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Despite its potentially high relevance for nitrate removal in freshwater environments limited in organic carbon, chemolithoautotrophic denitrification has rarely been studied in oligotrophic groundwater. Using thiosulfate and H2 as electron donors, we established a chemolithoautotrophic enrichment culture from groundwater of a carbonate-rock aquifer to get more insight into the metabolic repertoire, substrate turnover, and transcriptional activity of subsurface denitrifying consortia. The enriched consortium was dominated by representatives of the genus Thiobacillus along with denitrifiers related to Sulfuritalea hydrogenivorans, Sulfuricella denitrificans, Dechloromonas sp. and Hydrogenophaga sp., representing the consortium's capacity to use multiple inorganic electron donors. Microcosm experiments coupled with Raman gas spectroscopy demonstrated complete denitrification driven by reduced sulfur compounds and hydrogen without formation of N2O. The initial nitrate/thiosulfate ratio had a strong effect on nosZ transcriptional activity and on N2 formation, suggesting similar patterns of the regulation of gene expression as in heterotrophic denitrifiers. Sequence analysis targeting nirS and nosZ transcripts identified Thiobacillus denitrificans-related organisms as the dominant active nirS-type denitrifiers in the consortium. An additional assessment of the nirS-type denitrifier community in the groundwaterclearly confirmed the potential for sulfur- and hydrogen-dependent chemolithoautotrophic denitrification as important metabolic feature widely spread among subsurface denitrifiers at the Hainich Critical Zone Exploratory.

Thiosulfate- and hydrogen-driven autotrophic denitrification by a microbial consortium enriched from groundwater of an oligotrophic limestone aquifer