Fungi are the most important group of organisms contributing to degradation of lignocellulosic biomass

Fungi are the most important group of organisms contributing to degradation of lignocellulosic biomass, among which a particular group of fungi known as the white rot fungi have received much attention in recent years due to their efficient enzyme systems that effectively remove lignin. Out of several pretreatment methods available for removal of lignin, the biological pretreatment methods using lignolytic microorganisms (bacteria or fungi) or their enzymes are environmentally friendly. In the present study, wild basidiomycetous fungi (Ganoderma gibbosum, Lentinus sajor – caju, Trametes sp, Pleurotus sp, Peniophora sp and Lepiota sp) were collected from Rajiv Gandhi University (altitude 345msl, latitude 27° 09′ 00” N and longitude 93° 46′ 15”E), Arunachal Pradesh were screened for their lignocellulolytic enzyme production. After screening (qualitative) two isolates (Ganoderma gibbosum and Lentinus sajor – caju) were selected, for cellulolytic (FPase, CMCase, ?-glucosidase), and hemicellulolytic (Xylanase) and ligninolytic activities {Laccase, Lignin Peroxidase (LiP), Manganese peroxidase (MnP)} under solid state fermentation using two substrates, wheat bran and saw dust. Crude enzyme extract obtained was used for determination of various enzymatic activities (IU/g). Production optimization for lignocellulosic enzymes was carried by classical approaches (OFAT). In classical approach effect of carbon source, nitrogen source, temperature, pH, inoculum amount, inoculum age, moisture ratio, along with effect of metal ions (Cu2+ and Mn2+), surfactants (Tween 20, Tween 60, Tween 80 and Triton X), veratryl alcohol were studied. The lignocellulolytic potential of both fungi and the ratio of individual hydrolytic enzymes in their enzyme complex were significantly affected by the type of substrates used for SSF. Production of laccase, MnP, CMCase and ?-glucosidase by both white rot fungi was substantially more on wheat bran. They produced high amount of laccase on wheat bran (L. sajor-caju – 5580 IU/g and G. gibbosum – 13,560 IU/ g). On saw dust, decline in laccase production was reported by both isolates. G. gibbosum produced substantially good amount of xylanase only on wheat bran (218.9 IU/g). L. sajor-caju did not produce this enzyme on any substrate. Cellulase activity by both fungi was much lesser in comparison to their lignolytic activity. Except for MnP by G. gibbosum on wheat bran, activities of LiP and MnP in both fungi were suboptimal. Among ligninases laccase was produced in more amounts compared to LiP and MnP by both strains. On the basis of enzyme activity observed in the study, both white rot fungi appear to be selective lignin degraders. Addition of enhancers, metal ions (Cu2+ and Mn2+), aromatic compound (veratryl alcohol); and surfactants their laccase activity increases many folds. From the results obtained it was found that there existed a positive effect of metal ions, VA and surfactants on laccase production. Among surfactants, Tween 20 was the best, giving 39180 IU/g laccase in Ganoderma spp. and 37780 IU/g in Lentinus spp. On wheat bran corresponding to 2.9 – and 6.8 -fold increase over control condition. Triton X-100 was the best surfactant providing 29,380 and 19,877 IU/g laccase activity in Ganoderma spp. and Lentinus spp. on saw dust amounting to 2.5- and 3.9-fold increase over control condition, respectively. Ganoderma sp always produced more laccase than Lentinus sp on supplemented substrates. Between substrates, laccase production on supplementation was always more on wheat bran than saw dust. LiP and MnP activities were very low in comparison with laccase in both fungi under control conditions and upon treatment with surfactants; they did not show an appreciable change.
Based on optimization by OFAT, Ganoderma sp KX879638 turns to be potential laccase producer compared to Lentinus sp KYOO6984. It was found that Cu2+, surfactant (Tween 20) and inoculum amount had potential impact on laccase enzyme production by Ganoderma sp. These three independent variables were further evaluated by Box-Behnken method and optimum values for laccase enzyme production were evaluated. Compared to unoptimized conditions (13,560 IU/g), optimization process by RSM (45,877 IU/g) resulted into 3.4 fold increase in laccase production. Finally optimized concentrations were formulated and were used for further laccase enzyme production. The laccase enzyme produced was partially purified by acetone precipitation followed by gel filtration chromatography. Laccase had been partially purified 4.93 fold with a yield of 3.9%. A specific activity of 15,635.3 U/mg protein was found using guaicol as substrate under standard assay conditions. The approximate molecular mass of partially purified enzyme was estimated by Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS) and Laccase activity was visualized in NATIVE gel with guaiacol (10 mm) as substrate in 0.5 M citrate phosphate buffer (pH 5.0). Ph and temperature optima and stability of partially purified laccase were calculated. The laccase showed optimal activity at ph- (3.0 -5.0) and highest oxidation rates were obtained at 25°C. The enzyme was stable over a Ph range of (0.3 -0.5) and at a temperature up to 40°C. Apparent Km and Vmax (38.5mM and 0.25mM) were calculated using guaicol as substrate respectively. The application of crude laccase from Ganoderma sp and Lentinus sp were tested for decolorization of malachite green and it was found that Ganoderma sp turns to be more potent in decolorizing MG, compared to Lentinus sp and can nearly completely decolorize malachite green (100 mg/l) in the presence of 1mM 1-hydroxybenzotriazole (HBT) with 30U/ml of enzyme within 8 -12h at 25°C, pH 5.0 and 150 rpm. In the present work, an effort was made to compare the level of enzyme activity among both the fungi under the study along with between the two substrates used under solid state fermentation. Lignocellulosic enzymes are important in efficient bioconversion of plant residues and are promising for a great variety of biotechnological applications. The present work provides an insight to develop the best conditions for enhancing production of Ligninase. The results showed that wheat bran can be used effectively for laccase enzyme production. Ganoderma gibbosum is the best laccase producers. Laccase enzyme can be successfully be used in various places such as in pollution control (Deinking of various toxic dyes, delignification of agricultural wastes etc.