Science of the Total Environment 423 (2012) 176–184

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Biologically-induced precipitation of sphalerite–wurtzite nanoparticles bysulfate-reducing bacteria: Implications for acid mine drainage treatment

Julio Castillo a, Rafael Pérez-López a,b,⁎, Manuel A. Caraballo a,c, José M. Nieto a, Mónica Martins d,M. Clara Costa d, Manuel Olías e, Juan C. Cerón e, Rémi Tucoulou f

a Department of Geology, University of Huelva, Campus ‘El Carmen’, 21071, Huelva, Spainb Institute of Environmental Assessment and Water Research, IDÆA-CSIC, Jordi Girona 18, 08034, Barcelona, Spainc Department of Geosciences, Virginia Tech, Blacksburg, VA 24061, USAd Centro de Ciências do Mar, CCMAR, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugale Department of Geodynamic and Palaeontology, University of Huelva, Campus ‘El Carmen’, 21071 Huelva, Spainf European Synchrotron Radiation Facility, 6 Rue Jules Horowitz, Grenoble, France

⁎ Corresponding author at: Department of Geology, UCarmen’, 21071, Huelva, Spain. Tel.: +34 95 921 9819;

E-mail address: rafael.perez@dgeo.uhu.es (R. Pérez-

0048-9697/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.scitotenv.2012.02.013

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 December 2011Received in revised form 9 February 2012Accepted 9 February 2012Available online 11 March 2012

Keywords:Sulfate-reducing bacteria (SRB)Zinc toleranceZnS nanocrystalsTreatmentAcid mine drainage

Several experiments were conducted to evaluate zinc-tolerance of sulfate-reducing bacteria (SRB) obtainedfrom three environmental samples, two inocula from sulfide-mining districts and another inoculum from awastewater treatment plant. The populations of SRB resisted zinc concentrations of 260 mg/L for 42 days ina sulfate-rich medium. During the experiments, sulfate was reduced to sulfide and concentrations in solutiondecreased. Zinc concentrations also decreased from 260 mg/L to values below detection limit. Both decreaseswere consistent with the precipitation of newly-formed sphalerite and wurtzite, two polymorphs of ZnS,forming b2.5-μm-diameter spherical aggregates identified by microscopy and synchrotron-μ-XRD. Sulfateand zinc are present in high concentrations in acid mine drainage (AMD) even after passive treatmentsbased on limestone dissolution. The implementation of a SRB-based zinc removal step in these systemscould completely reduce the mobility of all metals, which would improve the quality of stream sediments,water and soils in AMD-affected landscapes.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Wastes from the mining of sulfide-bearing ores interact withwater and air to produce extremely acidic leachates containing highconcentrations of sulfate and heavy metals known as acid mine drain-age (AMD). AMD treatment technologies based on the addition ofchemicals in plants are available, but relatively expensive, in zoneswhere the mining industry is active. However, such technologiescould be impracticable at abandoned mine sites with unclear legal re-sponsibility for treatment. Instead, passive treatment technologiesprovide a viable low-cost and -maintenance alternative (Johnsonand Hallberg, 2005).

Most of the passive restoration strategies promote acidity neutral-ization andmetal removal in chemical reactors by the use of limestoneas alkaline reagent, which causes efficient precipitation of hydroxidesof trivalent metals such as Fe and Al. These secondary phases can re-tain trace elements by adsorption and/or co-precipitation processes(Bigham and Nordstrom, 2000). However, AMD waters can alsoshow high concentrations of divalent metals like Zn and Mn (tens to

niversity of Huelva, Campus ‘Elfax: +34 95 921 9810.López).

rights reserved.

hundreds of mg/L) and minor but toxic concentrations of Cd, Co andNi (tens to hundreds of μg/L). Chemically induced precipitation ofsuch divalent metals requires pH values ranging between 8 and 10,which is impossible to achieve in AMD by limestone dissolution be-cause, due to the high Ca concentration in these waters, equilibriumwith calcite is reached at pH between 6 and 7 (Cortina et al., 2003).

Biological passive treatment based on the use of sulfate-reducingbacteria (SRB) has recently emerged as an attractive solution forAMD treatment. SRB are a taxonomically diverse group of strictlyanaerobic bacteria, capable of reducing sulfate and oxidizing organ-ic substrates simultaneously (e.g., see review in Barton and Fauque,2009). Sulfate-reduction leads to the production of sulfide that caneasily react with divalent metals to form precipitates of insolublemetal sulfides (Gadd and White, 1993). Due to the ability to immo-bilize toxic metals via precipitation, SRB play an important role inthe remediation of polluted wastes by both natural attenuation pro-cesses (e.g., Sarmiento et al., 2009) and engineered treatment tech-nologies (e.g., Jong and Parry, 2003). Hence, SRB are one of themost important bacterial communities within the existing microbialbiodiversity due to their great economic, environmental and bio-technological importance.

Passive treatment technologies, based on the use of SRB, typicallyemploy a mixture of alkaline material (mainly limestone or other

177J. Castillo et al. / Science of the Total Environment 423 (2012) 176–184

carbonates to increase and maintain a neutral water pH) and an or-ganic substrate (as carbon source for the bacterial metabolism) to re-mediate AMD pollution. The common approach employed in the fieldsites is to submit the reactive mixture, and subsequently the bacterialcommunity, directly to the “raw” AMD without any previous pre-treatment to decrease or eliminate a portion of the metal load and di-versity. This design criterion subjects the SRB communities not onlyto the toxic effect of each metal but also to the synergic effect of thepresence of various metals in the same solution. The common conse-quence in SRB-based passive treatments dealing with moderate tohigh metal polluted AMDs is the absence of any significant reducingenvironment and metal removal by SRB after some weeks or monthsof operation (e.g., Caraballo et al., 2010). To solve this problem, someauthors have proposed the use of an alkaline pretreatment previousto the use of a SRB-based treatment (e.g., Pagnanelli et al., 2008). Pas-sive treatment technologies using alkaline reagents have been effi-ciently tested for the complete removal of high trivalent metalconcentrations in AMD (e.g., Caraballo et al., 2009). However, theuse of SRB to remediate the highly divalent metal polluted effluentsof the alkaline treatments still needs to be addressed.

The lack of tolerance of bacterial cells to high concentrations ofheavy metals is one of the main factors limiting their use in bioreme-diation (Utgikar et al., 2001). Divalent metals such as Zn and Mn oftenremain at high concentrations in mining effluents even after the useof an alkaline pretreatment (e.g., Ríos et al., 2008; Caraballo et al.,2009). The suppressive effect of Mn on SRB growth is weaker thanthat of Zn, and up to 200 mg/L can be totally removed from the solu-tions without affecting the microbial consortium (Yoo et al., 2004).However, some studies have reported concentrations of 210 mg/L ofZn to be lethal for SRB (Radhika et al., 2006). In this sense, the identi-fication of SRB strains resistant to high concentrations of zinc is cru-cial for the development of effective bioremediation processes to beimplemented in highly polluted waters. The main aim of this reportis to demonstrate the effectiveness of a biological treatment withSRB to deal with effluents containing high sulfate and zinc concentra-tions. To this end, some laboratory experiments are conducted to ob-tain consortia of SRB that can be even able to both: (1) tolerate higherzinc concentrations than 210 mg/L, and (2) remove zinc from solu-tions by precipitation of insoluble metal sulfides.

2. Materials and methods

2.1. Inocula and bacterial enrichment

The bacterial communities were obtained from two solid samplesthat acted as bacterial inocula and one from a mixed culture of SRB

Fig. 1. Field photographs of acidic streams from two abandoned mines at the IPB in which Cthe submerged entrance to Cueva de la Mora mine (white arrow) and (b) ME inoculum fro

previously obtained by Martins et al. (2009). The solid sampleswere collected from sediments in acidic streams draining two IberianPyrite Belt (IPB) mining districts in which water Zn concentrationscorrespond to the maximum and minimum contents commonlyfound in this region (Fig. 1): Cueva de la Mora (CM) and Mina Esper-anza (ME) with 400 and 30 mg/L of Zn, respectively. The zinc concen-trations in the discharge from Cueva de la Mora are reported to be apriori toxic to SRB (Azabou et al., 2007).

The mixed culture of SRB was selected because it contains highlyheavy metal resistant SRB which are able to tolerate Zn concentra-tions up to 150 mg/L (Martins et al., 2009). These resistance limitssuggest that the inoculum could be even tolerant to higher concentra-tions, typical of IPB mining environments. This bacterial communitywas grown, and maintained in Postgate B medium (Postgate, 1984)in anaerobic conditions at room temperature, from a sludge sample(SS) from the wastewater treatment plant of Montenegro located atthe Algarve region (S Portugal), relatively close to the IPB (around50 km).

The enrichment culture was developed in batch experimentsunder anaerobic conditions, as described by Martins et al. (2009).Serum bottles were filled with 100 mL of modified Postgate B culturemedium at pH of 6–7 and seeded with 5% of inoculum samples. Post-gate B medium with resazurin as redox indicator (30 mg/L) was an-aerobically prepared as described by Postgate (1984). Thus, themedium was gassed with a stream of O2-free N2 for 3 min and10 mL of sterile liquid paraffin was added to enclose it and to avoidthe oxygen diffusion. After inoculation, the bottles were sealed withbutyl rubber stoppers and aluminum crimps, and then incubated atroom temperature (±21 °C) for 35 days. During the experimentalrun, solutions were monitored once per week.

The SRB were enumerated by the most probable number (MPN)technique (n=3) utilizing Postgate E medium (Postgate, 1984) atthe middle and end of the experiment. After bacterial enrichment,SRB were collected by centrifugation at 4000 rpm for 10 min, washedwith the same culture medium and transferred to the zinc removalexperiments.

2.2. Zinc removal experiments

Experimental cultures were carried out over 42 days to investigateboth activity and tolerance of the previously enriched SRB in the pres-ence of an initial Zn concentration of 260 mg/L. The chemical of solu-tions in these experiments, i.e. neutral waters with highconcentrations of sulfate (ca. 2000 mg/L) and zinc (ca. 260 mg/L), re-flects exactly the real output leachates from an alkaline passive sys-tem installed to treat extremely polluted AMD in the IPB (Caraballo

M and ME inoculum samples were obtained: (a) CM inoculum from outflow pond andm outflow tunnel of Mina Esperanza mine.

178 J. Castillo et al. / Science of the Total Environment 423 (2012) 176–184

et al., 2009). To date, no study has published a SRB inoculum that hasbeen able to resist such high Zn concentrations. Cultivations wereconducted in 250 mL flasks containing 200 mL of minimal growthmedium (Martins et al., 2010). The minimal medium contains50 mg/L yeast extract, 1000 mg/L NH4Cl, 60 mg/L CaCl2·6H2O,1000 mg/L MgSO4·7H2O, 2000 mg/L Na2SO4 and 5000 mg/L sodiumlactate. Zinc hemisulfate salt (ZnSO4·7H2O; ca. 1100 mg/L) wasadded to the medium in each experiment to adjust the initial concen-tration to the desired value and Zn concentrations were monitoredwith time. The pH of the culture medium was adjusted to 7 with1 M NaOH solution, and the final solution was deaerated with a fluxof N2 and sealed with sterile liquid paraffin. Moreover, the experi-ments were carried out at room temperature inside a N2-purgedglove box to avoid contamination by atmospheric O2. Solutionswere sampled once per week. Moreover, the bacterial activity as anindirect assessment of the efficiency of zinc removal was once moremeasured by enumerating the SRB using the MPNmethod at the mid-dle and end of the experiments.

7.0

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0 5 10 15 20 25 30 350

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SSMECM

Fig. 2. Time evolution of (a) pH, (b) Eh and (c) sulfate concentration in the media in theenrichment experiments [SS: inoculum from treatment plant sludge samples, ME: in-oculum from Mina Esperanza mine, and CM: inoculum from Cueva de la Mora mine].Data are the mean of duplicates and error bars are smaller than the symbols, thereforenot shown.

2.3. Analytical methods

All glassware and plastic materials and culture medium were ster-ilized at 120 °C for 20 min using a Trade Raypa autoclave system(model AES-28) before use. The monitoring of each culture was car-ried out by sampling 5 mL for enrichment experiments and 10 mLfor tolerance experiments of medium solution with a syringe. Solu-tions in vials were centrifuged at 4000 rpm for 10 min and filteredthrough a 0.2-μm filter. Values of pH, redox potential (Eh) and sulfateconcentration were measured immediately to avoid dissolution ef-fects of CO2(g) and O2(g) on these parameters. The pH was measuredusing Crison instruments and Eh was measured using a Hanna mea-surer with Pt and Ag/AgCl electrodes (Crison). Eh measurementswere corrected to standard hydrogen electrode. Sulfate concentra-tions were analyzed by a Hach spectrophotometer (model DR/890colorimeter) according to the turbidimetric method described in theHach Procedures Manual — Method Sulfate 8051. In zinc removal ex-periments, the filtered sample was later acidified to pHb2 with HNO3

(2%) suprapur and stored at 4 °C until analysis of total sulfur and Znby inductively coupled plasma-atomic emission spectroscopy (ICP-AES) with a Jobin Yvon spectrometer (model JY ULTIMA 2).

After centrifugation, the final solid products were frozen and ly-ophilized to complete dryness using a VirTis benchtop freeze-dryer(Hucoa-Erlöss, Spain). The final solids were studied using an opticalmicroscope equipped with a digital camera (NIKON model EclipseLV100POL), a scanning electron microscope equipped with an energydispersive system (SEM-EDS; JEOL model JSM-5410), and a four-spectrometer electron microprobe (EMP; JEOL model JXA-8200Superprobe). Complementary, the mineralogy of newly-formed pre-cipitates was determined by X-ray diffraction (XRD). ConventionalXRD was used for the mineralogical characterization; however, bothsmall amount of sample and poor crystallinity due to its biogenic na-ture gave broad diffraction peaks that hampered the performance of adetailed mineral identification. Micro-X-ray diffraction (μ-XRD) usingsynchrotron-based high-flux beams with diameters of a few micro-meters helped to overcome this drawback at the ID18F beamline ofthe European Synchrotron Radiation Facility (ESRF, Grenoble,France). A 2-dimensional CCD based X-ray detector (refined detectordistance 112.1131 mm) was used to collect X-ray diffraction patternsin transmission mode. The X-ray wavelength of 0.443 Å was refinedusing a Si standard. Debye–Scherrer diffraction rings of the 2D-XRDpatterns were unwrapped and integrated versus the azimuthalangle to produce a 1D diffraction pattern using the ESRF packageFit2D (Hammersley et al., 1996). This software performs correctionsfor sample–detector distance, tilt angle of the detector with respectto the direction of the incident radiation and polarization.

3. Results

3.1. Trends in solution chemistry

In the bacterial enrichment experiments, the pH of the solutionincreased fast at the start from 7.1 to an average value of 7.6 andthen remained near-constant; while the Eh gradually decreasedfrom 400 mV to an average value of −220 mV (Fig. 2a,b). Sulfateconcentration in solution for all experiments also decreased frominitial contents of 2155 mg/L to values below detection limit(Fig. 2c). These values showed an inverse trend with regard tothe population growth of SRB, as monitored by the MPN method.In fact, SRB concentration increased from zero at the start to valuesof 4.7·105 and 4·105 CFU/mL (i.e. colony forming units per milli-liter) at the end of the experiments with ME and CM inocula, re-spectively. The SS inoculum is derived from a primary consortium

SSMECM

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Fig. 3. Time evolution of (a) pH, (b) Eh, and (c) sulfur/sulfate and (d) zinc concentrations in the media in zinc-removal experiments [SS: inoculum from treatment plant sludgesamples, ME: inoculum from Mina Esperanza mine, and CM: inoculum from Cueva de la Mora mine]. Data are the mean of duplicates and error bars are smaller than the symbols,therefore not shown.

Fig. 4. Transmitted-light optical microscope images of SRB biofilm from zinc-tolerance/removal experiments: from (a) to (d) spherical particles with dark outlines (black arrows)associated with groups of bacteria and organic polymers (white arrows). In (c) and (d), samples were stained with Gram's stain and Gram-negative bacilli were observed due to thereddish-pink color of decolorized organic polymers and cells.

179J. Castillo et al. / Science of the Total Environment 423 (2012) 176–184

Fig. 5. SEM images of ZnS spheroids from zinc-tolerance/removal experiments. Note that spherical particles are composed of nearly pure sulfur and zinc according to EDS spectrum.

180 J. Castillo et al. / Science of the Total Environment 423 (2012) 176–184

that initially showed a population of 5.3·105 CFU/mL; and subse-quently, this bacterial population favorably grew during the enrich-ment experiment until values of 1.3·106 CFU/mL. Thus, it seemsthat environmental conditions are likely suitable for bacterial sul-fate reduction.

The three SRB consortia previously enriched in the enrichmentexperiments were tested for both tolerance and metal-removal ex-periments in response to high zinc concentrations (approx.260 mg/L). Again, the final pH of solutions slightly increased withrespect to the initial value for all experiments, though with valuesranging between 5.5 and 8.0 (Fig. 3a); whereas Eh decreased from420 to −100 mV for the experiment with SS inoculum, and to55 mV for the experiments with CM and ME inocula (Fig. 3b).Total sulfur concentrations in solution also dropped, showing an av-erage removal percentage of 28% (from 467 to 337 mg/L), of whichonly sulfate concentrations decreased by 83% (from 1400 to 233 mg/L), as shown in the Fig. 3c. As observed in the enrichment experi-ments, the decreases in sulfate concentrations are inversely correlat-ed with an increase in the bacterial concentration. SRB countincreased in the experiments from 6.5·104, 2.4·104 and2·104 CFU/mL at the start to 1·107, 1.3·106 and 1.7·106 CFU/mLat the end for SS, ME and CM inocula, respectively. Nevertheless, itis worth noting that the populations of SRB slightly abated at theonset of the experiment with respect to its start. SRB count after14 days of experiment was 1.4·104, 3.7·103 and 1.6·103 CFU/mLfor SS, ME and CM inocula, respectively. Finally, the decrease of sul-fate concentration along with the final growth of SRB is concomitant

aS(220)W(110)

S(311)W(112)

W(103)

SS

CM

ME

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Inte

nsi

ty (

cou

nts

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Degree 2 ( = 0.443Å)

Figure 6b

Fig. 6. (a) Synchrotron-based μ-XRD patterns of ZnS particles from zinc-tolerance/removal eexperiment with SS inoculum in the 7.5–9° 2θ angular range. In (b), overlapping sphaleritesphalerite; W: wurtzite.

with a decrease of zinc concentration with time (Fig. 3d). After 14,35 and 42 days of the experiments with SS, ME and CM inocula, re-spectively, the zinc removal percentage in solution reached 100%,i.e. concentrations decreased from 260 mg/L to values below the de-tection limit.

3.2. Mineralogical characterization

The final solid products for all zinc-tolerance experiments corre-sponded to a white to light-brown biofilm perfectly visible in handsamples. According to the analysis, no differences were found be-tween the precipitates obtained from the different inocula. Opticalmicroscope images show the presence of spherical particles associat-ed with the groups of bacteria forming the biofilm (Fig. 4). SEM im-ages reveal that spheroids are typically b2.5-μm in diameter andcomposed exclusively of sulfur and zinc as shown by the EDS spectra(Fig. 5). Synchrotron-based μ-XRD patterns suggest that newly-formed precipitates correspond to two polymorphic forms of ZnS:sphalerite and wurtzite (Fig. 6). Most of the main XRD peaks of bothphases overlap or are close to each other. However, wurtzite is iden-tified by an exclusive peak at around 14.5° 2θ (for λ=0.443 Å) corre-sponding to the (103) plane (Fig. 6a). In order to ensure the presenceof sphalerite in spectra, the region between 7.5 and 9° 2θwas decom-posed into elementary Gaussian–Lorentzian curves using theDECOMPXR code according to Lanson (1993). The quality of spectrais high enough to decompose without smoothing and backgroundsubtraction. As evidenced by the decomposition, this broad region

W(002)W(100) W(101)

S(111)Fitted

SS

7.5 8.0 8.5 9.0

b

Degree 2 ( = 0.443Å)

Reliability factor: 99.6%

xperiments. (b) Example of decomposition of the XRD pattern from the zinc-toleranceand wurtzite peaks were separated numerically using DECOMPXR (Lanson, 1993). S:

181J. Castillo et al. / Science of the Total Environment 423 (2012) 176–184

results from the sum of four elementary reflections, which may be at-tributed to a major reflection of the (111) plane of sphalerite and tominor reflections of (100), (002), (101) planes of wurtzite (Fig. 6b).Reliability factors are higher than 99% for all the fits, which indicateda good agreement in the adjustments. The contribution of the (111)plane of sphalerite is absolutely necessary to explain and fit such re-gion. Moreover, both sphalerite and wurtzite typically occur togetherin spherical aggregates by action of SRB in similar conditions as dis-cussed below.

Crystallite size in the direction normal to any XRD-plane as an es-timation of grain size can be calculated using the FWHM (full width athalf maximum) and intensity data of integrated peak reflections bythe Scherrer's equation (Klug and Alexander, 1974). Accordingly,spheroids are composed of nanocrystalline ZnS particles in theorder of 3–6 nm in size. Note that aggregates of closely-spaced nano-particles could give the roughened appearance of the spheroid sur-face. Microscope observations also show that abundant amounts ofboth organic polymers (Fig. 7a–c) and spherical to rod-shaped bacte-rial cells up to 3 μm long (Fig. 7d) are closely associated with the

Fig. 7. SEM and EMP images of ZnS spheroids (black arrows) associated with organic polymof the same area: the comparison clearly reveals that ZnS precipitates occur as brighter partmagnified in (c). (c) SEM image of organic coating on ZnS spheroid. (d) SEM image of abundwith ZnS aggregates (white arrows).

spherical aggregates. These images even show that some bacteriacontain ZnS spheroids both around and incrusted within their cellwall (Fig. 7e,f).

4. Discussion

As stated in the Introduction, SRB catalyze the reduction of sulfateto sulfide in the presence of organic matter under anaerobic condi-tions (Heijs and van Gemerden, 2000). Although abiotic reductionprocesses are thermodynamically feasible, transformation of sulfateto sulfide would be notably slower in environmental conditions with-out bacterial catalysis (Trudinger et al., 1985). Bacterial reductionprocesses imply the use of sulfate as final acceptor of electrons fromthe oxidation of organic compounds.

Subsequently, sulfides react with divalent metals in solution andare capable to immobilize these metals as insoluble sulfides. For fur-ther information about metal removal by microbiologically-inducedsulfide precipitation the reader is referred to the numerous literature

ers and bacterial cells (white arrows). (a) Backscattered and (b) secondary SEM imagesicles and organic polymers as darker areas in (b) with respect to (a). Small rectangle isant bacterial cells. (e) and (f) EMP images of a cross-section of bacterial cells encrusted

182 J. Castillo et al. / Science of the Total Environment 423 (2012) 176–184

available on this subject (e.g., Gadd and White, 1993; Rickard andMorse, 2005). The following reactions describe the overall process:

1. During metabolic processes, SRB oxidize simple organic com-pounds (represented by CH2O), producing hydrogen sulfide andbicarbonate ions (Eq. (1)).

SO−24 þ 2CH2O→H2Sþ 2HCO

−3 ð1Þ

2. Hydrogen sulfide reacts with metallic cations (Me2+) to form pre-cipitates of insoluble metallic sulfides (MeS(s); Eq. (2)).

H2S þMe2þ↔MeSðsÞ þ 2H

þ ð2Þ

According to reactions (1) and (2), trend in solution chemistry inconjunction with growth of SRB population is indicative of bacterialsulfate reduction in enrichment experiments. Anaerobic conditionscause most of the sulfate to be reduced to sulfide and, subsequently,concentrations of both species decrease in solution after precipitationof the latter with metallic cations (Me2+). The transformation of sul-fate to sulfide releases bicarbonate ions into the solution, which ex-plains the increase in pH. The drop in the Eh values and sulfateconcentrations, and the coupled bacterial growth, in enrichment ex-periments with SS and CM inocula occur practically from the verystart. However, the growth of SRB population occurs relatively laterin the experiment with ME inoculum, but it ends up being as effectiveas SS and CM (Fig. 2). In these experiments, medium for bacterial en-richment contains Fe2+ as metallic cation in solution and solid parti-cles of FeS precipitate as insoluble sulfide (Postgate, 1984; Gramp etal., 2010). In sedimentary environments, iron salts are present inlarge amounts and the precipitation of FeS in anaerobic conditionsby catalytic action of SRB is widely known (e.g. Pósfai and Dunin-Borkowski, 2006). Even in AMD-affected environments, high ironconcentrations are not enough to eradicate these organisms, whichcan attenuate the contamination by FeS precipitation in either naturalsystems (Sarmiento et al., 2009) or bioremediation reactors(Caraballo et al., 2010). Indeed, iron species have been reported asnon-toxic to SRB for concentrations up to 400 mg/L (Tabak et al.,2004). Concerning Zn concentration and toxicity, the results reportedby Azabou et al. (2007) show that concentration exceeding 150 mg/Lproduces massive toxic response in SRB cultures. Therefore, the pre-sent work moves bacteria tolerance to Zn to new limits since differentcultures after enrichment are subjected to a culture medium whereFe2+ is replaced by Zn2+ at concentrations of 260 mg/L, hitherto con-sidered fatal to these microorganisms (Radhika et al., 2006).

In zinc-tolerance/removal experiments, time evolution of solu-tions, total bacterial growth and mineralogical characterization offinal solid products suggest that sulfate reduction successfully oc-curs by action of SRB. In this case, culture medium contains Zn2+

as metallic cation and solid particles of ZnS precipitate as nano-crystals of sphalerite and wurtzite (Fig. 6). Microscope images in-directly support the idea that precipitation of ZnS is exclusivelyinduced by a biological process since most aggregates are in closeassociation with the biofilm and sometimes embedded in bacteria(Fig. 7). Precipitation of spherical aggregates of sphalerite andwurtzite nanoparticles induced by metabolic activity has beendocumented in natural biofilms of SRB growing in waters with afew tens of μg/L Zn from Piquette Pb–Zn abandoned mine locatedat Tennyson, Wisconsin (Labrenz et al., 2000; Druschel et al.,2002; Moreau et al., 2004). Labrenz et al. (2000) showed how mi-crobes control metal concentrations in groundwater and wetland-based remediation systems and suggested biological routes for theformation of some low-temperature ZnS ore deposits. Druschel etal. (2002) developed a thermodynamic model to explain in situbiogenic precipitation of ZnS particles within these biofilms.Moreau et al. (2004) reported using data from high resolutiontransmission electron microscopy that nanocrystals and spherical

aggregates of sphalerite and wurtzite contain multiple stackingfaults on sphalerite {111}, which cause a disorder resulting inlayers of wurtzite intercalated between domains of sphalerite.These last authors also explained that ZnS aggregates are sphereswhose internal structure is in detail composed of fine concentricbands as a reflection of (1) post-precipitation flocculation and ag-gregation process, (2) different episodes of SRB activity and/or al-ternately (3) attractive forces experienced between unchargednanoparticles and hydrophobic moieties on organic polymers.

The consortia of SRB used in our experiments responded favorablydespite the extreme zinc concentration. At the start of the experi-ments, total sulfur concentration (467 mg/L) is equal to the sulfurconcentration as sulfate species (1400 mg/L), i.e. approx 14.6 mM, ifone considers molar concentrations. This is obvious because culturemedium was prepared with sulfate salts. As experiments run, the dif-ference between total sulfur and sulfate species, i.e. mainly sulfideions produced by sulfate reduction, increases up to an average valueof 8.1 mM for all inocula. At the end of the experiments, the precipi-tation of zinc is not sufficient to totally remove the reduced sulfur,and thus, an excess remains in solution. High concentrations of sul-fide species may also have toxic effects on bacteria, although SRBhave a relatively high resistance and maximum tolerance levelswere not achieved in these experiments (e.g. around 1000 mg/L offree H2S can inhibit SRB by only 50%; Isa et al., 1986). According tothe reaction stoichiometry, the amount of sulfur consumed by theprecipitation of ZnS plus the amount of total sulfur remaining in thesolution coincides at any time with the amount of initial sulfur input-ted at the start of the experiment. Therefore, the amount of volatilesulfur species that potentially can be lost is negligible.

At the end of the zinc-tolerance/removal experiments, the pop-ulation of SRB cultured from SS inoculum grew 6 and 8 times morethan those from CM and ME inocula, respectively, according to datafrom MPN. In addition, bacterial growth relative to the start wasalso higher in the experiments with SS inoculum, which coincideswith the most rapid decrease in Eh values and sulfate and zinc con-centrations in solution (Fig. 3). The decrease that occurs during thefirst weeks of experiment in the SRB populations of the three inoc-ula could be explained by Zn toxicity that slightly affected thegrowth and bacterial activity. It seems to be that earlier cell gener-ations were likely struggling to grow under high Zn conditions andresponded in distress by forming surface on which Zn could ad-sorb, and thus, be less bioavailable to harm later generations ofcells. In this sense, bacteria cells developed the biofilm as a strate-gy to survive and tolerate high Zn concentrations. Biofilm is an ag-gregate of microorganisms in which cells adhere on the surface ofextracellular polymeric substances (EPS) (White and Gadd,2000). The EPS is a mixture of polysaccharides, mucopolysaccha-rides and proteins which vary in composition between speciesand culture conditions (Zinkevich et al., 1996) and can take up sol-uble metals by complexation of fine particles. Later, the develop-ment of biofilm as a coping mechanism for adaptation of bacteriaalong with a decrease of zinc concentrations by precipitation asmetal sulfide helps to reduce toxicity effects and bacterial popula-tions increase again in the experiments. Nevertheless, adaptationonce more appears to be less traumatic in the population from SSsample due to the lower rate of death shown by the microorgan-isms in this inoculum. Therefore, the consortium of SRB from SS in-oculum seems to be the most tolerant and effective in the processof zinc removal from solutions.

According to the solution chemistry, bacterial sulfate-reductionwould be the main process that explains the depletion of zinc in thetolerance experiments. However, it is important to highlight otherminor bacterial processes that can also remove zinc from the solution.Metals or solid metallic particles can undergo biosorption on the cellwall surface (Jalali and Baldwin, 2000). Ngwenya (2007) alsoreported that bacterial death or autolysis involves the auto-

183J. Castillo et al. / Science of the Total Environment 423 (2012) 176–184

digestion of the cell wall by peptidoglycan hydrolases and the pro-duction mainly of proteins but also of nucleic acids, polysaccharidesand lipids that are themselves capable of complexing metals in solu-tion. These removal mechanisms are also supported by our observa-tions of zinc-sulfide spherical aggregates in close relationshipwith bacterial cells and extracellular organic polymers (see for exam-ple Fig. 7).

Precipitation of metal sulfides on bacterial surface can coat and in-sulate the SRB, protecting them from the toxic effects of heavy metals.However, this tolerance mechanism can inhibit the metabolic activityof SRB as well (Utgikar et al., 2002). Radhika et al. (2006) observed asuccessful growth of SRB in the presence of low zinc concentrations,however, microbes showed a decrease in the cell number at210 mg/L. These authors utilize the sulfide coating on cells as onepossible hypothesis to explain the inhibition of bacterial growth. Al-though in our experiments some bacteria can be in contact with ZnSspheroids (see for example Figs. 7e,f), it is evident that sulfide coatingdid not impede the metabolic activity of the bacteria by blanketingthe cells. To our knowledge, this is the first confirmation of toleranceand total zinc-removal by SRB at very high concentrations.

Extrapolation of lab experimental results to full scale treatmentsin the field requires control of some variables such as the source ofSRB and the temperature. To stimulate bioremediation in passivetreatment systems, reactors are often inoculated with SRB consor-tia obtained in sulfate-rich reducing environments such as thoseobserved in lacustrine and wetland sediments, cattle manure, andsediments from streams impacted by sewage or AMD (Doshi,2006). The SRB source may significantly affect the success of thetreatment, especially in very extreme environmental conditions.For this reason, it is strongly recommended to use the SRB consor-tia obtained from AMD-impacted sediments to contain bacteriapreviously exposed to contamination, and hence, with more toler-ance (Johnson and Hallberg, 2005). Among the experiments ofthe present study, the results obtained with the inoculum fromthe wastewater treatment plant were likely better than the resultsshown by the two inocula from the IPB because the initial SRBconcentration of the former was substantially higher than the oneobserved in the latter. However, the inocula from the IPB miningdistricts should be seriously considered for future full-scale appli-cations in the field, especially the sample from Cueva de la Moramine, to likely contain more metal-tolerant bacteria. As mentioned,temperature is another factor affecting the SRB growth. Low tem-peratures generally reduce SRB activity. Except a group of thermo-philic SRB, the remaining taxonomic groups typically grow inenvironmental conditions and have optimal growth temperaturesranging from 20 to 40 °C (Doshi, 2006). The SRB isolated in thisstudy should contribute to improve AMD remediation strategiesin the IPB where the average temperature is close to the optimalgrown range during most of the year and to the temperaturethat prevailed in the laboratory during the enrichment and Zn tol-erance experiments.

5. Conclusions

Some passive treatment systems of acid mine drainage (AMD)show a complete removal of Fe and Al in highly polluted discharges.However, these systems display serious deficiencies in the removalof zinc. This study strongly encourages the use of sulfate-reducingbacteria (SRB) to remediate a medium containing initial zinc concen-trations as high as 260 mg/L. Zinc concentration was completely re-moved from solution due to precipitation of sphalerite–wurtzite(ZnS) nanoparticles as identified by microscopy and synchrotron-based μ-XRD. This information highlights the potential environmentalimportance of the precipitation of insoluble sulfides in the uptake ofZn for treatment of contaminated waters.

Acknowledgments

This work was financed by the European Union POCTEP pro-gram (TRASAGUA project, Ref. 0432_I2TEP_5_E) and the SpanishMinistry of Education and Science (METODICA project, Ref.CGL2010-21956-C02). The authors are very grateful to ESRF (ID18F) for their assistance during our experiment (EC-724). Very spe-cial thanks go to Bruno Lanson from Laboratoire de Géodynamiquedes Chaînes Alpines (Université Joseph Fourier, France) for provid-ing us the DECOMPXR code. The analytical assistance of Rafael Car-rasco, María José Ruiz, Cristobal Cantero, María Paz Martín andother technicians from the Central Research Services of the Univer-sity of Huelva is also gratefully acknowledged. R. Pérez-López alsothanks the Spanish Minister of Science and Innovation and the‘Ramón y Cajal’ Subprogramme (MICINN-RYC 2011). Manuel A.Caraballo was financially supported by the Spanish Minister of Ed-ucation and the Post-doctoral International Mobility Sub-programme I+D+i 2008–2011. We would also like to thank Dr.Damià Barceló (Associate Editor) and an anonymous reviewer forthe support and comments that significantly improved the qualityof the original paper.

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