Cellulose Degrading Microorganisms.docx

Exercise No. 14

Date: ISOLATION OF CELLULOSE DEGRADING MICROORGANISMS FROM COMPOST

Cellulose is an organic compound with the formula (C6H10O5)n, a polysaccharide consisting of a linear chain of several hundred to many thousands of β(1→4) linked D-glucose units.[3][4] Cellulose is an important structural component of the primary cell wall of green plants, many forms of algae and the oomycetes. Some species of bacteria secrete it to form biofilms.[5] Cellulose is the most abundant organic polymer on Earth.[6] The cellulose content of cotton fiber is 90%, that of wood is 40–50%, and that of dried hemp is approximately 57%.Cellulose is mainly used to produce paperboard and paper. Smaller quantities are converted into a wide variety of derivative products such as cellophane and rayon. Conversion of cellulose from energy crops into biofuels such as cellulosic ethanol is under development as a renewable fuel source. Cellulose for industrial use is mainly obtained from wood pulp and cotton. Some animals, particularly ruminants and termites, can digest cellulose with the help of symbiotic micro-organisms that live in their guts, such as Trichonympha. In human nutrition, cellulose is a non-digestible constituent of insoluble dietary fiber, acting as a hydrophilicbulking agent for feces and potentially aiding in defecation.

Isolation of cellulose degrading bacteria:

1. Different sample sources were used for the isolation. 2. Czapeck agar medium(CMC) is used for isolation of bacteria 3. 1 g soil sample was dissolved in 9 ml of sterile water. 4. Using 1 ml of the suspension, serial dilution was performed upto 10-7. From each dilution about 100µl of sample was spread on plate containing starch nutrient agar. 5. Plates were incubated @ 30°c for 48 hours. 6. Different colonies observed on inoculated plate were selected and streaked onto fresh Czapeck agar medium(CMC) and colonies were repeatedly streaked to obtain pure culture. Conformational test for screening cellulolytic activity: Congo red test

1. Isolated colonies were screened by using congo red 2. Pure colonies were streaked on CMC agar containing and incubated @ 30°c for 48 hr. 3. Then plates were flooded with 1 % congo red and allowed to stand for 15 minutes at room temperature. 4. After that one molar NaCl was thoroughly used for counterstaining the plates. If a strain was cellulolytic then it started hydrolyzing the cellulose present in the surrounding, so in the zone of hydrolysis, there will be a clear zone. 5. The colonies showing clear zone were selected as cellulose degraders. 6. The selected isolates were maintained through subculturing and also maintained @ 4°c.

Cellulose Degrading Microorganisms Cellulolytic enzymes are synthesized by a number of microorganisms. Fungi and bacteria are the main natural agents of cellulose degradation. The cellulose utilizing population includes aerobic and anaerobic mesophilic bacteria, filamentous fungi, thermophilic and alkaliphilic bacteria, actinomycetes and certain protozoa, However, fungi are well known agents of decomposition of organic matter, in general, and of cellulosic substrate in particular. Cellulolytic microorganisms are, Fungi, Aspergillus niger, A. nidulans, A. oryzae, A. terreus; Fusarium solani, F.oxyspourm; Penicillium brasilianum, P.occitanis, P.decumbans,; Trichoderma reesei, T. harzianum; Bacteria, Aerobic bacteria Acinetobacter junii, A. amitratus; Acidothermus cellulolyticus; Anoxybacillus sp; Bacillus subtilis, B.pumilus, B. licheniformis, B. amyloliquefaciens, B. circulans, B. flexus Anaerobic bacteria Acetovibrio cellulolyticus; Butyrivibrio fibrisolvens; Clostridium thermocellum; C. cellulolyticum; C. acetobutylium; Experimental set up

Colony of cellulolytic Microorganism

Sample incubated ay 30°C in selected media which is CMC media for 48 hr

Congo red test of the sample look like this