Impacts of Shallow Geothermal Energy Production on RedoxProcesses and Microbial CommunitiesMatthijs Bonte,*,†,⊥ Wilfred F. M. Roling,‡ Egija Zaura,§ Paul W. J. J. van der Wielen,†

Pieter J. Stuyfzand,†,∥ and Boris M. van Breukelen∥

†KWR Watercycle Research Institute, P.O. Box 1072, 3430BB Nieuwegein, The Netherlands.‡Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam,The Netherlands§Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU UniversityAmsterdam, Gustav Mahlerlaan 3004, 1081LA Amsterdam, The Netherlands∥Critical Zone Hydrology Group, Department of Earth Sciences, VU University Amsterdam, De Boelelaan 1085, 1081HVAmsterdam, The Netherlands

*S Supporting Information

ABSTRACT: Shallow geothermal systems are increasinglybeing used to store or harvest thermal energy for heating orcooling purposes. This technology causes temperatureperturbations exceeding the natural variations in aquifers,which may impact groundwater quality. Here, we report theresults of laboratory experiments on the effect of temperaturevariations (5−80 °C) on redox processes and associatedmicrobial communities in anoxic unconsolidated subsurfacesediments. Both hydrochemical and microbiological datashowed that a temperature increase from 11 °C (in situ) to25 °C caused a shift from iron-reducing to sulfate-reducing andmethanogenic conditions. Bioenergetic calculations could explain this shift. A further temperature increase (>45 °C) resulted inthe emergence of a thermophilic microbial community specialized in fermentation and sulfate reduction. Two distinct maxima insulfate reduction rates, of similar orders of magnitude (5 × 10−10 M s−1), were observed at 40 and 70 °C. Thermophilic sulfatereduction, however, had a higher activation energy (100−160 kJ mol−1) than mesophilic sulfate reduction (30−60 kJ mol−1),which might be due to a trade-off between enzyme stability and activity with thermostable enzymes being less efficient catalyststhat require higher activation energies. These results reveal that while sulfate-reducing functionality can withstand a substantialtemperature rise, other key biochemical processes appear more temperature sensitive.

■ INTRODUCTION

Shallow subsurface geothermal or aquifer thermal energystorage (ATES) systems are increasingly being used to storeor harvest thermal energy for heating or cooling purposes in thebuilt environment.1−4 In summer, ATES systems abstractgroundwater to cool buildings, and subsequently, the heatedgroundwater is reinjected into the aquifer. In winter, the cyclereverses, and the stored heated groundwater from last summeris abstracted to heat buildings. In general, regulators allowinjection temperatures up to 20−25 °C in countries like TheNetherlands, Germany, and Austria.5,6 Systems with higheroperational temperatures (up to 110 °C) are less common,7,8

but interest in these systems is growing because of the potentialrelatively high energy savings. Because ATES systems are oftenrealized in aquifers used for drinking water production,questions are raised on the risks of ATES for water qualityand drinking water production.9,10 Also, interest is growing inthe combination of ATES and monitored natural attenuation in

contaminated urban aquifers, but the effects of temperaturechanges on biodegradation are still unclear.11,12

Previous research on ATES revealed that groundwaterquality is influenced by mixing of different water types,13,14

temperature changes,7,8 intrusion of oxygen, or degassing.15,16

Most of the earlier research focused on operational aspects inrelation to inorganic chemistry, such as well-clogging due tomineral precipitation occurring in high temperature systems(>25 °C).7,8,15,17 Two studies that are more relevant for theeffects of ATES on water quality reported increasedconcentrations of dissolved organic carbon (DOC) withincreasing temperature in laboratory experiments.18,19 In ourprevious research, we found that the mobility of arsenic isenhanced with a temperature increase, which was attributed to

Received: July 9, 2013Revised: November 20, 2013Accepted: November 22, 2013Published: November 22, 2013

Article

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either desorption from or reductive dissolution of iron oxides.20 Microbiological studies on ATES did not report evidence forgrowth or survival of pathogens21 or increasing cell counts 22

due to temperature elevation. However, the microbialcommunity structure and a selected number of groundwaterinvertebrates were reported to be significantly influenced bychanging temperature near an ATES system23 and in laboratoryexperiments. 24

None of these previous studies investigated the temperatureimpacts on both microbial functioning and associated hydro-chemical changes. In order to fill this void, we present alaboratory column study on the influence of temperature (5−80 °C) on the kinetics and competition of redox reactions andassociated changes in microbial communities in anoxicsediments. The novelty of this study is that we combinedconventional hydrochemical analyses to determine thepredominant redox processes, sulfate reduction rates as aproxy for microbial activity, and 454 pyrosequencing to mapthe changes in microbial community. We derived activationenergies for sulfate reduction, which was the dominant redoxreaction in the investigated sediments, providing a compre-hensive quantitative description of the temperature depend-ence, which can be incorporated in hydrochemical modelsdealing with temperature changes due to ATES.

■ MATERIALS AND METHODSSediment and Groundwater. Sediments and groundwater

used in the experiments were collected at two locations (70 kmdistance between sites) from an aquifer intensively used forboth drinking water production and ATES in The Netherlands:Helvoirt (derived from a depth between 32 and 34 m) andScherpenzeel (depth between 34 and 36 m). The uncon-solidated sandy aquifer is part of the fluvial Sterksel Formationranging in thickness between 5 and 75m, which was depositedduring the Early to Middle Pleistocene.25 Sediment cores werecollected with the percussion drilling method and transportedin an ice box filled with N2 gas to ensure cores remained anoxic.Geochemical analyses revealed that sediment samples consistedmainly of quartz sand (>90%), with minor fractions of K-feldspar and clay (details on geochemical analyses given in theSupporting Information). Reactive phases included organicmatter (0.15% and 0.06% for sediments Helvoirt andScherpenzeel, respectively), carbonates (0.5% and 0.7%), pyrite(0.06% and 0.09%), and iron oxides (0.6% and 0.5%).Influent for the column experiment was derived from

groundwater abstracted from monitoring wells constructed inthe boreholes for sediment sampling and collected in a stainlesssteel 60 L pressure barrel using a submersible sampling pump at20 m below the water table. Groundwater in this aquifer isanoxic, lacking O2 and NO3, with SO4 concentrationsdecreasing with depth13 from 80 mg L−1 at the top of aquiferto 3 mg L−1 at the bottom of the aquifer, which may be due tosulfate reduction or a change in the historic input of SO4 andNO3 with groundwater recharge.26 At the sediment samplingdepth, SO4 and Fe concentrations were 8.5 and 6.5 mg L−1 forHelvoirt and 6 and 0.3 mg L−1 for Scherpenzeel, respectively.At both sites, CH4 concentrations in the aquifer at the samplingdepth were generally <0.01 mg L−1.Experimental Setup. The cores were unpacked in the

laboratory under a N2 atmosphere, mixed thoroughly for 30min using stainless steel spatulas to obtain a homogeneoussample, and repacked in four 0.4 m long stainless steel coreswith an internal diameter of 0.066 m. The cores were then

maintained at temperatures of 5 °C (representing cold storage),11 °C (ambient temperature), 25 °C (maximum allowedregular ATES), and 60 °C (high temperature ATES).At the start of the experiments, the sediment cores were first

flushed for 25 days with a residence time of 1 day allowing themicrobiological community to acclimatize to the testingtemperatures of 5, 11, 25, and 60 °C. Following thisacclimatization period, the residence time of water in thesediments was stepwise increased to 30 days, to evaluate theoccurrence of redox processes by measuring the concentrationsof major redox species such as SO4, Fe, DOC, and CH4 asfunction of residence time. During each residence time step,water was stagnant in the columns until the required residenceperiod was achieved. Following this, water in the column wassampled for analysis and subsequently flushed with influent forsix pore volumes in two days, resetting the aqueousconcentrations in the column. This experiment (called hereafterthe increasing residence time (IRT) experiment) was carriedout for both sediments Helvoirt and Scherpenzeel.In order to gain a more detailed insight into the temperature

dependence of the redox kinetics, sediment Scherpenzeel wassubsequently subjected to an experiment where the temper-ature was increased from 5 to 80 °C in steps of 5 °C, each witha constant 5 day residence time (temperature-ramping (TR)experiment). The resulting data sets were used to calculate thesulfate reduction rates (SRRs) at different temperatures andactivation energies.

Chemical and Microbiological Analyses. Water samplescollected during the experiments were analyzed for the redoxrelevant species SO4, Fe, CH4, and DOC. Fe was analyzed astotal dissolved Fe (following filtration over a 0.45 μm filter),speciation calculations with PHREEQC v2.15 27 showed this is>99% Fe(II). Terminal restriction fragment length poly-morphism (T-RFLP) and 454 pyrosequencing of PCR-amplified bacterial 16S rRNA genes and denaturing gradientgel electrophoresis (DGGE) of PCR fragments of archaeal 16SrRNA genes followed by sequencing of excised bands were usedto detect changes in the microbial community. This was carriedout on both water samples and on DNA extracted fromsediment. Details on analytical procedures are provided in theSupporting Information.

Deriving Kinetic and Thermodynamic Parameters. Todetermine the temperature dependence of sulfate reductioncoupled to sedimentary organic matter (SOM) mineralization,we first derived the sulfate reduction rate using the Monodkinetics28−33

=+

T Tm

k mSRR( ) SRR ( )max

SO

SO SO

4

4 4 (1)

where SRR(T) is the sulfate reduction rate [M s−1] attemperature T (K), SRRmax(T) is the maximum rate constant[M s−1] at temperature T (K) not restricted by lacking sulfate,mSO4

is the sulfate concentration [M], and kSO4is the half

saturation constant [M]. The temperature dependence ofSRRmax can be expressed using the Arrhenius equation 34

= +−⎜ ⎟⎛

⎝⎞⎠T A

ER T

ln[SRR ( )] ln( )1

maxa

(2)

where Ea is the activation energy (J mol−1), R is the moleculargas constant (8.314 J K−1 mol−1), T is the absolute temperature(K), and A is the constant pre-exponential factor [M s−1]. Notethat only the activation energy and not the pre-exponential

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factor (A) is required to convert a rate from one temperature toanother. The temperature ramping experiment alloweddetermination of the optimum temperature for sulfatereduction (Topt, K) and SRR at this temperature (SRRTopt,[M s−1]), which can be compared to the SRR at in situtemperatures (SRRin situ, [M s−1]). A commonly used parameterto express the temperature dependence is Q10, which gives theincrease in reaction rate for a 10 K increase in temperature34

=+

⎡⎣⎢

⎤⎦⎥Q

ERT T

exp10

( 10)10a

(3)

Equations 1−3 were used to determine SRRTopt, SRRin situ, Ea,and Q10 values by minimizing the difference between observedand simulated SO4 concentrations using the universal modeloptimizer PEST. 35 The half saturation constant, kSO4

, wasbased on the transition from a linear to first-order sulfatereduction rate as observed in the SO4 versus time plots. Notethat both Monod kinetics and the application of the Arrheniusequation for temperature correction represent a high ordersimplification covering for a large number of steps within theturnover process of organic matterpresumably affecteddifferently by different factors changing in the system. Thisimplies that the derived Monod parameters cannot directly betranslated to other aquifer settings.

■ RESULTSIncreasing Residence Time (IRT) Experiments. SO4

depletion was not significant in sediment Helvoirt at in situtemperature (11 °C) and below (5 °C); however, at elevatedtemperature (25 and 60 °C), SO4 decreased with increasingresidence time indicating sulfate reduction (Figure 1A). For

sediment Scherpenzeel, SO4 depletion with increasingresidence times was observed over the entire temperaturerange (Figure 1B). The elevated Fe(II) concentration after thelongest incubation time in the 5 °C effluent and to a lesserextent in the 11 and 25 °C effluents of sediment Helvoirtindicated the occurrence of iron-reducing conditions (Figure1E). The Fe(II) concentration remained stable in the 60 °Ceffluent of sediment Helvoirt and for sediment Scherpenzeelover the entire temperature range (Figure 1F). Effluents weresampled and analyzed for CH4 only from sedimentScherpenzeel, and CH4 was generated in significant amountsonly at 25 °C, especially during residence times longer than 15days (Figure 1G). In the 60 °C column, no CH4 was produced.When the observed changes in redox species were expressed onthe basis of transferred electrons [by multiplying the change inthe concentration of electron acceptor with the number oftransferred electrons (expressed in electron equivalents, meqL−1):36 8 for SO4

2−, up to 8 for CH4 (exact number of electronsdepends on the mineralization pathway), and 1 for Fe(II)], thetotal electron transfer by sulfate reduction (0.57 meq L−1, at 60°C in sediment Helvoirt) was much higher than by ironreduction (0.03 meq L−1, at 5 °C in sediment Helvoirt) or CH4production (<0.02 meq L−1 at 25 °C in sedimentScherpenzeel). Because this approach did not consider thepossible precipitation of iron sulfides (quantification notpossible as H2S was not measured), the transferred electronsfor iron reduction should be considered as a minimum. Insediment Helvoirt, DOC increased at 60 °C, whereas insediment Scherpenzeel, an increase in DOC production wasobserved at both 25 and 60 °C (Figure 1C,D).

Temperature Ramping (TR) Experiments. The TRexperiments showed SO4 depletion occurring at all temper-atures (Figure 2A), indicating sulfate reduction to occur,whereas Fe(II) concentrations remained near the influentconcentration (Figure 2C), both of which are in line with theresults of the IRT experiments. The higher temperatureresolution did, however, reveal two distinctive maxima in SO4consumption at Topt = 40 °C and at Topt = 70 °C. Between 45and 50 °C, SO4 concentrations were very close to the influentconcentrations.CH4 production was greatest between 25 and 40 °C

corresponding to the first region of maximum sulfate reduction(Figure 2D). The DOC concentrations were close to theinfluent concentration up to 15 °C (Figure 2C). Beyond 20 °C,a gradual increase in DOC was observed from 0.5 mg L−1

(equal to influent) to 3.6 mg L−1 at 80 °C. It is noteworthy thatthe general pattern of steadily increasing DOC concentrationlacks distinct minima as observed for sulfate.

Microbial Community Changes. Bacterial 16S rRNAgene-based T-RFLP (Figure S4, Supporting Information) andpyrosequencing [Figure 3; on the basis of 1670 reads, whichallowed for good coverage of bacterial richness (Figure S5,Supporting Information)] revealed obvious differences betweenthe bacterial communities in effluents and sediments. Attemperatures between 5 and 25 °C, the bacterial communitiesin the effluent strongly resembled the bacterial community inthe influent. However, at 60 °C a clear change in communitystructure was observed, especially for communities derivedfrom sediment (Figure 3; Figure S4, Supporting Information).The bacterial community shifted from Proteobacteria dominated[50−92% of operational taxonomic units (OTUs)] toFirmicutes (26−72%), in particular putative anaerobic, moder-ate thermophilic, and spore-forming Thermoanaerobacteraceae

Figure 1. Observed concentrations of SO4, DOC, Fe(II), and CH4(symbols and dashed lines) for effluents of sediments Helvoirt (H)and Scherpenzeel (S) as a function of residence time. Observed SO4data were also fitted to Monod kinetic models (solid lines). Monodmodel parameters for both locations are shown in Table 1.

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and Peptococcaceae (both Clostridia) and Bacilliceae (Figure S6and Table S3, Supporting Information) strongly increased in

abundance. Furthermore, an OTU most strongly related toCaldilineaceae (Chloroflexi) increased to 14% of OTUs insediment. These families also became enriched in the effluent.Temperature-related shifts in the occurrence of OTUs that

can be linked to specific redox processes were evident. Adiversity of putative sulfate reducers, belonging to the generaDesulfovibrio, Desulfatiferula, Desulfobacula, Desulfarculus, Desul-fosalsimonas, Desulfobulbus, Desulfurivibrio, Desulfatibacillum,and Desulfobacca (all Proteobacteria), and Desulfosporosinus,and Desulfurispora (Firmicutes), was present at moderatetemperatures, but sulfate-reducing Proteobacteria were hardlydetectable at 60 °C (Table S3, Supporting Information). At thistemperature OTUs most closely related to putative spore-forming, sulfate-reducing, and thermophilic Desulfotomaculum(Firmicutes) became enriched, although it should be noted thatDNA similarities were very low (87−90%).Likewise, genera known for their iron-reducing capabilities

(Geothrix (Acidobacteria), Shewanella, Desulfuromonas, Geo-bacter, and Abideferax (all Proteobacteria) and Desulfitobacterium(Firmicutes), were no longer detected at the highest temper-ature (Table S3, Supporting Information). At 25 °C, inparticular Desulfuromonas and Geobacter were present at lowerabundance in sediment than at 5 and 11 °C, in line with theobserved lower iron reduction inferred from hydrochemicaldata.Furthermore, temperature had a large impact on Archaea

communities. While nearly all (9 out of 12) samples of theexperiments held at 5 to 25 °C revealed an Archaea-specificPCR product, and only one of the 4 samples from theexperiment at 60 °C was positive. DGGE analysis revealed asingle band, which was 97% similar to the ammonium-oxidizingThaumarchaeote Nitrosopumilis (Figure S7, Supporting In-formation). In contrast, sequencing of several bands fromDGGE profiles corresponding to samples taken from columnexperiments conducted at lower temperatures revealed putativemethanogenic Euryarchaea (Figure S6, Supporting Informa-tion), in line with the observed CH4 production.

Kinetics and Thermodynamics of Sulfate Reductionand SOM Mineralization. The Arrhenius equation can onlybe applied over a limited temperature range, depending on thetype of enzymes involved and their denaturation temperature,which was not a priori known. 34 We used the results of the TRexperiment (Figure 2) and the 454 pyrosequencing data(Figure 3) to constrain two temperature ranges (defined hereas a mesophilic range of 5−50 °C and a thermophilic range of50−70 °C) for which kinetic and thermodynamic parameterscould be derived. In Figures 1 and 2, the simulated SO4

concentrations are shown on the basis of the optimizedparameters for the Monod and Arrhenius equations (eqs 1 and2), and a visually derived half saturation concentration of 4 ×10−5 M. In situ SRRs of 4.8 × 10−12 to 4.3 × 10−11 M s−1 at 11°C and Ea values of 115 ± 268 and 54 ± 26 kJ mol−1 werecalculated for the IRT experiments on sediments Helvoirt andScherpenzeel, respectively (Table 1). The mesophilic Ea forsediment Helvoirt had a very large standard error, which is dueto the low SRR at 5 and 11 °C, causing the Ea to be poorlyconstrained. The TR experiment on sediment Scherpenzeelyielded maximum SRRopt values of 4.8 ± 6.1 × 10−10 and 5.3 ±11 × 10−10 M s−1 at Topt of 40 and 70 °C, respectively. DerivedEa values for the low and high temperature ranges of sedimentScherpenzeel were 41 ± 19 and 80 ± 44 kJ mol−1, respectively.

Figure 2. Temperature ramping experiment with sediment Scherpen-zeel. Observed concentrations of SO4, DOC, Fe(II), and CH4 (points)for effluent with 5 day residence time at different temperatures. Alsoshown are the influent concentrations (thin dashed line) and thesimulated SO4 concentrations.

Figure 3. Nonmetric multi-dimensional scaling plot of Bray−Curtissimilarities among bacterial communities in effluent and sediment incolumn experiments at various temperatures using 16S rRNA genepyrosequencing. Stress was 0.092. Coloring of symbols corresponds totemperature levels used in Figure 1. Circles represent water samples.Squares represent sediment samples. Sample codes provideinformation on temperature (5, 11, 25, or 60 °C), medium (effluentor sediment), sampling location (Helvort or Scherpenzeel), andincubation time (in days).

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■ DISCUSSION

Impact of Temperature on Prevailing Redox Reac-tions. Over the entire temperature range in sedimentScherpenzeel and at elevated temperature (25 and 60 °C) insediment Helvoirt, hydrochemical data, supported by microbialcommunity data, revealed sulfate reduction as the dominantredox reaction, whereas iron-reducing conditions only occurredat in situ temperature (11 °C) and below (5 °C) in sedimentHelvoirt. The prevalence of sulfate reduction in sedimentScherpenzeel and the shift in prevailing redox processes fromiron reduction to sulfate reduction with increasing temperatureobserved in sediment Helvoirt can be placed in a thermody-namic context by calculating the H2 threshold required for agiven redox reaction to occur.37−39 Microorganisms that gainenergy from redox reactions with a low H2 threshold have acompetitive advantage because they are able to oxidize H2produced by fermenting organisms at H2 levels sufficiently lowto impede the respiration of microorganisms that depend onredox processes having higher H2 thresholds. We calculated thetemperature-dependent H2 thresholds for specific redoxprocesses from the initial aqueous concentrations, standardGibbs free energy, and minimum free energy of −7 kJ mol−1 39

required for biochemical reactions to proceed. We assumed thisthreshold energy is constant for the considered redox processesand with changing temperature (Figure 4, details on thecalculation are presented in the Supporting Information). Notethat this minimum free energy cannot directly be compared tothe activation energies for SOM mineralization (Table 1) as thelatter are used to describe the full mineralization pathway[hydrolysis, fermentation, and the terminal electron acceptingprocess (TEAP)], whereas the minimum free energy is only

applicable to the TEAP. The relationship between temperatureand redox process-specific H2 thresholds shows that reductionof Fe(III) from lepidocrocite (a relatively reactive iron oxide)has the lowest H2 threshold at a temperature below 25 °C,while at higher temperature sulfate reduction proceeds with thelowest H2 threshold. This agrees with the observed shift towardmore strongly sulfate-reducing conditions with elevatedtemperature in sediment Helvoirt. Note that potentialoccurrence of iron oxide recrystallization at higher temperatureresulting in more crystalline and stable iron oxides40 wouldincrease the H2 threshold of iron reduction and might haveadded to this transition to sulfate-reducing conditions. Thisredox shift could be further investigated by combiningcalculated threshold H2 concentrations with measured H2concentrations;41 this was, however, not possible in our studydue to the constraints of our experimental setup.Although iron oxide content was similar for both sediments,

dissolved Fe(II) was considerably higher in groundwater atHelvoirt (6.5 and 0.3 mg L−1 at sites Helvoirt andScherpenzeel, respectively), potentially indicating more favor-able conditions for iron reduction at Helvoirt. Possibly ironoxides were more reactive at Helvoirt (e.g., presence oflepidocrocite) promoting iron reduction there. Figure 4 showsthat iron reduction with less reactive goethite is unlikely tooccur at ambient aqueous conditions and may explain whysulfate reduction is dominant in sediment Scherpenzeel. Thisaspect could be further investigated by determining the separateiron oxide phases present, for example, by the methoddescribed by Larsen and Postma.42 For the combination ofaqueous concentrations used to calculate H2 thresholds inFigure 4, methanogenesis is not thermodynamically favorable,but as concentrations of SO4 decrease with ongoing sulfatereduction, the H2 threshold for sulfate reduction increases.When it approaches the H2 threshold for methanogenesis, thisredox process becomes possible. This is also visible in Figure1B and G with the rate of methanogenesis increasing withdecreasing SO4 concentrations.

Thermophilic Redox Processes and Microbial Com-munities. Both sulfate depletion, sulfate reduction rates, andmolecular data indicate the emergence of a thermophilicfermenting and sulfate-reducing bacterial community attemperatures >45 °C. This shows that part of the microbialfunctionality was maintained despite great temperaturechanges. In contrast, hydrochemical data, supported by thedata on microbial community composition, indicated that ironreduction and methanogenesis were insignificant in thethermophilic range. A number of studies using cold (<15 °C)marine sediments showed a similar emergence of athermophilic-fermenting and sulfate-reducing bacterial com-munity in laboratory experiments.43−46 Hubert et al.45 studiedthe temperature impact on organic matter mineralization in

Table 1. Kinetic and Thermodynamic Sulfate Reduction and SOM Mineralization Parameters for the Monod Kinetic Modela

sediment type or experiment Topt (°C) Tmax (°C) SRRin situ (M s−1) SRRTopt (M s−1) Ea (kJ mol−1) Q10

A. IRT experimentssediment Helvoirt (5−25 °C) n.a.b n.a. 4.8 × 10−12 n.a. 115 ± 267 5.3

sediment Scherpenzeel (5−25 °C) n.a. n.a. 4.3 × 10−11 n.a. 54 ± 26 2.2B. TR experiments

sediment Scherpenzeel (5−40 °C) 40 47 4.3 × 10−11 4.8 ± 6.1 × 10−10 41 ± 19 1.9sediment Scherpenzeel (50−70 °C) 70 >80 n.a. 5.3 ± 11 × 10−10 80 ± 44 3.2

aShown are derived best fit values and the standard errors (±). bTopt, Tmax, and SRRTopt could only be determined in the temperature rampingexperiment: n.a., not available.

Figure 4. Hydrogen gas thresholds for different terminal electron-accepting processes as function of temperature using pH 7.0, Fe(II)=10−4 M, SO4

2− = 10−4 M, HS− = 10−12 M, CH4 = 10−11 M, andHCO3

− = 10−3 M at 11 °C (speciation at other temperatures wascalculated using the geochemical speciation model PHREEQCv2.1527), and assuming the ΔGmin of reaction is −7 kJ/mol.

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marine sediments and found two distinct maxima in SRRs at 32and 56 °C. On the basis of the analysis of cloned 16S rRNAgenes, Hubert et al.45 further showed that at higher temper-atures (50 °C), a thermophilic community developed in themarine sediments with Caminicella and Caloranaerobactor−Clostridiisalibacter−Thermohalobacter lineages (Firmicutes phy-lum) capable of producing volatile fatty acids. This productionstimulated the germination of spores and growth ofDesulfotomaculum species, which are sulfate-reducing spore-forming bacteria that were naturally present in these sedimentsat very low concentrations.45 The emergence of several OTUclosely related to spore-forming thermophilic members of theFirmicutes phylum was also evident in our sediments despite thevery different origin of sediments used by Hubert et al.45 andours.Accumulation of Dissolved Organic Carbon (DOC)

and Organic Carbon Turnover. Mineralization of SOMcomprises a number of steps,47 including hydrolysis,fermentation, and terminal oxidation, e.g., by sulfate reduction,while the intermediate reaction products contributing to DOCare generally kept at low concentrations at the in situtemperature.48 This is consistent with our observation that insediment Scherpenzeel sulfate reduction was occurring at 5, 11,and 15 °C, but no increase in DOC was observed (Figure 2).Only if the rate of one of the first two steps (hydrolysis andfermentation) exceeds the rate of terminal oxidation, or if DOCis less bioavailable at higher temperature, an increase in DOC isexpected.48 The changing reactivity with temperature ofmobilized DOC was also observed by Xu and Saiers49 inunsaturated soil column experiments and by Brons50 in batchexperiments with saturated sediments. Xu and Saiers49

explained this by the dependence of DOC mobility on boththe temperature and molecular weight. Relatively large(recalcitrant) organic molecules remain sorbed at lowertemperature implying that the accumulation of DOC withrising temperature is in fact a physical or abiotic process.49 Thefairly continuous DOC increase over the entire temperaturerange (Figure 2), without the clear transition of mesophilic tothermophilic activity as observed in the sulfate depletion,substantiates that the DOC accumulation is primarily an abioticprocess producing relatively recalcitrant DOC and is not readilyconsumed by the fermenting community.Kinetics and Thermodynamics of SOM Mineralization

and Sulfate Reduction. A comparison between in situ SRRsreported in the literature for aquifers33,51,52 (Figure 5A; SRRdata, Supporting Information) and our results shows that theSRR for sediment Helvoirt falls within the 50% central regionof SRR values reported in literature, while that of sedimentScherpenzeel is just above this. It is also evident from Figure 5Athat SRRs observed in aquifers span a large range with valuesranging 3 orders of magnitude. The SRRs in fresh aquifersediments are generally lower than those observed in bothsaline marine28,31,46,53−58 and geothermal sediments59−61

(Figure 5A), which may due to a combination of sedimentage,51,62 extent of oxygen exposure during deposition,63 lowerabundance of recalcitrant biomacromolecules (lignin) in marinesediments,63 and difference in sulfate concentrations.32

Activation energy for SOM mineralization by sulfatereduction in aquifers has to our knowledge only been reportedby Benner et al.33 for a reactive barrier comprising reactive C(Ea = 40 kJ mol−1; Figure 5B). This value is similar to valuesfound for the mesophilic range for sediment Scherpenzeel (54± 26 and 41 ± 19 kJ mol−1 for the IRT and TR experiments,

respectively) and sediment Helvoirt (115 ± 268 kJ mol−1),considering the standard errors of the found Ea values.Comparing the SRRs with Ea values (Figure 5) shows that in

sediments with relatively old and recalcitrant SOM, as used inour experiments, sulfate reduction rates are low, but temper-ature sensitivity is relatively high (high Ea and Q10 values). Insediments with relatively reactive SOM (most marine studies),SRR is relatively fast but less sensitive to temperature change.This agrees with the conceptual model for temperaturedependence of soil carbon mineralization by Davidson andJanssens,64 in which the mineralization rate of recalcitrant soilorganic matter is low but highly temperature dependent.Two studies on marine53 and estuarine sediments55 report Ea

values of sulfate reduction in the same range as observed for themesophilic range in sediment Scherpenzeel (50% of Ea valuesbetween 30 and 60 kJ/mol in Figure 5B). The Ea for sulfatereduction in geothermal systems (Figure 5B), however, revealsan Ea range between 100 and 160 kJ/mol (50% box) suggestinga different thermodynamic behavior of thermophilic comparedto mesophilic sulfate reduction. We observed a similar effect inthe TR experiment using sediment Scherpenzeel, where the Eawas considerably larger in the thermophilic (80 kJ mol−1) thanin the mesophilic range (44 kJ mol−1). A possible explanationfor the larger activation energy under thermophilic conditions isa trade-off in biochemical processes between the enzyme

Figure 5. Panel A shows sulfate reduction rates (SRR) for ourexperiments at Tin situ and Tmax (blue and red dots, respectively)compared with box plots of SRR for in situ and optimum temperaturesderived from literature for aquifer33,51,52 (in situ only), ma-rine,28,31,53−58 and geothermal sediments.59−61 Panel B shows theactivation energy (Ea) of sulfate reduction derived from ourexperiments (dots), a reactive permeable barrier in an aquifer,33 andbox plots for values for geothermal59−61 and marine sedi-ments.45,46,53,55 Ea values for a number of these studies45,46,59,61 werederived from SRR at different temperatures (details in the SupportingInformation). The SRR and Ea values found by Benner et al.33

(discussed in text) are indicated by the green point.

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stability and activity.65 The catalytic capacity of enzymesdepends on their molecular flexibility, which determines theircapacity to bind and catalyze substrates.66 Enzymes producedby thermophilic sulfate-reducing organisms require a higherthermostability, making them less flexible and less efficient,potentially resulting in higher Ea values for thermophilicenzymes.65 This phenomenon has not yet been reported formicrobial processes in aquifers but has been reported for a pureculture study using mesophilic and thermophilic homologues67

and in another study on a number of psychrophilic enzymes.68

■ ENVIRONMENTAL AND TECHNOLOGICALIMPLICATIONS

While realizing that we only investigated a limited number oflocations and temperatures, the observed temperature-inducedimpacts on redox processes are relevant for groundwaterquality, drinking water production, and biodegradation oforganic pollutants. First, the increasing mineralization rate andincreasing DOC concentrations with enhanced temperature cancause discoloration of the groundwater requiring additionaltreatment when used for drinking water production.69 Thisprocess can be especially problematic because DOC mobilizedat higher temperatures is potentially more recalcitrant making ithard to remove in water treatment. Second, the aquifer’s SOMis an important contributor to contaminant buffering capacity,70

for example, as a sorption medium for organic micro-pollutants.29 Thermal removal of SOM from aquifers can,therefore, increase their vulnerability. Third, a temperature-induced shift from iron-reducing to sulfate-reducing conditionsmay affect the biodegradation potential of organic pollutants invarious ways. On the one hand, a transition to more reducedconditions lowers the degradation rate of aromatic hydro-carbons.71 On the other hand, such a shift and subsequentlowering of sulfate levels may promote reductive dechlorinationof chlorinated ethenes. 72

The identified impacts are also relevant for the design andoperation of shallow geothermal systems. Sulfate reduction cancause anaerobic corrosion to, and subsequent failure of, ferrousmetals and to a lesser degree to stainless steel73,74 reducing thelifetime of the system. Spores of thermophilic sulfate reducersmight be omnipresent in the subsurface, and as such, this issueshould be considered during design and operation of shallowgeothermal systems. 45 Furthermore, the mobilized organiccarbon may provide substrate for microbial communitiespresent on the geothermal well screen, resulting in slimeformation and clogging of wells or in line filters.75,76 The latteraspect depends largely on the reactivity of mobilized DOCwhich, as discussed, is likely to decrease with increasingtemperature.

■ ASSOCIATED CONTENT

*S Supporting InformationDetailed description of the applied methods, additionalmicrobiological results (bacterial TRFLP plot, ArchaeanDGGE profiles, phyla distributions, and characteristic operatingtaxonomical units (spreadsheet)), and the results of theliterature compilation on sulfate reduction kinetics andthermodynamics. This material is available free of charge viathe Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: +31-(0)70-4472472. E-mail: matthijs.bonte@shell.com.

Present Address⊥Matthijs Bonte: Shell Global Solutions, Lange Kleiweg 40,2288 GK Rijswijk, The Netherlands.

Author ContributionsThe manuscript was written through contributions of allauthors.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe greatly acknowledge the help of Rob Stoevelaar, RudoVerweij, Julia Claas, Mark Hanemaaijer, Martin Braster, andValentina Chacon of VU University Amsterdam, and RonaldItaliaander, Sidney Meijerink and Harry van Wegen of KWRand Mark Buijs of ACTA. This study was funded by the Dutchwater sector’s joint research program B11168, effects of shallowgeothermal energy.

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