Isotopic and microbiological signatures of pyrite-driven denitrification in a sandy aquifer

Chemical Geology 300-301 (2012) 123–132

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Research paper

Isotopic and microbiological signatures of pyrite-driven denitrification in a sandy aquifer Yan-Chun Zhang a, Caroline P. Slomp a,⁎, Hans Peter Broers b, c, d, Benjamin Bostick e, Hilde F. Passier b, Michael E. Böttcher f, 1, Enoma O. Omoregie a, g, Jonathan R. Lloyd g, David A. Polya g, Philippe Van Cappellen a, h, i a

Department of Earth Sciences, Faculty of Geosciences, Utrecht University, Budapestlaan 4, 3584 CD Utrecht, The Netherlands Deltares Research Institute, Subsurface and Groundwater, Utrecht, The Netherlands c TNO, Geological Survey of the Netherlands, Utrecht, The Netherlands d VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands e Lamont-Doherty Earth Observatory of Columbia University, Palisades NY, USA f Max Planck Institute for Marine Microbiology, Bremen, Germany g School of Earth, Atmospheric and Environmental Sciences, University of Manchester, Manchester, UK h School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta, USA i Department of Earth and Environmental Sciences, University of Waterloo, Ontario, Canada b

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 11 September 2011 Received in revised form 23 December 2011 Accepted 14 January 2012 Available online 31 January 2012 Editor: B. Sherwood Lollar Keywords: Denitrification Pyrite Groundwater Isotopes Microbiology Sediment

Denitrification driven by pyrite oxidation can play a major role in the removal of nitrate from groundwater systems. As yet, limited information is available on the interactions between the micro-organisms and aqueous and mineral phases in aquifers where pyrite oxidation is occurring. In this study, we examine the groundwater and sediment composition along a well-characterized redox gradient in a heavily nitrate-polluted pyritic sandy aquifer in Oostrum (the Netherlands) to identify the sequence of steps involved in denitrification coupled to pyrite oxidation. Multi-isotope analyses (δ15N-NO3−, δ18O-NO3−, δ34S-SO42 −, δ18O-SO42 − and δ34Spyrite) confirm that pyrite is the main electron donor for denitrification at this location. Enrichment factors derived from the observed changes in nitrate isotopic composition range from − 2.0 to − 10.9‰ for ε15N and from − 2.0 to − 9.1‰ for ε18O. The isotopic data indicate that pyrite oxidation accounts for approximately 70% of the sulfate present in the zone of denitrification. Solid-phase analyses confirm the presence of pyrite- and organic matter-rich clay lenses in the subsurface at Oostrum. In addition, sulfur XANES and iron XAS results suggest the presence of a series of intermediate sulfur species (elemental sulfur and SO32 −) that may be produced during denitrification. Consistent with geochemical analysis, 16S rRNA gene sequencing revealed the presence of bacteria capable of sulfide oxidation coupled to nitrate reduction and that are tolerant to high aqueous metal concentrations. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Nitrate is a common groundwater pollutant especially in regions of intensive agriculture (Strebel et al., 1989). The major sources of nitrate are manure, fertilizers, atmospheric deposition, soils and plants (Kendall et al., 2007). Denitrification is a natural microbial attenuation process (Korom, 1992) in which microorganisms use organic matter or inorganic compounds as electron donors. When pyrite (FeS2) acts as the electron donor, the process can be represented by the following idealized overall reaction: −

þ

5FeS2 ðpyriteÞ þ 14NO3 þ 4H →5Fe

2þ

2−

þ 7N2 ðgÞ þ 10SO4 þ 2H2 O

ð1Þ

⁎ Corresponding author. Tel.: + 31 302535514; fax: + 31 302535302. E-mail address: [email protected] (C.P. Slomp). 1 Present address: Leibniz Institute for Baltic Sea Research, Warnemünde, Germany. 0009-2541/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.chemgeo.2012.01.024

Pyrite oxidation leads to sulfate production and trace metal release to the groundwater (Broers, 1998). This process can have a major impact on local and regional water quality. Additional (intermediate) products may include nitrite and nitrous oxide (Appelo and Postma, 2005). Several studies have provided compelling evidence for the occurrence of nitrate reduction coupled to pyrite oxidation at field sites (Broers and Buijs, 1997; Pauwels et al., 1998; Postma et al., 1991; Schwientek et al., 2008; Jørgensen et al., 2009; Tesoriero et al., 2000; Zhang et al., 2009). These studies further suggest that denitrification with pyrite can be the dominant pathway of nitrate removal from groundwater, even when organic matter is present. Recent laboratory experiments with sediment from an agricultural area have confirmed the microbial nature of the process (Jørgensen et al., 2009). However, sulfate production in pyritic aquifers is often found to be lower than predicted by Eq. (1) (e.g. (Postma et al., 1991; Miotliński1, 2008; Zhang et al., 2009). This may indicate that

Y.-C. Zhang et al. / Chemical Geology 300-301 (2012) 123–132

2.1. Study site and sampling Oostrum is located in an intensive agricultural area in the south of the Netherlands (Limburg) (Fig. 1). The site covers approximately 1 km 2. Multi-screen sampling wells were installed roughly along the direction of groundwater flow: wells 40, 41 and 42 are located in

.0 15

2. Materials and methods

14 .0

16

.0

38 17 .0

40 41

18 .0 1km

42 .0 20

other processes are active, such as incomplete pyrite oxidation to elemental sulfur (Zhang et al., 2009). An alternative explanation for the mismatch between the observed changes in groundwater nitrate and sulfate with depth is that inputs of nitrogen from agriculture have declined with time. As a consequence, the present-day input of nitrate to the zone of pyrite oxidation may not directly correspond to the amount of sulfate produced at depth. Stable isotope analysis is a powerful tool to identify the sources and sinks of bioactive compounds in natural environments, because the sources may have characteristic isotopic signatures and the isotopes are often strongly fractionated during biogeochemical transformations. This holds for the commonly studied δ 15N-NO3− and δ34S-SO42 −, as well as for the oxygen isotope signatures of nitrate and sulfate. The simultaneous determination of stable isotope ratios of multiple elements may significantly strengthen the interpretation in terms of provenance and transformation processes (Böttcher et al., 1990, 2001; Deutsch et al., 2006; Schwientek et al., 2008; Pauwels et al., 2010). For example, Böttcher and coworkers showed that groundwater δ15N-NO3− and δ18O-NO3− values in a catchment area with high nitrate input were consistent with denitrification coupled to pyrite oxidation (Böttcher et al., 1990). Similarly, isotope data of groundwater sulfate and sediment sulfur compounds for a carbonate-containing organic-poor aquifer suggest that pyrite oxidation is the dominant source of sulfate in the groundwater system (Schwientek et al., 2008). The latter study also illustrates the need for combined studies of the groundwater and solid phase composition for a correct understanding of spatial and temporal trends in microbially-mediated processes in aquifers. Within this context, detailed sediment speciation analyses with techniques such as X-ray spectroscopy are of particular value because they allow potential solid phase intermediates of pyrite oxidation to be identified (Ziegler et al., 2009). Detailed microbial analysis of aquifer sediments can provide complimentary information on biogeochemical processes by providing insight into the organisms that mediate these processes. Such studies have been performed for heterotrophic and autotrophic denitrifiers in marine sediments and laboratory incubations. For example, Brettar et al. (2006) identified autotrophic denitrifiers at the oxic-anoxic interface in the Baltic Sea (Brettar et al., 2006). Various laboratory studies suggest Thiobacillus denitrificans is capable of coupling denitrification to pyrite oxidation (Jørgensen et al., 2009; Torrentó et al., 2010). However, studies identifying the bacteria responsible for denitrification coupled to pyrite oxidation in aquifer sediments from field sites are not yet available. Here, we combine multi-isotope analyses (i.e., δ34S-SO42 −, δ18O-SO42 −, 15 δ N-NO3−, δ18O-NO3−, and sediment δ 34Spyrite) with microbial analysis of the sediment (16S rRNA gene amplification and sequencing) and detailed field data of the aqueous and solid phase geochemistry for a sandy aquifer. Our study site Oostrum (Netherlands) is characterized by a nitrate-rich (up to 8 mM) upper layer overlying deeper, sulfaterich (up to 4 mM) groundwater. Earlier work provides strong evidence for denitrification coupled to pyrite oxidation at this location. This conclusion was based mainly on observed changes in groundwater nitrate and sulfate with depth in the aquifer (Zhang et al., 2009). The goal of this current study is to better characterize the biogeochemical processes occurring along the redox gradient in the aquifer by means of isotopic analyses and to gain insight into the microorganisms that are potentially involved in the processes affecting nitrogen and sulfur in the aquifer.

19 .0

Forest Farmland Town

7649

124

Fig. 1. Well locations at Oostrum. Wells 40, 41 and 42 are located under farmland, whereas well 38 is located in a forested area. The arrow indicates the general groundwater flow direction (Zhang et al., 2009). Two sediment cores were drilled at a distance of ~ 3 m west (Core 1) and ~ 200 m northeast (Core 2) of well 41.

farmland, well 38 is located in a nearby forested area (Broers and Buijs, 1997). The investigated aquifer is unconfined and has an approximate thickness of 45 m. Impermeable clay layers at its bottom clearly define the base of the aquifer. The main aquifer is usually situated between 15 and 45 m depth and consists of coarse fluvial sands of Pliocene age with, in the upper part, unconnected clay lenses, which often have high organic matter contents. Across the area, there are several faults, which are revealed by horizontal discontinuities in hydraulic head. The depth, thickness and grain size distribution of the intermediate clay layer is spatially variable, indeed, the clay is sometimes absent in parts of the area. Thin centimeter thick lenses of clay are present in the top of the Pliocene aquifer, just below the clay layer which separates the Pliocene and Pleistocene sequence. The Pliocene sequence is overlain by ~10 m of coarse fluvial sands and gravels of Pleistocene age which were deposited by the Meuse river and ~5 m of eolian sand deposits. The groundwater table is about 3–5 m below the land surface. Groundwater sampling was carried out in 1996 and 2006 and is described in detail elsewhere (Zhang et al., 2009). Here, we focus on the results for 2006. Two sediment cores were drilled in 2007 at this site, at a distance of ~ 3 m west and ~ 200 m northeast of well 41. These cores are referred to as core 1 and 2, respectively. The cores were split and sub-sampled under Ar. Samples from 28 depth intervals from both cores were freeze-dried and analysed for total Fe with X-ray Fluorescence and for total C and S with a LECO SC 144DR. Solid phase samples from six depths in the aquifer for core 1 and 2 were studied in detail (~16 m, ~18 m and ~23 m in core 1 and 11, 13 and 15 m in core 2) for their microbiological characteristics and Fe and S chemistry. These samples are all from the part of the aquifer where major changes in groundwater nitrate and sulfate are observed. Subsampling was performed directly after drilling and opening of the cores in a glovebox under argon. Samples for microbial analysis were stored at −20 °C. The other samples were stored at 4 °C under argon. For solid Fe and S speciation analysis, small aliquots of homogenized sediments were mixed with mineral oil in a nitrogen-filled glovebox and refrigerated under nitrogen. To confirm that these preservation methods were successful, moist mackinawite (nominally FeS, which reacts rapidly with oxygen) was treated in the same manner, and its integrity following storage was confirmed by X-

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ray absorption spectroscopy. Subsamples for S-isotope analysis were freeze-dried and stored under argon. 2.2. Analytical methods 2.2.1. Isotope analysis Groundwater major anion and cation analyses and 3H/ 3He groundwater age dating methods are described elsewhere (Zhang et al., 2009). Samples for δ 15N-NO3− and δ 18O-NO3− analyses were prepared using the AgNO3 method (Chang et al., 1999; Silva et al., 2000; Xue et al., 2009). The samples were prefiltered with 0.45 μm membrane filters before passing through cation and anion exchange resin columns for nitrate purification and extraction. HCl and Ag2O were then added and AgCl was removed by filtration. The solution was freeze-dried producing solid AgNO3. AgNO3 was converted to N2 gas for δ 15N analysis. Finally, CO2 for δ 18O analysis was produced by combusting AgNO3 with graphite. Isotope analyses were carried out by C-irmMS, and stable N and O isotope results are presented in the usual δ-notation versus air, V-SMOW, and V-CDT, respectively. Dissolved sulfate was precipitated quantitatively from acidified samples as BaSO4 by the drop-wise addition of 5% BaCl2 solution for further determinations of δ 34S-SO42 − and δ 18O-SO42 −. The solid was filtered through a 0.45 μm membrane filter, washed and dried at 60 °C in a drying oven. For oxygen isotope analysis the BaSO4 precipitate was heated further in a porcelain crucible to 500 °C to remove potential contaminants. Reduced inorganic sulfur compounds (essentially pyrite) in freeze-dried drill core sediments from cores 1 and 2 were extracted by hot acidic Cr(II)Cl2 solution (Canfield et al., 1986; Fossing and Jørgensen, 1989). The liberated hydrogen sulfide was trapped quantitatively as Ag2S. Sulfur isotope measurements (δ 34S) were carried out using a Thermo Finnigan Delta plus gas isotope mass spectrometer coupled via a Thermo Conflo split interface to an Euro EA elemental analyzer. For oxygen isotope measurements barium sulfate was combusted in a Thermo Quest TC-EA. Sulfur and O isotope ratios are reported in the conventional delta-notation versus V-CDT and V-SMOW, respectively. Replicate measurements agreed within ±0.3‰ and ±0.5‰ for S and O isotope measurements, respectively. Several international intercomparison materials and in-house standards were measured to calibrate the mass scales. 2.2.2. Solid sulfur XANES analysis Solid S speciation was analyzed by S K-edge X-ray near-edge structure (XANES) spectroscopy at the National Synchrotron Light Source, Brookhaven National Laboratory, New York, on beamline X19A. Samples on filter paper were analyzed using a 1 × 7 mm beam, in a He-purged atmosphere. The sulfate white line peak was calibrated to 2483.1 eV. Sample fluorescence was measured using a PIPS (passivated implanted planar silicon) detector. Data was background-corrected and normalized prior to least-squares fitting with known standards in WinXAS, over the range 2466 to 2487 eV. Data were best fit using reference spectra for sulfate, sulfite, elemental sulfur, pyrite, and iron sulfide (mackinawite). Organic sulfur compounds also were considered but were not present in sufficient quantities to affect fitting and thus are omitted in final fits. Speciation of S by K-edge XANES is possible for sediment with S contents down to 0.01 wt.% (Prietzel et al., 2011). 2.2.3. Solid iron XAS analysis Iron speciation of solids was investigated by X-ray absorption spectroscopy at the Stanford Synchrotron Radiation Laboratory, beamline 11–2, which is configured with a Si(220) monochromator and a phi angle of 0°. Sample fluorescence was measured with a 30element Ge detector in combination with a 6 μm Mn filter. The beam was detuned as needed to reject higher-order harmonic frequencies and prevent detector saturation. Spectra were backgroundcorrected and normalized using linear pre- and post-edge functions,

125

and internally calibrated using a metallic Fe reference foil (7112.0 eV). The k 3-weighted iron EXAFS spectra (spline from k = 2.5–14) were fit over the k range 2.55–12.5 using the k3-weighted chi functions of reference spectra. Pyrite, magnetite, mackinawite, hematite, goethite, ferrihydrite, biotite and hornblende model compounds were used in the final fits. Biotite and hornblende are representative of Fe silicates rather than explicit mineral components. Other common Fe minerals were also considered, but were not necessary to fit the data. Iron XANES spectra were fit over the range 7105 to 7150 eV, to confirm results obtained by EXAFS fitting or where EXAFS spectra were not available, and were similarly accurate for distinguishing ferric from ferrous phases. 2.2.4. Microbial community analysis Sub-sections of mixed sediment (100 mg) within the sediment redox transition zone in both drill cores were used for DNA extractions. Total genomic DNA was extracted using the PowerSoil DNA extraction kit (MOBIO, Carlsbad, USA), and the crude extracts were further purified using the Wizard DNA Clean-Up Kit (Promega, Madison, USA). The 16S rRNA gene was amplified from bacteria using universal bacterial primers. PCRs, cloning and sequencing were performed according to the approach described by Niemann et al. (Niemann et al., 2006). Sequence analysis was carried out using the ARB (Ludwig et al., 2004) software package with the Silva 98 release database (Pruesse et al., 2007) and the Mothur software package (Schloss et al., 2009). Sequences generated in this study have been deposited in the Genbank database, and are accessible under the following accession numbers HM641428-HM641687. 3. Results and discussion 3.1. Pore water profiles of aqueous components The depth distributions of nitrate in the agricultural area at our study site are characterized by a concentration maximum between 10 and 15 m below the soil surface, as shown for well 41 in 2006 (Fig. 2). Most denitrification takes places below this maximum in nitrate. Here, a sharp decline in nitrate levels coincides with an increase in sulfate concentrations, down to a depth of ~23 m. The depth interval in which nitrate and sulfate exhibit opposing gradients is hereafter referred to as the “reaction zone”. As shown previously, aqueous concentrations of Fe 2 + and trace metals increase strongly within the reaction zone (Zhang et al., 2009). Below the reaction zone, nitrate concentrations are near or below detection, while sulfate concentrations progressively decrease with depth. Qualitatively, the compositional changes in the reaction zone are consistent with those expected for denitrification coupled to the oxidative dissolution of pyrite. 3.1.1. Isotopic analysis of coupled denitrification-pyrite oxidation Within the reaction zone under farmland where denitrification driven by pyrite oxidation occurs in the aquifer, the average values of δ 15N-NO3− and δ 18O-NO3− increase with depth from 7.2‰ to 18.2‰ and from − 2.1‰ to 7.4‰, respectively (Fig. 3). These increases are qualitatively consistent with the preferential utilization of lighter isotopes by denitrifying organisms. Isotope enrichment factors for nitrate can be calculated from the changes in δ 15N-NO3− and δ 18O-NO3−, assuming that denitrification is the only fractionating process, that water parcels can be treated as closed systems, and that the fractionation factor remained constant. The Rayleigh equation (Mariotti et al., 1988; Böttcher et al., 1990) can then be used: δr ¼ δ0 þ ε ln C=C0

ð2Þ

where δr and δ0 are initial and residual isotope values, ε is the isotopic enrichment factor and C/C0 is the remaining fraction of the nitrate.

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Fig. 2. Example of the depth distributions of a) dissolved nitrate and sulfate in groundwater; b) δ34S-SO42 −, δ18O-SO42 − and δ34S in pyrite in solid phase and c) δ15N-NO3− and δ18ONO3− in the agricultural area (well 41). In the “reaction zone” (grey), nitrate concentrations decrease sharply whereas sulfate concentrations increase, suggesting denitrification coupled to pyrite oxidation. The depth intervals above and below the “reaction zone” are referred to as the “shallow part” and “deeper part”, respectively.

As initial nitrate concentration (Co) we impose the maximal value at the top of the reaction zone. Then, using the pair-wise concentration and δ values measured in the reaction zone, we obtain values of −10.9‰ (well 41) and −2.0‰ (well 42) for ε 15N, and −9.1‰ (well 41) and −2.0‰ (well 42) for ε 18O (Note: well 40 lacked sufficient nitrate containing samples to perform the isotope analyses). Culture experiments with heterotrophic denitrifiers have yielded wide ranges of enrichment factors: from −10‰ to − 39‰ for ε 15N, and between − 15‰ and 40‰ for ε 18O (Lehmann et al., 2003; Toyoda et al., 2005). Field studies of denitrification suggest comparable ranges of ε 15N and ε 18O for autotrophic and heterotrophic denitrification. For example, Böttcher et al. (1990) reported values of −16‰ for ε 15N and −8‰ for ε 18O for autotrophic denitrification coupled to pyrite oxidation in a sandy aquifer in Germany, while Mariotti et al. (1988) estimated a ε 15N value of ~− 5‰ for heterotrophic denitrification in an aquifer of northern France. Although the enrichment factors exhibit large ranges, the δ 15N:δ 18O ratios reported in the literature for denitrification are mostly comprised between 0.9 and 2.1 (Böttcher et al., 1990). Our value for the wells located in the agricultural area (~1, Fig. S1) falls within this range. The isotopic composition of dissolved sulfate in groundwater is also quite variable, with δ 34S-SO42 − ranging from − 4.8 to 45.3‰

and δ 18O-SO42 − from −0.7‰ to 15.3‰ (Table S1). The two major sources of groundwater sulfate in the Oostrum area are sulfate leaching from the land surface (where it is introduced via atmospheric input, manure and fertilizers) and sulfate from pyrite oxidation, with no important sinks (Zhang et al., 2009). Sulfur isotopes undergo essentially no isotope fractionation during pyrite oxidation to sulfate (Stempvoort Van et al., 1994) Hence, the groundwater δ 34S-SO42 − signature should only reflect the relative contributions of the sulfate sources, according to the following mass balance (Moncaster et al., 2000; Smith et al., 2009): Csulfate

34

total

 δ Ssulfate

total

¼ Csulfate

34

input

þ Csulfate

 δ Ssulfate 34

input

pyrite  δ Spyrite

ð3Þ

where Csulfate_total = Csulfate_input + Csulfate_pyrite and Csulfate_total is the measured (total) sulfate concentration in a groundwater sample, Csulfate_input is the sulfate concentration derived from the land surface and Csulfate_pyrite is that derived from pyrite oxidation. δ 34S sulfate_total, δ 34Ssulfate_input and δ 34Spyrite are the δ values of the groundwater, the historical input derived from the land surface and sedimentary pyrite, respectively.

Fig. 3. Box plots of δ15N-NO3−, δ18O-NO3−, δ34S-SO42 − and δ18O-SO42 − in the three depth zones in the agricultural area (wells 40, 41 and 42) in 2006. See Fig. 1 for the definition of the depth zones. Thick line: median; upper and lower boundary of the boxes: 25 and 75 percentile; whiskers: range; bullets: outliers; n: number of observations.

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Assuming that the most shallow groundwater samples collected from wells 40, 41 and 42 are representative of surface input in the agricultural area, the average δ 34Ssulfate_input value is around 2.6‰ (Fig. 3). Then, assigning a value to δ 34Spyrite of −4.2‰, which is the mean value measured on the samples of drill core 1 which is located adjacent to well 41 (Table S2), we can calculate the relative contributions of pyrite oxidation to the measured groundwater sulfate concentrations (i.e., Csulfate_pyrite/Csulfate_total). Input of sulfate leaching from the land surface accounts for >95% of total sulfate in the groundwater above the reaction zone. Across the reaction zone, however, the average δ 34S sulfate_total drops to −2.1‰, implying a significant contribution of lighter sulfate produced during pyrite oxidation, on the order of 70%. Below the reaction zone, Eq. (3) predicts a decrease in the contribution of pyrite oxidation, which is due to microbial sulfate reduction (see below). Sulfur isotope data for pyrite can vary over a wide range (Canfield et al., 2005). For example, pyrite analyzed in core 2, which is located ~ 200 m to the north west of well 41 (Fig. 1), has a much heavier isotope composition at shallow depths (Table S2). The variation likely reflects heterogeneity of δ 34S-pyrite in this Pliocene aquifer. However, such high δ 34S values in pyrite are not reflected in the groundwater composition of the wells at our study site. In addition to nitrate, molecular oxygen is a common electron acceptor for pyrite oxidation in natural settings. The oxygen isotopic composition of sulfate can help distinguish between the potential electron acceptors (e.g., (Taylor et al., 1984; Tuttle et al., 2009). According to the following reaction stoichiometry 2FeS2 ðpyriteÞ þ 7O2 þ 2H2 O→2Fe

2þ

2−

þ

þ 4SO4 þ 4H

ð4Þ

7/8 of the oxygen in sulfate is derived from molecular oxygen whereas the remaining oxygen comes from water. We can then use an isotope balance for δ 18O-SO42 − to calculate the expected δ 18O-SO42 − values when pyrite is oxidized by O2. Based on literature data, we assign δ18OO2 and δ18OH2O values of 23‰ (Tuttle et al., 2009) and −7‰ (Clark and Fritz, 1997), respectively and enrichment factors of ε 18O(SO4-O2) and ε 18O(SO4-H2O) of −11‰ and 4‰ (Taylor et al., 1984; Toran and Harris, 1989). The predicted groundwater δ 18O-SO42 − in the reaction zone should then be on the order of 10‰. This value is well outside the range of our measurements in the reaction zone in the agricultural area, which show a maximum value of 2.8‰ (Table S1). Therefore, we can eliminate O2 as an important oxidant for pyrite at the Oostrum site. The isotopic evidence is entirely consistent with the presence of a 10 m thick upper aquifer with low to negligible oxygen concentrations above the reaction zone (Zhang et al., 2009). A similar calculation demonstrates that H2O molecules cannot be the main source of oxygen in sulfate either. If all sulfate oxygen was derived from H2O, δ 18O-SO42 − should be on the order of −1‰ in the reaction zone, compared to the observed mean value of 1.5‰ and a minimum value of 0.2‰. If we assume that nitrate is the sole oxygen source for sulfate produced by pyrite oxidation (Eq. (1)), the isotope balance yields a corresponding ε 18O(SO4-NO3) value of 3‰. There are currently no other experimental data on oxygen isotope fractionation during pyrite oxidation coupled to nitrate reduction to which this field-derived estimate of ε 18O(SO4-NO3) can be compared. 3.1.2. Isotopic evidence for microbial sulfate reduction Bacterial dissimilatory sulfate reduction generates a typical enrichment of δ 34S-SO42 − in the residual sulfate (δ 32S-SO42 −) (Kaplan and Rittenberg, 1964; Fritz et al., 1989; Böttcher et al., 2001; Habicht and Canfield, 2001). Such a trend is observed in the isotope data from deeper parts of the aquifer (>20 m) where the δ 34S-SO42 − values (average of 8.9‰ with a maximum of 45.3‰) markedly exceed those in the reaction zone (Fig. 3). The trend of increasing δ 34S-SO42 − values with depth corresponds to decreasing sulfate concentrations

127

(Fig. 2 and Table S1; Zhang et al., 2009). The mean value of δ18O-SO42 − in the deeper groundwater (4.9‰) also shows an increase compared to the reaction zone. Taken together, the decrease in sulfate concentration and the dual-isotope fractionation implies active sulfate-reducing microorganisms below the zone of denitrification (Mizutani and Rafter, 1973; Fritz et al., 1989; Böttcher et al., 1998). 3.1.3. Time scales Groundwater age data can provide important information on groundwater flow paths and the time scales of biogeochemical processes in the subsurface, especially when coupled to groundwater concentrations of key elements and the historical input of concentrations in recharge (Visser et al., 2009; Zhang et al., 2009). For instance, the simultaneous increase in δ 15N-NO3− and decrease in δ 34S-SO42 − is restricted to groundwater that entered the aquifer between 1985 and 1995 (Fig. 4). Thus, it takes a maximum of 10 years for denitrification to remove the incoming nitrate from the groundwater. In contrast, the general increase in δ 34S- SO42 − with age beyond 1985 spans at least three decades. Given the corresponding relatively small decrease in the sulfate concentration (Fig. 2), this points to much lower rates of sulfate reduction compared to denitrification. 3.2. Solid-phase analysis Depth profiles of total sulfur, carbon and iron for drill cores 1 and 2 confirm the strong heterogeneity of the sediment in the aquifer at Oostrum observed visually (Fig. 5). In drill core 1, sediment layers enriched in total carbon, sulfur and iron and in fine-grained material (b63 μm) are present at depths of ~ 14–15 and 21 m below the land surface. In drill core 2, similar layers are present at depths of ~12–14, 16 and 21 m. Both drill cores show the ~ 2 m thick clay layer at the top of the Pliocene Formation, which corresponds with the samples with the highest proportion of the size fraction b63 μm. The deeper samples with a larger fraction b63 μm coincide with intervals where thin clay lenses exist. The reaction zone identified from the water chemistry data coincides with the top of the Pliocene aquifer, in which the higher proportions of clay and silt material, organic matter and sulfur are concentrated. Sampling sediments from such a heterogeneous system implies that the samples cannot be treated as being representative for a gradient in solid phase chemistry. Instead, we consider the 6 samples that were used for XANES/XAS analysis to be illustrative rather than to be exhaustive. Insight in the mineralogical composition of the sediment from 6 selected depth intervals in the reaction zone (at depths of ~16, ~18 and ~23 m in core 1 and 11, 13 and 15 m in core 2) is obtained from sulfur XANES and iron XAS analysis (Table 1). In general, the fraction of Fe present in silicates is variable and reflects differences in primary mineralogy and sediment sources. In these samples, silicates (e.g. glaucophane, biotite or glauconite) represent a minor fraction (5–34%) of the total Fe. Here, we restrict our discussion to nonsilicate Fe phases to effectively focus on the reactants and products of redox reactions in the aquifer. The 6 samples contain between 0.1 and 0.7% of Fe (Fig. 6, Table 1). The amount of pyrite increases strongly at depth in both cores, which is in general agreement with the redox gradient. Note, however that there is substantial heterogeneity in the fraction b63 μm, in S and organic C between the individual samples (Fig. 5). The samples from the upper part of the reaction zone (16 and 11 m in cores 1 and 2, respectively) are dominated by reduced ferrous and ferric iron oxide phases and contain little pyrite Fe. The samples from intermediate depths (13 and 18 m in cores 1 and 2, respectively) have lower iron contents and show a mixture of ferric and ferrous iron and pyrite Fe. The deepest samples analysed (23 and 15 m in cores 1 and 2, respectively) are dominated by pyrite Fe and contain about 30% of ferric and ferrous oxides. In well 40, close to core 2, groundwater at 15 m depth still contained some nitrate in 1996 (Zhang et al., 2009) whereas

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Fig. 4. Values of δ15N-NO3− and δ34S-SO42 − plotted versus groundwater age for wells 40, 41 and 42, which are all located in an agricultural area. The increase in δ15N-NO3− and decrease in δ34S-SO42 − during the period 1985 to 1995 implies denitrification coupled to the oxidation of isotopically light pyrite. In older groundwater, the general increase in δ34S-SO42 − with age is interpreted to reflect sulfate reduction. The anomalously low δ34S-SO42 − values in the groundwater older than 1970 observed in well 42 just below the reaction zone are probably due to local heterogeneity of the aquifer.

nitrate was absent in 2006. Thus, in this part of the aquifer pyrite oxidation with nitrate likely occurred in the past. This may explain the presence of ferrous iron at this depth. The sample from 23 m depth in core 1, in contrast, is located well below the reaction zone where nitrate never has been present. Sulfur contents in the 6 samples roughly follow the pattern of the pyrite Fe contents, with almost no sulfur being present in samples 16 and 18 m depth in core 1 and from 11 m depth in core 2 (Fig. 6). The other 3 samples show significant amounts of sulfur, dominated by pyrite-S and SO4-S. The presence of sulfate mineral throughout the reaction zone was unexpected (Table 1) and may be explained by the partial oxidation of sulfide minerals in storage, but may also indicate the presence of solid phase sulfur (in adsorbed or mineral form) in the aquifer. The sulfur speciation also reveals the presence of previously unidentified sulfur species (elemental sulfur and SO32 −). For example, these intermediate oxidation-state species represent >90% of the total S in the sample from 18 m depth in core 1 (Table 1) However, total S in this sample is low (Table 1). Aqueous concentrations measured in the reaction zone suggest that the bulk of the denitrification coupled to pyrite oxidation is associated with an increase of Fe[II] and sulfate and decrease of nitrate

(Zhang et al., 2009). Together with the groundwater geochemistry data, the abundant presence of non-silicate Fe[III] mineral suggests that, besides pyrite oxidation producing sulfate and Fe[II] (Eq. (1)), further oxidation of Fe(II) by nitrate (Eq. (5)) to Fe(III) could occur: −

þ

5Fe½II þ NO3 þ 7H2 O→5FeOOH þ 0:5N2 þ 9H

ð5Þ

Previous field studies suggest that oxidation of Fe[II] to Fe[III] with nitrate (Eq. (5)) is less important for nitrate removal than sulfide (pyrite) oxidation (Postma et al., 1991; Zhang et al., 2009). In addition, the observed rise in pH in the reaction zone (Zhang et al., 2009) is consistent with a more important role for pyrite oxidation with nitrate following Eq. (1) at this depth. It is possible that the sulfur intermediates which are found in many of the 6 samples are formed as minor products through one or more separate reactions coupling pyrite oxidation and denitrification. Further work would be needed to quantify their occurrence over the whole depth profile in this heterogeneous aquifer and to assess their potential role in contributing to possible variations in the stoichiometry of the overall reactions as suggested for this site (Zhang et al., 2009). Alternatively, these intermediates also could be the major reaction products of

Fig. 5. Depth profiles of total sulfur, carbon and iron (in wt.%) and the grain size fraction b63 μm in sediment drill cores 1 and 2. The depths of samples used for sulfur XANES and iron XAS in core 1 and 2 are indicated with open circles. The lithology is indicated in the two borehole descriptions. A large fraction of material with a grain size b63 μm indicates clay and silt layers, some of which are 2 m thick, whereas others are only millimeters or a few cm thick.

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Table 1 Weight percentages of iron and sulfur and their speciation as determined with XAS and XANES in samples from 3 depth intervals in Core 1 and Core 2. Core

Depth (m)

Total Fe (wt.%)

Fe-silicate minerals (% of total Fe)

Fe (II) oxides (% of total Fe)

Fe (III) oxides (% of total Fe

Pyrite (% of total Fe)

Pyrite (wt.% based on total Fe)

1 1 1 2 2 2

16 18 23 11 13 15

0.20 0.09 0.42 0.73 0.16 0.43

26 21 18 34 14 5

43 18 25 26 32 0

16 59 2 37 26 32

15 2 55 3 28 63

0.03 0.00 0.23 0.02 0.04 0.27

Core

Depth (m)

Total S (wt.%)

FeS (% of total S)

Pyrite (% of total S)

S(0) (% of total S)

SO32 − (% of total S)

SO4 mineral (% of total S)

Pyrite (wt.% based on total S)

1 1 1 2 2 2

16 18 23 11 13 15

0.11 0.02 1.37 0.01 0.10 0.28

0 0 5 0 0 3

6 0 40 13 17 29

0 32 4 2 8 5

0 60 0 0 17 0

94 8 51 85 58 63

0.01 0.00 0.55 0.00 0.02 0.08

pyrite oxidation by nitrate, with the elemental sulfur being oxidized or disproportionated by autotrophs leading to sulfate as one endproduct (Bak and Cypionka, 1987; Thamdrup et al., 1994; Finster, 2008). The high contents of pyrite-Fe and pyrite-S in the samples at 15 m depth (Core 2) and 23 m depth (Core 1) coincide with the depth range where nitrate concentrations are below the detection limit and dissolved sulfate concentrations are near peak values (~3 mM, Fig.1 and Zhang et al., 2009). It is likely that no nitrate at this depth is consumed by pyrite oxidation, leaving all pyrite in the sediment matrix intact. The major redox reaction taking place below this depth range is likely sulfate reduction, as indicated by the depth trends in δ 34S-SO42 − in the groundwater of wells 40 and 41 (Fig. 2 and Table S1). 3.3. Linking geochemistry to microbial community composition The solid-phase and aqueous geochemistry indicate that the aquifer at this site shows a distinct reaction zone within an overall strongly heterogeneous sediment matrix. While in the samples from the upper and middle part of the reaction zone Fe oxides are the most abundant non-silicate Fe-phases, pyrite is quantitatively most important in the lower samples. Here, micro-organisms are linked to

geochemical processes by relating both sediment mineralogy and porewater geochemistry to genetic characterizations of the microbial community. Between 40 and 45 16S rRNA gene sequences were obtained from each of 6 samples in the reaction zone from drill cores 1 and 2 (Table 2). Organisms closely related to heterotrophic nitrate reducing bacteria, such as Massilia brevitalea, (Zul et al., 2008), Herbaspirillum autotrophicun (Ding and Yokota, 2004) and Cupriavidus metallidurans (Goris et al., 2001) were detected in the sediments from both cores. The presence of these bacteria suggests heterotrophic denitrification cannot be excluded completely and these bacteria are either carrying out heterotrophic denitrification at a low rate or are involved in other processes associated with the slow oxidation of recalcitrant sedimentary organic carbon. Note that heterotrophic denitrification would be expected to more favorable than autotrophic denitrification based on theoretical energy yields (Devlin et al., 2000). However, the pyrite in this system is likely more reactive than the organic matter allowing autotrophic denitrification to dominate”. The porewater and solid phase data indicate drastic changes in the chemical forms and concentrations of N, Fe and S with depth that are consistent with denitrification linked to pyrite oxidation (Zhang et al., 2009). While microbially mediated nitrate-dependent sulfide (pyrite) oxidation is well-known, microbial evidence for a similar process (by

Fig. 6. Distribution of Fe (non-silicate) minerals and S minerals in solid phase in fresh sediment from the drill cores 1 and 2. The fractions of Fe and S minerals were determined with XAS (for Fe) and XANES (for S). For the solid phase total Fe (wt.%) and the total S (wt.%) see Table 1.

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Table 2 Breakdown of 16S rRNA gene sequences for three different depths [m] in the drill core 1 and core 2. Phylogentic groupa

Janthinobacterium/ Oxalobacter Herbaspirillum Ralstonia/Cupriavidus Acidovorax Thiobacillus Thiothrix Pseudomonas Acinetobacter Sphingomonas Gallionella Other Gammaproteobacteria Methylobacterium Other Alphaproteobacteria Deltaproteobacteria Acidobacteria Nitrospirae Actinobacter Firmicutes Bacteriodetes Total number of sequences

Closest relativeb

% Identityc

Core 1

Core 1

Core 1

Core 2

Core 2

Core 2

16 m

18 m

23 m

11 m

13 m

16 m

2

13

11

13

6 5

7 10

8 13

1

3

3

Massilia brevitalea, EF54677

99–95

15

Herbaspirillum autotrophicum, AB074524 Cupriavidus metallidurans CH34, Y10824 BrG1, U51101 Thiobacillus denitrificans, AJ243144 Thiothrix nivea, L40993, Thiothrix unzii, L79961 Pseudomonas sp. QZ1, EF542804 Acinetobacter baumannii, EF672505 – Gallionella ferruginea, L078797 – Methylobacterium thiocyanatum, U58018 – – Acidobacteriaceae bacterium TAA48, AY587229 Thermodesulfovibrio thiophilus, AB231857 – – –

97–96 99–97 99–94 98 99,98 99–97 99–97 – 94–96 – 99–97 – – 87–72 86–85

2 16 4

2

1 4

1 12

1 1 1 1

8 8

2

4

2 1 2 3 23 2

0 2

3

– – 45

2 40

2 1 43

2 4 1 11 1 2

46

1 1 1

5 1

1

42

43

(a) Phylogenetic affiliation; (b) Closest type strain or cultured representative; (c) Sequence identity to closest type strain or cultured representative.

Thiobacillus denitrificans) with pyrite has only recently been published (Jørgensen et al., 2009). Bacteria closely related (99–98%) to sulfide-oxidizing, denitrifying species, such as Thiobacillus denitrificans (Kelly and Wood, 2000), Thiotrix nivea (Larkin and Shinabarger, 1983) and Thiotrix unzii (Polz et al., 1996) were present at a depth of 23 m in core 1. Furthermore, bacteria closely related to the sulfide-oxidizing, denitrifying bacterium Pseudomonas stutzeri (Mahmood et al., 2009) were detected within the same interval of core 1 and in all 3 intervals analysed for core 2. The presence of other solid-phase products in the reaction zone, i.e. elemental sulfur and sulfite suggests that some of the S is incompletely oxidized and that the complete oxidation of pyrite to sulfate involves an additional step (and possibly an additional microbe) that can oxidize these species. Organisms closely related to Methylobacterium thiocyanatum (Anandham et al., 2009) (99–97%) are present in many of the samples. This last organism and its relatives are known to oxidize intermediate nitrogen and sulfur compounds such as thiocyanate and thiosulfate (Anandham et al., 2009). These organisms may play a role in pyrite oxidation coupled to denitrification, by recycling reduced or intermediate sulfur compounds that are produced upon the interaction between aqueous solutions and sulfide minerals (Schippers and Jørgensen, 2002). Bacteria closely related to Gallionella ferruginea, which is known to oxidize Fe[II] (Hallbeck et al., 1993), were detected along with bacteria closely related to the iron oxidizer BrG1(Straub et al., 1996) in the samples from core 1 (Table 2). These organisms are likely responsible for Fe[II] oxidation, possibly with nitrate. Over 50% of the sequences recovered from these sediments were either closely related to those of bacterial genera that are often detected in environments with elevated metal/metalloid concentrations (such as observed in natural waters impacted by mining or waste water), or to those of genera with members that have shown a specific tolerance to elevated metal/metalloid concentrations, e.g. Acinetobacter, Cupriavidus, Herbaspirillum, Janthinobacterium, Methylobacterium, Oxalobacter, Pseudomonas, and Rahnella (Hery et al., 2003; Mergeay et al., 2003; Reardon et al., 2004; Idris et al., 2006; Palmroth et al., 2007; Zakaria et al., 2007). The presence of these organisms coincides with the high metal concentrations observed in groundwater from the reaction zone in this aquifer (e.g. Ni concentrations of ~200 μg/L; Zhang et al., 2009). These geochemical conditions are the result of the recent input of anthropogenic nitrate into the pyrite containing aquifer and the corresponding mobilization of trace metals.

4. Conclusions Denitrification coupled to pyrite oxidation is identified as the principal biogeochemical process causing large changes in nitrate and sulfate concentrations in groundwater below agricultural fields at our field site, at Oostrum (the Netherlands). Isotopic data for sulfate (δ 34S-SO42 −, δ 18O-SO42 −), nitrate (δ 15N-NO3−, δ 18O-NO3) and pyrite (δ 34Spyrite) confirm our earlier work (Zhang et al., 2009) that pyrite is the main electron donor for denitrification at this location and that sulfate reduction occurs at depth. Solid phase analyses confirm the highly heterogeneous distribution of organic matter and pyriterich clay lenses in the aquifer. Iron XAS and sulfur XANES confirm the presence of pyrite and suggest that various intermediate sulfur species (elemental sulfur and SO32 −) are formed in the zone of denitrification. Observed differences in microbial populations with depth are suggestive of a series of reactions along the redox gradient in this aquifer. Besides sulfide (pyrite) oxidation with nitrate to sulfate, these processes include, heterotrophic denitrification (at a very slow rate), incomplete oxidation of sulfide to elemental sulfur and Fe(II) oxidation with nitrate. The microbial populations are also related to metal-tolerant bacteria which is consistent with the high concentrations of metals in the groundwater at this location. Acknowledgments Financial support was provided by the Netherlands Organisation for Scientific Research (NWO-water and Vidi grant 86405.004 to CPS). The authors thank A. Visser, J. Rozemeijer, A. Dale, D. van de Meent, H. de Waard, G. Nobbe, G. Laruelle, M. Verheul and G. Klaver for help with the field work. We thank TNO for making available data from an earlier project. We also thank the UU-TNO lab for laboratory analysis. EO acknowledges support from the European Commission through the FP6 Mobility Actions AquaTRAIN (Contract no. MRTN-CT-2006-035420). MEB wishes to thank R. Rosenberg (IOW) for lab assistance and T. Max (MPI-MM) for mass spectrometric support. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10. 1016/j.chemgeo.2012.01.024.

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