Differential structure and functional gene response to geochemistry associated with the suspended and attached shallow aquifer microbiomes from the Illinois Basin, IL.

Despite the clear ecological significance of the microbiomes inhabiting groundwater and connected ecosystems, our current understanding of their habitats, functionality, and the ecological processes controlling their assembly have been limited. In this study, an efficient pipeline combining geochemistry, high-throughput FluidigmTM functional gene amplification and sequencing was developed to analyze the suspended and attached microbial communities inhabiting five groundwater monitoring wells in the Illinois Basin, USA. The dominant taxa in the suspended and the attached microbial communities exhibited significantly different spatial and temporal changes in both alpha- and beta-diversity. Further analyses of representative functional genes affiliated with N2 fixation (nifH), methane oxidation (pmoA), and sulfate reduction (dsrB, and aprA), suggested functional redundancy within the shallow aquifer microbiomes. While more diversified functional gene taxa were observed for the suspended microbial communities than the attached ones except for pmoA, different levels of changes over time and space were observed between these functional genes. Notably, deterministic and stochastic ecological processes shaped the assembly of microbial communities and functional gene reservoirs differently. While homogenous selection was the prevailing process controlling assembly of microbial communities, the neutral processes (e.g., dispersal limitation, drift and others) were more important for the functional genes. The results suggest complex and changing shallow aquifer microbiomes, whose functionality and assembly vary even between the spatially proximate habitats and fractions. This research underscored the importance to include all the interface components for a more holistic understanding of the biogeochemical processes in aquifer ecosystems, which is also instructive for practical applications.

Orenia metallireducens sp. nov. Strain Z6, a Novel Metal-Reducing Member of the Phylum Firmicutes from the Deep Subsurface.

A novel halophilic and metal-reducing bacterium, Orenia metallireducens strain Z6, was isolated from briny groundwater extracted from a 2.02 km-deep borehole in the Illinois Basin, IL. This organism shared 96% 16S rRNA gene similarity with Orenia marismortui but demonstrated physiological properties previously unknown for this genus. In addition to exhibiting a fermentative metabolism typical of the genus Orenia, strain Z6 reduces various metal oxides [Fe(III), Mn(IV), Co(III), and Cr(VI)], using H2 as the electron donor. Strain Z6 actively reduced ferrihydrite over broad ranges of pH (6 to 9.6), salinity (0.4 to 3.5 M NaCl), and temperature (20 to 60°C). At pH 6.5, strain Z6 also reduced more crystalline iron oxides, such as lepidocrocite (γ-FeOOH), goethite (α-FeOOH), and hematite (α-Fe2O3). Analysis of X-ray absorption fine structure (XAFS) following Fe(III) reduction by strain Z6 revealed spectra from ferrous secondary mineral phases consistent with the precipitation of vivianite [Fe3(PO4)2] and siderite (FeCO3). The draft genome assembled for strain Z6 is 3.47 Mb in size and contains 3,269 protein-coding genes. Unlike the well-understood iron-reducing Shewanella and Geobacter species, this organism lacks the c-type cytochromes for typical Fe(III) reduction. Strain Z6 represents the first bacterial species in the genus Orenia (order Halanaerobiales) reported to reduce ferric iron minerals and other metal oxides. This microbe expands both the phylogenetic and physiological scopes of iron-reducing microorganisms known to inhabit the deep subsurface and suggests new mechanisms for microbial iron reduction. These distinctions from other Orenia spp. support the designation of strain Z6 as a new species, Orenia metallireducens sp. nov. IMPORTANCE: A novel iron-reducing species, Orenia metallireducens sp. nov., strain Z6, was isolated from groundwater collected from a geological formation located 2.02 km below land surface in the Illinois Basin, USA. Phylogenetic, physiologic, and genomic analyses of strain Z6 found it to have unique properties for iron reducers, including (i) active microbial iron-reducing capacity under broad ranges of temperatures (20 to 60°C), pHs (6 to 9.6), and salinities (0.4 to 3.5 M NaCl), (ii) lack of c-type cytochromes typically affiliated with iron reduction in Geobacter and Shewanella species, and (iii) being the only member of the Halanaerobiales capable of reducing crystalline goethite and hematite. This study expands the scope of phylogenetic affiliations, metabolic capacities, and catalytic mechanisms for iron-reducing microbes.

Tepidibacillus decaturensis sp. nov., a microaerophilic, moderately thermophilic iron-reducing bacterium isolated from 1.7 km depth groundwater.

A Gram-stain-negative, microaerophilic rod-shaped organism designated as strain Z9T was isolated from groundwater of 1.7 km depth from the Mt. Simon Sandstone of the Illinois Basin, Illinois, USA. Cells of strain Z9T were rod shaped with dimensions of 0.3×(1-10) µm and stained Gram-negative. Strain Z9T grew within the temperature range 20-60 °C (optimum at 30-40 °C), between pH 5 and 8 (optimum 5.2-5.8) and under salt concentrations of 1-5 % (w/v) NaCl (optimum 2.5 % NaCl). In addition to growth by fermentation and nitrate reduction, this strain was able to reduce Fe(III), Mn(IV), Co(III) and Cr(VI) when H2 or organic carbon was available as the electron donor, but did not actively reduce oxidized sulfur compounds (e.g. sulfate, thiosulfate or S0). The G+C content of the DNA from strain Z9T was 36.1 mol%. Phylogenetic analysis of the 16S rRNA gene from strain Z9T showed that it belongs to the class Bacilli and shares 97 % sequence similarity with the only currently characterized member of the genus Tepidibacillus, T.fermentans. Based on the physiological distinctness and phylogenetic information, strain Z9T represents a novel species within the genus Tepidibacillus, for which the name Tepidibacillus decaturensis sp. nov. is proposed. The type strain is Z9T (=ATCC BAA-2644T=DSM 103037T).

Fe-oxide grain coatings support bacterial Fe-reducing metabolisms in 1.7-2.0 km-deep subsurface quartz arenite sandstone reservoirs of the Illinois Basin (USA).

The Cambrian-age Mt. Simon Sandstone, deeply buried within the Illinois Basin of the midcontinent of North America, contains quartz sand grains ubiquitously encrusted with iron-oxide cements and dissolved ferrous iron in pore-water. Although microbial iron reduction has previously been documented in the deep terrestrial subsurface, the potential for diagenetic mineral cementation to drive microbial activity has not been well studied. In this study, two subsurface formation water samples were collected at 1.72 and 2.02 km, respectively, from the Mt. Simon Sandstone in Decatur, Illinois. Low-diversity microbial communities were detected from both horizons and were dominated by Halanaerobiales of Phylum Firmicutes. Iron-reducing enrichment cultures fed with ferric citrate were successfully established using the formation water. Phylogenetic classification identified the enriched species to be related to Vulcanibacillus from the 1.72 km depth sample, while Orenia dominated the communities at 2.02 km of burial depth. Species-specific quantitative analyses of the enriched organisms in the microbial communities suggest that they are indigenous to the Mt. Simon Sandstone. Optimal iron reduction by the 1.72 km enrichment culture occurred at a temperature of 40°C (range 20-60°C) and a salinity of 25 parts per thousand (range 25-75 ppt). This culture also mediated fermentation and nitrate reduction. In contrast, the 2.02 km enrichment culture exclusively utilized hydrogen and pyruvate as the electron donors for iron reduction, tolerated a wider range of salinities (25-200 ppt), and exhibited only minimal nitrate- and sulfate-reduction. In addition, the 2.02 km depth community actively reduces the more crystalline ferric iron minerals goethite and hematite. The results suggest evolutionary adaptation of the autochthonous microbial communities to the Mt. Simon Sandstone and carries potentially important implications for future utilization of this reservoir for CO2 injection.

Halomonas sulfidaeris-dominated microbial community inhabits a 1.8 km-deep subsurface Cambrian Sandstone reservoir.

A low-diversity microbial community, dominated by the γ-proteobacterium Halomonas sulfidaeris, was detected in samples of warm saline formation porewater collected from the Cambrian Mt. Simon Sandstone in the Illinois Basin of the North American Midcontinent (1.8 km/5872 ft burial depth, 50°C, pH 8, 181 bars pressure). These highly porous and permeable quartz arenite sandstones are directly analogous to reservoirs around the world targeted for large-scale hydrocarbon extraction, as well as subsurface gas and carbon storage. A new downhole low-contamination subsurface sampling probe was used to collect in situ formation water samples for microbial environmental metagenomic analyses. Multiple lines of evidence suggest that this H. sulfidaeris-dominated subsurface microbial community is indigenous and not derived from drilling mud microbial contamination. Data to support this includes V1-V3 pyrosequencing of formation water and drilling mud, as well as comparison with previously published microbial analyses of drilling muds in other sites. Metabolic pathway reconstruction, constrained by the geology, geochemistry and present-day environmental conditions of the Mt. Simon Sandstone, implies that H. sulfidaeris-dominated subsurface microbial community may utilize iron and nitrogen metabolisms and extensively recycle indigenous nutrients and substrates. The presence of aromatic compound metabolic pathways suggests this microbial community can readily adapt to and survive subsurface hydrocarbon migration.