Advisor(s)

April Z. Gu

Contributor(s)

Slava S. Epstein, Ferdinand L. Hellweger, Philip Larese-Casanova

Date of Award

2011

Date Accepted

8-2011

Degree Grantor

Northeastern University

Degree Level

Ph.D.

Degree Name

Doctor of Philosophy

Department or Academic Unit

College of Engineering. Department of Civil and Environmental Engineering.

Keywords

environmental engineering, EBPR, intracellular polymers, phosphorus, polyphosphate accumulating organisms, raman microscopy, stability

Subject Categories

Sewage - Microbiology, Eutrophication - Control

Disciplines

Civil and Environmental Engineering

Abstract

Enhanced biological phosphorus removal (EBPR) process is a widely applied process to control eutrophication in the receiving water bodies. Compared to chemical precipitation-based phosphorus removal approach, EBPR is considered a more environmentally-sound approach because it employs the natural functions of biological processes with minimal chemical consumption and waste production. Although, there has been significant advances and research towards unfolding the EBPR mechanisms, important metabolic details still remain unclear due to the absence of any isolates of the key agents such as polyphosphate accumulating organisms or PAOs, and the lack of tools that allow for quantification of population and cellular level parameters in the EBPR system. These knowledge gaps hamper our ability to better design and control the EBPR process, as many full-scale EBPR facilities still experience unexplainable performance upsets. This study aims to develop a Raman microscopy method that enables quantitative evaluation of intracellular functional polymers in key populations, namely, PAOs and GAOs, in 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). We examined the associated kinetics, population activities and dynamics in the system and demonstrated the ability of IFAS-EBPR process to enable decoupled solids residence time (SRT) controls of slow-growing nitrifiers and the faster-growing PAOs (and other heterotrophs, e.g. denitrifiers) by allowing the former to be attached to fixed film carrier and the latter to be in the suspended mixed liquor and therefore lead to overall optimization of both N and P removal processes. It was further demonstrated that the two subgroups of Accumulibacter-like PAOs, namely the nitrate-utilizing PAOs and aerobic or nitrite-utilizing PAOs, are genetically different and can coexist in a continuous-flow EBPR system and their relative abundances are dynamically affected and can be controlled by operating conditions such as nitrate and biomass recycle flows in a continuous flow system. These findings have important implications in EBPR since denitrification by nitrate-reducing PAOs contributes to the optimization of the usage of external carbon source and electron acceptor (e.g. oxygen or nitrate) for removing both phosphorus and nitrogen and therefore, the phylogenetic identities of different denitrifying PAOs and the factors that govern their abundances are important for better understanding, designing, and controlling of EBPR processes.

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. We further proposed the identification of PAOs and GAOs relevant to EBPR based on the unique Raman spectrum of different combinations of intracellular polymers within a cell at a given stage of the EBPR cycle. Quantification of total PAOs and GAOs by Raman method was validated by comparison of the abundance results with those obtained with conventional polyphosphate staining and fluorescence in situ hybridization (FISH) methods. Quantitative single-cell intracellular polymers measurements among the population also revealed the distribution and metabolic diversity, possibly implied phenotypic diversity, among different sub-PAOs groups. 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 with conventional bulk measurements of EBPR mixed cultures. Analysis of intracellular polymers distribution among PAOs at different metabolic stages of EBPR 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. These results demonstrate that the newly developed Raman method provides an alternative approach to those conventional phylogenic- markers-based methods for quantifying metabolically active PAOs and, it also provides a possibly new method for quantifying total GAOs population abundances in EBPR process, which does not exist currently.

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.

Better and fundamental understanding of EBPR mechanism facilitated by cellular level evaluations through tools, such as the demonstrated Raman microscopy, contributes to our progress towards applying EBPR for more sustainable P removal from wastewater. Observations at single cellular level- resolution can help overcome the knowledge gap that existed so far in EBPR due to the absence of PAO/GAO isolates. For example, the quantitative observation of the heterogeneous and distributed storage level of polyphosphate, PHB and glycogen polymers in the functionally relevant populations in EBPR provide crucial information for improved framework of EBPR modeling (e.g. agent-based models versus conventional average state-based models) and parameters values in different EBPR systems. Moreover, evaluation of relative carbon distribution, abundance shifts and metabolic diversity among the populations in different EBPR systems enabled by Raman microscopy, can allow for the evaluation and identification of the links between the metabolic pathways employed by the key populations and performance efficiency of the system. This information, in complement to those knowledge that can be learned from current approaches, contributes towards better understanding of EBPR ecology and mechanism, therefore better design, operation and control of stability and performance of EBPR processes. Further development of Raman based technique in combination with other in situ identification methods to allow for simultaneous phylogenetic and intracellular metabolic state evaluation is desired.

Document Type

Dissertation

Rights Holder

Nehreen Majed


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