Subsurface Ecosystems Research Articles

New evidence for a hydroxylation pathway for anaerobic alkane degradation supported by analyses of functional genes and signature metabolites in oil reservoirs

Microbial degradation of recalcitrant alkanes under anaerobic conditions results in the accumulation of heavy oil fraction in oil reservoirs. Hydroxylation of alkanes is an important activation mechanism under anaerobic conditions, but the diversity and distribution of the responsible microorganisms in the subsurface environment are still unclear. The lack of functional gene polymerase chain reaction (PCR) primers and commercially available intermediate degradation chemical compounds are the major obstacles for this research. In this investigation, PCR primers for the ahyA gene (encoding alkane hydroxylase) were designed, evaluated, and improved based on the nucleotide sequences available. Using microbial genomic DNA extracted from oil-contaminated soil and production water samples of oil reservoirs, ahyA gene nucleotide sequences were amplified and retrieved successfully from production water sample Z3-25 of Shengli oilfield. Additionally, the signature biomarker of 2-acetylalkanoic acid was detected in both Shengli and Jiangsu oilfields. These results demonstrate that anaerobic hydroxylation is an active mechanism used by microorganisms to degrade alkanes in oxygen-depleted oil reservoirs. This finding expands the current knowledge of biochemical reactions about alkane degradation in subsurface ecosystems. In addition, the PCR primers designed and tested in this study serve as an effective molecular tool for detecting the microorganisms responsible for anaerobic hydroxylation of alkanes in this and other ecosystems.

Rock Surface Fungi in Deep Continental Biosphere-Exploration of Microbial Community Formation with Subsurface In Situ Biofilm Trap.

Fungi have an important role in nutrient cycling in most ecosystems on Earth, yet their ecology and functionality in deep continental subsurface remain unknown. Here, we report the first observations of active fungal colonization of mica schist in the deep continental biosphere and the ability of deep subsurface fungi to attach to rock surfaces under in situ conditions in groundwater at 500 and 967 m depth in Precambrian bedrock. We present an in situ subsurface biofilm trap, designed to reveal sessile microbial communities on rock surface in deep continental groundwater, using Outokumpu Deep Drill Hole, in eastern Finland, as a test site. The observed fungal phyla in Outokumpu subsurface were Basidiomycota, Ascomycota, and Mortierellomycota. In addition, significant proportion of the community represented unclassified Fungi. Sessile fungal communities on mica schist surfaces differed from the planktic fungal communities. The main bacterial phyla were Firmicutes, Proteobacteria, and Actinobacteriota. Biofilm formation on rock surfaces is a slow process and our results indicate that fungal and bacterial communities dominate the early surface attachment process, when pristine mineral surfaces are exposed to deep subsurface ecosystems. Various fungi showed statistically significant cross-kingdom correlation with both thiosulfate and sulfate reducing bacteria, e.g., SRB2 with fungi Debaryomyces hansenii.

Lithogenic hydrogen supports microbial primary production in subglacial and proglacial environments

Life in environments devoid of photosynthesis, such as on early Earth or in contemporary dark subsurface ecosystems, is supported by chemical energy. How, when, and where chemical nutrients released from the geosphere fuel chemosynthetic biospheres is fundamental to understanding the distribution and diversity of life, both today and in the geologic past. Hydrogen (H2) is a potent reductant that can be generated when water interacts with reactive components of mineral surfaces such as silicate radicals and ferrous iron. Such reactive mineral surfaces are continually generated by physical comminution of bedrock by glaciers. Here, we show that dissolved H2 concentrations in meltwaters from an iron and silicate mineral-rich basaltic glacial catchment were an order of magnitude higher than those from a carbonate-dominated catchment. Consistent with higher H2 abundance, sediment microbial communities from the basaltic catchment exhibited significantly shorter lag times and faster rates of net H2 oxidation and dark carbon dioxide (CO2) fixation than those from the carbonate catchment, indicating adaptation to use H2 as a reductant in basaltic catchments. An enrichment culture of basaltic sediments provided with H2, CO2, and ferric iron produced a chemolithoautotrophic population related to Rhodoferax ferrireducens with a metabolism previously thought to be restricted to (hyper)thermophiles and acidophiles. These findings point to the importance of physical and chemical weathering processes in generating nutrients that support chemosynthetic primary production. Furthermore, they show that differences in bedrock mineral composition can influence the supplies of nutrients like H2 and, in turn, the diversity, abundance, and activity of microbial inhabitants.

Culturing of "Unculturable" Subsurface Microbes: Natural Organic Carbon Source Fuels the Growth of Diverse and Distinct Bacteria From Groundwater.

Recovery and cultivation of diverse environmentally-relevant microorganisms from the terrestrial subsurface remain a challenge despite recent advances in modern molecular technology. Here, we applied complex carbon (C) sources, i.e., sediment dissolved organic matter (DOM) and bacterial cell lysate, to enrich groundwater microbial communities for 30 days. As comparisons, we also included enrichments amended with simple C sources including glucose, acetate, benzoate, oleic acid, cellulose, and mixed vitamins. Our results demonstrate that complex C is far more effective in enriching diverse and distinct microorganisms from groundwater than simple C. Simple C enrichments yield significantly lower biodiversity, and are dominated by few phyla (e.g., Proteobacteria and Bacteroidetes), while microcosms enriched with complex C demonstrate significantly higher biodiversity including phyla that are poorly represented in published culture collections (e.g., Verrucomicrobia, Planctomycetes, and Armatimonadetes). Subsequent isolation from complex C enrichments yielded 228 bacterial isolates representing five phyla, 17 orders, and 56 distinct species, including candidate novel, rarely cultivated, and undescribed organisms. Results from this study will substantially advance cultivation and isolation strategies for recovering diverse and novel subsurface microorganisms. Obtaining axenic representatives of “once-unculturable” microorganisms will enhance our understanding of microbial physiology and function in different biogeochemical niches of terrestrial subsurface ecosystems.

Euro-Mediterranean fauna of Campodeinae (Campodeidae, Diplura)

Campodeinae is the most diverse subfamily of Campodeidae diplurans and inhabits soils and subsurface ecosystems. These are distributed in the Palearctic area, clearly rarifying towards northern latitudes. The major taxonomic characters are chaetotaxy, shape and complexity of pretarsal structures, and secondary sexual characters. This monograph provides a taxonomic revision of the subfamily Campodeinae in the Euro-Mediterrnean region, based on extensive literature review and new material studied. It comprises detailed morphological descriptions and illustrations together with habitat and distribution data of 161 species and 14 subspecies from 10 genera: Campodea Westwood, 1842, Edriocampa Silvestri, 1933, Eutrichocampa Silvestri, 1902, Helladocampa Condé, 1984, Libanocampa Condé, 1955, Litocampa Silvestri, 1933, Oreocampa Condé, 1950, Podocampa Silvestri, 1932, Remycampa Condé 1952, and Spaniocampa Silvestri, 1933. With 116 species Campodea is the most diverse and widely distributed genus in the studied area.

How Thermodynamics Illuminates Population Interactions in Microbial Communities

In traditional population models of microbial ecology, there are two central players: producers and consumers (including decomposers that depend on organic carbon). Producers support surface ecosystems by generating adenosine triphosphate (ATP) from sunlight, part of which is used to build new biomass from carbon dioxide. In contrast, the productivity of subsurface ecosystems with a limited supply of sunlight must rely on bacteria and archaea that are able generate ATP solely from chemical or electric energy to fix inorganic carbon. These “light-independent producers” are frequently not included in traditional food webs, even though they are ubiquitous in nature and interact with one another through the utilization of the by-products of others. In this review, we introduce theoretical approaches based on population dynamics that incorporate thermodynamics to highlight characteristic interactions in the microbial community of subsurface ecosystems, which may link community structures and ecosystem expansion under conditions of a limited supply of sunlight. In comparison with light-dependent producers, which compete with one another for light, the use of Gibbs free energy (chemical energy) can lead cooperative interactions among light-independent producers through the effects of the relative quantities of products and reactants on the available chemical energy, which is termed abundant resource premium. The development of a population theory that incorporates thermodynamics offers fundamental ecological insights into subsurface microbial ecosystems, which may be applied to fields of study such as environmental science/engineering, astrobiology, or the microbial ecosystems of the early earth.

Event-driven dynamics of the total mobile inventory in undisturbed soil account for significant fluxes of particulate organic carbon

Considerable portions of the total mobile inventory of soil seepage are the diverse colloidal and larger suspended materials that essentially contribute to pedogenesis, soil functioning, and nutritional supply of subsurface ecosystems. However, the size- and material-spectra of the total mobile inventory, and field-scale factors controlling its long-term seasonal and episodic dynamics in undisturbed soil, are scarcely investigated so far. In a 4.5-year field-scale study, we utilized automated tension-controlled lysimeters optimized for in situ-sampling of total mobile inventory. Covering different land uses in a low-mountain groundwater recharge area in central Germany, seepage of top- and subsoil was collected at least biweekly and analyzed by hydrochemical and spectromicroscopic techniques (SEM/EDX, nanoparticle tracking analysis). In undisturbed soil, diverse mineral-, mineral-organic, organic, and bioparticles (microbial cells, biotic detritus) up to 75 μm was mobile. Atmospheric forcing was the major factor that governed transport of the total mobile inventory, causing considerable seasonality in seepage pH and certain solutes (e.g. sulphate), as well as episodic fluctuation of particulates. Especially episodic high-flow events, like those following snow melts and lasting rainstorms, primarily contributed to the export of inorganic/organic matter beyond the subsoil-regolith boundary. Individual infiltration events during winter accounted for up to 80% of annual fluxes of particulate organic carbon. On average, a significant proportion of 21% of the mobile organic carbon belonged to the >0.45 μm fraction. The pedological setting and land use mostly impacted the solute signature but were of minor importance for the particle load. Our ongoing monitoring provides evidence of significant episodic nutrient fluxes and unveiled pronounced temporal patterns of field-scale pH fluctuations. We conclude that dynamics of the total mobile inventory, including particulates >0.45 μm must be considered in approaches that budget carbon and elemental fluxes, but also in concepts and models on nutrient cycles and subsurface ecosystem functioning.

Spatial variability of ground water quality: a case study of Udupi district, Karnataka State, India

Groundwater is a reliable source of fresh water for domestic and agricultural water users. It supports subsurface ecosystem by balancing the geo-biological and bio-geochemical processes at micro- and macro-scales of the ecosystem. Overexploitation, anthropogenic activities and improper agricultural practices have contributed to the pollution of groundwater sources all around the globe. The water quality index (WQI) is the most extensively used indicator which transforms the water quality information derived from several parameters into a single value/rating to categorize and provide a general perception of water quality standard. Groundwater quality analysis and mapping via geographical information system (GIS) proves to be beneficial in identifying the locations where the groundwater quality is deteriorating. In the current study, the WQI of groundwater was determined for the samples collected from open and tube wells located within the Udupi district of Karnataka state, India. The groundwater quality parameters such as pH, hardness, calcium, chlorides, nitrates, iron, fluoride, sulfates, manganese, sodium, magnesium, potassium, turbidity, and phosphate were analyzed for water samples collected from 112 randomly chosen open/tube wells in order to determine the WQI. Interpolation approaches such as inverse distance weighting (IDW) and kriging were adopted in the GIS environment to quantify the spatial variability of groundwater quality over the entire geographical area. The groundwater quality maps were generated using the best fit models. The results portray that, the accuracy of interpolation using IDW and kriging methods was dependent on the measures of central tendency and variability of water quality data of different parameters. The kriging interpolation was much accurate for most of the groundwater quality parameters compared to IDW maps. The WQI maps, perhaps signposted the poor quality of groundwater quality in about 1.88% of the geographical area of Udupi district. Further, about 21.69% of the area was affected by poor quality of groundwater where suitable strategies for replenishment of groundwater resources should be taken up by the concerned authorities. The spatial distribution maps of groundwater quality aid to locate vulnerable places where immediate action is required.

Groundwater Microbial Communities in Times of Climate Change.

Climate change has a massive impact on the global water cycle. Subsurface ecosystems, the earth largest reservoir of liquid freshwater, currently experience a significant increase in temperature and serious consequences from extreme hydrological events. Extended droughts as well as heavy rains and floods have measurable impacts on groundwater quality and availability. In addition, the growing water demand puts increasing pressure on the already vulnerable groundwater ecosystems. Global change induces undesired dynamics in the typically nutrient and energy poor aquifers that are home to a diverse and specialized microbiome and fauna. Current and future changes in subsurface environmental conditions, without doubt, alter the composition of communities, as well as important ecosystem functions, for instance the cycling of elements such as carbon and nitrogen. A key role is played by the microbes. Understanding the interplay of biotic and abiotic drivers in subterranean ecosystems is required to anticipate future effects of climate change on groundwater resources and habitats. This review summarizes potential threats to groundwater ecosystems with emphasis on climate change and the microbial world down below our feet in the water saturated subsurface.

Bacterial community variations with salinity in the saltwater-intruded estuarine aquifer

Bacterial community has been significantly enrolled in the biogeochemical cycling of the coastal subsurface ecosystem. The bacterial community variations with salinity have been extensively investigated in the surface environment, such as lake, soil, and estuary, but not in the subsurface environment. Here we explore the responses of bacterial populations to the salinity and other environmental factors (EFs) by considering both the abundant and rare sub-community in a coastal Holocene groundwater system. Our study results indicate that the bacterial diversity was independent of the salinity in both the abundance and rare sub-community. Besides diversity, no flourishing of abundant bacteria relative abundance is observed with increasing or decreasing salinity. Yet the rare taxa exhibit a bio-growth with salinity, which has a significant correlation (p < 0.001) with sulfate concentration. The responses of the abundant sub-community taxa to nutrients, temperature, pH, and dissolved oxygen are insensitive. However, the correlation between δ18O, δD and the entire community diversity is significant, which demonstrates the bacterial community is affected by the groundwater origin. Besides, not all the species in one class or order are necessarily shaped by the same factor. To quantify the impact of EFs on the community properties, analyses in different taxonomic levels is suggested. These findings imply that the spatial organization of microbial communities is complicated and influenced by multiple factors on a regional scale. The investigated results are useful for understanding biogeochemical processes in the coastal groundwater.

Molecular and taxonomic characterization of arsenic (As) transforming Bacillus sp. strain IIIJ3\u20131 isolated from As-contaminated groundwater of Brahmaputra river basin, India

BackgroundMicrobe-mediated redox transformation of arsenic (As) leading to its mobilization has become a serious environmental concern in various subsurface ecosystems especially within the alluvial aquifers. However, detailed taxonomic and eco-physiological attributes of indigenous bacteria from As impacted aquifer of Brahmaputra river basin has remained under-studied.ResultsA newly isolated As-resistant and -transforming facultative anaerobic bacterium IIIJ3–1 from As-contaminated groundwater of Jorhat, Assam was characterized. Near complete 16S rRNA gene sequence affiliated the strain IIIJ3–1 to the genus Bacillus and phylogenetically placed within members of B. cereus sensu lato group with B. cereus ATCC 14579(T) as its closest relative with a low DNA-DNA relatedness (49.9%). Presence of iC17:0, iC15:0 fatty acids and menaquinone 7 corroborated its affiliation with B. cereus group, but differential hydroxy-fatty acids, C18:2 and menaquinones 5 & 6 marked its distinctiveness. High As resistance [Maximum Tolerable Concentration = 10 mM As3+, 350 mM As5+], aerobic As3+ (5 mM) oxidation, and near complete dissimilatory reduction of As 5+ (1 mM) within 15 h of growth designated its physiological novelty. Besides O2, cells were found to reduce As5+, Fe3+, SO42−, NO3−, and Se6+ as alternate terminal electron acceptors (TEAs), sustaining its anaerobic growth. Lactate was the preferred carbon source for anaerobic growth of the bacterium with As5+ as TEA. Genes encoding As5+ respiratory reductase (arr A), As3+ oxidase (aioB), and As3+ efflux systems (ars B, acr3) were detected. All these As homeostasis genes showed their close phylogenetic lineages to Bacillus spp. Reduction in cell size following As exposure exhibited the strain’s morphological response to toxic As, while the formation of As-rich electron opaque dots as evident from SEM-EDX possibly indicated a sequestration based As resistance strategy of strain IIIJ3–1.ConclusionThis is the first report on molecular, taxonomic, and ecophysiological characterization of a highly As resistant, As3+ oxidizing, and dissimilatory As5+ reducing Bacillus sp. IIIJ3–1 from As contaminated sites of Brahmaputra river basin. The strain’s ability to resist and transform As along with its capability to sequester As within the cells demonstrate its potential in designing bioremediation strategies for As contaminated groundwater and other ecosystems.

Microbial material cycling, energetic constraints and ecosystem expansion in subsurface ecosystems.

To harvest energy from chemical reactions, microbes engage in diverse catabolic interactions that drive material cycles in the environment. Here, we consider a simple mathematical model for cycling reactions between alternative forms of an element (A and Ae), where reaction 1 converts A to Ae and reaction 2 converts Ae to A. There are two types of microbes: type 1 microbes harness reaction 1, and type 2 microbes harness reaction 2. Each type receives its own catabolic resources from the other type and provides the other type with the by-products as the catabolic resources. Analyses of the model show that each type increases its steady-state abundance in the presence of the other type. The flux of material flow becomes faster in the presence of microbes. By coupling two catabolic reactions, types 1 and 2 can also expand their realized niches through the abundant resource premium, the effect of relative quantities of products and reactants on the available chemical energy, which is especially important for microbes under strong energetic limitations. The plausibility of mutually beneficial interactions is controlled by the available chemical energy (Gibbs energy) of the system. We conclude that mutualistic catabolic interactions can be an important factor that enables microbes in subsurface ecosystems to increase ecosystem productivity and expand the ecosystem.

Densely Populated Water Droplets in Heavy-Oil Seeps.

Most of the microbial degradation in oil reservoirs is believed to take place at the oil-water transition zone (OWTZ). However, a recent study indicates that there is microbial life enclosed in microliter-sized water droplets dispersed in heavy oil of Pitch Lake in Trinidad and Tobago. This life in oil suggests that microbial degradation of oil also takes place in water pockets in the oil-bearing rock of an oil leg independent of the OWTZ. However, it is unknown whether microbial life in water droplets dispersed in oil is a generic property of oil reservoirs rather than an exotic exception. Hence, we took samples from three heavy-oil seeps, Pitch Lake (Trinidad and Tobago), the La Brea Tar Pits (California, USA), and an oil seep on the McKittrick oil field (California, USA). All three tested oil seeps contained dispersed water droplets. Larger droplets between 1 and 10 μl revealed high cell densities of up to 109 cells ml-1 Testing for ATP content and LIVE/DEAD staining showed that these populations consist of active and viable microbial cells with an average of 60% membrane-intact cells and ATP concentrations comparable to those of other subsurface ecosystems. Microbial community analyses based on 16S rRNA gene amplicon sequencing revealed the presence of known anaerobic oil-degrading microorganisms. Surprisingly, the community analyses showed similarities between all three oil seeps, revealing common OTUs, although the sampling sites were thousands of kilometers apart. Our results indicate that small water inclusions are densely populated microhabitats in heavy oil and possibly a generic trait of degraded-oil reservoirs.IMPORTANCE Our results confirmed that small water droplets in oil are densely populated microhabitats containing active microbial communities. Since these microhabitats occurred in three tested oil seeps which are located thousands of kilometers away from each other, such populated water droplets might be a generic trait of biodegraded oil reservoirs and might be involved in the overall oil degradation process. Microbial degradation might thus also take place in water pockets in the oil-bearing oil legs of the reservoir rock rather than only at the oil-water transition zone.

Ecological Assembly Processes Are Coordinated between Bacterial and Viral Communities in Fractured Shale Ecosystems.

The ecological drivers that concurrently act upon both a virus and its host and that drive community assembly are poorly understood despite known interactions between viral populations and their microbial hosts. Hydraulically fractured shale environments provide access to a closed ecosystem in the deep subsurface where constrained microbial and viral community assembly processes can be examined. Here, we used metagenomic analyses of time-resolved-produced fluid samples from two wells in the Appalachian Basin to track viral and host dynamics and to investigate community assembly processes. Hypersaline conditions within these ecosystems should drive microbial community structure to a similar configuration through time in response to common osmotic stress. However, viral predation appears to counterbalance this potentially strong homogeneous selection and pushes the microbial community toward undominated assembly. In comparison, while the viral community was also influenced by substantial undominated processes, it assembled, in part, due to homogeneous selection. When the overall assembly processes acting upon both these communities were directly compared with each other, a significant relationship was revealed, suggesting an association between microbial and viral community development despite differing selective pressures. These results reveal a potentially important balance of ecological dynamics that must be in maintained within this deep subsurface ecosystem in order for the microbial community to persist over extended time periods. More broadly, this relationship begins to provide knowledge underlying metacommunity development across trophic levels.IMPORTANCE Interactions between viral communities and their microbial hosts have been the subject of many recent studies in a wide range of ecosystems. The degree of coordination between ecological assembly processes influencing viral and microbial communities, however, has been explored to a much lesser degree. By using a combined null modeling approach, this study investigated the ecological assembly processes influencing both viral and microbial community structure within hydraulically fractured shale environments. Among other results, significant relationships between the structuring processes affecting both the viral and microbial community were observed, indicating that ecological assembly might be coordinated between these communities despite differing selective pressures. Within this deep subsurface ecosystem, these results reveal a potentially important balance of ecological dynamics that must be maintained to enable long-term microbial community persistence. More broadly, this relationship begins to provide insight into the development of communities across trophic levels.

Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation.

The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are adapted to low nutrient availability or quality. Here, we use high-throughput dilution-to-extinction culturing to isolate shallow subsurface microbes from a conifer forest in Arizona, USA. We hypothesized that the concentration of heterotrophic substrates in microbiological growth medium would affect which microbial taxa were culturable from these soils. To test this, we diluted cells extracted from soil into one of two custom-designed defined growth media that differed by 100-fold in the concentration of amino acids and organic carbon. Across the two media, we isolated a total of 133 pure cultures, all of which were classified as Actinobacteria or Alphaproteobacteria The substrate availability dictated which actinobacterial phylotypes were culturable but had no significant effect on the culturability of Alphaproteobacteria We isolated cultures that were representative of the most abundant phylotype in the soil microbial community (Bradyrhizobium spp.) and representatives of five of the top 10 most abundant Actinobacteria phylotypes, including Nocardioides spp., Mycobacterium spp., and several other phylogenetically divergent lineages. Flow cytometry of nucleic acid-stained cells showed that cultures isolated on low-substrate medium had significantly lower nucleic acid fluorescence than those isolated on high-substrate medium. These results show that dilution-to-extinction is an effective method to isolate abundant soil microbes and that the concentration of substrates in culture medium influences the culturability of specific microbial lineages.IMPORTANCE Isolating environmental microbes and studying their physiology under controlled conditions are essential aspects of understanding their ecology. Subsurface ecosystems are typically nutrient-poor environments that harbor diverse microbial communities-the majority of which are thus far uncultured. In this study, we use modified high-throughput cultivation methods to isolate subsurface soil microbes. We show that a component of whether a microbe is culturable from subsurface soils is the concentration of growth substrates in the culture medium. Our results offer new insight into technical approaches and growth medium design that can be used to access the uncultured diversity of soil microbes.

Genome-inferred spatio-temporal resolution of an uncultivated Roizmanbacterium reveals its ecological preferences in groundwater.

Subsurface ecosystems like groundwater harbour diverse microbial communities, including small-sized, putatively symbiotic organisms of the Candidate Phyla Radiation, yet little is known about their ecological preferences and potential microbial partners. Here, we investigated a member of the superphylum Microgenomates (Cand. Roizmanbacterium ADI133) from oligotrophic groundwater using mini-metagenomics and monitored its spatio-temporal distribution using 16S rRNA gene analyses. A Roizmanbacteria-specific quantitative PCR assay allowed us to track its abundance over the course of 1 year within eight groundwater wells along a 5.4 km hillslope transect, where Roizmanbacteria reached maximum relative abundances of 2.3%. In-depth genomic analyses suggested that Cand. Roizmanbacterium ADI133 is a lactic acid fermenter, potentially able to utilize a range of complex carbon substrates, including cellulose. We hypothesize that it attaches to host cells using a trimeric autotransporter adhesin and inhibits their cell wall biosynthesis using a toxin-antitoxin system. Network analyses based on correlating Cand. Roizmanbacterium ADI133 abundances with amplicon sequencing-derived microbial community profiles suggested one potential host organism, classified as a member of the class Thermodesulfovibrionia (Nitrospirae). By providing lactate as an electron donor Cand. Roizmanbacterium ADI133 potentially mediates the transfer of carbon to other microorganisms and thereby is an important connector in the microbial community.

Fueling Diversity in the Subsurface: Composition and Age of Dissolved Organic Matter in the Critical Zone

Surface ecosystems are rapidly changing on a global scale and it is important to understand how this influences aquifers in the subsurface, as groundwater quality is a major concern for future generations. Dissolved organic matter (DOM) contains molecular and isotopic signals from surface-derived inputs and from the biotic and abiotic subsurface environment and is therefore ideal to study the connectivity between both environments. Therefore, we evaluated a 3-year time series of DOM composition using ultrahigh resolution mass spectrometry and age using 14C accelerator mass spectrometry along a hillslope well transect in the fractured bedrock of the Hainich Critical Zone Exploratory, Germany. We found a wide range of DOM 14C depletion, from Δ14C = -47.9 to Δ14C = 782.4, within different zones of the shallow groundwater. The 14C content of DOM mirrored the connectivity of the aquifers to the surface. The composition of DOM was highly interrelated with its 14C age. The proportions of surface-derived DOM components decreased with DOM age, whereas microorganism-derived DOM components increased. The intensity of surface-sourced DOM signals differed between the wells and likely reflected the hydrological complexity of fractured-rock environments. During recharge, DOM was more enriched in Δ14C, contained more surface-derived molecular components and was more diverse. As a potential response to the varying DOM substrate, bacterial 16S rRNA gene analysis revealed community evolution and increased bacterial diversity during recharge. The influx of diverse, surface-derived DOM potentially fueled evolution within the autochthonous bacterial communities, as in contrast to DOM, the bacterial community did not retreat to the initial diversity and community composition during the recession period. Our results demonstrate on the one hand that combined analyses of the composition and age of groundwater DOM strongly contribute to the understanding of interconnections, community evolution and the functioning of subsurface ecosystems and on the other hand that changes in surface ecosystems have an imprint on subsurface ecosystems.

Multi-directional flow dynamics shape groundwater quality in sloping bedrock strata

Multi-directional fluid flow and transport dynamics as intrinsic characteristics of hillslope flow regimes can strongly contribute to the quality evolution of groundwater resources and compartmentalization of subsurface ecosystems. However, their extent and importance in topographic highs (groundwater recharge areas) is typically less investigated, because productive groundwater bodies and thus monitoring activities are frequently lacking. In the Hainich Critical Zone Exploratory, we explored the hydrogeological functioning of the widely distributed setting of thin-bedded, alternating mixed carbonate-/siliciclastic bedrock, by using basic environmental timeseries. Up to 8-year records of weather parameters, ground temperatures, multi-depth hydraulic heads, and non-conservative tracers, were exploited applying a scheme for the exploratory analysis of dis-/continuous groundwater quality data. We identified transient, multi-directional flow dynamics, comprising fluctuating perched groundwater, localized recharge and groundwater mounding in the aeration zone that modify and partly reverse flow patterns in the phreatic zone. This interplay of flow dynamics within the hillslope aeration and phreatic zone causes significant re-distribution (e.g. oxygen, nitrate) that even overcome the “protective cover” of thick argillaceous strata by supplying surface-sourced substances to deep groundwater resources. As a factor for ecosystem compartmentalization, our results further suggest to carefully consider the hillslope multi-directional flow and matter exchange in biogeochemical models, as well as in resource protection and groundwater management practices.

Diversity, Distribution and Co-occurrence Patterns of Bacterial Communities in a Karst Cave System.

Caves are typified by their permanent darkness and a shortage of nutrients. Consequently, bacteria play an important role in sustaining such subsurface ecosystems by dominating primary production and fueling biogeochemical cycles. China has one of the world’s largest areas of karst topography in the Yunnan-Guizhou Plateau, yet the bacteriomes in these karst caves remain unexplored. In this study, bacteriomes of eight karst caves in southwest China were examined, and co-occurrence networks of cave bacterial communities were constructed. Results revealed abundant and diversified bacterial communities in karst caves, with Proteobacteria, Actinobacteria, and Firmicutes being the most abundant phyla. Statistical analysis revealed no significant difference in bacteriomes among the eight caves. However, a PCoA plot did show that the bacterial communities of 128 cave samples clustered into groups corresponding to sampling types (air, water, rock, and sediment). These results suggest that the distribution of bacterial communities is driven more by sample types than the separate caves from which samples were collected. Further community-level composition analysis indicated that Proteobacteria were most dominant in water and air samples, while Actinobacteria dominated the sediment and rock samples. Co-occurrence analysis revealed highly modularized assembly patterns of the cave bacterial community, with Nitrosococcaceae wb1-P19, an uncultured group in Rokubacteriales, and an uncultured group in Gaiellales, being the top-three keystone members. These results not only expand our understanding of cave bacteriomes but also inspires functional exploration of bacterial strains in karst caves.

In situ transformation of ethoxylate and glycol surfactants by shale-colonizing microorganisms during hydraulic fracturing.

In the last decade, extensive application of hydraulic fracturing technologies to unconventional low-permeability hydrocarbon-rich formations has significantly increased natural-gas production in the United States and abroad. The injection of surface-sourced fluids to generate fractures in the deep subsurface introduces microbial cells and substrates to low-permeability rock. A subset of injected organic additives has been investigated for their ability to support biological growth in shale microbial community members; however, to date, little is known on how complex xenobiotic organic compounds undergo biotransformations in this deep rock ecosystem. Here, high-resolution chemical, metagenomic, and proteomic analyses reveal that widely-used surfactants are degraded by the shale-associated taxa Halanaerobium, both in situ and under laboratory conditions. These halotolerant bacteria exhibit surfactant substrate specificities, preferring polymeric propoxylated glycols (PPGs) and longer alkyl polyethoxylates (AEOs) over polyethylene glycols (PEGs) and shorter AEOs. Enzymatic transformation occurs through repeated terminal-end polyglycol chain shortening during co-metabolic growth through the methylglyoxal bypass. This work provides the first evidence that shale microorganisms can transform xenobiotic surfactants in fracture fluid formulations, potentially affecting the efficiency of hydrocarbon recovery, and demonstrating an important association between injected substrates and microbial growth in an engineered subsurface ecosystem.