Biodegradation of cellulose-rich substrates is an indispensable process for soil carbon replenishment in various ecological niches. Biodegradation of cellulose has been studied extensively via an enzyme assay to quantify the amount of cellulase with a view to identify effective cellulose degraders. However, a bulk enzyme assay undermines the role of physiological heterogeneity between cells; it is therefore imperative to opt out for a more effective method such as single-cell Raman spectroscopy combined with heavy water (D2O) to reveal active cellulose degraders. Cellular incorporation of D2O-derived D produces a new C-D Raman band which can act as a quantitative indicator of microbial activity. In this study, metabolic responses of seven cellulose-degrading bacteria to carboxymethylcellulose (CMC) and glucose were evaluated via the C-D Raman band. On the basis of % C-D, CMC was demonstrated to be most efficiently metabolized by Bacillus velezensis 2a-9 and Providencia vermicola 5a-9(b). Metabolic activity between individual cells of B. velezensis and P. vermicola towards CMC ranged from approximately 8 to 27% and 6 to 16%, respectively, clearly indicating heterogeneous degradation activities among isogenic populations. Linear correlation between % C-D and specific endoglucanase activity validated Raman results on CMC-degrading activity. Also, % C-D obtained from bacteria cultivated with only glucose was around 60% higher than that obtained from CMC, indicating the preference of bacteria for simple sugar glucose than CMC. In conclusion, Raman spectroscopy combined with heavy water is a sensitive analytical technique to reveal cellulose degraders and their degrading activities.
Keywords: Cellulose biodegradation; Heavy water isotope labeling; Heterogeneous degradation activity; Raman spectroscopy; Single-cell microbiology.