Novel dichloromethane-fermenting bacteria in the Peptococcaceae family

Abstract

Dichloromethane (DCM; CH₂Cl₂) is a toxic groundwater pollutant that also has a detrimental effect on atmospheric ozone levels. As a dense non-aqueous phase liquid, DCM migrates vertically through groundwater to low redox zones, yet information on anaerobic microbial DCM transformation remains scarce due to a lack of cultured organisms. We report here the characterisation of DCMF, the dominant organism in an anaerobic enrichment culture (DFE) capable of fermenting DCM to the environmentally benign product acetate. Stable carbon isotope experiments demonstrated that the organism assimilated carbon from DCM and bicarbonate via the Wood-Ljungdahl pathway. DCMF is the first anaerobic DCM-degrading population also shown to metabolise non-chlorinated substrates. It appears to be a methylotroph utilising the Wood-Ljungdahl pathway for metabolism of methyl groups from methanol, choline, and glycine betaine. The flux of these substrates from subsurface environments may either directly (DCM, methanol) or indirectly (choline, glycine betaine) affect the climate. Community profiling and cultivation of cohabiting taxa in culture DFE without DCMF suggest that DCMF is the sole organism in this culture responsible for substrate metabolism, while the cohabitants persist via necromass recycling. Genomic and physiological evidence support placement of DCMF in a novel genus within the Peptococcaceae family, 'Candidatus Formimonas warabiya'.

Fig. 1. DCM degradation by culture DFE.

A DCMF growth was concomitant with the depletion of DCM and formation of acetate. Substrate and product concentrations are quantified on the left y-axis; DCMF and total bacterial 16S rRNA gene copies are quantified on the right y-axis. Error bars represent standard deviation, n = 2. B In MOPS-buffered medium, DCM consumption was only observed in the presence of bicarbonate (filled circles). Cumulative DCM consumption is from repeat amendment of 1 mM DCM. Empty circles represent cultures with no exogenous bicarbonate. Error bars represent standard deviation, n = 3.

Fig. 2. Degradation of methanol and quaternary amines by culture DFE.

DCMF growth correlated with the depletion of methanol and formation of acetate (A) and the depletion of choline (B) and glycine betaine (C) with formation of acetate and monomethylamine. Cultures amended with glycine betaine and hydrogen (D) did not produce trimethylamine, rather acetate and monomethylamine were once again the products. Substrate and product concentrations are quantified on the left y-axis; DCMF and total bacterial 16S rRNA gene copies are quantified on the right y-axis. Error bars represent standard deviation, n = 3.

Fig. 3. Temporal shifts in the community composition of culture DFE with different substrates.

Illumina 16S rRNA amplicon sequencing was used to determine DFE community composition (left y-axis) at timepoints across the growth experiments amended with A DCM, B choline, C glycine betaine, and D methanol reported in Figs. 1 and 2. ASVs are reported down to genus level where possible, otherwise taxonomic level is indicated in the legend ([F] = family, [P] = phylum, [C] = class, [O] = order). Reads with <1% abundance were filtered out in QIIME2. Unassigned reads and ASVs consistently <2% relative abundance were classed together as ‘Other’. Substrate concentration (black circles, right y-axis) and a line connecting the mean substrate concentration at each time point is overlaid on the community composition graphs. These are aligned with the time points written on the x-axis, not drawn to scale.

Fig. 4. Morphology of DCMF.

FISH microscopy images show DCMF cells stained red with the Cy3-labelled Dcm623 probe (A), all bacterial cells stained green with the 6-FAM-labelled Eub338i probe (B), and the overlay of Cy3- and 6-FAM-labelling in these images (C). The scale bars represent 10 µM.

Fig. 5. Assimilation of carbon from ¹³C-labelled DCM and bicarbonate to form acetate.

Cumulative [¹³C]DCM consumption with concomitant acetate production (A), the proportion of labelled and unlabelled acetate (B), and the ¹³C mass balance from [¹³C]DCM (C). Error bars represent standard deviation, n = 3. Cumulative DCM consumption and acetate production in cultures amended with H¹³CO₃⁻ (D). Total (labelled and unlabelled) aqueous HCO₃⁻ is also shown (i.e. gaseous CO₂ is not accounted for here). Values in (D) are from a single representative culture as all triplicates had similar dechlorination rates and product concentrations but began dechlorinating at different times. The proportion of labelled and unlabelled acetate (E) and ¹³C mass balance from H¹³CO₃⁻ (F) is again shown. All pie charts represent the average of triplicate cultures at the final time point.

Fig. 6. Proposed model for metabolism of DCM, methanol, and quaternary amines by DCMF.

The Wood–Ljungdahl pathway is central to transformation of all substrates into acetate. Proteins (with the exception of that catalysing the putative transformation of DCM to CH₂=FH₂, indicated by dotted arrow) are all identified in the DCMF genome and listed in Table S5. CODH/ACS carbon monoxide dehydrogenase/acetyl-CoA synthase, DCM dichloromethane, DMG dimethylglycine, GB glycine betaine, MT methyltransferase, ox oxidised, red reduced, THF tetrahydrofolate, Tr thioredoxin.