Available online at www.sciencedirect.com

www.elsevier.com/locate/gca

ScienceDirect

Geochimica et Cosmochimica Acta 156 (2015) 207–221

Spatial distributions of sulphur species andsulphate-reducing bacteria provide insights into

sulphur redox cycling and biodegradation hot-spotsin a hydrocarbon-contaminated aquifer

Florian Einsiedl a,⇑, Giovanni Pilloni b,1, Bettina Ruth-Anneser b,2,Tillman Lueders b, Christian Griebler b

a Technische Universitat Munchen, Arcisstrasse 21, Chair of Hydrogeology, Germanyb Institute of Groundwater Ecology, Helmholtz Zentrum Munchen – German Research Centre for Environmental Health,

Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany

Received 13 March 2014; accepted in revised form 13 January 2015; available online 16 February 2015

Abstract

Dissimilatory sulphate reduction (DSR) has been proven to be one of the most relevant redox reactions in the biodegra-dation of contaminants in groundwater. However, the possible role of sulphur species of intermediate oxidation state, as wellas the role of potential re-oxidative sulphur cycling in biodegradation particularly at the groundwater table are still poorlyunderstood. Here we used a combination of stable isotope measurements of SO4

2�, H2S, and S0 as well as geochemical pro-filing of sulphur intermediates with special emphasis on SO3

2�, S2O32�, and S0 to unravel possible sulphur cycling in the

biodegradation of aromatics in a hydrocarbon-contaminated porous aquifer. By linking these results to the quantificationof total bacterial rRNA genes and respiratory genes of sulphate reducers, as well as pyrotag sequencing of bacterial commu-nities over depth, light is shed on possible key-organisms involved.

Our results substantiate the role of DSR in biodegradation of hydrocarbons (mainly toluene) in the highly active plumefringes above and beneath the plume core. In both zones the concentration of sulphur intermediates (S0, SO3

2� and S2O32�)

was almost twice that of other sampling-depths, indicating intense sulphur redox cycling. The dual isotopic fingerprint of oxy-gen and sulphur in dissolved sulphate suggested a re-oxidation of reduced sulphur compounds to sulphate especially at theupper fringe zone. An isotopic shift in d34S of S0 of nearly +4& compared to the d34S values of H2S from the same depthlinked to a high abundance (�10%) of sequence reads related to Sulphuricurvum spp. (Epsilonproteobacteria) in the same depthwere indicative of intensive oxidation of S0 to sulphate in this zone. At the lower plume fringe S0 constituted the main inor-ganic sulphur species, possibly formed by abiotic re-oxidation of H2S with Fe(III)oxides subsequent to sulphate reduction.These results provide first insights into intense sulphur redox cycling in a hydrocarbon contaminant plume, which widensthe perspective of redox processes and microbial interactions ongoing in contaminated aquifers.� 2015 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.gca.2015.01.014

0016-7037/� 2015 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +49 8921833.E-mail address: f.einsiedl@tum.de (F. Einsiedl).

1 Current address: ExxonMobil Research and Engineering, 1545Route 22, Annandale, NJ, USA.

2 Current address: Munich University of Applied Sciences,Dachauer Str. 100a, 80636 Munchen, Germany.

1. INTRODUCTION

A wide variety of anthropogenic compounds such asBenzene, Toluene, Ethylbenzene and Xylenes (BTEX) andpolycyclic aromatic hydrocarbons (PAHs) cause pollution

208 F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221

of groundwater systems (e.g. Zamfirescu and Grathwohl,2001; Anneser et al., 2008). The degradation of such hydro-carbons by intrinsic microbial communities is a key-compo-nent of natural attenuation in groundwater, makesremediation schemes both more economical, and providesmore sustainable solutions than conventional remediationsuch as pump and treat methods (Lovley, 2003).Dissimilatory sulphate reduction (DSR) is often the domi-nant electron-accepting process in organically contaminat-ed aquifers (Bolliger et al., 2001; Spence et al., 2005;Knoller et al., 2006; Anneser et al., 2008). Hydrogen sul-phide (H2S) is formed during DSR, a reactive compoundwhich undergoes multiple chemical reactions under differ-ent redox conditions. Yet, the role of potential re-oxidativesulphur cycling in biodegradation of contaminants ingroundwater particularly near the water table is still poorlyunderstood.

Under oxic conditions hydrogen sulphide is rapidly re-oxidised to sulphate (SO4

2�) either by biotic or abiotic reac-tions (Jørgensen and Nelson, 2004). In contrast, H2S is notdirectly transformed to SO4

2� in oxygen-limited habitats,but forms different sulphur intermediates such as sulphite(SO3

2�) and thiosulphate (S2O32�) (Zhang and Millero,

1993). Under strictly anoxic conditions, H2S can be re-oxi-dised to elemental sulphur (S0) with iron- and manganeseoxides and to a lesser extent to polysulphides (Sn

2�),S2O3

2�, and SO32� (Moses and Herman, 1991; Peiffer et al.,

1992; Zopfi et al., 2004; Kamyshny and Ferdelman,2010). Elemental sulphur itself reacts with unreacted H2Sto Sn

2�, whereas the very reactive SO32� tends to react with

S0 to produce S2O32�, resulting in a rapid removal of

SO32� and the accumulation of S2O3

2�in sediments(Jørgensen and Bak, 1991). Under anoxic conditions andin the presence of ferrous iron, dissolved H2S precipitatesas monosulphide (FeS) and subsequently reacts with S0 topyrite (FeS2). In deep anoxic freshwater systems with lowFe(II) concentrations, however, Einsiedl et al. (2007) foundthat in the presence of dissolved organic matter H2S wasalso partly incorporated in fulvic acids. Pyrite is the mostabundant inorganic sulphur species in marine sedimentsand was also found to be dominant in gasoline contaminat-ed aquifers (Knoller and Schubert, 2010; Van Stempvoortand Kwong, 2010). In the presence of molecular oxygenFeS2 may be oxidised to sulphate, and to a lesser extent,to the sulphur intermediates S2O3

2� and SO32� (Moses and

Herman, 1991; Schippers and Sand, 1996).Hydrogen sulphide can also be rapidly oxidised to sul-

phate by microbes in the presence of oxygen or nitrate(Jørgensen and Nelson, 2004). Limited supply of these oxi-dants, however, also leads to the formation of sulphurintermediates. The role of chemolithoautotrophic hydrogensulphide oxidising bacteria such as Thiobacillus orThioploca, the latter uses nitrate that is stored in intracellu-lar vacuoles to oxidise H2S, was intensively studied in mar-ine sediments, but hardly in groundwater systems to date(Jørgensen and Nelson, 2004 and Ref. therein). Sulphurintermediates which are formed during incomplete oxida-tion of H2S can further undergo disproportionation, wheresulphur species are at the same time reduced to H2S andoxidised to SO4

2� (Bak and Cypionka, 1987).

Stable isotope fractionation has frequently been used toelucidate the biogeochemical pathways of sulphur cycling inpristine aquatic habitats (e.g. Jørgensen and Bak, 1991;Mayer et al., 1995; Habicht and Canfield, 1997; Einsiedland Mayer, 2005). In addition, individual biodegradationstudies on organically contaminated aquifers with DSR asthe dominant electron accepting process made use of theisotopic composition of d34S and d18O in the remaining dis-solved sulphate to qualitatively and quantitatively assessDSR (Schroth et al., 2001; Knoller et al., 2006; Gibsonet al., 2011; Druhan et al., 2014). Sulphur isotope enrich-ment factors which were obtained for toluene degradingsulphate–reducers in the field ranged from approximately�14 to �25&, with an average value of approximately�23& (Bolliger et al., 1999; Schroth et al., 2001; Knolleret al., 2006). Knoller and Schubert (2010) reported onstrong reservoir effects of H2S in sediments of a con-taminated aquifer leading to stronger isotopic shifts of upto 20& between observed d34S values and stable isotopicfractionation predicted by the Rayleigh equation.Moreover, Abe and Hunkeler (2006) showed that physical-ly heterogeneous systems can mask isotope distribution pat-terns and lead to an underestimation of the extent ofbiodegradation. It has also been suggested that sorptionprocesses in expanding plumes (Kopinke et al., 2005) aswell as mixing due to aquifer heterogeneity and Monod-type degradation kinetics may affect the extent of stable iso-tope fractionation (Van Breukelen et al., 2004).

Although many studies have focused on DSR in con-taminated aquifers and the effect of mixing of electrondonors and acceptors on microbial degradation at geo-chemical gradients (Cirpka et al., 1999; Prommer et al.,2006; Tuxen et al., 2006; Anneser et al., 2008), in situ sul-phur redox-cycling processes that consider the role of themicrobial community distribution in groundwater havenot been elucidated. In contrast, the formation of interme-diate oxidation products of H2S and sulphur redox cyclinghave intensively investigated for marine environments(Jørgensen and Bak, 1991; Zopfi et al., 2004; Holmkvistet al., 2011) and in laboratory experiments (Druhan et al.,2014).

We have previously investigated the spatial distributionof biodegradation processes and degrader communities at ahydrocarbon- contaminated aquifer in Germany (Anneseret al., 2008, 2010; Winderl et al., 2008; Pilloni et al.,2011). Sampling was performed at a single high-resolutionmulti-level well (HR-MLW) as well as from fresh sedimentcores taken during the installation of the well. The estab-lishment of a highly specialised and active toluene-degrad-ing microbial community driven by sulphate reductionwas observed at the lower plume fringe (Anneser et al.,2008; Winderl et al., 2008). A central role ofDesulfobulbaceae in toluene degradation at this con-taminated site with sulphate as the electron acceptor wasdemonstrated for this site by stable isotope probing (SIP)(Pilloni et al., 2011). The same SIP study excluded a directrole of Geobacter populations in hydrocarbon degradationcoupled to ferric iron reduction in situ. However, a poten-tially indirect role of Geobacter spp. in biodegradationinvolving the use of S0 as electron acceptor, as

F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221 209

demonstrated for other sites and pure-cultures (Caccavoet al., 1994; Lovley et al., 1995), has not been tackled todate. Also disproportionation of sulphur intermediatessuch as S0 could be of relevance in areas of contaminatedaquifers where electron acceptors are depleted.

In this study, we trace the formation and the spatial dis-tribution of the sulphur intermediates S0, SO3

2� and S2O32�

to elucidate potential microbial re-oxidation processes inthe aforementioned hydrocarbon-contaminated aquifer.Sulphur species were correlated to the general biogeochem-istry as well as to microbial community distribution acrossa high-resolution depth profile. Our approach aimed to (i)explore sulphur redox cycling processes by using stable sul-phur and oxygen isotopes of dissolved sulphate, hydrogensulphide and elemental sulphur, (ii) characterise oxidativesulphur cycling processes driven by groundwater table fluc-tuation leading to a zone of changing redox conditions atthe saturated/unsaturated interface, (iii) unravel microbialcommunity components potentially involved in these pro-cesses, and finally (iv) localise potential hot-spots ofbiodegradation involving sulphur intermediates.

2. MATERIALS AND METHODS

2.1. Study site

The investigated site is a well characterised porous aqui-fer located at a former gas work plant in Dusseldorf-Flingern, Germany (Anneser et al., 2008). Most of the sur-face at the former gas works site is sealed excluding that sig-nificant amounts of seepage water infiltrate from theunsaturated zone into the aquifer. A multi-level well galleryhas been installed at this field site in order to characterisethe distribution of contaminant concentrations in the aqui-fer (Fig. 1). The aquifer is of Ca–HCO3

� type and a plumewith a length of approximately 200 m, a width of 40 m, and

Contaminant plume

Fig. 1. Schematic view and cross-section through the investigated aquiferthe high resolution multi-level well (HR-MLW), and the adjacent convdistribution; bgs = below ground surface.

a thickness of 2 m was estimated (Anneser et al. 2008). Thecontaminant plume is composed of monoaromatic (BTEX)and polycyclic aromatic hydrocarbons (PAH), with concen-trations of up to 100 and 10 mg/L, respectively (Anneseret al. 2008). BTEX were mostly detected in the liquid phasenear the groundwater table, while significant amounts ofPAH were present in sediments stemming from deeperzones of the aquifer (Anneser et al., 2010). In 2005, onehigh-resolution multi-level well was installed (HR-MLW)in the centreline of the petroleum–hydrocarbon-con-taminated plume, about 15 m downstream of the con-taminant source, allowing sampling between 3 and12 m bgs. The HR-MLW was equipped with samplingports every 3–30 cm.

The aquifer, with a thickness between 10 and 15 m, ismainly composed of medium and coarse sand interruptedby distinct thin gravel layers at depths >10 m below groundsurface (bgs). The mean hydraulic conductivity of the sedi-ments is 1 � 10�3 m s�1 and a mean flow-velocity of about1 m day�1 was estimated (Wisotzki and Eckert, 1997). Ahydraulic gradient of 6& was calculated with a flow direc-tion from east to west (Wisotzki and Eckert, 1997). At adepth of approximately 15 m bgs, the aquifer is confinedby Tertiary sand layers with low permeability.

During our sampling campaign in 2007 the groundwatertable was found at approximately 6.5 m bgs. The redoxpotential, pH, and the electrical conductivity were mea-sured in-situ (WTW, Weilheim, Germany).

2.2. Groundwater sampling and sample preparation

In February 2007 a one-dimensional (1D) snapshot wastaken with respect to the sulphur species dissolved ingroundwater and 25 different depths (n = 25) were sampledat the HR-MLW (Fig. 1). Water was withdrawn throughstainless steel capillaries (inner diameter = 1 mm) and

Contami-na�on

depicting the geological situation and the approximate location ofentional multi-level well (C-MLW) in relation to the contaminant

210 F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221

collected in 100 mL glass bottles after the tubes and filterscreens of the HR.MLW were purged. In order to preventmixing of groundwater from different depths, 25 filterscreens were sampled simultaneously using a peristalticpump. Concentrations of dissolved sulphide in samples(n = 25) were measured immediately on site. Dissolvedhydrogen sulphide was determined in groundwater samplesby the colorimetric methylene blue method of Cline (1969)which was modified for extra small sample volumes of200 lL. The detection limit for this method was1 lmol L�1 and the analytical reproducibility was in gener-al better than ±5%. Groundwater samples dedicated for themeasurement of sulphate and hydrogen sulphide weredirectly fixed with 10 mL of a 20% (w/v) Zn acetate solutionin order to prevent re-oxidation of dissolved H2S to sul-phate. Thereafter, samples were filtered to remove hydro-gen sulphide as ZnS and sulphate concentrations weredetermined using a Dionex DX 100 ion chromatograph.Subsamples for S2O3

2� (n = 7) and SO32� (n = 6) measure-

ments were stabilized by the addition of formaldehyde toa final concentration of 20 lM. Sulphite was measured ashydroxymethanesulfonate within 36 h (Lindgren et al.,1982).

For sulphur stable isotope analysis in dissolved H2S(n = 23) hydrogen sulphide was preserved with Zn-acetateas ZnS and filtered through a 0.45 lm pore size filter(Millipore). For sulphur and oxygen stable isotope analysesin groundwater sulphate the filtrate was acidified to apH < 4 to remove HCO3

� and sulphate was precipitatedas BaSO4 with 3 mL of a 10% BaCl2 solution. The pre-cipitate was recovered by centrifuging or filtration and care-fully washed and dried prior to d34S and d18O isotopeanalyses. For determination of d34S in hydrogen sulphideZnS was precipitated with AgNO3 as Ag2S at a temperatureof approximately 60 �C as described in Schwientek et al.(2008). Finally, the Ag2S formed was carefully filtered,dried, and measured for d34S. Elemental sulphur stemmingfrom the uppermost few centimetres of the aquifer andfrom the lower plume fringe was recovered by Soxhlet dis-tillation using dichloromethane as a solvent (Hall et al.,1988). Subsequently, elemental sulphur was precipitatedon activated copper as copper sulphide and afterwardstransferred to AgS2 (Hall et al., 1988).

For BTEX analysis, a sample volume of 3 mL was trans-ferred into 10 mL headspace GC vials. Thereafter 300 lL of1 M NaOH (final concentration 0.1 M) was added to thewater sample to stop biological activity and vials weresealed with gas tight polytetrafluoroethylene (PTFE) septa.Samples dedicated for the analyses of polycyclic aromatichydrocarbons (PAH) were filled in 15 mL glass vials(Supelco/Sigma–Aldrich, Munich, Germany) and closedwith PTFE-coated screw caps. In the laboratory, PAH wereextracted from groundwater using cyclohexane. Furtherinformation on the concentration measurements of BTEXand PAH using GC–MS can be found in Anneser et al.(2008).

For microbiological investigations, in 2007approximately 750 mL of water was collected for differentdepths (6.5, 6.8, 7.1, 8 and 9 m bgs) in autoclaved 1 L glassbottles from Schott. Microbial biomass was collected on

0.22 lm cellulose acetate filters (Corning Inc., NY, USA)on site immediately after sampling and stored frozen ondry ice and later at �23 �C until DNA extraction. Watersamples from three depths (�6.5, 6.8, 7.1 m bgs) were alsochosen for detailed microbial community analyses.

2.3. Sediment sampling

For the determination of solid S0, sediment samplesfrom eleven depths were subsampled at a vertical resolutionof 0.1 m from sediment cores which were available after theinstallation of the HR-MLW in August 2005. Ten gram ali-quots of sediment material sampled at a vertical resolutionof 0.1 m were placed in plastic bags flooded with argon gas,fixed in 20% (w/v) zinc acetate solution and stored at�20 �C. Three out of 11 samples were taken for d34S mea-surements on S0 (6.50–6.55, upper plume fringe; 6.80–6.90,and 7.10–7.20 m bgs, lower plume fringe).

2.4. Identification of sulphur intermediates

Formaldehyde-fixed water samples were filtered through0.2 lm nylon filters and sulphite and thiosulphate weredetermined by ion chromatography (Metrohm IC-System,with Metrosep Anion Dual 3 column, 0.8 mL min�1 withchemical suppression). With an injection volume of100 lL the detection limits for S2O3

2� and SO32� were 0.5

and 1 lmol L�1, respectively. The precision for measure-ments of 10 lmol L�1 standards was better than ±3%.

Elemental sulphur (n = 11) was extracted with metha-nol/water solution (�5:1) from zinc acetate-fixed wet sedi-ment samples (2 g) over 12 h on a rotary shaker. For thisanalysis we used very fine sand, silt and clay material onlywhich was separated by sieving from coarser sedimentmaterial. Thereafter, the sediment was separated by cen-trifugation and subsequent filtration on 0.2 lm nylon filter.Elemental sulphur, measured as cyclo-S8, was determinedby HPLC using a C18-column at a flow rate of0.4 mL min�1 with a methanol/water solution (5:1) as theeluent and UV detection at 265 nm. Standard dilutionsfrom 1 to 1000 lM were prepared also in 5:1 methanol/wa-ter solution, from 2 mM stock solution of S0 in methanol(16 mg S0 dissolved in 25 mL methanol) and used ascalibration curve for the analyses. The detection limit forS0 was <0.4 lmol L�1 and the analytical precision of themethod was better than ±3%.

Contaminant concentrations (BTEX and PAH) weredetermined with a Trace DSQ GC–MS instrument(Thermo Electron, Dreieich, Germany) equipped with aCombi PAL autosampler (CTC Analytics, Zwingen,Switzerland). Details for analytical procedure can be foundby Anneser et al. (2008).

2.5. Stable isotope analysis

Stable isotope measurements were performed by isotoperatio mass spectrometry (IRMS Thermo Electron MAT253) after complete conversion of BaSO4 or AgS2 to SO2,via high temperature reaction with WO2 and V2O5. Ford18O measurements in sulphate, CO was produced through

F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221 211

pyrolysis of BaSO4 with pure graphite under vacuum at1450 �C and subsequent isotope analysis was conductedagain by IRMS, as described in Holt (1991).

Sulphur and oxygen isotope ratios are reported in partsper thousand (&) according to the conventional delta nota-tion (Eq. (1)):

d ¼ Rsample

Rstandard

� 1

� �� 1000 ð1Þ

where Rsample and Rstandard are the 34S/32S or 18O/16O ratiosof the sample and the standard, respectively. The d34S val-ues are reported relative to the Canyon Diablo Triolitestandard (V-CDT). The d18O values of groundwater sul-phate refer to Vienna Standard Mean Ocean Water (V-SMOW). Different international and internal lab standardswere repeatedly measured to calibrate the mass spec-trometer. The reproducibility for d34S measurements ondissolved sulphate and elemental sulphur was ±0.2& and±0.4& for d18O. However due to insufficient fine drillingmaterial it was not possible to repeat extraction procedurefor S0 to determine the overall uncertainty of the prepara-tion of the sediment material on d34S measurements.However pre-experiments with Tertiary sand and artificialadded S0 yielded a recovery of always >80% for S0. Theanalytical uncertainty of d34S measurements using thepreparation method of Hall et al. (1988) was 6±1&.

Stable isotope fractionation was assessed using theRayleigh equation (Eq. (2)).

lnRt

R0

¼ e=1000� lnCt

C0

ð2Þ

Rt and R0 denote the stable isotope ratios of sulphur attimes t and zero. Ct and C0 represent the respective sulphateconcentrations. In a closed system the enrichment factor (e),which is defined as (a � 1) * 1000, describes the relationshipbetween the initial isotope composition of a substrate (R0)relative to its isotopic composition at any given time pointduring the microbial degradation (Rt).

However, it is worth to be mentioned that the Rayleighequation is a description of the redistribution of the iso-topes of an element in a molecule that is undergoing micro-bial degradation in a fully mixed and closed reservoir only.Groundwater systems, in contrast, often represent open sys-tems where concentrations and isotope ratio gradients areaffected by hydrodynamic processes such as dispersionand diffusion (Abe and Hunkeler, 2006). In addition theRayleigh equation is only strictly applicable to a first orderirreversible rate law, whereas microbially mediated redoxreactions are commonly described by Monod orMichaelis–Menten kinetics (Van Breukelen et al., 2004;Druhan et al., 2014). As a result the authors stated thatthe Rayleigh approach doesn’t allow an adequate calcula-tion of the stable isotope enrichment factor for heteroge-neous groundwater system.

2.6. Microbial community analyses

Total DNA for groundwater microbial community ana-lysis was extracted from frozen filters as previouslydescribed (Brielmann et al., 2009). For quantifying total

bacterial 16S rRNA genes and catabolic sulphate reductiongenes, we used a real time PCR (qPCR) approach. 16SrRNA genes were analysed as described by Kunapuliet al. (2007), while for sulphate-reducing genes we usedthe primer pair dsrp2060fw/dsr4rv (Geets et al., 2006).The target DNA amplifies a 350 bp region on the dsrB sub-unit of the dsrAB gene of the dissimilatory sulphite reduc-tase (dsr) operon. The qPCR measurements wereperformed on a MX3000P qPCR cycler (Stratagene). Foreach depth, DNA extracts from filtered groundwater werequantified in two different dilutions (1:10 and 1:50). Each(final volume) 50 ll PCR reaction in nuclease-free H2Ocontained 1� PCR buffer, 3 mM MgCl2, 0.1 mM dNTPs,1 U Taq polymerase (all Promega), 10 mg BSA (Roche),0.5 mM of each primer (MWG Biotech), 0.1� Sybr Green(FMC Bio Products) and 2 ll of DNA template. Initialdenaturation (94 �C, 3 min) was followed by 45 cycles ofdenaturation (94 �C, 30 s), annealing (56 �C, 30 s), andelongation (70 �C, 30 s). Subsequently, a melting curvewas recorded between 56 �C and 94 �C to discriminatebetween specific and unspecific amplification products. Afull length bacterial dsrB gene amplicon from an environ-mental-cloned and sequenced DNA was quantified usingthe PicoGreen double-stranded DNA quantification kit(Molecular Probes) and applied as standard DNA forqPCR in a concentration ranging between 107 and 101

copies ll�1.MID-tagged PCR amplification and amplicon pyrose-

quencing were performed using the Titanium chemistryon a 454 GS FLX pyrosequencer (Roche), as recentlydescribed from the developer. For this study, three depths(�6.5: upper plume fringe zone; �6.8: centre of the plume;and �7.1 m bgs, lower plume fringe zone) selected on thebasis of their sulphur intermediates profiles, were sequencedin a pool of 26 MID-mixed amplicons on 1/4 of a FLXpicotitre plate. Quality filtering and data handling was per-formed as described (Pilloni et al., 2012).

3. RESULTS AND DISCUSSION

3.1. Concentrations of BTEX and sulphur species in

groundwater and in sediments

Physico-chemical parameters and vertical profiles of dis-solved sulphate at high spatial resolution, together with theconcentration of toluene determined in groundwater duringthe sampling campaign in February 2007 are shown inFig. 2. The redox potential (Eh) in the groundwater rapidlydeclined with depth from +300 mV at the uppermost sam-pling point to values between �89 and �42 mV in the cen-tre of the plume (6.81 m bgs) (Fig. 2). Below the plumecentre the redox potential was fairly constant and showedvalues close to 0 mV. The pH values ranged from 6.6 atthe uppermost sampling point to a maximum value of 7.7at 6.9 m bgs. The electrical conductivity ranging from1393 to approximately 1100 lS cm�1 showed the highestvalues at the uppermost few centimetres of the saturatedzone with decreasing values with increasing depth. Whilethe electrical conductivity in general follows the concentra-tion profiles of dissolved sulphate and BTEX, the pH

6.0

6.4

6.8

7.2

7.6

8.0

0 500 1000 1500D

epth

(m b

ls.)

PAH/ BTEX/ Toluene (µM/L), electr. conductivity [µs/cm]

6.00

6.40

6.80

7.20

7.60

8.00

0 1500 3000 4500

SO42- (µM)

6.0

6.4

6.8

7.2

7.6

8.0

6.8 7.0 7.2 7.4 7.6 7.8

pH

6.0

6.4

6.8

7.2

7.6

8.0

-100 0 100 200 300

Redox potential [mV]

6.80

7.20

6.8

7 2

6.8

7 2

Upper plumefringe

Plume coreLower plume

fringe

Groundwater table

A B C D

Fig. 2. High-resolution depth profiles stemming from the HR-MLW show concentrations of PAH (A, solid line), BTEX (A, open squares),toluene (A, open triangles), and electrical conductivity (A, diamonds), concentrations of dissolved sulphate (B), pH values (C), and redoxpotential (D). BTEX and toluene values represent mean of duplicate measurements ± SD (standard deviation). For dissolved sulphate andphysico-chemical parameters error bars were smaller than the symbols.

212 F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221

mirrors the generation of acid by e.g. FeS2 oxidation andmicrobial CO2 production through BTEX degradation withlowest pH values of 6.8 close to the groundwater table.

Dissolved sulphate concentrations in groundwater werefound of up to 2 mM with a mean of about 1.6 mM up-gra-dient and lateral from the hydrocarbon-contaminated area(Wisotzki and Eckert, 1997). Groundwater sampled fromthe HR-MLW had always low nitrate concentrations,mostly below 0.05 mM (Anneser et al., 2008).

The sulphate concentrations in groundwater are highestin the first few centimetres and, thereafter, strongly decreasein the upper 10 cm of the saturated zone starting from4.4 mM to values of around 0.5 mM. High sulphate con-centrations within the first few centimetres of the aquiferwere also found during former sampling campaigns, wheresulphate concentrations were approximately up to �0.5 and�1 mM in 2005 and 2006, respectively, but were significant-ly lower compared to those found in this study (�4.4 mM).Here it is worth to be mentioned that the unsaturated zoneof the investigated aquifer was partially filled with construc-tion waste (e.g. bricks, ash, bed ash, basalt). This construc-tion waste contains a significant amount of sulphate whichcould represent an additional sulphate source for theaquifer. However most of the surface at the former gasworks site is sealed and sulphate leaching from theunsaturated zone may be quite low. Nevertheless we cannotrule out that some leaching of sulphate takes place andaffects the sulphate concentration at the groundwater table.We assume that groundwater table fluctuations change theredox conditions near the groundwater table and mobilise ahigh amount of reduced sulphur species in the unsaturatedzone. The reduced sulphur species is formed during DSR athigher groundwater table and is left-behind in theunsaturated zone during decreasing groundwater tableconditions.

The uppermost 20 cm of the aquifer corresponded to theupper fringe of the BTEX plume, where BTEX (mainlytoluene) increased at the groundwater table from0.011 mM to 0.730 mM in the centre of the plume. Belowthe plume core, in the lower plume fringe, a steady increasein sulphate to approximately 1.34 mM was observed. Anopposing gradient was observed for BTEX, with concentra-tions declining from 0.730 mM to less than 0.019 mM at

7.31 m bgs. As a result, the lower plume fringe was charac-terised by a counter-gradient of dissolved sulphate andBTEX. Sampling revealed concentrations of 0.73 mM oftoluene and 0.5 mM of sulphate in the plume core, hintingat limitations for biodegradation others than the avail-ability of electron donors and acceptors. In the course of10 years of observation (from 2005 to 2014) of hydrocar-bons and electron acceptors at this site, it was found thatduring our sampling campaign in 2007 considerableamounts of dissolved sulphate were present in the plumecore, while during the sampling in 2006 (Anneser et al.,2008) the core of the contaminant plume was almost deplet-ed in sulphate. The HR-MLW was established with a seriesof sampling ports. By means of multi-channel pumps up to25 ports of the HR-MLW were sampled simultaneously.This should minimise the risk of mixing of groundwaterfrom different depths in this quite homogeneous aquifer.The observed counter-gradients of toluene and sulphate,as well as the pronounced appearance of hydrogen sulphidewhere toluene and sulphate meet were striking and weobserve clear chemical gradients. However, the preferentialsampling of those sections of the aquifer with higher perme-ability cannot be ruled out by our sampling strategy andcould lead to the observed concentrations of dissolved sul-phate in the centre of the plume. Two further possibilities,others than artificial effects, could explain the observed highsulphate concentrations within the plume core. Taking intoaccount all data on nitrogen (NO3

� and NH4+) as well as

phosphorus (soluble reactive phosphorus; SRP) we see thatat time points where sulphate is completely reduced traceamounts of nitrate were detectable; while at time pointswhere nitrate was below the detection limit (0.1mg/L = 1.65 lM) some dissolved sulphate (0.5 mM) waspresent in the plume core (Anneser et al., 2008).However, SRP was measureable at all-time points, whichcould indicate an intermittent nitrogen limitation.Another possible explanation for the temporarily high sul-phate concentration in the plume centre could be the kinet-ics of sulphate reduction. Since biodegradation coupled tosulphate reduction is rather slow compared to aerobicbiodegradation and contaminant transformation undernitrate reducing conditions, we speculate that it is a matterof time to fully reduce the sulphate upstream to the HR-

F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221 213

MLW. In combination with groundwater table fluctuationand increasing transversal mixing processes this couldexplain sulphate concentrations to be temporarily presentin the plume core.

The vertical concentration profiles of individual sulphurspecies in groundwater, i.e. hydrogen sulphide, sulphite,thiosulphate and elemental sulphur are depicted in Fig. 3.Within the first few centimetres of the upper plume fringezone (6.56 and 6.59 m bgs.) no dissolved H2S was detected.Further down, elevated levels of dissolved H2S were mea-sured in both the upper and lower fringe zones with max-ima of 0.15 and 0.06 mM, respectively. Below the lowerfringe zone (P7.8 m bgs), H2S concentrations dropped to0.01 mM. The gradients of total BTEX and sulphateaccompanied by elevated H2S concentrations clearly indi-cated zones of high microbial sulphate reduction and thushot-spots of contaminant biodegradation, particularly atthe upper and lower plume fringes. The intermediateSO3

2� followed similar patterns exhibiting highest values(26 lM) in the upper and lower fringes (15 mM), similarto the observed dissolved H2S concentrations (Fig. 3).Also two maxima of S2O3

2� were found in the upper(313 lM) and lower fringe (160 lM) at a depth of 6.7 mand between 7.1 and 7.3 m bgs, again corresponding withthe highest values for H2S and S0 (Fig. 3). Concentrationsof S0, as determined from sediment samples, ranged from0.313 mmol/g at the upper gradient zone to 0.068 mmol/gsediment at a depth of 7.7 m bgs (Fig. 3) with maximumconcentrations at depths of 6.7 m bgs and between 7.1and 7.3 m bgs. The concentration of S0 stemming fromthe unsaturated zone was 63 lM/g sediment.

The occurrence of elevated concentrations of H2S at theupper and lower plume fringe zones (Fig. 3) accompaniedby a local depletion of dissolved sulphate and decreasingBTEX concentrations (Fig. 2) hints at BTEX biodegrada-tion coupled to DSR. Locally elevated concentrations ofthe very reactive SO3

2� and S2O32� were linked with the

hydrogen sulphide peak located close to the unsaturated/saturated interface as well as the depth of the second hydro-gen sulphide peak with a depth of �7.3 m bgs.

Near the groundwater table the redox potential wasfound positive during our sampling campaign in 2007(Fig. 2). It is highly likely that oxygen deriving from theunsaturated zone occasionally diffused into the first fewcentimetres of the saturated zone. Since moderate but

6.0

6.4

6.8

7.2

7.6

8.0

0 10 20 30

SO32-(µM)

6.0

6.4

6.8

7.2

7.6

8.0

0 50 100 150 200

Dep

th (m

bls

.)

H2S (µM)

6.8

7.2

Groundwater table

Fig. 3. Mean concentrations of duplicate measurements of H2S (left), SOSD was smaller than the size of the data points.

frequent groundwater table fluctuations have beenobserved at this site reduced solid sulphur compoundsmay repeatedly have faced unsaturated conditions andexposure to O2. Wisotzki and Eckert (1997) reported onthe accumulation of FeS and FeS2 in the upper plumefringe probably as a product of the interaction betweenH2S and Fe(II), with the latter formed during bacterial ironreduction (Wisotzki and Eckert, 1997; Anneser et al., 2008).According to the conventional understanding of FeS/FeS2

oxidation and the oxidation of suphide under oxygen-limit-ed conditions (Zhang and Millero, 1993), diffusion of oxy-gen from the unsaturated zone as well as a lowering ofthe groundwater table and exposure to unsaturated oxicconditions may facilitate the oxidation of reduced sulphurphases at the unsaturated/saturated interface. Since oxida-tion of FeS2 and FeS was shown to be accompanied bythe formation of SO3

2� and S2O32� in marine sediments

(Moses and Herman, 1991; Schippers and Jorgensen,2001), the observed high thiosulphate concentrations inthe uppermost few centimetres of the aquifer may partiallybe the result of re-oxidation processes of FeS/FeS2. This issupported by pH measurements (Fig. 2). Acid is formed bythe FeS2/FeS oxidation lowering the pH together with theformation of acid by microbial production of CO2 and mix-ing with atmospheric pCO2 near the groundwater tablefrom 7.4 to about 6.8 in the uppermost few centimetres ofthe aquifer. As the saturated/unsaturated interface repre-sents a very dynamic zone of frequently changing redoxconditions, we also speculate that S0 and SO3

2� may beformed by the microbial reduction of sulphate to H2S underoxygen-limited conditions, followed by the incompleteoxidation of unreacted H2S to S0 and SO3

2�.Also a high abundance of Fe(III) phases was reported

for this aquifer (Wisotzki and Eckert, 1997; Anneseret al., 2008). Under strictly anoxic redox conditions, whenDSR can be assumed to dominate the uppermost few cen-timetres of the saturated zone, these Fe(III)oxides mayreact with dissolved H2S to S0 (Peiffer et al., 1992) andto a lower extent to SO3

2�, which again reacts with S0

resulting in a rapid removal of SO32� and the accumula-

tion of S2O32� in the sediments (Jørgensen and Bak,

1991; Zopfi et al., 2004). Similar sulphur cycling processeswere suggested for marine sediments (Jørgensen and Bak,1991), where S2O3

2� accumulated in the uppermost fewcentimetres, constituted about 70% of the immediate

6

6.4

6.8

7.2

7.6

8

0 100 200 300 400 500

S2O32- (µM), S0 (µM/ g)

40 50 60

6.8

7.2

Upper plume fringe

Lower plume fringe

Plume core

32� (crosses), S2O3

2� (diamonds) ± SD and S0 (triangles). For S2O32�,

214 F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221

H2S-oxidation products. The presence of sulphur interme-diates and H2S and the accumulation of S0 and FeS/FeS2

may be a consequence of frequently changing redox con-ditions within the first few centimetres of the aquifer(Fig. 4). Accumulation of S0 has also been found in sedi-ments beneath waste disposal sites in England (Bottrellet al., 1995) and Denmark (Crouzet et al., 2000), for ahydrocarbon contaminated site in Canada (VanStempvoort and Kwong, 2010), and in column experi-ments packed with porous media stemming from theOld Rifle aquifer (Druhan et al., 2014). Here, S0 andH2S were taken as clear evidence of DSR. High concen-trations of sulphur intermediates, formed during sulphurcycling processes, are also reported in highly active mar-ine environments, where non steady-state conditions mayhave led to their accumulation (Luther, 1991; Zopfiet al., 2008).

The second, less pronounced peak of SO32�, S2O3

2�, andS0 in the lower plume fringe zone cannot be related tothe interaction between molecular oxygen and H2S orFeS/FeS2. Several sampling campaigns documented strong-ly reduced conditions in this zone of the aquifer (Anneseret al., 2008; Anneser et al., 2010). Therefore, it can beassumed that H2S directly precipitates with Fe2+ to FeSas well as reacts with Fe(III) minerals to sulphurintermediates.

In general, the formation of sulphur intermediates dur-ing sulphide oxidation and their further microbial turnoverby disproportionation, oxidation or reduction may occur inparallel to abiotic transformation processes. For deeperinsights into the microbial components of these processesdepth-resolved bacterial community patterns and stable iso-tope measurements of dissolved sulphate, sulphide and S0

were performed here.

6.0

6.4

6.8

7.2

7.6

8.0

-400 -200 0 200 400

May 06 Feb 07

May 09 Jun 09

Redox potential [mV]

Dep

th (m

bls

.)

Groundwater tablefluctuation

Plume core

Fig. 4. Changing redox potential over an observation period of3 years.

3.2. Depth-resolved microbial communities and their potential

role in sulphur cycling

The total number of bacteria, as estimated via 16 rRNAgene qPCR for groundwater sampled in Feb. 2007, showedan abundant bacterial community with up to 1010 genescopies per litre of groundwater (Fig. 5), especially at theupper and lower plume fringes, where we also identifiedthe highest sulphide concentrations (Fig. 3). In contrast,minimum counts of total bacterial genes were detected inthe plume core. Similar patterns were observed for theabundance of dsrB genes over depth (Fig. 5), with generallyover one order of magnitude lower counts, but with evenmore marked distinctions over depth. The maximal ratioof dsrB was found within the lower fringe zone, where itaccounted for 90% of the bacterial rRNA gene counts.This is in agreement with previous observations identifyingthe lower plume fringe zone as hot-spot for biodegradationand DSR (Anneser et al., 2008, 2010; Winderl et al., 2008).

To relate the relative abundance of potential sulphatereducers to specific bacterial lineages, pyrotag sequencingof total bacterial communities for water samples collectedin 2007 was performed for three selected depths with astrong indication for intensive sulphur cycling. Prominentdifferences in the bacterial community composition of theanalysed samples were observed (Table 1). Proteobacteria

were well represented in all three examined depths (6.5 m,6.8 m, and 7.1 m bgs), with exceptionally high ratios at6.5 m bgs, which represents the upper plume fringe zone.This depth was the one characterised also by higherrelative abundance of Betaproteobacteria (19%), Deltapro-

teobacteria (�27%) and also Epsilonproteobacteria (24%).Especially amongst the latter, the detected reads relatedto Sulphuricurvum spp. (�10%) represent well-known aero-bic or nitrate-reducing oxidisers of sulphide, thiosulphate

dsrB 16S rRNA

dsrB/16S ratio

Genes abundance [copies/L]

Dep

th[m

bgs]

Fig. 5. Depth-distribution, as quantified via real time PCR, of 16SrRNA and dsrB genes and their ratio (broken line).

Table 1Pylogenetic assignment of 454 16S pyrotag reads at selected depths. Phylum- or division-level read abundances (bold) include genus- orlineage-specific read abundances (non bold).

Phylogenetic affiliation 6.5 m bgs 6.8 m bgs 7.1 m bgs

Reads % Reads % Reads %

Bacteria 9345 100 6999 100 7699 100

Unclassified Bacteria 281 3.0 618 8.8 539 7.0Proteobacteria 7765 83.1 1437 20.5 4574 59.4

Unclassified Proteobacteria 313 3.3 15 0.2 9 0.1Alphaproteobacteria 58 0.6 8 0.1 4 0.1

Betaproteobacteria 1741 18.6 348 5.0 110 1.4

Unclassified Betaproteobacteria 968 10.4 125 1.79 12 0.2Burkholderiales 288 3.1 97 1.4 48 0.6Comamonadaceae 272 2.9 96 1.4 45 0.6Rhodocyclales 467 5.0 96 1.4 36 0.5

Gammaproteobacteria 920 9.8 67 1.0 4 0.1

Pseudomonadales 847 9.06 53 0.76 1 0.0Deltaproteobacteria 2504 26.8 937 13.4 4440 57.6

Unclassified Deltaproteobacteria 28 0.3 69 1.0 53 0.7Geobacteraceae 109 1.2 101 1.4 18 0.2Desulfobulbaceae 2287 24.5 250 3.6 4074 52.9Desulfobacteraceae 27 0.3 190 2.7 100 1.3

Epsilonproteobacteria 2229 23.9 62 0.9 7 0.1

Helicobacteraceae 2093 22.4 61 0.9 3 0.0Sulphuricurvum 896 9.6 31 0.4 0 0.0

Firmicutes 530 5.7 1040 14.9 877 11.4

Unclassified Firmicutes 14 0.1 93 1.3 80 1.0Clostridia 514 5.5 941 13.4 755 9.8

Sedimentibacter 90 1.0 338 4.8 557 7.2Desulfosporosinus 289 3.1 368 5.3 9 0.1

Bacteroidetes 468 5.0 1998 28.5 1330 17.3

Unclassified Bacteroidetes 250 2.7 1233 17.6 868 11.3Porphyromonadaceae 212 2.3 753 10.8 452 5.9

Chloroflexi 259 2.8 1594 22.8 218 2.8

Unclassified Anaerolineaceae 258 2.8 1566 22.4 211 2.7Spirochaetes 16 0.2 116 1.7 42 0.5

OP11 8 0.1 128 1.8 73 0.9

F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221 215

or elemental sulphur under low oxygen availability (Wrightet al., 2013). It has been demonstrated for marine shelfwaters that chemolithoautotrophic Gamma- andEpsilonproteobacteria can counteract the expansion of deepsulfidic waters (Lavik et al., 2009). Higher ratios (�24%) ofsite-specific sulfidogenic toluene degraders within theDesulfobulbaceae (Pilloni et al., 2011) were also identifiedfor this depth. However, members of this lineage haverecently also been shown capable of aerobic sulphide oxida-tion in marine sediments (Pfeffer et al., 2012).

The sample from the plume core, localised at 6.8 m bgs,was characterised by a different community, mainly com-posed of Bacteroidetes and Anaerolineaceae (Chloroflexi) atrelative abundances of 28% and 23%, respectively. Whilethe Proteobacteria in general, and the site-specific degraderDesulfobulbaceae in particular, were strongly reduced at thisdepth, an increased relative abundance of Clostridia (13.4%),was observed. Among them, Desulfosporosinus spp. has beenas identified as secondary toluene degrader and sulphatereducer in sediments of the field-site (Pilloni et al., 2011). Ithas also been shown that representatives of the genusClostridium can be capable of reducing S2O3

2�, Sn2� and S0

associated with Sn2� (Takahashi et al., 2010).

The final depth screened, localised at 7.1 m bgs, corre-sponded to the lower plume fringe and was again highlyenriched in Desulfobulbaceae (up to �53% of the total com-munity), followed by Bacteroidetes (17%) and Clostridia

(�10%). The presence of Bacteroidetes at the lower gradientcould potentially be related to sulphur re-oxidation process-es in this zone, since some of these microbes carry genesrelated to sulphur oxidation (Friedrich et al., 2005). In par-ticular the Flavobacteriaceae, found to be presented by 6%of the reads, have been described as dimethylsulphideoxidisers in marine environments (Green et al., 2011), buttheir role in anoxic habitats is unclear to date.

3.3. Stable sulphur and oxygen isotope signatures in sulphate,

sulphide, and elemental sulphur

3.3.1. Bacterial sulphate reduction and microbial oxidation of

S0

In previous studies conventional multi-level wells wereused to collect water samples to identify the natural atten-uation potential of the test field site. In 1995, Wisotzkiand Eckert (1997) sampled a conventional multi-level well(MLW) with a depth-resolution between 0.5 and 1 m

216 F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221

approximately 200 m upstream of the HR-MLW and founda vertical distribution of d34S and d18O signatures in dis-solved sulphate with maxima of up to 41 and 15&, respec-tively, in the centre of the plume. In 2006, selected ports ofthe HR-MLW were sampled for dissolved sulphide, Fe(II),major anions and cations as well as aromatic hydrocarbons.In addition, stable isotope measurements on dissolved sul-phate were performed and compared with the results whichwere obtained from a conventional MLW located closest tothe HR-MLW (Fig. 1). The results showed clear evidence ofDSR coupled to biodegradation of toluene at the plumefringes with d34S values somewhat above 40& (Anneseret al., 2010). In February 2007 the d34S values of groundwa-ter sulphate (Fig. 6) increased from 6.6& at the capillaryfringe to values of up to 48& in the plume core. The iso-topic signature of the remaining dissolved sulphate in theplume core indicates that the differences in sulphate concen-trations between the plume centre and the deeper non-con-taminated zones are due to microbial sulphate reduction. Incontrast, our results from bacterial community analysis hintat the plume fringes to be the hotspots of biodegradationcoupled to DSR (Anneser et al., 2008, 2010; Winderlet al., 2008). This apparent contradiction could beexplained by three different scenarios. Firstly, isotope frac-tionation can be affected by transverse dispersion. Thelighter isotope shows a higher diffusion coefficient com-pared to the heavier one and results, therefore, in an enrich-ment of the lighter isotope at the plume fringes. This effectwas experimentally demonstrated with a mixture of non-la-belled ethylbenzene (H10) and fully labelled ethylbenzene(D10) (Rolle et al., 2010). The authors concluded thatneglecting the fractionation by dispersion leads to an over-estimation of biodegradation at the plume core and anunderestimation of biodegradation at the plume fringes.However, the authors also stated that the isotope effect thatis controlled by transverse mixing is expected to be muchsmaller when dealing with natural non-labelled compound,for which the differences of masses of the isotopologues aremuch smaller, resulting in more minute differences in theaqueous diffusion coefficients. A modelling study has beenpreviously performed for the Flingern site by Prommer

Fig. 6. Left: Isotopic profiles of groundwater sulphate (d34S, open squashown as mean values of duplicate measurements ± SD and d34S value(±1&), right: d34S (open symbols) and d18O values of dissolved groundwand hydrogen sulphide all measurement uncertainties were smaller thanisotopic difference between measured d34S values of dissolved sulphate a

et al. (2009). Their modelling results showed only little iso-tope effects of transverse dispersion on the isotope rationsof sulphate at the plume fringes. Secondly, transverse dis-persion may also control mixing of dissolved sulphate fromoutside the contaminant plume with sulphate deriving fromthe plume centre. As sulphate concentrations in the plumecentre are lower compared to those found closer to theplume fringes the isotopic signature of dissolved sulphatemay be stronger masked by mixing within the plume centre.Thirdly, we performed one additional sampling campaignat one HR-MLW. It is likely that most of the sulphate isalready reduced close to the contaminant source and theremaining dissolved sulphate that is enriched in the heavier34S isotope is transported down to the plume core. As aresult we observed highest d34S values upstream of theHR-MLW, since most of the sulphate has been reducedalong the pathway and observed a minor role of DSR inthe plume centre close to the HR-MLW. With increasingdepth d34S values decreased concomitantly with an increasein sulphate concentrations and decreasing BTEX (toluene)concentrations (Figs. 2 and 6). Below a depth of 7.2 m bgs,d34S values of groundwater sulphate were fairly constant atvalues of �19&, accompanied by toluene concentrations ofless than 0.01 mM. The elevated stable isotope values ofsulphate linked to the observed concentrations of very reac-tive sulphur intermediates such as SO3

2� as well as the pres-ence of sulphate reducers provide strong evidence that DSRis co-occurring with abiotic sulphur recycling processesacross the depth profile and especially at the plume fringes.As we have never detected molecular oxygen in the lowerplume fringe zone during our sampling campaigns, we sug-gest that sulphur intermediates are formed during the re-oxidation of H2S with iron oxides. Here S0 and SO3

2� areformed during the reaction (Moses and Herman, 1991;Peiffer et al., 1992; Zopfi et al., 2004; Kamyshny andFerdelman, 2010). The very reactive SO3

2� tends to reactwith S0 to produce S2O3

2�, resulting in a rapid removal ofSO3

2� and the accumulation of S2O32�in sediments

(Jørgensen and Bak, 1991), as observed in our study. Theformed monosulphides (FeS) may subsequently furtherreact with S0 to pyrite (FeS2), leading to a high abundance

res), measured d34S of dissolved hydrogen sulphide (open circles)s of S0 (triangles) with maximum uncertainty of the used methodater sulphate (filled squares). For d34S and d18O values in sulphatethe size of the data points. The enrichment factor e represents thend hydrogen sulphide.

Fig. 7. d34S against d18O isotope plot for the upper fringe zonebetween 6.56 and 6.70 m bgs (open circles) and the plume core andthe lower fringe zone (open squares) located below 6.70 m.

F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221 217

of reduced sulphur minerals at this site (Wisotzki andEckert, 1997).

The d34S values of S0 were between 2.8 and �12.2&. Inthe upper plume fringe the d34S values of sulphide closelyfollowed the d34S values of S0 from the same depth.Similar d34S values of S0 and H2S can be explained by theabiotic oxidation of H2S to S0 with iron oxides where noor only little stable isotope fractionation between H2Sand sulphate is expected (Bottcher et al., 1998). However,a significant isotopic difference between measured d34S val-ues of S0 and H2S of about 3.8& was observed in the upperplume fringe. If we assume that the isotopic composition ofd34S of S0 and H2S can directly be compared, S0 couldbe repeatedly further oxidise to sulphate with molecularoxygen as electron acceptor resulting in an enrichmentof the heavier 34S in the remaining S0. Particularly, weidentified members of the genus Sulphuricurvum and otherclosely related Epsilonproteobacteria, to be abundant inthis zone, bacteria well-known to be capable of oxidisingsulphur species. These organisms were recently also identi-fied within toluene-degrading sulfidogenic enrichmentsfrom contaminated aquifers (Kleinsteuber et al. 2008;Pilloni et al. 2011). Wright et al. (2013) reported a highabundance of Sulphurovum and Sulphuricurvum, bothEpsilonproteobacteria, in an S0 deposit of Borup FiordPass Glacier in the Canadian High Arctic. The authors sug-gested that Sulphurovum and Sulphuricurvum oxidise S0

with molecular oxygen. A sulphur isotope fractionationbetween S0 and SO4

2� of a few per mill was reported withan increase of the heavier isotopes in S0 (McCready andKrouse, 1982), quite similar to what was observed in ourstudy. On the basis of the isotopic shift in S0 to heavier iso-topes we speculate that Sulphuricurvum oxidise S0 to sul-phate leading to an extra isotopic shift in the d34S of S0

compared to the d34S of H2S.We are aware that our S0 data have to be interpreted

with caution, since S0 was determined in sediment samplesstemming from June 2005, 1.5 years. prior to this ground-water sampling campaign. Therefore we cannot directlycompare sedimentary sulphur with the concentration of sul-phur species found in groundwater. Nevertheless, similardistribution patterns of sulphide in groundwater, even atvalues of up to � 37% and 65% higher in the upper andlower plume fringe zones, respectively, support that S0

was present in groundwater during our sampling campaignin 2007 as well. Quite similar d34S values in sulphate werealso observed during the different sampling campaigns in2005 and 2006 (Winderl et al., 2008; Anneser et al., 2008).In addition, the calculated stable isotope fractionation fac-tor between S0 and sulphate of nearly 4& was close to thevalue published by McCready and Krouse (1982) for thisreaction and a direct role of Sulphuricurvum in oxidationof S0 was found for another site (Wright et al. (2013).

3.3.2. Re-oxidation processes of reduced sulphur compounds

at the upper and lower plume fringe

The contrasting sulphur isotopic composition and dif-ferences in sulphate concentrations in groundwater whichwere sampled from the uppermost 20 cm of the saturatedzone (6.61 m and 6.7 m bgs) and further down between

6.8 and 7.7 m are illustrated in Figs. 6 and 7. Plottingd34S against d18O may document a generally markedeffect of re-oxidation processes of reduced sulphur specieson d34S and d18O in groundwater sulphate. For ground-water which was sampled from the uppermost few cen-timetres of the upper fringe zone (6.61 and 6.75 m bgs)and between 6.8 and 7.8 m bgs (lower plume fringe), dif-ferent slopes in a d34S against d18O plot were observed(Fig. 7).

We hypothesise that an isotopic shift in d34S and d18O ofdissolved sulphate to lower values in the uppermost fewcentimetres of the saturated sediment zone linked to a posi-tive redox potential of up to +300 mV may derive mostlyfrom re-oxidation of sulphur minerals precipitated nearthe groundwater table and which are mobilised by ground-water fluctuations (Fig. 4). Balci et al. (2012) showed intheir laboratory experiments that d18O values in dissolvedsulphate can distinguish aerobic and anaerobic oxidationpathways of FeS and FeS2. As a result d18O values in dis-solved groundwater sulphate were used as indicator of theidentification of pyrite oxidation and the oxidation of ironmonosulphide at our field site. Under oxic conditions atmo-spheric oxygen (+23.5&) and oxygen from water molecules(�7.5&) as two oxygen sources in the aquifer can be incor-porated into newly formed sulphate stemming from re-oxidation processes of reduced sulphur species with a con-tribution of 13% and 87% and an oxygen fractionationeffect of �9.8& and 2.8&, respectively (Balci et al.,2012). The d18O values of dissolved sulphate ofapproximately �2.3& may indicate that sulphate derivesfrom re-oxidation processes of sulphur minerals underaerobic conditions. In our study d18O values in groundwa-ter sulphate of approximately up to �1& and +0.5& werefound in the uppermost 6 cm of the aquifer (Figs. 6 and 7).These values are close to the d18O values reported by Balciet al. (2012) for oxidation of FeS and FeS2 to dissolved sul-phate with oxygen. In addition, re-oxidation of dissolvedreduced sulphur compounds, which are enriched in thelighter 32S isotope, to sulphate are also generally accompa-nied by an isotopic shift to lower d34S values in the newlyformed sulphate, as observed in our study.

218 F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221

Numerical simulations performed for this aquifer haveshown that modelled d34S values for dissolved sulphate ingroundwater significantly overestimated the observed d34Svalues at the upper plume fringe if only DSR without anyre-oxidation processes are taken into account (Prommeret al., 2009). This modelling result is in excellent agreementwith our interpretation that re-oxidation processes of previ-ously formed reduced sulphur compounds reduce distinc-tions in d34S values between H2S and SO4

2� in this zone(Fig. 6).

In the gradient zone below the plume core, where kineticisotopic fractionation may dominate the d18O in remainingsulphate, only a slight increase of d18O values of dissolvedsulphate was found (Fig. 7). Under strictly anoxic condi-tions water-oxygen can be exchanged either during DSRby cell-internal formed sulphuroxy-intermediates as report-ed by Brunner et al., (2005), Mangalo et al. (2007, 2008)and Einsiedl (2009), or by secondary sulphur transforma-tion processes (Balci et al., 2012). Both processes cannotbe separated from each other and we suggest that bothre-oxidation of cell-internal sulphuroxy-intermediates andsulphur-intermediates such as SO3

2� which can exchangewith water–oxygen, may affect the d18O values in theremaining sulphate leading to a flat slope of 0.12 for thedata points from the lower plume fringe.

3.3.3. Sulphur isotope enrichment factor

There are two principal ways to determine the character-istic sulphur enrichment factor e for DSR. Under closedsystem conditions the enrichment factor e is derived fromlinearisation of the data in a logarithmic plot (Rayleighplot) or the value is directly obtained from the differencein measured d34S values of the sulphate and the sulphidepool. For the latter approach two extreme scenarios canbe assumed, a complete accumulation of dissolved sulphidein a product reservoir or a very fast precipitation of hydro-gen sulphide as metal sulphide leading to an instantaneous-ly formed product in a dissolved state. However, neitherclosed system conditions can be assumed in groundwatersystems nor should irreversible first order rate laws be takento occur when redox reactions are commonly described byMonod or Michaelis–Menten kinetics (Van Breukelenet al., 2004; Abe and Hunkeler, 2006; Druhan et al.,2014). Therefore the application of the Rayleigh equationto field data may mostly yield an apparent enrichment fac-tor e which often underestimates the real enrichment factorthat is associated with DSR at a field site (Knoller et al.,2006).

We observed sulphate concentrations of approximately4.4 mM in the first few centimetres of the saturated zonecompared to a background sulphate concentration of about2 mM in the aquifer. As outlined above the high concentra-tions of dissolved sulphate could be explained by mobilisa-tion of reduced sulphur in the unsaturated zone bygroundwater fluctuation and reoxidation processes. Bothprocesses do not allow for a closed system approach tothe upper plume fringe zone and the application of a simpleRayleigh approach to the field data. For the lower plumefringe, however, we suggest a quasi-closed systemapproach. As we assume a very low transversal dispersivity

in the aquifer, we conceptually separate the aquifer at theHR-MLW in distinct, effectively closed horizontal zones,where DSR is active for different time periods. Plottingthe sulphur data obtained from the lower plume fringe(from 6.75 m to 7.75 m bgs) in accordance to the Rayleighequation (C0 = 1.6 mM and R0 = 3&) yields an apparent

sulphur enrichment factor e of approximately �44&. Thisvalue is close to the sulphur isotopic enrichment factor cal-culated from the difference in measured d34S values of dis-solved hydrogen sulphide and sulphate (�40&) for thelower plume fringe and is close to the value of �40& thatwas found for sulfidogenic pure-cultures with toluene aselectron donor by Mangalo et al. (2007). At the upperplume fringe zone the d34S values in hydrogen sulphide ran-ged from approximately �5& to +6& and the d34S valuesin groundwater sulphate ranged from +23 to +34&. Theisotope effect that was obtained from the difference of theaverage of these isotopic d34S patterns was approximately�28&. (6.56–6.70 m bgs). This value is significantly lessnegative compared to �40& that was obtained from themeasured d34S difference between the substrate and the pro-duct pool stemming from the plume core and the lowerplume fringe zone. Based on measured d18O values in thegroundwater sulphate and the results shown in Figs. 6and 7 we argue that re-oxidation of the reduced sulphurspecies with molecular oxygen near the groundwater tableis particularly important for the sulphur cycling in theuppermost 15 cm of the aquifer. Re-oxidation of reducedsulphur compounds exhibits no or only little isotope frac-tionation (Bottcher et al., 1998) and sulphate that isenriched in the 32S is added to the existing sulphate pool.Consequently, high oxidation rates of reduced sulphurcompounds of around 67% can be estimated using thetwo end members of �4.7 and 8.4& for the reduced andoxidised sulphur sources, respectively, and a sulphurenrichment factor of �40&. This process causes an isotopicshift of d34S in groundwater sulphate to lower values andmask the extent of stable isotope fractionation controlledby DSR.

An overall estimated value of the isotopic differencesbetween sulphate and hydrogen sulphide of about �40&

is not unusual for DSR in groundwater systems con-taminated with BTEX. Secondary chemical processes suchas disproportionation may lead to a stronger enrichmentof the lighter sulphur isotope in the hydrogen sulphideand may shift the d34S values in hydrogen sulphide to lowervalues compared to those controlled by DSR only. Sulphurenrichment factors of up to approximately �70& may givesome evidence of disproportionation processes of sulphurintermediates (Canfield and Thamdrup, 1994). The enrich-ment factor that was calculated from our data set is farfrom the calculated sulphur enrichment factors for dispro-portionation reactions and can be explained by the currentknowledge of DSR metabolism.

4. CONCLUSIONS

On the basis of compound-specific stable isotope analy-sis, measurements of individual sulphur species, as well asbacterial community patterns over depth, we provide

F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221 219

congruent lines of evidence of effective abiotic and micro-bial sulphur cycling at redox gradients in a petroleum-con-taminated aquifer. This study shows that complementaryinformation on the microbial population distribution andsulphur isotopes facilitate the application of sulphur stableisotopes to qualitatively describe sulphur cycle in aquifersediments. In the future, biodegradation of contaminantslinked to the re-cycling of sulphur intermediates shouldbe considered as a process in advanced and more sustain-able site remediation schemes.

ACKNOWLEDGEMENTS

The authors thank M. Blank from the Technische UniversitatMunchen, Chair of Hydrogeology, for her support during thelaboratory work. We are grateful to two anonymous reviewersfor their thoughtful comments that significanly improved thispaper.

REFERENCES

Abe Y. and Hunkeler D. (2006) Does the Rayleigh equation applyto evaluate field isotope data in contaminant hydrogeology?Environ. Sci. Technol. 40, 1588–1596.

Anneser B., Einsiedl F., Meckenstock R. U., Richters L., WisotzkyF. and Griebler C. (2008) High-resolution monitoring ofbiogeochemical gradients in a tar oil-contaminated aquifer.Appl. Geochem. 23, 1715–1730.

Anneser B., Pilloni G., Bayer A., Lueders T., Griebler C., EinsiedlF. and Richters L. (2010) High resolution analysis of con-taminated aquifer sediments and groundwater – What can belearned in terms of natural attenuation? Geomicrobiol. J. 27,130–142.

Bak F. and Cypionka H. (1987) A novel type of energy metabolisminvolving fermentation of inorganic sulphur compounds.Nature 326, 891–892.

Balci N., Mayer B., Shanks Iii W. C. and Mandernack K. W.(2012) Oxygen and sulfur isotope systematics of sulfateproduced during abiotic and bacterial oxidation of sphaleriteand elemental sulfur. Geochim. Cosmochim. Acta 77, 335–351.

Bolliger C., Hohener P., Hunkeler D., Haberli K. and Zeyer J.(1999) Intrinsic bioremediation of a petroleum hydrocarbon-contaminated aquifer and assessment of mineralization basedon stable carbon isotopes. Biodegradation 10, 201–217.

Bolliger C., Schroth M. H., Bernasconi S. M., Kleikemper J. andZeyer J. (2001) Sulfur isotope fractionation during microbialsulfate reduction by toluene-degrading bacteria. Geochim.

Cosmochim. Acta 65, 3289–3298.Bottcher M. E., Smock A. M. and Cypionka H. (1998) Sulfur

isotope fractionation during experimental precipitation ofiron(II) and manganese(II) sulfide at room temperature.Chem. Geol. 146, 127–134.

Bottrell S. H., Hayes P. J., Bannon M. and Williams G. M. (1995)Bacterial sulfate reduction and pyrite formation in a pollutedsand aquifer. Geomicrobiol J. 13, 75–90.

Brielmann H., Griebler C., Schmidt S. I., Michel R. and Lueders T.(2009) Effects of thermal energy discharge on shallow ground-water ecosystems. FEMS Microbiol. Ecol. 68, 273–286.

Brunner B., Bernasconi S. M., Kleikemper J. and Schroth M. H.(2005) A model for oxygen and sulfur isotope fractionation insulfate during bacterial sulfate reduction processes. Geochim.

Cosmochim. Acta 69, 4773–4785.Caccavo, Jr., F., Lonergan D. J., Lovley D. R., Davis M., Stolz J.

F. and McInerney M. J. (1994) Geobacter sulfurreducens sp.

nov., a hydrogen- and acetateoxidizing dissimilatory metal-reducing microorganism. Appl. Environ. Microbiol. 60, 3752–3759.

Canfield D. E. and Thamdrup B. (1994) The production of 34S-depleted sulfide during bacterial disproportionation of elemen-tal sulfur. Science 266, 1973–1975.

Cirpka O. A., Frind E. O. and Helmig R. (1999) Numericalsimulation of biodegradation controlled by transverse mixing.J. Contam. Hydrol. 40, 159–182.

Cline J. D. (1969) Spectrophotometric determination of hydrogensulfide in natural waters. Limnol. Oceanog. 14, 454–458.

Crouzet C., Kedziorek M. A. M., Altmann R. S. and Bourg A. C.M. (2000) Speciation of inorganic sulphur in aquifer sedimentscontaminated by landfill leachate using chemical extractiontechniques. Environ. Sci. Technol. 21, 285–296.

Druhan J. L., Steefel C. I., Conrad M. E. and DePaolo D. J. (2014)A large column analog experiment of stable isotope variationsduring reactive transport: I. A comprehensive model of sulfurcycling and d34S fractionation. Geochim. Cosmochim. Acta 124,366–393.

Einsiedl F. (2009) Effect of NO2� on stable isotope fractionation

during bacterial sulfate reduction. Environ. Sci. Technol. 43, 82–87.

Einsiedl F. and Mayer B. (2005) Sources and processes affectingsulphate in a karstic groundwater system of the franconianAlb, Southern Germany. Environ. Sci. Technol. 39, 7118–7125.

Einsiedl F., Schafer T. and Northrup P. (2007) Combined sulfur K-edge XANES spectroscopy and stable isotope analyses of fulvicacids and groundwater sulfate identify sulfur cycling in a karsticcatchment area. Chem. Geol. 238, 268–276.

Friedrich C. G., Bardischewsky F., Rother D., Quentmeier A. andFischer J. R. (2005) Prokaryotic sulfur oxidation. Curr. Opin.

Microbiol. 8, 253–259.Geets J., Borremans B., Diels L., Springael D., Vangronsveld J.,

van der Lelie D. and Vanbroekhoven K. (2006) DsrB gene-based DGGE for community and diversity surveys of sulfate-reducing bacteria. J. Microbiol. Methods 66, 194–205.

Gibson B. D., Amos R. T. and Blowes D. W. (2011) 34S/32Sfractionation during sulfate reduction in groundwater treat-ment systems: reactive transport modeling. Environ. Sci.

Technol. 45, 2863–2870.Green D. H., Shenoy D. M., Hart M. C. and Hatton A. D. (2011)

Coupling of dimethylsulfide oxidation to biomass productionby a marine flavobacterium. Appl. Environ. Microbiol. 77, 3137–3140.

Habicht K. S. and Canfield D. E. (1997) Sulfur isotope frac-tionation during bacterial sulfate reduction in organic-richsediments. Geochim. Cosmochim. Acta 61, 5351–5361.

Hall G. E. M., Pelchat J.-C. and Loop J. (1988) Separation andrecovery of various sulphur species in sedimentary rocks forstable sulphur isotopic determination. Chem. Geol. 67, 35–45.

Holmkvist L., Ferdelman T. G. and Jørgensen B. B. (2011) Acryptic sulfur cycle driven by iron in the methane zone ofmarine sediment (Aarhus Bay, Denmark). Geochim.

Cosmochim. Acta 75, 3581–3599.Holt B. D. (1991) Oxygen Isotopes. Wiley, New York.Jørgensen B. B. and Bak F. (1991) Pathways and microbiology of

thiosulfate transformations and sulfate reduction in a marinesediment (Kattegat, Denmark). Appl. Environ. Microbiol. 57,847–856.

Jørgensen, B.B., Nelson, D.C., 2004. Sulfide oxidation in marinesediments: geochemistry meets microbiology, in SulfurBiogeochemistry - Past and present, edited by: Amend, J. P.,Edwards, K. J., and Lyons, T. W., Geol. Soc. of Am. SpecialPaper, 379, 63–81.

220 F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221

Kamyshny, Jr., A. and Ferdelman T. G. (2010) Dynamics ofzerovalent sulfur species including polysulfides at seep sites onintertidal sand flats (Wadden Sea, North Sea). Mar. Chem. 121,17–26.

Kleinsteuber S., Schleinitz K. M., Breitfeld J., Harms H., RichnowH. H. and Vogt C. (2008) Molecular characterization ofbacterial communities mineralizing benzene under sulfate-reducing conditions. FEMS Microbiol. Ecol. 66, 143–157.

Knoller K. and Schubert M. (2010) Interaction of dissolved andsedimentary sulfur compounds in contaminated aquifers. Chem.

Geol. 276, 284–293.Knoller K., Vogt C., Richnow H. H. and Weise S. M. (2006) Sulfur

and oxygen isotope fractionation during benzene, toluene, ethylbenzene, and xylene degradation by sulfate-reducing bacteria.Environ. Sci. Technol. 40, 3879–3885.

Kopinke F.-D., Georgi A., Voskamp M. and Richnow H. H.(2005) Carbon isotope fractionation of organic contaminantsdue to retardation on humic substances: implications fornatural attenuation studies in aquifers. Environ. Sci. Technol.

39, 6052–6062.Kunapuli U., Lueders T. and Meckenstock R. (2007) The use of

stable isotope probing to identify key iron-reducing microor-ganisms involved in anaerobic benzene degradation. ISME J. 1,643–653.

Lavik G., Stuhrmann T., Bruchert V., Van der Plas A., MohrholzV., Lam P., Muszmann M., Fuchs B. M., Amann R., Lass U.and Kuypers M. M. M. (2009) Detoxification of sulphidicAfrican shelf waters by blooming chemolithotrophs. Nature

457, 581–584.Lindgren M., Cedergren A. and Lindberg J. (1982) Conditions for

sulfite stabilization and determination by ion chromatography.Anal. Chim. Acta 141, 279–286.

Lovley D. R. (2003) Cleaning up with genomics: applyingmolecular biology to bioremediation. Nat. Rev. Microbiol. 1,35–44.

Lovley D., Phillips E., Lonergan D. J. and Widman P. K. (1995)Fe(III) and S0 reduction by Pelobacter carbinolicus. Appl.

Environ. Microbiol. 61, 2132–2138.Luther I. G. W. (1991) Pyrite formation via polysulfide com-

pounds. Geochim. Cosmochim. Acta 55, 2839–2849.Mangalo M., Meckenstock R. U., Stichler W. and Einsiedl F.

(2007) Stable isotope fractionation during bacterial sulfatereduction is controlled by reoxidation of intermediates.Geochim. Cosmochim. Acta 71, 4161–4171.

Mangalo M., Einsiedl F., Meckenstock R. U. and Stichler W.(2008) Influence of the enzyme dissimilatory sulfite reductase onstable isotope fractionation during sulfate reduction. Geochim.

Cosmochim. Acta 72, 1513–1520.Mayer B., Fritz P., Prietzel J. and Krouse H. R. (1995) The use of

stable sulfur and oxygen isotope ratios for interpreting themobility of sulfate in aerobic forest soils. Appl. Geochem. 10,161–173.

McCready R. G. L. and Krouse H. R. (1982) Sulfur isotopefractionation during the oxidation of elemental sulfur bythiobacilli in a solonetzic soil. Can. J. Soil Sci. 62, 105–110.

Moses C. O. and Herman J. S. (1991) Pyrite oxidation atcircumneutral pH. Geochim. Cosmochim. Acta 55, 471–482.

Peiffer S., Dos Santos Afonso M., Wehrli B. and Gaechter R.(1992) Kinetics and mechanism of the reaction of hydrogensulfide with lepidocrocite. Environ. Sci. Technol. 26, 2408–2413.

Pfeffer C., Larsen S., Song J., Dong M., Besenbacher F., Meyer R.L., Kjeldsen K. U., Schreiber L., Gorby Y. A., El-Naggar M.Y., Leung K. M., Schramm A., Risgaard-Petersen N. andNielsen L. P. (2012) Filamentous bacteria transport electronsover centimetre distances. Nature 491, 218–221.

Pilloni G., von Netzer F., Engel M. and Lueders T. (2011) Electronacceptor-dependent identification of key anaerobic toluenedegraders at a tar–oil-contaminated aquifer by Pyro-SIP.FEMS Microbiol. Ecol. 78, 165–175.

Pilloni G., Granitsiotis M., Engel M. and Lueders T. (2012) Testingthe limits of 454 pyrotag sequencing: reproducibility, quantita-tive assessment and comparison to T-RFLP fingerprinting ofaquifer microbes. PLoS ONE, e40467.

Prommer H., Tuxen N. and Bjerg P. L. (2006) Fringe-controllednatural attenuation of phenoxy acids in a landfill plume:integration of field-scale processes by reactive transport mod-eling. Environ. Sci. Technol. 40, 4732–4738.

Prommer H., Anneser B., Rolle M., Einsiedl F. and Griebler C.(2009) Biogeochemical and isotopic gradients in a BTEX/PAHcontaminant plume: model-based interpretation of a high-resolution field data set. Environ. Sci. Technol. 43, 8206–8212.

Rolle M., Chiogna G., Bauer R., Griebler C. and Grathwohl P.(2010) Isotopic fractionation by transverse dispersion: flow-through microcosms and reactive transport modeling study.Environ. Sci. Technol. 44, 6167–6173.

Schippers A. and Jorgensen B. B. (2001) Oxidation of pyrite andiron sulfide by manganese dioxide in marine sediments.Geochim. Cosmochim. Acta 65, 915–922.

Schippers A. and Sand W. (1996) Sulfur chemistry in bacterialleaching of pyrite. Appl. Environ. Microbiol. 62, 3424–3431.

Schroth M. H., Kleikemper J., Bolliger C., Bernasconi S. M. andZeyer J. (2001) In situ assessment of microbial sulfate reductionin a petroleum-contaminated aquifer using push–pull tests andstable sulfur isotope analyses. J. Contam. Hydrol 51, 179–195.

Schwientek M., Einsiedl F., Stichler W., Stogbauer A., Strauss H.and Maloszewski P. (2008) Evidence for denitrification regulat-ed by pyrite oxidation in a heterogeneous porous groundwatersystem. Chem. Geol. 255, 60–67.

Spence M. J., Bottrell S. H., Thornton S. F., Richnow H. H. andSpence K. H. (2005) Hydrochemical and isotopic effectsassociated with petroleum fuel biodegradation pathways in achalk aquifer. J. Contam. Hydrol. 79, 67–88.

Takahashi Y., Suto K. and Inoue C. (2010) Polysulfide reductionby Clostridium relatives isolated from sulfate-reducing enrich-ment cultures. J. Biosci. Bioeng. 109, 372–380.

Tuxen N., Albrechtsen H.-J. and Bjerg P. L. (2006) Identificationof a reactive degradation zone at a landfill leachate plume fringeusing high resolution sampling and incubation techniques. J.

Contam. Hydrol. 85, 179–194.Van Breukelen B. M., Griffioen J., Roling W. F. M. and van

Verseveld H. W. (2004) Reactive transport modelling ofbiogeochemical processes and carbon isotope geochemistryinside a landfill leachate plume. J. Contam. Hydrol. 70, 249–269.

Van Stempvoort D. R. and Kwong Y. T. (2010) Sulfur analyses astracers of microbial degradation of hydrocarbons in thecapillary fringe. J. Contam. Hydrol. 114, 1–17.

Winderl C., Anneser B., Griebler C., Meckenstock R. U. andLuders T. (2008) Depth-resolved quantification of anaerobictoluene degraders and aquifer microbial community patterns indistinct redox zones of a tar oil contaminant plume. Appl.

Environ. Microbiol. 74, 792–801.Wisotzki F. and Eckert P. (1997) Sulfat-dominierter BTEX-abbau

im grundwasser eines ehemaligen gaswerksstandortes.Grundwasser, 11–21.

Wright K. E., Williamson C., Grasby S. E., Spear J. R. andTempleton A. S. (2013) Metagenomic evidence for sulfurlithotrophy by Epsilonproteobacteria as the major energysource for primary productivity in a sub-aerial arctic glacialdeposit, Borup Fiord Pass. Front. Microbiol. 4.

F. Einsiedl et al. / Geochimica et Cosmochimica Acta 156 (2015) 207–221 221

Zamfirescu D. and Grathwohl P. (2001) Occurrence and atten-uation of specific organic compounds in the groundwater plumeat a former gasworks site. J. Contam. Hydrol. 53, 407–427.

Zhang J.-Z. and Millero F. J. (1993) The products from theoxidation of H2S in seawater. Geochim. Cosmochim. Acta 57,1705–1718.

Zopfi J., Ferdelman T. G. and Fossing H. (2004) Distribution andfate of sulfur intermediates-sulfite, tetrathionate, thiosulfate,

and elemental sulfur-in marine sediments. Geol. Soc. Am. 379,97–116.

Zopfi J., Bottcher M. E. and Jørgensen B. B. (2008)Biogeochemistry of sulfur and iron in Thioploca-colonizedsurface sediments in the upwelling area off central Chile.Geochim. Cosmochim. Acta 72, 827–843.

Associate editor: Jack J. Middelburg