Attached and Suspended Denitrifier Communities in Pristine Limestone Aquifers Harbor High Fractions of Potential Autotrophs Oxidizing Reduced Iron and Sulfur Compounds.

Oxygen and nitrate availability as well as the presence of suitable organic or inorganic electron donors are strong drivers of denitrification; however, the factors influencing denitrifier abundance and community composition in pristine aquifers are not well understood. We explored the denitrifier community structure of suspended and attached groundwater microorganisms in two superimposed limestone aquifer assemblages with contrasting oxygen regime in the Hainich Critical Zone Exploratory (Germany). Attached communities were retrieved from freshly crushed parent rock material which had been exposed for colonization in two groundwater wells (12.7 and 48 m depth). Quantitative PCR and amplicon pyrosequencing of nirK and nirS genes encoding copper-containing or cytochrome cd1 heme-type nitrite reductase, respectively, and of bacterial 16S ribosomal RNA genes showed a numerical predominance of nirS-type denitrifiers in both attached and suspended groundwater communities and a dominance of nirS-type denitrifiers closely related to the autotrophic thiosulfate- and hydrogen-oxidizing Sulfuritalea hydrogenivorans and the iron- and sulfide-oxidizing Sideroxydans lithotrophicus ES-1. Potential rates of nitrate reduction in association with exposed crushed rock material were higher with an inorganic electron donor (thiosulfate) compared to an organic electron donor (fumarate/acetate) in the upper aquifer assemblage but similar in the lower, oxic aquifer. Our results have clearly demonstrated that groundwater from pristine limestone aquifers harbors diverse denitrifier communities which appear to selectively attach to rock surfaces and harbor a high potential for nitrate reduction. Our findings suggest that the availability of suitable inorganic versus organic electron donors rather than oxygen availability shapes denitrifier communities and their potential activity in these limestone aquifers.