Wilmes, Paul

Abstract The candidate phylum Cloacimonadota is frequently detected in anoxic environments such as anaerobic digestion (AD) reactors, hydrothermal vents, and deep-sea sediments, yet its metabolism remains poorly understood. Metagenomic evidence suggests capacities for amino acid fermentation, carbohydrate degradation, as well as a potential role in syntrophic propionate oxidation (SPO), a key bottleneck in AD. However, a complete methylmalonyl-CoA (mmc) pathway, central to SPO, has not been previously identified in Cloacimonadota genomes. Here, we report results from an acidified lab-scale anaerobic baffled reactor fed with sugar beet pulp, where an increase in the relative abundance of Cloacimonadota correlated with recovery of methanogenesis, resulting in increased methane content in the produced biogas. Metagenomic and metatranscriptomic analyses enabled metabolic reconstruction of the dominant Cloacimonadota OTU. Furthermore, using a curated database of 204 genome-resolved Cloacimonadota species, we characterised the phylum-level metabolic potential. Comparative genomics revealed alternative proteins, including 2-oxoglutarate:ferredoxin oxidoreductase and aspartate aminotransferase, likely to substitute for missing enzymes in the classical mmc pathway. These proteins were widely distributed and highly conserved across the analysed Cloacimonadota genomes, suggesting that this variant of the SPO pathway could represent a phylum-specific trait. Moreover, we hypothesise that these alternative pathway steps may link propionate metabolism to protein degradation and poly-γ-glutamate biosynthesis. Network analysis identified the methanogenic archaeon Methanothrix as a potential syntrophic partner, an interaction further supported by propionate-fed enrichment cultures showing co-occurrence of Cloacimonadota and Methanothrix species. Our study sheds light on the Cloacimonadota metabolism, advancing our understanding of their ecological roles and potential for biotechnological applications.

Abstract The candidate phylum Cloacimonadota is frequently detected in anaerobic environments such as anaerobic digestion (AD) reactors, hydrothermal vents, and deep-sea sediments, yet its metabolism remains poorly understood due to the lack of cultured representatives. Metagenomic evidence suggests capacities for amino acid fermentation, cellulose degradation, and production of carbohydrate-active enzymes, with particular interest in their presumed role in syntrophic propionate oxidation (SPO), a key bottleneck in AD. However, a complete methylmalonyl-CoA (mmc) pathway, central to SPO, has not been previously identified in Cloacimonadota genomes. Here, we report results from a lab-scale anaerobic baffled reactor fed with sugar beet pulp, where a sharp increase in an uncultured Cloacimonadota OTU coincided with recovery of methanogenesis and enhanced methane production. Metagenomic and metatranscriptomic analyses enabled metabolic reconstruction of this OTU, complemented by a curated database of 47 genome-resolved Cloacimonadota species. Comparative genomics revealed conserved protein clusters indicative of an alternative mmc pathway, suggesting that this variant of the SPO pathway is a widespread, phylum-specific trait potentially linked to protein degradation and poly-γ-glutamate biosynthesis. Network analysis identified the methanogenic archaeon Methanothrix as a primary syntrophic partner, an interaction further supported by propionate-fed enrichment cultures showing co-occurrence of Cloacimonadota and Methanothrix species. Our study sheds light on the Cloacimonadota metabolism, advancing our understanding of their ecological roles and potential for biotechnological applications.

Abstract Microorganisms in biological wastewater treatment plants require adaptive strategies to deal with rapidly fluctuating environmental conditions. At the population level, the filamentous bacterium Candidatus Microthrix parvicella (Ca. M. parvicella) has been found to fine-tune its gene expression for optimized substrate assimilation. Here we investigated in situ substrate assimilation by single cells of Ca. M. parvicella using nano-scale secondary-ion mass spectrometry (nanoSIMS). NanoSIMS imaging highlighted phenotypic heterogeneity among Ca. M. parvicella cells of the same filament, whereby 13C-oleic acid and 13C-glycerol-3-phosphate assimilation occurred in ≈21–55% of cells, despite non-assimilating cells being intact and alive. In response to alternating aerobic–anoxic regimes, 13C-oleic acid assimilation occurred among subpopulations of Ca. M. parvicella cells (≈3–28% of cells). Furthermore, Ca. M. parvicella cells exhibited two temperature optima for 13C-oleic acid assimilation and associated growth rates. These results suggest that phenotypic heterogeneity among Ca. M. parvicella cells allows the population to adapt rapidly to fluctuating environmental conditions facilitating its widespread occurrence in biological wastewater treatment plants.

ABSTRACT “ Candidatus Microthrix” bacteria are deeply branching filamentous actinobacteria which occur at the water-air interface of biological wastewater treatment plants, where they are often responsible for foaming and bulking. Here, we report the first draft genome sequence of a strain from this genus: “ Candidatus Microthrix parvicella” strain Bio17-1.