Optimization of medium composition for alkali-stable xylanase production by Aspergillus fischeri Fxn 1 in solid-state fermentation using central composite rotary design

Abstract

Response surface methodology and central composite rotary design (CCRD) was employed to optimize a fermentation medium for the production of alkali-stable cellulase-free xylanase by Aspergillus fischeri in solid-state fermentation at pH 9.0 with wheat bran as substrate. The four variables involved in this study were sodium nitrite, potassium dihydrogen phosphate, magnesium sulphate and yeast extract. The statistical analysis of the results showed that, in the range studied, only sodium nitrite had a significant effect on xylanase production. The optimized medium containing (in g/l) NaNO₂—7.0, K₂HPO₄—1.0, MgSO₄—0.5 and yeast extract—5.0 resulted in 1.9-fold increased level of alkali-stable xylanase (1024 U/g wheat bran) production compared to initial level (540 U/g) after 72 h of fermentation, whereas its value predicted by the quadratic model was 931 U/g. The level of protease activity was considerably decreased in optimized medium, thus helping to preserve the xylanase activity and demonstrating another advantage of applying statistical experimental design.

Introduction

Xylan is the major hemicellulosic polysaccharide of wood and agricultural wastes, where it comprises up to 20–35% dry weight. Xylan is a hetero-polysaccharide and is composed of a xylose backbone, linked by β-1,4-xylosidic bonds, substituted with arabinosyl, glucoronosyl and acetyl residues. Among xylanolytic enzymes, endoxylanase and β-xylosidase are the key enzymes that cleave the xylan backbone into lower xylooligomers and xylose units. Tolerance to high pH and temperature are desirable properties of xylanases for effective use in pulp pre-treatment, which improves the efficiency of conventional chemical bleaching and therefore assists with pollution control. The alkali-stable xylanase can reduce the pollution by chloro-organo compounds in the paper-pulp industry due to usage of chlorine for bleaching, which is also a significant health hazard (Buchert et al., 1994). Fungal enzymes are commonly used in industries due to various technical reasons, including the feasibility of obtaining enzymes in high concentration by solid-state fermentation (Mitchell and Lonsane, 1992). A few fungal strains produce alkali-tolerant, cellulase-free xylanase when grown under alkaline conditions (pH 8–10) (Bansod et al., 1993). Aspergillus fischeri produces alkali-stable xylanase (40 U/ml) that is active at 60 °C under alkaline conditions (Anthony et al., 2003). A. fischeri grows well on wheat bran in solid-state culture conditions and produces a high level of xylanase. The optimal culture medium has not yet been developed for this strain for solid-state fermentation and designing such a medium would significantly improve the yield and quality of xylanase.

Culture medium optimization by the traditional ‘one-factor-at-a-time’ technique requires a considerable amount of work and time. An alternate strategy is a statistical approach, e.g. factorial experimental design and response surface methodology (RSM), involving a minimum number of experiments for a large number of factors, by which improvement in enzyme production has been successfully demonstrated (Ghanem et al., 2000, Bocchini et al., 2002). These methods have also been employed to improve the production of fungal xylanases in submerged culture (Haltrich et al., 1993). The present work describes the successful optimization of a culture medium for the production of alkali-stable xylanase by A. fischeri in solid-state fermentation (SSF).