Application of Raman microscopy for simultaneous and quantitative evaluation of multiple intracellular polymers...

Polyphosphate (poly-P), polyhydroxyalkanoates (PHAs), and glycogen are the key functionally relevant intracellular polymers involved in the enhanced biological phosphorus removal (EBPR) process. Further understanding of the mechanisms of EBPR has been hampered by the lack of cellular level quantification tools to accurately measure the dynamics of these polymers during the EBPR process. In this study, we developed a novel Raman microscopy method for simultaneous identification and quantification of poly-P, PHB, and glycogen abundance in each individual cell and their distribution among the populations in EBPR. Validation of the method was demonstrated via a batch phosphorus uptake and release test, in which the total intracellular polymers abundance determined via Raman approach correlated well with those measured via conventional bulk chemical analysis (correlation coefficient r = 0.8 for poly-P, r = 0.94 for PHB, and r = 0.7 for glycogen). Raman results, for the first time, clearly showed the distributions of microbial cells containing different abundance levels of the three intracellular polymers under the same environmental conditions (at a given time point), indicating population heterogeneity exists. The results revealed the intracellular distribution and dynamics of the functionally relevant polymers in different metabolic stages of the EBPR process and elucidated the association of cellular metabolic state with the fate of these polymers during various substrates availability conditions.