Incorporating microbial ecology into the metabolic modelling of polyphosphate accumulating organisms and glycogen accumulating organisms

In the enhanced biological phosphorus removal (EBPR) process, the competition between polyphosphate accumulating organisms (PAO) and glycogen accumulating organisms (GAO) has been studied intensively in recent years by both microbiologists and engineers, due to its important effects on phosphorus removal performance and efficiency. This study addresses the impact of microbial ecology on assessing the PAO-GAO competition through metabolic modelling, focussing on reviewing recent developments, discussion of how the results from molecular studies can impact the way we model the process, and offering perspectives for future research opportunities based on unanswered questions concerning PAO and GAO metabolism. Indeed, numerous findings that are seemingly contradictory could in fact be explained by the metabolic behaviour of different sub-groups of PAOs and/or GAOs exposed to different environmental and operational conditions. Some examples include the glycolysis pathway (i.e. Embden-Meyerhof-Parnas (EMP) vs. Entner-Doudoroff (ED)), denitrification capacity, anaerobic tricarboxylic acid (TCA) cycle activity and PAOs’ ability to adjust their metabolism to e.g. a GAO-like metabolism. Metabolic modelling may further yield far-reaching influences on practical applications as well, and serves as a bridge between molecular/biochemical research studies and the optimisation of wastewater treatment plant operation.     

Advancing post-anoxic denitrification for biological nutrient removal

The objective of this research was to advance a fundamental understanding of a unique post-anoxic denitrification process for achieving biological nutrient removal (BNR), with an emphasis on elucidating the impacts of surface oxygen transfer (SOT), variable process loadings, and bioreactor operational conditions on nitrogen and phosphorus removal. Two sequencing batch reactors (SBRs) were operated in an anaerobic/aerobic/anoxic mode for over 250 days and fed real municipal wastewater. One SBR was operated with a headspace open to the atmosphere, while the other had a covered liquid surface to prevent surface oxygen transfer. Process performance was assessed for mixed volatile fatty acid (VFA) and acetate-dominated substrate, as well as daily/seasonal variance in influent phosphorus and ammonia loadings. Results demonstrated that post-anoxic BNR can achieve near-complete (>99%) inorganic nitrogen and phosphorus removal, with soluble effluent concentrations less than 1.0 mgN L⁻¹ and 0.14 mgP L⁻¹. Observed specific denitrification rates were in excess of typical endogenous values and exhibited a linear dependence on the glycogen concentration in the biomass. Preventing SOT improved nitrogen removal but had little impact on phosphorus removal under normal loading conditions. However, during periods of low influent ammonia, the covered reactor maintained phosphorus removal performance and showed a greater relative abundance of polyphosphate accumulating organisms (PAOs) as evidenced by quantitative real-time PCR (qPCR). While GAOs were detected in both reactors under all operational conditions, BNR performance was not adversely impacted. Finally, secondary phosphorus release during the post-anoxic period was minimal and only occurred if nitrate/nitrite were depleted post-anoxically.     

The effect of free nitrous acid on the anabolic and catabolic processes of glycogen accumulating organisms

Nitrite/Free Nitrous Acid (FNA) has previously been shown to inhibit aerobic and anoxic phosphate uptake by polyphosphate accumulating organisms (PAOs). The inhibitory effect of FNA on the aerobic metabolism of Glycogen Accumulating Organisms (GAOs) is investigated. A culture highly enriched (92 ± 3%) in Candidatus Competibacter phosphatis (hereafter called Competibacter) was used. The experimental data strongly suggest that FNA likely directly inhibits the growth of Competibacter, with 50% inhibition occurring at 1.5 × 10⁻³ mgN-HNO₂/L (equivalent to approximately 6.3 mgN-NO₂⁻/L at pH 7.0). The inhibition is well described by an exponential function. The organisms ceased to grow at an FNA concentration of 7.1 × 10⁻³ mgN-HNO₂/L. At this FNA level, glycogen production, another anabolic process performed by GAOs in parallel to growth, decreased by 40%, while the consumption of polyhydroxyalkanoates (PHAs), the intracellular carbon and energy sources for GAOs, decreased by approximately 50%. FNA likely inhibited either or both of the PHA oxidation and glycogen production processes, but to a much less extent in comparison to the inhibition on growth. The comparison of these results with those previously reported on PAOs suggest that FNA has much stronger inhibitory effects on the aerobic metabolism of PAOs than on GAOs, and may thus provide a competitive advantage to GAOs over PAOs in enhanced biological phosphorus removal (EBPR) systems.     

Kinetic competition between phosphorus release and denitrification on sludge under anoxic condition

The kinetic behaviors of simultaneous phosphorus release and denitrification on sludge were investigated under anoxic condition. A phosphorus enriched sludge produced from Anaerobic-Anoxic-Oxic (AnAO) process under various SRT (5, 10 and 15 days) operation conditions was carried out in a series of batch tests. Experimental results indicated that the available organic substrate determined the kinetic behaviors of phosphorus release/uptake and denitrification. The simultaneous phosphorus release and denitrification demonstrated a kinetic competition under anoxic conditions in the presence of an available organic substrate. When the substrate was abundant, sludge was under “releasable-phosphorus-limited” condition; phosphorus release rate decreased slightly by nitrate inhibition. However, nitrate significantly inhibited phosphorus release when sludge was under “initial-substrate-limited” condition. Moreover, the sludge's phosphorus contents (as created by different SRT processes) dominated the kinetics of competition between phosphorus release and denitrification. The sludge with a high phosphorus content had a higher phosphorus release rate in accordance with a lower denitrification rate. Additionally, the substrate sequestrated rate markedly increased under the condition of simultaneous phosphorus release and denitrification. Finally, a preliminary metabolism model of denitrifying phosphorus removal bacteria was proposed, and found to be capable of adequately accounting for simultaneous phosphorus release and denitrification under anoxic conditions.     

Glycerol as a sole carbon source for enhanced biological phosphorus removal

Wastewaters with low organic matter content are one of the major causes of EBPR failures in full-scale WWTP. This carbon source deficit can be solved by external carbon addition and glycerol is a perfect candidate since it is nowadays obtained in excess from biodiesel production. This work shows for the first time that glycerol-driven EBPR with a single-sludge SBR configuration is feasible (i.e. anaerobic glycerol degradation linked to P release and aerobic P uptake). Two different strategies were studied: direct replacement of the usual carbon source for glycerol and a two-step consortium development with glycerol anaerobic degraders and PAO. The first strategy provided the best results. The implementation of glycerol as external carbon source in full-scale WWTP would require a suitable anaerobic hydraulic retention time. An example using dairy wastewater with a low COD/P ratio confirms the feasibility of using glycerol as an external carbon source to increase P removal activity. The approach used in this work opens a new range of possibilities and, similarly, other fermentable substrates can be used as electron donors for EBPR.     

Recent advances in removing phosphorus from wastewater and its future use as fertilizer (1997-2003)

Large quantities of phosphate present in wastewater is one of the main causes of eutrophication that negatively affects many natural water bodies, both fresh water and marine. It is desirable that water treatment facilities remove phosphorus from the wastewater before they are returned to the environment. Total removal or at least a significant reduction of phosphorus is obligatory, if not always fulfilled, in most countries. This comprehensive review summarizes the current status in phosphorus-removal technologies from the most common approaches, like metal precipitation, constructed wetland systems, adsorption by various microorganisms either in a free state or immobilized in polysaccharide gels, to enhanced biological phosphorus removal using activated sludge systems, and several innovative engineering solutions. As chemical precipitation renders the precipitates difficult, if not impossible, to recycle in an economical industrial manner, biological removal opens opportunities for recovering most of the phosphorus and beneficial applications of the product. This review includes the options of struvite (ammonium-magnesium-phosphate) and hydroxyapatite formation and other feasible options using, the now largely regarded contaminant, phosphorus in wastewater, as a raw material for the fertilizer industry. Besides updating our knowledge, this review critically evaluates the advantage and difficulties behind each treatment and indicates some of the most relevant open questions for future research.     

Obtaining highly enriched cultures of Candidatus Accumulibacter phosphates through alternating carbon sources

Candidatus Accumulibacter Phosphatis is widely considered to be a polyphosphate accumulating organism (PAO) of prime importance in enhanced biological phosphorus removal (EBPR) systems. This organism has yet to be isolated, despite many attempts. Previous studies on the biochemical and physiological aspects of this organism, as well as its response to different EBPR operational conditions, have generally relied on the use of mixed culture enrichments. One frequent problem in obtaining highly enriched cultures of this organism is the proliferation of glycogen accumulating organisms (GAO) that can compete with PAOs for limited carbon sources under similar operational conditions. In this study, Candidatus Accumulibacter Phosphatis has been enriched in a lab-scale bioreactor to a level greater than 90% as quantified by fluorescence in situ hyrbridisation (FISH). This is the highest enrichment of this organism that has been reported thus far, and was obtained by alternating the sole carbon source in the feed between acetate and propionate every one to two sludge ages, and operating the bioreactor within a pH range of 7.0–8.0. Simultaneously, the presence of two known groups of GAOs was eliminated under these operational conditions. Excellent phosphorus removal performance and stability were maintained in this system, where the phosphorous concentration in the effluent was below 0.2 mg/L for more than 7 months. When a disturbance was introduced to this system by adding sludge from an enriched GAO culture, Candidatus Accumulibacter Phosphatis once again became highly enriched, while the GAOs were out-competed. This feeding strategy is recommended for future studies focused on describing the physiology and biochemistry of Accumulibacter, where a highly-enriched culture of this organism is of high importance.     

A new method for characterizing denitrifying phosphorus removal bacteria by using three different types of electron acceptors

This study investigated the characteristics of denitrifying phosphorus removal bacteria by using three different types of electron acceptors as well as the positive role of nitrite in phosphorus removal process. Denitrifying phosphorous removal bacteria was enriched under anaerobic-anoxic (A/A) condition. To understand A/A sludge better, sludge from two other sources were also studied. These include sludges obtained from a lab-scale anaerobic-anoxic-aerobic (A/A/O) system and a local sewage treatment plant. Three types of possible electron acceptors (oxygen, nitrate and nitrite) were examined for their roles in phosphorus uptake. The results obtained indicated that oxygen, nitrate and nitrite were able to act as electron acceptors successfully. This observation suggested that in addition to the two well-accepted groups of phosphorus removal bacteria (one can only utilize oxygen to take up phosphorus, P(O), while the other can use both oxygen and nitrate, P(ON)), a new group of phosphorus removal bacteria, P(ON(n)), which could use oxygen, nitrate or nitrite to take up phosphorus was identified. The relative population of these three types of bacteria could be calculated from results obtainable from phosphorus uptake batch experiments with either oxygen or nitrate or nitrite as electron acceptor. The results obtained in this study showed that A/A sludge had similar phosphorus removal performance as the A/A/O sludge. However, it has better denitrifying phosphorus removal capability, which was demonstrated by the relative population of the three groups of bacteria. The results also suggested that nitrite was not an inhibitor to phosphorus removal process. Instead, it is an alternative electron acceptor to oxygen or nitrate.     

Microalgae growth-promoting bacteria as "helpers" for microalgae: a novel approach for removing ammonium and phosphorus from municipal wastewater

A combination of microalgae (Chlorella vulgaris or C. sorokiniana) and a microalgae growth-promoting bacterium (MGPB, Azospirillum brasilense strain Cd), co-immobilized in small alginate beads, was developed to remove nutrients (P and N) from municipal wastewater. This paper describes the most recent technical details necessary for successful co-immobilization of the two microorganisms, and the usefulness of the approach in cleaning the municipal wastewater of the city of La Paz, Mexico. A. brasilense Cd significantly enhanced the growth of both Chlorella species when the co-immobilized microorganisms were grown in wastewater. A. brasilense is incapable of significant removal of nutrients from the wastewater, whereas both microalgae can. Co-immobilization of the two microorganisms was superior to removal by the microalgae alone, reaching removal of up to 100% ammonium, 15% nitrate, and 36% phosphorus within 6 days (varied with the source of the wastewater), compared to 75% ammonium, 6% nitrate, and 19% phosphorus by the microalgae alone. This study shows the potential of co-immobilization of microorganisms in small beads to serve as a treatment for wastewater in tropical areas.     

A comparison of bacterial populations in enhanced biological phosphorus removal processes using membrane filtration or gravity sedimentation for solids-liquid separation

In an earlier phase of this study, we compared the performances of pilot scale treatment systems operated in either a conventional enhanced biological phosphorus removal (CEBPR) mode, or a membrane enhanced biological phosphorus removal (MEBPR) mode. In the present investigation, we characterized the bacterial community populations in these processes during parallel operation with the same municipal wastewater feed. The objectives of the study were (1) to assess the similarity of the bacterial communities supported in the two systems over time, (2) to determine if distinct bacterial populations are associated with the MEBPR and CEBPR processes, and (3) to relate the dynamics of the community composition to changes in treatment process configuration and to treatment process performance. The characteristics of the bacterial populations were first investigated with ribosomal intergenic spacer analysis, or RISA. To further understand the bacterial population dynamics, important RISA phylotypes were isolated and identified through 16S RNA gene sequencing.The parallel MEBPR and CEBPR systems developed bacterial communities that were distinct. The CEBPR community appeared to exhibit greater diversity, and this may have been the primary reason why the CEBPR treatment train demonstrated superior functional stability relative to the MEBPR counterpart. Moreover, the more diverse bacterial population apparent in the CEBPR system was observed to be more dynamic than that of the MEBPR process.Several RISA bands were found to be characteristic of either the membrane or conventional biological system. In particular, the MEBPR configuration appeared to be selective for the slow-growing organism Magnospira bakii and for the foam-associated Microthrix parvicella and Gordonia sp., while gravity separation led to the washout of M. parvicella. In both pilot trains, sequence analysis confirmed the presence of EBPR-related organisms such as Accumulibacter phosphatis. The survey of the CEBPR system also revealed many uncultured organisms that have not been well characterized. The study demonstrated that a simple replacement of a secondary clarifier with membrane solids-liquid separation is sufficient to shift the composition of an activated sludge microbial community significantly.     

Biochemical model for enhanced biological phosphorus removal

Enhanced biological phosphorus (bio-P) removal from wastewater is a promising technology for which the fundamental mechanisms are still unclear. The purpose of this paper is to present a biochemical model that explains bio-P removal mechanisms occurring under anaerobic, aerobic and anoxic conditions of the process. A bio-P bacterium is referred to as one that can store both polyphosphate and carbon (as poly-β-hydroxybutyrate for example). In this communication, observations from the literature are first reviewed and mechanisms of bacterial bioenergetics and membrane transport are summarized. The model for bio-P metabolism under anaerobic, aerobic and anoxic conditions is then presented. The role of polyphosphate under anaerobic conditions is suggested to be as a source of energy both for the reestablishment of the proton motive force, which would be consumed by substrate transport and for substrate storage. The role of the anaerobic zone is to maximize the storage of organic substrates in bio-P bacteria. For this purpose the supply of readily available substrates should be maximized and the presence of electron acceptors (molecular oxygen or oxidized nitrogen) minimized. Under subsequent aerobic or anoxic conditions, bio-P bacteria will accumulate polyphosphates in response to the availability of electron acceptors (oxygen or oxidized nitrogen) for energy production. Carbon reserves in bio-P bacteria should provide energy for growth and for soluble phosphate accumulation as polyphosphate reserves.