Identification of glucose-fermenting bacteria in a full-scale enhanced biological phosphorus removal plant by stable isotope probing

• All WWTPs studied had stable nitrogen and phosphorus removal at the time of sampling.

• SIP experiments confirmed glucose fermentation kinetics in Aalborg East WWTP, at 0.122 ± 0.027 mmol glucose/(g VSS)/h.

• Glucose was fermented into SCFAs: formic, acetic, propionic, butyric, and lactic acids.

• RNA-SIP revealed active uptake of 13C-labeled glucose, with effective RNA extraction and clear density gradients separating labeled RNA.

• Phylogenetic analysis of labeled RNA identified major groups: Firmicutes, Actinobacteria, and Proteobacteria.

• Designed specific FISH probes for Lactococcus, Propionicimonas, and Streptococcus; optimized across 10 full-scale WWTPs.

• MAR-FISH confirmed that all FISH-positive bacteria from these genera actively fermented glucose.

• Incubation with glucose did not alter microbial composition, confirming probe stability and specificity.

• Most fermenting bacteria were Firmicutes and Actinobacteria, with Tetrasphaera, Propionicimonas, and Streptococcus as key fermenters.

• Quantification across 10 WWTPs showed:

  • Tetrasphaera: up to 44% of total biovolume (avg. 33%)

  • Propionicimonas: present in all plants (avg. 4–5%)

  • Streptococcus & Lactococcus: generally low abundance (mostly <1%)

• Tetrasphaera clades 1–3 were all capable of anaerobic glucose fermentation, confirmed by MAR-FISH.

• The new FISH probes identified ~85% of all glucose-fermenting bacteria in the samples.

The study successfully achieved its initial objectives:

• Identified key glucose-fermenting bacteria in activated sludge using rRNA-based Stable Isotope Probing (SIP) and validated their physiology with MAR-FISH.

• Confirmed the in situ fermentation ability of Tetrasphaera, Streptococcus, and newly identified Propionicimonas as dominant glucose fermenters.

• Quantified the fermenters’ abundance, revealing they comprise up to 38.9 ± 7% of total bacterial biovolume across 10 full-scale EBPR wastewater treatment plants.

• Demonstrated the functional diversity of fermenters, most of which are facultative and belong to the phyla Actinobacteria and Firmicutes.

• Revealed new functional roles for known and previously unassociated genera (e.g. Propionicimonas, Lactococcus, Tolumonas, and Burkholderia) in glucose fermentation.

• Designed and validated new FISH probes for accurate detection and quantification of fermenting populations in situ.

• Highlighted the ecological and operational importance of fermentation, showing it as a widespread and significant process in activated sludge systems.

• Provided actionable insight into improving biological N and P removal through better understanding and monitoring of fermenter communities.

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