Identification of enriched hyperthermophilic microbial communities from a deep-sea hydrothermal vent chimney under electrolithoautotrophic culture conditions

Archaeoglobales as systematic (electro)lithoautotrophs of the community

We have evidenced the development of microbial electrotrophic communities and metabolic activity supported by current consumption (Fig. 1), product production (Fig. 2), and qPCRs (Fig. 3). These data suggest that growth did occur from energy supplied by the cathode. Our study is the first to show the possibility of growth of biofilm from environments harboring natural electric current in the total absence of soluble electron donors. To further discuss the putative mechanism, it is necessary to have a look at our conditions unfavorable for water electrolysis (see Supplementary Fig. S2). The equilibrium potential for water reduction into hydrogen at 80 °C, pH 7, and 1 atm was calculated at − 0.490 V vs SHE in pure water. The operational reduction potential is expected to be lower than the theoretical value due to internal resistances (from electrical connections, electrolytes, ionic membranes, etc.)¹⁶ and overpotentials (electrode material). This was confirmed with the on-set potential of H₂ evolution measured at − 0.830 mV vs SHE in both experimental condition and abiotic control, indicating the absence of catalytic effect of putative hydrogenases secreted by the biofilm or metals from inoculum. Also, during preliminar potentials screening, the increase in current consumption and H₂ production was observed only below − 0.7 V vs SHE (Supplementary Figs. S1 and S2). In addition, the presence of catalytic waves observed by CV with midpoint potentials between − 0.217 V to − 0.639 V indicate the implication of enzymes directly connected to the surface of the electrode (see Supplementary Fig. S2). Finally, the fixation of 267–1596 Coulombs day⁻¹ into organics (Fig. 1) exceeds the maximum theoretical abiotic generation of hydrogen from abiotic current (~ 3 C day⁻¹) 90- to 530-fold¹⁷.

Therefore, under our experimental conditions, the biofilm growth should be largely ensured by a significant part of a direct transfer of electrons from the cathode, thereby demonstrating the presence of electrolithoautotroph microorganisms. This is supported by obtaining a similar biodiversity on sulfate with the cathode poised at − 300 mV [compared to − 590 mV vs SHE (Fig. 3)], whose potential is 190 mV more positive than the Equilibrium potential of H₂ evolution (− 490 mV vs SHE), with then no electrochemical possibility of H₂ production, even at molecular level.

Taxonomic analysis of the enriched microbial communities at the end of the experiments showed the systematic presence of Archaeoglobales on cathodes. Moreover, the qPCR and MiSeq data (Fig. 3) highlighted a strong correlation between current consumption and density of Archaeoglobales in the biofilm (Supplementary Fig. S4, R² = 0.945).

The OTUs were related to some Archaeoglobales strains with 95–98% identities. Thus, we assume that under our experimental conditions new specific electrotrophic metabolisms or new electrolithoautotrophic Archaeoglobaceae species were enriched on the cathode. They were retrieved in all conditions and belonged to the only order in our communities exhibiting autotrophic metabolism. Autotrophic growth in the Archaeoglobales order is ensured mainly through using H₂ as energy source and requires both branches of the reductive acetyl-CoA/Wood-Ljungdahl pathway for CO₂ fixation¹⁸. Terminal electron acceptors used by this order include sulfate, nitrate, poorly crystalline Fe (III) oxide, and sulfur oxyanions¹⁹. Moreover, Archaeoglobus fulgidus has been recently shown to grow on iron by directly snatching electrons under carbon starvation during the corrosion process²⁰. Furthermore, Ferroglobus and Geoglobus species were shown to be exoelectrogens in pure culture in a microbial electrosynthesis cell¹² and have been enriched within a microbial electrolysis cell^11,13. Given these elements, the identified Archaeoglobales species could be, under our electrolithoautotrophic conditions, the first colonizers of the electrode during the first days of growth. This hypothesis was confirmed into a more detailed study focusing on the enrichment on nitrate²¹.

The growth of Archaeoglobales species in presence of oxygen is a surprising finding. Archaeoglobales have a strictly anaerobic metabolism, and the reductive acetyl-CoA pathway is very sensitive to the presence of oxygen²². This can be firstly explained by the low solubility of oxygen at 80 °C. Secondly, carbon cloth mesh reduces oxygen in the environment, allowing for anaerobic development of microorganisms into a protective biofilm²³. This observation was supported by the near absence of Archaeoglobales in the liquid medium (Fig. 3). One of the hypotheses concerns direct interspecies electron transfer (DIET)^24,25, with Archaeoglobales transferring electrons to another microorganism as an electron acceptor. Research into DIET is in its early stages, and further investigations are required to better understand the diversity of microorganisms and the mechanism of carbon and electron flows in anaerobic environments²⁵ such as hydrothermal ecosystems.

Electrosynthesis of organic compounds

Accumulation of pyruvate, glycerol and acetate was measured, while another set of compounds that appeared transiently were essentially detectable in the first few days of biofilm growth (Supplementary Table S1). They included amino acids (threonine, alanine) and volatile fatty acids (formate, succinate, lactate, acetoacetate, 3-hydroxyisovalerate) whose concentrations did not exceed 0.1 mM. Despite their thermostability, this transient production suggests they were used by microbial communities developing on the electrode in interaction with the primary producers during enrichment.

On the other hand, in presence of nitrate, sulfate and oxygen as electron acceptors, the liquid media accumulated mainly acetate, glycerol, and pyruvate (Fig. 1). Coulombic efficiency calculations (Fig. 2) showed that electron content of the carbon products represented 60–90% of electrons consumed, the rest being potentially used directly for biomass or transferred to an electron acceptor. This concurs with the energy yield from the Wood-Ljungdahl pathway of Archaeoglobales, with only 5% of carbon flux directed to the production of biomass and the other 95% diverted to the production of small organic end-products excreted from the cell²⁶.

Pyruvate is a central intermediate of CO₂ uptake by the reducing pathway of the acetyl-CoA/WL pathway²⁷. It can be used to drive the anabolic reactions needed for biosynthesis of cellular constituents. Theoretically, the only explanation for improved production and accumulation of pyruvate (up to 5 mM in the liquid media of sulfate experiment) would be that pyruvate-consuming enzymes were inhibited or that pyruvate influx exceeded its conversion rate. Here we could suggest that in-cell electron over-feeding at the cathode leads to significant production of pyruvate when the electron acceptor runs out.

In an ecophysiological context, similar pyruvate and glycerol production could occur on hydrothermal chimney walls into which electric current propagates²⁸. The electrotroph biofilms would continually receive electrons, leading to an excess of intracellular reducing power which would be counterbalanced by overproduction of glycerol and pyruvate^29,30. Furthermore, these products can serve as carbon and energy sources for heterotrophic microorganisms or for fermentation. In our experiments, pyruvate and glycerol concentrations varied over time, suggesting they were being consumed by heterotrophic microorganisms. Acetate production would thus result from the fermentation of pyruvate or other compounds produced by electrotrophic Archaeoglobales.

Enrichment of rich heterotrophic biodiversity from electrotrophic Archaeoglobales community

During our enrichment experiments, the development of effective and specific biodiversity was dependent on the electron acceptors used (Fig. 3). Heatmap analyses (Supplementary Fig. S3) showed four distinct communities for the three electron acceptors and the initial inoculum. Thus, at the lower taxonomic level of the biodiversity analysis, most OTUs are not common to multiple enrichments, except for one OTU of Thermococcales that was found in both the nitrate and sulfate experiments. This suggests a real specificity of the communities and a specific evolution or adaptation of the members of the shared phyla to the different electron acceptors available in the environment. However, the various enrichments also showed the presence of Thermococcales regardless of the electron acceptors used, thus demonstrating a strong interaction between Thermococcales, assumed to be heterotrophs, and Archaeoglobales, the only demonstrated autotrophs. Moreover, members of these two groups have frequently been found together in various hydrothermal sites^4,5,31,32, where they are considered potential primary colonizers^{33,34,35,36,37}. After Thermococcales, the rest of the heterotrophic biodiversity was specific to each electron acceptor.

On nitrate, two additional phylogenetic groups were retrieved: Desulfurococcales and Thermales. OTUs of Desulfurococcales are mainly affiliated to Thermodiscus or Aeropyrum species, which are hyperthermophilic and heterotrophic Crenarchaeota growing by fermentation of complex organic compounds or sulfur/oxygen reduction (Huber and Stetter, 2015). Concerning Thermales, a new taxon was enriched on cathode and only affiliated to Vulcanithermus mediatlanticus with similarity of 90%. This new taxon of Thermales (OTU 15, Supplementary Fig. S3) was also enriched up to 2% on the cathode of sulfate enrichment. Thermales are thermophilic (30–80 °C) and heterotrophic bacteria whose only four genera (Marinithermus, Oceanithermus, Rhabdothermus, and Vulcanithermus) are all retrieved in marine hydrothermal systems. They can grow under aerobic, microaerophilic and some anaerobic conditions with several inorganic electron acceptors such as nitrate, nitrite, Fe (III) and elemental sulfur³⁸. All of the Thermales species can utilize the pyruvate as carbon and energy source with the sulfate or nitrate as electron acceptors.

Pseudomonadales and Bacillales were found in the oxygen experiment. Most Pseudomonas are known to be aerobic and mesophilic bacteria, with a few thermophilic species (up to 65 °C)^39,40. There have already been some reports of mesophilic Pseudomonas species growing in thermophilic conditions in composting environments⁴¹. Moreover, some Pseudomonas sp. are known to be electroactive in microbial fuel cells through long-distance extracellular electron transport^42,43,44, and were dominant on the cathodes of a benthic microbial fuel cell on a deep-ocean cold seep⁴⁵. In Bacillales, the Geobacillus spp. and some Bacillus sp. are known to be mainly (hyper)thermophilic aerobic and heterotrophic Firmicutes⁴⁶.

Hydrothermal electric current: a new energy source for the development of primary producers

The presence of so many heterotrophs in an initially autotrophic condition points to the hypothesis of a trophic relationship inside the electrotrophic community (Fig. 5). This suggests that the only autotrophs retrieved in all communities, the Archaeoglobales, might be the first colonizer of the electrode, using CO₂ as carbon source and the cathode as energy source. Models using the REACT module of the Geochemist’s Workbench (GWB) and based on electron donor acceptor availability predicted low abundances of Archaeoglobales (< 0.04%) due to low concentration of H₂³⁶ whereas in-situ detection found abundances of more than 40% in the inner section of the studied hydrothermal chimney⁴⁷. Dahle et al. concluded on a probable H₂ syntrophy, with hydrogen being produced by heterotrophic microorganisms such as fermentative Thermococcales species. Our study showed that Archaeoglobales can also grow electrolithoautotrophically, feeding on the natural electric current through the chimney walls. This new energy source, which is not considered in the models, would gap this model/observation difference and raises the question of the importance of this metabolism in the primary colonization of hydrothermal vents. It would allow a long-range transfer between the electron donor (H₂S oxidized on the inner surface of the chimney wall) and the electron acceptors (O₂, sulfur compounds, nitrate, metals) covering the external surface of the chimney. This electrical current would thus allow primary colonizers to grow, releasing organic compounds which are then used by the heterotrophic community, as observed in our experiments. Moreover,this would provide a constant source of electron donor to all over the surface of the chimney, allowing to meet a wider range of physiological conditions through pH, temperature, and oxidoreduction gradients. This allows a wider diversity of growth patterns than through chemolithoautotrophy, which is restricted to unstable and limited contact zones between reduced compounds (H₂, H₂S) in the hydrothermal fluid and electron acceptors around the hydrothermal chimneys (O₂, SO₄, NO₃), which often precipitate together.

Source: Ecology - nature.com