Chemosphere 90 (2013) 1366–1371

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Chemosphere

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Sulfur and oxygen isotope tracing in zero valent iron based In situremediation system for metal contaminants

Naresh Kumar a,e,⇑, Romain Millot a, Fabienne Battaglia-Brunet b, Philippe Négrel a, Ludo Diels c,e,Jérôme Rose d, Leen Bastiaens c

a BRGM, Metrology Monitoring Analysis Department, Orléans, Franceb BRGM, Environment and Process Division, Orléans, Francec VITO, Flemish Institute for Technological Research, Mol, Belgiumd CEREGE, UMR-7330, CNRS-Aix Marseille University, Aix-en-Provence, Francee Department of Biology, University of Antwerp, Antwerp, Belgium

h i g h l i g h t s

" We followed sulfur and oxygen isotope fractionation of dissolved sulfate molecule in groundwater." Sediment was incubated with zero valent iron in flow through columns." Microbial sulfate reduction was observed." A good relationship between d34S and sulfate reduction rate was obtained." A linear relationship is between d34S and d18O was observed.

a r t i c l e i n f o

Article history:Received 31 May 2012Received in revised form 25 July 2012Accepted 26 July 2012Available online 21 September 2012

Keywords:Sulfur isotopesOxygen isotopesZero valent ironIn situ remediation

0045-6535/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.chemosphere.2012.07.060

⇑ Corresponding author at: BRGM, Metrology MoniE-mail address: kumar@cerege.fr (N. Kumar).

a b s t r a c t

In the present study, controlled laboratory column experiments were conducted to understand the bio-geochemical changes during the microbial sulfate reduction. Sulfur and oxygen isotopes of sulfate werefollowed during sulfate reduction in zero valent iron incubated flow through columns at a constant tem-perature of 20 ± 1 �C for 90 d. Sulfur isotope signatures show considerable variation during biological sul-fate reduction in our columns in comparison to abiotic columns where no changes were observed. Themagnitude of the enrichment in d34S values ranged from 9.4‰ to 10.3‰ compared to initial value of2.3‰, having total fractionation dS between biotic and abiotic columns as much as 6.1‰. Sulfur isotopefractionation was directly proportional to the sulfate reduction rates in the columns. Oxygen isotopes inthis experiment seem less sensitive to microbial activities and more likely to be influenced by isotopicexchange with ambient water. A linear relationship is observed between d34S and d18O in biotic condi-tions and we also highlight a good relationship between d34S and sulfate reduction rate in biotic columns.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction relatively larger available specific surface area makes Fe� a suitable

In situ groundwater remediation of metal contaminants hasemerged as a sustainable option in recent years for various reasons,e.g. economic feasibility and energy consumption in pump andtreats methods, site accessibility, etc. (Farhadian et al., 2008). Forin situ remediation, various possibilities have also been exploredfor instance by enhancing natural attenuation, providing electrondonors (Satyawali et al., 2010) or by using reactive barrier materi-als (Benner et al., 1999; Waybrant et al., 2002). Zero valent iron(Fe�) is getting large attention lately as a reactive material forin situ applications (Wilkin and McNeil, 2003; Dries et al., 2005;Burghardt and Kassahun, 2005). The highly reducing nature and

ll rights reserved.

toring Analysis Department, Orléan

medium for groundwater contaminant removal. There have beenalready many successful installation of Fe� based remediation sys-tem in last decade (Gu et al., 1998; Rowland, 2002; Liang et al.,2003; Phillips et al., 2010). Fe� has been successfully used in labscale as well as field scale applications, dealing with wide rangeof groundwater contaminants e.g. chlorinated compounds andmetals (Dries et al., 2005; Doong and Lai, 2006; Habekost and Aris-tov, 2012), radioactive material (Burghardt and Kassahun, 2005;Klimkova et al., 2011), metalloid, e.g. arsenic (Su and Puls, 2003;Lien and Wilkin, 2005; Biterna et al., 2010), pharmaceuticals andpesticides (Keum and Li, 2004; Bautitz et al., 2012). However,the biogeochemical dynamics and contaminants behavior in

s, France. Tel.: +33 4 42 97 15 48; fax: +33 4 42 97 15 59.

N. Kumar et al. / Chemosphere 90 (2013) 1366–1371 1367

subsurface environments are still poorly understood, as real fieldsites often encounter problems due to little or no control overfluxes and changes in subsurface processes with time or seasons.Regular chemical analysis and monitoring may not be a practicalor economic option in many isolated sites. These complications of-ten make it difficult to understand the actual processes (biotic/abi-otic) contributing in contaminant removal, needless to say that thisdistinction is rather very important in designing realistic remedia-tion strategy for any particular site.

Stable isotopes have emerged as a potential tool in understand-ing the dynamics of pollutants in natural systems (Fritz et al.,1989; Slater et al., 2002; Banas et al., 2009). Relatively easy analy-sis and bulk information makes isotope study a practical and viableoption. The characterization and quantification of electron-accept-ing processes, like nitrate, iron and sulfate reduction are extremelyvaluable in estimating the sustainability and longevity of degrada-tion processes in any subsurface system (Knöller et al., 2006). Thischaracterization is of primary importance in case of in situ treat-ment process, where there is little control over changing processesin subsurface environments. Biological sulfate reduction has beenreported earlier in similar Fe� based treatments systems (Guet al., 2002; Van Nooten et al., 2007; Xin et al., 2008). Quantifica-tion of sulfate reduction by following dissolved concentrations ofsulfate and co-existing sulfide in groundwater is often a challengedue to possible dilution, mixing or mineral precipitation processes.Precipitation of dissolved sulfide is particularly important in caseof Fe� based systems where abundant Fe(II) is available in ground-water for possible FeS precipitation. It has been shown that sulfatereducing bacteria (SRB) preferentially remove lighter isotopes ofsulfur and oxygen from the sulfate molecule resulting into isotopicenrichments of both heavier isotopes i.e. 18O and 34S in residualsulfate (Fritz et al., 1989).

In this study, we followed the evolution of d34S and d18O fromdissolved sulfate in groundwater within lab scale Fe� based flowthrough columns, which were primarily designed for heavy metalremoval from groundwater. The aim of this study was to character-ize the isotope fractionation and changes during a long-term treat-ment processes and also to determine the impact of microbiology onisotopic signature in subsurface redox environment. We summarizehere the results of sulfur and oxygen isotope fractionation in Fe�based lab scale in situ removal systems over 90 d of experiment.

2. Material and methods

2.1. Column design

Four double jacketed flow through glass columns (30 cmlength � 4.5 cm id, total liquid volume = 480 mL) were setup inlaboratory for �30 week at controlled temperature (20 ± 1 �C),with the primary aim of groundwater contaminant removal usingFe� as a reactive material. Columns were filled with sediment ob-tained from a heavy metal contaminated site in Belgium from adepth of �32 m, more description about this site is given else-where (Vanbroekhoven et al., 2008). Efforts were made to designa lab scale concept of in situ reactive barrier using two types ofFe� differing in particles size and source, i.e. granular zero valentiron (gFe�, Gotthard Maier, Germany) and micro zero valent iron(mFe�, Högenäs, Sweden) with an average particle size of 0.25–2 mm and 20–40 lm, respectively. For each column, the first bot-tom half (�240 mL) was filled with an aquifer/Fe� mixture with ra-tios of 80:20 and 98:2 v/v for gFe� and mFe�, respectively. Thesecond (upper) half of all columns was filled only with aquifer(in Supplementary Material (SM), Fig. SM-1). Filling of columnswas performed under nitrogen atmosphere in a glove box. Simu-lated groundwater, which was prepared in lab conditions corre-

sponding to the site characteristics (Table SM-1), was injected inparallel through the columns using a peristaltic pump at a constantflow rate of 1 ± 0.2 mL h�1. A slight over pressure (1 kPa) of N2 wasmaintained in the feeding bottles to avoid air contact during col-umn feeding. All tubes and fittings used in the experiment wereacid washed and flushed with nitrogen before use.

For each Fe� type, two columns were set-up, of which one wasfed with a small dose of glycerol (0.1% v/v of inlet water) to en-hance indigenous microbial activity and the other columns was ex-posed to gamma radiation (Ionisos, Dagneux, France), withminimum absorbed radiation dose of 25 kGy, before injectinggroundwater to restrict all microbial activities.

2.2. Analytical methods

Defined volumes of samples (from 50 to 1000 lL) were ex-tracted from columns using a nitrogen filled plastic syringe, byinjecting the nitrogen and extracting equal amount of liquid fromcolumn prior to observation and counting of bacterial cells. Sam-ples were immediately diluted in deionized water and filtered ontoa black polycarbonate filter, 0.22 lm (Nuclepore, Whatman, Kent,UK). The filter was incubated 15 min in the dark with 1 mL filtereddeionized water mixed with 1 lL DAPI (40, 6-diamidino-2-phenyl-indole) solution (1 mg mL�1, Sigma). This mixture was removed byfiltration, and the filter was rinsed two times with 1 mL filtereddeionized water. The filter was then mounted on a glass slide withCitifluor (Biovailey, France), and observed with an optical micro-scope (Zeiss Axio Imager Z1) equipped with Filter Set 49 for DAPI,UV HBO lamp and a digital camera. Bacteria were enumerated on10 independent fields (each of 5800 lm2). Cell counts were calcu-lated considering the volume of the sample used and filter surfacearea calculations on an average basis. Sulfate concentration wasanalyzed with a spectrophotometer operating at k 540� using spe-cific analysis kits (Merck Spectroquant kit 1.14548.001, Germany).

Samples for sulfur and oxygen isotope analysis were collected atthe outlet of columns using 250 mL pre-acid washed plastic perplexbottles. Cd-Acetate was already added in the bottles (5% v/v) priorto sample collection, to fix sulfur as CdS, and then the aliquot wasfiltered through a 0.2 lm nitrocellulose filter before chemicaldetermination of residual sulfate. The amount of sample collectedvaried at different time points during the experiment as the sulfateconcentration in the outlet solution changed over the time. How-ever, in any case, a minimum of 5 mg of SO4 was collected for everysampling point. The analysis was performed as described by (Fritzet al., 1989).

Dissolved sulfate was precipitated as BaSO4 at pH < 4 (in orderto remove HCO�3 and CO2�

3 species) by adding a BaCl2 solution.The isotopic analyses on BaSO4 were carried out using a Delta + XPmass spectrometer coupled in continuous-flow mode to a ThermoElemental Analyzer in BRGM laboratories. Sulfate-isotope compo-sitions are reported in the usual d-scale in ‰ with reference toV-CDT (Vienna Canyon Diablo Troilite) and V-SMOW (Vienna Stan-dard Mean Ocean Water) according to dsample (‰) = {(Rsample/Rstandard) � 1}1000, where R is the 34S/32S and 18O/16O the atomicratios. Sulfate-isotope compositions (d34S (SO4) and d18O (SO4))were measured with a precision of ±0.3‰ vs. CDT for d34S (SO4)and ±0.8‰ vs. VSMOW for d18O (SO4), respectively.

3. Results and discussion

3.1. Sulfate reduction

Sulfate reduction is a common phenomenon observed in Fe�based PRB’s due to favorable growth environment i.e. close toneutral pH and a very low ORP conditions (Gu et al., 2002). The

Fig. 1. Sulfate concentration (mM) as a function of time (d) in column outlets.

1368 N. Kumar et al. / Chemosphere 90 (2013) 1366–1371

efficiency of Fe� in increasing pH, decreasing ORP and productionof water born H2 (reaction 1) is well known (Johnson et al.,2008). This water born H2 can also be a potential electron donorfor SRB (Karri et al., 2005). In the present study, dissolved sulfateconcentration, pH and ORP (Tables SM-2 and SM-3) in the columnoutlet solution was analyzed regularly to follow behavior andactivity of SRB.

Fe� þ 2H2O! Fe2þ þH2 þ 2OH� ð1Þ

Dissolved sulfate concentrations in the column outlet solutiondecreased from initial inlet concentrations of 3.95 to 0.03 and0.09 mM in gFe� and mFe� biotic columns respectively (Fig. 1).Appearance of black patches (believed to be FeS precipitation) inboth biotic columns was in agreement with microbial sulfatereduction. Providing glycerol probably also stimulated bacterialgrowth by acting as carbon source along with availability of H2.In this experiment no extra microbial culture was added, so onlynatural population of sulfate reducers were expected to grow.However, in the abiotic columns, sulfate concentrations were unaf-fected throughout the experiment (Fig. 1), which was expected dueto absence of viable microbial cells after gamma radiation expo-sure to sediment and the small dose of formaldehyde (0.1 mL L�1)that was added with inlet water to avoid any possibility of micro-bial growth.

Sulfate reduction rates (SRR, nmol cm�3 h�1) were calculatedusing equation described by Stam et al. (2011). SRR values in thisexperiment are consistent with the sulfate reduction as the maxi-mum rate was achieved after 30 d of incubation, after which sim-ilar values of SRR were obtained (value of 4–4.1 nmol cm�3 h�1

Fig. 2a) for both granular and microbiotic columns. Equal and stea-dy values of SRR throughout the experiment are consistent withthe equal and limited supply of sulfate in the system. In generalthe SRR is also believed to influence the isotope fractionation,and in this study SRR was directly related to the sulfur isotope frac-tionation (Fig. 2b). It is also argued in literature that the substratetype may also influence the fractionation, considering H2 gas pro-ducing lower fractionation than organic substrate (Kaplan andRittenberg, 1964; Kemp and Thode, 1968). As in the present studyH2 as well as glycerol were available as electron donor, the individ-ual contribution cannot be established in this experimental setup.

3.2. Cell counting and microscopic observations

Bacterial cell counting was performed using DAPI imaging.Changes in cell numbers during the experiment can give generaltrend of microbial activity; increase or decrease in bacterial cellcount can be related to the overall SRR. Normally, if the sulfate isnot limiting and energy source is available, the cell count could in-crease with time, which would further enhance the sulfatereduction.

Images of DAPI-stained samples (Fig. 3) shows the presence ofdominant rod shaped bacterial cells with an average length of5 lm. All cells were visually similar in shape and sizes, suggestingthe growth of a dominant species. No efforts were made at thisstep of the experiment to further identify the bacterial species.Bacterial cells were counted at 30 d of column test, when dissolvedsulfate was completely reduced in biotic columns. An average cellcount of 1.9 � 107 and 1.2 � 107 cells mL�1 was obtained in gFe�biotic and mFe� biotic column, respectively. At the end of theexperiment (90 d), the cell concentrations were 1.7 � 107 and6.5 � 107 cell mL�1 in gFe� and mFe� biotic column respectively.These observations suggest that the cell concentration during theexperimental period did not change significantly, which is consis-tent with limited and uniform supply of sulfate and almost con-stant SRR in the columns. These observations also suggest that

due to limited sulfate doses, the SRB population would continueto consume sulfate in columns, so the cell concentrations are morelikely to be related to the dissolved sulfate supply in this case.

3.3. d34S variations and origin of sulfur isotope fractionation

Although isotopes of the same element behave the same phys-ically and chemically, reaction rates differ due to the mass differ-ence between the isotopes. This mass difference causes apreferential partitioning, namely isotope fractionation, that resultsin varying isotopic compositions during reaction. On the one hand,in the biotic columns d34S enrichment is evident (Fig. 4a), and it isexplicable by a preferred use of lighter sulfur element of SO4 (32S)by SRB, which results into abundance of heavier element 34S in theremaining sulfate molecules. On the other hand, no sulfate reduc-ers were active in the abiotic columns, so d34S values remain al-most the same as the initial value (Fig. 4a). There is nomechanism reported till date for abiotic sulfate reduction.

A large range of variation in d34S values (from 4‰ up to 46‰)has been observed in pure bacterial cultures where sulfate wasavailable abundantly (Kaplan and Rittenberg, 1964; Detmerset al., 2001). In the present study, the maximum values of d34Swere observed equal to 10.3‰ in the mFe� biotic columns with fi-nal enrichment values of 9.4‰ (Table SM-2). A total DS was ob-served as much as 6.1‰, while compared between biotic andabiotic processes, where DS is the difference of d34S (‰) betweenbiotic and abiotic columns. The isotope measurement was only fol-lowed until 90 d of column operation, after this period SRR wasconstant so no further fractionation was expected; also sample col-lection was not possible for isotopes measurement as the dissolvedsulfate concentration was nearly zero.

Sulfide produced by SRB was not considered to be associatedwith isotope fractionation in this study as sulfide was very likelyto be precipitated with Fe(II) available in the plume to form insol-uble iron sulfide and this phenomenon is known to not be associ-ated with sulfur isotope fractionation effect (Canfield et al., 1992).Fig. 5 shows that the dissolved sulfate concentration and d34S (‰)follows an inverse trend in both biotic columns, where dissolvedsulfate concentration decreases as microbial activity increases, onthe other hand d34S (‰) value increases through time. This obser-vation confirms that the sulfur isotope fractionation only

Fig. 2. (a) Sulfate reduction rate (SRR, nmol cm�3 h�1) in biotic columns as afunction of time (d). (b) Sulfur isotope values (d34S, ‰) as a function of sulfatereduction rate (SRR, nmol cm�3 h�1) in biotic columns.

Fig. 3. Photographs of DAPI staining (a) gFe� biotic column and (b) mFe� bioticcolumn.

N. Kumar et al. / Chemosphere 90 (2013) 1366–1371 1369

originates from microbial sulfate reduction avoiding any contribu-tion of sulfate from sediment.

3.4. d18OSO4 variations and origin of the oxygen isotope fractionation

Although significant enrichment of 34S was evident in residualsulfate, a corresponding enrichment of 18O was not observed inthis study. Similar observations were previously reported by otherresearchers (Spence et al., 2005). Sulfur and oxygen isotope fol-lowed a different isotopic pattern during sulfate reduction proba-bly because of fundamental differences in the enrichmentmechanism itself. Sulfur isotope fractionation is a purely kinetic ef-fect, whereas oxygen isotope fractionation is influenced by cataly-sis of isotopic exchange between water and sulfate during sulfatereduction (Fritz et al., 1989).

An identical pattern shift was observed in d18O behavior in bothbiotic and abiotic processes (Fig. 4b), so it is very likely that the rel-

atively small variation observed in d18O could be due to shift in iso-topic equilibrium with ambient water. This leads to the widelybelieved assumption that the oxygen isotope exchange dominatedover kinetic isotope fractionation. d18O of ambient water was notanalyzed in this experiment, so we were not able to confirm thishypothesis. Direct chemical or microbial oxidation of H2S to sulfuris accompanied by much smaller oxygen isotope effect. The d18Ofractionation is normally controlled by ambient surface water. Thishypothesis is considered in many studies recently, however noestablished explanation of this process has been reported yet inliterature (Knöller et al., 2006).

3.5. Isotope variation: d18O vs. d34S

In Fig. 6, linear relationships were observed between d34S andd18O in the biotic experiments irrespective of the type of Fe� (eithergranular or micro Fe), although the slopes are slightly different foreach in Fig. 6. These results suggest that the particle size may alsoaffect the overall sulfate reduction process in some cases.

It is also argued in literature that there is no simple relationshipbetween SRR and isotope fractionation (Detmers et al., 2001), how-ever in the present study we observed a linear relationship (Fig. 6).Brunner et al. (2005) proposed a combined investigation of theinfluence of SRB on the sulfur and oxygen isotopic composition

Fig. 4. (a) Sulfur isotope variation (d34S, ‰) as a function of time (d) in biotic (red)and abiotic columns (blue). (b) Oxygen isotope (d18O, ‰) values in columns as afunction of time (d). (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)

Fig. 5. Comparison of dissolved sulfate (mM) and d34S (‰) as a function of time forboth biotic columns (micro mFe� and granular gFe�).

Fig. 6. d34S (‰) vs. d18O (‰) in sulfate for biotic columns.

1370 N. Kumar et al. / Chemosphere 90 (2013) 1366–1371

of residual sulfate could be the key to a better understanding ofSRR. Böttcher et al. (1998) hypothesized that d34S vs. d18O relation-ship reflected SRR in marine sediments, the steeper the slope theslower the SRR. In the present study, we observed not sosteep slope for d34S vs. d18O relationship, which is consistent withthe experimental condition where SRR was stable after initialincrease.

It is also noteworthy that the d34S vs. d18O relationship does notreflect the bulk SRR but rather cell-specific SRR. So basically, a largenumber of bacteria with slow cell specific SRR or a small number ofbacteria with high cell specific SRR, both can achieve a high bulkSRR (Brunner et al., 2005). But in the present study, the numberof bacterial cell in both the biotic columns did not increase signif-icantly during the experimental period.

4. Conclusions

In the present study, we report results from a long-term exper-iment, designed for groundwater treatment using real site sedi-ment. Sulfur isotope analysis is a good and practically viableoption to characterize sulfate reduction activities and SRR in anysubsurface system without going for microbial analysis and char-acterization. Oxygen isotope analysis is also important but needsto be considered in light of ambient water oxygen isotope ex-change, as it is more likely to be controlled by ambient water. Sul-fur and oxygen isotopes both provide important information andsupports the actual practical data obtained during the experiment.However, it is important to see the long term effect of isotopebehavior as very less knowledge is available in literature for thesekinds of system for long time operations, which is very typical forin situ remediation treatment systems.

Isotope geochemistry can also be used for precise identificationof pollution sources, effectiveness of remediation process, and canprovide crucial insight into contaminants fate and transport. Con-sidering the simple methods for isotopic analysis and less analyti-cal cost, these standard analytical tools can easily be incorporatedinto typical field sampling. As the standard geochemical analysisalways retain certain level of uncertainty at times important ques-tion might left unanswered. In combination with other chemicaland biogeochemical techniques, isotopic analysis can be used forbetter understanding the processes.

N. Kumar et al. / Chemosphere 90 (2013) 1366–1371 1371

Acknowledgements

This is a contribution of the AquaTRAIN MRTN (Contract No.MRTN-CT-2006-035420) funded under the European CommissionSixth Framework Programme (2002–2006) Marie Curie actions,human resources and mobility activity area-research training net-works. We also thank the Research Division of BRGM for additionalfunding. This work is collaborative effort of Metrology MonitoringAnalysis and Environment and Process Division teams of BRGM. C.Fléhoc is acknowledged for S and O isotope analysis. This is BRGMcontribution n� 724794.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chemosphere.2012.07.060.

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