Insights into the Metabolic Pathways and Diversity of Functionally Relevant Populations in Enhanced Biological Phosphorus Removal Processes using Raman Microscopy

This study demonstrated the application of Raman microscopy method for metabolic-state based identification and quantification of functionally relevant populations namely, polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs)in enhanced biological phosphorus removal (EBPR) process. Analysis of unique Raman spectrum of different combinations of multiple intracellular polymers including polyphosphate, glycogen and polyhydroxybutyrate within a cell at a given stage of the EBPR cycle allows for its identification as PAO, GAO or neither. Quantification of total PAOs and GAOs by Raman method was validated by comparison of the abundance results with those obtained with poly-P staining and fluorescence in situ hybridization (FISH). Quantitative single-cell intracellular polymers measurements among the population also revealed the distribution of different sub-PAOs groups among the total PAO populations based on their different intracellular polymer inclusions, which indicated varying phenotypes and metabolic diversity. These results provided evidence for the hypothesis that different PAOs may employ different extents of combination of glycolysis and TCA cycle pathways for anaerobic reducing power and energy generation and it is possible that some PAOs may rely on TCA cycle solely without glycolysis. Sum of cellular level quantification of the internal polymers associated with different population groups showed differentiated and distributed trends of glycogen and PHB level between PAOs and GAOs, which could not be elucidated with conventional bulk measurements of EBPR mixed cultures.