1. INTRODUCTIONLignocellulosic biomass comprises cellulose, hemicellulose and lignin, of which lignin is the most recalcitrant to biodegradation and only higher fungi are capable of degrading this polymer via an oxidative process involving an array of oxidases, peroxidases and hydrolytic enzymes [1,2]. Cellulose, hemicellulose and lignin form structures called microfibrils, which are organized into macrofibrils that mediate structural stability in the plant cell wall [2]. The complex lignin polymer forms a cementing envelop around cellulose in lignocellulosic wood; thus the success of the bioconversion of lignocelluloses into fermentable sugars lies in the removal of lignin wrap around the cellulose fibers packed inside. Cellulose is a fibrous, insoluble and crystalline polysaccharide consisting of D-glucose residues linked by β-1, 4-glucosidic bonds; owing to its highly ordered crystalline structure, cellulose is more resistant to hydrolysis than hemicellulose. Hemicellulose macromolecules are often polymers of pentoses (xylose and arabinose), hexoses (mostly mannose) and a number of sugar acids, while cellulose is a homogenous polymer of glucose [3]. Cellulose is the most abundant biopolymer in nature, being degraded to glucose through the synergistical hydrolysis of three classes of cellulases, viz., including endo-β-1,4-glucanase (EC 3.2.1.4), exoglucanase or cellobiohydrolase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21). Glucose from the hydrolysis of cellulose can easily be fermented to useful industrial products such as ethanol, lactic acid, single cell protein and other value-added products [4]. There are different methods for the production of lowcost enzymes by the activity of microorganisms.
Some members of all classes of microorganisms, viz., fungi, actinomycetes and bacteria are known to produce cellulase isoforms [5,6]. Among them, mycelial fungi constitute a group unique of microorganisms showing prominent lignocellulolytic activity. Lignocellulolytic fungi are often divided into three groups, viz., white rot, brown rot and soft rot fungi. Trichoderma spp., the ascomycetous green-spored having cosmopolitan distribution are classified under white rot fungi. The surface colony colour is white and scattered greenish patches become visible as the conidia are formed and may form concentric rings. The highly branched conidiophores bear phialides singly or in groups. Trichoderma spp. are ubiquitous colonizers of cellulosic materials and can thus often be found wherever decaying plant material is available as well as in the rhizosphere of plants, where they can also induce systemic resistance against pathogens [6]. At present, the various strains of Trichoderma spp. are recoganized by the researchers as the steadiest and safest fungi for the production of cellulase and hemicellulases. Mostly T. reesei and its variants are widely employed for the commercial production of hemicellulases and cellulases. T. reesei might be a good producer of hemi-cellulolytic and cellulolytic enzymes but is unable to degrade lignin. The ability of fungi to degrade lignocellulosic materials is due to their highly efficient enzymatic system [7]. T. viride have mostly been studied for cellulase production. There exist many studies describing the optimization of process parameters for the production of cellulase using submerged fermentation from T. viride [8]. Species of Trichoderma are shown to have lignolytic, hemicellulolytic and cellulolytic activities, but no organism is reported yet having the embodiment of all these enzyme complexes together. Based upon this background, the specific objectives of this study are to: 1) Confirm the novel isolate as T. viride by morphological characteristics; 2) Check the lignocellulolytic activities of T. viride; 3) Analyze cellulase production potential of T. viride in detail; and 4) Check the nature of pigment produced by T. viride.
2. MATERIALS AND METHODS2.1. MicroorganismPure T. viride culture procured from the Kerala Agricultural University, Mannuthi, Kerala (Receipt No.17 dated 15-12-2011) was used for the present study. Stock cultures were maintained on potato dextrose agar (PDA) slants at 4˚C. Analytical grade chemicals from Merck India Ltd. and Himedia were used.
2.2. Morphological CharacterizationMorphological characteristics of T. viride were determined by lactophenol cotton blue staining under binocular microscope attached with Image Analyzer (Nikon Eclipse 400). Phase-contrast images of T. viride spores were taken using Leica M80 (Germany).
2.3. Screening for Lignocellulosic Activity6 day old T. viride on PDA slants was cultured on basal mineral salt medium (BSM) containing (g/L) NaNO3—2, K2HPO4—1, KCl—0.5, MgSO4·7H2O—0.5, proteose peptone—2, agar—20 containing 0.5% CMC (for cellulase activity), xylan (for hemicellulase activity) or lignin (for ligninase activity) was used for preliminary screening of cellulase, hemicellulase or ligninase activities, respectively. The agar plates were incubated at 28˚C. Growth after incubation indicates lignocellulolytic activity of T. viride.
2.4. Screening for Cellulolytic Activity on CMC MediumCellulolytic activity of T. viride on CMC medium was determined qualitatively by iodine plate assay. 6 day old T. viride from PDA slant were cultured on BSM-agar medium containing CMC for enrichment and incubated for 4 days, then the plates were flooded with iodine solution (containing 1% iodine crystals and 2% potassium iodide), incubated at 30˚C for 15 min, and excess stain was washed off for visual observation. Cellulolytic activity was indicated by the clear zone around the colonies.
2.5. Screening for Hemicellulase Activity on Xylan MediumHemi-cellulolytic activity of T. viride on xylan medium was determined qualitatively by congo-red plate assay. 6 day old T. viride on PDA slant was cultured on BSM agar containing xylan and incubated for 4 days, then the plate were flooded with 10 ml Congo-red (0.1%) solution, incubated for 15 min, then excess stain was drained off, and 10 ml of 1 M NaCl was added for destaining; opaque clear zone formation around the colonies indicates positive result.
2.6. Screening for Ligninase Activity on Lignin Medium6 day old T. viride from PDA slant was cultured on BSM containing lignin for enrichment. T. viride culture was grown for 5 - 6 days on BSM-lignin medium; growth on lignin medium indicates positive result.
2.7. Pigment ExtractionPigmented biomass of five days old culture on PDA agar plate was collected in various solvents (distilled water, acetone, hexane, chloroform or methanol) and stirred well on a magnetic stirrer (10 min), followed by centrifugation (9000 × g for 15 min, 4˚C). The supernatant was pooled and its absorption spectrum was determined using UV-visible spectrophotometer (Elico BL 200 double beam biospectrophotometer).
2.8. Cellulase Production Characteristics of T. virideFor making the inoculum uniform; spore suspension in sterile ddH2O was stirred well on a magnetic stirrer aseptically for 5 min, and number of spores in 1 mL of suspension was calculated by counting the spores using Neubauer haemocytometer. Different parameters like effects of incubation period, pH, temperature, CMC concentration, synthetic carbon sources, natural carbon sources and nitrogen sources were optimized.
For initial studies, BSM supplemented with CMC (0.5%) was used. In all studies, 100 µL of spore suspension (~7.2 × 106 spores/mL) was used for 10 mL medium. All flasks were incubated in an environmental shaker (Scigenics Biotech) at 150 rpm, 28˚C for 10 days. Various culture parameters studied for cellulase production were: pH (1 to 8 using different buffers), temperature (25˚C, 28˚C, 31˚C, 33˚C, 35˚C and 37˚C), CMC concentrations (0.75%, 1.0%, 1.25% and 1.5%), various synthetic carbon sources (0.5% glucose, lactose, maltose, dextrose and sucrose), natural carbon sources (0.5% banana flour, banana peel flour, tapioca flour, potato flour and jack seed flour) and nitrogen sources (0.2% peptone, beef extract, yeast extract, sodium nitrate and ammonium nitrate). Growth and yield were monitored up to 7 days. The culture broth was centrifuged (9000 × g for 15 min at 4˚C) and the supernatant was used for cellulase assay.
2.9. Cellulase AssayEnzyme production was quantitatively measured by DNS (3,5-dinitrosalicylic acid) spectrophotometric assay by the method of Miller [9]. Fermented culture broth was centrifuged at 9000 × g for 10 min at 4˚C and supernatant was used as crude enzyme solution for assays. Reaction mixture contains 0.5 mL of 1% CMC in 0.1 M sodium citrate buffer (pH, 4.8) and 0.5 mL of crude enzyme solution added, then incubated at 50˚C for 30 min. The reaction was stopped by adding 3 mL DNS and incubated in a boiling water bath for 5 min and cooled. The absorbance was measured at 540 nm (Elico double beam bl 200 bio spectrophotometer). One unit (U/mL) of cellulase activity is defined as the amount of protein (cellulase) required to liberate 1 micromole of reducing sugar (D-glucose) from CMC per min under the assay conditions. Cellulase activity was calculated using the formula,
. Where, ΔE = absorbance at 540 nm, Vf = final volume of reaction mixture including DNS, Vs = crude supernatant (ml) containing cellulase used, Δt = incubation time for hydrolysis, ∑ = extinction coefficient of glucose (0.0026), d = diameter of cuvette.
2.10. StatisticsAll studies were repeated at least three times to check the reproducibility of the results, and average values were given with standard deviation. Microsoft Excel was used to draw the figures.
3. RESULTS AND DISCUSSIONResults shown here are the average values of at least 3 independent experiments. The fungal culture started to grow as white mycelia on PDA plate, then spread all over the surface of the medium as a dark-green mat. It was stained using lactophenol cotton blue and observed under Image Analyzer. The mycelia, conidiophores bearing warted phialides singly or in clusters and numerous conidiospores were observed (Figure 1A). Usually the phialides were ellipsoidal or flask shaped inflated at the base dark-green coloured spores with smooth outer wall were found to be spherical and biconcave, resembling human erythrocytes (Figures 1B and C). This preparation was observed under phase-contrast microscope. Bissette [10] described that the species in Trichoderma (Ascomycetes, Hypocreales) have narrow and flexuous conidiophores and branches, with branches and phialides uncrowded, frequently paired, and seldom with more than three elements in a whorl. Its type species viride typically produced warted conidia [11], as we described (Figure 1A). Thus, the novel fungal culture used in this study can be confirmed as T. viride.
3.1. Lignocellulolytic ActivityFigure 2 clearly shows that the fungus harbours an en-