STUDIES ON ENZYMATIC ASPECTS OF MICROBIAL ORIGIN AND BIO-BLEACHING OF HARDWOODS

Abstract

In view of wood-fiber crises and preservation of forests, the pulp and paper industry is moving towards fast growing wood species that can grow on waste land. The pulp and paper industry is under constant pressure for reducing the pollution load due to stringent rules of the governments; hence it is adopting the eco-friendly technologies. Therefore, the present investigation aims to produce crude enzyme preparations having high xylanase activity from the isolated fungal strain using cheap agro-residues as the substrate, and to develop an eco-friendly technology by using crude xylanase as prebleaching agent in different bleaching sequences of kraft pulps produced from two fast growing hardwood trees growing on waste land i.e. L. leucocephala and C. equisetifolia. The ultimate objective of this research work is to evaluate the feasibility of reducing the chemical consumption and pollution load while preserving the strength properties of L. leucocephala and C. equisetifolia kraft pulps through enzymatic treatment. To achieve this, an investigation is undertaken for isolating, screening and identifying a potent fungal strain comprising the potential ability of secreting xylanase. A thorough investigation of a variety of natural sites has led to isolation of xylanase producing fungal strain RM-1. This strain has notably higher xylanase activity along with moderate cellulase activity and is identified as white rot basidiomycete Coprinopsis cinerea from National Fungal Culture Collection of India, Agharkar Research Institute, Pune (India). Enzyme production from fungal strain RM-1 is evaluated under SSF and SmF conditions, and SSF results into higher levels of xylanase as well as cellulase production. Various agro-residues are evaluated for their xylanase and cellulase producing ability under SSF and the combination of wheat bran and corn cob (7:3) is selected as the substrate for crude xylanase production based on its privilege of providing high xylanase along with minor cellulase contamination. On the other hand, sugarcane bagasse is selected for crude cellulase production as the highest cellulase activity by C. cinerea RM-1 is obtained on this substrate under SSF. The different components of NSS are also optimized for crude xylanase and crude cellulase production. The eight operating parameters are screened under SSF through Plackett–Burman experimental design for achieving the optimum level of xylanase secretion. Out of eight variables studied through Plackett–Burman experimental design, three variables namely, initial pH, incubation temperature and incubation time show significant influence on xylanase and cellulase ii production, and are selected for further optimization by central composite experimental design. Under optimal conditions, the predicted responses agree very well with the experimental data, and this confirms the fitness and applicability of the models obtained for crude xylanase and crude cellulase production. Statistical analysis confirms the significance of all the data. The biochemical characterization of crude xylanase and crude cellulase produced by fungal strain RM-1 confirms that the enzymes produced by C. cinerea RM-1 are thermo-alkali-tolerant. Therefore, this test strain C. cinerea RM-1 is chosen for the further bio-bleaching studies of L. leucocephala and C. equisetifolia kraft pulps. The detailed anatomical and proximate chemical analysis of L. leucocephala and C. equisetifolia are determined in order to assess their suitability for pulp and paper making. The studies indicate that these two plants can satisfactorily be used as the raw materials for pulp and paper production. Kraft pulping process is used for pulping of L. leucocephala and C. equisetifolia wood chips. The three operating cooking variables for kraft pulping i.e. active alkali (as Na2O), maximum cooking temperature and cooking time are optimized using CCD. Statistical analysis confirms the significance of all the models. Under optimal conditions, the predicted responses agree very well with the experimental data. The mechanical strength properties are also optimized at different beating levels and beating level of 40±1 °SR is found optimum for L. leucocephala and 45±1 °SR for C. equisetifolia. The strength properties of L. leucocephala kraft pulp are found superior than that of C. equisetifolia. Bauer-McNett fiber classifier is used for fiber length distribution of kraft pulps. It confirms that kraft pulp of L. leucocephala has more percentage of long sclerenchymatous fiber. It indicates that the kraft pulp of L. leucocephala is stronger than that of C. equisetifolia. The crude xylanase enzyme produced by C. cinerea RM-1 is analyzed for its application in bio-bleaching of L. leucocephala and C. equisetifolia kraft pulps and reduction of toxicity in effluents generated, in terms of AOX while preserving mechanical strength properties during various bleaching sequences. Xylanase prebleaching stage (X) is optimized in terms of xylanase dose, retention time and pulp consistency, and the pulp filtrates are analyzed for the release of reducing sugars and chromophores from the kraft pulps of L. leucocephala and C. equisetifolia. The effect of crude xylanase preparation is observed on different bleaching sequences i.e. conventional (CEHH and CEHHP), ECF (ODED and ODEP) and TCF (O(EOP)P). Xylanase pretreatment results in small gain in viscosity over controls for conventional, ECF and TCF bleaching sequences of kraft pulps of L. leucocephala and C. equisetiolia. This indicates that the iii cellulase contamination in the crude enzyme extract has no adverse effect on the pulps of two raw materials. A gain in COD and color values of bleach effluents from all bleaching sequences is noticed by xylanase pretreatment. This indicates the solubilization of residual lignin carbohydrate complexes. Xylanase pretreatment of L. leucocephala and C. equisetifolia kraft pulps also shows the reduction in refining energy in terms of PFI revolutions for achieving the fixed beating level as compared to their respective controls during all bleaching sequences. The xylanase pretreatment of L. leucocephala and C. equisetifolia kraft pulps reduces the total chlorine demand and the AOX formation in bleach effluents while still attaining the high brightness and slightly improving the mechanical strength properties as compared to their respective controls.