https://www.nature.com/articles/s41396-019-0527-4

The subject of our work was an association of two anaerobic alkaliphilic bacteria, both isolated from a soda lake of Central Asia, growing at pH 9.5. The obligate syntroph Candidatus “Contubernalis alkalaceticum” can oxidize ethanol to acetate with the release of hydrogen that needs to be consumed by a partner organism to make “Contubernalis” grow. We used Geoalkalibacter ferrihydriticus as such a partner. This is an iron-reducing bacterium capable of using molecular hydrogen, ethanol, acetate and other substrates as electron donors. Recently, Geoalkalibacter was also found to possess the whole set of Wood-Ljungdahl pathway enzymes, that allows it to grow lithoautotrophically by acetogenesis from carbonate using an insoluble form of ferrous iron as the electron donor. Simultaneous cultivation of two microorganisms revealed unusual diversity of their interrelation modes. Three different types of cultivation conditions for the growth of these two organisms on ethanol were tested: those with ferric iron (amorphous mineral ferryhydrite Fe(OH)₃), without any solid electron donors or acceptors, and with ferrous iron (mineral siderite FeCO₃).

In the presence of ferrihydrite, both organisms were competing for ethanol, and initially, the free-living Geoalkalibacter clearly outcompeted (“Situation 1” on the cartoons). However, upon ferrihydrite exhaustion due to the coverage of its surface with a layer of magnetite (the product of ferrihydrite reduction), “Contubernalis” entered the play, while Geoalkalibacter moved backwards and switched its catabolism from ferric iron reduction to homoacetogenesis (the reduction of carbonate to acetate). In the absence of iron minerals, only the second process took place, namely, syntrophic ethanol oxidation to acetate by “Contubernalis” with interspecies transfer of the released H₂ to Geoalkalibacter which utilizes it as the electron donor for homoacetogenic reduction of carbonate.

In the presence of Fe(II) mineral siderite (“Situation 2” on the cartoons), syntrophic ethanol oxidation was accompanied by acetogenesis coupled to ferrous iron oxidation performed by Geoalkalibacter. Here, several interspecies interactions might occur: electron transfer from “Contubernalis” via the product of siderite oxidation (electrically conductive magnetite), via electrically conductive pili which production is genomically predicted in Geoalkalibacter, or via interspecies hydrogen transfer.

 Alternatively, the presence of xap genes in Geoalkalibacter and the observed formation of extracellular polymeric matrix in co-cultures allow us to propose the formation of an electron transfer network comprised of secreted multiheme cytochromes embedded into exopolysacharides. Also, production of conductive cellular appendages by “Contubernalis” could not be excluded and will be evaluated upon availability of its genome. In general, in the presence of redox-active iron minerals sustaining the growth of Geoalkalibacter, interspecies electron transfer between syntrophic partners could involve both indirect and direct strategies simultaneously.

It should also be mentioned that we could not demonstrate acetogenic growth of G. ferrihydriticus with ethanol or molecular hydrogen as the energy substrates. This could be explained either by much lower concentrations of hydrogen necessary for acetogenic growth, or by direct electron transfer from C. “C. alkalaceticum” to G. ferrihydriticus via electrically conductive pili or diffusible multiheme cytochromes inside the extracellular polymeric matrix of the co-culture. The undisputed proofs are absent in both cases.

Our work demonstrates the feasibility of syntrophic interactions based on the redox transformations of iron minerals and lithotrophic acetogenesis. Such relations could occur in Proterozoic biosphere with the predominance of iron cycle. Coupling of acetogenesis with Fe(III) minerals oxidation could be one of the main reaction leading to the accumulation of magnetite – typomorphic mineral of banded iron formations (BIF) – global iron sedimentary deposits with yet poorly understood genesis formed at anoxic conditions of the Precambrian period.