Global warming readiness: Feasibility of enhanced biological phosphorus removal at 35 °C.

Recent research has shown enhanced biological phosphorus removal (EBPR) from municipal wastewater at warmer temperatures around 30 °C to be achievable in both laboratory-scale reactors and full-scale treatment plants. In the context of a changing climate, the feasibility of EBPR at even higher temperatures is of interest. We operated two lab-scale EBPR sequencing batch reactors for > 300 days at 30 °C and 35 °C, respectively, and followed the dynamics of the communities of polyphosphate accumulating organisms (PAOs) and competing glycogen accumulating organisms (GAOs) using a combination of 16S rRNA gene metabarcoding, quantitative PCR and fluorescence in situ hybridization analyses. Stable and nearly complete phosphorus (P) removal was achieved at 30 °C; similarly, long term P removal was stable at 35 °C with effluent PO43-_P concentrations < 0.5 mg/L on half of all monitored days. Diverse and abundant Candidatus Accumulibacter amplicon sequence variants were closely related to those found in temperate environments, suggesting that EBPR at this temperature does not require a highly specialized PAO community. A slow-feeding strategy effectively limited the carbon uptake rates of GAOs, allowing PAOs to outcompete GAOs at both temperatures. Candidatus Competibacter was the main GAO, along with cluster III Defluviicoccus members. These organisms withstood the slow-feeding regime, suggesting that their bioenergetic characteristics of carbon uptake differ from those of their tetrad-forming relatives. Comparative cycle studies revealed higher carbon and P cycling activity of Ca. Accumulibacter when the temperature was increased from 30 °C to 35 °C, implying that the lowered P removal performance at 35 °C was not a direct effect of temperature, but a result of higher metabolic rates of carbon (and/or P) utilization of PAOs and GAOs, the resultant carbon deficiency, and escalated community competition. An increase in the TOC-to-PO43--P ratio (from 25:1 to 40:1) effectively eased the carbon deficiency and benefited PAOs. In general, a slow-feeding strategy and sufficiently high carbon input benefited a high and stable EBPR at 35 °C, representing basic conditions suitable for full-scale treatment plants experiencing higher water temperatures.

Recent advances in understanding the ecophysiology of enhanced biological phosphorus removal.

Enhanced biological phosphorus removal (EBPR) is an efficient, cost-effective, and sustainable method for removing excess phosphorus from wastewater. Polyphosphate accumulating organisms (PAOs) exhibit a unique physiology alternating between anaerobic conditions for uptake of carbon substrates and aerobic or anoxic conditions for phosphorus uptake. The implementation of high-throughput sequencing technologies and advanced molecular tools along with biochemical characterization has provided many new perspectives on the EBPR process. These approaches have helped identify a wide range of carbon substrates and electron acceptors utilized by PAOs that in turn influence interactions with microbial community members and determine overall phosphorus removal efficiency. In this review, we systematically discuss the microbial diversity and metabolic response to a range of environmental conditions and process control strategies in EBPR.

Metabolic Traits of Candidatus Accumulibacter clade IIF Strain SCELSE-1 Using Amino Acids As Carbon Sources for Enhanced Biological Phosphorus Removal.

Despite recent evidence from full-scale plants suggesting that Candidatus Accumulibacter may be capable of using amino acids, this metabolic trait has never been confirmed in a bioreactor experiment. Here we show that an enriched culture of Ca. Accumulibacter clade IIF strain SCELSE-1 could metabolize 11 of 20 α-amino acids, with aspartate, glutamate, asparagine, and glutamine resulting in the highest phosphorus removal. The anaerobic uptake of aspartate and glutamate was achieved through a glutamate/aspartate-proton symporter fully powered by the proton motive force (PMF). Under anaerobic conditions aspartate was deaminized and routed into core carbon metabolic pathways to form polyhydroxyalkanoates (PHA). The lack of genes encoding NADH dependent isocitrate dehydrogenase in the Ca. Accumulibacter genome resulted in a kinetic barrier for glutamate to be channelled to the TCA cycle. Glutamate was stored as glutamate polymer. When amino acids (aspartate or glutamate) and acetate were supplied together, Ca. Accumulibacter took up both carbon sources simultaneously, with the uptake rate of each carbon source largely preserved. Overall energy savings (up to 17%) were achieved under mixed carbon scenarios, due to the ability of Ca. Accumulibacter to rearrange its anaerobic carbon metabolism based on the reducing power, PMF and ATP balance.

Parameter Selection for a Microvolume Electrochemical Escherichia coli Detector for Pairing with a Concentration Device.

Waterborne infections are responsible for health problems worldwide and their prompt and sensitive detection in recreational and potable water is of great importance. Bacterial identification and enumeration in water samples ensures water is safe for its intended use. Culture-based methods can be time consuming and are usually performed offsite. There is a need to for automated and distributed at-source detectors for water quality monitoring. Herein we demonstrate a microvolume Escherichia coli (E. coli) detector based on a screen printed electrode (SPE) bioelectroanalytical system and explore to what extent performance can be improved by coupling it with a filtration device. To confidently benchmark detector performance, we applied a statistical assessment method to target optimal detection of a simulated concentrated sample. Our aim was to arrive at a holistic understanding of device performance and to demonstrate system improvements based on these insights. The best achievable detection time for a simulated 1 CFU mL-1 sample was 4.3 (±0.6) h assuming no loss of performance in the filtration step. The real filtered samples fell short of this, extending detection time to 16-18 h. The loss in performance is likely to arise from stress imposed by the filtration step which inhibited microbial growth rates.

Polyphosphate-accumulating organisms in full-scale tropical wastewater treatment plants use diverse carbon sources.

Enhanced biological phosphorus removal (EBPR) is considered challenging in the tropics, based on a great number of laboratory-based studies showing that the polyphosphate-accumulating organism (PAO) Candidatus Accumulibacter does not compete well with glycogen accumulating organisms (GAOs) at temperatures above 25 °C. Yet limited information is available on the PAO community and the metabolic capabilities in full-scale EBPR systems operating at high temperature. We studied the composition of the key functional PAO communities in three full-scale wastewater treatment plants (WWTPs) with high in-situ EBPR activity in Singapore, their EBPR-associated carbon usage characteristics, and the relationship between carbon usage and community composition. Each plant had a signature community composed of diverse putative PAOs with multiple operational taxonomic units (OTUs) affiliated to Ca. Accumulibacter, Tetrasphaera spp., Dechloromonas and Ca. Obscuribacter. Despite the differences in community composition, ex-situ anaerobic phosphorus (P)-release tests with 24 organic compounds from five categories (including four sugars, three alcohols, three volatile fatty acids (VFAs), eight amino acids and six other carboxylic acids) showed that a wide range of organic compounds could potentially contribute to EBPR. VFAs induced the highest P release (12.0-18.2 mg P/g MLSS for acetate with a P release-to-carbon uptake (P:C) ratio of 0.35-0.66 mol P/mol C, 9.4-18.5 mg P/g MLSS for propionate with a P:C ratio of 0.38-0.60, and 9.5-17.3 mg P/g MLSS for n-butyrate), followed by some carboxylic acids (10.1-18.1 mg P/g MLSS for pyruvate, 4.5-11.7 mg P/g MLSS for lactate and 3.7-12.4 mg P/g MLSS for fumarate) and amino acids (3.66-7.33 mg P/g MLSS for glutamate with a P:C ratio of 0.16-0.43 mol P/mol C, and 4.01-7.37 mg P/g MLSS for aspartate with a P:C ratio of 0.17-0.48 mol P/mol C). P-release profiles (induced by different carbon sources) correlated closely with PAO community composition. High micro-diversity was observed within the Ca. Accumulibacter lineage, which represented the most abundant PAOs. The total population of Ca. Accumulibacter taxa was highly correlated with P-release induced by VFAs, highlighting the latter's importance in tropical EBPR systems. There was a strong link between the relative abundance of individual Ca. Accumulibacter OTUs and the extent of P release induced by distinct carbon sources (e.g., OTU 81 and amino acids, and OTU 246 and ethanol), suggesting niche differentiation among Ca. Accumulibacter taxa. A diverse PAO community and the ability to use numerous organic compounds are considered key factors for stable EBPR in full-scale plants at elevated temperatures.

Non-denitrifying polyphosphate accumulating organisms obviate requirement for anaerobic condition.

Enhanced biological phosphorus removal (EBPR) is a widely used process in wastewater treatment that requires anaerobic/aerobic or anaerobic/anoxic cycling. Surprisingly, phosphorus (P) release was observed in the presence of nitrate in the anoxic compartment of the activated sludge tank in a full-scale treatment plant with the Modified Ludzack Ettinger configuration. We therefore studied the potential of this full-scale activated sludge community to perform EBPR under anoxic/aerobic cycling. The polyphosphate accumulating organism (PAO) Candidatus Accumulibacter represented 3.3% of total bacteria based on 16S rRNA gene amplicon sequencing, and metagenome analysis suggested it was likely to be dominated by Clade IIC. Using acetate as the carbon source in batch experiments, active denitrifying organisms (DPAOs) were estimated to comprise 39-44% of the total PAO population in the sludge, with the remaining 56-61% unable to utilize nitrate. When propionate was provided as the organic carbon source, 95% of the PAO population was unable to denitrify. EBPR occurred under defined anoxic/aerobic conditions, despite the presence of DPAOs, when synthetic wastewater was supplemented with either acetate or propionate or when primary effluent was supplied. In addition, the P release and subsequent uptake rates under anoxic/aerobic conditions were comparable to those observed under anaerobic/aerobic conditions. In contrast, a significant reduction in P release rate was observed when acetate was provided under oxic conditions. We postulate that non-DPAOs that recognize the anoxic condition as pseudo-anaerobic were the key players in anoxic/aerobic EBPR.

Integrative microbial community analysis reveals full-scale enhanced biological phosphorus removal under tropical conditions.

Management of phosphorus discharge from human waste is essential for the control of eutrophication in surface waters. Enhanced biological phosphorus removal (EBPR) is a sustainable, efficient way of removing phosphorus from waste water without employing chemical precipitation, but is assumed unachievable in tropical temperatures due to conditions that favour glycogen accumulating organisms (GAOs) over polyphosphate accumulating organisms (PAOs). Here, we show these assumptions are unfounded by studying comparative community dynamics in a full-scale plant following systematic perturbation of operational conditions, which modified community abundance, function and physicochemical state. A statistically significant increase in the relative abundance of the PAO Accumulibacter was associated with improved EBPR activity. GAO relative abundance also increased, challenging the assumption of competition. An Accumulibacter bin-genome was identified from a whole community metagenomic survey, and comparative analysis against extant Accumulibacter genomes suggests a close relationship to Type II. Analysis of the associated metatranscriptome data revealed that genes encoding proteins involved in the tricarboxylic acid cycle and glycolysis pathways were highly expressed, consistent with metabolic modelling results. Our findings show that tropical EBPR is indeed possible, highlight the translational potential of studying competition dynamics in full-scale waste water communities and carry implications for plant design in tropical regions.

Population changes in a biofilm reactor for phosphorus removal as evidenced by the use of FISH.

Induction of denitrification was investigated for a lab-scale phosphate removing biofilm reactor where oxygen was replaced with nitrate as the electron acceptor. Acetate was used as the carbon source. The original biofilm (acclimatised with oxygen) was taken from a well-established large-scale reactor. During the first run, a decrease in the denitrifying bio-P activity was observed after 1 month following a change in the anaerobic phase length. This was initially interpreted as a shift in the microbial population caused by the changed operation. In the second run, biomass samples were regularly collected and analysed by fluorescent in situ hybridisation (FISH) and confocal laser scanning microscopy (CLSM). Concurrently, samples were taken from the original reactor with oxygen as electron acceptor in order to investigate natural microbial fluctuations. A similar decrease in the activity as in the first run was seen after one month, although the phase lengths had not been varied. Hence, the decrease after 1 month in the first and second run should be seen as a start-up phenomenon. FISH could detect a noticeable shift in the microbial population mainly within the first 2 weeks of operation. Almost all bacteria belonging to the alpha subclass disappeared and characteristic clusters of the beta and gamma subclasses were lost. Small clusters of gram-positive bacteria with a high DNA G + C content (GPBHGC) were gradually replaced by filamentous GPBHGC. Most of the bacteria in the denitrifying, phosphate removing biofilm belonged to the beta subclass of Proteobacteria. The applied set of gene probes had been selected based on existing literature on biological phosphate removing organisms and included a recently published probe for a Rhodocyclus-like clone. However, none of the specific probes hybridised to the dominant bacterial groups in the reactors investigated. No noticeable changes were detected in the aerobic bench-scale reactor during this period, indicating that the observed changes in the lab-scale reactor were caused by the changed environment.