Isolation of cellulose-degrading bacteria and determination of their cellulolytic potential

Abstract

Eight isolates of cellulose-degrading bacteria (CDB) were isolated from four different invertebrates (termite, snail, caterpillar, and bookworm) by enriching the basal culture medium with filter paper as substrate for cellulose degradation. To indicate the cellulase activity of the organisms, diameter of clear zone around the colony and hydrolytic value on cellulose Congo Red agar media were measured. CDB 8 and CDB 10 exhibited the maximum zone of clearance around the colony with diameter of 45 and 50 mm and with the hydrolytic value of 9 and 9.8, respectively. The enzyme assays for two enzymes, filter paper cellulase (FPC), and cellulase (endoglucanase), were examined by methods recommended by the International Union of Pure and Applied Chemistry (IUPAC). The extracellular cellulase activities ranged from 0.012 to 0.196 IU/mL for FPC and 0.162 to 0.400 IU/mL for endoglucanase assay. All the cultures were also further tested for their capacity to degrade filter paper by gravimetric method. The maximum filter paper degradation percentage was estimated to be 65.7 for CDB 8. Selected bacterial isolates CDB 2, 7, 8, and 10 were co-cultured with Saccharomyces cerevisiae for simultaneous saccharification and fermentation. Ethanol production was positively tested after five days of incubation with acidified potassium dichromate.

Figure 1

Zone of clearance on cellulose Congo Red agar plates for isolate CDB 10 after 48 hrs of incubation. The formation of clearing zone around the colonies confirms the secretion of extracellular cellulase.

Figure 2

Extracellular cellulase activity of two enzymes (FPCase and endoglucanase) of all CDBs isolates. The activities ranged from 0.012 to 0.196 IU/mL for FPCase and 0.1622 to 0.400 IU/mL for endoglucanase assay. Values in figure are means of three replicates with standard deviation.

Figure 3

Filter paper degradation by isolates CDB 2, 3, 6, 7, 8, 9, and 10 cultured in basal salt medium supplemented with Whatman filter paper no.1 (1 × 6 cm strip × 2, 0.05 g per 20 mL) at the end of 96 hrs of incubation. Flask FP C is the control for this experimental set up and does not show any filter paper degradation.

Figure 4

Percent filter paper degradation by various bacterial isolates obtained from termite, snail, bookworm, and caterpillar by gravimetric method. Maximum percentage of filter paper degradation was found to be 65.7% by CDB 8. Values in figure are means of three replicates with standard deviation.