Electrochemical Characteristics of the Oxidation of Sulfur- and Iron-Containing Compounds by Acidophilic Microorganisms

Abstract

The electrochemical features of the interactions of sulfur- and iron-containing compounds (ferrous sulfate, elemental sulfur, pyrite tailings, cysteine, sodium thiosulfate) with a model acidophilic consortium, including the genera Leptospirillum, Sulfobacillus, Acidithiobacillus, Ferroplasma, and Acidiplasma, were studied. The method of cyclic voltammetry recorded redox processes at the electrode/solution interface in the presence of the studied sulfur- and iron-containing compounds. In general, the modeling consortium led to the intensification of these processes. The characteristics of the diffuse layer near the electrode/solution interface were studied using electrochemical impedance spectroscopy. The introduction of microorganisms and/or substrates into the supporting electrolyte led to a decrease in the slope and can be interpreted as evidence of their effect on the diffusion part of the double layer. Its contribution decreases in favor of ion transport. All this, in general, does not contradict the assumption of cell adsorption on the electrode surface. Confocal laser scanning microscopy confirmed this assumption and showed cell adhesion to the surface. The data obtained confirm the importance of bioelectrochemical processes of the studied group of microorganisms in biotechnological processes associated with the leaching of metals from sulfide ores.

Introduction

The cost of extracting metals from low-grade and off-balance sulfide ores, as well as from mining waste using traditional technologies (enrichment and pyrometallurgical processing) is often too high to be profitable. Microbiological leaching is one of the promising alternatives to traditional methods of extracting metals from such raw materials [1–3]. In the by-leaching process carried out by acidophilic microorganisms, metals are released into solution due to the destruction of sulfide minerals by enzymes. At the same time, redox reactions are catalyzed with the participation of various metals and sulfur compounds. These properties of acidophiles underlie biohydrometallurgy. There is a lot of uncertainty about the mechanisms of interaction between microorganisms and sulfide minerals [4–6].

There are several hypotheses about the interaction of minerals and microbial cells. But it is obvious that the biooxidation of sulfide minerals is a complex bioelectrochemical process. In it, the biooxidation of iron and sulfur compounds, catalyzed by microorganisms, is of paramount importance. In this case, the adhesion of microbial cells to the surface of minerals also plays a significant role. In addition, the rate of biooxidation of sulfide minerals is determined by the interaction between various minerals that are always simultaneously present in natural ores. [4–8]. It should also be noted that acidophilic microorganisms involved in the oxidation of sulfide minerals include species with different metabolic abilities (oxidation and reduction of iron and sulfur compounds in combination with carbon dioxide fixation or oxidation of organic compounds). Acidophiles are able to grow in a wide range of conditions (for example, temperature and salinity) and carry out the biooxidation of sulfide ores and concentrates under various technological parameters [9–12].

Understanding the mechanisms of the bioelectrochemical interaction of acidophilic microorganisms with sulfide minerals and the role of their adhesion in the intensity of these processes is extremely important in studying the ability of these microorganisms to carry out bioleaching. Research in this area should help develop the theoretical foundations for the use of acidophiles in biohydrometallurgy. The purpose of this work was to study the electrochemical processes of oxidation of sulfur- and iron-containing compounds by acidophilic microorganisms.

Materials and Methods

Strains and Culture Conditions

A mixed culture of acidophilic microorganisms was used as a bioagent. It included the strains Leptospirillum ferriphilum INMI 2010, Sulfobacillus thermosulfidooxidans SH-1, Sulfobacillus thermotolerans Kr1T, Acidithiobacillus thiooxidans INMI 2017, Acidithiobacillus caldus MBC-1, Ferroplasma acidiphilum Y1T, Acidiplasma sp. MBA-1, Acidithiobacillus ferrooxidans TFBK. which were in a 1:1 ratio in equal volumes of eight cultures. The strains were provided by Ph.D. Bulaev A.G. (Laboratory of Chemolithotrophic Microorganisms, Scientific Research Center for Biomedical Research, Russian Academy of Sciences, Moscow, Russia). Some properties of strains are presented in Table 1.

Table 1.

Physiological and biochemical properties of acidophilic strains included in a mixed culture

Strain	Property							Source of isolation
	Morphology	Topt (Tmin−Tmax), °C	pHopt (pHmin−pHmax)	Oxidized inorganic substrate	Carbon nutrition	Fe3+ reduction	Resistance to NaCl, mM
Leptospirillum ferriphilum INMI 2010	Comma-shaped cells, spirilla	35–37 (45)	1.3–1.8 (n.d.)	Fe2+	Autotroph	−	n.d	Reactors for biooxidation of pyrite-arsenopyrite-pyrrhotite concentrate [13]
Acidithiobacillus ferrooxidans TFBK	Rods	25–35 (37)	2.0–2.2	Fe2+, S	Autotroph	+	> 347	Pyrite-arsenopyrite ore sample [14]
Acidithiobacillus thiooxidans INMI 2017	Rods	38 (40)	n.d	S	Autotroph	−	n.d	Reactors for biooxidation of pyrite-arsenopyrite-pyrrhotite concentrate [13]
Acidithiobacillus caldus MBC-1	Rods	45 (53)	n.d	S	Autotroph	−	n.d	Reactors for biooxidation of pyrite-arsenopyrite concentrate [15]
Sulfobacillus thermotolerans Kr1T	Rods	40 (20–60)	2.0–2.5 (1.2–5.0)	Fe2+, S	Mixotroph	+	250	Reactors for biooxidation of pyrite-arsenopyrite concentrate [16]
Sulfobacillus thermosulfidooxidans SH-1	Rods	50 (55)	1.5–1.7	Fe2+, S	Mixotroph	+	100	Sample of copper-nickel ore [17]
Ferroplasma acidiphilum Y1T	Pleomorphic cells	35 (15–45	1.7 (1.3–2.2)	Fe2+	Mixotroph	+	n.d	Reactors for biooxidation of pyrite-arsenopyrite concentrate [18]
Acidiplasma sp. MBA-1	Pleomorphic cells	50–55 (15–65)	0.8–1.2 (0.0–2.0)	Fe2+, S	Heterotroph	+	250	Reactors for biooxidation of pyrite-arsenopyrite concentrate [15]

Microbial strains selected for the study are representatives of acidophilic bacteria and archaea, which predominate in microbial communities associated with acid mine drainages (AMD) formation [19–22], mining wastes oxidation [23, 24] as well as in biotechnological processes of sulfide ore and concentrate oxidation [13–18]. As mixed culture included strains with different physiological properties (temperature and pH optima, capability to oxidize different inorganic compounds and utilize different carbon sources), it had potential to adapt to various conditions that in turn may allow performing technological processes in required mode depending on necessity (presence of different inorganic and organic substances, as well as wide range of environmental conditions).

Experiments and cultivation of microorganisms were carried out in Leathen’s medium containing (mg/L): (NH4)₂SO₄ −750; KCl—50; MgSO₄ × 7H₂O—120; K₂HPO₄—120. The initial pH value adjusted to 10 N H₂SO₄ was 2.0 [25]. In different variants of the experiment, iron sulfate, elemental sulfur, pyrite tailings, cysteine, sodium thiosulfate (20 g/L of each compound) were added to the medium. In one of the variants of the experiment, a mixture of the above components was also used at a concentration of 20 g/L (i.e., 4 g/L of each component). Pyrite tailings were obtained from the Uchalinsky Mining and Processing Plant (Tables 2).

Table 2.

Chemical and mineral composition of pyrite tailings

	Fe	Cu	Zn	Stotal	Ssulfate	S0	Ssulfide
Element
Content, %	29.5	0.12	0.25	29.2	3.5	0.3	25.4

	Quartz	Feldspar	Chlorites	Pyrite	Gypsum	Chalcopyrite	Sphalerite
Mineral
Content, %	29	8	6	49	8	< 1	< 1

Electrochemical Measurements

The processes of oxidation and reduction of sulfur and iron-containing compounds by a mixed culture of acidophilic microorganisms were reflected in the cyclic voltammograms (CV curves). To obtain anaerobic conditions, before measuring the CV curves, the solution in the electrochemical cell was purged with argon for 5 min. CV curves were measured using an IPC Pro potentiostat (Russia) controlled by the ICP 2000 software. All CV data were measured at a scan rate of 50 mV/s.

All glassware and the electrochemical cell were treated with a hot mixture of concentrated sulfuric acid and 30% hydrogen peroxide solution. After treatment with the detergent mixture, the glassware was thoroughly washed, first with hot, then with cold tap water, and then three times with bidistilled water.

CV curves and Nyquist diagrams were recorded in a three-electrode cell with a volume of 25 cm3. To study the processes of oxidation and reduction of sulfur and ironcontaining compounds, a mixed culture of the studied acidophilic microorganisms with a volume of 25 cm3, obtained by mixing cultures in equal proportions (by volume of the suspension), was introduced into the electrochemical cell. In the inoculum, the final number of strains was 7 × 10⁷ cells/mL. Cyclic voltammetry (CV) working electrodes are made from glassy carbon (GC-2000) cut with 2 × 38 × 6 mm plates. The conductivity value for this material is 200 Ohm⁻¹ cm⁻¹. The reference electrode was Ag | AgCl | NaCl 3 M (BAS RE1, Japan). The platinum grid was used as a counter electrode.

An Autolab PGSTAT 128 N potentiostat (MetrohmAG, Switzerland) equipped with an FRA 32 M fnd NOVA 1.10 impedance module was used to measure voltammetry and impedance spectra, as well as to analyze and simulate electrochemical impedance spectrometry (EIS) data. To assess the effect of the potential sweep on the characteristics of the electrode/solution interface, the impedance spectra were recorded twice: before and after voltammetric testing. The potential sweep rate in all measurements was 50 mV/s. The frequency range in the EIS was 1–10⁴ Hz.

Biofilm Formation Experiment

A mixed culture of the studied acidophilic strains was obtained by mixing individual strains in equal proportions (according to the volume of the suspension). It was added in a volume of 3 ml to sterile Erlenmeyer flasks (250 ml) with Leathen’s medium (30 ml). 0.1 g of carbon fabric "URAL T-22P A" and carbon fiber felt Karbopon-B-22 (JSC "Svetlogorsk Khimvolokno", Republic of Belarus) were added to the medium with inoculated microorganisms. Ferrous sulfate, cysteine, sodium thiosulfate, elemental sulfur, pyrite tailings were used as energy sources. Ferrous sulfate, cysteine and sodium thiosulfate were dissolved in the medium and sterilized by filtration through a membrane filter (pore size 0.22 μm); flotation tailings of elemental sulfur, pyrite and old pyrite were sterilized at 105 °C. for 3 h. In the control variant, only the suspension of strains and tissue samples were added to the medium. The flasks were incubated at ambient temperature at 100 rpm on an orbital shaker for 3 days.

Microbial cells adhered to the electrode surface were visualized using confocal laser scanning microscopy (CLSM). An Eclipse Ti microscope with an A1 confocal module (Nikon Corporation, Japan) was used. To obtain images with a resolution of 1024 × 2014, an Apo TIRF 60 × /1.49 oil lens was used. The pinhole was installed according to the manufacturer's instructions. Samples were stained with Sybr Green I (Sigma-Aldrich, USA) by placing them in a dye solution for 15 min. The resulting images were processed using the Nikon NIS-Elements software (Nikon Corporation, Japan).

Results and Discussion

When studying the processes of oxidation and reduction of sulfur- and iron-containing compounds, CV curves were used (Fig. 1, two consecutive cycles are shown). The data obtained allow us to say that the use of a mixed culture of acidophiles during incubation with the studied substrates in Leathen’s medium intensifies redox processes. For elemental sulfur and sulfur-containing substrates, the addition of microorganisms resulted in significant shifts in the potentials of the current peaks. The anode peak shifted towards more positive potential values, and the cathode peak shifted towards more negative ones. It should be noted that the voltammetric response of elemental sulfur in the absence of microorganisms was very insignificant. No reduction or oxidation peaks were observed in the studied potential range (Fig. 1.1, curve a). But the redox processes of ferrous sulfate proceeded both in the presence and in the absence of microorganisms. Microorganisms practically did not affect the values of current and potential in peaks (Fig. 1.2).

Fig. 1.

Cyclic voltammograms measured in Leathen’s medium in the presence of acidophilic microorganisms (in ratio of 1:1) and/or elemental sulfur (1), ferrous sulfate (2), pyrite tailings (3), sodium thiosulfate (4), cysteine (5) and mixture of substrates (6); a—Leathen’s medium supplemented with the studied compounds; b—Leathen’s medium incubated with acidophilic microorganisms supplemented with the studied compounds; Leathen’s medium incubated with acidophilic microorganisms without studied compounds (7, green CV curve) (Color figure online)

When using pyrite tailings, the effect of sulfur and iron on redox reactions was demonstrated (Fig. 1.3). An additional quasi-reversible redox pair occurs at potential values of 0.4 V (cathode process) and 0.15 V (anodic process). In the case of pyrite tailings, incubation in the presence of microbial culture resulted in a significant increase in peak current values. Taking into account these observations, it can be assumed that ferric iron ions formed during the interaction of pyrite with microorganisms acted as a mediator in the process of electron transfer to the electrode [26]. Voltamperograms measured in the presence of sodium thiosulfate (Fig. 1.4) showed its irreversible oxidation at E values from 0.9 to 1.5 V. The current values obtained for this process in the presence of microorganisms showed a slight decrease compared to the control without microorganisms. A possible explanation for this fact may be based on the consumption of the substrate by microorganisms.

Heterotrophic oxidation of thiosulfate to tetrathionate is widespread in beta- and gammaproteobacteria, especially in obligate chemolithotrophic genera, including Acidithiobacillus. In these microorganisms, this is an intermediate pathway for the metabolism of polythionates. [27, 28]. Thiosulfate also plays an important role in sulfate formation in sulfur-oxidizing chemolithotrophs. Some of the thiosulfate can be converted to sulfate by the sulfate hydrolase encoded by the soxBYZ genes. Another part of thiosulfate can be converted into sulfite in accordance with the detected thiosulfate serotransferase encoded by cysA3 genes [29]. However, this process was most likely caused by changes at the electrode-solution interface due to the adsorption of microorganisms on the electrode surface.

Similar behavior was observed in the experiment with cysteine (Fig. 1.5). But in this case, the voltammetric response of the system containing cysteine and microorganisms was insignificant and no reduction or oxidation peaks were recorded. Cysteine sulfhydryl group as a nucleophile, is capable of binding several electrophilic groups in protein molecules. This property of cysteine is commonly seen as an explanation for its toxicity to bacteria. In addition, cysteine forms poorly soluble compounds with metal cations necessary for microorganisms, such as iron, manganese, zinc, etc. [30]. On the other hand, the appearance of insoluble products such as cystine and elemental sulfur may result from the oxidation of cysteine or the decomposition of thiosulfate in an acidic medium, respectively. These products reduce the electrochemically active surface of the electrode and lead to its degradation. Thus, these irreversible processes can be the reason for the absence of cathodic peaks on CV curves measured in the presence of the studied sulfur-containing substrates (Fig. 1.4 and 5).

The results of electrochemical impedance spectroscopy are presented in the form of Nyquist diagrams in Fig. 2. The behavior of the dependences on the Nyquist diagrams can be used as a “diagnostic tool” for the characteristics of the diffuse layer near the electrode/solution interface. Thus, it is stated [31] that the steep slope z’ vs. z'' is due to the formation of a double layer, while the low slope, with ionic diffusion. The introduction of microorganisms and/or substrates into the supporting electrolyte (Leathen’s medium) leads to a decrease in the slope and can be interpreted as evidence of their effect on the diffusion part of the double layer. Its contribution decreases in favor of ion transport. All this, in general, allows us to speak about the ongoing process of adsorption of cells on the electrode surface.

Fig. 2.

Nyquist plots obtained from EIS data in the studied solutions before (a, c, e) and after (b, d, f) cyclic voltammetry: a, b—Leathen’s medium supplemented with the studied compounds; c, d—Leathen’s medium incubated with acidophilic microorganisms without studied compounds; e, f—Leathen’s medium incubated with acidophilic microorganisms supplemented with the studied compounds. 1—acidophilic microorganisms and/or elemental sulfur, 2—ferrous sulfate, 3—pyrite tailings, 4—sodium thiosulfate, 5—cysteine, 6—a mixture of substrates

On the other hand, the sorption of microorganism cells on the electrode surface should increase the electrode impedance, and the death of these cells as a result of external influences leads to a decrease in the impedance [32]. From this point of view, Fig. 2.6 (mixture of substrates). In the presence of cells before measuring cyclic voltammograms, the impedance has maximum values, and after cyclic voltammetry it strongly decreases (both in the presence and in the absence of cells in the system under study), but, at the same time, in the experiment with microorganisms, the impedance continues to remain higher. than in the blank experiment. The observed can be explained as follows: the studied microorganisms are sorbed on the electrode surface, increasing its impedance characteristics. Potential cycling in a wide range of values (essentially, up to the boundaries of the water stability region) can lead to the death of microorganisms, but at least part of them, even after that, can be present on the electrode surface. Similar changes were observed both in the presence of microorganisms and in sterile control samples. However, under certain conditions, acidophilus microorganisms exacerbated these impedance differences, in particular in the case of pyrite tailings.

It is generally accepted that the simplest model of the electrode/solution interface can be represented by the solution resistance connected in series to the capacitance–resistance pair. The last two elements are connected in parallel to each other and depend on the electrical properties of the double layer. However, for correct modeling of complex systems, an equivalent circuit may be suitable. Thus, for the systems studied in the work, the best agreement between the experimental data and the simulation results was achieved with the equivalent circuit, taking into account the possible growth of the adsorbed layer on the electrode surface (Fig. 3). The values of the parameters of the equivalent circuit were estimated by modeling the EIS spectra using the NOVA 1.10 software.

Fig. 3.

Equivalent circuit used to simulate the electrical parameters of the investigated electrode / solution interface; Rs is the stability of the solution; R1 and C1 are the resistance and capacitance of the double layer, respectively; R2 is the resistance of the adsorbed layer on the electrode surface; Q is a constant phase element that simulates the capacity of the adsorbed layer and is characterized by phase deviation (N) and electrical conductivity (Y0)

The values of the equivalent circuit parameters corresponding to the best simulation results are shown in Table 3. It was assumed that an increase in resistance and a simultaneous decrease in capacitance were evidence of adsorption processes occurring on the electrode surface. Based on this, the data obtained allowed us to select the systems that are most active in interaction with the electrode. Noticeable changes in the parameters of the equivalent circuit were demonstrated by solutions obtained after incubation of microorganisms in the presence of cysteine, pyrite tailings, and mixed of the studied compounds (Table 3). It should be noted that the last two cases were also the most electrochemically active in voltammetry experiments.

Table 3.

Equivalent circuit parameters

Experimental conditions	RS, Ohm	R1, mOhm	C, nF	R2, kOhm	Y0, µMho	N
Leathen’s medium + sulfur (before CV)	125	4.49	242	67.3	1.13	0.871
Leathen’s medium + sulfur (after CV)	132	1.95	533	43.2	2.4	0.818
Leathen’s medium + mixed culture (before CV)	107	1.81	233	76.3	0.91	0.886
Leathen’s medium + mixed culture (after CV)	105	0.56	396	62.7	1.02	0.867
Leathen’s medium + sulfur + mixed culture (before CV)	110	0.65	405	36.8	1.66	0.895
Leathen’s medium + sulfur + mixed culture (after CV)	112	0.14	926	37.5	1.51	0.884
Leathen’s medium + ferrous sulfate (before CV)	87.2	1.2	268	57.0	1.13	0.878
Leathen’s medium + ferrous sulfate (after CV)	87.6	0.186	957	106	0.935	0.875
Leathen’s medium + ferrous sulfate + mixed culture (before CV)	89.9	0.30	432	48.9	1.02	0.901
Leathen’s medium + ferrous sulfate + mixed culture (after CV)	89.2	0.05	1680	55.3	0.94	0.891
Leathen’s medium + pyrite tailings (before CV)	117	1.99	272	52.5	1.36	0.876
Leathen’s medium + pyrite tailings (after CV)	116	0.19	811	56.0	1.27	0.863
Leathen’s medium + pyrite tailings + mixed culture (before CV)	99.3	0.97	266	72.4	0.76	0.898
Leathen’s medium + pyrite tailings + mixed culture (after CV)	101	0.196	571	67.8	0.78	0.881
Leathen’s medium + thiosulfate (before CV)	85.5	3.29	256	67.2	1.00	0.874
Leathen’s medium + thiosulfate (after CV)	81.7	0.01	12,800	1,100,000,000	25.7	0.767
Leathen’s medium + thiosulfate + mixed culture (before CV)	82.6	3.37	278	54.9	1.3	0.866
Leathen’s medium + тиocyльфaт + mixed culture (after CV)	82.9	0.09	14,700	8.7	26.3 ↑	0.766
Leathen’s medium + cysteine (before CV)	198	4.89	231	69	1.14	0.867
Leathen’s medium + cysteine (after CV)	184	0.65	1540	27.6	4.92	0.767
Leathen’s medium + cysteine + mixed culture (before CV)	151	4.02	247	65.6	1.11	0.860
Leathen’s medium + cysteine + mixed culture (after CV)	150	1.65	643	35.9	2.86	0.809
Leathen’s medium + mixture of compounds (before CV)	126	3.43	274	42.9	1.61	0.862
Leathen’s medium + mixture of compounds (after CV)	125	0.071	4110	33.1	3.35	0.811
Leathen’s medium + mixture of compounds + mixed culture (before CV)	113	3.83	191	77.3	0.80	0.872
Leathen’s medium + mixture of compounds + mixed culture (after CV)	112	0.13	2140	41.4	2.21	0.816

Quite similar in electrochemical studies were carried out by Peng et al. A microbial consortium dominated by Acidithiobacillus spp. and Sulfobacillus spp. led to accelerated leaching of chalcopyrite. CV curves, potentiodynamic polarization and EIS have shown that the mixed culture enhances dissolution reactions, reduces the corrosion potential and increases the corrosion current, as well as reduces the charge transfer resistance and impedance of the chalcopyrite electrode passivating layer compared to pure culture. This study revealed the mechanisms by which mixed culture contributed to the bioleaching of chalcopyrite [33].

CLSM showed that when the studied community of microorganisms grew in the presence of all the studied compounds (iron sulfate, elemental sulfur, pyrite tailings, cysteine, sodium thiosulfate) on carbon fabric and carbon fiber felt, cell sorption occurred. At the same time, in the control experiment, when sulfur- and iron-containing compounds were not added to the medium, cell adhesion on the surface of carbon materials was not observed (Fig. 4).

Fig. 4.

Visualization of cell adhesion on the surface of carbon fiber (left) and carbon felt (right) in the presence of ferrous sulfate, elemental sulfur, pyrite flotation tailings, cysteine and sodium thiosulfate using the CLSM method. A-cells stained with SYBR Green I, B-cells stained with SYBR Green I on the substrate surface in transmitted light. Bar scale—50 µm (Color figure online)

Conclusions

The processes of bioelectrochemical interaction of a model consortium of acidophilic microorganisms, including the genera Leptospirillum, Sulfobacillus, Acidithiobacillus, Ferroplasma and Acidiplasma, with various sulfur- and iron-containing substrates (ferrous sulfate, sodium thiosulfate, elemental sulfur, pyrite tailings). The combination of electrochemical methods and confocal microscopy suggested that acidophils were not only actively involved in the biooxidation of the used substrates, but were also sorbed on the electrode surface. New materials have been obtained on bioelectrochemical processes occurring between acidophilic chemolithotrophic microorganisms and solid surfaces associated with the leaching of metals from sulfide ores.