Identification of Functionally Relevant Populations in Enhanced Biological Phosphorus Removal Processes Based On Intracellular Polymers Profiles and Insights into the Metabolic Diversity and Heterogeneity

Abstract

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.

Fluorometric Quantification of Polyphosphate in Environmental Plankton Samples: Extraction Protocols, Matrix Effects, and Nucleic Acid Interference

“…PolyP breakdown allows cells to survive during P limitation (12, 13), although some microbes transiently accumulate polyP upon nitrogen and P stress (8,14,15). Massive accumulation of polyP by certain bacteria sequesters dissolved P during biological wastewater treatment (16)(17)(18).While polyP is studied in a range of microbiological and environmental fields, the lack of easy quantitative methods has hampered progress. For example, polyP has very rarely been measured in the oceans despite increasing interest in the physiological responses of marine microbes to the ultralow P concentrations frequently found at sea and their biogeochemical implications (14,(19)(20)(21)(22)(23)(24)(25).…”

“…PolyP breakdown allows cells to survive during P limitation (12, 13), although some microbes transiently accumulate polyP upon nitrogen and P stress (8,14,15). Massive accumulation of polyP by certain bacteria sequesters dissolved P during biological wastewater treatment (16)(17)(18).…”

A Review of Phosphorus Removal Technologies and Their Applicability to Small-Scale Domestic Wastewater Treatment Systems

“…and it has since been shown that PAO are capable of utilizing both the TCA cycle and Glycolysis (Zhou et al, 2009). Some have suggested that some PAO may switch between metabolic pathways according to internal or external conditions (Acevedo et al, 2012(Acevedo et al, , 2014Majed et al, 2012;Lanham et al, 2013). Utilisation of glycolysis has been shown to yield more efficient P-removal rates than the TCA cycle, within the context of an enhanced CAS system (Lanham et al, 2013).…”

Mathematical Modeling of Microbial Community Dynamics: A Methodological Review

“…Taking individual cells as the modeling units is justified considering that internal states and phenotypic traits of individual bacterial cells can be highly heterogeneous in populations, even in isogenic ones [22]. For example, a recent experimental study [23] showed that the internal state (i.e., the level of internally stored nutrients) of cells can vary significantly in a population.…”