Candidate Brocadiales dominates C, N and S cycling in anoxic groundwater of a pristine limestone-fracture aquifer

Groundwater-associated microorganisms are known to play an important role in the biogeochemical C, N and S cycling. Metaproteomics was applied to characterize the diversity and the activity of microbes to identify key species in major biogeochemical processes in the anoxic groundwater of a pristine karstic aquifer located in Hainich, central Germany. Sampling was achieved by pumping 1000 L water from two sites of the upper aquifer assemblage and filtration on 0.3 μm glass filters. In total, 3808 protein groups were identified. Interestingly, the two wells (H4/2 and H5/2) differed not only in microbial density but also in the prevalence of different C, N and S cycling pathways. The well H5/2 was dominated by the anaerobic ammonia-oxidizing (anammox) candidate Brocadiales (31%) while other orders such as Burkholderiales (2%) or Nitrospirales (3%) were less abundant. Otherwise, the well H4/2 featured only low biomass and remarkably fewer proteins (391 to 3631 at H5/2). Candidate Brocadiales was affiliated to all major carbon fixation strategies, and to the cycling of N and S implying a major role in biogeochemical processes of groundwater aquifers. The findings of our study support functions which can be linked to the ecosystem services provided by the microbial communities present in aquifers. Significance Subsurface environments especially the groundwater ecosystems represent a large habitat for microbial activity. Microbes are responsible for energy and nutrient cycling and are massively involved in the planet's sustainability. Microbial diversity is tremendous and the central question in current microbial ecology is “Who eats what, where and when?”. In this study, we characterize a natural aquifer inhabiting microbial community to obtain evidence for the phylogenetic diversity and the metabolic activity by protein abundance and we highlight important biogeochemical cycling processes. The aquifer was dominated by Candidatus Brocadiales while other phylotypes such as Burkholderiales, Caulobacterales and Nitrospirales were less abundant. The candidate comprised all major carbon fixation strategies, ammonification, anammox and denitrification as well as assimilatory sulfate reduction. Our findings have broad implications for the understanding of microbial activities in this aquifer and consequently specific functions can be linked to the ecosystem services provided by the microbial communities present in aquifers.