Himanshi Garg1, Neha Nagar1, Prerna Sharma1, Jyoti Kanwar1, Sadiqa Zaki1, Chandra Sekhar Gahan 1,2*
1Department of Microbiology, School of Life Sciences, Central University of Rajasthan, NH-8, Bandarsindri, Kishangarh, Ajmer-305817, Rajasthan, India
2Department of Sports Biosciences, School of Sports Sciences, Central University of Rajasthan, NH-8, Bandarsindri, Tehsil Kishangarh, Dist-Ajmer-305817, Rajasthan, India
*Corresponding author: Chandra Sekhar Gahan, Department of Microbiology, School of Life Sciences, Central University of Rajasthan, NH-8, Bandarsindri, Kishangarh, Ajmer-305817, Rajasthan, India. Tel: +91 7727805067; Email: email@example.com@gmail.com
Received Date: 25 March, 2019; Accepted Date: 04 April, 2019; Published Date: 12 April, 2019
The present study investigates effect of anions such as chloride (Cl-), nitrate (NO32-), and sulfate (SO42-) with their counter cations like sodium (Na+), potassium(K+), and ammonium (NH4+) on sulfur oxidizing microorganisms. Bioprocess parameters such as pH and viable cell count were considered to understand the toxic effect of the anions as well as cations on the sulfur-oxidizing microorganism. The pH and viable cell count data reveals that Cl-and NO32- had more inhibitory effects at lower concentrations compared to SO42- ions. Effect of the same concentration of Cl- ion as Na+, K+, and NH4+ counter cations showed, NaCl as more inhibitory with less viable cell count compared to KCl and NH4Cl However, all the three salts such as NaCl, KCl and NH4Cl showed inhibitory effects at 4 g/L Cl- with an order of toxicity of NaCl> NH4Cl>KCl. Similar studies conducted on NO32- ions with similar counter cations resulted with an order of inhibition for NO32- ion as KNO3>NaNO3>Ca(NO3)2 indicating a more inhibitory effect of monovalent cations (Na+ and K+) then divalent (Ca2+) ion. The viable cell count data for the study supports the pH patterns with time followed by viable cell count for Ca(NO3)2 supporting the less inhibitory effect of Ca2+ ion. While for the higher concentration of NO32-the Na+ ion and K+ ion showed a similar pattern for pH and viable cell count. The higher concentration of SO42- 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 SO42- concentrations. Na2SO4 and K2SO4 showed almost similar effects at their lower concentrations.
Bio-Oxidation; Chloride; Nitrate; Potassium; Sulphate; Sulphur Oxidising
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 . 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 . 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-, NO32-, SO42-) and cations (K+, Na+, Ca2+) that may affect the growth and hence the bioleaching activity of culture . 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 . 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- . Iron oxidizers are proved more sensitive in comparison to sulfur oxidizers as they prefer low pH 1-2 and have less tolerance for Cl- . The activity of S oxidizers gets stimulated in the range of 10-50 mM of the anions, i.e., Cl-, PO42-, K2SO4, and Na2SO4, above which a complete inhibition of cell growth has been observed .
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 . There are many studies which suggest that for the same anionic concentrations SO42- (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-, SO42- 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 (SO42-) 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 NO32-, Cl- and sulphate ions along with their counter cations was carried out to check their various inhibitory effects. The NO32- 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 NO32-. KNO3 and NaNO3 for 0.85 g/L to 1.16 KNO3 and 0.68 g/L to 1.30 of NaNO3 having same NO32- 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 SO42- (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 SO42- concentration. The pH for the negative control without microorganisms remained constant at pH 3 during the whole experiment. The pH for elevated concentrations of, Na2SO4, 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 NO32- 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 Na2SO4, 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 SO42- 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 SO42- ion has a less harmful effect than the other two foresaid anions. SO42-has adverse effects at comparatively high concentrations while the other two ions (Cl- and NO32-) 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 SO42- 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.
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 NO32- had more
inhibitory effects at lower concentrations than SO42-. 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 NO32-ion was as follows Ca (NO3)2<< NaNO3
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 KNO32- on pH.
Figure 2: Effect of various concentration of NaNO3.
Figure 3: Effect of calcium NO32- 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)2SO4.
Figure 10: Comparative plot of the change in viable cell count with varying concentration of KNO3.
Figure 11: Comparative plot of change in viable cell count with varying concentration of NaNO3.
Figure 12: Comparative plot of change in viable cell count with varying concentration of (Ca)2NO3.
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.