Removal of Arsenic Using Acid/Metal-Tolerant Sulfate Reducing Bacteria: a New Approach for Bioremediation of High-Arsenic Acid Mine Waters

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Communication Removal of Arsenic Using Acid/Metal-Tolerant Sulfate Reducing Bacteria: A New Approach for Bioremediation of High-Arsenic Acid Mine Waters

Jennyfer Serrano 1,2 and Eduardo Leiva 1,2,3,4,* ID

1 Departamento de Química Inorgánica, Facultad de Química, Pontiﬁcia Universidad Católica de Chile, Avenida Vicuña Mackenna 4860, Macul, Santiago 7820436, Chile; [email protected] 2 Facultad de Ciencias de la Salud, Universidad Católica Silva Henríquez, General Jofré 462, Santiago 8330225, Chile 3 Departamento de Ingeniería Hidráulica y Ambiental, Pontiﬁcia Universidad Católica de Chile, Avenida Vicuña Mackenna 4860, Macul, Santiago 7820436, Chile 4 CEDEUS, Centro de Desarrollo Urbano Sustentable, El Comendador 1916, Providencia, Santiago 7520245, Chile * Correspondence: [email protected]; Tel.: +56-2-2354-7224

Received: 3 October 2017; Accepted: 15 December 2017; Published: 19 December 2017

Abstract: Fluvial sediments, soils, and natural waters in northern Chile are characterized by high arsenic (As) content. Mining operations in this area are potential sources of As and other metal contaminants, due to acid mine drainage (AMD) generation. Sulfate Reducing Bacteria (SRB) has been used for the treatment of AMD, as they allow for the reduction of sulfate, the generation of alkalinity, and the removal of dissolved heavy metals and metalloids by precipitation as insoluble metal sulﬁdes. Thus, SRB could be used to remove As and other heavy metals from AMD, however the tolerance of SRB to high metal concentrations and low pH is limited. The present study aimed to quantify the impact of SRB in As removal under acidic and As-Fe-rich conditions. Our results show that SRB tolerate low pH (up to 3.5) and high concentrations of As (~3.6 mg·L−1). Batch experiments showed As removal of up to 73%, Iron (Fe) removal higher than 78% and a neutralization of pH from acidic to circum-neutral conditions (pH 6–8). In addition, XRD analysis showed the dominance of amorphous minerals, while Scanning Electron Microscopy/Energy Dispersive X-ray Spectroscopy (SEM-EDX) analysis showed associations between As, Fe, and sulfur, indicating the presence of Fe-S-As compounds or interaction of As species with amorphous and/or nanocrystalline phases by sorption processes. These results indicate that the As removal was mediated by acid/metal-tolerant SRB and open the potential for the application of new strains of acid/metal-tolerant SRB for the remediation of high-As acid mine waters.

Keywords: Arsenic; sulfate reducing bacteria; acid mine drainage; acidic waters; acid-tolerant; metal-tolerant

1. Introduction Arsenic (As) is a ubiquitous and toxic trace metalloid, which is widely distributed in natural environments [1]. Several groundwaters, sediments and minerals of Northern Chile are enriched in As [2–5]. Speciﬁcally, the Chilean Altiplano has high concentrations of As and sulfate in ﬂuvial waters, seriously affecting the quality of water resources and limiting the use of water for consumption, agricultural, and industrial purposes [4]. In the Azufre River sub-basin, the release of contaminants from natural and anthropogenic sources negatively impact the quality of rivers and surface waters. In this area, hydrothermal waters emerge with high concentrations of dissolved As (>3 mg·L−1), iron (Fe) (>80 mg·L−1), and a pH lower than

Water 2017, 9, 994; doi:10.3390/w9120994 www.mdpi.com/journal/water Water 2017, 9, 994 2 of 12

3 [4,5]. The As occurrence in fluvial waters is linked to the presence of ferric Fe(III)-oxyhydroxide ores, because As is sorbed onto these minerals [6]. In addition, arid and semiarid climates strongly contribute to the generation of As-rich waters, due to high evaporation rates that concentrate surface runoff [7]. Despite this, little is known about the microbial speciation, precipitation/dissolution, or sorption processes that are involved in the fate of As in these systems and how they could be optimized in treatment systems. The main source of As in the environment comes from its release from As-rich minerals (e.g., arsenopyrite (FeAsS)) [1], but mining operations can accelerate their release to the aqueous phase [8]. Particularly, acid mine drainage (AMD) can enhance the release of As from different minerals [9–11]. AMD is characterized by the release of acidic waters (pH < 4) with high dissolved metals (e.g., Fe and As, among others) and sulfate concentrations [12,13]. AMD has a negative impact on the mobilization of As and can promote the release of As directly through the oxidation of As-bearing sulfide or by dissolution of As-rich mineral phases (i.e., As-rich Fe oxyhydroxides) [10,11]. The removal of As from acid waters is a great challenge, because acidic conditions favor the dissolution of As species and prevent its removal. Nevertheless, microbial communities that are selected under acidic conditions may result in an efficient treatment for As removal. Conventional treatment technologies for As removal, acid neutralization, and salt removal, such as co-precipitation, adsorption, chemical neutralization, or ion exchange processes have several limitations, both economic and operational [14]. Sulfate reduction processes mediated by Sulfate Reducing Bacteria (SRB) allow for the reduction of sulfate concentrations, precipitation of metals, and potentially increase the pH [15]. However, most strains of SRB grow and reduce sulfate optimally at a circum-neutral pH (6–8) [16]. Low pH severely affects the growth of bacteria and sulfate removal rates [17]. Likewise, metal and metalloid concentrations (e.g., Fe and As) have a negative effect on the sulfate removal rates [18]. Particularly, the growth of SRB can decline significantly under high As concentrations, due to its toxicity. Even so, several studies have shown that bacteria can tolerate low pH and can be an alternative for the treatment of acid mine waters [19–22]. Thus, bioremediation systems that are based on acid/metal-tolerant SRB could be used for the treatment of acidic metallic waters, but it is still necessary to study its feasibility of operation under specific conditions. The present study shows the removal of As from AMD waters by biological sulfate reduction processes under acidic and As-rich conditions. The relevance of these findings lies in that new strains of acid/metal-tolerant SRB can potentially be used to optimize As removal and AMD treatment simultaneously through its bacterial activity.

2. Materials and Methods

2.1. Study Site: Aguas Calientes Area in the Upper Section of the Azufre River Sub-Basin The Aguas Calientes area is located in the Azufre River sub-basin near the Tacora volcano, in the Lluta River Watershed (LRW) in northern Chile (18◦000–18◦300 S and 70◦200–69◦220 W). The area is characterized by water scarcity, high altitude (>4000 m a.s.l.), and high evaporation rates (4.9 ± 0.5 mm d−1). The Azufre River originates in the upper section of the LRW in the Altiplano with high concentrations of [As] (3.6 ± 0.46 mg·L−1), [Fe] (81.6 ± 13.5 mg·L−1), [Al] (205.3 ± 25.7 mg·L−1), [Zn] (7.4 ± 1.2 mg·L−1), 2- −1 [SO4 ](4,678 ± 16.9 mg·L ), and a pH less than 2.0 [4,5] due to contributions from hydrothermal springs and acidic metal-rich runoffs. The Aguas Calientes area is impacted by a legacy sulfur mine site on the slopes of the Tacora volcano, where there are mineral deposits of elemental sulfur and at the base of the volcano there is an accumulation of ﬁne elemental sulfur (S0) tailings (Figure1). In addition, there is AMD generation that is associated with S0 tailings, characterized by an acidic pH (1.9 to 4.0), high speciﬁc conductivity (1.0 to 5.7 mS·cm−1), and elevated concentrations of dissolved Fe (>10 mg·L−1) and As (>0.4 mg·L−1). The S0 tailings are disposed near a natural wetland, where the decomposition of plant organic Water 2017, 9, 994 3 of 12 matter occurs and generates anaerobic environments, enabling sulfate reduction processes to occur continuously (Figure1). Water 2017, 9, 994 3 of 12

Tacora Elemental Sulfur Deposits Volcano 2- 2-SO4 SO4 S0 Tailings

Surface runoff H+ Surface runoff

O Azufre Tailings river

Wetland Area

Symbology Sampling points Azufre river 0 200m Tacora Volcano Flow Direction

(a) (b)

Figure 1. (a) Schematic representation of the study site in the Aguas Calientes area, Azufre River sub- Figure 1. (basina) Schematic in northern representation Chile. (b) Photograph of the study of the sitewetland in the area Aguas with a Calientes representative area, sulfate Azufre reduction River sub-basin in northern Chile;sediment (b) profile. Photograph of the wetland area with a representative sulfate reduction sediment profile.

2.2. Sampling 2.2. Sampling To evaluate sulfate reduction processes, sediment profiles were sampled at different points that To evaluatewere located sulfate at the interface reduction of S0processes, tailings with the sediment wetland growth profiles zone. were These sampled sediments atreceive different an points that wereAMD located runoff at and the have interface Fe and As of concentrations S0 tailings of withup to 187.2 the wetlandg·kg−1 and 9.5 growth g·kg−1, respectively. zone. These Five sediments receive ansampling AMD points runoff were and selected have for Fe hydrogeochemical and As concentrations analysis, elemental of up analysis to 187.2 in solid g·kg phase,−1 and and 9.5 g·kg−1, cultures of microorganisms in anaerobic reactors. Sampling points were selected based on monitoring respectively.of on-site Five geochemical sampling pointsparameters were (pH, selected Eh, cond foructivity, hydrogeochemical and dissolved oxygen analysis, (DO)) elementalin surface analysis in solid phase,waters andand saturated cultures sediments of microorganisms in the Aguas Caliente in anaerobics area carried reactors. out in May Sampling 2012. The points presence were of selected based on monitoringa sulfate reduction of on-site process geochemicalwas evidenced by parameters black spots on (pH, gray Eh, surface conductivity, sediments, which and dissolvedbecomes oxygen (DO)) in surfacecompletely waters black at and 5 cm saturated of depth. Sediment sediments profiles in the were Aguas collected Calientes using polyethylene area carried cores out(25 cm in May 2012. depth) that were inserted by hand in sediments. Immediately after collection, the cores were sliced The presenceinto 5 of cm a sulfatedeep layers. reduction The undisturbed process layers was evidencedwere stored in by 300 black mL High spots Density on gray Polyethylene surfacesediments, which becomes(HDPE) completely bottles and were black kept at in 5 the cm dark of depth. at 4 °C until Sediment processing profiles and microbial were collected culture. using polyethylene cores (25 cm depth)Surface water that weresamples inserted were collected by hand near ineach sediments. sediment profile Immediately for elemental after analysis. collection, Water the cores were slicedsamples into were 5 cm collected deep layers.in 300 mL TheHDPE undisturbed bottles and were layers filtered were in the stored field using in 3000.22 μ mLm nylon High Density filters. Furthermore, on-site water analyses included temperature◦ (PHC301, HACH), pH (PHC301, PolyethyleneHACH), (HDPE) dissolved bottles oxygen and (DO) were (LDO101, kept in HACH), the dark and at electrical 4 C until conductivity processing (CDC401, and microbialHACH) culture. Surface(Hq40d water Multi, samples HACH, Loveland, were collectedCO, USA). near each sediment profile for elemental analysis. Water samples were collected in 300 mL HDPE bottles and were filtered in the field using 0.22 µm nylon 2.3. Batch Experiments filters. Furthermore, on-site water analyses included temperature (PHC301, HACH), pH (PHC301, HACH), dissolvedBatch reactors oxygen were (DO) inoculated (LDO101, with HACH), SRB-enriched and electricalcultures that conductivity were obtained (CDC401, from HACH) Winogradsky columns prepared with high concentrations of dissolved As, Fe, and sulfate (10 mg·L−1 (Hq40d Multi, HACH, Loveland, CO, USA). of As and 100 mg·L−1 of Fe and 3070 mg·L−1 of sulfate). Acid tolerance was evaluated through microbial growth in culture media with variable pH. Each anaerobic batch reactor consisted of 250 2.3. BatchmL Experiments glass bottle inoculates (in duplicate), with actively growing planktonic SRB cell suspensions and SRB biofilms (50–100 mg wet weight) in a working volume of 250 mL (without empty space volume). Batch reactors were inoculated with SRB-enriched cultures that were obtained from Winogradsky The initial cell suspension was set at ~5.5 × 108 cells·mL−1. The planktonic SRB growth was quantified −1 columns preparedthrough optical with density high concentrationsat 600 nm. The culture of dissolved media that As, was Fe, used and in sulfatethe experiments (10 mg was·L a of As and −1 −1 100 mg·L modified-Postgateof Fe and 3070 C mgmedium·L composedof sulfate). of 0.5 Acid g KH tolerance2PO4, 4.5 wasg Na2 evaluatedSO4, 1 g NH through4Cl, 0.06 g microbialMgSO4 growth in culture7H media2O, 0.06 with g CaCl variable2 6H2O, 0.3 pH. g sodium Each citrate, anaerobic 0.004 g batchFeSO4 7H reactor2O, and consistedcompleted with of 250H2O mLto 1 L. glass bottle Additionally, 5 g·L−1 of fresh plant organic matter (wet weight, preserved at 4 °C) that was extracted inoculates (in duplicate), with actively growing planktonic SRB cell suspensions and SRB biofilms

(50–100 mg wet weight) in a working volume of 250 mL (without empty space volume). The initial cell suspension was set at ~5.5 × 108 cells·mL−1. The planktonic SRB growth was quantified through optical density at 600 nm. The culture media that was used in the experiments was a modified-Postgate C medium composed of 0.5 g KH2PO4, 4.5 g Na2SO4, 1 g NH4Cl, 0.06 g MgSO4 7H2O, 0.06 g CaCl2 −1 6H2O, 0.3 g sodium citrate, 0.004 g FeSO4 7H2O, and completed with H2O to 1 L. Additionally, 5 g·L Water 2017, 9, 994 4 of 12 of fresh plant organic matter (wet weight, preserved at 4 ◦C) that was extracted from a wetland located in the Aguas Calientes area was added to the culture medium to favor microbial growth. Decomposition of this plant organic matter occurs naturally in the proximity of sediment profiles that were sampled for this study. The pH was adjusted to 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5 (seven reactors and one control reactor [pH = 7.0]), and every three days 5 mL samples were taken and the pH evolution and chemical oxygen demand (COD) consumption were monitored. The reactors were operated without agitation (static state) at 25 ◦C in the dark. The reactors had teflon-lined septa at the top for periodic sampling and to bubble N2 gas every three days to remove oxygen and generate an anaerobic environment. Sulfate reduction experiments were conducted with the same SRB-enriched culture, under the same operating conditions mentioned above (static state, 25 ◦C in the dark), and with the same culture media (modified-Postgate C medium) enriched with plant organic matter (5 g·L−1 wet weight), but with natural water from the Azufre River as culture medium (See Section 2.1). Four batch reactors (two replicates) were installed in the laboratory and the initial pH of the natural water was adjusted using 2M NaOH to 3.5, according to acid tolerance assays. Similarly, reactors were bubbled with N2 gas to remove oxygen and were kept in the dark at room temperature (23 ± 1 ◦C). The batch reactors were kept for 30 days without sampling to allow for the adaptation and acclimatization of acid tolerance in SRBs.

2.4. Geochemical Analyses Water samples from the reactors were taken every seven days, for a total of 80 days. The bioreactor performance was assessed by monitoring pH, electrical conductivity, sulfate, and by As and Fe removal. Measurements of pH (PHC301, HACH), DO (LDO101, HACH), and electrical conductivity (CDC401, HACH) were determined immediately after the collection of the sample. Sulfate was analyzed by ion chromatography (882 compact IC plus, Metrohm, Herisau, Switzerland), whereas total dissolved metal concentrations (As and Fe) were carried out by Inductively Coupled Plasma Optical Emission Spectrometry (IC-OES). Solid phases that precipitated during sulfate reduction experiments (with natural water from the Azufre River) were examined by X-ray diffraction (XRD) and Scanning Electron Microscopy/Energy Dispersive X-ray Spectroscopy (SEM-EDX). XRD analyses were performed on a Bruker D8 Advance (40 kV, 40 mA Cu Kα radiation, λ = 1.5406 Å) and surface characterization of solid phases were conducted using a scanning electron microscope (JSM-IT300LV; JEOL Ltd., Tokyo, Japan), equipped with an Oxford EDX AZtec detector (Oxford Instruments, High Wycombe, UK). Prior to the observation by SEM-EDX, the samples were fixed with 2% glutaraldehyde solution in a sodium cacodylate buffer (0.2 M, pH 7.2, at 4 ◦C) and dehydrated in water/ethanol solutions (50, 70, 90 and 100%). The samples were then treated for 45 minutes with a critical point dryer (Autosamdri-815; Tousimis, Rockville, MD, USA), and were coated with carbon in a desktop sputterer (Desk V; Denton Vacumm, Moorestown, NJ, USA).

3. Results and Discussion

3.1. Metal-Tolerant Sulfate Reducing Bacteria Sediments from sampling site were used to evaluate sulfate reduction processes under extreme conditions (Low pH, high concentration of heavy metals). From these sediments, metal-tolerant SRB were enriched in Winogradsky columns. We observed the precipitation of sulﬁde minerals inside the Winogradsky columns through the increase of black areas over time in the external face of the acrylic column and sulfate removal of up to 76%. Metal-tolerant bacterium can successfully grow under these conditions, withstanding highly toxic As concentrations. These results suggest that SRB can tolerate high concentrations of As and Fe in solid and dissolved phases, and can develop metal resistance mechanisms to protect their cellular components. High concentrations of dissolved and precipitated metals have a negative effect on bacterial growth, and cause a reduction of sulfate removal rates in SRB [18]. Arsenic, Fe, and other metals compete with Water 2017, 9, 994 5 of 12 essentialWater cations2017, 9, 994 and may inactivate enzymes and other proteins [18]. The toxicity depends on5 of the 12 type of metal and the type of bacteria or the exposed microbial community, ranging from a few mg·L−1 to depends on the type of metal and the type of bacteria or the exposed microbial community, ranging concentrations above 10 mg·L−1 [18]. Interestingly, our results show that SRB tolerate concentrations to from a few mg·L−1 to concentrations above 10 mg·L−1 [18]. Interestingly, our results show that SRB the order of g·kg−1 in the solid phase, and higher than 10 mg·L−1 in the aqueous phase. The mechanisms tolerate concentrations to the order of g·kg−1 in the solid phase, and higher than 10 mg·L−1 in the for tolerationaqueous phase. of high The concentrations mechanisms for of toleration metals may of high be associated concentrations with of processes metals may of surface be associated adsorption, oxidation/reductionwith processes of reaction, surface enzymaticadsorption, speciation,oxidation/re cellularduction respiration, reaction, enzymatic bioaccumulation, speciation, biosynthesis cellular of metal-bindingrespiration, proteins bioaccumulation, or extracellular biosynthesis polymers, of amongmetal-binding others [proteins23,24]. Operationally, or extracellular for polymers, SRB, the metal toleranceamong is others more relevant[23,24]. Operationally, because it does for not SRB, allow the metal for precipitated tolerance is more metal relevant sulfides because to alter it their does metabolic not activity.allow Even for precipitated so, the increase metal in sulfides metal to precipitates alter their metabolic may reduce activity. the growth Even so, kinetics the increase of SRB in andmetal sulfate removalprecipitates rates when may Fe reduce and As the precipitates growth kinetics reach of values SRB thatand aresulfate higher removal than thoserates when reported Fe inand sediments. As However,precipitates field conditions reach values in that the Aguasare higher Calientes than those area reported suggest in that sediments. SRB strains However, can tolerate field conditions high dissolved in the Aguas Calientes area suggest that SRB strains can tolerate high dissolved and solid phase metal and solid phase metal concentrations. Therefore, the isolation and growth of these microorganisms under concentrations. Therefore, the isolation and growth of these microorganisms under controlled controlledconditions conditions can enhance can enhance the efficiency the efficiency of trea oftment treatment systems systems for waters for waters with withextremely extremely high high concentrationsconcentrations of As of andAs and Fe. Fe.

3.2. Metal-Tolerant3.2. Metal-Tolerant Sulfate Sulfate Reducing Reducing Bacteria Bacteria AreAre Tolerant to to Low Low pH pH SRBSRB tolerance tolerance to low to pHlow conditionspH conditions was evaluatedwas evaluated in batch in batch reactors. reactors. The resultsThe results showed showed microbial growthmicrobial in pH growth values in thatpH values were higherthat were than higher 3.5 than (Figure 3.5 (Figure2). This 2). growthThis growth is evidenced is evidenced by by the the COD consumptionCOD consumption (−∆30–78%), (−Δ30–78%), pH increase, pH increase, and and by theby the counting counting of of total total cells cells inin thethe culture (Data (Data not shown).not shown). Under Under pH 3.5, pH no3.5, increase no increase in in cell cell number number waswas observed and and organic organic matter matter degradation degradation activityactivity (COD (COD consumption) consumption) was was not not reported, reported, this suggests suggests that that under under pH pH 3.5, 3.5,the acidity the acidity was lethal was lethal for microbial activity (associated to SRB). Conversely, in reactors with a pH of greater than pH 3.5, for microbial activity (associated to SRB). Conversely, in reactors with a pH of greater than pH 3.5, an increase in the pH that is associated with the COD consumption was observed, which is explained an increase in the pH that is associated with the COD consumption was observed, which is explained by by the increase in the total alkalinity of the culture medium, mediated by sulfate reduction activity. the increaseIn the sulfate in the reduction total alkalinity process, ofSRB the reduce culture sulfate medium, to sulfide mediated and oxidize by sulfate the electron reduction donor activity. (organic In the sulfatecarbon reduction source) process, into bicarbonate SRB reduce [25,26]. sulfate The bicarb to sulﬁdeonate and increases oxidize the the alkalini electronty causing donor pH (organic increase carbon source)[25,26]. into bicarbonate [25,26]. The bicarbonate increases the alkalinity causing pH increase [25,26].

9 pH 2,5 pH 4,5 1.0 8 pH 3,0 pH 5,0 pH 3,5 pH 5,5 7 pH 4,0 0.8 0

6 /C i 0.6 pH 5

COD C 0.4 4 pH 2,5 pH 4,5 0.2 pH 3,0 pH 5,0 3 pH 3,5 pH 5,5 pH 4,0 2 0.0 0 1020304050607080 0 1020304050607080 Time (d) Time (d) (a) (b)

Figure 2. Sulfate Reducing Bacteria (SRB) acid tolerance. (a) Variation of pH and chemical oxygen Figure 2. Sulfate Reducing Bacteria (SRB) acid tolerance. (a) Variation of pH and chemical oxygen demand demand (COD) consumption (b) in anaerobic batch reactors. The COD consumption is expressed as (COD) consumption (b) in anaerobic batch reactors. The COD consumption is expressed as the relative the relative concentration (Ci/C0) related to the initial COD concentration of the culture medium (Ci concentration (Ci/C0) related to the initial COD concentration of the culture medium (Ci = concentration = concentration at time i, C0 = initial COD concentration). C0 in the aqueous phase of all reactors was −1 at time i, C = initial−1 COD concentration).2 C−1 in the aqueous−1 phase of all reactors was 1040 ± 5 mg·L . 1040 ± 50 mg·L . Initial SO4 = 3067.9 mg·L 0, Fe = 0.8 mg·L . A linear regression was fitted to the data 2 −1 −1 Initialin SOdifferent4 = 3067.9 pH conditions. mg·L , Fe = 0.8 mg·L . A linear regression was fitted to the data in different pH conditions. Typically, optimal growth conditions for SRB in different natural systems occur at pH values of greaterTypically, than optimal 5.0, because growth SRB conditionsactivity is reduced for SRB under in different this pH [16]. natural Acidophilic systems SRB occur can atsurvive pH values in of natural waters with pH < 3.0, but the sulfate reduction rates are significantly reduced [27,28]. This greater than 5.0, because SRB activity is reduced under this pH [16]. Acidophilic SRB can survive occurs because SRB must maintain a higher/circum-neutral internal cell pH, which would produce a in natural waters with pH < 3.0, but the sulfate reduction rates are significantly reduced [27,28]. high consumption of energy, and therefore they would have less energy to grow [19]. Nevertheless, Thisalthough occurs because acidic SRBconditions must maintainare not preferable, a higher/circum-neutral the Gibbs free internalenergy calculations cell pH, which show would that the produce a highreaction consumption is more favorable of energy, at a and lower therefore pH, obtaining they would a higher have energy less gain energy [19]. to Sánchez-Andrea, grow [19]. Nevertheless, et al. although acidic conditions are not preferable, the Gibbs free energy calculations show that the reaction is more favorable at a lower pH, obtaining a higher energy gain [19]. Sánchez-Andrea, et al. [19] Water 2017, 9, 994 6 of 12 propose that the extra energy that is produced at low pH could compensate the higher energy required to pump the protons out of the cell. Other studies support the presence of SRB at low pH based on the use of non-ionic substrates, such as glycerol, hydrogen, alcohols, or sugars [29–31]. However, it is also necessary to consider that at acidic pH, H2S is found almost completely non-dissociated (pKa = 7, 30 ◦ C), and undissociated sulfide (H2S) has an inhibitory effect on growth [28–30]. These mechanisms support theWater lack 2017,of 9, 994 growth observed in reactors under pH 3.5. However, at a pH higher6 of 12 than 3.5, the sulfate-reducing[19] propose metabolicthat the extra activity energy that may is produced generate at total low pH alkalinity, could compensate raising the higher pH and energy neutralizing the inhibitoryrequired effect, to pump thus increasingthe protons out the of growth the cell. ratesOther andstudies the support sulfate the reduction presence of rates SRB whenat low approachingpH circum-neutralbased conditions.on the use of non-ionic substrates, such as glycerol, hydrogen, alcohols, or sugars [29–31]. However, it is also necessary to consider that at acidic pH, H2S is found almost completely non- AMD treatment has been extensively studied in recent years [25,31–34]. Typically, AMD waters dissociated (pKa = 7, 30 °C), and undissociated sulfide (H2S) has an inhibitory effect on growth [28– have a pH30]. below These 4.0mechanisms [12,35]. support SRB growthe lack optimally of growth ob underserved in circum-neutral reactors under pH conditions, 3.5. However, thereforeat a the AMD treatmentpH higher that than is mediated3.5, the sulfate-re by SRBducing requires metabolic pH activity neutralization. may generate However, total alkalinity, many raising researchers the have studied acid-tolerantpH and neutralizing and acidophilic the inhibitory SRB effect, [21 thus,36 increasing–40]. Kimura, the growth et al. rates [41 and] have the sulfate reported reduction a reduction of rates when approaching circum-neutral conditions. sulfate at pH 3.8, and Bijmans, et al. [42] reported a high rate of sulfate reduction at pH 4.0 and 4.5 AMD treatment has been extensively studied in recent years [25,31–34]. Typically, AMD waters using H2 andhave formatea pH below as 4.0 electron [12,35]. donorsSRB grow in optimally laboratory under experiments. circum-neutral Despiteconditions, this, therefore few studiesthe have linked SRBAMD acid treatment tolerance that with is mediated tolerance by SRB to highrequires concentrations pH neutralization. of heavyHowever, metals many andresearchers their potential impact on Ashave removal studied acid-tolerant from acid mine and acidophilic drainage SRB waters [21,36,37–40]. [22,43]. WeKimura, found et SRBal. [41] to have be capable reported of a tolerating reduction of sulfate at pH 3.8, and Bijmans, et al. [42] reported a high rate of sulfate reduction at pH pH up to 3.5 and concentrations of As higher than 10 mg·L−1, and the interaction between Fe and As 4.0 and 4.5 using H2 and formate as electron donors in laboratory experiments. Despite this, few may be relevantstudies have for thelinked removal SRB acid process. tolerance Thewith nativetolerance acid/metal-tolerant to high concentrations of SRB heavy may metals be adequateand for removal oftheir As concentrationspotential impact on from As removal contaminated from acid mine waters. drainage waters [22,43]. We found SRB to be capable of tolerating pH up to 3.5 and concentrations of As higher than 10 mg·L−1, and the interaction 3.3. Arsenicbetween and Iron Fe and Are As Removed may be relevant in Sulfate for the Reduction removal process. Reactors The native acid/metal-tolerant SRB may be adequate for removal of As concentrations from contaminated waters. Batch tests were conducted to probe the removal of As and Fe under acidic conditions. The pH neutralization3.3. Arsenic wasobserved and Iron Are over Removed time in Sulfate in two Reduction reactors. Reactors The pH increased rapidly from 3.5 to between 6.0 and 7.5 (FigureBatch 3tests). Additionally, were conducted anto probe increase the removal in the of total As and alkalinity Fe under wasacidic observed conditions. (fromThe pH 5 mg ·L−1 neutralization−1 was observed over time in two reactors. The pH increased rapidly from 3.5 to between to 500 mg·L CaCO3), suggesting that the processes of acid neutralization may be due to biological 6.0 and 7.5 (Figure 3). Additionally, an increase in the total alkalinity was observed (from 5 mg·L−1 to sulfate reduction.500 mg·L−1Interestingly, CaCO3), suggesting both that of the the processes reactors of showedacid neutralization a constant may pH be due above to biological 6.5 after 35 days of operation,sulfate suggesting reduction. Interestingly, that microbial both consortiaof the reactors are showed able to a constant overcome pH above the acclimatization 6.5 after 35 days of stage and maintain circum-neutraloperation, suggesting conditions. that microbial The consortia pH values are able reported to overcome during the theacclimatization batch experiments stage and suggest that SRB aremaintain active circum-neutral during the operation conditions. andThe pH tolerate values the reported initial during acidity. the Additionally,batch experiments in suggest the two reactors that SRB are active during the operation and tolerate the initial acidity. Additionally, in the two SRB activityreactors was SRB confirmed activity was by confirmed sulfate removal by sulfate of removal more of than more 60% than(data 60% (data not not shown). shown).

12 11 R1 10 R2 9 Control 8 7 6 pH 5 4 3 2 1 0 0 7 14 21 28 35 42 49 56 63 70 77 84 Time (d) Figure 3. pH evolution in batch reactors inoculated with SRBs enriched from anerobic sediments. Figure 3. pHInitial evolution SO42 = 3067.9 in mg·L batch−1, Fe reactors = 0.8 mg·L inoculated−1.The data are with presented SRBs as enrichedthe mean ± SEM from (error anerobic bars). sediments. 2 −1 −1 Initial SO4 = 3067.9 mg·L , Fe = 0.8 mg·L .The data are presented as the mean ± SEM (error bars). To reveal if pH neutralization and the sulfate reduction process promote As removal, we evaluated the change in Fe and As concentrations in natural water from the Azufre River that was To revealused ifas pH culture neutralization medium in sulfat ande reduction the sulfate batch reduction reactors. The process results promote showed around As removal, 90% and 80% we evaluated the changeremoval in Fe and of Fe As and concentrations As, respectively. inFigure natural 4 shows water that the from highest the Azufreremoval Riverof As and that Fe wasoccurred used in as culture medium in sulfate reduction batch reactors. The results showed around 90% and 80% removal of Fe and As, respectively. Figure4 shows that the highest removal of As and Fe occurred in correlation Water 2017, 9, 994 7 of 12

Water 2017, 9, 994 7 of 12 with thecorrelation increase ofwith pH the (Figure increase3 ),of suggestingpH (Figure 3), an su initialggesting pH-dependent an initial pH-dependent mechanism mechanism that is that associated is with theassociated sulfate reduction. with the sulfate reduction.

100 100

90 90

80 80

70 70

60 60

50 50

40 40 Fe removal % 30 As removal30 %

20 20 R1 R2 Control R1 R2 Control 10 10

0 0 0 7 14 21 28 35 42 49 56 63 70 77 84 0 7 14 21 28 35 42 49 56 63 70 77 84 Time (d) Time (d) (a) (b)

Figure 4. Total dissolved Fe (a) and As (b) removal during batch reactor operation. A marked removal Figure 4. Total dissolved Fe (a) and As (b) removal during batch reactor operation. A marked removal of As and Fe was observed after 14 days of operation. The metal removal data are the mean of two of As andreactors Fe was (average observed of two after replicates) 14 days. Initial of concentrations operation. The in batch metal reactors removal were data As = are3.6 mg·L the mean−1, Fe = of two −1 reactors81 (average mg·L−1 and of SO two42− = replicates) 4678 mg·L−1.. TheInitial data concentrations are presented as the in mean batch ± reactorsSEM (error were bars). As = 3.6 mg·L , −1 2− −1 Fe = 81 mg·L and SO4 = 4678 mg·L . The data are presented as the mean ± SEM (error bars). X-ray diffraction that was performed on the precipitated solids (lyophilized) recovered at the end of batch sulfate reduction experiments shows a strongly bulging amorphous baseline (Figure 5). X-rayPeaks diffraction of crystalline that minerals was performed for As sulfides on were the precipitated not observed, solidsbut weak (lyophilized) peaks were detected recovered for at the end of batchsulfate sulfate phases,reduction such as Gibsum experiments (CaSO4 2H shows2O) and a Fe strongly such as bulgingJacobsite amorphous(MnFe2O4) (Figure baseline 5). Poor (Figure 5). Peaks ofcrystallization crystalline of minerals minerals forcould As be sulfidesa consequence were of not early observed, stages of sulfide but weak precipitation. peaks wereElectron detected microscopy images confirmed the presence of minerals that are associated with microorganisms for sulfate phases, such as Gibsum (CaSO4 2H2O) and Fe such as Jacobsite (MnFe2O4) (Figure5). Poor crystallization(Figure 6a) and of mineralsEDX microanalysis could be revealed a consequence Fe, As, ofand early S signals stages for of different sulfide precipitation.surface particles Electron indicating the presence of Fe-S-As, Fe-As or Fe-S compounds in the precipitated solids (Figure 6b). microscopy images confirmed the presence of minerals that are associated with microorganisms Thus, solid phase analysis indicates that the removal of As and Fe is associated with compounds (Figure6precipitateda) and EDX in sulfate microanalysis reduction reactors. revealed Fe, As, and S signals for different surface particles indicating theRemoval presence of As of Fe-S-As,may be associated Fe-As or with Fe-S the compounds formation inof theFe precipitatedsulfides, which solids in anoxic (Figure 6b). Thus, solidenvironments phase analysis are capable indicates of sorbing that dissolved the removal As [44]. of In As anoxic and environm Fe is associatedents, the formation with compounds of precipitatedsulfide in mineral sulfate phases reduction control reactors. the mobility and speciation of As [44]. Arsenic can coprecipitate with Removalsulfide ofminerals As may (realgar, be associated orpiment) with [45] theor sorb formation onto sulfide of Fe minerals sulfides, (makinawite which in anoxicor troilite) environments [46]. Changes in species distribution of As and chemical associations with Fe and S minerals are are capabledetermined of sorbing by variations dissolved in pH As and [44 redox]. In potential anoxic environments,[46–48]. Recent studies the formation have shown of that sulfide As can mineral phases controlbe removed the as mobility orpiment and (As2 speciationS3) or realgar of (AsS) As [under44]. Arsenic anerobic canconditions coprecipitate from acidic with waters sulfide [43,49– minerals (realgar,52]. orpiment) In turn, Le [45 Pape] or et sorb al., [22] onto showed sulfide the mineralscomplete removal (makinawite of As from or troilite) AMD waters [46]. by Changes an in speciesindigenous distribution SRBof consortium. As and Using chemical Extended associations X-ray Absorption with FeFine and Structure S minerals (EXAFS) arespectroscopy, determined by III III variationsthey in showed pH and that redox As prec potentialipitated as [46 thiol-bound–48]. Recent As , studies amorphous have orpiment shown (am-As that As2S can3) and be realgar removed as (AsIIS). This supports the idea that As removal could be explained by the formation of amorphous orpiment (As S ) or realgar (AsS) under anerobic conditions from acidic waters [43,49–52]. In turn, Fe-bearing2 3 sulfides and amorphous As-baring sulfides and can occur by precipitation, and/or Le Papecoprecipitation et al., [22] showed processes. the However, complete As removal speciation of analysis As from at the AMD molecular waters level by is an necessary indigenous to SRB consortium.confirm Using that this Extended mechanism X-ray is relevant Absorption for the As Fine removal, Structure as mediated (EXAFS) by acid/metal-tolerant spectroscopy, they SRBs. showed III III II that As precipitatedAlso, it is not as possible thiol-bound to rule As out As, amorphous removal by an orpiment indirect mechanism (am-As 2ofS 3adsorption) and realgar of As (As S). This supportsspecies theonto idea Fe thatminerals As removal(amorphous could and/or beexplained nanocrystaline by the phases). formation The As of amorphoussoption has been Fe-bearing sulfidesextensively and amorphous studied for As-baring Fe oxides sulfidesas ferrihydrite and or can goethite occur and by Fe precipitation, sulfides as mackinawite and/or coprecipitationor pyrite. [53–55]. For these processes to occur, tolerance of SRB to high concentrations of dissolved metals (As processes. However, As speciation analysis at the molecular level is necessary to confirm that this and Fe) must be a prerequisite. Our results indicate that acid/metal-tolerant SRB could be decisive for mechanismAs removal is relevant from forpolluted the As waters, removal, and the as integrat mediatedion of by both acid/metal-tolerant resistance processes SRBs. (acid and metal Also,tolerance) it is not would possible allow to for rule the out treatment As removal of acidic by waters an indirect enriched mechanism with As and of other adsorption heavy metals. of As species onto Fe minerals (amorphous and/or nanocrystaline phases). The As soption has been extensively studied for Fe oxides as ferrihydrite or goethite and Fe sulfides as mackinawite or pyrite. [53–55]. For these processes to occur, tolerance of SRB to high concentrations of dissolved metals (As and Fe) must be a prerequisite. Our results indicate that acid/metal-tolerant SRB could be decisive for As removal from polluted waters, and the integration of both resistance processes (acid and metal tolerance) would allow for the treatment of acidic waters enriched with As and other heavy metals. Water 2017, 9, 994 8 of 12 WaterWater 2017 2017, 9,, 9949, 994 8 of8 of 12 12

5000 5000

JacobsiteJacobsite 4000 4000 Gypsum Gypsum

3000 3000 Lin (Counts) Lin (Counts) 2000 2000

1000

2 10 20 30 40 50 60 70 880 0

2 10 20 30 2-Theta40 - Scale 50 60 70 880 File: KL_P15_5-40.raw - Type: 2Th/Th locked - Start: 2.00 ° - End: 80.00 ° - Step: 0.02 ° - Step ti 00-010-0319 (I)( ) - Jacobsite, syn - MnFe2O4 ( )( ) Operations: X Offset 0.040 | Background 1.000,1.000 | Import 2-Theta - Scale00-019-0932 (I) - Microcline, intermediate - KAlSi3O8 File: KL_P15_5-40.raw00-041-1480 (I) - Albite, - Type: Ca-rich, 2Th/Th ordered locked - -(Na,Ca)Al(Si,Al)3O8 Start: 2.00 ° - End: 80.00 ° - Step: 0.02 ° - Step ti 00-010-031900-021-0816 (I)((*)) -- Jacobsite,Gypsum - CaSO4·2H2Osyn - MnFe2O4 ( )( ) Operations:00-046-1045 X Offset (*) -0.040 Quartz, | Background syn - SiO2 1.000,1.000 | Import 00-019-093200-038-1477 (I)(*) -- Microcline, Massicot -Pb intermediateO - KAlSi3O8 00-041-148000-041-1481 (I) - Albite,(I) - Anor Ca-rich,thite, Na-rich, ordered disordered - (Na,Ca)Al(Si,Al)3O8 - (Ca,Na)(Si,Al)4O8 00-021-0816 (*) - Gypsum - CaSO4·2H2O 00-046-1045 (*) - Quartz, syn - SiO2 00-038-1477 (*) - Massicot -PbO Figure00-041-1481 5. (I ) Representative- Anorthite, Na-rich, disordered X-ray- (Ca,Na)(Si, diffractionAl)4O8 (XRD) pattern for biologically produces solid precipitates Figure 5. Representative X-ray diffraction (XRD) pattern for biologically produces solid precipitates Figurecollected 5. Representative at the end of sulfateX-ray diffractionreduction experiments (XRD) pattern (80 fordays). biologically Peaks marked produces by arrows solid precipitateshave been collected at the end of sulfate reduction experiments (80 days). Peaks marked by arrows have been collectedidentified at theas Jacobsite end of sulfate and Gypsum. reduction The experiments formation of (80 amorphous days). Peaks precipitates marked is by shown arrows as ahave bulking been identiﬁed as Jacobsite and Gypsum. The formation of amorphous precipitates is shown as a bulking of identifiedof the baseline as Jacobsite (Black and line). Gypsum. The vertical The formationbar shows ofthe amorphous scale of relative precipitates counts. Initialis shown concentrations as a bulking −1 −1 2− −1 theof baseline thein batch baseline (Blackreactors (Black line). were line). TheAs =The vertical3.6 verticalmg·L bar, Febar shows = 81shows mg·L the the scale and scale SO of of4 relative relative= 4678 counts.mg·L counts.. Initial Initial concentrations concentrations in batch reactors were As = 3.6 mg·L−1, Fe = 81 mg·L−1 and SO 2− = 4678 mg·L−1. in batch reactors were As = 3.6 mg·L−1, Fe = 81 mg·L−1 and SO442− = 4678 mg·L−1.

0 5 um

0 10 um

(a) 0 10 um

(a)

Figure 6. Cont.

Water 2017, 9, 994 9 of 12 Water 2017, 9, 994 9 of 12

(b)

Figure 6. (a) Scanning electron micrographs of SRBs and solid phases precipitates inside the sulfate Figurereduction 6. (a) reactors, Scanning carbon electron was micrographs used as a surface of SRBs coating; and solid (b) Energy phases dispersive precipitates X-ray inside spectrum the sulfate of reductionprecipitates reactors, solids. carbon Intensity was of As, used Fe, as and a surfaceS emission coating; lines are (b) shown Energy in dispersivethe Energy X-rayDispersive spectrum X-ray of precipitatesSpectroscopy solids. (EDX) Intensity spectrum. of As, Other Fe, andelements S emission such as lines Mn, are Zn, shown Ni, Co., in thePb, Energyand Al have Dispersive also been X-ray Spectroscopyidentified. The (EDX) natural spectrum. water of Otherthe Azufre elements river suchhas concentrations as Mn, Zn, Ni, of these Co., Pb,elements, and Al which have can also also been identiﬁed.precipitate The during natural sulfate water reduction of the processes. Azufre river Initial has concentrations concentrations in ofbatch these reactors elements, were whichAs = 3.6 can

alsomg·L precipitate−1, Fe = 81during mg·L−1 and sulfate SO4 reduction2− = 4678 mg·L processes.−1 (The natural Initial water concentrations of Azufre river in batch has others reactors heavy were −1 −1 2− −1 Asmetals = 3.6 mg identified·L , Fe in = the 81 EDX mg· Lspectrum).and SO 4 = 4678 mg·L (The natural water of Azufre river has others heavy metals identified in the EDX spectrum). 4. Conclusions 4. ConclusionsOur results show that SRB tolerate low pH and can grow under high concentrations of As and Fe.Our pH resultsneutralization show that that SRB is mediated tolerate lowby microbial pH and canactivity grow derived under from high concentrationsacid/metal-tolerant of AsSRB and Fe.was pH observed neutralization in batch that reactors. is mediated pH values by microbial remain in activity circum-neutral derived ranges from acid/metal-tolerant due to the increase in SRB alkalinity during the sulfate reduction process and this promotes the precipitation of metal was observed in batch reactors. pH values remain in circum-neutral ranges due to the increase in carbonates and hydroxides in oxic microenvironments. Fe and As removal is influenced by the pH alkalinity during the sulfate reduction process and this promotes the precipitation of metal carbonates increase that is mediated by acid/metal-tolerant SRB. The increase in pH and sulfide concentration and hydroxides in oxic microenvironments. Fe and As removal is influenced by the pH increase that produces Fe removal associated with the precipitation of Fe-ores. Conversely, As removal may be is mediated by acid/metal-tolerant SRB. The increase in pH and sulfide concentration produces Fe associated with the precipitation of amorphous Fe-bearing sulfides and/or amorphous As-baring removal associated with the precipitation of Fe-ores. Conversely, As removal may be associated sulfides (i.e., amorphous orpiment; am-AsIII2S3) or by an indirect mechanism of adsorption onto Fe withminerals the precipitation (Fe-sulfides). ofThese amorphous results are Fe-bearing a step forward sulfides in understanding and/or amorphous sulfate reduction As-baring systems sulfides III (i.e.,under amorphous acidic and orpiment; high metal am-As conditions,2S3) or by and an indirectsuggest mechanismthe potential of future adsorption applications onto Fe for minerals the (Fe-sulfides).bioremediation These of resultsacidic waters are a step enriched forward in As. in understanding sulfate reduction systems under acidic and highThese metal findings conditions, contribute and to suggest the understanding the potential of future biological applications sulfate reduction for the bioremediationprocesses under of acidicextreme waters conditions enriched of in low As. pH and high concentrations of As and Fe. Simultaneous tolerance to low pHThese and high findings concentrations contribute of tometals the understanding can be transformed of biological into an effective sulfate alternative reduction for processes As removal under extremeand could conditions be expanded of low towards pH and the high treatment concentrations of other of heavy As and metals Fe. Simultaneousderived from natural tolerance sources to low or pH andmining high concentrationsoperations. In the of metals same way, can be the transformed study and intoapplication an effective of these alternative processes for could As removal improve and couldtraditional be expanded SRB-mediated towards AMD the treatment treatment of systems, other heavy which metals have derivedthe constraints from natural of the initial sources operating or mining operations.conditions In (i.e., the samelow pH, way, dissolved the study heavy and applicationmetals). Additional of these processesefforts are couldneeded improve to describe traditional the SRB-mediatedmechanisms AMDthat are treatment involved systems,in the tolerance which to have As, Fe the and constraints low pH, and of the the initial tolerance operating to other conditions metals. (i.e.,Future low pH,research dissolved should heavy include metals). the study Additional of these processes efforts are and needed the interaction to describe of acid/metal-tolerant the mechanisms that areSRB involved with other in the acidophilic tolerance or to acid-tolerant As, Fe and low microorganisms pH, and the toleranceliving in AMD to other waters. metals. Future research should include the study of these processes and the interaction of acid/metal-tolerant SRB with other Acknowledgments: This research was funded by PIA-UC Eduardo Leiva/DDA-VRA and CEDEUS acidophilicCONICYT/FONDAP/15110020. or acid-tolerant microorganisms living in AMD waters.

Water 2017, 9, 994 10 of 12

Acknowledgments: This research was funded by PIA-UC Eduardo Leiva/DDA-VRA and CEDEUS CONICYT/FONDAP/15110020. Author Contributions: The manuscript was written by Eduardo Leiva and Jennyfer Serrano, both authors contributed to its preparation and review. Experiments were performed by Eduardo Leiva. Data analyses were carried out by Eduardo Leiva and Jennyfer Serrano. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

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