Cardona, Laëtitia

Abstract Enhanced biological phosphorus removal (EBPR) relies on polyphosphate-accumulating organisms that experience dynamic fluctuations in redox conditions and substrate availability. How these organisms regulate metabolism across molecular layers under cyclic operation remains incompletely understood. Here, we investigated molecular regulation in a long-term, stable aerobic granular sludge system dominated by Candidatus Accumulibacter . We combined metagenomics, time-resolved metatranscriptomics, and matched metaproteomics across three consecutive operational cycles. Genome-resolved metagenomics revealed an Accumulibacter -dominated community encoding canonical EBPR functions. Time-resolved metatranscriptomics revealed extensive and reproducible transcriptional dynamics affecting hundreds of genes. These dynamics did not align with discrete EBPR phases or instantaneous environmental transitions, but instead unfolded gradually over the cycle. Despite pronounced transcript-level heterogeneity, aggregation at the functional level revealed strong consolidation, with only a limited number of KEGG orthologs exhibiting coherent temporal regulation. Quantitative metaproteomics revealed a stable proteome across EBPR cycles, with no individual proteins or functional categories showing significant differential abundance despite direct transcript-protein mapping and time-matched sampling. This indicated that transcriptional dynamics did not translate into measurable changes in protein pool size on the time scale of individual cycles, consistent with slow, growth-coupled proteome turnover relative to cycle duration. Together, these results support a hierarchical model of regulation in Candidatus Accumulibacter , in which dynamic transcriptional responses are buffered at both the functional and protein levels. This organization provides a plausible basis for stable EBPR performance under predictable cyclic operation and demonstrates how time-resolved multi-omics can be used to resolve regulatory dynamics across molecular layers in engineered microbial ecosystems.

Polyphosphate-accumulating organisms (PAOs) play a crucial role in enhanced biological phosphorus removal (EBPR) processes. In addition to biosynthesis, they rely on phosphate for energy generation. However, Candidatus Accumulibacter, a model PAO, has been shown to adapt to low phosphate conditions by switching to a glycogen-accumulating metabolism (GAM), with variable success across genus members and experiments. This study aimed to explore the metabolic shift of several Accumulibacter species subjected to low-phosphate concentration in different operating conditions using metatranscriptomics analysis. Furthermore, the study enabled a comparison of the transcriptomic profiles of Accumulibacter with those of Propionivibrio , a glycogen-accumulating organism typically found in EBPR plants. Two sequencing batch reactors were operated with different carbon sources to enrich for different populations of Accumulibacter . After decreasing the influent phosphate concentration, carbon removal performance was maintained while anaerobic phosphate release dropped dramatically, suggesting a shift from a phosphate-accumulating to a glycogen-accumulating metabolism. Analysis of metatranscriptomics data indicated that Accumulibacter regalis (type I) and Propionivibrio aalborgensis remained the most abundant species after the phosphate decrease in the reactor with acetate-propionate and allylthiourea, while Accumulibacter delftensis (type I) and Accumulibacter phosphatis (type II) remained active in the reactor with acetate-glucose and no allylthiourea. Transcription of the genes from the ethylmalonyl-CoA pathway involved in the production of propionyl-CoA and regulation of the anaerobic redox balance was enhanced under low-phosphate conditions, especially for type I Accumulibacter . Conversely, the transcription of the methylmalonyl-CoA pathway was enhanced under low-phosphate conditions in Propionivibrio and type II Accumulibacter .