Diversity and functional significance of cellulolytic microbes living in termite, pill-bug and stem-borer guts

Abstract

Arthropods living on plants are able to digest plant biomass with the help of microbial flora in their guts. This study considered three arthropods from different niches - termites, pill-bugs and yellow stem-borers - and screened their guts for cellulase producing microbes. Among 42 unique cellulase-producing strains, 50% belonged to Bacillaceae, 26% belonged to Enterobacteriaceae, 17% belonged to Microbacteriaceae, 5% belonged to Paenibacillaceae and 2% belonged to Promicromonosporaceae. The distribution of microbial families in the three arthropod guts reflected differences in their food consumption habits. Most of the carboxymethylcellulase positive strains also hydrolysed other amorphous substrates such as xylan, locust bean gum and β-D-glucan. Two strains, A11 and A21, demonstrated significant activity towards Avicel and p-nitrophenyl-β-D-cellobiose, indicating that they express cellobiohydrolase. These results provide insight into the co-existence of symbionts in the guts of arthropods and their possible exploitation for the production of fuels and chemicals derived from plant biomass.

Figure 1. Pictures of termites, pill-bugs and rice stem-borers collected from various ecological niches.

Figure 2. Classification and phylogenetic analysis of natural isolates from the guts of termites, pill-bugs and yellow stem-borers.

(A) 16s rDNA sequences of 42 isolates screened for their ability to produce cellulolytic enzymes obtained from the guts of termites (), pill bugs () and yellow stem borers () were used to perform blast searches to identify their nearest neighbors, and MEGA5 was used to construct the phylogenetic tree. The natural isolates were found to belong to five families. (B) Relative abundance of various bacterial families in the guts of these three arthropods.

Figure 3. Qualitative assessment of hydrolytic enzymes produced by various natural isolates.

Enzyme production was tested in the extracellular (A) and cell-bound fractions (B) against amorphous, crystalline and chromogenic substrates. Black and white boxes represent significant and non-significant activities, respectively, in the liquid culture assay. (C) Venn diagram for natural isolates exhibiting cellulase, hemicellulase and other glycosidase activities. Cellulase positive strains were considered to be those that hydrolysed CMC, Avicel, rice straw, pNPC or pNPG. Hemicellulase positive strains were those that hydrolysed xylan or pNPX. Strains that hydrolysed locust bean gum or β-D-glucan were considered to be positive for other glycosyl hydrolases.

Figure 4. Quantitative assessment of the enzymatic activities of extracellular fractions against amorphous polysaccharides.

All bacterial strains that were found to be cellulase positive by the agar plate assay were grown in liquid medium, and their extracellular fractions were tested for the hydrolysis of various substrates. Each graph indicates the substrate. Black bars represent strains that exhibited significantly higher activities compared to the negative controls, while white bars represent negative strains. The data represent the average and standard deviation of two different assays.

Figure 5. Quantitative assessment of the enzymatic activities of extracellular fractions against chromomeric substrates and rice straw.

All bacterial strains that were found to be cellulase positive by the agar plate assay were grown in liquid medium, and their extracellular fractions were tested for the hydrolysis of various substrates. Each graph indicates the substrate. Black bars represent strains that exhibited significantly higher activities compared to the negative controls, while white bars represent negative strains. The data represent the average and standard deviation of two different assays.

Figure 6. Quantitative assessment of the enzymatic activities of the cell-bound fractions.

All bacterial strains that were found to be cellulase positive by the agar plate assay were grown in liquid medium, the cells were lysed by sonication, and the lysed cell samples were tested for the hydrolysis of various substrates. Each graph indicates the substrate. Black bars represent strains that exhibited significantly higher activities compared to the negative controls, while white bars represent negative strains. The data represent the average and standard deviation of two different assays.

Figure 7. Zymograms to determine the enzymatic activities of the extracellular fractions of natural isolates against (A) CMC and (B) xylan.

Figure 8. Kinetic properties of enzyme preparations from strains A11 and A21.

Substrate (para-nitrophenyl cellobiose (pNPC)) saturation kinetics of the (A) A11 and (B) A21 strains. (C) Hanes-Woolf graph for the calculation of K_m and V_max. The data represent the average of two different assays.