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Isolation, Identification and Screening of Aspergilli as a Cellulolytic Fungus from Cellulosic Wastes

M. F. Begum
Department of Botany, University of Rajshahi, Rajshahi-6205

M. S. Alam
Institute of Food Science & Technology, BCSIR, Dhaka

F. Begum
Institute of Food Science & Technology, BCSIR, Dhaka

Abstract/ Executive Summary:

In the present study a total of one hundred and three samples were collected from different cellulosic wastes for isolation of cellulolytic fungi and 970 colonies/ strains were isolated and divided into 54 groups on the basis of their morphological characteristics. These 54 groups were screened depending on their growth, vigour and zone of clearance around the colony on czapek’s medium with 2% CMC. Out of 54 groups 23 strains were highly cellulolytic and identified as Aspergillus tubengensis, A. niger, A. ficuum, A. awamori str. 1. & 2, A. foetidus, A. japonicus str. 1 & 2, A. aculeatus str. 1 & 2, A. flavus str. 1, 2, 3 & 4, A. parasiticus, A. oryzae, A. fumigatus, A.cervinus, A. ochraceus, A. crustosus, A. terreus and A. cremeus as new recod as cellulolytic fungi in this experiment. Selected 24 strains were further screened for identification of cellulase by TLC plate examination and estimation of their reducing sugar level by DNS method. A. oryzae (+ + +), A. ochraceus (+ + +), A.cervinus (+ + +) and A. flavus str. 3 (+ + +) were highly cellulolytic and their reducing sugar level were 520, 492, 423 and 415 mg/ml obtained from TLC and DNS experiment, respectively.

Keywords: Islolation, Aspergilli, TLC, Reducing sugar, Cellulase

Location: Department of Botany, University of Rajshahi, Rajshahi

Start Date:

End Date:

Program Area: Risk Management in Agriculture

Non-commodity: Adoption of technology

Objectives:

1. To isolation of Aspergilli as cellulolytic fungi and characterizes their morphology for species identification. Further, they were screened to select the most potent cellulase producing Aspergillus strains for commercial purpose.

Methodology:

Sample collection - For isolation of Aspergilli, a total of 103 samples were collected from different sources such as agricultural wastes, kitchen wastes, fruit wastes, poultry feed wastes; sugar mill, paper mill and jute mill wastes; soil, sewages and municipal wastes (Table 1). The samples were kept in sterile polythene bags and stored at 4⁰C until use. Isolation - For the isolation of Aspergilli, Czapek’s medium (Composition g/l as sourcrose-30, sodium nitrate NaNO₃-2, potassium bi phosphate KH₂PO₄-1, magnesium sulphate MgSO4 7H₂O-0.5, potassium chloride KCl-0.5, ferrous sulphate FeSO₄ 7H₂O-0.01) was used. Isolation was done by two methods viz., agar platemethod and dilution plate method. Aspergilli, those grew upon Czapek’s medium were transferred o another plates containing selective medium which was prepared as the same procedure used Czapek’s medium supplemented with 2% CMC (Carboxylmethyl cellulose). The organisms those grow well upon the selective medium were considered as cellulolytic fungi and they were selected for further study and preserved at 40C. Identification of the strains - Isolates of Aspergillus were identified. Identification of isolates was based on the morphological characteristics of asexual reproductive organs in majority cases. This has been done because the genus Aspergillus is an amorphic, but in some cases, species identification was done with the help of teleomorphs available. Preparation of crude enzyme solution for cellulase activity - In order to prepare of crude enzyme, selected cellulolytic fungi were inoculated in the broth of Czapek’s medium containing 2% CMC) and allowed to grow at 28°C with shaking for 7 days. Then fungal mat was removed by filtering and filtrate was centrifuged at 10,000 rpm for 10 minutes at room tempreture. The debris was remove and clear supernatant was used as crude enzyme solution for further experiment. Confirmation of cellulase activity by TLC - Cellulase is the enzyme which release sugar from cellulosic substrate. After applying the enzyme on cellulose powder, the released sugar can be identified by thin layer chromatography (TLC), following the method. Measurement of cellulase activity by DNS method - Cellulase activity was measured by detecting the release of reducing sugars from the used substrates. The amount of reducing sugar present was determined by dinitro-salicylic acid (DNS) method (Miller, 1959) in brief, four ml of substrate solution, 1 ml of buffer and 2 ml of crude enzyme were taken in different test tubes for sample and control while a reagent blank tube was used by adding 4 ml of substrate solution and 3 ml of distilled water. After mixing, sample tubes were incubated at 37 °C for 120 minutes in a water bath (GFL-1083) with manual shaking. Tubes were then taken out and heated on boiling water for 5 minutes then cooled and filtered using filter paper (Whatman No. 1). Three ml of filtrates was taken in different test tubes and 3 ml of DNS reagent was added to each of the test tube and the tubes were heated on boiling water bath (GFL-1083) for 5 minutes. One ml of 40% Rochelle salt was added in each of the warmed test tube. The optical density of these solutions was taken at 575 nm by a colorimeter (Spectronic 21).

Source of Information: J. Environ. Sci. & Natural Resources, 3(1): 39-46, 2009; ISSN 1999-7361

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In the present investigation, the cellulolytic activities of primarily screened 24 strains were identified by comparing of sugar spot with standard on TLC plate and confirmed that only 23 strains had cellulytic ability, and four strains such as A. oryzae A. ochraceus, A. cervinus and A. flavus str. 3) were identified as highly potent strain. Sultana (1997) studied comparative cellulolytic activities of 9 fungal strains by TLC plate method and observed that among them two had no cellulase activities and seven strains showed glucose spot positive.

Knowledge Management: Journal