Journal of Plant and Pest Science (ISSN: )

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1 Open Access (ISSN: ) Vol. 1, Issue.2: (October 2014) journals.sfu.ca/jpps/index.php/jpps/index Original Research Article Received: 29 July 2014, Accepted: 15 Aug 2014, Published: 20 Oct 2014 Isolation and Cellulase production of Aspergillus niger Bitty BM and Nair N N Author Info Post Graduate and Research Department of Botany Mar Thoma College, Tiruvalla *Corresponding author s bittybyju1305@gmail.com Key Words Aspergillus niger, Cellulase, Cowpea, Seed borne pathogen Abstract Aspergillus niger was isolated from the seed of Vigna unguiculata var.sesquipedalis(l) Verdcourt. Biomass as well as cellulase production of isolate were quantitatively determined in five different broth media. The selected broth media were Asthana Hawker s, Potato Dextrose, Richards s, Sabouraud Dextrose and Yeast Extract. Aspergillus niger produced more biomass in Richards s broth followed by Sabouraud Dextrose broth, Potato Dextrose broth, Yeast Extract broth and Asthana Hawker s broth whereas cellulase production was the highest in Potato Dextrose broth and Sabouraud Dextrose broth followed by Richadrs s broth, Asthana Hawker s broth and Yeast Extract broth. Introduction Cellulases are inducible enzymes which are synthesized by microorganisms during their growth on cellulosic materials. They are studied extensively due to their application in the hydrolysis of cellulose, the most abundant organic polymer and potential source of utilizable sugars, which serves as a raw material in the production of chemicals and fuel (Lee and Koo, 2001). Cellulases have a wide range of industrial applications. The main applications include textiles, paper and pulp, food, animal feed, fuel and chemical industry. They can also be used in waste management, pharmaceutical industry, protoplast production and genetic engineering (Bhat, 2000). In addition to these industrial applications, it has some negative roles in agricultural field. The parasitic fungi utilize these enzyme machineries to penetrate the host plant body and develop pathogenesis (Wolfgang et al., 1977). Penicillium and Aspergillus are among the most studied cellulolytic fungi (Sukumaran et al., 2005; Favela Torres et al., 2006; Adeleke et al., 2012). It is well established that Aspergillus niger produces number of cellulolytic enzymes. Ikeda have reported the isolation of a homogeneous cellulolytic enzyme from Aspergillus niger with an unusually low P H (2.5) (Ikeda et al., 1967, 1973). ). A wide range of Aspergillus species have been identified to possess all component of cellulase enzyme system (Vries and Visser, 2001). In a study by Al Hindi et al. (2011) Xylanase, cellulase, and α-amylase were detected in the cell-free broth of Fusarium oxysporum, Aspergillus flavus, Aspergillus niger, Rhizopus stolonifer. Thus the present study was undertaken to measure the enzyme producing ability of Aspergillus niger in different broth media. MATERIALS AND METHODS Source of organism Lola variety of Cowpea (Vigna unguiculata var. Cite this article: Bitty BM and Nair N (2014) Isolation and Cellulase production of Aspergillus niger, Journal of Plant and Pest Science, 1 (2):

2 sesquipedalis (L) Verdcourt) was selected for the isolation of seed borne pathogen Aspergillus niger. Isolation of Aspergillus niger Seeds were surface sterilized with 0.01% aqueous Mercuric chloride solution and thoroughly washed thrice with sterile distilled water and then transferred to culture tubes containing Water-Agar medium under aseptic condition (Neergaard, 1977). It is kept for seven days of incubation at 30 0 C. After seven days of incubation the inoculated tubes were tested for the presence of internal seed borne pathogen. Then the pure culture of isolate was prepared and it was identified by the reference of Gilman (1945). The isolate were maintained in PDA slants for further study. Determination of biomass production The isolates maintained on PDA medium were cut into small pieces with the help of sterile cork borer of 1cm width. These pellets of isolates were cultured in different broth media and kept for 7 days incubation at 30 0 C. The initial P H of the medium was maintained at 4.5. After 7 days of incubation the mycelial mats were collected on a pre weighed Whatman No. 1 filter paper, washed thrice with distilled water and oven dried at 70 o C for 24hrs and reweighed. The mycelial weights were calculated by subtracting the final and initial weights (Nair, 1995). Determination of cellulase (endoglucanase) activity The isolate was cultured in five different broth media at 30 0 C with an initial p H of 4.5. After seven days of incubation, the culture filtrates were collected which served as crude enzyme source. Liberation of sugar, as Glucose was measured by DNS method (Gascoigne and Gascoigne, 1960). For endoglucanase activity, the reaction mixture consisting of 1ml of enzyme sample and 3.5ml of 0.55% CMC in Sodium Citrate buffer (p H 5.4) was incubated at30 0 C for one hour. After removing the reaction mixture, 1ml of aliquot from each tube was taken out in another tube to which 1ml of Dinitrosalicylic acid reagent was added. The mixture was then heated at water bath for five minute, cooled and then 8ml of distilled water was added to make up the volume up to 10ml. Control was prepared with ten minute boiled enzyme. The absorption of the sample was measured on UV-Visible Spectrophotometer at 540nm and the amount of reducing sugar liberated was obtained by referring a standard graph prepared by using an aqueous solution of D-glucose (Nair, 1995). One unit of cellulase enzyme activity is defined as the amount of enzyme required to liberate reducing sugar equivalent to 10µg of glucose (Olutiola, 1976). RESULTS AND DISCUSSION Aspergillus niger was isolated from the seeds of Lola variety of Cowpea (Vigna unguiculata var. sesquipedalis (L) Verdcourt). Aspergillus niger colony appeared to be brown black in colour on PDA medium and they are colourless on the reverse side. The hyphae are well developed, profusely branched, septate, and hyaline, multinucleate. The mycelium produces an abundance of conidiophores and they arise singly from the somatic hyphal cell called foot cell. Conidiophores are long erect hyphae, each terminating in a bulbous head, the vesicle on which bottle shaped sterigmata arise all over the surface and produce chains of conidia at the tips. The conidia are typically globose. The colour of the conidiophores and the conidia impart brown black colour to the colony. Vesicle is globose and are 43.5µm in diameter and the primary sterigmata reach up to 60X8µm. Secondary sterigmata are short and are 10X3.5µm. B. Fawole et al., (2006) isolated Aspergillus niger from Cowpea seeds. Raj et al., (2007) also recorded Aspergillus spp., Cercospora spp., Penicillium spp. from farmers saved Cowpea seed. The isolate was grown in five different broth media for testing their effect on biomass and cellulase production. All the five media showed narrow to wide range in biomass production. Richards s broth showed the highest biomass production (311mg) whereas medium biomass production observed in 75

3 Table 1 - Effect of broth media on the growth of fungal isolate Isolates Biomass in mg Broth media AH PD RH SD YE Aspergillus niger Cellulase (Cx) Cellulase (Cx) AH PD RH SD YE Fig.1: In vitro production of cellulase- endo (1,4)-beta-D-glucanase (endoglucanase) in five different broth media AH Asthana Hawker s broth PD Potato Dextrose broth RH Richards s broth SD Sabouraud Dextrose broth YE - Yeast Extract broth 76

4 Sabouraud Dextrose broth and Potato Dextrose broth. Least biomass (52mg) obtained in Yeast Extract and Asthana Hawker s broth (Table.1). A number of microorganisms particularly fungi possessing cellulose degrading enzymes have been isolated and ACKNOWLEDGEMENT We express our sincere thanks to Dr. Mary Chacko, Head of the Department of Botany, Mar Thoma College, Thiruvalla for giving me an opportunity and providing necessary laboratory facilities. I wish to express my studied extensively (Kim et al., 2003). Many profound gratitude to Dr. Sajeena, Scientist of Journal research of Plant being and Pest Science Kerala achieved in cellulase production and their characterization Agricultural University Research Centre, Kallunkal, during receny years (Khan and Husaini, 2006; Milala et al., 2009; Roslan et al., 2011). Aspergillus are among the most Thiruvalla for providing seed samples for this work. REFERENCES studied cellulolytic fungi (Sukumaran et al., 2005; Adeleke et al., 2012). It is well established that Aspergillus niger produces number of cellulolytic enzymes. Ikeda have reported the isolation of a homogeneous cellulolytic enzyme from Aspergillus niger with an unusually low P H (2.5) (Ikeda et al., 1973). In a study by Al Hindi et al. (2011) Xylanase, cellulase, and α-amylase were detected in the cell-free broth of Fusarium oxysporum, Aspergillus flavus, Aspergillus niger, Rhizopus stolonifer. Out of the five different broth media Potato Dextrose broth and Sabouraud Dextrose broth were found to be the best for cellulase production. Richards s broth showed moderate cellulase production whereas Asthana Hawker s and Yeast Adeleke AJ, Odunfa SA, Olanbiwonninu A and Owoseni MC (2012) Production of Cellulase and Pectinase from Orange Peels by Fungi, Nature and Science., 10 (5): Al Hindi RR, Al Najada AR and Mohamed SA (2011) Isolation and identification of some fruit spoilage fungi: Screenings of plant cell wall degrading enzymes. African journal of Microbiology Research, 5 (4): Bhat M K (2000) Cellulases and related enzymes in biotechnology. Biotechnol. Adv., Favela Torres E, Volke Sepulveda T and Viniegra Extract broth showed least production of cellulase.cellulase Gonzalez G (2006). Production of hydrolytic production in Potato Dextrose broth, Sabouraud Dextrose depolymerising pectinases. Food Technol. broth and Richards s broth may be due to the effect of more Biotechnol., 44 (2) : sporulation or the effect of broth media. Fawole B and Balogun S (2006) Pathogenicity and cellwall SUMMARY AND CONCLUSION Seed-borne diseases have been found to affect the growth and productivity of crop plants. This must be degrading enzyme activities of some fungal isolates from Cowpea (Vigna unguiculata (L.) Walp). J. Biokemistri., 18 (1) : addressed properly because high seed quality is Gascoigne JA and Gascoigne MM (1960) Biological essential in any crop production venture to attain higher degradation of cellulose. Butterworths, London, P. yield and good quality products. Thus the present study 264. revealed the necessity of isolation and identification of internal seed borne fungi of crop plants especially legumes. Seed borne fungi induce infection by producing Ikeda R, Yamamato T and Funatsu M (1973a) Agric. Biol. Chem., 27 : hydrolytic enzymes. So it is also necessary to determine the hydrolytic enzyme production of fungal isolates. 77

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