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Article Bioleaching of Pyrrhotite with Bacterial Adaptation and Biological Oxidation for Recovery

Bong-Ju Kim, Yong-Kwon Koh and Jang-Soon Kwon *

Korea Atomic Research Institute, Deadeok-daero 989, Yuseong-Gu, Daejeon 34057, Korea; [email protected] (B.-J.K.); [email protected] (Y.-K.K.) * Correspondence: [email protected]; Tel.: +82-42-868-2904

Abstract: The microbially mediated recovery of valuable metals contained in mining waste presents an economical alternative to conventional hydrometallurgical processes. In order to investigate the effect of bacterial adaptation and biological oxidation on bioleaching, the microbially mediated bioleaching of a pyrrhotite sample from mine waste, with indigenous bacteria existing in , was studied. The indigenous bacteria were sub-cultured repeatedly for iron adaptation, and ferrooxidans was identified as the dominant member of the microbial consortium. The point of zero charge (PZC) of pyrrhotite sampled from mine waste was determined as 3.0. The performance of bioleaching by contact and non-contact biological oxidation was compared by

conducting bioleaching under different initial pH (pHini) conditions (2.8 and 3.2). Negatively charged bacteria could be attached onto the pyrrhotite, which has a positive surface charge at lower pHini (2.8) than the PZC (3.0). Bacteria attachment and corrosion pits on the surface of the pyrrhotite residues

were observed at pHini of 2.8. Under bacteria-adapted conditions, the concentration of Fe (44.2 mg/L) at pHini of 2.8 was 2.1 times greater than that (21.3 mg/L) at pHini of 3.2. Under  non-adapted bacteria conditions, the extent of Fe leaching was not significantly different between  the pHini of 2.8 and 3.2. This could be attributed to the fact that the adapted bacteria could more Citation: Kim, B.-J.; Koh, Y.-K.; easily attach onto the pyrrhotite surfaces at pHini 2.8, allowing contact biological oxidation during Kwon, J.-S. Bioleaching of Pyrrhotite the bioleaching experiments. We demonstrate here that the bioleaching of pyrrhotite could increase with Bacterial Adaptation and Fe recovery through bacterial adaptation and contact biological oxidation. Biological Oxidation for Iron Recovery. Metals 2021, 11, 295. Keywords: iron recovery; pyrrhotite; bioleaching; point of zero charge; bacterial adaptation; contact https://doi.org/10.3390/ biological oxidation met11020295

Academic Editor: Anna H. Kaksonen Received: 6 January 2021 1. Introduction Accepted: 4 February 2021 Published: 8 February 2021 Bioleaching is a technique used to recover valuable metals from sulfide . Sulfide minerals are sources of valuable metals such as Fe, Pb, Zn, Cd, and Cu, and the

Publisher’s Note: MDPI stays neutral valuable metals are available through the separation and decomposition of sulfide minerals. with regard to jurisdictional claims in Traditionally, physicochemical treatment methods such as roasting, flotation, and acid or published maps and institutional affil- base leaching have been used to decompose sulfide minerals. Currently, the bioleaching iations. method is attracting interest as an alternative method due to its environmental friendliness and cost effectiveness. Ever since it became a known fact that acidophilic bacteria can survive in a strongly acidic environment [1], bioleaching has been widely used for the recovery of valuable metals from slime or low-grade minerals and the removal of heavy metals from polluted Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. soils. Various studies using the bioleaching technique to recover valuable metals (Fe, This article is an open access article Cu, Pb, Zn, etc.) from sulfide minerals including [2], arsenopyrite [3,4], distributed under the terms and molybdenite [5,6], [7–9], and sphalerite [9,10] have been reported. In addition, conditions of the Creative Commons indigenous acidophilic bacteria have been already used for the recovery of Cu, Au, and U Attribution (CC BY) license (https:// in slime [11–13]. creativecommons.org/licenses/by/ The bioleaching of sulfide minerals for recovery can be regarded as a type of 4.0/). bio-oxidation that can be classified as contact oxidation or non-contact oxidation [14,15].

Metals 2021, 11, 295. https://doi.org/10.3390/met11020295 https://www.mdpi.com/journal/metals Metals 2021, 11, 295 2 of 11

Contact oxidation means oxidation by microbes that are placed onto the surface of the sulfide , while non-contact oxidation means the oxidation of sulfides by ferric (Fe3+), which occurs through the microbial catalytic oxidation of ferrous ion. In the case of contact oxidation, bacteria are able to select sites on which they can easily obtain energy through oxidization [16]. Generally, these sites are concentrated on the surfaces of sulfide minerals where they are characterized as mechanically and chemically weak crystallization, dislocation, and corrosion pits [17–19]. In the non-contact mechanism, the bacterial oxidation of ferrous ion (Fe2+) to ferric ion (Fe3+) regenerates the oxidant necessary for further reaction with the mineral. This mech- anism has been shown to proceed via two major routes: the and polysulfide pathways [17–19]. Acid-insoluble sulfides such as FeS2, MoS2, and WS2 are non-contact bioleaching by the ferric , generating thiosulfate, which is further oxidized to form . In contrast, the dissolution of a soluble metal sulfide involves a proton attacking the mineral surface. Bioleaching might be also affected by various factors including bacterial strain, pH, oxidation/reduction potential (ORP), temperature, and heavy metals in leachate. During the bioleaching processes, bacterial viability can be especially affected by toxic heavy metal ions, resulting in decreased recovery of the available elements from sulfide minerals. Therefore, bacterial adaptation to toxic heavy metals can play a significant role in enhancing the efficiency of bioleaching [20,21]. The aim of this study was to investigate the effects of bacterial adaptation and biologi- cal oxidation on the bioleaching of pyrrhotite using an indigenous bacterium. Bacterial adaptation was performed to examine the effect of the adaptation of indigenous bacteria. Bi- oleaching experiments were conducted to estimate the effect contact biological oxidation on bioleaching efficiency at different using non-adapted and adapted indigenous bacteria.

2. Materials and Methods 2.1. Mine Waste and Pyrrhotite In order to prepare the pyrrhotite sample used in the experiment, mine waste was acquired from an abandoned iron mine located in Ul-Jin, Korea. In the laboratory, the mine waste was crushed using a jaw crusher and a cone crusher, ground, and then sieved with a 20 mesh (841 micron, ASTM) sieve to exclude accessory minerals, quartz especially. The sieved mine waste sample was separated into magnetic and non-magnetic fractions under the magnetic field intensity of 0.2 T using a magnetic separator (Frantz magnetic separator, L-ICN, Frantz, Sterling, IL, USA) due to the highly magnetic properties of pyrrhotite. The effect of the magnetic separation process on the magnetic properties was evaluated using a vibration sample magnetometer (VSM, BHV-50HTI, Riken, Keiki, Tokyo, Japan). The chemical composition of the mine waste was analyzed using an atomic absorption spectrophotometer (AAS, AA-7000, Shimadzu, Kyoto, Japan) through aqua regia digestion. The powdered form of the pyrrhotite sample magnetic fraction was characterized using a powder X-ray diffractometer (XRD, D8ADVANCE, Bruker, Billerica, MA, USA) with Cu-Kα radiation (1.5406 Å) at a scanning speed of 2◦/min. The point of zero charge (PZC) of the pyrrhotite sample was determined by measuring the zeta potential (Zetasizer, Nano-ZS MPT-2, Malvern, PA, USA) at various pH values, which were controlled using HNO3 and NaOH.

2.2. Microbes 2.2.1. Collection and Cultivation of Microbes The indigenous microbes used in this bioleaching experiment were collected from the mine drainage (pH: 4.62 and ORP: 365 mV) located 82 m under the ground at the Ul-Jin Mine cave. The microbes were cultured in a mineral-salt medium (MSM). The MSM was prepared by dissolving 0.2 g (NH4)2SO4, 0.5 g MgSO4·7H2O, 0.25 g CaCl2, 3.0 g KH2PO4, and 0.05 g FeSO4 in 1.0 L of distilled [10]. The growth medium was prepared by adding elemental with a concentration of 1.0 g/L to the MSM. After 7 days of Metals 2021, 11, 295 3 of 11

incubation at 32 ◦C, the enrichment cultures were plated onto MSM agar supplemented with 0.5 wt % FeSO4 to isolate the iron-resistant bacteria. Of the colonies isolated, a single colony was used for bioleaching investigation.

2.2.2. Identification of the Bacterial Strain The 16S rRNA gene of the iron-resistant bacteria was amplified by polymerase chain reaction using a HotStarTaq®® Plus Master Mix Kit (Qiagen, Valencia, CA, USA), with the universal primers 27F (50-AGAGTTTGATCMTGGCTCAG-30) and 1492R (50- GGYTACCTTGTTACGAC-TT-30), which were sequenced at Macrogen Corp. (Daejeon, Korea). The sequenced gene was compared with the sequences available in the Gen- Bank databases using the BlastN program from the National Center for Information homepage.

2.3. Adaption of Iron-Resistant Bacteria The iron-resistant bacteria from the bacterial culture were enriched by culturing in the MSM with 1% FeSO4 for 21 days, and this culturing was repeated several times as necessary to enhance the iron resistivity (adaptation). Then, we adapted the enriched bacteria to iron ions using the same adaptation medium (MSM with 1% iron) [13]. During enrichment and adaptation, the pH and ORP in the media were measured using a Thermo Scientific Orion 3Star portable meter (Orion 3Star Portable, Thermo Scientific, Beverly, MA, USA) to confirm the ability of the bacteria to oxidize the pyrrhotite sample. This meter was equipped with a pH probe (Triode gel-filled epoxy-body LM, 9107WMMD) and ORP electrode (Sure-flow combination redox/ORP electrode, 9678BNWP, Ag/AgCl), and all electrodes were calibrated daily using standard procedures during the experiments.

2.4. Bioleaching Experiments Bioleaching experiments were conducted to evaluate the effect of bacterial adaptation on the bioleaching efficiency. Batch-type bioleaching experiments were prepared in 500-mL Erlenmeyer flasks containing 300 mL of culture medium (290 mL of fresh MSM + 10 mL of adapted bacteria medium) amended with 3 g of 20 mesh sieved pyrrhotite sample particles. A control experiment was also carried out simultaneously under abiotic conditions to check the contribution of simple chemical leaching from the growth medium. After the leaching experiments, the iron (Fe) concentration in the leachates was analyzed using an atomic absorption spectrophotometer (AAS). The morphological features of the residues were observed using a scanning microscopy (SEM, S4800, Hitachi, Tokyo, Japan). The mass of Fe leached per unit mass of the pyrrhotite sample was applied to the following Equation (1): -kt E = EI (1 − e ) (1)

where E is the leaching concentration at time t, EI is the maximum leaching concentration, and k is the leaching rate constant.

3. Results and Discussion 3.1. Pyrrhotite in the Mine Waste The chemical analysis shows that the mine waste of 1 kg in mass obtained from the abandoned Ul-Jin Fe mine contained 5670 mg of Fe, 140 mg of Pb, 479 mg of Cu, and 238 mg of Zn. The XRD result of two classified fractions after magnetic separation of the mine waste (Figure1) demonstrated that the magnetic fraction is composed of pyrrhotite with quartz as a minor impurity, whereas the non-magnetic fraction includes various minerals such as galena, , pyrrhotite, quartz, and sphalerite. Metals 2021, 11, x FOR PEER REVIEW 4 of 11

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The XRD result of two classified fractions after magnetic separation of the mine The XRDwaste result (Figure of two1) de classifiedmonstrated fractions that the after magnetic magnetic fraction separation is composed of the of minepyrrhotite with waste (Figurequartz 1) de asmonstrated a minor impurity, that the whereas magnetic the fraction non-magnetic is composed fraction of pyrrhotite includes various with minerals such as galena, pyrites, pyrrhotite, quartz, and sphalerite. Metals 2021, 11, 295 quartz as a minor impurity, whereas the non-magnetic fraction includes various minerals 4 of 11 such as galena, pyrites, pyrrhotite, quartz, and sphalerite.

Figure 1. XRD results of the mine waste after magnetic separation: (a) magnetic fraction; (b) non-magnetic fraction. Figure 1. XRDFigure results 1. XRD of the results mine of waste the mine after waste magnetic after separation: magnetic separation: (a) magnetic (a) fraction; magnetic (b fraction;) non-magnetic (b) fraction. non-magnetic fraction. TheThe distribution distribution of of magnetization magnetization along along the the mineral mineral composition composition is is illustrated illustrated by by The distributioncomparingcomparing ofthe the magnetization magnetic magnetic hysteresis hysteresis along theloops loops mineral of of the the compositionmagnetic magnetic and and is non non-magneticillustrated-magnetic by fractions fractions in in comparing thethethe mmagnetic mineine waste waste hysteresis (Figure (Figure 2). 2loops). The The maximizedof maximized the magnetic magnetization magnetization and non-magnetic of of the the two two fractions fractions fractions in was was 3.1 3.1 and and 2 2 the mine waste69.869.8 Am(Figure Am/kg/kg 2).in intheThe the non maximized non-magnetic-magnetic magnetization and and magnetic magnetic of fractions, the fractions, two fractions respectively. respectively. was 3.1In In addition, and addition, in in the the 69.8 Am2/kgcase casein the of of non magnetically magnetically-magnetic separatedand separated magnetic samples, samples,fractions, magnetic magneticrespectively. minerals minerals In have addition, ferromagnetic have in the ferromagnetic properties, case of magneticallyproperand non-magneticties, and separated non- mineralsmagnetic samples, haveminerals no magnetic magnetism. have no minerals magnetism. have ferromagnetic properties, and non-magnetic minerals have no magnetism.

FigureFigure 2. 2.MagneticMagnetic hysteresis hysteresis loops loops of of (non (non-)-) magnetic magnetic fractions fractions in in the the mine mine waste waste sample. sample. Figure 2. Magnetic hysteresis loops of (non-) magnetic fractions in the mine waste sample. TheThe zeta zeta potential of of the the pyrrhotite pyrrhotite sample sample (magnetic (magnetic fraction) fraction) measured measured under undervarious pH conditions is shown in Figure3. As the pH increased from 1.0 to 9.0, the zeta potential The zetavarious potential pH conditions of the pyrrhotite is shown samplein Figure (magnetic 3. As the fraction)pH increased measured from 1.0 under to 9.0, the zeta of the pyrrhotite decreased from +8.1 to −25.6 mV. The pH value of the pyrrhotite was various pHpotential conditions of isthe shown pyrrhotite in Figure decreased 3. As the from pH +8.1 increased to −25.6 from mV. pzc1.0 The to pH9.0,pzc the value zeta of the pyr- rhotitedetermined was determined to be 3.0, which to be is3.0, similar which to is the similar result to of the Widler result and of SewardWidler and [22], Seward who reported [22], potential of the pyrrhotite decreased from +8.1 to −25.6 mV. The◦ pHpzc value of the pyr- that the pHpzc of artificial pyrrhotite produced at 230 C and 100 atm was 2.7. This indicates rhotite waswho determined reported to that be 3.0, the whichpHpzc isof similarartificial to pyrrhotitethe result ofproduced Widler and at 230 Seward °C and [22 100], atm was 2.7.that This the indicates pyrrhotite that collected the pyrrhotite from the collected mine waste from was the effectively mine waste separated was effectively for the purpose sep- who reportedof that these th experiments.e pHpzc of artificial It is obvious pyrrhotite that solidproduced material at 230 is positively °C and 100 charged atm was when the pH is 2.7. This indicatesarated for that the the purpose pyrrhotite of these collected experiments. from the It mine is obvious waste thatwas solideffectively material sep- is positively lower than the pHpzc, while it is negatively charged when the pH is higher than the pHpzc. arated for thecharge purposed when of thesethe pH experiments. is lower than It theis obvious pHpzc, while that solid it is negatively material is charged positively when the pH is higher than the pHpzc. charged when the pH is lower than the pHpzc, while it is negatively charged when the pH is higher than the pHpzc.

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FigureFigure 3.3. ZetaZeta potentialspotentials ofof pyrrhotitepyrrhotite usedused inin thethe experiments.experiments.

3.2.3.2. BacterialBacterial AdaptationAdaptation TheThe resultsresults ofof thethe phylogeneticphylogenetic analysisanalysis showshow thatthat A.A. ferrooxidansferrooxidansferrooxidans isisis the the the dominantdominantdominant A. ferrooxidans membermember among the the four four-step--stepstep adaptedadapted adapted bacteriabacteria bacteria (Figure(Figure (Figure 4).4).4). A. ferrooxidans,, knownknown, known asas anan as Fe anFe-- Fe- and S-oxidizing acidophile, has a well characterized genetic system and has been and S--oxidizing acidophile,, hashas aa wellwell characterizedcharacterized geneticgenetic systemsystem andand hashas beenbeen previ-previ- previously shown to be a ubiquitous member of the acid mine drainage ecosystem [23]. ously shown to be a ubiquitous member of the acid mine drainage ecosystem [23]. The The presence of A. thiooxidans and A. thermosulfidooxidans also indicates the potential for presence of A. thiooxidans and A. thermosulfidooxidans also indicates the potential for thermophilic enrichment [24]. thermophilicthermophilic enrichmentenrichment [2[24].].

FigureFigure 4.4. MicrobialMicrobial profilingprofiling ofof thethe four-stepfour--stepstep adapted adaptedadapted bacteria. bacteria.bacteria.

TheThe adaptation adaptation of ofA. A. ferrooxidans ferrooxidanswas was performed performed in four-step in four- adaptation-stepstep adaptation adaptation cycles, cycles, cycles,based onbased the on report the ofreport Tuovinen, of Tuovinen, Niemelä, Niemelä, and Gyllenberg and Gyllenberg [24]. The [2 variations4].]. TheThe variationsvariations of pH and ofof pH ORPpH andand in theORP culture inin thethe mediumcultureculture mediummedium during the duringduring adaptation thethe adaptationadaptation cycles are cyclescycles presented areare presentedpresented in Figure inin5. FigureFigure 5.5.

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Figure 5. VariationsFigure 5. ofVariations pH (a) andof pH oxidation/reduction (a) and oxidation/reduction potential potential (ORP) ( (ORP)b) in the (b) culturein the culture medium medium during different adaptation cycles.during different adaptation cycles.

The estimatedThe results estimated show that results the show pH in that the theculture pH inmedium the culture decreased medium with decreased the with the increase of adaptationincrease oftime, adaptation whereas time, ORP whereasincreased ORP with increased time. This with might time. be This due mightto the be due to the biological oxidationbiological of energy oxidation sources of energy (Fe2+) sourcesby A. ferrooxidans (Fe2+) by A. along ferrooxidans with thealong inorganic with the inorganic oxidation of oxidativeoxidation energy of oxidative sources energy (Fe2+ sources) partially (Fe 2+during) partially the adaptation. during the adaptation.Differences Differences in in pH decreasespH and decreases ORP increases and ORP increasesin adaptation in adaptation cycles were cycles observed. were observed. In the first In the ad- first adaptation, aptation, pH decreasedpH decreased from from2.6 to 2.6 1.8, to whereas 1.8, whereas ORP increased ORP increased from 390 from to 390470 tomV. 470 In mV. the In the fourth fourth adaptation,adaptation, pH decreased pH decreased,, starting starting from 2.6 from to 1.3 2.6 after to 1.3 21 after days, 21 whereas days, whereas ORP in- ORP increased creased from 400from to 400 580 to mV. 580 In mV. the In third the third and fourth and fourth adaptations, adaptations, there there was wasno significant no significant difference difference in thein the results results of pH of pH and and ORP ORP estimated estimated with with the the adaptation adaptation time. time. It was It was ex- expected that pected that A.A. ferrooxidans ferrooxidans sufficientlysufficiently adapted adapted to to iron iron ions ions at least byby thethe thirdthird adaptation.adapta- Moreover, tion. Moreover,in in the the third third and and fourth fourth adaptations, adaptations, thethe pHpH showedshowed an equilibrium equilibrium con- constant after 15 stant after 15 days of inoculation, which indicates indicates that that the the optimum optimum adaptation adaptation of of A.A. fer- ferrooxidans was rooxidans wasreached reached by by day day 15. 15. Previous Previous studies studies have have reported reported optimum optimum adaptation adaptation periods of one periods of oneweek week [25 [,25,26],26], two two weeks weeks [27 –[2729],–29], and and four four weeks weeks [30,31 [30,31].]. Reference Reference [20] also [20] mentioned that also mentionedthe that repetition the repetition of adaptation of adaptation performance performance to leads a reduction to a reduction in the lag in phasethe of bacteria lag phase of bacteriaand improved and improved biological biological oxidation oxidation capacity capacity of bacteria. of bacteria. 3.3. pH and ORP Changes through Pyrrhotite Bioleaching 3.3. pH and ORP Changes through Pyrrhotite Bioleaching The variations of pH and ORP during bioleaching of the pyrrhotite under different ini- The variations of pH and ORP during bioleaching of the pyrrhotite under different tial pH conditions (pHini 2.8 and 3.2) are presented in Figure6. At the initial pH ini of 2.8, the initial pH conditions (pHini 2.8 and 3.2) are presented in Figure 6. At the initial pHini of 2.8, pH remained relatively constant regardless of bioleaching time in bacterial adaptation con- the pH remained relatively constant regardless of bioleaching time in bacterial adapta- ditions, although a slight pH fluctuation was observed at 41 days (Figure6a ). pH increased tion conditions,gradually although with a slight time pH up tofluctuation 3.9 under was the abioticobserved condition at 41 days and (Figure up to 4.1 6a). in pH the non-adapted increased graduallybacterial with condition. time up The to 3.9 ORP under values the decreased abiotic condition in all three and experimental up to 4.1 in conditions, the and the non-adapted variances bacterial condition. were intensive The inORP the values order of decreased adaptation in < all non-adaptation three experimental < abiotic conditions conditions, and(Figure the variances6b). The were reason intensive for the in opposite the order pH of and adaptation ORP changes < non- duringadaptation the oxidation of < abiotic conditionspyrrhotite (Figure could 6b). be The explained reason for by the inorganic/biologicalopposite pH and ORP oxidation changes processduring occurring as the oxidationdescribed of pyrrhotite in Reactions could be (2) explained and (3). by the inorganic/biological oxidation process occurring as described in Reactions (2) and (3).

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Figure 6. VariationsFigure of6. pHVariations and ORP of duringpH and bioleaching ORP during of bioleaching the pyrrhotite of the under pyrrhotite different under initial different pH conditions: initial pH (a) and (b), conditions: (a) and (b), pHini. 2.8; (c) and (d) pHini. 3.2. pHini. 2.8; (c) and (d) pHini. 3.2.

The pH valuesThe in pH the valuesnon-adaptation in the non-adaptation condition were condition slightly were lower slightly than those lower in than the those in the abiotic conditionabiotic (Figure condition 6c). The (Figure ORP6c). values The ORPdecreased values up decreased to day 10 up of tothe day experim 10 of theents experiments and then fluctuatedand then thereafter fluctuated in thereafter all three experimental in all three experimental conditions. conditions. The ORP values The ORP were values were in in the order theof adaptation order of adaptation > non-adaptation > non-adaptation ≈ abiotic≈ conditionabiotic condition (Figure 6d). (Figure The6d). pH The in- pH increase crease in thein abiotic the abiotic condition condition was was affected affected by hydrogen by hydrogen ions ions being being consumed consumed by by the the chemical chemical oxidationoxidation of ofpyrrhotite. pyrrhotite. The The decrease decrease of of ORP ORP and and increase increase of of pH pH are caused byby the chemical the chemicalcharacteristics characteristics of of pyrrhotite pyrrhotite in in dissolution dissolution [32 [32,33].,33]. Dissolution Dissolution reactions reactions of of pyrrhotite can pyrrhotite canbe be expressed expressed by by Reactions Reactions (2) (2) and and (3) (3) [34 [34]:]:

+ + 2+ 3+2+ 3+ 퐹푒1−푥푆 + 2Fe퐻1− x→S +(12−H3푥)→퐹푒 (1+−23푥x퐹푒)Fe ++ 퐻22xFe푆 + H2S (2) (2)

+ 2+ 3+ 0 2Fe1−+xS + O2 + 4H 2+→ (2 − 63x+)Fe 0+ 4xFe + 2S + 2H2(O3). (3) 2퐹푒1−푥푆 + 푂2 + 4퐻 → (2 − 6푥)퐹푒 + 4푥퐹푒 + 2푆 + 2퐻2푂. Reaction (2) represents the non-oxidative dissolution of pyrrhotite. Reaction (3) indi- Reaction (2) represents the non-oxidative dissolution of pyrrhotite. Reaction (3) indi- cates the oxidative dissolution of pyrrhotite in the presence of . Although indigenous cates the oxidative dissolution of pyrrhotite in the presence of oxygen. Although indige- bacteria were present in the non-adaptation condition, the same patterns of pH and ORP nous bacteria were present in the non-adaptation condition, the same patterns of pH and changes for the abiotic condition were observed. This indicates that indigenous bacteria do ORP changes for the abiotic condition were observed. This indicates that indigenous bac- not have a major role in biological oxidation in the non-adaptation condition, but chemical teria do not have a major role in biological oxidation in the non-adaptation condition, but oxidation could contribute to changes of pH and ORP. Biological oxidation by indigenous chemical oxidation could contribute to changes of pH and ORP. Biological oxidation by bacteria using pyrrhotite S and Fe ions can be expressed by Reactions (4) and (5): indigenous bacteria using pyrrhotite S and Fe ions can be expressed by Reactions (4) and (5): 0 2− + S + 1.5O2 + H2O → SO4 + 2H (4) 0 2− + 푆 + 1.5푂2 +2+ 퐻2푂 → 푆푂4 ++ 2퐻 3+ (4) 4Fe + O2 + 4H → 4Fe + 2H2O. (5)

As mentioned2+ above, the+ values3 of+ pH in the case of the adaptation condition(5) were 4퐹푒 + 푂2 + 4퐻 → 4퐹푒 + 2퐻2푂. lower than those in the abiotic and non-adaptation conditions, whereas the ORP values As mentionedin the adaptationabove, the values condition of pH were in the higher case thanof the those adaptation in the abioticcondition and were non-adaptation lower than thoseconditions. in the abiotic This could and non be- ascribedadaptation to biologicalconditions, oxidation whereas the by activeORP values adapted in indigenous the adaptationbacteria condition in the were adaptation higher than condition, those resultingin the abiotic in the and discontinuation non-adaptation of con- pH increase and

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Metals 2021, 11, 295 ditions. This could be ascribed to biological oxidation by active adapted indigenous 8 of 11 bacteria in the adaptation condition, resulting in the discontinuation of pH increase and ORP decrease caused by the chemical reactions (non-oxidation and direct oxidation of pyrrhotite) described in Reactions (2) and (3). ORP decrease caused by the chemical reactions (non-oxidation and direct oxidation of Biological attachment and bio-oxidation of the indigenous acidophilic adapted bac- pyrrhotite) described in Reactions (2) and (3). teria onto the pyrrhotite residues after the bioleaching under different pHini conditions Biological attachment and bio-oxidation of the indigenous acidophilic adapted bacteria was presented in Figure 7. The rod-shaped bacteria were highly attached onto the resi- onto the pyrrhotite residues after the bioleaching under different pHini conditions was due under thepresented pHini 2.8 in compared Figure7. The to the rod-shaped pHini 3.2. bacteria Moreover, were corrosion highly attached pits by onto the the residue adapted bacteria were observed on the surface of pyrrhotite residue under the pHini 2.8 under the pHini 2.8 compared to the pHini 3.2. Moreover, corrosion pits by the adapted (Figure 7a). Under the acidic condition, the negatively charged bacteria could be easily bacteria were observed on the surface of pyrrhotite residue under the pHini 2.8 (Figure7a ). attached at theUnder positively the acidic charged condition, places the of negatively minerals, wh chargedich are bacteria characterized could be by easily the attached at crystallographicthe imperfection positively charged [17–19 places]. The ofmeasurement minerals, which of zeta are-potential characterized of the by adapted the crystallographic bacteria wouldimperfection have been [helpful17–19]. Theto interpret measurement the electrostatic of zeta-potential attachment of the between adapted the bacteria would pyrrhotite andhave the beenA. ferrooxidans, helpful to interpretbut the measurement the electrostatic was attachment not performed between in this the study. pyrrhotite and the Nevertheless,A. several ferrooxidans, previousbut thestudies measurement [15,19,34] washave not reported performed that in the this pHs study.pzc of Nevertheless, the A. several ferrooxidans areprevious about studies2.0, and [ 15 these,19,34 results] have could reported support that the the pHs electrostaticpzc of the A. interaction ferrooxidans are about between the A.2.0, ferrooxidans and these resultsand the could pyrrhotite support used the electrostaticin this study interaction under the betweenacidic condi- the A. ferrooxidans tion (less thanand the the pH pyrrhotitepzc (3.0) of used the in pyrrhotite). this study In under addition the acidic to the condition electrostatic (less attach- than the pHpzc (3.0) ment, the secretionof the pyrrhotite). of slime layer, In addition protein- tobinding the electrostatic receptors, attachment, physical the secretion, and of slime layer, hydrophobic protein-binding interactions of receptors,microbes couldphysical be adsorption, also a function and hydrophobic of bacteria attachment interactions of microbes onto mineralscould [14–20 be]. also a function of bacteria attachment onto minerals [14–20].

Figure 7. SEMFigure images 7. ofSEM pyrrhotite images residuesof pyrrhotite after residues completion after of completion bioleaching of under bioleaching different under initial different pH conditions: initial (a) pHini pH conditions: (a) pHini 2.8; (b) pHini 3.2. 2.8; (b) pHini 3.2.

3.4. Fe recovery from Pyrrhotite Bioleaching 3.4. Fe recovery from Pyrrhotite Bioleaching The variations of iron (Fe) concentrations in the leachates through bioleaching of the pyrrhotite under differentThe variations initial pH of conditions iron (Fe) concentrations(pHini 2.8 and 3.2) in are the presented leachates in through Figure 8 bioleaching. of At pHini 2.8 (Figurethe pyrrhotite 8a), small under amounts different of iron initial (Fe) pHwere conditions dissolved (pHthroughoutini 2.8 and the 3.2) experi- are presented in ment under theFigure abiotic8 . Atcondition pHini 2.8 and (Figure remained8a), small low amountsduring the of experiments, iron (Fe) were with dissolved a con- throughout centration of the1.9 experimentmg/L by day under 41. Under the abiotic the non condition-adaptation and remained condition, low 23.8 during mg/L the of experiments,dis- with solved Fe wasa leached concentration by day of41. 1.9 In mg/Lcase of by the day adaptation 41. Under condition, the non-adaptation dissolved Fe condition,reached 23.8 mg/L of dissolved Fe was leached by day 41. In case of the adaptation condition, dissolved 47.4 mg/L by day 41. At the pHini of 3.2 (Figure 8b), iron (Fe) was rarely dissolved under the abiotic condition,Fe reached with just 47.4 0.7 mg/Lmg/L at by the day end 41. of Atleaching. the pH Inini theof 3.2non (Figure-adaptation8b), ironand (Fe)ad- was rarely aptation conditions,dissolved Fe concentrations under the abiotic increased condition, gradually with justwith 0.7 time, mg/L arriving at the at end 19.4 of and leaching. In the non-adaptation and adaptation conditions, Fe concentrations increased gradually with 21.9 mg/L, respectively, by day 41. This indicated that Fe recovery from the pyrrhotite bi- time, arriving at 19.4 and 21.9 mg/L, respectively, by day 41. This indicated that Fe recovery oleaching might be increased through the bacterial adaptation of indigenous bacteria. from the pyrrhotite bioleaching might be increased through the bacterial adaptation of indigenous bacteria.

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Figure 8. VariationFigure of8. FeVariation concentrations of Fe concentrations during bioleaching during experiments bioleaching underexperiments different under initial different pH conditions: initial pH (a ) pHini 2.8; (b) pHini 3.2.conditions: (a) pHini 2.8; (b) pHini 3.2.

Elzeky and AttiaElzeky [27] and performed Attia [27 bioleaching] performed experiments bioleaching using experiments (FeS using2), pyritechal- (FeS2), chal- copyrite (CuFeScopyrite2), and (CuFeS arsenopyrite2), and arsenopyrite (FeAsS) using (FeAsS) non-adapted using non-adapted and adapted and bacteria, adapted bacteria, and and they reportedthey reporteda higher aperformance higher performance of metal of recovery metal recovery (3.43 times (3.43 timesfor Cu for and Cu 1.65 and 1.65 times for times for Fe) underFe) under the bacteria the bacteria adaptation adaptation environment environment than than that that of ofnon non-adaptation.-adaptation. The The Fe-adapted Fe-adapted microbesmicrobes d demonstrateemonstrate higher higher oxidizing oxidizing power thanthan non-adaptednon-adapted microbes, microbes, which resulted which resultedfrom from the the increased increased tolerance tolerance to to the the heavy heavy metal metal [12 [12].]. Throughout theThroughout bioleaching the of bioleaching the pyrrhotite, of the the pyrrhotite, maximum the leaching maximum concentration leaching concentration (EI) and leaching(EI) and rate leaching constant rate (k) were constant calculated (k) were (Table calculated 1). At (Table the pH1).ini Atof 2.8,the pH the iniEI of 2.8, the EI value (44.2 mg/L)value under (44.2 the mg/L) bacterial under adaptation the bacterial condition adaptation was condition2.1 times greater was 2.1 than times that greater than that (21.6 mg/L) of(21.6 the mg/L)non-adaptation of the non-adaptation condition, whereas condition, the whereas k values the werek values similar. were At similar. the At the pHini pHini of 3.2, theof E 3.2,I value the E andI value k value and kwerevalue the were same the or same showed or showed little difference. little difference. This in- This indicated that bacterial adaptation enhanced the bioleaching of Fe at pH 3.0 because the pH of dicated that bacterial adaptation enhanced the bioleaching of Fe at pHini 3.0ini because the pzc the pyrrhotite used here was positively charged under pH conditions lower than pH 3.0. pHpzc of the pyrrhotite used here was positively charged under pH conditions lower than In addition, previous studies have shown that the pH of A. ferrooxidans is in the range pH 3.0. In addition, previous studies have shown that the pHpzc of A.pzc ferrooxidans is in the of 2.1 to 2.4 [35]. Therefore, it is expected that at the pH of 2.8, the negatively charged range of 2.1 to 2.4 [35]. Therefore, it is expected that at the pHini of ini2.8, the negatively charged A. ferrooxidansA. ferrooxidans attachesattaches well wellonto onto the positively the positively charged charged surfaces surfaces of pyrrhotite of pyrrhotite due to an due to an electricalelectrical attraction; attraction; that that is, is, the the contact contact biological biological oxidation ofof thethe pyrrhotitepyrrhotite could coincide at the pHini of 2.8, whereas non-contact biological oxidation is the dominant reaction at could coincide at the pHini of 2.8, whereas non-contact biological oxidation is the domi- pHini 3.2. nant reaction at pHini 3.2.

Table 1. The maximum leaching concentration (EI) and leaching rate constant (k) calculated using Table 1. The maximum leaching concentration (EI) and leaching rate constant (k) calculated using Equation (1). Equation (1). 2 Experimental Conditions E (mg/L) k (1/Day) 2 R Experimental Conditions EI (mg/L) I k (1/Day) R non-adaptation non-adaptation21.6 21.60.08 0.080.93 0.93 pHini 2.8 pHini 2.8 adaptation adaptation44.2 44.20.09 0.090.93 0.93 non-adaptation 19.8 0.14 0.91 nonpH-adaptation3.2 19.8 0.14 0.91 pHini 3.2 ini adaptation adaptation21.3 21.30.14 0.140.90 0.90

Xia et al. [20] Xiaand etKim al. et [20 al.] and[21] Kimconducted et al. [bioleaching21] conducted of pyrites bioleaching using ofadapted pyrites A. using adapted ferrooxidans andA. ferrooxidans reported thatand the reported adapted that bacteria the adapted increase bacteria the recovery increase of the valuable recovery of valuable metals from spentmetals refinery from spent catalysts refinery because catalysts the because adapted the bacteriaadapted bacteriahave increased have increased re- resistance sistance to theto the toxicity of heavy of heavy metals. metals. They They also also reported reported that that the adaptation the adaptation could could improve improve the abilitythe ability of bacteria of bacteria to adhere to adhere to mineral to mineral surfaces surfaces,, as the asadapted the adapted bacteria bacteria has a has a lower lower interactioninteraction energy energywith the with surfaces the surfaces than the than non the-adapted non-adapted one. one. 4. Conclusions Various bioleaching experiments characterized by bio-adaptation and bio-oxidation were carried out using a pyrrhotite sample (pHpzc of 3.0) obtained from an abandoned mine and indigenous bacteria (A. ferrooxidans) inhabiting the acid mine drainage in order

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to enhance the recovery of valuable metal (iron) from mine waste. During the bioleaching reaction, the leachate pH and ORP under the iron adapted bacteria condition were lower and higher, respectively, than those of the non-adapted and abiotic conditions. Iron re- covery was also enhanced under the adapted bacteria condition. The maximum leaching concentration of iron at the pHini of 2.8 was 44.2 mg/L, which is 2.1 times greater than that at the pHini of 3.2. Bacteria attachment and corrosion pits on the surface of the pyrrhotite residues were observed at pHini of 2.8 under the adapted condition. These phenomena could be attributed to the fact that the adapted bacteria could more easily attach to the pyrrhotite surface at pH 2.8; therefore, contact biological oxidation occurred during the bioleaching experiments. Under the non-adapted bacteria condition, iron leaching was not significantly enhanced compared to the abiotic condition. We demonstrated that the bioleaching of pyrrhotite could be enhanced through bacterial adaptation and contact biological oxidation.

Author Contributions: B.-J.K. carried out the bioleaching experiments, participated in the reference research, and drafted the manuscript. Y.-K.K. participated in the design of the study and carried out the model analysis. J.-S.K. revised and edited the manuscript. J.-S.K. conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Research Foundation of Korea (NRF) funded by the Korean government, Ministry of Science and ICT (No. 2017M2A8A5014859). Data Availability Statement: Data sharing not applicable. Acknowledgments: This work was supported by the National Research Foundation of Korea (NRF) funded by the Korean government, Ministry of Science and ICT (No. 2017M2A8A5014859), and Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea government ministry of trade, industry and energy (No. 20171510300670, 20193210100120). Conflicts of Interest: The authors declare no conflict of interest.

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