2000 Kluwer Academic Publishers. Printed in the Netherlands.

Short note

World Journal of Microbiology & Biotechnology 16: 111112, 2000.

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Production of xylanases by immobilized Aspergillus sp. using lignocellulosic waste

P.V. Gawande and M.Y. Kamat* Food and Fermentation Technology Division, Department of Chemical Technology, University of Mumbai, Matunga, Mumbai-400 019, India *Author for correspondence: Tel.: +91 22 4145616, Fax: + 91 22 4145614, E-mail: mykamat@t.udct.ernet.in
Received 8 July 1999; accepted 29 September 1999

Summary In shake asks immobilized Aspergillus terreus and Aspergillus niger produced 29 IU/ml, 26.7 IU/ml xylanases at 10 mg/ml, 14 mg/ml wheat bran concentration after 48 and 60 h of incubation at 37 C respectively. In repeated batch fermentation of immobilized Aspergillus sp. the same biocatalyst could be used for three successive cycles. D-Xylan, the most abundant noncellulosic polysaccharide present in nature in large amounts, is a major product of the farming industry. The basic structure of xylans is a main chain of (1 4)-linked b-D-xylopyranosyl residues, which is cleaved by xylanases. Interest in these enzymes has increased greatly in the last decade due to their potential biotechnological applications. For commercial applications, xylanases should ideally be produced from simple and inexpensive substrates. Abundantly available agro-residues are an obvious source as substrate. (Wong et al. 1988). We have isolated Aspergillus sp. 5 (Aspergillus terreus) and Aspergillus sp. 44 (Aspergillus niger) from compost manure and these strains were successfully used for xylanase production in immobilized state using nylon bolting cloth (400 mesh) as a carrier (Gawande & Kamat 1998a); xylanase was immobilized on Eudragit S100 (Gawande & Kamat 1998b) and puried by anity precipitation (Gawande & Kamat 1999). Aspergillus sp. immobilization on nylon bolting cloth, xylanase production and assay was carried out as described by Gawande & Kamat (1998a). For immobilization 5 5 cm2 pieces of nylon bolting cloth (400 mesh) were pretreated by boiling for 10 min in distilled water and washed throughly three times with the same. Immobilization was carried out by placing a preweighed bolting cloth support in the 50 ml of growth medium (Mandels and Strenberg solution + 1% glucose) prior to inoculation. For growth, 0.5 ml of spore suspension (1 106)1 107) was inoculated and the asks were incubated at 37 1 C on an orbital shaker incubator (100 rev/min). In the present work agricultural waste (wheat bran, rice straw, bagasse and soybean hull) as a substrate was used for xylanase production by immobilized Aspergillus. For the xylanase production by immobilized Aspergillus sp., biolms were developed on a carrier by incubating for 15 h in a growth medium. After 15 h, 27 and 35 mg (dry weight basis) A. terreus and A. niger growth was obtained per bolting cloth piece, receptively. The biolm was washed twice in sterile distilled water as mentioned above and transferred to production medium (Mandels and Strenberg solution with dierent carbon sources at 2 mg/ml) and production was monitored after 48 h. As shown in Table 1, low xylanase activity was observed after growth on glucose, xylose and lactose. High xylanase activities were observed when the organisms were grown on commercial xylan (oatspelt) or on lignocellulosic residues (wheat bran, rice straw, sugarcane bagasse and soybean hulls). Kang et al. (1995) reported signicant xylanase activities in a bubble column when A. niger KKS immobilized on celite was grown on rice straw. The inductive nature of the production of xylanases by Aspergillus sp. was suggested by the high levels of these enzymes in cultures with xylan or lignocellulosic residues as carbon sources whereas the xylanases activity was low on glucose-, xylose- and lactose-containing cultures. However, the fact that xylanases could be formed in the absence of xylan

Table 1. Eect of carbon source on xylanase production by immobilized Aspergillus sp. after 48 h incubation. Carbon source (2 mg/ml) Glucose Xylose Lactose Xylan Wheat bran Surgarcane bagasse Soybean hull Rice straw Enzyme activity (IU/ml) Aspergillus terreus 4.5 7.5 5.3 25 20.1 10.1 12.3 8.9 Aspergillus niger 3.2 6.8 4.2 10.9 18.3 7.5 10.7 11.2

Xylanase assay was carried out as described by Gawande & Kamat (1998a).

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Table 2. Xylanase production (IU/ml) in repeated batch experiments by immobilized Aspergillus sp. grown in Mandels and Strenberg solution containing wheat bran as a carbon source. Activity (IU/ml) Organism Batch no. I II III IV Aspergillus terreus 24 48 72 96 120 144 168 192 h h h h h h h h 15.5 28.9 12.5 27.0 13.1 27.7 10.1 11.2 Aspergillus niger

11.2 23.0 10.5 22.8 9.7 22.2 9.2 10.1

10 and 14 mg/ml wheat bran was used for Aspergillus terreus and Aspergillus niger in Mandels and Strenberg solution respectively.

Figure 1. Time course of xylanases production by immobilized Aspergillus terreus (h) and Aspergillus niger (j). Activity was measured as outlined in Table 1.

A. terreus and A. niger respectively. During the fourth batch xylanase production by the two Aspergillus spp. decreased considerably. The immobilized biomass of the two Aspergillus spp. could be used for three successive cycles. References
Gawande, P.V. & Kamat, M.Y. 1998a Immobilization of Aspergillus sp. on nylon bolting cloth for production of xylanase. Journal of Fermentation and Bioengineering 86, 243246. Gawande, P.V. & Kamat, M.Y. 1998b Preparation, characterization and application of Aspergillus sp. xylanase immobilized on Eudragit S100. Journal of Biotechnology 66, 165175. Gawande, P.V. & Kamat, M.Y. 1999 Purication of Aspergillus sp. xylanase by precipitation with an anionic polymer Eudragit S100. Process Biochemistry 34, 577580. Kang, S.W., Kim, S.W. & Lee, J.S. 1995 Production of cellulase and xylanase in bubble column using immobilized Aspergillus niger KKS. Applied Biochemistry and Biotechnology 53, 101106. Wong, K.K.Y., Tan, L.U.L. & Saddler, J.N. 1988 Multiplicity of b-1, 4-xylanase in microorganisms: functions and applications. Microbiological Reviews 52, 305317.

seems to indicate that a fraction of the enzyme was formed constitutively. The eect of varying substrate concentrations (wheat bran or oatspelt xylan) on the production of xylanases was investigated (data not shown). Wheat bran at 10 and 14 mg/ml was found to be optimal for xylanase production by A. terreus and A. niger. At optimal wheat bran concentration, xylanase production by Aspergillus sp. was monitored for 84 h (Figure 1). A. terreus (29 IU/ ml) and A. niger 26.7 IU/ml) produced maximum xylanases after 48 and 60 h of incubation. Xylanase production by surface immobilized Aspergillus sp. on bolting cloth in repeated batch fermentation is shown in Table 2. During the rst batch, xylanases activities increased to 28.9 and 23.0 IU/ml in 48 h by