Enhancement of Biofilm Formation on Pyrite by Sulfobacillus

minerals

Article Enhancement of Bioﬁlm Formation on Pyrite by Sulfobacillus thermosulﬁdooxidans

Qian Li, Wolfgang Sand and Ruiyong Zhang *

Bioﬁlm Centre, Aquatische Biotechnologie, Universität Duisburg—Essen, Universitätsstraße 5, 45141 Essen, Germany; [email protected] (Q.L.); [email protected] (W.S.) * Correspondence: [email protected]; Tel.: +49-201-183-7076; Fax: +49-201-183-7088

Academic Editor: W. Scott Dunbar Received: 29 April 2016; Accepted: 4 July 2016; Published: 9 July 2016

Abstract: Bioleaching is the mobilization of metal cations from insoluble ores by microorganisms. Biofilms can enhance this process. Since Sulfobacillus often appears in leaching heaps or reactors, this genus has aroused attention. In this study, biofilm formation and subsequent pyrite dissolution by the Gram-positive, moderately thermophilic acidophile Sulfobacillus thermosulfidooxidans were investigated. Five strategies, including adjusting initial pH, supplementing an extra energy source or ferric ions, as well as exchanging exhausted medium with fresh medium, were tested for enhancement of its biofilm formation. The results show that regularly exchanging exhausted medium leads to a continuous biofilm development on pyrite. By this way, multiply layered biofilms were observed on pyrite slices, while only monolayer biofilms were visible on pyrite grains. In addition, biofilms were proven to be responsible for pyrite leaching in the early stages.

Keywords: bioleaching; Sulfobacillus thermosulﬁdooxidans; bioﬁlm formation

1. Introduction The term of bioleaching refers to mobilization of metal cations from insoluble ores by microorganisms [1,2]. Leaching can be done by microorganisms via two ways: contact and non-contact leaching. Ferric ions are of great importance in both of these two ways. As an oxidizing agent, ferric ions in acidic environments readily oxidize metal sulfides and cause the release of ferrous ions and reduced inorganic sulfur compounds (RISCs). Ferrous ions can be re-oxidized to ferric ions by iron-oxidizing bacteria, and RISCs can be oxidized to sulfuric acid by sulfur-oxidizing bacteria. Thus, minerals get dissolved [3]. Comparing the two ways, contact leaching is seen to be more efficient. This may be because extracellular polymeric substances (EPS), secreted by bacteria, accumulate ferric ions via complexation and decrease the reaction distance between the cells and the mineral surface [4]. During the last several decades, bioleaching has already been applied worldwide industrially for recovering metals from low grade ores [5]. Instead of a sole species, microbial populations exhibited substantial diversity in stirred-tank, mineral-processing bioreactors and ore leaching heaps. Microbial community analysis of the leachate has shown that the microbial bioleaching communities varied in commercial mineral leaching operations, but the genus of Sulfobacillus can be detected frequently [6–8]. The genus Sulfobacillus was first reported by Golovacheva and Karavaiko in 1978 [9]. Until now, there have been five species classified into this genus based on phylogenetic and physiological characteristics: Sb. thermosulfidooxidans, Sb. acidophilus, Sb. thermotolerans, Sb. sibiricus and Sb. benefaciens. Among them Sb. thermosulfidooxidans is the most studied species. The sulfobacilli are Gram-positive, generally non-motile, rod shaped, endospore-forming and moderately thermophilic acidophiles. They can oxidize ferrous iron, RISCs such as tetrathionate, thiosulfate and elemental sulfur, as well as sulfide minerals in the presence of 0.02% yeast extract.

Minerals 2016, 6, 71; doi:10.3390/min6030071 www.mdpi.com/journal/minerals Minerals 2016, 6, 71 2 of 10

Because of the versatile metabolism and tolerance to a hostile environment characterized by low pH and high metal concentrations, the genus Sulfobacillus has been considered for optimization of microbial consortia [10], and they are crucial in mixed cultures during leaching. It is reported that the smaller proportion of Sulfobacillus in leaching consortia under continuous conditions was correlated with the reduction of chalcopyrite leaching kinetics than in batch mode. Spolaore et al. [11] compared chalcopyrite leaching efficiency of an undefined leaching consortia under continuous condition with that in batch mode. The results show that the leaching kinetics in the continuous test were lower than in the batch test, which can be attributed to a smaller proportion of Sulfobacillus, in agreement with Foucher et al. [12]. Extensive oxidization of metal sulfides by biofilms of Sb. thermosulfidooxidans was indicated by the etch pits appearing within 94 h on pyrite surfaces [13]. All of these studies indicate that the biofilm formation by Sulfobacillus probably leads to a high mineral dissolution rate. Thus, it is worth studying the adhesion of Sulfobacillus to mineral surfaces and enhancing their biofilm formation on them. Initial adhesion of planktonic cells to a surface is the first step in biofilm formation [14]. It is induced by biological and physicochemical interactions between cells and substratum [15,16]. Cells interact with the environment by their surface, and the environment affects cell surface properties. The energy source can influence the chemical composition of EPS and cause changes of cell surface hydrophobicity and charge. As a consequence, bacterial cells grown on different energy sources exhibit different attachment ability to the same substratum [4,17]. In view of the effect from the culturing environment, five strategies were chosen to test their effects on enhancement of biofilm formation by Sb. thermosulfidooxidans DSM 9293T on pyrite: changing medium pH, supplementing an extra energy source, adding ferric ions, phosphate starvation and exchanging exhausted medium with fresh medium. Epifluorescence microscopy (EFM), confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) were used for monitoring cell attachment and biofilm formation as well as microorganism-pyrite interface structures. This study first demonstrates a method for enhancing biofilm formation by Gram-positive acidophiles and provides a hint of interfacial interactions between these bacteria and pyrite.

2. Materials and Methods

2.1. Strain and Growth Conditions Sb. thermosulﬁdooxidansT was grown on 3% pyrite (m/v) with 0.02% yeast extract in a medium at pH 2.5, the medium was prepared according to Mackintosh et al. [18]. The culture was incubated at 45 ˝C on a rotary shaker at 150 rpm. Cells were harvested by centrifugation at 11,270 g for 10 min. Cell numbers were determined by directly counting with a Thoma chamber (Assistent®, Sondheim v. d. Rhön, Germany).

2.2. Preparation of Pyrite Grains and Pyrite Slices Pyrite grains with a size of 100–200 µm were used after wet-sieving ﬂoatation pyrite from Romania. Pyrite slices were cut off from pyrite cubes with a size of 1 cm ˆ 1 cm ˆ 1 cm (Museum grade, Freiburg, Germany). These pyrite cubes were naturally crystallized, and their six sides were smooth and shiny. After cutting, slices with one shiny side and one rough side were obtained, and this study only focused on the shiny side. No clear cell-shaped pits were observed by atomic force microscopy (AFM) (Supplementary Materials, Figure S1). Pyrite grains and slices were cleaned and sterilized as described before [19].

2.3. Strategies for Enhancing Bioﬁlm Formation

Different initial medium pH was adjusted with 1 M H2SO4 or KOH. In addition, 10 mM ferric chloride, 50 mM potassium tetrathionate, and sodium thiosulfate were prepared as a standard solution and sterilized by ﬁltration. Phosphate starvation was conducted by using phosphate free MAC medium. Minerals 2016, 6, 71 3 of 10

Medium replacement was performed by exchanging fresh MAC medium containing 0.02% yeast extract with exhausted medium every two days. The experiments were conducted in 300 mL ﬂasks with 3% pyrite in 150 mL MAC medium. The initial cell density for all experiments was 2 ˆ 108 cells/mL, which was achieved by inoculating a calculated volume of the cell suspension. The cells were inoculated prior to pyrite addition, and the precise initial cell density could be determined. Regular medium exchange without cell inoculation was set as an abiotic control. All assays were done in triplicate.

2.4. AFM, EFM and CLSM For visualization of the cell distribution on pyrite surfaces, nucleic acid staining was applied. The staining procedures described as follows. Pyrite grains and pyrite slices were first rinsed with sterile MAC medium and then with MilliQ water (Merck Millipore, Schwalbach, Germany). Afterwards, they were immersed in 6 µM SYTO® 9 for 15 min. Another rinsing with MilliQ water three times was done after staining in order to remove unbound dye. A laser scanning module (LSM 510 Carl Zeiss® Jena, Jena, Germany) combined with an inverted Axiovert100 M BP microscope (Zeiss®, Hallbergmoos, Germany) was used for biofilms visualization. MosaiX module was especially applied for visualizing biofilm development on pyrite slices. This program can provide information in one image by integrating several images. In this study, 25 (5 ˆ 5) images were integrated. In order to further observe architecture of biofilms, a NanoWizard II AFM (JPK Instruments, Berlin, Germany) in contact mode in liquid was used. Experiments were conducted in pH 2.5 MAC medium. A CSC38/NO AL (Mikromasch, Tallinn, Estonia) probe was also used and cantilever B with the following parameters were chosen for scanning: typical length, 350 µm; width, 32.5 µm; thickness, 1.0 µm; resonance frequency, 10 kHz; and force constant, 0.03 N/m. The applied setpoint was 1 V and the scan rate was 0.5 Hz.

3. Results and Discussion

3.1. Biofilm Formation of Sb. thermosulfidooxidans on Pyrite under Standard Conditions After 4 h of incubation, only 30% of the cells attached to pyrite grains (Figure1A). This indicates a low attachment ability of Sb. thermosulfidooxidans to pyrite. A few cells can be visualized on pyrite grain surface after 1 d incubation, and their distribution was heterogeneous (Figure1B). It became more and more difficult to find attached cells in the following days, and, after 20 d, no attached cells remained (Figure1C,D). The same results were obtained when pyrite slices were used as substratum (Figure2). Although there were many cells or big flocs on the surface after 1 d incubation, cells were all detached after 14 d incubation. A lack of motility might be one reason for the low amounts of attached cells and the badly formed biofilm on pyrite. Redistribution of attached cells by surface motility, binary division of attached cells, and recruitment of cells from the bulk fluid are the mechanisms of biofilm formation [14]. It is well known that leaching bacteria prefer to colonize on imperfections of mineral surface, where the crystal structure can be easily destroyed [3,20]. Because of a lack of motility, Sb. thermosulfidooxidans attached to the pyrite surface by chance, thus not all the attached sites were suitable for further colonization. Cell detachment might occur, which leads to the poor biofilm formation. In addition, many publications emphasized the importance of motility for the biofilm formation [21,22]. When cells were incubated with pyrite slices, large flat surfaces and reduced shearing forces provide a chance to facilitate their attachment on the surface. However, these cells detach during the course of the experiment, and, finally, almost no cells were detectable. Minerals 2016, 6, 71 4 of 10 Minerals 2016, 6, 71 4 of 10 Minerals 2016, 6, 71 4 of 10

Figure 1. Amounts of attached cells of Sb. thermosulfiooxidans to pyrite grains within 4 h (A) and their Figure 1. AmountsAmounts of of attached attached cells cells of ofSb.Sb. thermosulfiooxidans thermosulﬁooxidans to pyriteto pyrite grains grains within within 4 h (A 4) h and (A) their and biofilm development on pyrite grains after one day (B); 20 days (C) and 40 days (D) incubation. Scale theirbiofilm bioﬁlm development development on pyrite on pyrite grains grains after one after day one (B day); 20 ( Bdays); 20 ( daysC) and (C 40) and days 40 ( daysD) incubation. (D) incubation. Scale bar, 20 µm. Scalebar, 20 bar, µm. 20 µm.

Figure 2. Biofilms of Sb. thermosulfiooxidans on pyrite slice after one day (A); seven days (B) and Figure 2. Biofilms of Sb. thermosulfiooxidans on pyrite slice after one day (A); seven days (B) and 14Figure days 2.(CBiofilms) incubation. of Sb. Scale thermosulfiooxidans bar, 20 µm. on pyrite slice after one day (A); seven days (B) and 14 days (14C )days incubation. (C) incubation. Scale bar, Scale 20 µ bar,m. 20 µm. 3.2. Effect of Different Strategies on Enhancement of Biofilm Formation by Sb. thermosulfidooxidans on Pyrite 3.2. Effect of Different Strategies on Enhancement of Biofilm Formation by Sb. thermosulfidooxidans on Pyrite 3.2. Effect of Different Strategies on Enhancement of Biofilm Formation by Sb. thermosulfidooxidans on Pyrite The initial medium pH can regulate sporulation. Even changes of one unit of pH may increase The initial medium pH can regulate sporulation. Even changes of one unit of pH may increase sporulationThe initial [23]. medium Sb. thermosulfidooxidans pH can regulate was sporulation. reported Evento have changes the best of oneferrous unit ion of pHoxidation may increase at pH sporulation [23]. Sb. thermosulfidooxidans was reported to have the best ferrous ion oxidation at pH 1.4.sporulation A significant [23]. Sb.reduction thermosulfidooxidans of ferrous ionwas oxidation reported rate to havewas observed the best ferrous if the pH ion varied oxidation in the at pHrange 1.4. 1.4. A significant reduction of ferrous ion oxidation rate was observed if the pH varied in the range ofA significant1.0–3.0 [24]. reduction Thus, we of studied ferrous and ion compared oxidation the rate biofilm was observed formation if the of pHSb. thermosulfidooxidans varied in the range at of of 1.0–3.0 [24]. Thus, we studied and compared the biofilm formation of Sb. thermosulfidooxidans at initial1.0–3.0 pH [24 1.5]. Thus, and 3.5. we studiedThe epifluorescence and compared microsco the biofilmpy (EFM) formation images of Sb. (Figure thermosulfidooxidans 3A,B) indicateat that initial no initial pH 1.5 and 3.5. The epifluorescence microscopy (EFM) images (Figure 3A,B) indicate that no cellspH 1.5 were and attached 3.5. The on epifluorescence pyrite grains microscopyeither at pH (EFM) 1.5 or images3.5 after (Figure one week.3A,B) Although indicate that some no green cells weredots cells were attached on pyrite grains either at pH 1.5 or 3.5 after one week. Although some green dots onattached pyrite onsurface pyrite were grains detectable, either at when pH 1.5 cells or 3.5 were after grown one week. at pH Although1.5, these somedots were green most dots likely on pyrite not on pyrite surface were detectable, when cells were grown at pH 1.5, these dots were most likely not cellssurface as indicated were detectable, by shape when and cells size. were The grownfinding at demonstrates pH 1.5, these that dots variation were most of initial likely notpH cellscannot as cells as indicated by shape and size. The finding demonstrates that variation of initial pH cannot stimulateindicated cell by shapeadhesion and and size. biofilm The finding formation. demonstrates that variation of initial pH cannot stimulate stimulate cell adhesion and biofilm formation. cell adhesion and biofilm formation.

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Figure 3. Biofilms of Sb. thermosulfiooxidans on pyrite grains after one week of incubation with initial Figure 3. Biofilms of Sb. thermosulfiooxidans on pyrite grains after one week of incubation with initial pH pH 1.5 (A) or 3.5 (B); or with 2 mM K2S4O6 (C); 2 mM Na2O3S2·5H2O (D) or 1 mM FeCl3 (E) 1.5 (A) or 3.5 (B); or with 2 mM K S O (C); 2 mM Na O S ¨ 5H O(D) or 1 mM FeCl (E) supplanted, supplanted, or under condition2 of4 phosphate6 starvation2 (3F).2 Scale2 bar, 20 µm. 3 or under condition of phosphate starvation (F). Scale bar, 20 µm. A recent study indicated that not planktonic but biofilm cells of Acidithiobacillus ferrooxidansT Amade recent contributions study indicated to pyrite that dissolution not planktonic within the but first biofilm 4–5 days cells after of Acidithiobacillus inoculation [25]. ferrooxidans Accordingly,T made contributionswe may speculate to pyrite that dissolution biofilms withinof Sb. thermosulfidooxidans the first 4–5 days on after pyrite inoculation grains did [25 not]. Accordingly,have a sufficient we may speculatesupply that of biofilmsenergy, which of Sb. resulted thermosulfidooxidans in starvation triggeringon pyrite sporulation grains did notor dormancy. have a sufficient Thus, 2 supplymM of tetrathionate or thiosulfate were supplemented as extra energy source. However, neither of them energy, which resulted in starvation triggering sporulation or dormancy. Thus, 2 mM tetrathionate or showed a positive effect on enhancement of biofilm formation (Figure 3C,D). thiosulfateFerric were ions supplemented contribute to leaching as extra as energy oxidizing source. agent However, [26], but also neither play a ofrole them in initial showed adhesion. a positive effectThe on complexed enhancement iron ofions biofilm in EPS formationprovide a positive (Figure charge3C,D). on the cell surface inducing cells to adhere Ferricto negatively ions contribute charged pyrite to leaching surfacesas via oxidizing electrostatic agent attract [26 ],forces but also[3,27]. play It has a role been in shown initial that adhesion. a The complexedsupplement ironof 1 ionsmM inferric EPS ions provide causes a a positive 10 fold chargeincrease on of thethe cellcolonization surface inducingof pyrite by cells cells to of adhere to negativelyA. ferrooxidans charged R1 [25]. pyrite In our surfaces study, ferric via electrostatic ions did not increase attract forcesadhesion [3 ,27of ].Sb. It thermosulfidooxidans has been shown that a supplementon and colonization of 1 mM of ferric pyrite ions surfaces causes (Figure a 10 fold3E). increaseThis could of be the attributed colonization to low ofamounts pyrite of by the cells of A. ferrooxidansferric ironR1 complexing [25]. In our uronic study, acids ferric in ionsthe tightly-bound did not increase EPS adhesion of cells of of Sb.Sb. thermosulfidooxidans thermosulfidooxidans on and colonization(unpublished ofdata). pyrite surfaces (Figure3E). This could be attributed to low amounts of the ferric It is accepted that EPS are a prerequisite for leaching bacteria to attach to solid substrata such as iron complexing uronic acids in the tightly-bound EPS of cells of Sb. thermosulfidooxidans (unpublished data). pyrite or elemental sulfur [4]. Thus, inducing EPS production may enhance cell adhesion. ItPhosphorus is accepted is a thatvery EPSimportant are a element prerequisite for organisms, for leaching but the bacteria response to attachof bacteria to solidto limited substrata suchphosphate as pyrite orcan elemental be different. sulfur Phosphorus [4]. Thus, inducinglimitation EPS can production increase attachment may enhance in cellcase adhesion. of PhosphorusAgrobacterium is a verytumefaciens important [28]. elementBellenberg for et organisms,al. also described but the that response growth ofunder bacteria phosphate to limited phosphatestarvation can conditions be different. resulted Phosphorus in an enhancement limitation of can EPS increase production attachment and well-formed in case of biofilmsAgrobacterium of tumefaciensA. ferrooxidans[28]. Bellenberg [29]. However, et al. Pseudomonas also described fluorescens that growth detached under from phosphate its substratum starvation surface conditionswhen resultedgrowing in an under enhancement low phosphate of EPSconditions production [30]. From and our well-formed own images biofilms(Figure 3F), of itA. is obvious ferrooxidans that [29]. However,after onePseudomonas week of incubation fluorescens underdetached phosphate from star itsvation substratum conditions, surface no well-formed when growing biofilms under exist low phosphateon the conditionspyrite grain [30surfaces]. From and our only own a few images single (Figurecells occur,3F), itwhich is obvious indicates that that after phosphate one week starvation does not significantly enhance biofilm formation. of incubation under phosphate starvation conditions, no well-formed biofilms exist on the pyrite Exchanging exhausted medium with fresh medium is the only strategy showing a positive graineffect surfaces (Figure and 4). only This a enhancement few single cells was occur,reproducible which when indicates pyrite that slices phosphate were applied starvation (Figure does 5). not significantlyHowever, enhance the architecture biofilm formation.of biofilms grown on pyrite slices is different from the one grown on Exchangingpyrite grains. exhaustedComparing mediumthe images, with we can fresh see medium that biofilms is the grown only on strategy a pyrite showing slice display a positive a effectmultiple (Figure 4layer). This structure enhancement (Figure 6), was while reproducible biofilms grown when on pyrite pyrite slices grains were display applied monolayer (Figure 5). However,structure the (Figure architecture 4B,C). Strong of biofilms shear forces grown in onthe pyritecase of slicespyrite grains is different may be from the main the onereason grown for on pyritethe grains. different Comparing biofilm phenotype. the images, we can see that biofilms grown on a pyrite slice display a multipleEnhancement layer structure of biofilm (Figure formation6), while by biofilms Sb. thermosulfidooxidans grown on pyrite due grains to exchanging display monolayer spent medium structure may be due to several reasons. One is that the organic supplement facilitates the growth of (Figure4B,C). Strong shear forces in the case of pyrite grains may be the main reason for the different biofilm phenotype. Enhancement of biofilm formation by Sb. thermosulfidooxidans due to exchanging spent medium may be due to several reasons. One is that the organic supplement facilitates the growth of Minerals 2016, 6, 71 6 of 10

Minerals 2016, 6, 71 6 of 10 Sb. thermosulfidooxidans. It is well known that Sulfobacilli have a mixotrophic metabolism on Fe2+, Sb. thermosulfidooxidans. It is well known that Sulfobacilli have a mixotrophic metabolism on Fe2+, S0, 0 Minerals2´ 2016, 6, 712´ 6 of 10 S ,S4O2−6 ,S22−O3 and sulfide minerals in the presence of low amounts of organic compounds. S4O6 , S2O3 and sulfide minerals in the presence of low amounts of organic compounds. Their Their autotrophic and heterotrophic growth are poor. The dramatic decrease of planktonic cell autotrophicSb. thermosulfidooxidans and heterotrophic. It is well growth known are that poor. Sulfobacilli The dramatic have decreasea mixotrophic of planktonic metabolism cell on numbers Fe2+, S0, of numbers of subcultures of 1 d biofilms of Sb. thermosulfidooxidans (Figure7) after 50 h may also indicate subculturesS4O62−, S2O of32− 1and d biofilms sulfide ofminerals Sb. thermosulfidooxidans in the presence of (Figure low amounts 7) after of50 organic h may alsocompounds. indicate Theirthat the thatconsumption theautotrophic consumption andof the heterotrophic of yeast the yeastextract growth extract caused are caused poor.the decre theThe decreasedramaticase in cell decrease in celldensity. density. of planktonic When When fresh cell fresh numbersmedium medium ofwas was supplied,supplied,subcultures growth growth of continued.1 continued.d biofilms Theof The Sb. second secondthermosulfidooxidans possibilitypossibility is is(Figure the the removal removal 7) after of 50 of harmful h harmful may also factors factors indicate in inthe that the growth the growth medium,medium,consumption thereby thereby of ensuring ensuringthe yeast biofilm biofilmextract formation.formation.caused the A decre hi highghase cell cell in densitycell density density. may may Whentrigger trigger fresh sporulation sporulation medium and was and the the formationformationsupplied, of of persister growth persister continued. cells cells [31 [31].]. The second possibility is the removal of harmful factors in the growth medium, thereby ensuring biofilm formation. A high cell density may trigger sporulation and the formation of persister cells [31].

Figure 4. Biofilms of Sb. thermosulfiooxidans on pyrite grains after one day (A); 20 days (B); 40 days (C) Figure 4. Bioﬁlms of Sb. thermosulﬁooxidans on pyrite grains after one day (A); 20 days (B); 40 days andFigure 60 days 4. Biofilms (D) incubation. of Sb. thermosulfiooxidans Scale bar, 20 µm. on pyrite grains after one day (A); 20 days (B); 40 days (C) (C) andand 60 60 daysdays (D) incubation. incubation. Scale Scale bar, bar, 20 20µm.µ m.

Figure 5. Biofilms of Sb. thermosulfiooxidans on pyrite slice after one month (A); five months (B) and FigureFigure 5. 5.Biofilms Biofilms of ofSb. Sb. thermosulfiooxidans thermosulfiooxidans on pyrite slice slice after after one one month month (A (A); );five five months months (B ()B and) and 10 months (D) incubation; (C) shows the enlarged area from the frame in (B); (E) is the cross-section 1010 months months (D ()D incubation;) incubation; ( C(C)) shows shows the the enlargedenlarged area from the the frame frame in in (B (B);); (E (E) is) is the the cross-section cross-section view of surface topography in the area indicated by the red dashed line in (C); Formation of pits up view of surface topography in the area indicated by the red dashed line in (C); Formation of pits up viewto of 35 surface µm in topographydepth can be inseen the inarea (E); Scale indicated bar in by(A, the B and red D dashed) is 100 µm line and in( inC); (C Formation) is 20 µm. of pits up to 35 toµm 35 in µm depth in depth can becan seen be seen in ( Ein); ( ScaleE); Scale bar bar in (inA ,(BA,and B andD) D is) 100is 100µm µm and and in in (C (C) is) is 20 20µ µm.m.

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Figure 6. 3D3D AtomicAtomic force force microscopy microscopy images images showing showing the biofilmsthe biofilms of Sb. of thermosulfiooxidans Sb. thermosulfiooxidanson pyrite on pyritesliceFigure after slice 6. one3D after Atomic month one month ( Aforce); three (microscopyA); months three months ( Bim) andages (B five ) showingand months five monthsthe (C )biofilms incubation. (C) incubation. of Sb. thermosulfiooxidans on pyrite slice after one month (A); three months (B) and five months (C) incubation.

Figure 7. Cell numbers of subcultures of one day biofilms of Sb. thermosulfiooxidans on pyrite in suspension. FigureFigure 7. Cell 7. numbersCell numbers of subcultures of subcultures of one day of one biofilms day biofilmsof Sb. thermosulfiooxidans of Sb. thermosulfiooxidans on pyrite inon suspension. pyrite 3.3. Bioleaching of Sb. thermosulfidooxidans under Biofilm-Favoring Conditions in suspension. 3.3. BioleachingBioleaching of efficiencySb. thermosulfidooxid of Sb. thermosulfidooxidansans under Biofilm-Favoring, when the Conditions medium was regularly exchanged, was3.3. BioleachingevaluatedBioleaching by of efficiency Sb.comparing thermosulfidooxidans of with Sb. thermosulfidooxidans the results under of Biofilm-Favoring cultivation, when under the Conditions medium standard was conditions regularly (without exchanged, any exchange). Figure 8A shows the cell growth and the leached iron as a function of time under was evaluatedBioleaching by efficiency comparing of withSb. thermosulfidooxidans the results of cultivation, when under the medium standard was conditions regularly (without exchanged, any standard conditions. A fluctuation of cell numbers was observed in the first 2 d, indicating cell wasexchange). evaluated Figure by comparing8A shows the with cell the growth results and of cultivation the leached under iron standardas a function conditions of time (without under attachment and detachment. Afterwards, the cell density kept decreasing, probably related to the anystandard exchange). conditions. Figure A8 Afluctuation shows the of cell cell growth numbers and was the leachedobserved iron in asthe a first function 2 d, ofindicating time under cell reduced organic nutrition. The concentration of iron ions remained constant during the first 7 d of standardattachment conditions. and detachment. A fluctuation Afterwards, of cell the numbers cell density was observedkept decreasing, in the firstprobably 2 d, indicatingrelated to cellthe the experiment. The largest iron leaching rate was measured between day 7 and 18. Afterwards, the attachmentreduced organic and detachment.nutrition. The Afterwards, concentration the of cell iron density ions remained kept decreasing, constant probably during the related first to7 d the of iron ion concentration remained constant again, which indicates that the bioleaching stopped. In reducedthe experiment. organic The nutrition. largest Theiron concentrationleaching rate was of iron measured ions remained between constant day 7 and during 18. Afterwards, the first 7 d the of total, 195 mg iron ions were leached within 35 days. Figure 8B,C show the cell density and the theiron experiment. ion concentration The largest remained iron co leachingnstant again, rate was which measured indicates between that the day bioleaching 7 and 18. Afterwards,stopped. In concentration of iron ion in the spent medium before exchanging with fresh medium, respectively. thetotal, iron 195 ion mg concentration iron ions were remained leached constant within 35 again, days. which Figure indicates 8B,C show that the bioleachingcell density stopped.and the The results indicate that the cell numbers in the leachate started to decrease considerably from day Inconcentration total, 195 mg of ironiron ionsion in were the leachedspent medium within be 35fore days. exchanging Figure8B,C with show fresh the medium, cell density respectively. and the 18, but the concentration of iron ion decreased only slowly. Only 4.1 mg iron ions in total were concentrationThe results indicate of iron that ion the in thecell spentnumbers medium in the before leachate exchanging started to with decrease fresh considerably medium, respectively. from day dissolved within 35 d under these conditions, whereas, under the standard conditions, 195 mg iron The18, but results the indicateconcentration that the of cell iron numbers ion decreased in the leachate only slowly. started Only to decrease 4.1 mg considerably iron ions in from total day were 18, ions in total were dissolved. It is noticeable that the bioleaching stopped under the standard butdissolved the concentration within 35 d ofunder iron ionthese decreased conditions, only wherea slowly.s, Onlyunder 4.1 the mg standard iron ions conditions, in total were 195 dissolved mg iron conditions after 18 d, whereas bioleaching continued if the medium kept being exchanged. In withinions in 35 total d under were these dissolved. conditions, It is whereas, noticeable under that the the standard bioleaching conditions, stopped 195 under mg iron the ions standard in total addition, iron precipitates formed and covered the surface of biofilms and minerals (unpublished wereconditions dissolved. after It18 is noticeabled, whereas that bioleaching the bioleaching continued stopped if the under medium the standard kept being conditions exchanged. after 18 In d, data). The degree of oxidation also provides information about the leaching efficiency. As it shown whereasaddition, bioleachingiron precipitates continued formed if the and medium covered kept the beingsurface exchanged. of biofilms In and addition, minerals iron (unpublished precipitates in Figure 5D,E, many pits on the pyrite slices incubated for 10 months can be seen. The confocal laser formeddata). The and degree covered of theoxidation surface also of biofilms provides and information minerals (unpublished about the leaching data). Theefficiency. degree As of oxidationit shown scanning microscopy images of the pyrite slice surfaces indicate an average depth of the pits of alsoin Figure provides 5D,E, information many pits on about the thepyrite leaching slices efficiency.incubated Asfor it10 shown months in can Figure be seen.5D,E, The many confocal pits on laser the 25 ± 9 µm. It is reported that after four months of bioleaching by A. ferrooxidans, many etch pits with pyritescanning slices microscopy incubated images for 10 monthsof the pyrite can be slice seen. surf Theaces confocal indicate laser an scanningaverage depth microscopy of the imagespits of an average depth of 320 nm occurred [32]. Sulfobacilli have a high leaching efficiency if the pit of25 the± 9 µm. pyrite It is slice reported surfaces that indicate after four an months average of depth bioleaching of the pitsby A. of ferrooxidans 25 ˘ 9 µm., many It is reportedetch pits with that formation rates are compared with those of A. ferrooxidans: 2500 nm/month versus 80 nm/month. In afteran average four months depth ofof bioleaching320 nm occurred by A. ferrooxidans [32]. Sulfobaci, manylli have etch pitsa high with leaching an average efficiency depth ofif 320the nmpit the abiotic control, pits with a depth lower than 2 µm were measured (Supplementary Materials, occurredformation [32 rates]. Sulfobacilli are compared have with a high those leaching of A. efficiencyferrooxidans if: the 2500 pit nm/month formation versus rates are 80 compared nm/month. with In Figurethe abiotic S1). control, pits with a depth lower than 2 µm were measured (Supplementary Materials, Figure S1).

Minerals 2016, 6, 71 8 of 10

those of A. ferrooxidans: 2500 nm/month versus 80 nm/month. In the abiotic control, pits with a depth Mineralslower than2016, 6 2, 71µm were measured (Supplementary Materials, Figure S1). 8 of 10

FigureFigure 8. 8. CellCell numbers numbers of of Sb.Sb. thermosulfiooxidans thermosulﬁooxidans andand leached leached iron iron concentration concentration as as function function of of time time duringduring bioleaching bioleaching under under standard standard conditions conditions ( (AA)) or or regularly regularly changing changing medium medium ( (BB andand CC).).

4.4. Conclusions Conclusions TheThe low low initial initial adhesion adhesion rate rate of Sb. thermosulfidooxidans thermosulfidooxidans toto pyrite pyrite and and its its fast fast detachment detachment demonstratedemonstrate thatthat thisthis organism organism is notis anot good a biofilmgood biofilm former underformer the under standard the conditions.standard Strategiesconditions. of Strategieschanging of the changing initial pH the of initial the medium, pH of the supplementing medium, supplementing an extra energy an extra source, energy adding source, ferric adding ions or ferricphosphate ions or starvation phosphate do starvation not show do a not significantly show a significantly positive effect positive on biofilm effect on formation. biofilm formation. However, However,if regularly if exchangingregularly exchanging exhausted exhausted medium withmedium fresh with medium, fresh aftermedium, one monthafter one of incubation,month of incubation,robust biofilms robust can biofilms be observed can be on observed pyrite surfaces, on pyrite and surfaces, bioleaching and bioleaching of pyrite continues. of pyrite Incontinues. addition, Insubstratum addition, substratum size has an effectsize has on an the effe architecturect on the architecture of the biofilms. of the biofilms.

SupplementarySupplementary Materials: Materials: TheThe following following are are available available online online at at www.mdpi.com/2075-163X/6/3/71/s1. www.mdpi.com/2075-163X/6/3/71/s1. Acknowledgments: Qian Li gratefully acknowledges the China Scholarship Council (CSC) for financial funding. Acknowledgments: Qian Li gratefully acknowledges the China Scholarship Council (CSC) for financial We especially thank the two anonymous reviewers for their constructive suggestions that improved this funding.contribution We especially considerably. thank the two anonymous reviewers for their constructive suggestions that improved this contribution considerably. Author Contributions: Qian Li performed the laboratory tasks and carried out the experiments. Qian Li wrote Authorthe manuscript. Contributions: Wolfgang Qian Sand Li performed and Ruiyong the Zhanglaboratory assisted tasks in and preparing carried theout manuscript,the experiments. and all Qian authors Li wrote read theand manuscript. approved the Wolfgang final version. Sand and Ruiyong Zhang assisted in preparing the manuscript, and all authors read andConflicts approved of Interest: the finalThe version. authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References

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