Isolation of Cellulase producing organisms from soil

 Aim

To isolate cellulose producing organisms (cellulolytic) from the soil

 Principle 

Cellulose is a predominant component of higher plants and most abundant biopolymer and polysaccharide found on earth. It forms the main constituent of cell wall of plants, most algae and some fungi. Cellulose is a linear homologous polymeric chain consisting of D-glucose residues that make up to 10,000 glucose residues, linked by β-1,4 glycosidic bonds. 

Though cellulose is plentiful, it is not utilized completely due to the fact of being insoluble. It is the food for ruminant mammals and termites which have bacteria that secrete cellulose digesting enzymes-cellulases. A cellulosic enzyme system consists of three major components: endo-ß-glucanase (CMCase) exo-ß-glucanase (cellobiohydrolase) and ß-glucosidase (cellobiase). The co-operative action of these three enzymes result in the complete hydrolysis of cellulose to glucose.

 The organisms which produces cellulase are known as cellulolytic organisms. Soil contains numerous microorganisms which produce cellulase. Eg. Cellulomonas, Clostridium, Pseudomonas and Trichoderma, Aspergillus etc. Fungi are the main cellulase-producing microorganisms, though a few bacteria and actinomycetes have also been recently reported to produce cellulase. 

Cellulase production can be detected using agar containing cellulose. Cellulose agar is a medium where if we grow microorganisms which produce cellulase, a clear zone develops around the growth, which indicates cellulolysis.

 Cellulose is the most abundant and renewable feedstock for energy source and can be used for the production of alternative fuels by the conversion of lignocellulosic biomass to biofuel. It has a wide range of applications such as food and beverage, pulp and paper, textile, animal feed, detergent and agriculture. 

Materials Required

 Soil sample, Petriplates, Carboxymethyl cellulose agar,  Tubes for serial dilution, L- rod

 

Procedure

1.      1g fertile soil was dissolved in 100 ml sterile distilled water to obtain 10^-2 dilution. It was then serially diluted to obtain dilutions till 10^-6.

2.    Aliquots of I ml from 10^-4, 10^-5, 10^-6 dilutions were transferred into Carboxymethyl cellulose plates and spread plate performed.

3.      Inoculated Carboxymethyl cellulose plates were then incubated at 37⁰C for 2-5 days.

4.      After incubation, the plates were flooded with Gram’s Iodine and observed

 

Result

Presence of colourless zones or clear halo was detected around cellulolytic colonies on the plates, which indicated cellulose hydrolysis.

 

Media Composition (Don't write in record sheet)

 Carboxymethyl Cellulose Agar (pH 7.0)

Carboxymethyl Cellulose 10 g

NaCl 0.1 g

Yeast extract 0.4 g

NH 4 H 2 PO 4 0.1 g

MgSO 4 7H 2 O 0.2 g

Agar 15 g

Distilled water 1000 ml