Candidatus Thiothrix phosphatis SCUT-1: A novel polyphosphate-accumulating organism abundant in the enhanced biological phosphorus removal system.

A novel coccus Thiothrix-related polyphosphate-accumulating organism (PAO) was enriched in an acetate-fed enhanced biological phosphorus removal (EBPR) system. High EBPR performance was achieved for an extended period (>100 days). A high-quality draft genome (completeness 97.2 %, contamination 3.26 %) was retrieved, representing a novel Thiothrix species (with similarity<93.2 % to known Thiothrix species), and was denoted as 'Candidatus Thiothrix phosphatis SCUT-1'. Its acetate uptake rate (6.20 mmol C/g VSS/h) surpassed most Ca. Accumulibacter and known glycogen-accumulating organisms (GAOs), conferring their predominance in the acetate-fed system. Metatranscriptomic analysis suggested that Ca. Thiothrix phosphatis SCUT-1 employed both low- and high-affinity pathways for acetate activation, and both the conventional (PhaABC) pathway and the fatty acid ß-oxidation pathway for PHA synthesis; additionally, a much more efficient FAD-dependent malate: quinone oxidoreductase (MQO) were encoded and employed than the traditional malate dehydrogenase (MDH) to oxidize malate to oxaloacetate in the TCA and glyoxylate cycle, collectively contributing to a higher acetate utilization and processing rate of this microorganism. Batch tests further demonstrated the versatile ability of this PAO in using VFA (acetate, propionate, and butyrate), lactate, amino acids (aspartate and glutamate), and glucose as carbon sources for EBPR, showing a partially overlapped but unique ecological niche of this microorganism comparing to Ca. Accumulibacter and known GAOs. A metabolic model was built for Ca. Thiothrix phosphatis SCUT-1 using the above-mentioned carbon sources for EBPR. Overall, this study represents the first comprehensive characterization of the physiology and metabolic characteristics of representative coccus Thiothrix-related PAOs, which are expected to provide new insights into PAO microbiology in EBPR systems.

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.

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.