Fermentative Spirochaetes mediate necromass recycling in anoxic hydrocarbon-contaminated habitats

Abstract

Spirochaetes are frequently detected in anoxic hydrocarbon- and organohalide-polluted groundwater, but their role in such ecosystems has remained unclear. To address this, we studied a sulfate-reducing, naphthalene-degrading enrichment culture, mainly comprising the sulfate reducer Desulfobacterium N47 and the rod-shaped Spirochete Rectinema cohabitans HM. Genome sequencing and proteome analysis suggested that the Spirochete is an obligate fermenter that catabolizes proteins and carbohydrates, resulting in acetate, ethanol, and molecular hydrogen (H₂) production. Physiological experiments inferred that hydrogen is an important link between the two bacteria in the enrichment culture, with H₂ derived from fermentation by R. cohabitans used as reductant for sulfate reduction by Desulfobacterium N47. Differential proteomics and physiological experiments showed that R. cohabitans utilizes biomass (proteins and carbohydrates) released from dead cells of Desulfobacterium N47. Further comparative and community genome analyses indicated that other Rectinema phylotypes are widespread in contaminated environments and may perform a hydrogenogenic fermentative lifestyle similar to R. cohabitans. Together, these findings indicate that environmental Spirochaetes scavenge detrital biomass and in turn drive necromass recycling at anoxic hydrocarbon-contaminated sites and potentially other habitats.

Fig. 1

Metabolic pathway reconstruction for Spirochaetes in hydrocarbon- and organohalide-contaminated environments. Genes for the illustrated pathways were detected in the genomes of R. cohabitans, uncultured Spirochete bacterium bdmA 4, and uncultured Spirochete bacterium SA-8. The predicted functions of the putative proteins indicated by the numbers in the figure can be found in Table S1

Fig. 2

Determinants of H₂ metabolism in Spirochaetes in hydrocarbon- and organohalide-contaminated environments. a Phylogenetic tree of the [FeFe]-hydrogenase catalytic subunit sequences detected in R. cohabitans, uncultured Spirochete bacterium bdmA 4, and uncultured Spirochete bacterium SA-8. b Phylogenetic tree of the [NiFe]-hydrogenase catalytic subunit sequences detected in Desulfobacterium N47. c Genetic organization of hydrogenases from R. cohabitans. d Genetic organization of the hydrogenase operon detected in Desulfobacterium N47. The illustrated tree for Figure 2a is condensed, with the full tree including all hydrogenases in Figure S1. The genetic organization of the Group 1a [NiFe] hydrogenase gene is not shown due to incomplete sequence coverage. Genetic organization diagrams are shown to scale and genes/domains are color-coded as follows: green, catalytic site; blue, secondary subunit; yellow, electron acceptor or donor; light orange, maturation factor. Redox-active centers are shown in circles, where yellow indicates [2Fe2S] cluster, green [3Fe4S] cluster, and red [4Fe4S] cluster

Fig. 3

H₂ oxidation and evolution under different growth conditions. a Interspecies hydrogen exchange between R. cohabitans and Desulfobacterium N47. Arrows indicate injection of sodium molybdate. b Hydrogen evolution with various substrates as carbon sources for R. cohabitans. c Hydrogen evolution with dead biomass and metabolites as carbon sources for R. cohabitans. Desulfobacterium in the figure represents biomass from the highly enriched Desulfobacterium culture as a substrate. Values are means of two or three individual incubations. Error bars indicate SD of biological replicates

Fig. 4

Quantitative proteomic analysis of the highly enriched Desulfobacterium culture (without R. cohabitans) and enrichment culture N47 grown in the presence of naphthalene. The volcano plot was generated using the PERSEUS software. The calculated Log2 (fold change) (x-axis) was then plotted against the corresponding p values (y-axis). Proteins to the left and above the significance line are significantly depleted in enrichment culture N47 and proteins to the right and above the significance line are significantly enriched in enrichment culture N47. The filled squares indicate proteins from R. cohabitans; the filled circles represent proteins from Desulfobacterium N47. The different colors highlight functional groups (for details see legend within figure)

Fig. 5

Condensed maximum likelihood tree of partial 16S rRNA gene sequences of Spirochaetes showing the phylogenetic affiliation of R. cohabitans, uncultured Spirochete bacterium bdmA 4, and uncultured Spirochete bacterium SA-8. Phylogenetic trees were constructed by the maximum likelihood method and are bootstrapped with 500 replicates. The expanded tree is shown in Figure S3

Fig. 6

A proposed conceptual model of a subsurface microbial loop for nutrient recycling at anoxic hydrocarbon- and organohalide-contaminated habitats. Hydrocarbons and organohalides serve as exogenous energy, electron, and carbon inputs, leading to primary biomass production in the absence of sunlight. Bacteria like environmental Spirochaetes use extracellular hydrolases and fermentative pathways to recycle biomass. In turn, they release usable electron donors such as hydrogen and potentially nutrients like phosphorus and nitrogen to the system. This in turn may stimulate microbial degradation processes performed by key players, e.g., sulfate-reducing bacteria and organohalide-respiring bacteria, and other community members like methanogenic archaea