Metagenomic and metaproteomic analyses of Accumulibacter phosphatis-enriched floccular and granular biofilm.

Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here we operated two laboratory-scale sequencing batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal. Reactors formed two distinct biofilms, one floccular biofilm, consisting of small, loose, microbial aggregates, and one granular biofilm, forming larger, dense, spherical aggregates. Using metagenomic and metaproteomic methods, we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. To understand biofilm differences, we compared protein abundances that were statistically enriched in both biofilm states. Floccular biofilms were enriched with pathogenic secretion systems suggesting a highly competitive microbial community. Comparatively, granular biofilms revealed a high-stress environment with evidence of nutrient starvation, phage predation pressure, and increased extracellular polymeric substance and cell lysis. Granular biofilms were enriched in outer membrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter-enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.

High-temperature EBPR process: the performance, analysis of PAOs and GAOs and the fine-scale population study of Candidatus "Accumulibacter phosphatis".

The applicability of the enhanced biological phosphorus removal (EBPR) process for the removal of phosphorus in warm climates is uncertain due to frequent reports of EBPR deterioration at temperature higher than 25 °C. Nevertheless, a recent report on a stable and efficient EBPR process at 28 °C has inspired the present study to examine the performance of EBPR at 24 °C-32 °C, as well as the PAOs and GAOs involved, in greater detail. Two sequencing batch reactors (SBRs) were operated for EBPR in parallel at different temperatures, i.e., SBR-1 at 28 °C and SBR-2 first at 24 °C and subsequently at 32 °C. Both SBRs exhibited high phosphorus removal efficiencies at all three temperatures and produced effluents with phosphorus concentrations less than 1.0 mg/L during the steady state of reactor operation. Real-time quantitative polymerase chain reaction (qPCR) revealed Accumulibacter-PAOs comprised 64% of the total bacterial population at 24 °C, 43% at 28 °C and 19% at 32 °C. Based on fluorescent in situ hybridisation (FISH), the abundance of Competibacter-GAOs at both 24 °C and 28 °C was rather low (<10%), while it accounted for 40% of the total bacterial population at 32 °C. However, the smaller Accumulibacter population and larger population of Competibacter at 32 °C did not deteriorate the phosphorus removal performance. A polyphosphate kinase 1 (ppk1)-based qPCR analysis on all studied EBPR processes detected only Accumulibacter clade IIF. The Accumulibacter population shown by 16S rRNA and ppk1 was not significantly different. This finding confirmed the existence of single clade IIF in the processes and the specificity of the clade IIF primer sets designed in this study. Habitat filtering related to temperature could have contributed to the presence of a unique clade. The clade IIF was hypothesised to be able to perform the EBPR activity at high temperatures. The clade's robustness most likely helps it to fit the high-temperature EBPR sludge best and allows it not only to outcompete other Accumulibacter clades but coexist with GAOs without compromising EBPR activity.

Evidence for bacteriophage activity causing community and performance changes in a phosphorus-removal activated sludge.

Bacteria are known to play important roles in biogeochemical cycles and biotechnology processes, but little is known about the influence of bacteriophage on these processes. A major impediment to the study of host-bacteriophage interactions is that the bacteria and their bacteriophage are often not available in a pure culture. In this study, we detected an unexpected decline in the phosphorus-removal performance of a granular laboratory-scale wastewater treatment reactor. Investigations by FISH, transmission electron microscopy and proteomics led us to hypothesize that a bacteriophage infection of the uncultured Candidatus 'Accumulibacter phosphatis' was responsible for the decline in performance. Further experiments demonstrated that the addition of a putative bacteriophage-rich supernatant, obtained from the previous failed reactor to phosphorus-removal reactors, caused a decrease in the abundance of Accumulibacter in both granular and floccular activated sludges. This coincided with increases in bacteriophage-like particles and declining phosphorus-removal performance. The granular sludge did not recover after the attack, but the floccular sludge regained Accumulibacter numbers and phosphorus-removal performance. These findings suggest that bacteriophage may play a significant role in determining the structure and function of bacterial communities in activated sludges.

Effect of pH reduction on polyphosphate- and glycogen-accumulating organisms in enhanced biological phosphorus removal processes.

We investigated the effect of pH reduction on polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) in the enhanced biological phosphorus removal (EBPR) process. Three laboratory-scale EBPR reactors were used. Initially, the reactors were operated at pH 7.9±0.1 and 6.5±0.1, and after 27 days, the pH was lowered to 6.5±0.1 and 6.0±0.1, respectively. PAOs and GAOs were monitored using real-time quantitative polymerase chain reaction and/or fluorescent in situ hybridization. Phosphorus removal performance was also monitored. During the start-up period, high EBPR activity and increases in Candidatus 'Accumulibacter phosphatis' (Accumulibacter) and Candidatus 'Competibacter phosphatis' (Competibacter) were observed. In all runs, Accumulibacter and Competibacter were the dominant PAO and GAO, respectively. Accumulibacter began to decline 10-18 days after lowering the pH to 6.5±0.1. After lowering the pH to 6.0±0.1, the Accumulibacter population decreased immediately. Contrastingly, an obvious adverse effect of pH reduction on Competibacter was not observed. In all runs, EBPR activity began to deteriorate 6-12 days after Accumulibacter decline began. Thus, our results show that pH reduction had an immediate or delayed effect on Accumulibacter decline. Moreover, the time lag between the start of Accumulibacter decline and that of EBPR deterioration implies that EBPR deterioration by pH reduction went through unknown process.