Large Fractions of CO ₂ -Fixing Microorganisms in Pristine Limestone Aquifers Appear To Be Involved in the Oxidation of Reduced Sulfur and Nitrogen Compounds

Abstract

The 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 CO 2 via the Calvin cycle, with abundances of cbbM and cbbL genes, encoding RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) forms I and II, ranging from 1.14 ؋ 10 3 to 6 ؋ 10 6 genes liter ؊1 over 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 CO 2 -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 CO 2 fixation in environments with strong oxygen and CO 2 fluctuations. The genera Sulfuricella and Nitrosomonas were represented by read fractions of up to 78 and 33%, respectively, within the cbbM and cbbL transcript 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 CO 2 fixation, with energy most likely provided by the oxidation of reduced sulfur and nitrogen compounds. Due to the lack of light-driven primary production, groundwater ecosystems were originally believed to be controlled by surface-derived allochthonous organic matter input (1-3) and to be dominated by heterotrophic prokaryotes adapted to nutrient limitation. However, there is increasing evidence of the important role of lithoautotrophy for carbon flow in aquifers (4-7). A large proportion of drinking water originates from groundwater resources (8), with karstic aquifers providing ϳ25% of the drinking water sources on a global scale (9). Despite the crucial role of microbial activity in shaping groundwater geochemistry (10-12), the links between microbial diversity and function in groundwater ecosystems, especially with regard to chemolithoautotrophy, are still poorly understood (7). Recent studies suggest that microbial CO 2 assimilation in aquifers could be fueled by energy conserved by nitrification, oxidation of ferrous iron and reduced sulfur compounds (6, 7), or oxidation of H 2 or methane (13, 14). Oxidation of electron donors present as solid minerals such as pyrite can even yield highly reactive dissolved ions that might affect other minerals and dissolved ions in the aquifer, leading to changes to the makeup of rocks and groundwater.Today, there are six known autotrophic CO 2 fixation pathways (reviewed in refere...

Carbon isotopes of dissolved inorganic carbon reflect utilization of different carbon sources by microbial communities in two limestone aquifer assemblages

“…Within the time period of monitoring there was no significant change from previous reported hydrochemical patterns (Table 1) (Herrmann et al, 2015;Küsel et al, 2016). Water chemistry reflects the limestone environment of the catchment.…”

“…Chemolithoautotrophic microorganisms, i.e., microbes that metabolize CO 2 instead of organic carbon, have been shown to be key players and important primary producers in groundwater microbial communities (Alfreider et al, 2012;Hutchins et al, 2016;Kellermann et al, 2012). A high potential for microbial CO 2 fixation has already been demonstrated in our studied aquifers by molecular analyses (Herrmann et al, 2015;Lazar et al, 2016a;Schwab et al, 2017b). We hypothesized that turnover of OM derived from chemoautotrophic microorganisms should be reflected in DIC isotopes, since OM derived from CO 2 fixation should be isotopically distinct from other sources like surface-derived OM or sedimentary organic matter.…”

“…Chemolithoautotrophs can use six different metabolic pathways to fix DIC, of which the Calvin-Benson cycle and the acetyl-CoA pathway are the two most important ones (Fuchs, 2011). Herrmann et al (2015) found that up to 17 % of the microbial community in well H-43 has the potential for chemolithoautotrophic CO 2 fixation via the Calvin-Benson cycle linked to the oxidation of reduced sulfur and nitrogen compounds. The CalvinBenson cycle can function under anoxic and oxic conditions, whereas microorganisms using the acetyl-CoA pathway are restricted to strictly anoxic conditions (Fuchs, 2011;Berg, 2011).…”

How Deep Can Surface Signals Be Traced in the Critical Zone? Merging Biodiversity with Biogeochemistry Research in a Central German Muschelkalk Landscape

“…Initial investigations have identified two factors which appear to be key drivers of microbial community composition and thus also strongly influence the potential for biogeochemical processes to take place: (i) the strong differences in oxygen availability between the two aquifers, and (ii) the distance to surface soils and thus the likelihood of the input and potential establishment of soil-derived microbial groups in the aquifer microbial communities (Opitz et al, 2014;Herrmann et al, 2015). Comparative investigations of microbial community structure in the groundwater using next generation amplicon sequencing of 16S rRNA genes and transcripts revealed that members of the Betaproteobacteria and the CytophagalesFlavobacteria-Bacteroidetes group dominate in the upper, anoxic aquifer while members of the Deltaproteobacteria and Nitrospira dominate the communities in the oxic, lower aquifer .…”

Sulfurirhabdus autotrophica gen. nov., sp. nov., isolated from a freshwater lake

“…The type strain of Sulfuricella denitrificans was isolated from a cold anoxic hypolimnion of a freshwater lake, and was characterized as a psychrotolerant sulfur oxidizer (Watanabe et al, 2012). Based on nucleotide sequences, its close relatives have been found in freshwater lake sediments (Nelson et al, 2007;Song et al, 2012;Watanabe et al, 2013), Thioploca samples from two freshwater lakes (Nemoto et al, 2011), drinking water distribution systems (Li et al, 2010;Sun et al, 2014a, b), the sediment of a drinking water reservoir (Cheng et al, 2014), soil (Field et al, 2010), a bioreactor (Wang et al, 2015), subglacial sediment (Boyd et al, 2014), wetland sediment (Liu et al, 2014), a limestone aquifer (Herrmann et al, 2015), groundwater (Hong et al, 2013), black shale (Li et al, 2014), a horizontal subsurface flow system constructed in wetlands (Zhong et al, 2015) and the freshwater layer of a meromictic lake (Kubo et al, 2014). Members of the genus Sulfuricella are likely to contribute to sulfur cycling in a wide range of freshwater ecosystems, but only two cultured strains of the genus Sulfuricella have, so far, been described (Watanabe et al, 2015b).…”