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
Cellulose is a linear polysaccharide of glucose residues with
Cellulolysis is basically the biological process controlled and processed by the enzymes of cellulase system. Cellulase enzyme system comprises three classes of soluble extracellular enzymes: 1, 4-
Source for cellulase system extraction is best suitable from microbial system found in the gut of organisms thriving on cellulosic biomasses as their major feed. Insects like termites (
Cellulase due to its massive applicability has been used in various industrial processes such as biofuels like bioethanol [
The present work concentrates on the isolation of cellulose-degrading bacteria from invertebrates such as termites, snails, caterpillars, and bookworms and assessment of their cellulolytic activity. The coculturing of cellulose-degrading bacteria and yeast was also carried out for simultaneous saccharification and fermentation of cellulose into ethanol.
Cellulose feeding organisms like termite, caterpillar, bookworm, and snail were collected for isolation of cellulose-degrading bacteria from woody habitats. Guts of the collected organism were separately crushed in 0.9% saline solution under sterile condition.
The macerated gut of the collected organisms was inoculated in a basal salt media (NaNO3 2.5 g; KH2PO4 2 g; MgSO4 0.2 g; NaCl 0.2 g; CaCl2·6H2O 0.1 g in a liter) containing filter paper (Whatman filter paper no. 1 of area 70.541 cm2) for the isolation of cellulolytic bacteria. These cultures were incubated for 7 days in a shaker incubator at 37°C at 100 rpm. Bacterial colonies capable of utilizing cellulose as sole source of carbon were isolated on cellulose agar media composed of KH2PO4 0.5 g MgSO4 0.25 g cellulose 2.0 g agar 15 g gelatin 2 g and distilled water lL and at pH 6.8–7.2.
Confirmation of cellulose-degrading ability of bacterial isolates was performed by streaking on the cellulose Congo-Red agar media with the following composition: KH2PO4 0.5 g, MgSO4 0.25 g, cellulose 2 g, agar 15 g, Congo-Red 0.2 g, and gelatin 2 g; distilled water 1 L and at pH 6.8–7.2. The use of Congo-Red as an indicator for cellulose degradation in an agar medium provides the basis for a rapid and sensitive screening test for cellulolytic bacteria. Colonies showing discoloration of Congo-Red were taken as positive cellulose-degrading bacterial colonies [
The selected CDB isolates were cultured at 37°C at 150 rpm in an enzyme production media composed of KH2PO4 0.5 g, MgSO4 0.25 g, and gelatin 2 g, distilled water 1 L and containing Whatman filter paper No.1 (1 × 6 cm strip, 0.05 g per 20 mL) and at pH 6.8–7.2. Broth culture after three days of incubation period was subjected to centrifugation at 5000 rpm for 15 min at 4°C. Supernatant was collected and stored as crude enzyme preparation at 4°C for further enzyme assays. Pellet recovered after centrifugation of broth culture was subjected to gravimetric analysis in order to determine the residual cellulose of filter paper [
Total cellulose activity was determined by measuring the amount of reducing sugar formed from filter paper. Endoglucanase (
A total of four isolates CDB 2, 7, 8, and 10 were grown in mixed culture using basal salt medium in two different sets, one containing filter paper and the other containing cellulose powder as substrate for production of cellulolytic enzyme and to initiate saccharification process. Culture was incubated at 37°C with mixing at 100 rpm for 3 days. After completion of three days of incubation, the above culture broth was conditioned for coculturing of
Cellulose degrading bacteria were enriched and isolated by inoculating filter paper in liquid medium with macerated guts from termite, bookworm, snail, and caterpillar separately. All bacterial culture showed growth as the medium turned cloudy and the filter paper became macerated. Cellulolytic bacteria were also isolated from gut of insects by R. J. Dillon and V. M. Dillon. [
Maximum clearing zone and hydrolytic capacity (HC) value of CDB on cellulose Congo red agar media. This table shows the assessment of bacterial isolates from the different source organism for cellulose decomposition via measurement of clear zone around the colony and calculation of hydrolytic value in cellulose Congo Red media. Maximum clearing zone of 50 mm and HC value of 9.8 were estimated for CDB 10.
Source organism | Isolate number | Maximum clearing zone (mm) | Average hc value | Maximum HC value |
---|---|---|---|---|
Termite | CDB1 | 30 | 5.49 | 6.77 |
CDB2 | 42 | 4.29 | 8.4 | |
CDB8 | 45 | 5.36 | 9 | |
CDB9 | 28 | 4.32 | 4.39 | |
Snail | CDB6 | 40 | 3.45 | 6.45 |
CDB10 | 50 | 5.96 | 9.8 | |
Bookworm | CDB3 | 30 | 3.51 | 4.3 |
Caterpillar | CDB7 | 50 | 5.35 | 8.2 |
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.
A total of eight positive isolates (CDB1, 2, 3, 6, 7, 8, 9, and 10) were selected for enzyme production and their respective cellulolytic activity was estimated. Enzyme assay for cellulase activity on filter paper was found to be highest for CDB 10 with 0.194 IU/mL, while for endoglucanase assay maximum activity was determined to be 0.400 IU/mL by CDB 8. The activities ranged from 0.012 to 0.196 IU/mL for FPCase and 0.1622 to 0.400 IU/mL for endoglucanase assay. The two isolates CDB8 and CDB10 exhibited the highest extracellular cellulase activities compared to other isolates as shown in activity assay performed for all isolates in Figure
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.
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.
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.
The experiment setup for simultaneous saccharification and fermentation of mixed bacterial culture (CDB, 2, 7, 8, and 10) with
The bacterial isolates showed a potential to convert cellulose into reducing sugars which could be readily used in many applications like feed stock for production of valuable organic compounds; for example in the present study this has been demonstrated by simultaneous saccharification and fermentation of cellulose into ethanol.