Fungal utilisation of pulp mill waste water for xylanase production

English: The application of xylanolytic enzymes in the pulp and paper industry is considered one of the most important new large-scale biotechnological applications of enzymes. Incorporation of these enzymes in the bleaching process has resulted in the decreased use of chlorinebased chemicals as well as the production of less hazardous waste waters. One of the factors limiting the widespread commercial application of xylanases is the cost of production, which may be decreased by the use of lignocellulosic materials as inducer as well as carbon substrate. The main objective of this study was to evaluate spent sulphite liquor (SSL) that had been concentrated five-fold at a South African pulp mill as an alternative inexpensive carbon feedstock for xylanase production by strains of Aspergillus and Gliocladium. These fungal strains were evaluated for xylanase production with SSL, xylan and D-xylose as carbon substrates initially in shake flask cultures. Aspergillus oryzae NRRL 3485 and A. phoenicis ATCC 13157 yielded higher xylanase activities with SSL than with xylan as carbon substrate, although xylan induced a higher xylanase activity per g cell mass. Xylose induced little to no xylanase activity in the fungal strains evaluated. Apart from the sugars in the SSL, the above Aspergillus strains also utilised a non-sugar component in the SSL as additional carbon substrate, as indicated by total organic carbon analysis. The utilisation of the nonsugar component in the SSL resulted in high biomass concentrations which gave high biomass yield coefficients if calculated in terms of only the sugars present in the SSL. Characterisation of the crude xylanase preparations of A. oryzae and A. phoenicis indicated three and two xylanase isozymes with molecular weights of approximately 32, 22, 19 and 25 and 21 kDa, respectively. The A. oryzae xylanase preparation exhibited unusually high pH and temperature optima of 6.5 and 65 °C, respectively, therefore being better suited for biobleaching applications in the pulp and paper industry, whereas the properties of the xylanase preparation from A. phoenicis were typical of fungal xylanases. In bioreactor cultures of A. oryzae using SSL as carbon substrate, the cultivation pH had a significant effect on xylanase production, with pH 7.5 resulting in the highest xylanase activity. The agitation rate in the range of 400 to 800 rpm had no effect on xylanase production by Aspergillus oryzae NRRL 3485. Xylose repression of xylanase production was alleviated in fed-batch culture operating at a constant feed rate of 2.38 g xylose h-1, where a xylanase activity of up to 210 U ml-1 was reached. Xylanase production was further increased up to 260 U ml-1 by increasing the (NH4)2SO4 concentration in the medium. These values rank amongst the highest xylanase activities reported for Aspergillus strains. Pretreatment of the SSL using ultrafiltration and overliming resulted in decreased xylanase activities. Although both pretreatment procedures resulted in the removal of compounds inhibitory to microbial growth, they may have also resulted in the removal of the inducing compound/s, hence the low xylanase activities obtained with the pretreated SSL. SSL, a waste water of the pulp and paper industry, therefore could serve both as carbon substrate and inducer for xylanase production, yielding high xylanase activities as well as biomass concentrations. Furthermore, these xylanase preparations were cellulase-free and in laboratory trials with hardwood pulp their application decreased the usage of chlorine bleaching chemicals by 20 to 30% without sacrificing brightness, thus demonstrating their potential for application as biobleaching agents in the pulp and paper industry.