A Cell Extraction Method for Oily Sediments

Hydrocarbons can be found in many different habitats and represent an important carbon source for microbes. As fossil fuels, they are also an important economical resource and through natural seepage or accidental release they can be major pollutants. DNA-specific stains and molecular probes bind to hydrocarbons, causing massive background fluorescence, thereby hampering cell enumeration. The cell extraction procedure of Kallmeyer et al. (2008) separates the cells from the sediment matrix. In principle, this technique can also be used to separate cells from oily sediments, but it was not originally optimized for this application. Here we present a modified extraction method in which the hydrocarbons are removed prior to cell extraction. Due to the reduced background fluorescence the microscopic image becomes clearer, making cell identification, and enumeration much easier. Consequently, the resulting cell counts from oily samples treated according to our new protocol are significantly higher than those treated according to Kallmeyer et al. (2008). We tested different amounts of a variety of solvents for their ability to remove hydrocarbons and found that n-hexane and – in samples containing more mature oils – methanol, delivered the best results. However, as solvents also tend to lyse cells, it was important to find the optimum solvent to sample ratio, at which hydrocarbon extraction is maximized and cell lysis minimized. A volumetric ratio of 1:2–1:5 between a formalin-fixed sediment slurry and solvent delivered highest cell counts. Extraction efficiency was around 30–50% and was checked on both oily samples spiked with known amounts of E. coli cells and oil-free samples amended with fresh and biodegraded oil. The method provided reproducible results on samples containing very different kinds of oils with regard to their degree of biodegradation. For strongly biodegraded oil MeOH turned out to be the most appropriate solvent, whereas for less biodegraded samples n-hexane delivered best results

A System for Incubations at High Gas Partial Pressure

High-pressure is a key feature of deep subsurface environments. High partial pressure of dissolved gasses plays an important role in microbial metabolism, because thermodynamic feasibility of many reactions depends on the concentration of reactants. For gases, this is controlled by their partial pressure, which can exceed 1 MPa at in situ conditions. Therefore, high hydrostatic pressure alone is not sufficient to recreate true deep subsurface in situ conditions, but the partial pressure of dissolved gasses has to be controlled as well. We developed an incubation system that allows for incubations at hydrostatic pressure up to 60 MPa, temperatures up to 120°C, and at high gas partial pressure. The composition and partial pressure of gasses can be manipulated during the experiment. To keep costs low, the system is mainly made from off-the-shelf components with only very few custom-made parts. A flexible and inert PVDF (polyvinylidene fluoride) incubator sleeve, which is almost impermeable for gases, holds the sample and separates it from the pressure fluid. The flexibility of the incubator sleeve allows for sub-sampling of the medium without loss of pressure. Experiments can be run in both static and flow-through mode. The incubation system described here is usable for versatile purposes, not only the incubation of microorganisms and determination of growth rates, but also for chemical degradation or extraction experiments under high gas saturation, e.g., fluid–gas–rock-interactions in relation to carbon dioxide sequestration. As an application of the system we extracted organic compounds from sub-bituminous coal using H2O as well as a H2O–CO2 mixture at elevated temperature (90°C) and pressure (5 MPa). Subsamples were taken at different time points during the incubation and analyzed by ion chromatography. Furthermore we demonstrated the applicability of the system for studies of microbial activity, using samples from the Isis mud volcano. We could detect an increase in sulfate reduction rate upon the addition of methane to the sample

From exploration to remediation: A microbial perspective for innovation in mining

As society transitions to low-carbon, renewable energy resources, the demand for metals and minerals is set to increase. Massive quantities of base metals and mineral materials (for example, silica and concrete) along with smaller quantities of precious metals will be required for the construction of wind turbines, solar panels and battery storage facilities to meet the demands of the ‘Electric Planet’ of the future. Harnessing microbe-mineral-metal interactions may offer many opportunities to improve some mining practises and support the long-term sustainability of mining. As easily exploitable, high-grade deposits are becoming increasingly depleted there is a need for new technologies to improve exploration and mining strategies. Microorganisms are ubiquitous and diverse, surviving in almost all environments in the Earth's crust and recent advances in molecular techniques have enabled scientists to study these communities is extraordinary detail. Microorganisms also interact directly with their environment; both responding to and changing the environment around them. These responses and their influences on the surrounding environment are preserved within their genome (a complete set of the DNA of the microorganism). Here, we discuss using state-of-the-art sequencing techniques to identify key microbial genes that have been demonstrated to correlate with metal concentrations. These genetic-based bioindicators may provide additional tools to guide and improve the success rate of mineral exploration programmes. Advances in molecular techniques will also improve existing biohydrometallurgical techniques and expand the commodity range for which biohydrometallurgy are currently economically viable. Finally, microorganisms may be used in a number of strategies for mine remediation; specifically, we review in detail microbially accelerated carbon capture and storage strategies and mine waste stabilisation

Global Distribution of Microbial Abundance and Biomass in Subseafloor Sediment

The global geographic distribution of subseafloor sedimentary microbes and the cause(s) of that distribution are largely unexplored. Here, we show that total microbial cell abundance in subseafloor sediment varies between sites by ca. five orders of magnitude. This variation is strongly correlated with mean sedimentation rate and distance from land. Based on these correlations, we estimate global subseafloor sedimentary microbial abundance to be 2.9⋅1029 cells [corresponding to 4.1 petagram (Pg) C and ∼0.6% of Earth’s total living biomass]. This estimate of subseafloor sedimentary microbial abundance is roughly equal to previous estimates of total microbial abundance in seawater and total microbial abundance in soil. It is much lower than previous estimates of subseafloor sedimentary microbial abundance. In consequence, we estimate Earth’s total number of microbes and total living biomass to be, respectively, 50–78% and 10–45% lower than previous estimates

Hydrogen Utilization Potential in Subsurface Sediments

Subsurface microbial communities undertake many terminal electron-accepting processes, often simultaneously. Using a tritium-based assay, we measured the potential hydrogen oxidation catalyzed by hydrogenase enzymes in several subsurface sedimentary environments (Lake Van, Barents Sea, Equatorial Pacific, and Gulf of Mexico) with different predominant electron-acceptors. Hydrogenases constitute a diverse family of enzymes expressed by microorganisms that utilize molecular hydrogen as a metabolic substrate, product, or intermediate. The assay reveals the potential for utilizing molecular hydrogen and allows qualitative detection of microbial activity irrespective of the predominant electron-accepting process. Because the method only requires samples frozen immediately after recovery, the assay can be used for identifying microbial activity in subsurface ecosystems without the need to preserve live material. We measured potential hydrogen oxidation rates in all samples from multiple depths at several sites that collectively span a wide range of environmental conditions and biogeochemical zones. Potential activity normalized to total cell abundance ranges over five orders of magnitude and varies, dependent upon the predominant terminal electron acceptor. Lowest per-cell potential rates characterize the zone of nitrate reduction and highest per-cell potential rates occur in the methanogenic zone. Possible reasons for this relationship to predominant electron acceptor include (i) increasing importance of fermentation in successively deeper biogeochemical zones and (ii) adaptation of H2ases to successively higher concentrations of H2 in successively deeper zones

High Microbial Diversity Despite Extremely Low Biomass in a Deep Karst Aquifer

Despite the importance of karst aquifers as a source of drinking water, little is known about the role of microorganisms in maintaining the quality of this water. One of the limitations in exploring the microbiology of these environments is access, which is usually limited to wells and surface springs. In this study, we compared the microbiology of the Madison karst aquifer sampled via the potentiometric lakes of Wind Cave with surface sampling wells and a spring. Our data indicated that only the Streeter Well (STR), which is drilled into the same hydrogeologic domain as the Wind Cave Lakes (WCL), allowed access to water with the same low biomass (1.56–9.25 × 103 cells mL-1). Filtration of ∼300 L of water from both of these sites through a 0.2 μm filter allowed the collection of sufficient cells for DNA extraction, PCR amplification of 16S rRNA gene sequences, and identification through pyrosequencing. The results indicated that bacteria (with limited archaea and no detectable eukaryotic organisms) dominated both water samples; however, there were significant taxonomic differences in the bacterial populations of the samples. The STR sample was dominated by a single phylotype within the Gammaproteobacteria (Order Acidithiobacillales), which dramatically reduced the overall diversity and species richness of the population. In WCL, despite less organic carbon, the bacterial population was significantly more diverse, including significant contributions from the Gammaproteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Planctomycetes, Fusobacter, and Omnitrophica phyla. Comparisons with similar oligotrophic environments suggest that karst aquifers have a greater species richness than comparable surface environs. These data also demonstrate that Wind Cave provides a unique opportunity to sample a deep, subterranean aquifer directly, and that the microbiology of such aquifers may be more complex than previously anticipated

Microbial methane cycling in sediments of Arctic thermokarst lagoons

Thermokarst lagoons represent the transition state from a freshwater lacustrine to a marine environment, and receive little attention regarding their role for greenhouse gas production and release in Arctic permafrost landscapes. We studied the fate of methane (CH4) in sediments of a thermokarst lagoon in comparison to two thermokarst lakes on the Bykovsky Peninsula in northeastern Siberia through the analysis of sediment CH4 concentrations and isotopic signature, methane-cycling microbial taxa, sediment geochemistry, lipid biomarkers, and network analysis. We assessed how differences in geochemistry between thermokarst lakes and thermokarst lagoons, caused by the infiltration of sulfate-rich marine water, altered the microbial methane-cycling community. Anaerobic sulfate-reducing ANME-2a/2b methanotrophs dominated the sulfate-rich sediments of the lagoon despite its known seasonal alternation between brackish and freshwater inflow and low sulfate concentrations compared to the usual marine ANME habitat. Non-competitive methylotrophic methanogens dominated the methanogenic community of the lakes and the lagoon, independent of differences in porewater chemistry and depth. This potentially contributed to the high CH4 concentrations observed in all sulfate-poor sediments. CH4 concentrations in the freshwater-influenced sediments averaged 1.34 ± 0.98 μmol g−1, with highly depleted δ13C-CH4 values ranging from −89‰ to −70‰. In contrast, the sulfate-affected upper 300 cm of the lagoon exhibited low average CH4 concentrations of 0.011 ± 0.005 μmol g−1 with comparatively enriched δ13C-CH4 values of −54‰ to −37‰ pointing to substantial methane oxidation. Our study shows that lagoon formation specifically supports methane oxidizers and methane oxidation through changes in pore water chemistry, especially sulfate, while methanogens are similar to lake conditions

Solute Reservoirs Reflect Variability of Early Diagenetic Processes in Temperate Brackish Surface Sediments

Coastal marine sediments are a hotspot of organic matter degradation. Mineralization products of early diagenetic processes accumulate in the pore waters of the sediment, are subject of biological uptake and secondary biogeochemical processes and are released back into the water column via advective and diffusive fluxes across the sediment-water interface. Seven representative sites in the shallow coastal area of the southern Baltic Sea (15–45 m water depth), ranging from permeable sands to fine grained muds, were investigated on a seasonal basis for their key mineralization processes as well as their solid phase and pore water composition to identify the drivers for the variability of early diagenetic processes in the different sediment types. The sandy sediments showed about one order of magnitude lower organic carbon contents compared to the muds, while oxygen uptake rates were similar in both sediment types. Significantly higher oxygen uptake rates were determined in two near-shore muddy sites than in a deeper coastal muddy basin, which is due to higher nutrient loads and the corresponding addition of fresh algal organic matter in the near-shore sites. Pore water concentration profiles in the studied sediments were usually characterized by a typical biogeochemical zonation with oxic, suboxic, and sulfidic zones. An up to 15 cm thick suboxic zone was sustained by downward transport of oxidized material in which dissolved iron and phosphate indicate an intensive reduction of reactive Fe with the release of adsorbed phosphorus. While the geochemical zonation was stable over time in the muds of the studied deeper basin, high variability was observed in the muds of a near-coastal bay probably mainly controlled by sediment mixing activities. The sediments can be characterized by essentially two factors based on their near-surface benthic solute reservoirs: (1) their organic matter mineralization and solute accumulation efficiency and (2) their redox-state. Benthic solute reservoirs in the pore waters of the top decimeter were generally higher in the muddy than in the sandy sediments as the more permeable sands were prone to an intensive exchange between pore water and bottom water. The three studied muddy sites showed great dissimilarities with respect to their predominating redox-sensitive metabolites (dissolved iron, manganese, and sulfide). Surface-near advective transport like irrigation of permeable sands and rearrangement of cohesive muds had a particularly strong influence on early diagenetic processes in the studied sediments and were probably the most important cause for the spatiotemporal variability of their benthic solute reservoirs

Microbial community composition and abundance after millennia of submarine permafrost warming

Warming of the Arctic led to an increase in permafrost temperatures by about 0.3 �C during the last decade. Permafrost warming is associated with increasing sediment water content, permeability, and diffusivity and could in the long term alter microbial community composition and abundance even before permafrost thaws. We studied the long-term effect (up to 2500 years) of submarine permafrost warming on microbial communities along an onshore–offshore transect on the Siberian Arctic Shelf displaying a natural temperature gradient of more than 10 �C. We analysed the in situ development of bacterial abundance and community composition through total cell counts (TCCs), quantitative PCR of bacterial gene abundance, and amplicon sequencing and correlated the microbial community data with temperature, pore water chemistry, and sediment physicochemical parameters. On timescales of centuries, permafrost warming coincided with an overall decreasing microbial abundance, whereas millennia after warming microbial abundance was similar to cold onshore permafrost. In addition, the dissolved organic carbon content of all cores was lowest in submarine permafrost after millennial-scale warming. Based on correlation analysis, TCC, unlike bacterial gene abundance, showed a significant rank-based negative correlation with increasing temperature, while bacterial gene copy numbers showed a strong negative correlation with salinity. Bacterial community composition correlated only weakly with temperature but strongly with the pore water stable isotopes �18O and �D, as well as with depth. The bacterial community showed substantial spatial variation and an overall dominance of Actinobacteria, Chloroflexi, Firmicutes, Gemmatimonadetes, and Proteobacteria, which are amongst the microbial taxa that were also found to be active in other frozen permafrost environments. We suggest that, millennia after permafrost warming by over 10 �C, microbial community composition and abundance show some indications for proliferation but mainly reflect the sedimentation history and paleoenvironment and not a direct effect through warming

Enabling large-scale hydrogen storage in porous media – the scientific challenges

Expectations for energy storage are high but large-scale underground hydrogen storage in porous media (UHSP) remains largely untested. This article identifies and discusses the scientific challenges of hydrogen storage in porous media for safe and efficient large-scale energy storage to enable a global hydrogen economy. To facilitate hydrogen supply on the scales required for a zero-carbon future, it must be stored in porous geological formations, such as saline aquifers and depleted hydrocarbon reservoirs. Large-scale UHSP offers the much-needed capacity to balance inter-seasonal discrepancies between demand and supply, decouple energy generation from demand and decarbonise heating and transport, supporting decarbonisation of the entire energy system. Despite the vast opportunity provided by UHSP, the maturity is considered low and as such UHSP is associated with several uncertainties and challenges. Here, the safety and economic impacts triggered by poorly understood key processes are identified, such as the formation of corrosive hydrogen sulfide gas, hydrogen loss due to the activity of microbes or permeability changes due to geochemical interactions impacting on the predictability of hydrogen flow through porous media. The wide range of scientific challenges facing UHSP are outlined to improve procedures and workflows for the hydrogen storage cycle, from site selection to storage site operation. Multidisciplinary research, including reservoir engineering, chemistry, geology and microbiology, more complex than required for CH4 or CO2 storage is required in order to implement the safe, efficient and much needed large-scale commercial deployment of UHSP.This work was stimulated by the GEO*8 Workshop on “Hydrogen Storage in Porous Media”, November 2019 at the GFZ in Potsdam (Germany). NH, AH, ET, KE, MW and SH are funded by the Engineering and Physical Sciences Research Council (EPSRC) funded research project “HyStorPor” (grant number EP/S027815/1). JA is funded by the Spanish MICINN (Juan de la Cierva fellowship-IJC2018-036074-I). JM is co-funded by EU INTERREG V project RES-TMO (Ref: 4726 / 6.3). COH acknowledges funding by the Federal Ministry of Education and Research (BMBF, Germany) in the context of project H2_ReacT (03G0870C).Peer reviewe