A Raman microscopy method was developed and successfully applied to evaluate the dynamics of intracellular polyphosphate in polyphosphate-accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) processes. Distinctive Raman spectra of polyphosphates allowed for both identification of PAOs and quantification of intracellular polyphosphate during various metabolic phases in a lab-scale EBPR process. Observation of polyphosphate at individual cell level indicated thatthere are distributed states of cells in terms of polyphosphate content at any given time, suggesting that agent-based distributive modeling would more accurately reflect the behavior of an EBPR process than the traditional average-state based modeling. The results, for the first time, showed that the polyphosphate depletion or replenishment observed at the overall population level were collective results from shifts/transition in the distribution of abundance of PAOs with different amounts of polyphosphate inclusions during EBPR. Imaging construction based on simultaneous quantification of intracellular polyphosphate and protein revealed the spatial distribution of polyphosphate inside cells and showed that the polyphosphates accumulate in smaller or larger aggregates, rather than being evenly distributed within the cytoplasm. The results demonstated that Raman microscopy will allow for detailed cellular-level evaluation of polyphosphate metabolism and dynamics in EBPR processes and revealed mechanism insights, which otherwise would not be obtained using a traditional bulk measurement-based approach.
This study proposed and demonstrated the application
of a new Raman
microscopy-based 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) system via
simultaneous detection of multiple intracellular polymers including
polyphosphate (polyP), glycogen, and polyhydroxybutyrate (PHB). The
unique Raman spectrum of different combinations of intracellular polymers
within a cell at a given stage of the EBPR cycle allowed for its identification
as PAO, GAO, or neither. The abundance of total PAOs and GAOs determined
by Raman method were consistent with those obtained with polyP staining
and fluorescence in situ hybridization (FISH). Different combinations
and quantities of intracellular polymer inclusions observed in single
cells revealed the distribution of different sub-PAOs groups among
the total PAO populations, which exhibit phenotypic and metabolic
heterogeneity and diversity. These results also 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
before with conventional bulk measurements of EBPR mixed cultures.
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.
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