Evaluation of Intracellular Polyphosphate Dynamics in Enhanced Biological Phosphorus Removal Process using Raman Microscopy

Abstract

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.

Dynamics of Microbial Community Structure of and Enhanced Biological Phosphorus Removal by Aerobic Granules Cultivated on Propionate or Acetate

“…We hypothesized that Accumulibacter in the acetate reactor channeled large amounts of energy to poly-P formation which eventually "saturated" the cells. Indeed, based on Raman imaging, a recent report shows that poly-P inclusions can occupy as much as 75% of the Accumulibacter cell body (37). This metabolic state of Accumulibacter might in turn have allowed Competibacter to proliferate, as assessed here by two independent methods.…”

On the pathway of mineral deposition in larval zebrafish caudal fin bone

“…Crane et al [17] identified a complex mixture of amorphous calcium phosphate and disordered OCP-like mineral phases. No evidence for a polyphosphate phase (reported in murine bone forming cells (16)), which is expected to have peaks at 700 and 1180 cm − 1 [ [62], was observed. The mineral aggregates that we observed between bone hemi-cylinders are presumably intra-cellular, and represent the earliest stage of mineralization.…”

Heterogeneity of Intracellular Polymer Storage States in Enhanced Biological Phosphorus Removal (EBPR) – Observation and Modeling

“…Note that validation of the Raman analysis through comparison and correlation with conventional bulk measurements were demonstrated in our previous studies. 12 −14 The filtered samples through 0.45 μm filter papers were analyzed for Orthophosphate (orthoP, (PO 4 3 ) − ) and acetate (CH 3 COO − ) using DX-120 ion chromatograph (Dionex Benelux, Belgium). All phosphorus fractions were measured according to the standard method (4500-P).…”