Large Fractions of CO2-Fixing Microorganisms in Pristine Limestone Aquifers Appear To Be Involved in the Oxidation of Reduced Sulfur and Nitrogen Compounds

Abstract

ABSTRACTThe traditional view of the dependency of subsurface environments on surface-derived allochthonous carbon inputs is challenged by increasing evidence for the role of lithoautotrophy in aquifer carbon flow. We linked information on autotrophy (Calvin-Benson-Bassham cycle) with that from total microbial community analysis in groundwater at two superimposed—upper and lower—limestone groundwater reservoirs (aquifers). Quantitative PCR revealed that up to 17% of the microbial population had the genetic potential to fix CO2via the Calvin cycle, with abundances ofcbbMandcbbLgenes, encoding RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) forms I and II, ranging from 1.14 × 103to 6 × 106genes liter−1over a 2-year period. The structure of the active microbial communities based on 16S rRNA transcripts differed between the two aquifers, with a larger fraction of heterotrophic, facultative anaerobic, soil-related groups in the oxygen-deficient upper aquifer. Most identified CO2-assimilating phylogenetic groups appeared to be involved in the oxidation of sulfur or nitrogen compounds and harbored both RubisCO forms I and II, allowing efficient CO2fixation in environments with strong oxygen and CO2fluctuations. The generaSulfuricellaandNitrosomonaswere represented by read fractions of up to 78 and 33%, respectively, within thecbbMandcbbLtranscript pool and accounted for 5.6 and 3.8% of 16S rRNA sequence reads, respectively, in the lower aquifer. Our results indicate that a large fraction of bacteria in pristine limestone aquifers has the genetic potential for autotrophic CO2fixation, with energy most likely provided by the oxidation of reduced sulfur and nitrogen compounds.