Microbial degradation and assimilation of veratric acid in oxic and anoxic groundwaters.

Microbial communities are key players in groundwater ecosystems. In this dark environment, heterotrophic microbes rely on biomass produced by the activity of lithoautotrophs or on the degradation of organic matter seeping from the surface. Most studies on bacterial diversity in groundwater habitats are based on 16S gene sequencing and full genome reconstructions showing potential metabolic pathways used in these habitats. However, molecular-based studies do not allow for the assessment of population dynamics over time or the assimilation of specific compounds and their biochemical transformation by microbial communities. Therefore, in this study, we combined DNA-, phospholipid fatty acid-, and metabolomic-stable isotope probing to target and identify heterotrophic bacteria in the groundwater setting of the Hainich Critical Zone Exploratory (CZE), focusing on 2 aquifers with different physico-chemical conditions (oxic and anoxic). We incubated groundwater from 4 different wells using either 13C-labeled veratric acid (a lignin-derived compound) (single labeling) or a combination of 13CO2 and D-labeled veratric acid (dual labeling). Our results show that heterotrophic activities dominate all groundwater sites. We identified bacteria with the potential to break down veratric acid (Sphingobium or Microbacterium). We observed differences in heterotrophic activities between the oxic and anoxic aquifers, indicating local adaptations of bacterial populations. The dual labeling experiments suggested that the serine pathway is an important carbon assimilation pathway and that organic matter was an important source of hydrogen in the newly produced lipids. These experiments also yielded different labeled taxa compared to the single labeling experiments, showing that there exists a complex interaction network in the groundwater habitats.

14C-Free Carbon Is a Major Contributor to Cellular Biomass in Geochemically Distinct Groundwater of Shallow Sedimentary Bedrock Aquifers.

Despite the global significance of the subsurface biosphere, the degree to which it depends on surface organic carbon (OC) is still poorly understood. Here, we compare stable and radiogenic carbon isotope compositions of microbial phospholipid fatty acids (PLFAs) with those of in situ potential microbial C sources to assess the major C sources for subsurface microorganisms in biogeochemical distinct shallow aquifers (Critical Zone Exploratory, Thuringia Germany). Despite the presence of younger OC, the microbes assimilated 14C-free OC to varying degrees; ~31% in groundwater within the oxic zone, ~47% in an iron reduction zone, and ~70% in a sulfate reduction/anammox zone. The persistence of trace amounts of mature and partially biodegraded hydrocarbons suggested that autochthonous petroleum-derived hydrocarbons were a potential 14C-free C source for heterotrophs in the oxic zone. In this zone, Δ14C values of dissolved inorganic carbon (-366 ± 18‰) and 11MeC16:0 (-283 ± 32‰), an important component in autotrophic nitrite oxidizers, were similar enough to indicate that autotrophy is an important additional C fixation pathway. In anoxic zones, methane as an important C source was unlikely since the 13C-fractionations between the PLFAs and CH4 were inconsistent with kinetic isotope effects associated with methanotrophy. In the sulfate reduction/anammox zone, the strong 14C-depletion of 10MeC16:0 (-942 ± 22‰), a PLFA common in sulfate reducers, indicated that those bacteria were likely to play a critical part in 14C-free sedimentary OC cycling. Results indicated that the 14C-content of microbial biomass in shallow sedimentary aquifers results from complex interactions between abundance and bioavailability of naturally occurring OC, hydrogeology, and specific microbial metabolisms.

Nitrogen Loss from Pristine Carbonate-Rock Aquifers of the Hainich Critical Zone Exploratory (Germany) Is Primarily Driven by Chemolithoautotrophic Anammox Processes.

Despite the high relevance of anaerobic ammonium oxidation (anammox) for nitrogen loss from marine systems, its relative importance compared to denitrification has less been studied in freshwater ecosystems, and our knowledge is especially scarce for groundwater. Surprisingly, phospholipid fatty acids (PLFA)-based studies identified zones with potentially active anammox bacteria within two superimposed pristine limestone aquifer assemblages of the Hainich Critical Zone Exploratory (CZE; Germany). We found anammox to contribute an estimated 83% to total nitrogen loss in suboxic groundwaters of these aquifer assemblages at rates of 3.5-4.7 nmol L-1 d-1, presumably favored over denitrification by low organic carbon availability. Transcript abundances of hzsA genes encoding hydrazine synthase exceeded nirS and nirK transcript abundances encoding denitrifier nitrite reductase by up to two orders of magnitude, providing further support of a predominance of anammox. Anammox bacteria, dominated by groups closely related to Cand. Brocadia fulgida, constituted up to 10.6% of the groundwater microbial community and were ubiquitously present across the two aquifer assemblages with indication of active anammox bacteria even in the presence of 103 µmol L-1 oxygen. Co-occurrence of hzsA and amoA gene transcripts encoding ammonia mono-oxygenase suggested coupling between aerobic and anaerobic ammonium oxidation under suboxic conditions. These results clearly demonstrate the relevance of anammox as a key process driving nitrogen loss from oligotrophic groundwater environments, which might further be enhanced through coupling with incomplete nitrification.

Archaeal Diversity and CO2 Fixers in Carbonate-/Siliciclastic-Rock Groundwater Ecosystems.

Groundwater environments provide habitats for diverse microbial communities, and although Archaea usually represent a minor fraction of communities, they are involved in key biogeochemical cycles. We analysed the archaeal diversity within a mixed carbonate-rock/siliciclastic-rock aquifer system, vertically from surface soils to subsurface groundwater including aquifer and aquitard rocks. Archaeal diversity was also characterized along a monitoring well transect that spanned surface land uses from forest/woodland to grassland and cropland. Sequencing of 16S rRNA genes showed that only a few surface soil-inhabiting Archaea were present in the groundwater suggesting a restricted input from the surface. Dominant groups in the groundwater belonged to the marine group I (MG-I) Thaumarchaeota and the Woesearchaeota. Most of the groups detected in the aquitard and aquifer rock samples belonged to either cultured or predicted lithoautotrophs (e.g., Thaumarchaeota or Hadesarchaea). Furthermore, to target autotrophs, a series of 13CO2 stable isotope-probing experiments were conducted using filter pieces obtained after filtration of 10,000 L of groundwater to concentrate cells. These incubations identified the SAGMCG Thaumarchaeota and Bathyarchaeota as groundwater autotrophs. Overall, the results suggest that the majority of Archaea on rocks are fixing CO2, while archaeal autotrophy seems to be limited in the groundwater.