Introduction

The use of micro-organisms to facilitate the extraction and recovery of the base as well as precious metals via bioleaching or bio-oxidation processes from primary ores/concentrates, and secondary resources referred generically as ‘biomining’, has developed into a successful and expanding area of biotechnology [1]. The microorganisms play an essential role in bioleaching are autochemolithotrophic, and they grow by oxidizing reduced forms of sulfur or ferrous iron (or both) which are found in extreme natural conditions (acid mine drainage and acid rock drainage) of low pH and high salt and metal ion concentrations [2]. The microbially mediated mineral dissolution processes operate in an aqueous medium, where the process water becomes essential,as the microbial culture has to oxidize the ferrous iron or reduced sulfur species and replicate rapidly to have sufficient microbial population for successful bioleaching. There are many factors including pH and the toxicity of anions (Cl^-, NO₃^2-, SO₄^2-) and cations (K⁺, Na⁺, Ca²⁺) that may affect the growth and hence the bioleaching activity of culture [3]. Many of these ions (KCl, Ca(NO₃)₂, and (NH₄)₂SO₄ are required as micronutrients for the microbial growth and an essential part of microbial growth medium but their elevated concentrations may consequent deleterious effects [4]. An earlier study on the impact of anions on sulfur oxidation by A. thiobacillus have reported the more negative effect of anions at lower pH due to the destruction of positive inside membrane potential was in the series of SCN^-> NO₃^->Cl^->H₂PO₄>HSO₄^- [5]. Iron oxidizers are proved more sensitive in comparison to sulfur oxidizers as they prefer low pH 1-2 and have less tolerance for Cl^- [6]. The activity of S oxidizers gets stimulated in the range of 10-50 mM of the anions, i.e., Cl^-, PO₄^2-, K₂SO_4, and Na₂SO₄, above which a complete inhibition of cell growth has been observed [7].  

The mesophile and thermophile bacteria respond varyingly for saline conditions. In a comparative study on tolerance of different salt concentration reported less viability of acidophilic thermophiles in comparison to mesophile and moderate thermophiles among which the exceeded tolerance limit of mesophiles was 70g/L for NaCl and 350g/L of MgSO₄^.7H₂O [8]. There are many studies which suggest that for the same anionic concentrations SO₄^2- (Sulfate) is less inhibitory in comparison to Cl^- and NO₂^- which shows low microbial growth, low Fe and S oxidation rates with increased microbial growth lag phase [9-11]. The present study reveals the effect of various anions with their counter cations on the growth of S oxidizing microorganisms. The multiple salts used in the study are based on the fact that these ions are required as a minimal salt medium for microbial growth. The effect of Cl^-, NO₃^-, SO₄^2- with their counter cations K⁺, Na⁺, and NH₄⁺ with increasing concentrations was observed on the microbial growth pattern.

Material and Methods

Microorganisms and growth culture

The microbial culture for the present study was collected from Lulea University of Technology, Lulea, Sweden. Q-PCR analysis conducted by Bio clear B.V., Netherlands, revealed that the mixed culture of chemolithotrophic, mesophilic acidophilic Fe & S oxidizers, was dominated by Leptospirillum ferriphilum (Fe oxidizer) followed by Acidithiobacillus caldus (S-oxidiser), and with approximately the same amount of Acidithiobacillus thiooxidan (S-oxidiser), Sulphobacillus sp. (Fe-oxidiser) and Ferroplasma (Archaeal species, Fe-oxidiser). Inoculum for the study was prepared by subculturing the parental Fe & S mixed microbial culture in a Fe free 0K medium supplemented with 3 g/L elemental Sulphur S⁰ to provide selective growth conditions for S oxidizing microorganisms with a working volume of 100 ml (v/v). After several times of sub-culturing microbial culture dominated by Acidithiobacillus caldus and Sulphobacillus sp. was used as inoculum.

Bio-oxidation experiments with anionic effects

The three different anions selected for the bio-oxidation studies were Nitrate (NO₃^-), Chloride (Cl^-) and Sulphate (SO₄^2-) with their counter cations Na⁺, NH₄⁺ and K⁺. The selection was based on their presence in bacterial growth medium (Table 1) and essential role during bacterial bio-oxidation. All bio-oxidation experiments were carried out for three different concentrations of anions and the concentration of cations were calculated accordingly (Table 2,3,4).

The experiments were conducted in 3 parallel sets for each of the anionic salt. It was a bench scale study conducted with a working volume of 50 ml (v/v), 90% (v/v) 0K medium (supplemented with S⁰), 10% (v/v) inoculum in a 100ml Erlenmeyer flask. The inoculum pH was 1.8, and 0 K medium pH was 3 when after inoculation the final value of experimental pH was 2.2. The experiments were carried out in an orbital shaker with a rotation speed of 110 rpm, and at a temperature of 30°C supports mesophilic bacterial growth. A positive control contains S oxidizing microorganisms with no anionic salts added in the growth medium and a negative control flask without S oxidizing microorganisms (without MO) to ensure the existence of any other organism during the study were maintained. Two parameters viz., pH and viable cell count were analyzed a day thrice to follow the bio-oxidation trend. Water lost due to evaporation was compensated by regular addition of water based upon flask weight. No efforts were put to maintain sterile conditions. The above experiments were continued until the pH of positive control reached 1 as below pH 1.0 these microbes start to get stressed due to high acidic concentration.

Analytical and instrumentation techniques

A benchtop pH meter (Riviera Eutech pH-150) was used for regular pH measurement. Three-point calibration of pH meter was carried out daily by the standard buffers of pH 1.68, 4.0 and 7.0 and the slope obtained ranged from 95 to 100 which ensures good working conditions for pH meter. The Viable cell count was conducted on a bright field compound microscope with a 10× eyepiece and 100× objective lenses by loading the microbial culture sample on an improved Neubauer hemocytometer. The hemocytometer with Neubauer rulings, where the main divisions separate the grid into nine large squares with each square having a surface area of one square mm and a depth of the chamber was 0.1 mm. The counting grid lies under a volume of 0.9 mm³. Microbial culture samples or bioleaching solution samples were diluted enough whenever required to avoid overlapping of the cells with uniform distributed over the counting grid while sometimes vortex of the solution was done to liberate the attached cells. The magnification varies with different microbial cell sizes and types. A systematic counting of the cells was done in selected squares so that the total count of 100 square cells, while the four large squares (1/25 sq. mm) present in the four corners were considered together with one in the middle central square. Precautions were taken not to count the cells lying > 50% outwards the border lines of the square cells. The calculation of the cells per ml of solution was done by adding the total number cells counted in 5 different small squares, which has an area of 1/25 mm² (i.e., 0.04 mm²) and depth of 0.1 mm each. So, the total volume in each square would be 0.04 multiplied by 0.1 resulting as 0.004 mm³.

Result and Discussion

Comparative assessment of the effect of anions on pH profile

S oxidation is an acid producing process, so the pH profile studies show the bio-oxidation kinetics of the bacterial growth medium with time. The present study with cations such as NO₃^2-, Cl^- and sulphate ions along with their counter cations was carried out to check their various inhibitory effects. The NO₃^2- with its counterions sodium (Na⁺), Potassium (K⁺) and Calcium (Ca²⁺) indicate the more inhibitory effect of monovalent cations (Na⁺ and K⁺) then divalent (Ca²⁺) ion for the same concentration of NO₃^2-. KNO₃ and NaNO₃ for 0.85 g/L to 1.16 KNO₃ and 0.68 g/L to 1.30 of NaNO₃ having same NO₃^2- content showed almost similar patterns with a slight increase in lag phase and some extent to pH (1.5-1.8), in respect of control (no salts added) with pH 1.0. The further increase in concentration up to 2.44 g/L for KNO₃ showed an increment of pH>3 which is higher than the pH-3 of the negative control (without MO) (Figure 1-3).

The higher concentrations also increased the bacterial growth lag phase from 2 days (in case of positive control) to 7 days, while Ca(NO₃)₂ showed an utterly different pattern with a slight increase in pH and an almost similar pattern for all increased concentrations (Figure 1-3).  The control experiment where no inoculum has added no growth and sulfur oxidation was observed. The pH for the operation was constant to pH-3. The result ensures that the absence of laboratory contamination it also shows lower sulfur oxidation in the absence of sulfur-oxidizing microorganisms. In the positive control experiment, the pH goes below 1 which ensures the complete S oxidation (Figure 1-3).

The pH profile for Cl^- ions also followed an interesting pattern. As the Cl^- concentration was kept constant 2, 3 and 4 g/L similar for all cations there was an overall exciting correlation between Na⁺ ion and K⁺ ion with the same Cl^- concentration. It was found that 4.2 and 6.3 g/L of KCl showed faster kinetics compared to NaCl with 3.3 & 5.0 g/L Cl^- concentration of 2 and 3 g/L respectively. The lag phase in case of NaCl with the concentration of 5.0g/L was increased from 2 to 7 days while it was shorter for KCl. At Cl^- ion concentration 4g/L, all cations showed complete inhibition as at this level of concentration no growth was observed (Figures 4,5) in case of NH₄Cl there was a slight increase in pH for 4 g/L of Cl^- (Figure 6).

The comparative assessment of bacterial bio oxidation resulted that for the same concentration of SO₄^2- (45 g/L) the NH₄⁺ ion showed maximum inhibition effect at a concentration of 64.8 g/L (NH₄)₂SO₄ compared to 66.5 g/L of Na₂SO₄ and 81.6 g/L of K₂SO₄. The NH₄⁺ ion was inhibitory at lower concentrations then Na⁺ and K⁺ ions for the same SO₄^2- concentration. The pH for the negative control without microorganisms remained constant at pH 3 during the whole experiment. The pH for elevated concentrations of_, Na₂SO_4, K₂SO₄ and (NH₄)₂SO₄ was increased up to 3.03 to 3.49 respectively (Figure 7,8,9). Concentration of 51.7 g/L and 59.1 g/L of Na₂SO₄, 63.5 g/L of K₂SO₄ and 51.1g/L and 58.0g/L was adapted by the bacterial culture after a 2 days lag phase after which a gradual decrease in pH to the value of 1.5-1.8 was observed (Figure 7,8,9). These observations indicate that although bacteria can tolerate the salt concentration up to some extent a higher salt concentration exerts a negative effect on bacterial growth and viability.

Microbial cell viability analysis

The control flask having S Oxidizing microorganisms without any salt inserts showed the complete S Oxidation with a viable cell count 2.8×10⁵ viable cells/ml. Increase in concentration of KNO₃ and NaNO₃ showed a direct effect on cell viability (Figure 10, 11). A steep decrease had observed at 2.44 g/L of KNO₃ and 2.05 g/L of NaNO₃. Due to a less inhibitory effect of calcium NO₃^2- as compared to other anions, it doesn’t seem to be any toxic effect on cells the decrease in number was possibly due to its inhibitory effect for cell growth and replication rate (Figure 12). The cell count observed was 2.54 × 10⁵ cells/ ml for 0.67 g/L, 2.07×10⁵ cells/ml and 1.97×10⁵ cells/ml for 1.33 g/L and 1.99 g/L of Ca(NO₃)₂ which was very close to the cell count of control flask 2.85 × 10⁵ cells/ml.

Considering the effect of Cl^- ion on viable cells dynamics it was observed that the viable population increase was relatively faster in case of KCl compared to NaCl and it was also supported by the trend obtained in the change in pH (Figure 13,14,15). The exciting thing observed was the viable cell count for NH₄Cl at a concentration of 3 g/L Cl- had the highest increase in the population compared to NaCl and KCl.

The inhibitory concentration for microbial growth was 66.5 g/L, 81.6 g/L and 64.8 g/L of Na₂SO_4, K₂SO₄ and (NH₄)₂SO₄ respectively. The viable cell count data strengthen the pH profile data for bio-oxidation which states that (NH₄)₂SO₄ has more inhibitory effects at lower concentrations of NH₄⁺ in comparison to Na⁺ and K⁺ for the same concentration of SO₄^2- ion (Figure 16,17,18).

The viable cell count for 51.7 g/L, 59.1 g/L of Na₂SO₄ 63.5 g/L of K₂SO₄ regained after a short period of lag phase. The present data also reveals the fact that SO₄^2- ion has a less harmful effect than the other two foresaid anions. SO₄^2-has adverse effects at comparatively high concentrations while the other two ions (Cl^- and NO₃^2-) are harmful effects even at lower concentrations. The possible reason behind this differential effect can be explained through cell membrane potential and ion transport into the cell. The high concentration of anions in the growth medium leads to the entry of anions into the cell cytoplasm which disturbs the cell homeostasis by disrupting the inside positive potential. The cell membrane permeability for the different anions determines the extent of inhibition.

On the other hand, the more negative potential inside attracts the anions which again moves into the cytoplasm based on their permeability. The order of cell permeability (H⁺> K⁺>Na⁺> NH₄⁺) of anions gives a possible reason why the same anion with varying counter ions showed varying results. The divalent cations have less permeability for cell membrane that can be observed in our study as Ca²⁺ which showed almost negligible effects and SO₄^2- ion which was only inhibitory at its higher concentration. The other possible reasons for the inhibitory effects are a disturbance in osmotic gradient and destruction of the bacterial cell membrane.

Conclusion

The present study reveals the effect of various concentrations of ions on S bio-oxidation. The effect of different counterions with the same anion showed complete inhibition at higher concentrations. The pH and viable cell count data reveals that Cl^- and NO₃^2- had more inhibitory effects at lower concentrations than SO₄^2-. Effect of various anions for the same concentration of Cl^- was NaCl> NH₄Cl>KCl, the Na⁺ ion was more inhibitory with less viable cell count and higher pH then K⁺ and NH₄⁺. K⁺ ion was less inhibitory then Na⁺ and NH₄⁺ while all three cations showed similar toxicity at the concentration, i.e. 4 g/L of Cl^-. The order of inhibition for NO₃^2-ion was as follows Ca (NO₃)₂<< NaNO₃3 indicates the more inhibitory effect of monovalent cations (Na⁺ and K⁺) then divalent (Ca²⁺) ion for the same concentration of NO₃^2-. The viable cell count data for the study supports the pH patterns with time. The viable cell count for Ca (NO₃)₂ was close to the control experiment’s reading which helps the less inhibitory effect of Ca²⁺ ion. While for the higher concentration of NO₃^2- the Na⁺ ion and K⁺ ion showed a similar pattern for pH and viable cell count. The higher concentration of SO₄^2-was chosen for the study which reveals that (NH₄)₂SO₄ was more deleterious at lower concentrations. The order of inhibition observed was (NH₄)₂SO₄> Na₂SO₄> K₂SO₄ for the higher SO₄^2- concentrations. Na₂SO₄ and K₂SO₄ showed almost similar effects at their lower concentrations.

Acknowledgment

Authors are thankful to the DST, Govt. India for funding the research from SERB for Young Scientist YSS/2014/000895 and the DST-Inspire Fellowship and UGC Fellowship is gratefully acknowledged.

Figure1: Effect of KNO₃^2- on pH.

Figure 2: Effect of various concentration of NaNO_3.

Figure 3: Effect of calcium NO₃^2- on pH.

Figure 4: Comparative plot of the change in pH with varying concentration of NaCl.

Figure-5:  Comparative plot of the change in pH with varying concentration of KCl.

Figure 6: Comparative plot of the change in pH with varying concentration of NH₄Cl.

Figure 7:  Comparative assessment of pH for the various concentration of Na₂SO₄.

Figure 8: Comparative assessment of pH for the various concentration of K₂SO₄.

Figure 9: Comparative assessment of pH for the various concentration of (NH₄)₂SO_4.

Figure 10: Comparative plot of the change in viable cell count with varying concentration of KNO_3.

Figure 11: Comparative plot of change in viable cell count with varying concentration of NaNO_3.

Figure 12:  Comparative plot of change in viable cell count with varying concentration of (Ca)₂NO_3.

Figure 13: Comparative plot of change in viable cell count with varying concentration of NaCl.

Figure 14: Comparative plot of change in viable cell count with varying concentration of KCl.

Figure 15: Comparative plot of change in viable cell count with varying concentration of NH₄Cl.

Figure 16: Comparative plot of change in viable cell count with varying concentration of Na₂SO₄.

Figure 17: Comparative plot of change in viable cell count with varying concentration of K₂SO_4.

Figure 18: Comparative plot of change in viable cell count with varying concentration of (NH₄)₂SO_4.

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