Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Isolation and Screening of Cellulase Producing Fungal Isolates from Sea Sands

M Hasan*
Department of Pharmaceuticals and Industrial Biotechnology, Sylhet Agricultural University, Sylhet-3100, Bangladesh

J Ahmed
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

M H Sohag
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

Al-Hakim
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

A K Azad
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet-3114, Bangladesh

Abstract/ Executive Summary:

Cellulases are the enzymes which hydrolyse cellulosic biomass and are being produced by the microorganisms grown over cellulosic substrates. The cellulase producing fungal isolates were obtained from sea sands and cellulosic waste materials. Four fungal species were isolated and screened by using Carboxy Methyl Cellulose (CMC) agar medium as a selective medium. Production of clear zones by the fungal isolates on CMC agar medium supplemented with 1% CMC was considered as indicative of extracellular cellulase activity. The size of transparent zone diameter was considered as proportional to the level of cellulase production. These fungal isolates were identified as Aspergillus sp., Penicillium sp. and Fusarium sp. by studying their microscopic and macroscopic characters. A basal medium containing CMC, KH₂PO₄, K₂HPO₄, MgSO₄, (NH₄)₂SO₄, CaCl₂ and FeSO₄ at pH 7.0 was used for cellulase production. The assay of cellulase in term of CMCase was performed by measuring the release of reducing sugar. The crude cellulase produced by these fungal isolates was partially characterized. Optimum temperature for maximum cellulytic activity was 35°C and was active at 35°-40°C. The cellulase activity was significantly active over a broad pH range from 5.0 to 8.0 having maximum activity at pH 6.0.

Keywords: Cellulase, Carboxy Methyl Cellulose (CMC), Marine fungi, CMCase activity.

Location: Cox’s bazar sea coast in Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Fungal Disease

Objectives:

The present study was aimed to isolate and screen potential cellulase producing native fungal species from sea sands. A novel methodology of isolation and primary screening of cellulolytic fungi was adopted by exposing carboxymethyl cellulose agar, a selective media for cellulolytic microorganisms and enzyme activity was determined.

Methodology:

Isolation and identification of marine fungi: Different marine samples were collected from Cox’s bazar sea coast in Bangladesh. Isolation was done on sand dilution (serial dilution) plate method. Serial dilution was done till 10-4 . One ml of desired dilution (10-3 and 10-4 ) was transferred aseptically into a potato dextrose agar (PDA) prepared from marine water adding one drop of 20% lactic acid to suppress fungal growth. Plates were incubated at 30°C temperature for 3 days. After incubation, small portion mycelium from each fungal colony was transferred into CMC media. The sporulated fungi were identified based on the colony morphology and microscopical features using standard taxonomic keys and monographs (Carmichael et al., 1980). Production of crude cellulase: The culture was grown under optimal condition for cellulase production. The strain cultured in basal medium containing 1% CMC and incubated for 3-10 days on a rotary shaker (110 rpm min^-1) at 30°C. Each of the cultures was centrifuged at 8,000 rpm min^-1 for 15 min. The clear supernatants were collected aseptically as extracellular cellulase preparations. Enzyme assay: Cellulase activity was determined by CMCase enzyme assay. CMCase activity was assayed using a method described by Mandels and Weber (1969). The reducing sugar released from carboxymethyl cellulose per ml per min at 575nm was determined by dinitrosalicylic acid (DNS) method. One unit of total cellulase activity was defined as the amount of enzyme releasing 1μmole of reducing sugar per minute. Effects of temperature and pH on cellulase activity and stability: To investigate the effects of temperature and pH on cellulase activity, 500 μL of the crude enzyme was added to 500 μL of 1% CMC in 50 mM citrate buffer (pH 4.8). The reaction mixture was incubated for 30 min at various temperatures 30, 35, 40, 45, 50, 55 and 60°C. Cellulase activity was then measured as described above. To study the effects of pH on cellulase activity, different buffers such as 50 mM of sodium citrate (pH 4.0 and 5.0), potassium phosphate (pH 6.0 - 7.0) and Tris- HCl (pH 8.0 - 9.0) were used to assay the CMCase activity. To 0.5 ml of 1% CMC prepared in a suitable buffer of a particular pH (pH 4 - 9), 0.5 ml of crude enzyme was added. To investigate the temperature stability, 1 ml of crude enzyme was treated for 1 h at various temperatures 30, 35, 40, 45, 50, 55 and 60°C. The residual CMCase activity of crude cellulase was measured according to the standard assay procedure. To study the pH on cellulase stability, the crude cellulase enzyme was treated for 1 h at room temperature with buffers of different pH 4 - 9 as described above. Standard assay procedure was followed to measure the residual activity of crude cellulase.

Source of Information: J. Sylhet Agril. Univ. 3(2):281-289, 2016 ISSN: 2308-1597

Article Link (Online Journal): www.jsau.com.bd

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Fungi are key agents for degradation of cellulosic materials. The present study has isolated four cellulolytic fungi that have been identified as Aspergillus sp., Penicillium sp. and Fusarium sp. based on morphological, cultural and biochemical characteristics. The crude cellulase of these identified isolates was characterized in terms of effects of temperature and pH on the CMCase activity and stability. The collection of more fungal isolates from cellulosic solid wastes and genetic engineering approach would provide more paces to degrade the organic wastes as well as xenobiotic compounds. It is now a very important concern to develop ecologically sound and health promoting ways to manage cellulosic waste material for the sustainable development of the society.

Knowledge Management: Journal