Functional diversity of microbial communities in pristine aquifers inferred by PLFA- and sequencing-based approaches

Abstract

Microorganisms in groundwater play an important role in aquifer biogeochemical cycles and water quality. However, the mechanisms linking the functional diversity of microbial populations and the groundwater physicochemistry are still not well understood due to the complexity of interactions between surface and subsurface. Here, we used phospholipid fatty acids (PLFAs) relative abundances to link specific biochemical markers within the microbial communities to the spatio-temporal changes of the groundwater physicochemistry. PLFAs were isolated from groundwater of two physicochemically distinct aquifer assemblages in central Germany (Thuringia). The functional diversities of the microbial communities were mainly correlated with groundwater chemistry, including dissolved O2, Fet and NH4+ concentrations. Abundances of PLFAs derived from eukaryotes and potential nitrite oxidizing bacteria (11MeC16:0 as biomarker for Nitrospira moscoviensis) were high at sites with elevated O2 concentration where groundwater recharge supplies both bioavailable organic substrates and NH4+ needed to sustain heterotrophic growth and nitrification processes. In anoxic groundwaters more rich in Fet, PLFAs abundant in sulphate reducing bacteria (SRB), iron-reducing bacteria and fungi increased with Fet and HCO3− concentrations suggesting the occurrence of active iron-reduction and the possible role of fungi in meditating iron solubilisation and transport in those aquifer domains. In NH4+ richer anoxic groundwaters, anammox bacteria and SRB- derived PLFAs increased with NH4+ concentration further evidencing the dependence of the anammox process on ammonium concentration and potential links between SRB and anammox bacteria. Additional support of the PLFA-based bacterial communities was found in DNA and RNA-based Illumina MiSeq amplicon sequencing of bacterial 16S rRNA genes, which evidenced high predominance of nitrite-oxidizing bacteria Nitrospira e.g. Nitrospira moscoviensis in oxic zones of the aquifers and of anammox bacteria in NH4+ richer anoxic groundwater. Higher relative abundances of sequence reads in the RNA-based data sets affiliated with iron-reducing bacteria in Fet richer groundwater supported the occurrence of active dissimilatory iron-reduction. The functional diversity of the microbial communities in these biogeochemically distinct groundwater assemblages can be largely attributed to the redox conditions linked to changes in bioavailable substrates and input of substrates with the seepage. Our results demonstrate the power of complementary information derived from PLFA-based and sequencing-based approaches

Reply to comments of referee #2

“…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.…”

“…The bottle was rinsed two times and subsequently filled to the brim, closed quickly with a gastight screw cap, cooled at 4 • C in the dark, and transported to the lab for further analyses. To collect material for POC and DNA, a high-filtration campaign was conducted in May 2015; 1000 L of water were pumped from wells H 5.1, H 5.2, and H 4.3 through pre-combusted (500 • C) glass fiber filters with 0.2 µm pore size placed on a custom filtration unit (Schwab et al, 2017b). After pumping 1000 L, filters were removed, packed into aluminum foliation and cooled on dry ice for POC and DNA analyses.…”

“…Because this model suggests the formation of high amounts of methane, which was not observed in the aquifer (T. Behrendt, unpublished data), we ran a second model in-cluding more depleted values for POC. These values were based on PLFA data from Schwab et al (2017b), who measured 14 C activity and δ 13 C of the main bacterial biomarker C16:0. Measured vales were −39.83 ± 0.72 ‰ for δ 13 C and 7 ± 1 pMC for radiocarbon.…”

Aquifer configuration and geostructural links control the groundwater quality in thin-bedded carbonate–siliciclastic alternations of the Hainich CZE, central Germany

“…This is for instance applied to surface-sourced organic matter and microorganisms (Küsel et al, 2016;Schwab et al, 2017;Lazar et al, 2017) to further investigate subsurface connectivity, surfacesubsurface interactions and functions of microbial life in groundwater environments. Further CZ exploration should also aim at the investigation of deeper strata connection by regional groundwater flow, hydraulic properties and proportions of unsaturated zones and matter processing within.…”

Phylogenetic and metabolic diversity have contrasting effects on the ecological functioning of bacterial communities

“…The twenty-seven bacterial strains used in this study (Table S1) were isolated from pristine groundwater (Geesink et al 2018), obtained from the Hainich Critical Zone Exploratory located in Thuringia, Germany (Küsel et al 2016). The isolates were selected from a pool of more than 340 groundwater isolates as representatives of the most abundant taxonomic families within the total groundwater bacterial population, based on 16S rRNA gene MiSeq amplicon sequencing data (Schwab et al 2017, Kumar et al 2017), throughout the taxonomic classes of Actinobacteria , Bacilli , Flavobacteriia , Sphingobacteria , Alpha - and Gammaproteobacteria . The isolates were maintained on S2P medium agar (Geesink et al 2018).…”