MICROBIAL PRODUCTION OF CELLULASES FOR BIOCONVERSION OF LIGNOCELLULOSIC WASTES

Abstract

The prime emphasis of the present investigation was to define the factors allowing maximum bioconversion of the major, high cellulose containing lignocellulosic waste materials, sugarcane bagasse and water hyacinth {Eicchornia crassipes) biomass into fermentable sugars. Sugarcane bagasse is generated in large amounts from the sugarcane industries, has no further application and is burnt; whereas, water hyacinth (Eicchornia crassipes) an aquatic biomass, found in ponds, lakes and rivers is a serious concern for the water resources. In an attempt to isolate a strain with potential bioconversion ability, twenty two (fungal and bacterial) strains were isolated from decomposing substrates, among themAspergillus nigerRK-3 was found to have maximum cellulase producing ability. To further ascertain its bioconversion capability, the levels of cellulases produced were evaluated using the commercially available synthetic cellulosic substrates under submerged state fermentation. Production of higher cellulase levels was detected with these substrates. The wild type isolate Aspergillus niger RK-3 thus appeared as an attractive choice for bioconversion of lignocellulosic substrates. Analysis of cellulase production with sugarcane bagasse and Eicchornia crassipes biomass showed that high pressure steaming followed by alkali treatment of the sugarcane bagasse and Eicchornia crassipes biomass led to significant increase in the cellulase production as compared to the untreated substrates. Evaluation of physical and biological factors affecting the cellulase production denoted that 5% inoculum, 130 rpm as the agitation rate along with the (ii) pH at 4.8 and a temperature of 30°C were suitable for enzymatic production, however, temperature required for measuring the activity was 50°C. Among the medium nutrients and other chemical factors derived, use of 1% cellulosic substrates, ammonium sulfate, 2.1 gl"1 along with ammonium chloride, 0.4 gl"; potassium dihydrogen phosphate, 2.45 gl'1; vanillin, 0.5 gl"1 ; succinic acid, 1.0 gl" and cellobiose 2.0 gl"1 led to maximum production of the cellulases. The production medium denoted as R6m containing the sugarcane bagasse and the constituents as defined above led to significant increase in CMCase (83%), FPase (59%) and (3- glucosidase (288%) levels with respect to the enzymatic production with Mandels and Weber's medium. Similar induction in activities of CMCase (112%), FPase (79%) and P-glucosidase (316%) were observed with Eicchornia crassipes biomass as the substrate. A further improvement in cellulolytic activities of wild type Aspergillus niger RK-3 was attempted by genetic manipulation using physical (UV), chemical (NTG, sodium azide and colchicine) and mixed mutagenic treatments. A high yielding and end product resistant mutant UNSC-442 was finally selected having substantially higher cellulase production abilities. The mutant UNSC-442 resulted into CMCase (7.18 IU ml"1), FPase (6.0 IU ml"1) and p-glucosidase (2.55 IU ml"1) activities with treated sugarcane bagasse as the substrate and the activities respectively were 89%, 130% and 54.5% higher as compared to the cellulase production with wild type Aspergillus niger RK-3 strain under similar conditions. A similar level increase in cellulases was also observed with Eicchornia crassipes biomass. The extent of cellulose degradation observed was 61.5% - 67% higher with the mutant strain. The

protein profile alongwith the zymogram einalysis indicated increasing levels of CMCase (91.2 kD), FPase (68 kD) and p-glucosidase (52.4 kD) components in the mutant UNSC-442 strain as compared to the activities detected in the Aspergillus niger RK-3 wild type strain. Scanning electron microscopy demonstrated entirely distinct morphological features of the mutant UNSC-442 strain than its parental strain. Incubation under co-cultured condition using Aspergillus niger RK-3 and Trichoderma reesei MTCC-164 resulted into 23-33% increase in CMCase and FPase activities and 13% increase in P-glucosidase activity over the respective maximum activities observed under single culture condition with sugarcane bagasse as the substrate. Similarly, a 20-24% increase in CMCase and FPase and a 13% increase in P-glucosidase activity were observed with Eicchornia crassipes biomass as the substrate. On the other hand the co-cultivation of mutant UNSC-442 along with Trichoderma reesei MTCC-164 showed a further increase (8-23%) in CMCase and FPase activities and around 24% increase in p-glucosidase activity as compared to the activities obtained with mutant UNSC-442 strain with sugarcane bagasse in single culture conditions. Similar results were obtained with Eicchornia crassipes biomass under the conditions as referred for the sugarcane bagasse substrate. Further, the sugars generated from the enzymatic hydrolyzates of sugarcane bagasse and Eicchornia crassipes biomass was found to be a cheaper and excellent substrate for the single cell protein, gluconic acid and citric acid production.