PFI revolutions were decreased by 20% than control pulp. Paper properties improved for tensile index by 15%, burst index by 13% and tear index reduced by 14% than control pulp. These fungal enzymes were found to be active in application conditions (temperature, pH and consistency) and showed interesting results for enzyme-assisted refining of pulp (Chapter 4b). There was a refining energy reduction (based on PFI mill revolutions) for LA by 7% and 3% than revolutions for CA and LB, respectively. Tensile index was improved by 4% for LA and 1% for LB; burst index was improved by 3% for LA and 1% for LB; Tear index was reduced slightly less by 1% for LA and LB, when compared to CA and 4% to CB (Chapter 4b). These fungal enzymes were found to be active in application conditions (temperature, pH, consistency). Overall, in the present research work, a successful reduction in refining energy is achieved with a positive impact on pulp/paper quality. As the results for refining efficiency showed better results for fungal enzyme in crude form than CB and comparable results commercial enzyme made of monocomponent (CA), it can be concluded that these enzymes are efficient in refining of bleached hardwood pulp, eliminating a need to purify enzyme. Also, the work for fungal enzyme produced by the specific genera of Rhizopus and Penicillium for Indian pulp conditions is first time performed for refining application. Hence, these fungal enzymes can be used as effective refining agents, beneficial to pulp and paper industry