Optimization of the Medium for the Production of Cellulases by Aspergillus Terreus and Mucor Plumbeus
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Available online a t www.pelagiaresearchlibrary.com Pelagia Research Library European Journal of Experimental Biology, 2012, 2 (4):1161-1170 ISSN: 2248 –9215 CODEN (USA): EJEBAU Optimization of the medium for the production of cellulases by Aspergillus terreus and Mucor plumbeus *Padmavathi. T, Vaswati Nandy and Puneet Agarwal Department of Microbiology, Centre of PG Studies, Jain University, 18/ 3, 9 th Main, Jayanagar 3rd Block, Bangalore, India _____________________________________________________________________________________________ ABSTRACT Fungal cellulases are well-studied, and have various applications in industry, health or agriculture. The present paper investigates the isolation of marine fungi (Aspergillus terreus and Mucor plumbeus) for the production of cellulase using submerged fermentation technique. Ten different substrates such as rice bran, wheat bran, bamboo leaves, banana leaves, peepal leaves, sugar cane leaves, lantana leaves, ragi straw, maize leaves and eucalyptus leaves were collected from different parts of rural Bangalore (India) and were used as substrates for the cellulase production; of which lantana leaves gave best result. The fermentation experiments were carried out in Erlenmeyer flasks using pretreated Lantana leaves. Lantana leaves gave best enzyme activity of 213. 3IU/ ml and 206 IU/ ml by Aspergillus terreus and Mucor plumbeus respectively. Various parameters such as carbon source, nitrogen source, pH and incubation temperatures were studied for the production of cellulases. Incorporation of lantana as carbon source (660 IU/ ml), ammonium sulphate as nitrogen source, pH 3 (240. 07 IU/ ml) and incubation temperature at 37 o C (100 IU/ ml) gave good enzyme yield with Aspergillus terreus and Mucor plumbeus respectively. The degree of saccharification was also assayed on the basis of amount of reducing sugar released. The percentage of saccharification with respect to lantana leaves in presence of Aspergillus terreus and Mucor plumbeus were found to be 56% and 28% respectively. Keywords: Cellulases, Lantana, Marine fungi, Aspergillus terreus, Mucor plumbeus _____________________________________________________________________________________________ INTRODUCTION In a time of rising energy prices, utilization of abundantly available biomass as an alternative resource is one of the thrust area of research. Utilization of renewable, economical, abundantly available agro waste for the production of useful products is an increasing trend in recent years. Cellulose is the most abundantly available biomass on earth. It is the main product of photosynthesis in terrestrial environments, and most abundant renewable biosource produced in biosphere. Cellulose is commonly degraded by an enzyme cellulase. Cellulases are widely used in the food, textile, laundry, baking, brewing, pulp and paper industries from biomass and genetic engineering [1- 5]. Microbial conversion of cellulose/ lignocellulosic biomass into useful byproducts is a complex process involving combined action of 3 enzymes namely endogluconase, exogluconase and β- glucosidase. Although cellulases are distributed throughout the biosphere, they are manifested in fungi, bacteria and a few actinomyctes [6, 7]. These microorganisms produce cellulases to release sugar for cell growth and product formation under certain 1161 Pelagia Research Library Padmavathi. T et al Euro. J. Exp. Bio., 2012, 2 (4):1161-1170 ______________________________________________________________________________ environmental conditions. More than 14,000 species of fungi have been found to be active in cellulose degradation [8- 11]. Among all the fungi, marine fungi are most common in decomposing wood and plant detritus in coastal water [12- 15]. Marine fungi are also common in calcareous animal shells, algae and corals. Most of the marine fungi belong to ascomycetes and few belong to basidiomycetes. Marine fungi usually influence the biotechnological production because of their special adaptations to their environment. The physical factors that usually influence the marine fungi are salinity, pH, low water potential, high concentration of sodium ions, low temperature, oligotrophic nutrient conditions and high hydrostatic pressure. Many agricultural by products from agricultural activities and agro based processing litter the environment and constitute waste problems. Agro wastes such as rice straw, wheat bran, corn stover, sugar cane bagasse, pomace, corn cobs etc are used as substrate in solid state fermentation [16, 17]. The use of agro wastes as the basis for the cultivation media is a matter of great interest, aiming to decrease the costs of energy production and meeting the increase in awareness on energy conservation and recycling. Cellulase production by different organisms in submerged fermentation state has received more attention and is found to be cost prohibitive because of high cost of process engineering [18- 20]. Twenty fungal isolates were isolated from marine water. Most of the isolates, obtained from marine source, were Aspergillus spp., Mucor plumbeus which was very dominant in marine environment. The present paper investigates the isolation of marine fungi, effect of different substrates, pH and different incubation temperature on the production of cellulase by fungi. MATERIALS AND METHODS Isolation: Marine fungi were isolated from marine source (Pondicherry and Mangalore) using standard microbiological techniques. A sample of 10 gram was placed in a graduated cylinder, sterilized distilled water was added to make a total of 100 ml. Serial dilution was done till 10 -4. 1 ml of desired dilution (10 -3and10 -4) was transferred aseptically into a potato dextrose agar (PDA) prepared from marine water adding one drop of 20% lactic acid to suppress bacterial growth. Plates were incubated at room temperature for 5 days. After incubation, small portion of mycelium from each fungal colony was transferred into PDA slants. Identification: Identification of the tested fungal isolates was done depending on the morphological characters of fungi and comparing them with those that are present in the identification references [21]. Twenty fungal isolates were isolated from marine water. Most of the isolates, obtained from marine source, were Aspergillus spp. which was very dominant in marine environment. Among all the fungi Aspergillus terreus and Mucor plumbeus were selected as it showed high cellulase production on liquid state fermentation. The potential fungal isolates were assayed enzymatically for cellulases. The assay methods employed were: (a) Cellulolytic enzyme Assay: Test fungi were cultivated on basal medium supplemented with 0.4 % glucose and solidified with 1.6 % w/v agar. A single agar disc cut from the actively growing colony margin of culture was used to inoculate each assay medium. Basal media with concentration of 0.1- 0.2% w/v are sufficient to support cellulose degrading fungi . (b) Filter paper degrading method: Filter paper used in this assay was be almost 100% cellulose. Cellulolytic basal medium (CBM) was prepared, 10 ml aliquots was transferred to glass culture bottles and was autoclaved. One 25x 5mm strip of sterile filter paper was added aseptically to each bottle making sure that all filter paper stripes were completely submerged. The test fungus was inoculated and uninoculated bottles were retained as control. Care was taken that bottle caps were loosely fitted to allow adequate gas exchange. The bottles were incubated at 25 o C in darkness and were examined daily for 10 days. Degradation of filter paper was assessed based on increased opacity and physical degradation in comparison with uninoculated controls [22]. (c)Cellulase agar clearance (cellulose agar) method: This assay uses ball- milled acid swollen or microcrystalline cellulose. Generally microcrystalline cellulose is degraded more slowly than ball-milled or acid swollen cellulose. CBM medium was prepared by incorporating 4% w/v cellulose, 1.6% w/v agar and was autoclaved. It was transferred aseptically to petri dishes (agar was cooled until viscous and was mixed gently before pouring to ensure 1162 Pelagia Research Library Padmavathi. T et al Euro. J. Exp. Bio., 2012, 2 (4):1161-1170 ______________________________________________________________________________ uniform distribution of cellulose in the agar medium). The test fungus was inoculated. The culture was incubated at 25 o C in darkness and was examined daily for 10 days. Cellulolysis was assessed based on clearance zones of the opaque agar around growing colonies. (d)Dye staining of Carboxy methyl cellulase (CMC): CMC is a substrate for endogluconase and so can be used as a test for β glucanase activity. This assay is a good indicator to check the Cellulolytic ability of fungi. CBM was prepared, supplemented with 2% w/v of low viscosity CMC and 1. 6 % w/v agar, autoclaved and transferred to petri dishes aseptically. Test fungus was inoculated and incubated at 25 o C in darkness. When the colony diameter is approximately 30 mm, agar plates were stained using Congo red and left for 15 mins. Plates were decanted and agar surface was washed with distilled water. The plates were flooded with 1M Nacl for 15 mins to destain. Inoculum Preparation: The spore suspension was used as inoculum in the present studies. It was prepared from a 7 days old slant by adding 10 ml of sterilized marine water to it. The spores were scratched with the help of a sterilized wire loop to make a homogeneous suspension of spores. Spore count