Influence of Various Parameters on Cellulase and Xylanase Production by Different Strains of Trichoderma Species

Abstract

The main purpose of this study is to reduce the production cost of cellulase by using alternative carbon source such as wheat bran residue and optimized fermentation medium for high yields. In the present investigation, to isolate the novel cellulase producing fungi, Trichoderma viride and T. reesei from the soil samples and to optimize the physicochemical and nutritional parameters for cellulase and xylanase production. Wheat bran residue was found to be the best substrate source for the production of cellulase and xylanase by Trichoderma spp. Optimal concentration of wheat bran residue for the production of cellulase and xylanase was 1.0% (w/v). Optimum temperature, pH and incubation time of the medium for the cellulase and xylanase production by T. viride and T.reesei was 28° C, 5.5 and 120 h respectively. Cellulase production from T. reesei can be an advantage as the enzyme production rate is normally higher as compared to other fungi.

Keywords: Trichoderma spp. Cellulase; Xylanase; Wheat bran; Cellulose powder; Bio fuel

Introduction

Cellulases are multi-enzyme complex proteins secreted by diverse microorganisms that catalyze the conversion of most abundant organic polymer i.e. cellulose to smaller sugar units. These enzymes are considered as third largest industrial enzymes because of their tremendous application in cotton processing, paper recycling as well as feed additives. Currently due to rising oil prices and global warming, cellulase enzymes gained much importance as it plays a pivotal role in producing sustainable biofuel by the degradation of cellulosic biomass. Trichoderma species is widely studied fungi for cellulose degradation [1]. The cellulase produced by Trichoderma is resistant to chemical inhibitors and stable in stirred tank reactors at pH 4.8, 50° C for 48h or longer [2]. Majority of the cellulolytic microorganisms (Trichoderma spp.) produce complete cellulase enzymes that acts synergistically on native cellulose [3] besides producing other protein byproducts that helps in cellulose degradation [4]. T.reesei ß-glucosidase (BGL) is subject to product inhibition and though it is sufficient to support growth on cellulosic material; it is often supplemented with Aspergillus BGLs for complete biomass saccharification at industrial level [5]. Nevertheless, it is the best cellulase available today.

The major bottle neck against comprehensive application of cellulases in biofuel industry is the high cost of the enzyme production and source of substrate. Therefore, extensive research is needed to enhance the enzyme activity so that less enzyme is needed for the complete hydrolysis of biomass. In this investigation, cellulase producing strains of Trichoderma spp. isolated from soil samples were subjected to optimization of media and cultivation parameters for cellulase and xylanase production.

Materials and Methods

Chemical reagents

All chemicals and reagents used were of analytical grade. Different carbon sources such as birch wood xylan, Salicin, carboxymethyl cellulose (CMC) and glucose were obtained from Hi-media, Mumbai India; whereas wheat bran was purchased from local market of Srinagar, Kashmir, India.

Isolation of fungal culture

To isolate cellulase producing microorganisms, soil sample from decomposing sites of Botanical garden of Kashmir University was taken. The decomposing residue brought in the laboratory was kept at 4^°C ± 1. The cellulose-hydrolytic fungus was isolated by direct agar plate method of Warcup [6] using Czapek Dox Agar medium.

Purification of fungal isolate

The contaminated cultures of fungal isolates were purified and derived through single spores, using dilution plating [7]. To ensure purity, cultures were derived from single spore in each case. The bacterial contamination was prevented by using antibiotic streptomycin (50mg/100 ml) in the medium during isolation and purification of fungi. After ensuring purity, the cultures were maintained in potato dextrose agar (PDA) slants at 4°C.

Screening for cellulolytic activity

Screening for cellulolytic activity was made using PDA media supplemented with 5% carboxymethyl cellulose (CMC). After certain incubation, the plates were stained by Congo red followed discolouration by 1M NaCl. The cellulase activity of each culture was determined by measuring the zone of clearance on agar plate [8].

Preparation of enzyme

Isolated fungal cultures were grown in 500 ml flask containing Mandel and Reese medium [9] supplemented with different carbon sources (1% of wheat bran, cellulose powder) at 28^° C for 7 days. 10 ml samples were withdrawn on 7^th day of inoculation and mycelia was separated by centrifugation at 10000 RPM for 20 min. Supernatant obtained was used as source of crude enzyme [10].

Quantitative enzyme assays

Assay of endoglucanase activity (CMCase assay): Endoglucanase activity was measured as per method described by Mandels et al. [11] with slight modification. Reaction mixture consisting of 0.5 ml of 1% CMC in citrate buffer (pH 4.8), 0.5 ml of enzyme sample and the reaction mixture is incubated at 50^°C for 30 minutes. The reaction was stopped by adding 3 ml of DNS in the boiling water. After cooling down, the absorbance was taken at 540 nm [12].

Assay of exo-glucanase (FPase): Fpase activity was assayed by measurement of reducing sugars in the reaction mixture containing Whatman No. 1 filter paper (1.0 cm × 6.0 cm ~ 50.0 mg) as substrate in 1ml sodium citrate buffer (pH 4.8) and 0.5 ml enzyme sample and the reaction mixture is incubated at 50°C for 60 minutes and finally reaction is stopped by adding 3 ml of DNS reagent. The suspension is mixed well and the tubes are transferred to a boiling water bath for exactly 5 min. Tubes are immediately cooled in the cold water and absorbance is then measured at 540nm [11].

Assay of β–glucosidase (BGL) activity: BGL activity in measured as per the method described by Mandels [13]. Reaction mixture consisting of 0.5 ml of 1% salicin prepared in citrate buffer (pH 4.8), 0.5 ml of enzyme sample and then incubated at 50° C for 30 minutes. After appropriate period of incubation, the reaction is stopped by adding 3 ml of DNS reagent and the reaction mixture is boiled for exactly 5 minutes in vigorously boiling water bath. The tubes are cooled immediately in cold water and the absorbance is then measured at 540 nm. In accord with the International Union of Biochemistry, one enzyme unit (U) equals to 1μmol of glucose released per minute.

Xylanase activity: Xylanase activity was determined according to method described by Bailey et al. [14]. Reaction mixture consisting of 1.0 ml of crude extracellular enzyme sample, 1 ml of 1% birch wood xylan (prepared in 0.05 M Na-citrate buffer, pH 5.3) and 1 ml of 0.05 M citrate buffer. The mixture was incubated at 55°C for 10 min. The reaction was stopped by the addition of 3.0 ml of 3, 5- dinitrosalicylic acid (DNS) and the contents were boiled for 15 min [15]. After cooling, the color developed was read at 540 nm.

Optimization of culture conditions for enzyme production

Effect of incubation period in enzyme production: Fermentation period is important parameter for enzyme production by Trichoderma spp. In this study, experiments fermentation was carried out up to 6days and production rate measured at 24 h intervals.

Effect of pH and temperature on enzyme production: The most suitable pH of the fermentation medium was determined by adjusting the pH of the culture medium at different levels in the range of pH 4 to 5.5 using different buffers. In order to determine the effective temperature for cellulase production by the Trichoderma spp., fermentation was carried out at 28 to 32 ± 2°C.

Effect of various carbon sources on enzyme production: Effect of various carbon compounds viz., cellulose powder, wheat bran were used for studying. The broth was distributed into different flasks and 1% of each carbon sources were then added before inoculation of the strain and after culture inoculation, the flasks were incubated for 7 days at 28 ± 2°C.

Statistical analysis

Data presented on the average of three replicates (±SE) obtained from there independent experiments.

Results and Discussion

Isolation and identification of fungal strain

A total of two isolates of fungi were isolated and identified. Identification of fungal strain was made on the basis of morphological and microscopic characteristics by using standard reference manuals [16, 17] (Figure1, 2).