Functional microbial diversity explains groundwater chemistry in a pristine aquifer
© Flynn et al.; licensee BioMed Central Ltd. 2013
Received: 4 December 2012
Accepted: 3 June 2013
Published: 24 June 2013
The diverse microbial populations that inhabit pristine aquifers are known to catalyze critical in situ biogeochemical reactions, yet little is known about how the structure and diversity of this subsurface community correlates with and impacts upon groundwater chemistry. Herein we examine 8,786 bacterial and 8,166 archaeal 16S rRNA gene sequences from an array of monitoring wells in the Mahomet aquifer of east-central Illinois. Using multivariate statistical analyses we provide a comparative analysis of the relationship between groundwater chemistry and the microbial communities attached to aquifer sediment along with those suspended in groundwater.
Statistical analyses of 16S rRNA gene sequences showed a clear distinction between attached and suspended communities; with iron-reducing bacteria far more abundant in attached samples than suspended, while archaeal clones related to groups associated with anaerobic methane oxidation and deep subsurface gold mines (ANME-2D and SAGMEG-1, respectively) distinguished the suspended community from the attached. Within the attached bacterial community, cloned sequences most closely related to the sulfate-reducing Desulfobacter and Desulfobulbus genera represented 20% of the bacterial community in wells where the concentration of sulfate in groundwater was high (> 0.2 mM), compared to only 3% in wells with less sulfate. Sequences related to the genus Geobacter, a genus containing ferric-iron reducers, were of nearly equal abundance (15%) to the sulfate reducers under high sulfate conditions, however their relative abundance increased to 34% when sulfate concentrations were < 0.03 mM. Also, in areas where sulfate concentrations were <0.03 mM, archaeal 16S rRNA gene sequences similar to those found in methanogens such as Methanosarcina and Methanosaeta comprised 73–80% of the community, and dissolved CH4 ranged between 220 and 1240 μM in these groundwaters. In contrast, methanogens (and their product, CH4) were nearly absent in samples collected from groundwater samples with > 0.2 mM sulfate. In the suspended fraction of wells where the concentration of sulfate was between 0.03 and 0.2 mM, the archaeal community was dominated by sequences most closely related to the ANME-2D, a group of archaea known for anaerobically oxidizing methane. Based on available energy (∆GA) estimations, results varied little for both sulfate reduction and methanogenesis throughout all wells studied, but could favor anaerobic oxidation of methane (AOM) in wells containing minimal sulfate and dihydrogen, suggesting AOM coupled with H2-oxidizing organisms such as sulfate or iron reducers could be an important pathway occurring in the Mahomet aquifer.
Overall, the results show several distinct factors control the composition of microbial communities in the Mahomet aquifer. Bacteria that respire insoluble substrates such as iron oxides, i.e. Geobacter, comprise a greater abundance of the attached community than the suspended regardless of groundwater chemistry. Differences in community structure driven by the concentration of sulfate point to a clear link between the availability of substrate and the abundance of certain functional groups, particularly iron reducers, sulfate reducers, methanogens, and methanotrophs. Integrating both geochemical and microbiological observations suggest that the relationships between these functional groups could be driven in part by mutualism, especially between ferric-iron and sulfate reducers.
Microbial life thrives in natural waters, including those found deep in the terrestrial subsurface . Groundwater there may contain little or no dissolved oxygen, and in such cases microbial activity is dominated by populations that can respire using other electron acceptors such as ferric iron, sulfate, or carbon dioxide. By catalyzing a diverse array of oxidation and reduction reactions, microorganisms (i.e., bacteria and archaea) exert strong influence over the chemistry of groundwater , thereby controlling the rates of mineral weathering in aquifers , the fate and transport of metals and organic compounds , and the geological sequestration of greenhouse gases . Knowledge of the activity and composition of groundwater microbial communities across different spatial scales is therefore critical to the understanding of subsurface biogeochemistry.
Rather than being segregated into distinct zones where a single functional group predominates, molecular analyses commonly show diverse microbial populations coexisting in aquifers, regardless of how the bulk groundwater is classified by geochemical criteria. For example, molecular studies in an aquifer near Cerro Negro (New Mexico, U.S.) have demonstrated the presence of sulfate-reducing, iron-reducing, and denitrifying bacteria in groundwater systems where geochemical indicators point to sulfate reduction alone as the predominant form of respiration [6–9].
Currently there is limited knowledge of how microbial diversity relates to biogeochemical processes on an ecosystem scale . Studies of microbial ecology in aquifers are frequently confined to specific taxa of interest, such as groups known to degrade a particular contaminant or to comparisons of pristine and contaminated areas [4, 11]. Furthermore, most molecular characterizations of aquifer ecosystems have focused on microbiota suspended in pumped groundwater, which at least partially ignores the microbial fraction attached to sediment particles [12, 13]. While it is known that attached populations constitute the majority of cells in the subsurface and there are physiological differences between attached and suspended microbial communities, few studies have examined differences between these two fractions [14, 15]. One such difference associated with a specific group involves the iron-reducing bacteria, which are usually associated with a solid substrate  and therefore are expected to be underrepresented in the bulk groundwater.
The Mahomet aquifer in east-central Illinois hosts distinct zones of high and low sulfate groundwater . This aquifer contains a diverse community of iron-reducing and sulfate-reducing bacteria in which sulfate has been proposed as a key discriminant of bacterial community structure . Specifically, in high sulfate wells, sulfate reducers have been shown to co-exist with iron reducers throughout the aquifer , contrary to previous notions that sulfate reduction is excluded under iron-reducing conditions [19–21]. Previous studies focused exclusively on bacterial populations, leaving the distribution of archaeal populations such as methanogens unexplored. Dissolved methane exists at significant concentrations in this aquifer and isotopic studies indicate that it is of microbial origin , suggesting methanogenesis has occurred in the Mahomet aquifer alongside iron reduction and sulfate reduction. In this study, we examined in greater detail than previous studies the community composition of bacteria and archaea in both groundwater and Mahomet aquifer sediment using partial-length 16S rRNA gene sequence analysis. Furthermore, we calculate the available thermodynamic energy for microbial respiration and compare this available energy with the distribution of phylotypes with which a particular mode of respiration is associated.
Before filtering suspended cells from groundwater or deploying in situ “traps” of sterilized sediment to sample attached microbes, stagnant water was pumped out of the well at a rate of 8 L min-1 using a Grundfos® Redi-Flo II electric submersible pump. During pumping, the pH, temperature and electrical conductivity were monitored using an Oakton pH/CON 300 Meter (Oakton Instruments, Vernon Hills, IL) and recorded at three minute intervals. No samples were taken until readings for all three parameters stabilized for three consecutive measurements. All groundwater samples for geochemical analyses were filtered in the field using a 0.2 μm pore size Supor-200® polyethersulfone membrane (Pall Life Sciences). For analysis of dissolved inorganic carbon (DIC), 3 mL of groundwater was collected using a degassed syringe, then injected into a stoppered 70 mL serum bottle previously purged using O2-free, ultrapure N2 gas and 2 g of crystalline phosphoric acid (H3PO4). Samples for dissolved organic carbon (DOC) analyses were stored in amber glass bottles and preserved using sulfuric acid (0.5% v/v). Samples were stored on water ice in a sealed cooler for transport to the lab and kept refrigerated until they could be analyzed.
Microbial cells suspended in groundwater were filtered from two liters of groundwater using a 90 mm Supor-200® filtration membrane. After collecting duplicate filters and immediately freezing them on dry-ice, an in situ sediment trap containing sterilized aquifer solids (see Additional file 1) was lowered into each well where it could be colonized by native microbes . Sediment traps were lowered to the depth of the screened interval of each well and retrieved after 98 to 137 days of incubation, allowing active microbial populations to colonize the initially-sterile solids . Upon retrieval, sediment samples were immediately placed into separate sterile Whirl-Pak® bags and stored in coolers filled with dry ice. All microbiological samples (filters and sediments) were transported to the laboratory within four hours whereupon they were transferred to a -80°C freezer and stored awaiting further analysis.
Aqueous concentrations of methane and hydrogen in groundwater were determined using passive diffusion sampling . In situ gas samplers were equilibrated in an individual well for at least one week and then retrieved. Triplicate samples of dissolved gases were immediately injected into stoppered, N2-purged serum bottles for storage. The concentrations of major anions (F–, Cl–, Br–, NO3–, PO43–, SO42–) in groundwater samples were measured using a Metrohm Advanced ion chromatograph with a detection limit of 10 μM (Metrohm USA, Houston, TX). DOC analyses were performed at the Illinois Sustainable Technology Center using a Shimadzu TOC-VCPN carbon analyzer with a detection limit of 0.4 mg kg–1.
Methane and DIC concentrations were measured using an SRI 8610 gas chromatograph (SRI International, Menlo Park, CA) coupled to a thermal conductivity detector (TCD) and a flame ionization detector (FID). TCD measurements were used to determine DIC and dissolved methane concentrations greater than >100 μM, while the FID was used to measure methane <100 μM. Hydrogen concentrations were determined using the same GC equipped with a reducing gas detector (RGD). The RGD detector produced reliable concentration measurements down to 0.5 nM. Gas phase concentrations of CO2, methane and hydrogen within the passive diffusion samplers were converted to aqueous phase concentrations using the temperature-corrected Ostwald coefficient , taking into account the total dissolved gas pressure in the system as measured using a Hydrolab MiniSonde 4a® (Hach Hydromet, Loveland, CO).
Energy available for microbial respiration
Where ∆G°T is the standard state free energy change at temperature T (K), R is the universal gas constant, and y i , m i , and v i are the activity coefficients, molal concentrations, and reaction coefficients of the species involved in the redox reaction. The ∆GA for a particular functional group of microbes is equal to the amount of free energy released by that group’s respiratory reaction (∆Gr). The amount ∆Gr is equal to the sum of the electron donating (∆Gdon) and electron accepting (∆Gacc) half-reactions, and the available energy (∆GA) is therefore the inverse of the energy released (-∆Gr) . The energy available from electron donating and accepting half-reactions was calculated in The Geochemist’s Workbench® using the “thermo.dat” database of thermodynamic data compiled by Lawrence Livermore National Laboratory . Activity coefficients (y i ) were calculated from the overall chemical composition of the groundwater using the extended Debye-Hückel equation .
Molecular assays and sequence analyses
Total DNA was extracted from each sediment trap and each filter membrane collected from the wells following the method of Tsai and Olson  with some minor modifications (see Additional file 1). DNA extracts were used to amplify 16S rRNA genes using bacterial (i.e., 8 F and 787R) and archaeal (i.e., 25 F and 958R)-specific primers (see Additional file 1). Amplification products were cloned into pCR4.1 TOPO TA vector following the manufacturer’s instructions (Invitrogen™, Carlsbad, CA). Clones were sequenced using the BigDye® Terminator sequencing chemistry (Applied Biosystems, Foster City, CA) as described elsewhere . A minimum of 192 clones per sample were processed in this study. Raw sequence data was checked for quality and assembled into contigs using Sequencher® v4.10.1 (Gene Codes Corp, Ann Arbor, MI), and then screened for chimeras using Bellerophon . For the phylogenetic analyses bacterial and archaeal sequences were aligned using the algorithm implemented in the program Mothur  against a high-quality reference alignment selected from the Greengenes 16S rRNA gene database . Unique, chimera-free reference sequences were chosen from the 12 October 2010 release of Greengenes using ARB . Cloned sequences from the Mahomet that aligned poorly to the reference database or contained ambiguous base calls were discarded. The phylogeny of archaeal and bacterial 16S rRNA gene sequences was classified in Mothur using the “Hugenholtz” taxonomic nomenclature in Greengenes . Phylogenetic trees were constructed in ARB by adding cloned sequences to the Greengenes reference tree  using the ARB parsimony algorithm .
The community richness of bacteria and archaea in the Mahomet was estimated using Mothur . 16S rRNA gene sequences were clustered into operational taxonomic units (OTUs) based on an average nucleotide similarity at fixed cutoffs. Sequences with an average nucleotide similarity of 97% were binned together into a single OTU. The similarity of individual communities of bacterial and archaeal members across the Mahomet was quantified using the Bray-Curtis coefficient . Archaeal and bacterial communities were grouped together for these analyses on the basis of sample type (attached or suspended) and geochemical zone [15, 17, 18].
Bacterial and archaeal community composition differences were quantified by calculating the Bray-Curtis similarity coefficient and using multidimensional scaling (MDS) to create a two dimensional visualization of the extent to which communities differ from one another. MDS graphs were plotted using a non-metric configuration in which the distance between any two points is inversely proportional to their similarity. All MDS analyses were performed using the Primer-6 software package (Primer-E Ltd., Plymouth, UK). The overall similarity of the bacterial and archaeal communities within groups of wells was calculated using the analysis of similarity (ANOSIM) . Specifically, R-values (RANOSIM) were used to establish the dissimilarity of different paired-groups of microbial communities (e.g. communities from no sulfate vs. high sulfate groundwater). RANOSIM > 0.75 indicate two microbial communities (i.e. the attached and suspended communities from various wells in an aquifer) have characteristic structures largely distinct from one another . A value of RANOSIM between 0.25 and 0.75 indicates communities within each group cluster separately from those in the other, with some overlap, while an RANOSIM < 0.25 indicates communities in one group are almost indistinguishable from those in the other. SIMPER (similarity percentage) was used to calculate the extent to which individual OTUs contribute to the dissimilarity groups sets and to rank the populations from most to least responsible for the differences between groups [40, 41]. Representative sequences from each OTU were identified using Mothur and identified using the Greengenes reference taxonomy as described above. Representative sequences were deposited in GenBank under accession numbers KC604413 to KC604575 and KC604576 to KC607489.
Geochemistry of groundwater in Mahomet aquifer wells
sp. Cond. (μS.cm-1)
High sulfate (HS) wells
Low sulfate (LS) wells
Negligible sulfate (NS) wells
We evaluated the available energy (∆GA), which is equal to the –∆Gr, for different metabolisms using H2 as an electron donor based on the geochemical data shown in Table 1. In contrast to what might be expected from previous work [43, 44], H2 concentrations did not increase as methanogenic conditions became predominant in the NS wells and therefore had little impact on the available energy calculation. Thus the ∆GA varied little for both sulfate reduction and methanogenesis throughout all wells, despite changes in the concentration of key chemical species sulfate and methane (Additional file 1: Table S1). Based on the concentrations of the substrates involved, sulfate reduction via the oxidation of H2 yielded on average calculated ∆GA values between 43 and 102 kJ mol-1, while methanogenesis yielded ∆GA values between 17 and 56 kJ mol-1 in all the wells sampled. In contrast to these energetically favored reactions, anaerobic oxidation of methane (AOM), which proceeds via a reversal of the methanogenic pathway , was not energetically favorable (∆GA < 0) at the bulk concentrations of dihydrogen (H2) measured in the Mahomet aquifer groundwater. Since bulk H2 concentrations may differ from local sites in the aquifer we calculated the threshold concentration where AOM would yield a positive ∆GA and found it to be less than 0.2 nM (Additional file 1: Figure S3). It is therefore possible that, if coupled with H2-oxidizing organisms such as sulfate reducers or iron reducers, AOM could occur in LS wells, where 16S rRNA sequences most closely related to archaea capable of anaerobically oxidizing methane predominate (see below). The direct coupling of methane oxidation to sulfate reduction by a single organism where H2 is not an intermediate would also yield a positive ∆GA in the samples collected (Additional file 1: Table S1).
Microbial composition and diversity analysis
A total of 16,952 clones (8,786 bacteria, 8,166 archaea) were sequenced. Chimeric sequences detected by Bellerophon represented less than 3% of all sequences and were discarded before any further analyses were performed. At a sequence similarity cutoff of 97%, the bacterial community contains 2,681 unique operational taxonomic units (OTUs). Collectors curves showed how the observed richness increased with greater sequencing depth, indicating that the total richness of Mahomet bacterial community is likely to be even greater than quantified here (Additional file 1: Figure S1). Archaeal sequence diversity showed one order of magnitude less OTU richness than their bacterial counterparts, containing 271 unique OTUs. In contrast with the bacterial sequences, the collectors curves indicated that our depth of sequencing accounted for most of the richness of the archaeal community attached to the sediment samplers, but suggested the suspended archaea were undersampled in groundwater (Additional file 1: Figure S2). This may be due to insufficient sediment exposure time to the archaeal community or reflects a preference for most archaea to remain suspended in the groundwater.
Comparison of attached and suspended communities
Unique and shared richness of microbial communities
% OTUs shared
% Sequences in Shared OTUs
The archaeal community was considerably less diverse than the bacterial community, even though we analyzed a comparable number of sequences. The 4,870 archaeal sequences analyzed from ATT samples contained 60 total OTUs, while the 3,143 sequences from SUS archaea contained 266 OTUs. Seventeen OTUs were observed in both ATT and SUS archaeal fractions and 90% of ATT archaeal sequences fell within the shared OTUs, compared to only 22% of SUS archaea (Table 2).
Correlation of microbial community structure with groundwater chemistry
Results of analysis of similarity (ANOSIM) a between HS, LS, and NS wells b
HS - LS
HS - NS
LS - NS
The distinct physical and geochemical niches within the Mahomet aquifer harbour characteristic populations of bacteria and archaea. The microbial communities attached to the in situ sediment samplers (ATT) differed distinctly from those suspended in groundwater (SUS) (Figure 3), both in composition (Figures 4 & 5) and, in the case of archaea, species richness (Additional file 1: Figure S2). While the number of OTUs we observed varied little between ATT and SUS bacteria and the two groups shared only one-third of their phylogenetic diversity, the archaeal community that colonized our in situ samplers was a distinct subset of the suspended community. Over 90% of ATT archaeal sequences were from OTUs that were also detected in the SUS fraction, yet 78% of SUS archaeal sequences were not detected in ATT samples (Table 2). This provides strong evidence that the most active and fastest-growing archaeal populations colonized the initially-sterile sediment contained in our in situ samplers. The phylogenetic distinction between ATT and SUS samples (Figure 3) provides further evidence that this is the case, because no such differentiation of ATT from SUS would be expected if the attachment of cells to the in situ samplers was driven purely by neutral factors such as random adhesion rather than selective colonization [15, 48].
Sequences related to iron-reducing and sulfate-reducing bacteria are much more predominant among the ATT communities when compared to their corresponding SUS communities (Figure 6). Geochemical evidence also supports concurrent iron reduction and sulfate reduction processes in this area of the Mahomet aquifer [17, 22]. The near-absence of these functional populations from SUS groundwater samples suggests that their niche is likely localized to the surface of mineral grains. This makes sense since available ferric iron was associated with the sediment sand used in the traps. This result is not surprising in the case of iron reducers, due to the highly insoluble nature of ferric iron minerals expected in the Mahomet (pH = 7.1–7.9). Iron reducers such as Geobacter require some mechanism of physical attachment to ferric minerals in order to respire . Sulfate, conversely, is highly soluble, meaning sulfate reducers do not necessarily require attachment to aquifer sediment in order to respire. The greater abundance of apparent sulfate-reducing bacteria in ATT samples relative to SUS may occur because these organisms benefit from proximity to iron reducers, whose generation of ferrous iron prevents toxic sulfide from accumulating in solution [2, 42]. When ferrous iron and sulfide are produced simultaneously, they precipitate as the minerals mackinawite (FeS) and greigite (Fe3S4) , limiting the buildup of both reaction products in groundwater and maintaining the thermodynamic drive for each group’s metabolism . Iron reducers have also appeared to benefit from the presence of active sulfate reduction perhaps for the same reason . The predominance of sulfate reducers along with iron reducers in aquifer sediment over groundwater suggests that the two groups may benefit from concurrent respiration.
This simultaneous reduction of both iron and sulfate exemplifies mutualistic behavior, which exists when two distinct groups of organisms benefit from each other’s activity . So despite sulfate reducers and iron reducers competing for the same electron donors in the Mahomet aquifer, by working together they prevent product inhibition. Therefore, rather than being excluded due to thermodynamic constraints by iron reducers as is often suggested [19, 20], sulfate reducers seem to be thriving alongside them in the Mahomet aquifer. The relative richness of iron-reducing bacteria as a proportion of total OTUs only exceeded that of sulfate reducers when sulfate concentrations were below 0.2 mM. Although the relative abundance of an OTU does not necessarily correlate with the cell numbers of a particular functional group, the data do suggest that both metabolisms are maintained in the presence of sulfate. What appears to change is the relative proportion of each functional group as the sulfate concentration changes. Indeed, the primary discriminant of microbial community structure in the Mahomet was the concentration of sulfate in groundwater as indicated by ANOSIM (Table 3) and MDS analyses (Additional file 1: Figures S4 and S5). This is in agreement with results from recent studies which suggest that in the presence of sulfate-reducing bacteria, iron reducers will modify their rate of respiration in order to effectively remove sulfide to the benefit of both groups .
The availability of sulfate also appeared to control archaeal community structure within the Mahomet aquifer. MDS plots comparing archaeal community structure across the aquifer show a distinct clustering of wells with similar amounts of sulfate in the groundwater (Additional file 1: Figures S4 and S5). This differentiation is largely driven by differences in the relative abundance of methanogens compared to other archaea under high and low sulfate conditions. SIMPER analysis showed methanogen-like taxa to comprise a lower proportion of the total archaea in wells where the concentration of sulfate was > 0.03 mM (HS and LS wells), but the same sequences made up nearly 80% of all those obtained from NS wells (Figure 7). These results were commensurate with the concentration of methane detected in groundwater, which was nearly two orders of magnitude higher in NS wells than in HS or LS wells (Figure 2).
The relative abundance of methanogen 16S rRNA gene sequences correlates well with the inverse relationship between sulfate and methane concentrations that was observed in the wells sampled. This has also been observed in other aquifers, where it has been interpreted as a result of sulfate-reducing bacteria outcompeting methanogens and maintaining concentrations of H2 too low for the latter to respire [53, 54]. Assuming a minimum of 10 kJ mol-1 (∆Gmin) is required for these microorganisms to gain energy via respiration , our thermodynamic calculations show that sufficient energy exists for both methanogens and sulfate reducers to respire in every well sampled. In addition, despite the dynamic range of methane and sulfate concentrations shown in Figure 1, H2 concentrations show no correlation to the relative abundance of sulfate reducers or methanogens as would be expected if thermodynamics controlled which type of metabolism could occur [53, 56]. The very low relative abundance of methanogens in HS and LS wells can instead be explained by the kinetics, rather than the thermodynamics, of microbial metabolism. Methanogenesis provides organisms less energy per mole of substrate consumed than sulfate reduction, and kinetic theory suggests methanogens are not able to respire quickly enough to maintain a viable population in the presence of active sulfate reduction [2, 57]. Laboratory studies of co-cultured methanogens and sulfate reducers indicate that methanogenesis ceases following the addition of sulfate to an active biofilm . Even after switching back to a sulfate-free medium, the biofilm required two months to reach its previous level of activity, suggesting the methanogens had died off rather than simply being inhibited by sulfate. The relative low abundance of sulfate reducers observed in NS wells (Figure 6) despite sufficient available energy (Additional file 1: Table S1), conversely, provides further evidence that thermodynamics is not necessarily the ultimate control on the distribution of microbial activity. Rather, because sulfate enters the Mahomet aquifer mainly via leakage from the bedrock in a limited area of east-central Illinois , the flux of sulfate into NS areas of the Mahomet aquifer is likely too low to support a stable population of sulfate reducers.
In addition to controlling the abundance of methanogens, the concentration of sulfate also controls the abundance of Mahomet Arc 1 sequences, a group most closely related to the clade ANME-2D (Figure 5). Specifically Mahomet Arc 1 sequences match most closely archaea shown to anaerobically oxidize methane (AOM) [46, 47]. In this aquifer system, Mahomet Arc 1 archaea are present in nearly every well and were the most abundant member of the archaeal community in LS wells (Figure 7). Archaea in the ANME-2D clade have been implicated as the methane-oxidizing, hydrogen-producing half of a syntrophic partnership that works in tandem with hydrogen-consuming microbes such as sulfate reducers or denitrifiers . These hydrogenotrophs keep H2 concentrations low enough to allow anaerobic methane oxidation to remain thermodynamically favorable for the ANME organisms . Mahomet Arc 1 sequences are 99% similar to those found in an ecosystem confirmed to be anaerobically oxidizing methane , therefore it appears reasonable to hypothesize that this group is also serving this function in the Mahomet.
Despite the abundance of Mahomet Arc 1 sequences in our LS well samples, AOM via reverse methanogenesis remains endergonic at the bulk concentration of H2 measured in Mahomet groundwater (Additional file 1: Table S1). If methane oxidation occurs in the Mahomet, it must happen within aggregates or biofilms which allow a lower local concentration of H2 to be maintained by a hydrogen-consuming partner. AOM becomes energetically favorable in LS wells at concentrations of H2(aq) of less than roughly 0.2 nM (Additional file 1: Figure S3), which is 1–2 orders of magnitude less than the bulk concentration of H2 in groundwater. Depending upon the kinetics of H2 consumption, such a gradient would be feasible inside a biofilm . Alternatively, recent studies have demonstrated direct electron transfer between cells without the intermediate formation of H2[60, 61]. If this occurs close cell contact would still be required for AOM to be feasible. Our study, however does not resolve whether such specific close cell associations occur in the Mahomet aquifer or whether these are specifically associated with AOM in this system. We hope to address this more fundamentally in a future study.
The discovery of Mahomet Arc 1, which appears to be associated with AOM, in a pristine aquifer suggests the anaerobic oxidation of methane may be an additional important metabolic pathway in this system. The heterogeneity of aquifer sediments also leads to numerous microenvironments whose redox chemistry can differ greatly from the bulk groundwater . Molecular diffusion and advective transport can transport methane from the highly reduced zones where it is produced into areas where it might be consumed through an AOM-mediating syntrophic partnership. Because the rates at which CH4 is produced and potentially consumed are difficult to quantify in situ, anaerobic methane oxidation is frequently overlooked in groundwater ecosystems . The abundance of Mahomet Arc 1 sequences and their correlation to the concentration of sulfate then not only suggests the potential importance of AOM as a biogeochemical pathway in the Mahomet, but underscores the largely-untapped potential provided by molecular microbial ecology to better define redox processes in pristine aquifers.
While this study greatly increases our understanding of the microbial communities that catalyze the biogeochemical cycling of carbon and metals in the Mahomet aquifer, additional studies are needed to shed light on the dynamics of microbial activities of this and other subsurface systems over time. Moreover, molecular surveys represent an important foundation for studies trying to understand how changes in subsurface chemistry may impact subsurface communities exposed to anthropogenic perturbations such as geological carbon sequestration and hydrologic fracturing of gas-rich strata, both of which may lead to changes in groundwater flows and chemistries. Further, our results indicate that appropriate monitoring schemes must consider the assessment of the microbial fraction associated with subsurface biofilm communities as their composition and activities might not be easily predicted by targeting the suspended fraction extracted with groundwater.
Operational taxonomic unit
(nonmetric) Multidimensional scaling
Analysis of similarity
Anaerobic oxidation of methane.
The U.S. Environmental Protection Agency, through its Office of Research and Development and the RARE program, funded, managed, and collaborated in the research described herein. This work has been subjected to the agency’s administrative review and has been approved for external publication. Any opinions expressed in this paper are those of the authors and do not necessarily reflect the views of the agency; therefore, no official endorsement should be inferred. Any mention of trade names or commercial products does not constitute endorsement or recommendation for use. The authors thank B. Iker, M. Kyrias, D. Strattan, B. Farrell, E. Luber, M. Nolan, C. Salvatori, J. Shelton, and P. Bermudez for their assistance in the laboratory and the field. H. Ryu received funding through a fellowship from the National Research Council. This work was also supported in part through funding from the Department of Energy grant DE-FG02-02ER15317, a Director’s Postdoctoral Fellowship from Argonne National Laboratory to T. Flynn, and the SBR SFA at Argonne National Laboratory which is supported by the Subsurface Biogeochemical Research Program, Office of Biological and Environmental Research, Office of Science, U.S. Department of Energy (DOE), under contract DE-AC02-06CH11357.
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