Multiple extracellular polymeric substance pathways transcribed by Accumulibacter and the flanking community during aerobic granule formation and after influent modification

Abstract

Aerobic granular sludge is a biological wastewater treatment process in which a microbial community forms a granular biofilm. The role of Candidatus Accumulibacter in the production of a biofilm matrix composed of extracellular polymeric substances was studied in a sequencing batch reactor enriched with polyphosphate-accumulating organisms. The metabolisms of the microbial populations were investigated using de novo metatranscriptomics analysis. Finally, the effect of decreasing the influent phosphate concentration on the granule stability and microbial activity was investigated. A few weeks after the reactor start-up, the microbial community was dominated by Accumulibacter with up to nine species active in parallel. However, the most active species differed according to sampling time. Decreasing drastically the influent phosphate concentration led to a dominance of the glycogen-accumulating organism Propionivibrio, with some Accumulibacter species still abundant. De novo metatranscriptomics analysis indicated a high diversity of potential extracellular substances produced mainly by Accumulibacter, Azonexus, Candidatus Contendobacter, and Propionivibrio. Moreover, the results suggest that Azonexus, Contendobacter, and Propionivibrio recycle the neuraminic acids produced by Accumulibacter. Changes in the microbial community did not cause the granules to disintegrate, indicating that a Propionivibrio-dominated community can maintain stable granules.IMPORTANCEOne of the main advantages of the aerobic granular sludge wastewater treatment process is the higher settling velocities compared to the conventional activated sludge-based process. In aerobic granular sludge, the biomass is concentrated into a biofilm matrix composed of biopolymers, providing micro-niches to different types of microbial populations. We demonstrate with the help of de novo metatranscriptomics analysis that the formation of granules is a highly dynamic microbial process, even when enriching for a microbial guild, such as phosphate-accumulating organisms. Often underestimated, the flanking community of the main phosphate-accumulating organisms population enriched in the reactor is nonetheless active and transcribing genes related to different extracellular polymeric substance pathways. The multiplicity of the extracellular polymeric substances produced probably helped the matrix to remain stable, thanks to their specific properties. Moreover, the results suggest microbial interactions in extracellular polymeric substance recycling between different microbial populations that can be helpful to prevent a disruption of the granules while stressing out the microbial community.

Keywords: 16S rRNA gene amplicon sequencing; Propionivibrio; enhanced biological phosphate removal; metatranscriptomics; phosphate-accumulating organisms; sequencing batch reactor.

Fig 1

Nutrient removal and efficiencies during the 183 days of reactor operation. (A) Carbon removal efficiency during the anaerobic phase. (B) Phosphate concentration at the different phases: influent, end of anaerobic, and end of aerobic phase. The dashed line at day 103 indicates the change in the influent composition from COD/P of 12 to 200.

Fig 2

Relative abundance of the most abundant microorganisms. Relative abundance of the most abundant genera (minimum 3% in at least one sample) based on the 16S rRNA gene amplicon sequencing. The gray dashed line at day 103 indicates the change in the medium composition (from COD/P of 12 to COD/P of 200). Genera are colored based on the class level, with the exception of Accumulibacter and Propionivibrio highlighted in purple and yellow, respectively.

Fig 3

Active microbial population at different stages of granulation and influent composition. (A) Relative proportion of the gene expression per genus from the de novo metatranscriptomics analysis. Only the 20 most abundant genera are represented. (B) Shannon diversity index calculated using the level of expression of each genus. (C) Expression profile at the species level of the most abundant microorganisms using mOTUs (minimum of 3% in one sample).

Fig 4

Level of transcription of genes (log(cpm)) of metabolisms involved in the enhanced biological phosphate removal process at the different time points of sampling taken during the feeding phase. Differential gene expression analysis was done between two time points (26 vs 13, 103 vs 26, and 182 vs 103), and the significant differences (log-2 fold change > 2 and adjusted P value < 0.01) are represented by a triangle (up-pointing for higher expressed genes compared to the day before and down-pointing triangle for lower expression).

Fig 5

Transcription of genes related to extracellular polymeric substances (EPS) production at different time point sampling. Level of expression (log(cpm)) of genes per day for different genera expressing genes involved in EPS production. Differential gene expression analysis was done between two time points (26 vs 13, 103 vs 26, and 182 vs 103), and the significant differences (log-2 fold change > 2 and adjusted P value < 0.01) are represented by a triangle (up-pointing for higher expressed genes compared to the day before and down-pointing triangle for lower expression).