MICROBIAL PRODUCTION AND APPLICATION OF ALKALITOLERANT XYLANASES BY PENICILLIUM OXALICUM

Abstract

The present investigation was undertaken to isolate a potent microorganism for production of xylanase stable at alkaline pH and deficient in undesirable cellulase activity. The process waseconomized by employing agro-horticultural residual resources. The potent fungal strain secreting xylanase was isolated by selecting samples from soda ash and paper industry waste sites. The isolated strain SA-8 had remarkable levels of xylanase production and identified as Penicillium oxalicum by Indian Agricultural Research Institute, New Delhi. To achieve an improvement in enzyme production ability, the selected strain was subjected to mutagenesis. Mutagenesis by UV-irradiation and ethidium bromide led to the development of strain SAUE-3.510 having notably higher levels of xylanase production. A detectable level of p-xylosidase production was also observed. Mutagenized strain P. oxalicum SAUe-3.510 had discrete morphological features. The mycelia of mutant SAUE-3.510 were coiled, rounded and had rough surface as compared to wild type SA-8 strain which were flattened, shiny, smooth surfaced and straight. Similarly, spores from the wild type were rounded with rougher surface while those from mutant strain were larger, smooth, flattened and crescent shaped. Xylanase activity from mutant strain SAUE-3.510 was evaluated byemploying non food, weedy plant biomass (Parthenium sp. and Eichhornia crassipes) and other agroresidual materials, which are abundantly available and grow wildly to reduce the cost of production under submerged fermentation condition. An almost similar level of xylanase production was achieved by using congress grass as to that obtained with commercial oat spelt xylan. To develop an enzyme preparation possibly with a better ability for biobleaching applications, attempts were made to develop a xylanase-laccase mixture by co-cultivation of mutant P. oxalicum SAUE-3.510 and P. ostreatus MTCC 1804. Production of mixed enzyme was evaluated by co-cultivation of P. oxalicum SAUE-3.510 and P. ostreatus MTCC 1804 under surface, submerged and solid-state fermentation systems. Solid-state fermentation led to maximum level ofproduction. Among various solid supports used, combination ofbagasse and black gram husk, in a ratio of 3:1, was found to be the most ideal for fungal colonization and enzyme production during co-cultivation. In order to further enhance the enzyme production levels, amount ofsubstrate, moisture level and incubation period were evaluated. Mixed enzyme preparation with both xylanase and laccase activity was evaluated for its bleach enhancing ability ofmixed wood pulp in XCEPHHP and XODED sequences. Process of enzymatic bleaching was further ascertained by analysing the changes occurring in polysaccharide and lignin by HPLC and FTIR. The UV absorption spectrum ofthe compounds released during enzymatic treatment had denoted acharacteristic peak at 280 nm, indicating the presence oflignin in released colouring matter. Variations in morphology and crystallinity of pulp were evaluated by scanning electron microscopy and X-ray diffraction analysis. The enzyme pretreatment led to decrease in kappa number, yellowness, AOX and COD. Additionally, ithad improved mechanical and optical properties ofpaper along with reduction in chlorine consumption which in turn diminished the pollution load denoting it therefore, to be an ecofriendly and benign bleaching technology. An attempt to scale up the production of mixed enzyme preparation was made by designing an intermittent rotating drum bioreactor using the fungal co-culture under derived conditions. Acomparable level of scaled up SSF process was accomplished and significantly higher levels of xylanase and laccase were produced.