PRACTICAL BIOTECHNOLOGY

Cellulase production
Kasing Apun, Sarawak, Malaysia

Practical details
THERE IS GREAT INTEREST in utilising cellulose wastes as feedstocks for fermentation processes, thereby converting low cost starting materials into products of greater value. Most commercial cellulases are produced by submerged fermentation of the fungus Trichoderma reesei. However, the bacterium Cellulomonas grows more rapidly on a Petri dish and the production of extra-cellular cellulases by it is easy to measure.
1. Dip a 5 mm diameter cork borer into alcohol. Set re to the alcohol and let it burn away. Take care to hold the borer horizontally while doing this so that ames do not travel up the centre of the borer and burn your hand. Hold the lid of a CMC agar plate slightly to one side and use the borer to cut a well in the agar. Remove the agar plug from the borer if necessary using a mounted needle. Repeat steps 1 and 2 so that you have two wells in the agar. Label each well on the base of the Petri dish. A suitable code would be: C (Cellulomonas); W (sterile water, a control). Into the appropriate well place 0.2 cm3 of either microbial culture or sterile water, using a separate sterile syringe for each. Place the syringes, as they are used, in a beaker of disinfectant. Incubate the plates for up to a week at 2530C. Cellulomonas will produce clear zones up to 16 mm in diameter after 48 hours at 30C.

2.

Materials
Slope culture of Cellulomonas sp. (available from the NCBE) The slope culture should be used to prepare several nutrient broth cultures in McCartney bottles for classroom use. This should be done two or three days before it is intended to carry out this practical work, and the bottles should be incubated at 25 30C. Sterile Petri dishes For each Petri dish, 15 cm3 of CMC agar, made with 0.5 g carboxymethylcellulose (a soluble form of cellulose); 0.1 g NaNO3; 0.1 g K2HPO4; 0.1 g KCl; 0.05 g MgSO4; 0.05 g yeast extract; and 0.1 g glucose in 100 cm3 of water. The medium should be solidied using 1.7 % w/v agar. Sterile water (dispensed into a McCartney bottle) Sterile 1 cm3 syringes (without needles), 2 Discard beaker containing 5% solution of Chlorate I solution Congo red solution (made with 1 mg per cm3 of water) 1M sodium chloride solution Ethanol (for aming cork borer) Cork borer, 5 mm diameter Bunsen burner Incubator set at 2530C Pen for marking Petri dishes

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After incubation
1. Flood the plates with Congo red solution for 15 minutes, then de-stain with the salt solution for 1015 minutes. Unstained areas indicate where the CMC has been broken down to 14 glucans that contain seven or fewer glucose residues. The diameter of the clear zone can be measured to provide a quantitative comparison of cellulolytic activity.

Cellulomonas sp. growing on filter paper (electron micrograph taken after 3 days of incubation).

Safety
Standard microbiological safety procedures, including aseptic techniques, must be observed by teachers, technicians and students when carrying out this work. Teachers in England and Wales are referred to: Microbiology. An HMI guide for schools and further education (1990) HMSO [Second Edition] as well as any safety guidelines produced by their LEA and / or school governing body.

Further activities
1. Soil suspensions can be pipetted into wells on the plates to screen their microbial ora for cellulase activity. The same technique can be used to assay cellulase enzyme activity e.g. Novo Nordisk Celluclast. The course of cellulase breakdown can be followed over several days by using duplicate plates.

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Cellulase production Cellulose digest

Cut and remove a plug of agar from a plate, forming a well

1
Sterilize a cork borer by flaming it with alcohol

3
Make another well in the same way Inoculate one well with culture and the other with water

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Stain and observe the plate after 2 days
Measure diameter of zone in which cellulose has been broken down

as on om re ul tu ll ul Ce c

Incubate the plate the right way up

National Centre for Biotechnology Education, 1995