Genome-centric Insights into Full-scale EBPR Captured During a Biomonitoring Campaign

Phosphorus removal from wastewater remains a core component of modern wastewater treatment strategies critical for protecting aquatic ecosystems and capturing valuable nutrients. Enhanced biological phosphorus removal (EBPR) is attractive since it reduces the need for chemical precipitating agents and facilitates phosphorus recovery. However, instability can limit the reliable application of EBPR for meeting stringent effluent phosphorus limits. As part of an ongoing biomonitoring campaign, samples were collected before and during an EBPR process stability episode and sequenced for paired metagenomes and metatranscriptomes. Analysis revealed that shifts in carbon and phosphorus loads coupled with changes in the population sizes of non-fermenting polyphosphate accumulating organisms (PAOs; Candidatus Accumulibacter spp.), as identified in the metagenomic data, were associated with poor P-uptake. Metatranscriptomic data from these PAO populations revealed they were indeed substrate limited, as hypothesized from process data, with key genes related to carbon storage appearing down regulated during the instability event with respect to the stable control despite having a greater cell load during the instability period. Accordingly, several gene families related to carbon uptake, VFA metabolism, and PHA synthesis were found to be sensitive to the underlying carbon-limited conditions of the process instability. Our findings illustrate how combined metagenomic and metatranscriptomic sequencing can facilitate interpretation of process data and aid in evaluating the root cause of instability. These data also indicate potential gene targets suitable for developing EBPR performance markers as part of longer term or more frequent biomonitoring efforts.