Production of Xylanase from Corn Stover by Arachniotus sp.

Xylanase enzyme production was attempted in shake flask through fermentation of corn stover by Arachniotus sp. in the presence of optimum concentrations of (NH₄)₂SO₄, CaCl₂.2H₂O, MgSO₄.7H₂O, KH₂PO₄, Yeast extract and molasses (cane). Maximum production of xylanase was obtained at 48 hours from the medium containing corn stover, 7.5%; (NH₄)₂SO₄; 0.3%; CaCl₂.2H₂O, 0.05% and molasses (cane) 2.0% at pH 4 and 30°C.

Introduction

Efficient enzymatic degradation and complete utilization of agricultural and forestry wastes for single cell protein (SCP) production by micro-organisms requires the conversion of hernicelluloses to their respective sugar components before being incorported into microbiaal biomass (Chahal et al., 1979). Corn stover has high fibre and low energy contents and the availability of energy is limited due to lignocellulosic bonds present in fibre (Morison, 1959). Fermentation of corn stover with Arachniotus sp. would not only reduce the pollutants but will also serve as vital source of energy for production of fuels. Microbial production of xylanase is preferred because of easier availability and structural stability (Biely, 1985). The present study was undertaken to find out potimum cultural conditions for maximum production of xylanse by Arachniotus sp. when grown on corn stover.

Materials and Methods

Organism: Pure culture of Arachniotus sp. was obtained from the Department of Plant Pathology, University of Agriculture, Faisalabad. The stock culture was maintained on agar-corn stover slants (Table 1).

Inoculum Preparation: The inoculum was prepared by transferring the spores of Arachniotus sp. from slants to autoclaved inoculum medium (Table 1). The flask was incubated on orbital shaker at pH 4.0 and 30°C for 72 h. The mature inoculum was filtered through sterilized cotton. The filtrate containing homogenous fungal spore suspension (10⁶-10⁷ spores/mL) was used as inoculum.

Optimum Conditions: The growth media containing corn ’stover as substrate were prepared to study different conditions like fermentation period, substrate: water ratio, conc. of (NH₄)₂S0₄, CaCl₂.2H₂0, MgSO₄.7H₂0, KH₂PO₄, molasses (cane) and yeast extract for the maximum production of xylanase at pH 4 and 30°C temperature. All the experiments were conducted in a sequence after one another so that the most suitable ionic concentration of one experiment was used in next one.

Table 1:	Composition of sporulation and inoculum media (g/100 mL)*
*pH of the media was adjusted at 4

After each experiment the fermented broths were filtered through milipore filter and filtrates thus obtained were tested for the activity of xylanase.

Assay Method: Xylanase activity was determined by the spectrophotometric method of Miller (1959).

Results and Discussion

The production of xylanase by Arachniotus sp. Was optimized under following conditions and results have been given as under:

Fermentation Period: Fermentation of corn stover was carried out for different time periods viz., 24, 48, 72 and 96 hours to determine the best incubation period for the production of maximum xylanase activity. Maximum xylanase activity (0.647 IU/mL/min) was recorded after 48 h incubation. The results of present study are in line with Balakrishnan et al. (1992) who produced xylanase by alkalophilic bacillus grown on a wheat bran/yeast extract medium for 48 h.

Substrate: Water Ratio: Xylanase activity was determined (Table 2) in the culture supernatant of Arachniotus sp. grown in the medium containing different substrate:water ratios (2.5, 5, 7.5 and 10%) and it was found that 7.5 percent substrate:water ratio resulted in maximum xylanase production (0.673 IU/mL).

Table 2:	Effect of different levels of substrate water ratio (NH4)2SO4, CaCl2.2H20, MgSO4.7H20, KH2PO4, Yeast extract and molasses (cane) on the production of xylanase by Arachniotus sp.

Findings of present study are comparable with Smith and Wood (1991) who obtained optimum enzyme activity when Aspergilus awamori was grown on 4 percent oat spelt xylan.

(NH₄)₂S0₄: The xylanase activity was assayed at four concentrations of (NH₄)₂ SO₄ i.e. 0.1, 0.2, 0.3 and 0.4% and the results revealed that 0.3per cent (NH₄)₂SO₄ facilitated the maximum xylanase production (0.963 IU/mL) (Table 2). Dubeau et al. (1987) observed that 0.05% (NH₄)₂SO₄ in growth medium containing 1% xylan as a carbon source fermented with Chaetornium cellulolyticum maximum gave xylanase (0.92 IU/mL). The difference in xylanase activity is attributed to the difference of substrate and organsim.

CaCI₂.2H₂O: Xylanase production was studied at 0.01, 0.05, 0.1 and 0.5% CaC1₂.2H₂0 alongwith potimum (7.5%) substrate: water ratio and (NH₄)₂SO₄ (Table 2). It was found that 0.05% CaCl₂.2H₂0 was the best of all the levels tried. Sinha and Sengupta (1995) also reported 0.05% as the optimum concentration of CaCl₂.2H₂0 in growth medium of xylan fermented with Termitomyces clypeatus showing 4 1U/mL xylanase activity.

MgSO₄.7H₂0: Addition of 0.01, 0.015, 0.02 and 0.025%, MgSO₄.7H₂0 resulted in 2.17, 2.43, 3.29 and 2.92 IU/mL/min of xylanase. It was found that 0.02% MgSO₄.7H₂0 facilitated the highest production of xylanase. Dubeau et al. (1987) observed 0.15 percent as the optimum concentration of MgS0₄.7H₂0 in the growth medium containing 1 percent xylan used for xylanase production by Chaetomium cellulolyticum.

KH₂PO₄: To study the effect of KH₂PO₄ on xylanase production Arachniotus sp. was grown in basal medium containing 7.5 per cent corn stover initially adjusted at various levels of KH₂PO₄ i.e. 0.1, 0.2, 0.3 and 0.4% (Table 2). It was observed that at 0.1 % KH₂PO₄ showed maximum xylanase activity (3.53 IU/mL). Rajoka and Malik (1984) recovered maximum xylan6se activity by fermenting 1% kallar grass with Cellulomonas flavigena NIAB 441 after 5 days of incubation in the presence of 0.05% KH₂PO₄. The difference in optimum KH₂PO₄ level was due to the use of kallar grass and C. lavigena in place of corn stover and Arachniotus sp. respectively.

Yeast Extract: For the maximum xylanase production different concentrations (0.05, 0.1, 0.15 and 0.2%) of yeast extract were added to the medium (Table 2). It was found that 0.15% yeast extract facilitated the maximum xylanase activity. Purkarthofer et al. (1993) produced maximum xylanase from xylan by Thermomyces lanuginosus with 1.75% yeast extract in medium. The difference may be attributed to difference of substrate and the organism.

Molasses (Cane): Xylanase production was investigated by the addition of 0.5, 1.0, 1.5 and 2.0 percent molasses (cane) alongwith optimum levels of substrate (7.5%), (NH₄)₂SO₄ (0.3%), CaCl₂.2H₂0 (0.05%), MgSO₄.7H₂0 (0.02%), KH₂PO₄ (0.1%) and yeast extract (0.15%) (Table 2). The maximum xylanase activity (5.94 IU/mL) was recorded in the medium containing 2% molasses. Smith and Wood (1991) also observed an increase in xylanase production by A. awamori by the addition of molasses and also recovered maximum xylanase with 2 percent molasses in the xylan growth medium.