Dark Fermentation in the Dark Biosphere: The Case of <i>Citrobacter</i> sp. T1.2D-1²

Microbial diversity that thrives in the deep subsurface remains largely unknown. In this work, we present the characterization of <i>Citrobacter</i> sp. T1.2D-1, isolated from a 63.6 m-deep core sample extracted from the deep subsurface of the Iberian Pyrite Belt (IPB). A genomic analysis was performed to identify genes that could be ecologically significant in the IPB. We identified all the genes that encoded the formate–hydrogen lyase and hydrogenase-2 complexes, related to hydrogen production, as well as those involved in glycerol fermentation. This is particularly relevant as some of the substrates and byproducts of this process are of industrial interest. Additionally, we conducted a phylogenomic study, which led us to conclude that our isolate was classified within the <i>Citrobacter telavivensis</i> species. Experimentally, we verified the strain’s ability to produce hydrogen from glucose and glycerol and, thus, of performing dark fermentation. Moreover, we assessed the activity of the nitrate and tetrathionate reductase complexes and the isolate’s ability to tolerate high concentrations of heavy metals, especially Zn. These results suggest that <i>C. telavivensis</i> T1.2D-1 can play a role in the carbon, hydrogen, iron, nitrogen, and sulfur cycles that occur in the deep subsurface of the IPB, making it a candidate worthy of further study for possible biotechnological applications.