PRODUCTION OF MICROBIAL CELLULASE AND XYLANASE ENZYMES AND THEIR ROLE IN WASTE PAPER RECYCLING

Abstract

Pulp and paper worldwide is today looking at, to gain the reputation of being environmental friendly through innovative process technologies, state of art of modem plant & machinery and compliance of stringent government policies on corporate responsibilities of environmental problems. Water pollution through discharge of treated effluents from paper industry is a matter of great apprehension. The second burning issue is the scarcity of the raw material for pulp and paper making. The paper consumption is increasing day-by-day and available resources are declining every day which has made the pulp and paper industry to search for new avenues in order to bridge the gap. Since the wood is not much available for pulp and paper making so the future of Indian industry looks to be dependent on either agro-based raw materials or the waste paper recycling. Thirdly, the pulp and paper manufacturing, unfortunately, is energy intensive. Energy is therefore a vital subject affecting the economy of any pulp and paper operations. In integrated Indian pulp and paper mills energy cost accounts for up to 20 to 30% of the total cost of productions. This is an alarming situation and we should focus all of our attention toward these above discussed problems, otherwise it will be difficult for us to survive in the globally competitive market. The global concerns about energy, preservation of forests and elimination of pollution from pulp and paper making processes have led us towards exploration of alternate fibrous resources other than wood that are compassionate with the environment without sacrificing product quality. Keeping in view the above problems in mind, the raw material selection for pulp and paper making becomes important. If we look back at the problems, the waste paper (secondary fibers) as a pulp and making raw material seems to be one of the promising raw materials for pulp and paper making process. Deinking is an important operation while using waste paper as a raw material for paper making. Toner particles produced during conventional repulping process with high cost chemicals are too large to be removed by the ink flotation and washing processes and are a major technical obstacle for greater use of these varieties. The use of micro-organisms and their enzymes to replace or reduce chemical consumption during deinking of office waste paper in the pulp and paper industry is gaining utmost interest. ii The industries are using chemicals for this purpose and they are costly and have adverse effect on the environment. Therefore an effort has been to utilize. In past two decades, microbial enzymes have been used extensively in pulp and paper industry in various sections like pulp bleaching, pulp refining and bio-de-inking. Commercial enzyme preparations can replace conventional deinking chemicals to remove toner inks. The enzyme for deinking and the intensive research studies have been made to develop such enzymes in the laboratory to make the process economically viable and environmental friendly the pulp and paper industry. As mentioned above and keeping upon requirements in today's industrial scenario, the major outline of the work were structured as given below: Isolation screening and evaluation of fungal strains for cellulase and xylanase production. A total of 22 fungal strains were isolated from different sites (lignocellulosic waste, decomposing manure, sugarcane dumping site, fruiting body and paper industry waste). On the basis of primary (CMC-agar and xylan-agar plate assays) and secondary screening (enzyme production under S SF), AT-2 and AT-3 were selected as the best cellulase producers while the strain AT-1 held prominent xylanase production ability among all the isolates. The test isolates AT-2 and AT-3 were identified as different strains of Aspergillus species i.e. Aspergillus flavus AT-2 and Aspergillus niger AT-3 respectively from Indian Agricultural Research Institute (IARI), New Delhi (India). The selected fungal isolate AT-1 was morphologically acknowledged as a wild mushroom from Indian Type Culture Collection (ITCC), Plant Pathology Division, IARI, New Delhi (India). For the identification of wild mushroom AT-1 up to species level, molecular identification (ITS) was done at MTCC (IMTECH, Chandigarh). The NCBI-BLAST search analysis of the DNA sequence-data indicated that the fungus was a member of Coprinus cinereus. The Scanning electron microscopy (SEM) was carried out to study the finer structural of strains AT-1, AT-2 and AT-3. Analysis of cellulase production under submerged (SmF) and solid-state fermentation (SSF) system and selection of fermentation system yielding higher levels of cellulase and evaluation of critical parameters for achieving maximum production levels iii Production of cellulase by fungal isolates AT-2 and AT-3 was analyzed under SmF and SSF. Among the different carbon sources (rice straw, wheat bran, wheat straw, sorghum and bagasse) used during the SSF of strains AT-2 and AT-3, rice straw led to the maximum level of cellulase production. XRD and SEM studies valorized rice straw (pretreated) as the carbon substrate to be used for further enzyme (cellulases) optimization studies. SEM photomicrographs had exposed that untreated rice straw had compact and rigid structure compared to the treated rice straw, which was loose and fibrous. The maximum level of cellulase production by AT-2 and AT-3 occurred on 5th day of incubation, pH 4.8 and 5.3 respectively, temperature 32°C and a solid substrate: moisture content ratio of 1:3. Nitrogen sources (NH4)2 SO4 (inorganic) and yeast extract (organic) were found to be the best for both the strains. Both the strains were biochemically characterized by determining their temperature and pH stabilities and based on their pH and thermal stabilities, AT-3 was chosen for the bio-deinking studies and SDS PAGE analysis shows molecular weight of AT-3 29kDa. Analysis of xylanase production under SmF and SSF system and selection of fermentation system yielding higher levels of xylanase and evaluation of critical parameters for achieving maximum production levels Production of xylanase by fungal isolate AT-1 was analyzed under SmF and SSF. SSF appeared to be a suitable and led to relatively higher production levels. Among the different carbon sources (rice straw, wheat bran, wheat straw, sorghum and bagasse) used during the SSF of strain AT-1, wheat bran led to the maximum level of xylanase production. The maximum level of xylanase production by AT-1 occurred on 7th day of incubation. The maximum xylanase production by a strain AT-1 was obtained at optimum conditions like pH 6.4, temperature 37°C and a solid substrate: moisture content ratio of 1:3. Among organic nitrogen sources tested, yeast extract facilitated the highest xylanase production in strain AT-1. The strain AT-1 was biochemically characterised for temperature and pH stabilities and based on its thermo-alkali tolerant behaviour, strain AT-1 was chosen for the bio-deinking studies. SDS PAGE analysis shows molecular weight of AT-1 29kDa. Analysis and evaluation of the crude enzyme preparations for its application in bio-deinking of SOP iv The crude enzyme (cellulase) preparation from AT-3 was used in enzymatic deinking trial of SOP. Enzymes were added before pulping, during pulping and after pulping. It was observed that out of three distinct addition points of enzyme and surfactant, excellent results were obtained in case when enzyme and surfactant were added after pulping Optimum conditions for SOP pulping were: pulping time 20 min, pulping temperature 65°C, conditions for enzymatic treatments were: cellulase dose of 6 IU/g (0.D. pulp basis), temperature 55 °C, pH 5.3, surfactant dose 0.05%, pulp consistency 12% and reaction time 60 min and floatation deinking was carried out for 10 min. At these optimized conditions, maximum reductions in ERIC values and dirt counts and maximum improvement in brightness and deinkability factors were observed. Deinking of SOP with different natural and commercial enzymes concoctions under optimized conditions The enzyme concoctions used on SOP pulps improved deinking efficiency in the following ascending order of enzyme doses: Control<C (61U/mL)<CX (3 and 3IU/mL) < CX (6 and 3IU/mL) < CX (6 and 6IU/mL) < CXA (6, 3 and 1.5 IU/mL) < CXA (6, 3 and 3 IU/mL) < CXA (6, 3 and 6 IU/mL) <CXAL ( 6, 3, 1.5 and 1.5 IU/mL) < CXAL ( 6, 3, 1.5 and 3 IU/mL) <CXAL ( 6, 3, 1.5 and 6 IU/mL) [C= cellulase, X=)- cylanase, A=amylase, L=lipase]. Evaluation and comparison of various deinking processes i.e. conventional, enzymatic and chemi-enzymatic deinking Chemical deinking of SOP showed hike in brightness (10.35%), DB (24.67%) and DE (78.71%) along with reduction in ERIC value (64.27%) and dirt count (83.74%) compared to control. SOP pulp when treated with crude cellulase (A. niger AT-3) prior to ink flotation, mitigated ERIC value and dirt counts by 61.84 and 82.29% where as brightness, DB and DE improved by 8.13, 23.10 and 76.30% respectively compared to control. In chemi-enzymatic deinking trials, the chemical dosages were reduced from 100% to 75, 50 and 0% while keeping the dosage of cellulase constant. Brightness, DB, and DE increased with increasing chemical dosing and conversely, dirt count and ERIC values decreased accordingly during chemi-enzymatic deinking compared to enzymatic deinking. Similarly, all the strength properties mitigated as a result of increasing chemical dosing and total solids, COD and BOD of combined effluent increased in chemi-enzymatic deinking compared to enzymatic deinking. Effect of repeated recycling on paper properties during enzymatic and conventional deinking processes As a result of repeated recycling mechanical strength properties like burst index, tensile index and double fold numbers decreased whereas tear index increased up to 3rd recycling. The 4th recycling showed an insignificant increase in tear index. Bulk and opacity increased up to 3'd recycling and then remained almost constant. Effect of enzymatic and conventional deinking on removal of micro-stickies Chemical, cellulase, cellulase+xylanase, cellulase xylanase+amylase and cellulase xylanase+amylase+lipase deinking processes removed micro-stickies by 42.02, 46.01, 46.07, 46.24 and 48.59% respectively compared to control (3060 no./kg). Effect of enzymatic and conventional deinking on ink particle size distributions Ink particles size in terms of number of specks and number of specks/cm3 in chemical and enzymatic deinking processes reduced top to bottom in the following order: chemical>cellulase>cellulase+xylanase>cellulase+xylanase+amylanase>cellulase+xylanas e+amylase+lipase. SEM, AFM and XRD, TGA and FTIR studies of different deinked pulps AFM and SEM studies indicated various morphological changes in fiber surface brought about during conventional, enzymatic and chemi-enzymatic deinking trials. The surface roughness increased in the following descending order of enzymatic concoction: control< cellulase< cellulase+xylanase< cellulase+xylanase+amylase< cellulase+xylanase+amylase. The introduction of lipase to the mixture of cellulase, xylanase and amylase caused surface roughness increased by 159% compared to control SOP FTIR studies of SOP pulp showed the appearance of band at 875 cm-1. The disappearance of this band at 875.82 cm1 showed the dissolution of xylan by crude enzymes during enzymatic deinking and by peeling reactions during chemical deinking. The peak cantered at 1770-1800 cricl corresponds to vinyl ester group, which comes under strong intensity region. The band at a wavelength of 1797.80 cm-li was observed in FT-IR spectrum of SOP (control) which disappeared in chemical and enzymatic deinked pulp. XRD analysis of deinked pulp showed that the crystalline behaviour increased with enzyme concoction and found to be maximum in case of deinldng process having cellulase+xylanase+amylase+lipase. There was an insignificant increase in crystallinity of vi pulps with increasing chemical dosing i.e. from 41.3 to 42% in chemi-enzymatic deinking process. TGA analysis indicated the weight loss in SOP at a temperature between 27-100 °C was 5.40% and weight losses decreased with increasing crystallinity of deinked pulps in the following order: chemical deinked pulp>cellulase deinked pulp> cellulase+chemical (50%)>cellulase+xylanase deinked pulp>cellulase+chemical (100%) deinked pulp. In case of enzymatic and chemi-enzymatic deinking the weight losses decreased in the following order: cellulase>cellulase+xylanase>cellulase+chemical (50%)>cellulase+ chemical (100%). At 500 °C, SOP, chemically and cellulase deinked pulps showed a residual mass of 11.86, 2.10 and 0.11% respectively. The fist DTG peak temperatures observed for SOP, cellulase, chemical, cellulase+xylanase, cellulase+chemical (100%) and cellulase+chemical (50%) deinked pulps were 344, 343, 342, 344, 341 and 345 °C and their degradation rate per min was 1.83, 2.29, 1.73, 1.16, 0.95 and 0.17 mg/min respectively. Whereas, second DTG peaks were observed at 476, 482, 475, 477, 450 and 496 °C with degradation rate per min as 0.11. 0.15, 0.13, 0.09, 0.08 and 0.17 mg/min respectively for the above mentioned pulps. Deinking model equations and statistical analysis All experiments were done in triplicates and standard deviation was given in each table. For deinking experimental runs, statistical analysis was done and empirical model equations developed with the help of mathematical polynomial regression analysis program for estimation of deinkability factors i.e. DE and DB respectively. vii