Genome streamlining in Parcubacteria transitioning from soil to groundwater.

BACKGROUND: To better understand the influence of habitat on the genetic content of bacteria, with a focus on members of Candidate Phyla Radiation (CPR) bacteria, we studied the effects of transitioning from soil via seepage waters to groundwater on genomic composition of ultra-small Parcubacteria, the dominating CPR class in seepage waters, using genome resolved metagenomics. RESULTS: Bacterial metagenome-assembled genomes (MAGs), (318 total, 32 of Parcubacteria) were generated from seepage waters and compared directly to groundwater counterparts. The estimated average genome sizes of members of major phyla Proteobacteria, Bacteroidota and Cand. Patescibacteria (Candidate Phyla Radiation - CPR bacteria) were significantly higher in soil-seepage water as compared to their groundwater counterparts. Seepage water Parcubacteria (Paceibacteria) exhibited 1.18-fold greater mean genome size and 2-fold lower mean proportion of pseudogenes than those in groundwater. Bacteroidota and Proteobacteria also showed a similar trend of reduced genomes in groundwater compared to seepage. While exploring gene loss and adaptive gains in closely related CPR lineages in groundwater, we identified a membrane protein, and a lipoglycopeptide resistance gene unique to a seepage Parcubacterium genome. A nitrite reductase gene was also identified and was unique to the groundwater Parcubacteria genomes, likely acquired from other planktonic microbes via horizontal gene transfer. CONCLUSIONS: Overall, our data suggest that bacteria in seepage waters, including ultra-small Parcubacteria, have significantly larger genomes and higher metabolic enrichment than their groundwater counterparts, highlighting possible genome streamlining of the latter in response to habitat selection in an oligotrophic environment.

Iron coatings on carbonate rocks shape the attached bacterial aquifer community.

Most studies of groundwater ecosystems target planktonic microbes, which are easily obtained via water samples. In contrast, little is known about the diversity and function of microbes adhering to rock surfaces, particularly to consolidated rocks. To investigate microbial attachment to rock surfaces, we incubated rock chips from fractured aquifers in limestone-mudstone alternations in bioreactors fed with groundwater from two wells representing oxic and anoxic conditions. Half of the chips were coated with iron oxides, representing common secondary mineralization in fractured rock. Our time-series analysis showed bacteria colonizing the chips within two days, reaching cell numbers up to 4.16 × 105 cells/mm2 after 44 days. Scanning electron microscopy analyses revealed extensive colonization but no multi-layered biofilms, with chips from oxic bioreactors more densely colonized than from anoxic ones. Estimated attached-to-planktonic cell ratios yielded values of up to 106: 1 and 103: 1, for oxic and anoxic aquifers, respectively. We identified distinct attached and planktonic communities with an overlap between 17 % and 42 %. Oxic bioreactors were dominated by proteobacterial genera Aquabacterium and Rhodoferax, while Rheinheimera and Simplicispira were the key players of anoxic bioreactors. Motility, attachment, and biofilm formation traits were predicted in major genera based on groundwater metagenome-assembled genomes and reference genomes. Early rock colonizers appeared to be facultative autotrophs, capable of fixing CO2 to synthesize biomass and a biofilm matrix. Late colonizers were predicted to possess biofilm degrading enzymes such as beta-glucosidase, beta-galactosidase, amylases. Fe-coated chips of both bioreactors featured more potential iron reducers and oxidizers than bare rock chips. As secondary minerals can also serve as energy source, they might favor primary production and thus contribute to subsurface ecosystem services like carbon fixation. Since most subsurface microbes seem to be attached, their contribution to ecosystem services should be considered in future studies.

A glimpse of the paleome in endolithic microbial communities.

BACKGROUND: The terrestrial subsurface is home to a significant proportion of the Earth's microbial biomass. Our understanding about terrestrial subsurface microbiomes is almost exclusively derived from groundwater and porous sediments mainly by using 16S rRNA gene surveys. To obtain more insights about biomass of consolidated rocks and the metabolic status of endolithic microbiomes, we investigated interbedded limestone and mudstone from the vadose zone, fractured aquifers, and deep aquitards. RESULTS: By adapting methods from microbial archaeology and paleogenomics, we could recover sufficient DNA for downstream metagenomic analysis from seven rock specimens independent of porosity, lithology, and depth. Based on the extracted DNA, we estimated between 2.81 and 4.25 × 105 cells × g-1 rock. Analyzing DNA damage patterns revealed paleome signatures (genetic records of past microbial communities) for three rock specimens, all obtained from the vadose zone. DNA obtained from deep aquitards isolated from surface input was not affected by DNA decay indicating that water saturation and not flow is controlling subsurface microbial survival. Decoding the taxonomy and functional potential of paleome communities revealed increased abundances for sequences affiliated with chemolithoautotrophs and taxa such as Cand. Rokubacteria. We also found a broader metabolic potential in terms of aromatic hydrocarbon breakdown, suggesting a preferred utilization of sedimentary organic matter in the past. CONCLUSIONS: Our study suggests that limestones function as archives for genetic records of past microbial communities including those sensitive to environmental stress at modern times, due to their specific conditions facilitating long-term DNA preservation. Video Abstract.

Organic Matter from Redoximorphic Soils Accelerates and Sustains Microbial Fe(III) Reduction.

Microbial reduction of Fe(III) minerals is a prominent process in redoximorphic soils and is strongly affected by organic matter (OM). We herein determined the rate and extent of microbial reduction of ferrihydrite (Fh) with either adsorbed or coprecipitated OM by Geobacter sulfurreducens. We focused on OM-mediated effects on electron uptake and alterations in Fh crystallinity. The OM was obtained from anoxic soil columns (effluent OM, efOM) and included-unlike water-extractable OM-compounds released by microbial activity under anoxic conditions. We found that organic molecules in efOM had generally no or only very low electron-accepting capacity and were incorporated into the Fh aggregates when coprecipitated with Fh. Compared to OM-free Fh, adsorption of efOM to Fh decelerated the microbial Fe(III) reduction by passivating the Fh surface toward electron uptake. In contrast, coprecipitation of Fh with efOM accelerated the microbial reduction, likely because efOM disrupted the Fh structure, as noted by Mössbauer spectroscopy. Additionally, the adsorbed and coprecipitated efOM resulted in a more sustained Fe(III) reduction, potentially because efOM could have effectively scavenged biogenic Fe(II) and prevented the passivation of the Fh surface by the adsorbed Fe(II). Fe(III)-OM coprecipitates forming at anoxic-oxic interfaces are thus likely readily reducible by Fe(III)-reducing bacteria in redoximorphic soils.

Well-defined poly(ethylene glycol) polymers as non-conventional reactive tracers of colloidal transport in porous media.

HYPOTHESIS: A prominent fraction of mobile organic matter in natural aqueous soil solutions is formed by molecules in sizes that seamlessly exceed the lower end of what is defined as a colloid. The hydrodynamics and the functional diversity of these molecules result in a transport behavior that is fundamentally different from smaller compounds. However, there is a lack of "reactive tracers" that allow for the study of colloidal transport phenomena appropriately. We hypothesize that tailor-made and well-defined synthetic polymers can overcome this limitation. EXPERIMENTS: We prepared and characterized the hydrodynamic properties of water-soluble poly(ethylene glycol)s (PEG) and studied their adsorption to mixtures of quartz, illite, and goethite in batch and column experiments. FINDINGS: We used this information to independently predict the transport of PEG with striking agreement to the observed mean breakthrough times in all porous media. As PEG transport can be comprehensively and quantitatively reconstructed, we conclude that functionalized PEGs are promising candidates to be used as tailorable and non-toxic tracers available in the size range of natural organic (macro-)molecules.

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.

Superimposed Pristine Limestone Aquifers with Marked Hydrochemical Differences Exhibit Distinct Fungal Communities.

Fungi are one important group of eukaryotic microorganisms in a diverse range of ecosystems, but their diversity in groundwater ecosystems is largely unknown. We used DNA-based pyro-tag sequencing of the fungal internal transcribed spacer (ITS) rDNA gene to investigate the presence and community structure of fungi at different sampling sites of two superimposed limestone aquifers ranging from 8.5 to 84 m depth in the newly established Hainich Critical Zone Exploratory (Hainich CZE). We detected a diversity of fungal OTUs in groundwater samples of all sampling sites. The relative percentage abundance of Basidiomycota was higher in the upper aquifer assemblage, whilst Ascomycota dominated the lower one. In parallel to differences in the hydrochemistry we found distinct fungal communities at all sampling sites. Classification into functional groups revealed an overwhelming majority of saprotrophs. Finding taxa common to all analyzed groundwater sites, point to a groundwater specific fungal microbiome. The presence of different functional groups and, in particular plant and cattle pathogens that are not typical of subsurface habitats, suggests links between the surface and subsurface biogeosphere due to rapid transportation across the fracture networks typical of karstic regions during recharge episodes. However, further studies including sampling series extended in both time and space are necessary to confirm this hypothesis.

Synthesis of cryptomelane- and birnessite-type manganese oxides at ambient pressure and temperature.

Cryptomelane-type octahedral molecular sieves (K-OMS-2) were successfully synthesized at ambient pressure and temperature by a simple one-step reaction pathway. We designed three synthesis mixes based on redox reactions of either MnSO4 or MnCl2 together with KMnO4 in aqueous solution. The synthesis products were characterized structurally (XRD, FTIR spectroscopy), morphologically (SEM, BET surface area), and chemically (SEM-EDX, ICP-OES). For all mixes, a precursor octahedral layered K-birnessite (K-OL-1) was formed after 1d that subsequently transformed into K-OMS-2. This transformation process depends on the pH of the reaction solution, the respective Mn(II) salt and time. We obtained K-OMS-2 materials with BET surface areas between 50.4 and 104.5 m(2) g(-1) and different crystallinities. The described method is reliable, reproducible, easy to handle and may be the basis to produce well defined Mn oxides that could be used for remediation and catalysis purposes.

Kinetic control of contaminant release from NAPLs--information potential of concentration time profiles.

Release of contaminants from non-aqueous phase liquids (NAPLs) is often limited by the dynamic exchange with aqueous solutions governed by a priori unknown kinetic laws. Release experiments require a thorough evaluation of the potential and limitations of kinetic models to reveal release processes. In this study, we investigated the characteristic concentration-time profiles of various models for the release of contaminants from an organic phase into an aqueous solution under no flow conditions. Criteria have been tested that allow for distinction of a first order one domain, a first order two domain, a spherical diffusion model, a spherical diffusion model with a time variable diffusion coefficient, a model for diffusion in a sphere with organic film, and a model for diffusion in a sphere with an aqueous film. The results can serve to evaluate the processes potentially governing release of organic contaminants from non-aqueous liquid phases.

STXM and NanoSIMS investigations on EPS fractions before and after adsorption to goethite.

Extracellular polymeric substances (EPS) are expected to be an important source for the formation of mineral-organic associations in soil. Because such formations affect the composition of mobile and immobile organic matter as well as the reactivity of minerals, we investigated the composition of EPS before and after adsorption to goethite. Raman measurements on EPS extracted from Bacillus subtilis distinguished four fractions rich in proteins, polysaccharides, lipids, or lipids and proteins. Scanning transmission X-ray microscopy identified three different EPS-fractions that varied in their composition in proteins, nonaromatic proteins, and polysaccharides. Reaction of EPS with goethite led to a preferential adsorption of lipids and proteins. The organic coverage was heterogeneous, consisting of ~100 × 200 nm large patches of either lipid-rich or protein-rich material. Nanoscale secondary ion mass spectrometry showed a strong S enrichment in aggregates of ~400 nm in the goethite adsorbed EPS. From our simplified model system, we learned that only a small portion (<10%) of EPS was immobilized via adsorption to goethite. This fraction formed a coating of subµm spaced protein-rich and lipid-rich domains, i.e., of two materials which will strongly differ in their reactive sites. This will finally affect further adsorption, the particle mobility and eventually also colloidal stability.

Arsenic strongly associates with ferrihydrite colloids formed in a soil effluent.

Arsenic mobility may increase in liquid phase due to association with colloidal Fe oxides. We studied the association of As with Fe oxide colloids in the effluent from water-saturated soil columns run under anoxic conditions. Upon exfiltration, the solutions, which contained Fe2+, were re-aerated and ferrihydrite colloids precipitated. The entire amount of effluent As was associated with the ferrihydrite colloids, although PO4(3-), SiO4(4-), CO3(2-) and dissolved organic matter were present in the effluent during ferrihydrite colloid formation. Furthermore, no subsequent release of As from the ferrihydrite colloids was observed despite the presence of these (in)organic species known to compete with As for adsorption on Fe oxides. Arsenic was bound via inner-sphere complexation on the ferrihydrite surface. FTIR spectroscopy also revealed adsorption of PO4(3-) and polymerized silica. However, these species could not impede the quantitative association of As with colloidal ferrihydrite in the soil effluents.

Fractionation of organic matter due to reaction with ferrihydrite: coprecipitation versus adsorption.

In soil and water, ferrihydrite frequently forms in the presence of dissolved organic matter. This disturbs crystal growth and gives rise to coprecipitation of ferrihydrite and organic matter. To compare the chemical fractionation of organic matter during coprecipitation with the fractionation involved in adsorption onto pristine ferrihydrite surfaces we prepared ferrihydrite-organic matter associations by adsorption and coprecipitation using (i) a forest-floor extract or (ii) a sulfonated lignin. The reaction products were studied by (13)C CPMAS NMR, FTIR, and analysis of hydrolyzable neutral polysaccharides. Relative to the original forest-floor extract, the ferrihydrite-associated organic matter was enriched in polysaccharides, especially when adsorption took place. Moreover, mannose and glucose were bound preferentially to ferrihydrite, while fucose, arabinose, xylose, and galactose accumulated in the supernatant. This fractionation of sugar monomers was more pronounced during coprecipitation and led to an enhanced ratio of (galactose + mannose)/(arabinose + xylose). Experiments with lignin revealed that the ferrihydrite-associated material was enriched in its aromatic components but had a lower ratio of phenolic C to aromatic C than the original lignin. A compositional difference between the adsorbed and coprecipitated lignin is obvious from a higher contribution of methoxy C in the coprecipitated material. Coprecipitated organic matter may thus differ in amount and composition from adsorbed organic matter.

Model study on sorption of polycyclic aromatic hydrocarbons to goethite.

A systematic DFT study of interactions between a set of mono- and polyaromatic hydrocarbons (PAHs) and the (110) goethite surface have been performed in this work. It was found that PAHs form relatively weak surface complexes having their molecular plane practically parallel to the surface plane. The origin of the interactions is in the polarization of the pi-system by polar OH groups and in the formation of weak hydrogen bonds where the pi-system acts as a proton acceptor. The computed perpendicular distances of the molecular plane of PAHs to the hydrogen atoms of the surface OH groups range from 2.3 to 2.7 A. Computed interaction energies regularly increase for the linearly shaped molecules from benzene to anthracene. Two other PAHs with a nonlinear shape, phenanthrene and pyrene, are less strongly bound to the surface although they have a similar (phenanthrene) or even larger size (pyrene) than anthracene. These differences were explained by the specific configuration of the surface hydroxyl groups of goethite. The three types of OH groups, mu-OH, mu(3)-O(II)H, and OH, form a valley, the width of it fits very well the molecular shape of the linear PAHs. It was found that with anthracene as example the linear PAHs can easily slide along the valley of OH groups with practically no barrier. In summary it is concluded from our results that the (110) goethite surface will withhold linear PAHs significantly better than nonlinear ones.

Characterization of ferrihydrite-soil organic matter coprecipitates by X-ray diffraction and Mössbauer spectroscopy.

In soils and sediments ferrihydrite often precipitates from solutions containing dissolved organic matter, which affects its crystallinity. To simulate this process we prepared a series of 2-line ferrihydrite-organic matter coprecipitates using water extractable organic matter (OM) from a forest topsoil. The products were characterized byX-ray diffraction, Mössbauer spectroscopy, N2-gas adsorption and transmission electron microscopy. With increasing C/Fe ratios of the initial solution the d-spacings of the two major XRD peaks increased, while peak shoulders at 0.22 and 0.16 nm weakened. The asymmetry of the 0.26 nm peak decreased and disappeared at a C/Fe ratio of 0.78. The quadrupole splitting of the Mössbauer spectra at 300 K increased from 0.78 to 0.90 mm s(-1), the mean magnetic hyperfine field at 4.2 K dropped from 49.5 to 46.0 T, and the superparamagnetic collapse of the magnetic hyperfine splitting was shifted toward lower temperatures. These data reflect a strong interference of OM with crystal growth leading to smaller ferrihydrite crystals, increased lattice spacings, and more distorted Fe(O,OH)6 octahedra. Even small amounts of OM significantly change particle size and structural order of ferrihydrite. Crystallinity and reactivity of natural ferrihydrites will therefore often differ from their synthetic counterparts, formed in the absence of OM.