Mechanisms of the System and Performance Stability in Enhanced Biological Phosphorus Removal (EBPR) Process -Insights from Functionally Relevant Microbial Populations and Intracellular Polymers Dynamics

This study aims to develop a Raman microscopy method that enables quantitative evaluation of intracellular functional polymers in key populations, namely, polyphosphate accumulating organisms (PAOs) and GAOs, in enhanced biological phosphorus removal (EBPR) systems, and then to apply the tool to help elucidate the details of metabolic pathways and states within key populations and, to gain better understanding of the association between microbial population and intracellular status with system performance.;Recognizing that sequencing batch reactor (SBR), as employed in most EBPR studies, can not reflect and reveal the potential operational factors, such as recycling ratio, staging effect, reactor configuration etc. on the microbial ecology and metabolic states, we chose to establish both SBRs and a continuous flow EBPR process hybrid with Integrated fixed film activated sludge (IFAS) process. This IFAS-EBPR process not only more accurately represented the full-scale EBPR process that is mostly continuous flow system, but also incorporated more updated technology of IFAS for enhancing simultaneous nitrogen removal, as more full-scale facilities are trying to target effective simultaneous removal of both phosphorus (P) and nitrogen (N).;Recognition of the limitations associated with current methods and approaches for studying EBPR process and the need to develop new tools that can enable us to obtain more detailed information regarding cell metabolic states, we developed a new Raman microscopic method for single cellular level polymeric evaluation in the two key functionally relevant populations in EBPR system, namely PAOs and GAOs. Methods for simultaneous and quantitative evaluation of the intracellular polymers that play crucial roles in EBPR process, including polyphosphate, polyhydroxybutyrate (PHB) and glycogen, at both cellular and population level were developed and validated in both SBR and IFAS-EBPR system, via comparison of the results obtained with Raman with those retrieved via conventional chemical analysis. Intracellular identification and quantification of the polymers, as shown in this study, allowed for the first time, the observation of distributed states of storage polymers at cellular level in functionally relevant microbial populations pertaining to EBPR under different operational conditions. The information can reveal details such as the range and variation of relative content ratio of polyhydroxyvalerate (PHV) and polyhydroxybutyrate (PHB) at single cell level in PAOs, and the changes in the cellular polymers content levels under various system conditions.;The application of the newly developed Raman microscopy method was further demonstrated by applying it to evaluate the effect of influent loading conditions (biodegradable COD (bCOD) to P ratio) on the intracellular polymers dynamics in PAOs and GAOs and to gain insights into EBPR biochemical pathways and mechanisms. Influent bCOD to P ratio has been shown to affect relative populations' abundance and EBPR performance stability. Both Raman measurements and molecular assessment of total PAOs and total GAOs, as well as the phylogenetic subgroups under each population, consistently showed lowering of PAOs and increase in GAOs abundance at higher COD/P ratios. Significant variations in intracellular dynamics of storage polymers at different loading were revealed at cellular as well as different population levels. Lowering of P release at higher COD/P ratios was shown to be attributable not only to the lowering of PAOs abundance, but also decrease of polyphosphate content at individual PAO cells, which is different from current understanding that assumes a constant saturated polymer level in individual cell. Differentiated PHB and glycogen inclusion levels in PAO and GAO populations revealed the relative carbon storage distribution and shifts with changing loading conditions, concurrently with the relative population abundance changes of PAOs and GAOs. Intracellular polymeric analysis also elucidated that EBPR kinetic and stoichiometric parameters could vary within the same populations possibly due to the flexibility in and /or varying utilization of metabolic pathways within the subgroups at varying loading conditions. Employment of glycolysis (as compared to TCA cycle only) by greater fractions of PAOs possibly took place at higher COD/P ratios. These findings provided insights into the metabolic diversity in the PAO populations and the roles of different biochemical pathways under various system conditions. It also demonstrated the potential of application of Raman method as a powerful tool for the fundamental understanding of EBPR mechanism. (Abstract shortened by UMI.)...