Two new clades recovered at high temperatures provide novel phylogenetic and genomic insights into Candidatus Accumulibacter.

Candidatus Accumulibacter, a key genus of polyphosphate-accumulating organisms, plays key roles in lab- and full-scale enhanced biological phosphorus removal (EBPR) systems. A total of 10 high-quality Ca. Accumulibacter genomes were recovered from EBPR systems operated at high temperatures, providing significantly updated phylogenetic and genomic insights into the Ca. Accumulibacter lineage. Among these genomes, clade IIF members SCELSE-3, SCELSE-4, and SCELSE-6 represent the to-date known genomes encoding a complete denitrification pathway, suggesting that Ca. Accumulibacter alone could achieve complete denitrification. Clade IIC members SSA1, SCUT-1, SCELCE-2, and SCELSE-8 lack the entire set of denitrifying genes, representing to-date known non-denitrifying Ca. Accumulibacter. A pan-genomic analysis with other Ca. Accumulibacter members suggested that all Ca. Accumulibacter likely has the potential to use dicarboxylic amino acids. Ca. Accumulibacter aalborgensis AALB and Ca. Accumulibacter affinis BAT3C720 seemed to be the only two members capable of using glucose for EBPR. A heat shock protein Hsp20 encoding gene was found exclusively in genomes recovered at high temperatures, which was absent in clades IA, IC, IG, IIA, IIB, IID, IIG, and II-I members. High transcription of this gene in clade IIC members SCUT-2 and SCUT-3 suggested its role in surviving high temperatures for Ca. Accumulibacter. Ambiguous clade identity was observed for newly recovered genomes (SCELSE-9 and SCELSE-10). Five machine learning models were developed using orthogroups as input features. Prediction results suggested that they belong to a new clade (IIK). The phylogeny of Ca. Accumulibacter was re-evaluated based on the laterally derived polyphosphokinase 2 gene, showing improved resolution in differentiating different clades.

Relating the carbon sources to denitrifying community in full-scale wastewater treatment plants.

Carbon source is a key factor determining the denitrifying effectiveness and efficiency in wastewater treatment plants (WWTPs). Whereas, the relationships between diverse and distinct denitrifying communities and their favorable carbon sources in full-scale WWTPs were not well-understood. This study performed a systematic analysis of the relationships between the denitrifying community and carbon sources by using 15 organic compounds from four categories and activated sludge from 8 full-scale WWTPs. Results showed that, diverse denitrifying bacteria were detected with distinct relative abundances in 8 WWTPs, such as Haliangium (1.98-4.08%), Dechloromonas (2.00-3.01%), Thauera (0.16-1.06%), Zoogloea (0.09-0.43%), and Rhodoferax (0.002-0.104%). Overall, acetate resulted in the highest denitrifying activities (1.21-4.62 mg/L/h/gMLSS), followed by other organic acids (propionate, butyrate and lactate, etc.). Detectable dissimilatory nitrate reduction to ammonium (DNRA) was observed for all 15 carbon sources. Methanol and glycerol resulted in the highest DRNA. Acetate, butyrate, and lactate resulted in the lowest DNRA. Redundancy analysis and 16S cDNA amplicon sequencing suggested that carbon sources within the same category tended to correlate to similar denitrifiers. Methanol and ethanol were primarily correlated to Haliangium. Glycerol and amino acids (glutamate and aspartate) were correlated to Inhella and Sphaerotilus. Acetate, propionate, and butyrate were positively correlated to a wide range of denitrifiers, explaining the high efficiency of these carbon sources. Additionally, even within the same genus, different amplicon sequence variants (ASVs) performed distinctly in terms of carbon source preference and denitrifying capabilities. These findings are expected to benefit carbon source formulation and selection in WWTPs.

Blind spots of universal primers and specific FISH probes for functional microbe and community characterization in EBPR systems.

Fluorescence in situ hybridization (FISH) and 16S rRNA gene amplicon sequencing are commonly used for microbial ecological analyses in biological enhanced phosphorus removal (EBPR) systems, the successful application of which was governed by the oligonucleotides used. We performed a systemic evaluation of commonly used probes/primers for known polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs). Most FISH probes showed blind spots and covered nontarget bacterial groups. Ca. Competibacter probes showed promising coverage and specificity. Those for Ca. Accumulibacter are desirable in coverage but targeted out-group bacteria, including Ca. Competibacter, Thauera, Dechlorosoma, and some polyphosphate-accumulating Cyanobacteria. Defluviicoccus probes are good in specificity but poor in coverage. Probes targeting Tetrasphaera or Dechloromonas showed low coverage and specificity. Specifically, DEMEF455, Bet135, and Dech453 for Dechloromonas covered Ca. Accumulibacter. Special attentions are needed when using these probes to resolve the PAO/GAO phenotype of Dechloromonas. Most species-specific probes for Ca. Accumulibacter, Ca. Lutibacillus, Ca. Phosphoribacter, and Tetrasphaera are highly specific. Overall, 1.4% Ca. Accumulibacter, 9.6% Ca. Competibacter, 43.3% Defluviicoccus, and 54.0% Dechloromonas in the MiDAS database were not covered by existing FISH probes. Different 16S rRNA amplicon primer sets showed distinct coverage of known PAOs and GAOs. None of them covered all members. Overall, 520F-802R and 515F-926R showed the most balanced coverage. All primers showed extremely low coverage of Microlunatus (<36.0%), implying their probably overlooked roles in EBPR systems. A clear understanding of the strength and weaknesses of each probe and primer set is a premise for rational evaluation and interpretation of obtained community results.

Candidatus Accumulibacter use fermentation products for enhanced biological phosphorus removal.

Previous research suggested that two major groups of polyphosphate-accumulating organisms (PAOs), i.e., Ca. Accumulibacter and Tetrasphaera, play cooperative roles in enhanced biological phosphorus removal (EBPR). The fermentation of complex organic compounds by Tetrasphaera provides carbon sources for Ca. Accumulibacter. However, the viability of the fermentation products (e.g., lactate, succinate, alanine) as carbon sources for Ca. Accumulibacter and their potential effects on the metabolism of Ca. Accumulibacter were largely unknown. This work for the first time investigated the capability and metabolic details of Ca. Accumulibacter cognatus clade IIC strain SCUT-2 (enriched in a lab-scale reactor with a relative abundance of 42.8%) in using these fermentation products for EBPR. The enrichment culture was able to assimilate lactate and succinate with the anaerobic P release to carbon uptake ratios of 0.28 and 0.36 P mol/C mol, respectively. In the co-presence of acetate, the uptake of lactate was strongly inhibited, since two substrates shared the same transporter as suggested by the carbon uptake bioenergetic analysis. When acetate and succinate were fed at the same time, Ca. Accumulibacter assimilated two carbon sources simultaneously. Proton motive force (PMF) was the key driving force (up to 90%) for the uptake of lactate and succinate by Ca. Accumulibacter. Apart from the efflux of proton in symport with phosphate via the inorganic phosphate transport system, translocation of proton via the activity of fumarate reductase contributed to the generation of PMF, which agreed with the fact that PHV was a major component of PHA when lactate and succinate were used as carbon sources, involving the succinate-propionate pathway. Metabolic models for the usage of lactate and succinate by Ca. Accumulibacter for EBPR were built based on the combined physiological, biochemical, metagenomic, and metatranscriptomic analyses. Alanine was shown as an invalid carbon source for Ca. Accumulibacter. Instead, it significantly and adversely affected Ca. Accumulibacter-mediated EBPR. Phosphate release was observed without alanine uptake. Significant inhibitions on the aerobic phosphate uptake was also evident. Overall, this study suggested that there might not be a simply synergic relationship between Ca. Accumulibacter and Tetrasphaera. Their interactions would largely be determined by the kind of fermentation products released by the latter.

CRISPR-Cas phage defense systems and prophages in Candidatus Accumulibacter.

Candidatus Accumulibacter plays a major role in enhanced biological phosphorus removal (EBPR) from wastewater. Although bacteriophages have been shown to represent fatal threats to Ca. Accumulibacter organisms and thus interfere with the stability of the EBPR process, little is known about the ability of different Ca. Accumulibacter strains to resist phage infections. We conducted a systematic analysis of the occurrence and characteristics of clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR-Cas) systems and prophages in Ca. Accumulibacter lineage members (43 in total, including 10 newly recovered genomes). Results indicate that 28 Ca. Accumulibacter genomes encode CRISPR-Cas systems. They were likely acquired via horizontal gene transfer, conveying a distinct adaptivity to phage predation to different Ca. Accumulibacter members. Major differences in the number of spacers show the unique phage resistance of these members. A comparison of the spacers in closely related Ca. Accumulibacter members from distinct geographical locations indicates that habitat isolation may have resulted in the acquisition of resistance to different phages by different Ca. Accumulibacter. Long-term operation of three laboratory-scale EBPR bioreactors revealed high relative abundances of Ca. Accumulibacter with CRISPSR-Cas systems. Their specific resistance to phages in these reactors was indicated by spacer analysis. Metatranscriptomic analyses showed the activation of the CRISPR-Cas system under both anaerobic and aerobic conditions. Additionally, 133 prophage regions were identified in 43 Ca. Accumulibacter genomes. Twenty-seven of them (in 19 genomes) were potentially active. Major differences in the occurrence of CRISPR-Cas systems and prophages in Ca. Accumulibacter will lead to distinct responses to phage predation. This study represents the first systematic analysis of CRISPR-Cas systems and prophages in the Ca. Accumulibacter lineage, providing new perspectives on the potential impacts of phages on Ca. Accumulibacter and EBPR systems.

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.

Carbon uptake bioenergetics of PAOs and GAOs in full-scale enhanced biological phosphorus removal systems.

This work analyzed, for the first time, the bioenergetics of PAOs and GAOs in full-scale wastewater treatment plants (WWTPs) for the uptake of different carbon sources. Fifteen samples were collected from five full-scale WWTPs. Predominance of different PAOs, i.e., Ca. Accumulibacter (0.00-0.49%), Tetrasphaera (0.37-3.94%), Microlunatus phosphovorus (0.01-0.18%), etc., and GAOs, i.e., Ca. Competibacter (0.08-5.39%), Defluviicoccus (0.05-5.34%), Micropruina (0.17-1.87%), etc., were shown by 16S rRNA gene amplicon sequencing. Despite the distinct PAO/GAO community compositions in different samples, proton motive force (PMF) was found as the key driving force (up to 90.1%) for the uptake of volatile fatty acids (VFAs, acetate and propionate) and amino acids (glutamate and aspartate) by both GAOs and PAOs at the community level, contrasting the previous understanding that Defluviicoccus have a low demand of PMF for acetate uptake. For the uptake of acetate or propionate, PAOs rarely activated F1, F0- ATPase (< 11.7%) or fumarate reductase (< 5.3%) for PMF generation; whereas, intensive involvements of these two pathways (up to 49.2% and 61.0%, respectively) were observed for GAOs, highlighting a major and community-level difference in their VFA uptake biogenetics in full-scale systems. However, different from VFAs, the uptake of glutamate and aspartate by both PAOs and GAOs commonly involved fumarate reductase and F1, F0-ATPase activities. Apart from these major and community-level differences, high level fine-scale micro-diversity in carbon uptake bioenergetics was observed within PAO and GAO lineages, probably resulting from their versatilities in employing different pathways for reducing power generation. Ca. Accumulibacter and Halomonas seemed to show higher dependency on the reverse operation of F1, F0-ATPase than other PAOs, likely due to the low involvement of glyoxylate shunt pathway. Unlike Tetrasphaera, but similar to Ca. Accumulibacter, Microlunatus phosphovorus took up glutamate and aspartate via the proton/glutamate-aspartate symporter driven by PMF. This feature was testified using a pure culture of Microlunatus phosphovorus stain NM-1. The major difference between PAOs and GAOs highlights the potential to selectively suppress GAOs for community regulation in EBPR systems. The finer-scale carbon uptake bioenergetics of PAOs or GAOs from different lineages benefits in understanding their interactions in community assembly in complex environment.

Glycine adversely affects enhanced biological phosphorus removal.

Enhanced biological phosphorus removal (EBPR) is used extensively in full-scale wastewater treatment plants for the removal of phosphorus. Despite previous evidence showing that glycine is a carbon source for a certain lineage of polyphosphate accumulating organisms (PAOs) such as Tetrasphaera, it is still unknown whether glycine can support EBPR. We observed an overall adverse effect of glycine on EBPR using activated sludge from both full-scale wastewater treatment plants and lab-scale reactors harboring distant and diverse PAOs and glycogen accumulating organisms (GAOs), including Candidatus Accumulibacter, Thiothrix, Tetrasphaera, Dechloromonas, Ca. Competibacter, and Defluviicoccus, among others. Glycine induced phosphorus (P) release under anaerobic conditions without being effectively taken up by cells. The induced P release rate correlated with glycine concentration in the range of 10 to 50 mg C/L. PAOs continued to release P in the presence of glycine under aerobic conditions without any evident P uptake. Under mixed carbon conditions, the occurrence of glycine did not seem to affect acetate uptake; however, it significantly reduced the rate of P uptake in the aerobic phase. Overall, glycine did not appear to be an effective carbon source for a majority of PAOs and GAOs in full-scale and lab-scale systems, and neither did other community members utilize glycine under anaerobic or aerobic conditions. Metatranscriptomic analysis showed the transcription of glycine cleavage T, P and H protein genes, but not of the L protein or the downstream genes in the glycine cleavage pathway, suggesting barriers to metabolizing glycine. The high transcription of a gene encoding a drug/metabolite transporter suggests a potential efflux mechanism, where glycine transported into the cells is in turn exported at the expense of ATP, resulting in P release without affecting the glycine concentration in solution. The ability of glycine to induce P release without cellular uptake suggests a way to effectively recover P from P-enriched waste sludge.