Viability and Adaptation Potential of Indigenous Microorganisms from

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Viability and Adaptation Potential of Indigenous Microorganisms from Natural Gas Field Fluids in High Pressure Incubations with Supercritical CO2 Janin Frerichs,† Jana Rakoczy, Christian Ostertag-Henning, and Martin Krüger* Federal Institute for Geosciences and Natural Resources (BGR), Stilleweg 2, 30655 Hannover, Lower Saxony, Germany S Supporting Information *

ABSTRACT: Carbon Capture and Storage (CCS) is currently under debate as large-scale solution to globally reduce emissions of the greenhouse gas CO2. Depleted gas or oil reservoirs and saline aquifers are considered as suitable reservoirs providing sufficient storage capacity. We investigated the influence of high CO2 concentrations on the indigenous bacterial population in the saline formation fluids of a natural gas field. Bacterial community changes were closely examined at elevated CO2 concentrations under near in situ pressures and temperatures. Conditions in the high pressure reactor systems simulated reservoir fluids i) close to the CO2 injection point, i.e. saturated with CO2, and ii) at the outer boundaries of the CO2 dissolution gradient. During the incubations with CO2, total cell numbers remained relatively stable, but no microbial sulfate reduction activity was detected. After CO2 release and subsequent transfer of the fluids, an actively sulfate-respiring community was re-established. The predominance of spore-forming Clostridiales provided evidence for the resilience of this taxon against the bactericidal effects of supercritical (sc)CO2. To ensure the long-term safety and injectivity, the viability of fermentative and sulfate-reducing bacteria has to be considered in the selection, design, and operation of CCS sites.

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mineralization activities of the organic carbon content.7,8 The influence of these microbial populations on the geochemical conditions of their environment could enhance the mineral trapping mechanism in the reservoir for the injected CO2.9 Kirk10 reported that acetate-dependent microbial iron(III) reduction would be thermodynamically favored under CCS conditions, but the available energy from respiration processes for sulfate-reducing bacteria and methanogenic archaea remained unaffected. Additionally, urea-hydrolyzing cultures of Sporosarcina pasteurii decreased the porosity of the sandstone core due to biofilm formation and precipitation of calcium− carbonate11,12 which was postulated as a potential mechanism to seal leakage pathways in the sandstone matrix. Instead of bioaugmentation of microorganisms and substrates,12 the indigenous microbial population presumably can be stimulated by mobilized organic compounds (e.g., acetate) dissolved from the sandstone matrix in direct contact with scCO2.6,13 Bactericidal effects of scCO2 have also been documented, e.g. the reduction of the germination indexes of fruit juices and meat products.14 Supercritical CO2 permeabilizes microbial cell membranes, decreases intracellular pH, inhibits protein synthesis,15 and thus leads to cell death.16 These effects were

INTRODUCTION The increasing atmospheric CO2 concentration during the last 150 years is related to the anthropogenic usage of fossil fuels as energy source.1 One currently discussed technique to reduce these emissions, Carbon Capture and Storage (CCS), suggests the separation of the produced CO2 and the subsequent injection into suitable storage reservoirs.2 Potential storage sites are deep saline aquifers and depleted gas or oil reservoirs that meet the criteria in storage capacity and cap rock integrity to prevent the upward-migration of stored CO2.3 Furthermore, only storage reservoirs below 0.8 km depth provide the physical conditions to store CO2 in its supercritical state to minimize the storage volume.3 After the injection, supercritical (sc)CO2 accumulates at the top boundary of the reservoir due to the density differences of the scCO2-phase and the formation fluids (physical trapping).4 With the dissolution of the CO2 (solubility trapping) in the formation fluids the pH will decrease5 leading to mineral dissolution and finally to the long-term mineralization of the CO2 as carbonate (mineral trapping).4−6 In consequence, these trapping mechanisms will lead to the formation of concentration gradients comparable with contaminant plumes in groundwater with the highest dissolved CO2 concentrations (saturation) at the injection point and the top boundary.5 The currently discussed storage sites, especially the hydrocarbon reservoirs, often represent hotspots within the terrestrial and marine deep biosphere with elevated population sizes and © 2013 American Chemical Society

Received: Revised: Accepted: Published: 1306

June 28, 2013 December 3, 2013 December 9, 2013 December 9, 2013 dx.doi.org/10.1021/es4027985 | Environ. Sci. Technol. 2014, 48, 1306−1314

Environmental Science & Technology

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Scheme 1. Experimental Design and Conditions of Saturation and Stimulation Experiment Including the CO2 Incubations, Control Setups, and the Reactivation Approacha

The sulfate reduction rates (SRR in nmol mL−1 day−1 (±SD n = 2−3) were calculated from significant time-dependent-correlation of sulfate concentrations (coefficient R2 >0.65)); (-) no significant time depended reduction of sulfate concentrations observed. Fluid samples used for bacterial community analysis were indicated in correspondence to the label used in Table 1; i.e. a-f for the saturation experiment and a-g for the stimulation experiment.

a

at the well head facility of the gas reservoir. For each experiment 10 L fluid were sampled sterile and immediately transported to the lab.20 To determine the cell density (CD), replicates of the formation fluids were fixed overnight in 2% (v/v) formaldehyde in PBS (130 mM NaCl, 5 mM Na2HPO4, 5 mM NaH2PO4) at 4 °C. The fixed material was sonicated (20 s at 20% amplitude in 2 cycles), filtered onto 0.22 μm black polycarbonate filters (GTBP, Millipore), and stained with SybrGreenI (Invitrogen; embedded with Moviol/glycerol22). At least 800 cells per replicate were counted and calculated as cells mL−1 (SD; n = 2−3). Samples for water chemistry were filtered (0.45 μm pore size) and stored at 4 °C. The concentrations of sulfate and major ions (Na+, K+, Ca2+, Cl−) were determined by ion chromatography with a DX-500 system (Dionex, Germany). Prior to this work the reservoir was well described.23 It is located in a geologic horizon feasible for CCS applications.24The microbial community was monitored in detail since 2006 (Frerichs unpublished data and Ehinger et al.20) Preparation of Fluid Samples. Two separate experiments were conducted with freshly sampled formation fluids. The experimental conditions represented the assumed environmental conditions in different compartments of the CO2 gradient that would evolve after the injection of scCO2 in the reservoir system:5,21 a) fully CO2 saturated fluid close to the injection point (saturation experiment) and b) a CO 2 undersaturated fluid distant from the injection point that got additionally stimulated with an organic carbon source and was supplemented with sulfate as an electron acceptor (stimulation experiment). For the saturation experiment (a) the formation fluid was immediately upon arrival subdivided into two setups (see Scheme 1): i) pressurized control (10 replicates of 10 mL each) and ii) one experimental batch for the incubation with scCO2 (80 mL). The pressurized controls without addition of CO2 were incubated using a hydrostatic pressure system.25 The pressurized control was sampled at a few selected time points (after 16 and 30 days) to minimize the negative effect of repeated decompression and pressurization.26 For DNA extraction, determination of cell density, sulfate concentration, and pH measurement, two to three replicates were withdrawn from the system at each of the selected time points. A detailed description of the hydrostatic pressure system used for the control incubation is given in the Supporting Information. A

shown to act stronger on planktonic cells than on sessile biofilms,17 and spore-forming microorganisms seem to tolerate the CO2 stress due to their gram-positive cell wall structure.18 Besides the latter studies, which were conducted using facultative anaerobic microorganisms typically present in soil or groundwater,17,18 the effect of scCO2 has been mainly studied for food-borne pathogens. Therefore the knowledge about the effects of scCO2 on an indigenous reservoir community is still scarce.19 Particularly, the adaptation potential of a complex environmental microbial community stressed with scCO2 is not known. Consequently, this study investigated the viability potential of indigenous microorganisms in saline formation fluids of a natural gas field (North German Plain, Germany) during and after simulated injections of scCO2. Prior to this study, the selected gas field formation, i.e. the water chemistry, the microbial activity, and the reservoir community, were monitored over almost 6 years (Frerichs unpublished data and Ehinger et al.20). The original production fluids contained about 984 ± 32 mg L−1 sulfate (∼10 mM), 2200 mg L−1 HCO3−, considerable amounts of dissolved organic material (acetate, alcohols, etc.), and about 28 g L−1 total dissolved solids (TDS − with 0.39 M NaCl dissolved).20 The microbial biosphere in the formation fluids consisted mainly of methylotrophic archaea20 and a diversity of sulfur-utilizing and fermenting bacterial species, i.e. Desulfovibrio, Desulfomonadales, Desulfotomaculum, Thermoanaerobacter, and Petrotoga (Frerichs unpublished data). Especially sulfate-reducing bacteria were active, using a variety of organic substrates and showing considerable sulfate-reducing activity at the estimated reservoir temperature of about 80 °C. Our experiments simulated the dissolution process of CO2 in the formations fluid phase and mimicked the environmental conditions in different compartments of the CO 2 gradient. 5,21 The identification of resistant or sensitive bacterial taxa from the indigenous community and their viability potential after release of the scCO2 allows us to understand the adaptation potential of the reservoir biosphere and its potential consequences for the application of CCS.

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METHODS General Processing of Formation Fluids and Microbiological Characteristics of the Formation. Produced fluid was sampled from the surface gas/water-separation system 1307

dx.doi.org/10.1021/es4027985 | Environ. Sci. Technol. 2014, 48, 1306−1314


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coefficient were considered as microbial respiration process (significance level R2 >0.65). The geochemical modeling software PHREEQCi Version 2.1829 was used to calculate saturation indices, species distributions, and activities and to estimate the pH in situ for the reservoir and in vitro within the reactor systems. Reactivation Approach. The activity of surviving microorganisms and possible viability of the bacterial sulfate-reducing community was assessed using transfer microcosms inoculated with fluid at selected time points (see Scheme 1). All transfers were prepared anaerobically using sterilized formation fluid (0.1 μm filtered) with 10 Vol% final inoculum. The filtered media were effectively sterilized as no cells, microbial activity, or extractable DNA were observed/detected (data not shown). Fluid samples of the saturation experiment were transferred in duplicates after 16 and 30 days of incubation with scCO2. Transfers were supplemented with sodium sulfate (final 20 mM) and covered with a hydrogen gas phase (H2/CO2 80/20%). Microcosms of original fluid were directly prepared after the sampling approach to assess the initial microbial sulfate reduction activity. These original fluid microcosms were also stimulated with hydrogen (triplicates) to verify and quantify the magnitude of the sulfate reduction activity in the reactivation approach after the saturation experiment. All reactor transfers and control microcosms were incubated at 60 °C for 40 days (ambient pressure) and then prepared for DNA extraction by pooling the replicate incubations. For the stimulation experiment the final time point after releasing CO2 and pressure (after 40 days) was transferred and supplemented with TMA and sodium sulfate (5 and 20 mM final, respectively). All transfers were prepared anaerobically in triplicates from the control setups (both pressurized and unpressurized) and the high pressure CO2 system. The microcosms were incubated at 50 °C for 60 days (ambient pressure) and then prepared for DNA extraction to compare the reestablished bacterial community in the reactivation approach. During the reactivation approach microbial sulfate reduction activity was followed densitometrically by measuring the precipitation of copper sulfide from solved sulfide ions (HS−) in the media.30 Rates were calculated as nmol mL−1 day−1 (SD of n = 2−3 microcosms; R2 > 0.65). Methane accumulation in the headspace was monitored weekly (gas chromatographically25), but no methane formation was detected at any time point (data not shown). Molecular Biological Analyses. For DNA extractions fluid samples of the original formation fluid (200−300 mL), of several time points during the incubation (about 1−3 mL sample), and from the pooled transfer microcosms (40−60 mL) were filtered onto a 0.22 μm polycarbonate filter (GTTP, Millipore) and stored at −20 °C until further processing. The filter-retarded cells were lysed in phosphate buffer (100 mM; pH 8.5) using bead-mill treatment (FastPrep instrument; MP Biomedicals 10−20 s), followed by an enzymatic lysis (Lysozym [50 mg/L]; ProteinaseK [10 mg/ L]; 15 min at 37 °C). DNA was purified from the aqueous phase using a phenol-chloroform-isoamyl solution (Roth PCI; 24:23:1). The extract was washed twice with chloroformisoamyl alcohol (Roth; 24:1) to remove the phenol residues. DNA was precipitated with 0.3 M sodium acetate and 2propanol (−20 °C for about 12 h). The salted DNA was dissolved in 100 μL of ultrapure PCR water (Fluka) and stored at −20 °C.

description of the system used for high pressure CO2 incubations is given in the paragraph below. For the stimulation experiment (b) the production fluid was preincubated for two weeks prior to the experiment to enhance microbial growth rates and sulfate reduction activity. For this preincubation the fluid was filled anaerobically into heat sterilized glass bottles, sealed with butyl stoppers, and repeatedly flushed with N2 to remove residual O2. Microcosms were amended with sodium sulfate (final concentration 20 mM20) and trimethylamine (TMA; 5 mM) and incubated for 14 days at 50 °C. The TMA induced a strong sulfate reduction activity during the preincubations monitored by the produced sulfide (data not shown), further, TMA provided a slight buffering capacity feasible for the acidification caused by the increased CO2. Before starting the experiment, the preincubated batch culture was subdivided anaerobically into the following setups (see Scheme 1): i) nonpressurized control (3 replicates of 30 mL each), ii) pressurized control (10 replicates of 10 mL each), and iii) one experimental batch for the incubation with scCO2 (100 mL) (see paragraph below). Sampling of the pressurized control was again restricted to selected time points (sampling after 9, 30, 37, and 40 days; see above). For both experiments the formation fluid (i.e., preincubated, stimulated culture) was subsampled for DNA extraction (community analysis), measurements of cell density (cell mL−1), sulfate concentration, and pH at the beginning of the experiment. Installation and Sampling of High Pressure CO2 Incubation Systems. All high pressure CO2 incubations were conducted in a cylindrically shaped gold bag of about 100 mL volume closed with a titanium cap.27 Prior to use, the assemblage was heat sterilized to prevent contamination and to oxidize the titanium surface (5 h at 400 °C). The gold bag was filled under anaerobic conditions with the fluid and sealed with the titanium cap. The closed assembly was placed into a pressure vessel (Parr Instrument), which was filled with demineralized water and heated to 50−60 °C. After reaching temperature equilibrium, the system was pressurized using a syringe pump (Teledyne ISCO, Inc. US) holding the pressure continuously at 100 bar. In a second pump, gaseous CO2 was pressurized to 110 bar to reach supercritical state before dispersing it into the fluid within the gold bag. The CO2 solubility in the formation brine at the given temperature, pressure, and fluid’s salinity20 was calculated using the online available NaCl-Saline-CO2 algorithm.28 Temperature and pressure were constantly recorded showing less than 0.1% variance over both experiments (data not shown). Samples were extracted via sampling ports directly from the fluid phase within the gold bag without pressure loss or temperature changes. At each sampling point the pH was measured at ambient pressure and room temperature (VWR, pH100). Samples for cell density determination and community analysis were extracted under sterile conditions (sample volume between 1 and 3 mL per time point). To minimize the sample volume, a dilution was prepared for the sulfate measurement using 1% HNO3. Dilution errors were excluded as sulfate concentrations were normalized against sodium concentration in the undiluted, original fluid sample. Microbial sulfate reduction was calculated from the decrease of sulfate over time and given in nmol mL−1 day−1. The rate was evaluated using the time-dependent linear regression of sulfate concentration, and only rates with a sufficient correlation 1308



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Figure 1. Development of microbial and chemical parameters of scCO2 reactor and pressurized control during the saturation (left panel) and stimulation experiment (right panel). Top: pH values measured under ambient conditions; center: sulfate concentration normalized with the sodium concentration of the original formation fluid; bottom: Cell density (CD) and dsrA gene copies. Arrow heads indicate the first sample taken after CO2 injection and after releasing pressure and CO2 at the end of the experiment (about 45 min after the last sample under pressure was taken). Error bars represent the standard deviation.

dissolved CO2. Sulfate concentrations were stable over time and decreased only in the pressurized control without CO2 (Figure 1). During the stimulation experiment the dissolved CO2 concentration was about 7-fold increased (0.35 mol CO2 per kg formation fluid). The pH decreased to 6.3 within the first 6 h after CO2 injection (Figure 1) and reached equilibrium at 5.9 ± 0.1 after 24 h (average pH from day 7−14). The dissolution of CO2 within the vessel was slightly buffered by the TMA (pH 4.3), without TMA addition the pH would have decreased to 4.129 under the applied conditions. The sulfate concentration of the high pressure CO2 incubation was stable over time, while the pressurized control showed a strong decrease during the stimulation experiment (Figure 1). Under the in situ reservoir conditions (80 °C, 200 bar, 0.39 M NaCl) saturation would be reached at 1.04 mol CO2 per kg formation fluid.28 The modeled pH under in situ conditions (pH 3.2) is similar to the value reached in the saturation experiment. However, this estimated pH is much lower than previously reported values for other reservoir formations with scCO2 injection.6,35 Kihm et al.5 presented a model showing that only in close proximity of the injection point the fluid will be saturated with CO2, thus generating highly acidic conditions only around the injection well. Therefore, the CO2 concentrations in our experiments mimicked the assumed conditions in the reservoir: i) in direct proximity to the interface of brine and scCO2 phase and ii) in greater distance from the injection well following the diffusion gradient of the CO2. For natural reservoir systems a considerable buffering capacity is likely, including mineral dissolution processes4 and the dissolution of organic com-

Since sulfate reduction was the dominant microbial process, sulfate-reducing prokaryotes were quantified by targeting the alpha subunit of the dissimilatory sulfite reductase (dsrA) in a SybrGreen-based quantitative PCR (qPCR) approach31 using the primers DSR1F+/DSR-R.32 The specifically amplified dsrA gene of Desulfobacterium autotrophicum DSM 3382 was used as internal standard, and each run was finalized with a melting curve to check for unspecific byproducts or primer dimers. For terminal restriction fragment length polymorphism (TRFLP) bacterial 16S rDNA amplicons were prepared with Ba27f 5’-flourochrom/907r using described PCR protocols.33 Fluorochrome marked amplicons were not obtained from day 16 of the saturation experiment due to poor DNA recovery (data not shown). The resulting T-RFLP electropherograms of each fluid sample were analyzed using the t-REX software for denoizing the data sets and aligning the peaks into defined terminal restriction fragments (T-RFs).34 A more detailed description of quality controls for qPCR runs and the 16SrRNA based analysis of the bacterial community are given in the Supporting Information.

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RESULTS AND DISCUSSION CO2 Concentration and pH Values during High Pressure Incubations. In the saturation experiment the CO2 concentration was 0.95 mol CO2 per kg formation fluid (4.4 g kg−1 brine), representing a 19-fold increase of the dissolved carbonate concentration. The dissolution of CO2 decreased the pH to 5.4 (Figure 1). However, this value was measured outside the reactor under ambient conditions, while the pH inside the pressure vessel would be lower (calculated pH 3.2, using PHREEQCi29) due to the large amount of 1309



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pounds6,13 (here mimicked by the supplemented TMA). Thus, the acidification will be further limited in natural reservoir systems. Effects of Hydrostatic Pressure and scCO2 on Microbial Sulfate Reduction. During the incubation under CO2saturated conditions no significant time-dependent decrease of the sulfate concentration was found (R2 95% the closest uncultivated relative is given; (−) not detected in the sample. 2 Environmental conditions of the habitat, here temperature optimum and isolation source, were retrieved if possible from the NCBI GenBank entry or the taxonomic description. 3Transfer microcosms of the reactivation approach were supplemented with hydrogen for the saturation experiment (d−f) and TMA for stimulation experiment (e−g).

effect of scCO2,16 the reactivation of the sulfate reduction activity even after 30 days under CO2 saturated conditions was very surprising. The low microbial activity of the pressurized control transfer indicated an inhibitory effect of the repeated depressurization26 or a general substrate limitation for the microbial population. For the stimulation experiment the sulfate reduction rates of the reactivation approach showed only minor differences (Scheme 1, the produced sulfide over time is given in Figure S4), e.g. about 146 ± 5.8 and 155 ± 19 nmol sulfate mL−1 day−1 was reduced in the scCO2 and pressurized control transfer, respectively. This leads to the conclusion that a considerable proportion of microbial sulfate reducers tolerated the high CO2 concentrations in the stimulation experiment without significant bactericidal effects even after 40 days. Considering the CO2 diffusion gradient under realistic CCS conditions, the buffering capacity of the potential storage reservoir,5 and the possibly mobilized organic material6,13 the indigenous bacterial community seemed to be much more resistant toward CO2 stress than previously postulated for Bacteria.15,16,18,37 A significant part of the microbial population will survive especially in the outer areas of the diffusion plume. Consequently, in terms of CCS applications, microbiological parameters have to be closely evaluated to verify the proposed influence of the microbial population on the reservoir capacity.9,11

Microbial Community Changes during and after the Saturation Experiment. The following community analysis assessed the development during and after the incubation with scCO2 under saturated conditions. The identification of the OTUs, detected in the T-RFLP analysis, revealed microorganisms able to survive the CO2 stress, as indicated by the cell density and sulfate reduction activity of the reactivation approach. For better visualization the relative T-RF abundance profile and a statistic comparison of the abundance matrix were included in the Supporting Information (Figures S1 and S2). The community structure of the saturation experiment showed only minor variations between the pressurized control and scCO2 community (after 30 days of incubation). However, both incubations showed a relatively pronounced shift in comparison to the “original fluid” community (Figure S1). This became even more apparent in the comparison of the community structure in the reactivation approach with the hydrogen supplemented original fluid (Figures S1 and S2). These analyses revealed a relatively high similarity for the community of the pressurized control and scCO2 reactor, indicating that the detected organisms were able to withstand a variety of stress factors (e.g., substrate limitation, temperature variation, repeated decompression, etc.). In detail (Table 1), the community structure of the original fluid was dominated by Gammaproteobacteria, uncultered Peptococcaceae 2, and uncultured Bacteriodetes (T-RFs 490, 1311



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enriched which affiliated with Desulfobotulus sp. and Thermoanaerobacter sp. (T-RF 132 and T-RF 138, respectively; Table 1). Like the saturation experiment, Desulfotomaculum spp. (TRFs 209 and 230/231) and the uncultured Peptococcaceae 2 were relatively unaffected from the CO2. Furthermore, Petrotoga halophila was decreased, as has been observed for the community of the saturation experiment. In contrast to the saturation experiment, the number of detected T-RFs increased during the reactivation approach. In the scCO2 transfers several organisms were further enriched or reappeared, e.g. Thermovirga lienii (T-RF 64) and Geotoga petraea. Furthermore, both putatively spore-forming Desulfotomaculum spp. were increased. Generally, almost all identified organisms listed in Table 1 were affiliated with taxa documented from high temperature environments and likely represent indigenous members of the subsurface biosphere. 7,8,38,39 In total the comparison of community composition of the scCO2 transfers with the control setups showed high similarities with a few expectations, like Petrotoga halophila which was reduced in the scCO2 transfer. In all setups the proportion of thermophilic and putatively spore-forming Clostridales was high, e.g. the sulfate-reducing genera Desulfotomaculum. The predominance of several Clostridiales in the scCO2 transfer indicated a higher viability potential for this part of the starting community during the prolonged application of CO2 of the stimulation experiment. Both the saturation and the stimulation experiment revealed a pronounced dominance of putatively spore-forming Clostridiales. These organisms presumably possess the highest tolerance under reservoir conditions for the increasing CO2 concentrations and its environmental and physiological effects. Previously, it was shown that spores of Firmicutes (including Bacilli species) exhibit a higher resilience for scCO2.16 The thick spore coat and cortex structure were shown to considerably slow down the diffusion even of small molecules, and the low water content and the already acidic spore protoplasm could mitigate the intracellular acidification caused by CO2.40 However, the resistance of spores was mainly evaluated using pure cultures (and only for food-borne pathogens) showing that sterilization was only effective in combination with temperature increase.14,16,37 Since the surviving organisms were predominantly associated with thermophilic taxa, their resistance even under increased temperature is likely. Furthermore, also the vegetative cells of spore-forming Firmicutes were shown to have a higher resilience potential for high CO2 concentrations.18 The gram-positive cell wall might reduce the diffusion of CO2 into the membrane and cytoplasm, thus preventing the disturbance of the membrane integrity and intercellular pH.37 In consequence, the selection of these organisms by the effects of scCO2 will influence the distribution of microorganisms and microbial activity in the potential CO2 storage reservoir structures, which could possibly even affect the storage capacity.9 The viability of sulfate reducers was previously shown by Morazova et al.19 albeit no sulfate reduction activity was documented and the identity and origin of these organisms remained unclear. Microorganisms affect the carbonate concentration and pH of their environment thus presumably also affecting CCS, e.g. via increasing carbonate mineralization rates securing the storage of CO 2. 9,17 Also, microbially induced mineral precipitates could seal leakage pathways as shown previously with a Sporosarcina sp. pure culture.12 On the other hand the abundance of sulfate-reducing organisms could represent a

204, and 156/157, respectively). Next to these, Petrotoga halophila and Desulfotomaculum sp. were abundant in the formation fluid (T-RFs 264 and 209, respectively). Overall, the bacterial community composition was highly similar to the previously reported descriptions of the formation fluids.20 After 30 days of CO2 saturation (Table 1), the community showed a relative increase of T-RFs identified as putative sporeforming bacteria, e.g. two Desulfotomaculum species (T-RF 209 and 230/231). Also a Pseudomonas sp. (T-RF 488) was relatively increased. Slightly increased in the scCO2 reactor were Thermoanaerobacter spp. (T-RF 138) and Geotoga subterranean (T-RF 472). An organism relatively inhibited under CO2 saturation was identified as Anaerobaculum sp. (TRF 279/280). Furthermore, Petrotoga halophila and Geotoga petraea (T-RF 164/165) were decreased in comparison to the original fluid. The development of the microbial community in the pressurized control was similar to the scCO2 incubation. However, Thermoanaerobacter sp., Anaerobaculum sp., and Geotoga subterranea were increased compared to the CO2 saturated incubation and the original fluid (Table 1). The H2-amended original fluid showed several T-RFs that were not detected in the other setups (Figure S2). For the scCO2 transfer microcosms two T-RFs were predominantly enriched. These organisms represented together more than 93% of the community. Both fragments were identified as thermophilic spore-forming Clostridiales,38,39 i.e. Desulfotomaculum kucznetsovii (T-RF 209) and an uncultured Peptococcaceae 2 (T-RF 204). In comparison, the pressurized control transfer showed the enrichment of the T-RF of Petrotoga halophila (Table 1). The organism was already inhibited during the high pressure incubation in the CO2-saturated system and did not recover in the transfers of scCO2 reactor fluids. In summary, the number of detected T-RFs was decreased during the reactivation approach (Figure S2). Like our other evaluations these results implied the reminiscence of several organisms after the saturation experiment but not necessarily their survival. For example, Petrotoga halophila and Anaerobaculum spp. could be interpreted as negatively affected by the high CO2 concentrations. These organisms were increased in the pressurized control but relatively decreased after CO2 stress. On the other hand, the increased abundance of thermophile spore-forming Clostridiales was predominant for the scCO2 incubation. In general, the Clostridiales revealed a surprisingly high capability to withstand severe environmental changes, which needs closer examination of this widespread and abundant microbial group8 for CCS applications in deep geological reservoirs. Development of the Stimulated Microbial Community under CO2 Constraints. In contrast to the saturation experiment, the stimulation experiment used a sulfate-reducing enrichment culture obtained from the formation fluids. Thus, the community of the starting point was already changed due to the stimulation phase prior to the experiment (Figure S1). However, the stimulated organisms were already present in the original fluid, albeit at lower abundance levels confirming that these microorganisms represent indigenous habitants of the reservoir (Figure S3). The different setups of the stimulation experiment showed only minor shifts in the overall community composition (Figures S1 and S3). The statistic comparison (Figure S1) indicated a higher similarity of the scCO2 reactor to the pressurized than to the unpressurized control. In detail, during the high pressure incubation with CO2 organisms were 1312



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severe risk for the long-term safety of the reservoir. The produced HS− is highly corrosive,41 and its precipitates decrease the injectivity as described by Morazova et al.19 In conclusion, this study revealed for the first time a pronounced persistence of spore-forming Clostridiales originating from the formation fluids of a potential storage site. Our results highlight the importance of the indigenous bacterial community of the selected storage sites in the final consideration of the CCS applications. The survival of Clostridiales provides first insights into the mechanisms acting on the microbial population with i) bactericidal effects for vegetative cells (but possibly not spores) close to the injection point and ii) bacteriostatic effects within the CO2 enriched diffusion plume with lower CO2 concentrations.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details of molecular biological analysis, results of IPCA analysis, experimental design of hydrostatic pressure incubations, and Figures S1−S4. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 0511 6433102. E-mail: [email protected]. Present Address †

Molecular Microbiology & Bioenergetics, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval of the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The present work was conducted within the national framework of the BMBF-GEOTECHNOLOGIEN program in the projects RECOBIO-2 (FKZ-03G0697A) and CO2BioPerm (FKZ-03G0782A). The authors would also like to thank Daniela Zoch, Holger Probst, Annette Wurtmann (all BGR), Giovanni Pilloni, Frederick von Netzer, and especially Katrin Hörmann and Dr. Tillmann Lüders (all IGÖ Munich).

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ABBREVIATIONS CCS Carbon Capture and Storage T-RFLP terminal restriction fragment length polymorphism T-RF terminal restriction fragment qPCR quantitative PCR dsrA gene for the dissimilatory sulfite reductase subunit A SRR sulfate reduction rate species sp. (pl. spp.)

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REFERENCES

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