3.1 Characteristic of acid tolerance strain
Analysis of the 16S rDNA sequences and construction of the phylogenetic tree based on neighbor-joining analysis were performed for molecular identification of isolated strains. The partial 16S rDNA sequence of isolated strains displayed 100% identity with corresponding sequences of Leptospirilum ferriphilum DX-m (Fig. 1). And the isolated strain was therefore named Leptospirilum ferriphilum DX-m-ALE. Surface groups analysis based on FTIR revealed that the peak intensity in 1080 cm-1、1545 cm-1、1640 cm-1 and 2080 cm-1 increased, while in 1190 cm-1、1290 cm-1、1470 cm-1、1570 cm-1 and 3000 cm-1 decreased (Fig. 2). These data indicating more polysaccharide like substances, -C-O-C-, -C=C- and heterocyclic compound in Leptospirilum ferriphilum DX-m-ALE.
3.2 Iron oxidation and biomass production of Leptospirilum ferriphilum DX-m-ALE in wider conditions
As illustrated in Fig 3A, the iron oxidation capacity of Leptospirilum ferriphilum DX-m gradually decreased with the decrease of pH value and no biological iron ion oxidation was observed at pH 0.6. These results indicated serious inhibition of acid on the iron oxidation of Leptospirilum ferriphilum (Zhang et al., 2010). In comparison, Leptospirilum ferriphilum DX-m-ALE completely oxidized 10 g/L iron within 48 hours in the range of pH 0.6-1.8. Meanwhile, the production of biomass showed the same trend with ferrous iron oxidation (Fig.3B). These results indicating that the acid resistance of the evolved strain Leptospirilum ferriphilum DX-m-ALE was improved.
The effect of temperature on iron oxidation of both the starting strain Leptospirilum ferriphilum DX-m and the evolving strain Leptospirilum ferriphilum DX-m-ALE was presented in Fig 3C. In the case of pH 1.6, the iron oxidation rate for the evolved strain was 250 mg L-1h-1 at 30℃, while that of the starting strain was 80 mg L-1h-1. Even the temperature was at 25℃, the evolved strain showed 150 mg L-1h-1 iron oxidation rate. It is obvious that the evolved strain had stronger adaptability at low temperature. In the case of pH 0.7 , the evolved strain showed the maximum iron oxidation rate of 350 mgL-1h-1 at 45℃. However, the iron oxidation rate in low pH culture was slightly lower than that in high pH culture when pH below 37℃. For biomass production, the lower the temperature, the less biomass. It is not hard to conclude that the adaptive evolutionary strain Leptospirilum ferriphilum DX-m-ALE showed a wider range of temperature adaptability and could maintain this superiority under the low pH environment, but the double stress of low temperature and acidity still affects the activity of the evolved strain.
The combined effect of ferrous iron and pH on the iron oxidation for Leptospirilum ferriphilum DX-m-ALE and Leptospirilum ferriphilum DX-m was also determined (Fig 3D-F). At pH 1.6, both strains oxidized all ferrous iron within 48 hours below 5-10 g/L Fe2+. However, high concentrations of ferrous iron seriously inhibit the growth of starting strain. In the case of 30g/L Fe2+, little ferrous iron was observed to be biologically oxidized with 72 hours. In contrast, the evolved strain completely oxidized ferrous iron to ferric iron. These results demonstrated the evolved strain has a greater tolerance to Fe3+ compared to the starting strain. At pH 0.7, the evolved strain showed higher iron oxidation ability when the ferrous iron concentration below 20 g/L, which may due to Leptospirilum ferriphilum under low pH need more energy to resist acid stress (Matsumoto et al., 2004). However, dual stress of acid and high concentration of iron ions still inhibited the iron oxidation function of the evolved strain Leptospirilum ferriphilum DX-m-ALE, the time to oxidize 30 gL-1 Fe2+ extended to 84 hours. Based on the above results, it is concluded that Leptospirilum ferriphilum DX-m-ALE showed greater capacity for ferrous iron oxidation under different conditions
3.3 The adaptation for the basic salt in 9K medium
During the bioleaching process, the constituent lack of 9K basic salt seriously inhibited the growth of microorganisms, but the excessive chemicals in 9Kmeidum not only caused energy waste but also lead to the production of secondary precipitates and limited the growth of cells (Gramp et al., 2008). The reasonable component and concentration of basic salt is allimportant for the application of bioleaching techniques. It was observed that the iron oxidation capacity and biomass production of the evolved Leptospirilum ferriphilum in different concentration of basic salt was different from the starting strain.
Little influence on the iron oxidation rate of the starting strain was observed at 1-3 g/L ammonium salt, and the high concentration of ammonium salt may not be beneficial to the growth of starting strain (Fig. 4A). In comparison, the evolved strain showed a higher iron oxidation rate in the wider concentration of ammonium sulfate. In addition, the culture of the evolved L. ferriphilum in the low pH condition showed similar trends. These results indicated little dependency on ammonium salt concentration for starting strain and evolved strain, but the evolved strain showed higher growth capacity under different ammonium salt concentrations.
The negligible effect of KCl on iron oxidation rate was observed for both evolved and starting strains (Fig. 4B). Even in the absence of potassium chloride, the iron oxidation rate achieved for 250 mgL-1h-1. It was noted that the high concentration of potassium chloride improved the biomass production. Particularly in the case of pH 0.7, the higher concentration of potassium chloride, the more biomass was detected. This is possible that a higher concentration potassium ion could improve the acid resistance stress of microorganisms(Guan and Liu, 2020). It is demonstrated that the evolved strain could adapt wide range of KCl and thus would improve the growth.
The concentration of K2HPO4 had a great influence on iron oxidation and biomass of strains, and the effect of K2HPO4 on the iron oxidation capacity for starting strain and evolution was similar (Fig 4C). The maximum iron oxidation rate and biomass was obtained at 0.2g/L K2HPO4. However, the evolved strain showed higher iron oxidation rate at pH 0.7. And the biomass gradually increased with the increase of the concentration of K2HPO4. This may because more potassium ions improved the acidity resistance of microorganisms (Baker-Austin and Dopson, 2007) and low pH eliminated the inhibition effect of jarosite on cells. Therefore, the 0.2 g L-1 K2HPO4 is more suitable for the culture of the strain at high pH, but the increase of K2HPO4 concentration at low pH is beneficial to increase the growth rate of microorganisms. In all, the evolved strain could grow in a wider range of 9K basic salt.
3.4 The tolerance for high concentration salts, organic matter and heavy metals
After acid adaptation, L. ferriphilum showed the improved tolerance for higher concentrations of magnesium sulfate (Fig.5A). And the average iron oxidation rate of evolved L. ferriphilum reached 341 mg L-1h-1 under 10 g/L MgSO4 condition. Even at 20 g/L MgSO4, the average iron oxidation of 310 mg L-1h-1 was observed. In contrast, the average iron oxidation rate of starting L. ferriphilum reached the maximum of 291 mg L-1h-1 at 5 g L-1 MgSO4. At pH 0.7, the iron oxidation rate of the evolved L. ferriphilum still showed 320 mg L-1h-1 at 20 gL-1 MgSO4. It was worthy to note that the higher concentration of MgSO4, the smaller the biomass produced by both the evolved strain and the starting strain, indicating that high concentration of MgSO4 is detrimental to the production of biomass, previous studies also demonstrated that high concentration of Mg2+ reduced the biofilm quantity (Tang et al., 2018).Therefore, the enhanced iron oxidation capacity may due to high ionic conductivity environment that improved electron transfer (Li et al., 2014).
The improved tolerance for high concentration Na2SO4 were also observed (Fig. 5B). However, the iron oxidation rate and biomass decreased gradually with the increase of sodium sulfate concentration. In case of 20 gL-1, the iron oxidation rate of the starting strain declined to 250 mg L-1h-1, while the iron oxidation rate of the evolved strain maintained to 308 mgL-1h-1. Accordingly, the enhanced performance on acid resistance was beneficial for the improvement of adaptation for sulfate. During bioleaching process, high concentration of sodium sulfate seriously limited the bioleaching efficiency through the formation of Fe(III)-precipitates (Liu et al., 2018) and inhibition effect for microorganisms (Bevilaqua et al., 2013), the adaptation of L. ferriphilum in high concentration of sodium sulfate and low pH conditions indicated significant synergetic relationship between acid tolerance and environmental adaptation.
In the absence of organic matter and high pH, the iron oxidation rate of the starting L. ferriphilum was 270 mg L-1h-1 and that of the evolved L. ferriphilum was 325 mgL-1h-1 (Fig 5C) However, the iron oxidation rate of the starting strain gradually decreased and only 163 mg L-1h-1 was observed under 1g/L organic matter condition. Meanwhile, the total biomass decreased to 0.6×108 cells/mL. These results indicated that the organic matter greatly inhibited the iron oxidation and microbial growth of the starting strain. In contrast, the iron oxidation rate of the evolved strain maintained over 280 mg L-h-1 at same range of organic matter. Especially at low pH condition, the 310 mg L-1h-1 IOR and 1.7×108 cells/mL biomass was observed. These results indicated that, although organic compounds still had a strong inhibitory effect on the evolving strains, the performance on adaptation was improved. Therefore, adaptive evolution may be an available strategy to improve adaptation of L. ferriphilum for organic matter. Previous research revealed that the addition of galactose in the medium can significantly improve the adhesion performance of EPS, strengthen the adsorption effect of strain (Aguirre et al., 2018), the evolutionary strain of L. ferriphilum increase in sensitivity to organic matter for the application of the bacteria also provides a new train of thought. Besides, no double inhibition effect of organic matter and acidity also revealed the probability of a similar mechanism of tolerance for acidity and organic matter.
Great challenges for bioleaching deriving from the inhibition effect of heavy metals, such as Ni, Cu, Co and Cr seriously limited iron oxidation and cell yields of microorganism. After adaptation, this phenomenon is still serious (Fig 5D-F). In the case of nickel-containing medium, it was observed that the iron oxidation rate of both the starting strain and the evolving strain declined to 138 mg L-1h-1 at pH 1.6, indicating that nickel ion had a serious inhibitory effect on the growth of both the evolving strain and the originating strain, and no great difference in the nickel tolerance was observed between the evolving strain and the starting strain, indicating that only the improvement of acidity tolerance can’t realize the adaptation for nickel ion. In contrast, the evolved L. ferriphilum showed 166 mgL-1h-1 IOR at pH 0.7. Although nickel ion still has serious influence on the growth of microorganism inhibition, the IOR increased by 24.48% compared to the high pH cultivation. The reason for the enhanced nickel ions resistance at low condition may due to acid activated the nickel operon (Tian et al., 2007), implying that the nickel resistance may due to the transcriptional control. In the presence of Cu and Co ions, it took 72 hours for both the starting strain and the evolved strain to completely oxidize 10g/L ferrous iron at pH 1.6, and 96 hours for the evolved strain in pH 0.7. The results indicated the double inhibitory effect of these two metals for L. ferriphilum. It is demonstrated the resistance pathway of the strains to these two metals was not consistent with the acid-resisting pathway. No obvious ferrous iron oxidation and biomass increase were observed in the presence of Cr ions, indicating that Cr not only inhibited the iron oxidation activity of the strain but also inhibited the proliferation of the bacteria. It is concluded that the adaptability to heavy metals was not improved after acid adaptation. Therefore, it is necessary for the improvement of adaptation to heavy metals before the industrial application of acidity adapted L. ferriphilum.
3.5 Bioleaching of metals from PCB and pyrite
The dissolution of pyrite is observed to vary greatly under bioleaching by starting and evolved strain (Fig. 6A). At 45°C, the leaching efficiency of the evolved L. ferriphilum is observed to be 58% at pH 1.6. In comparison, that of the evolved L. ferriphilum displayed superior pyrite leaching at pH 1.0, the soluble iron achieved for 8.43 g/L (79.52%). When the temperature of the leach environment was 30°C, the low pH leaching performance of the evolved L. ferriphilum was inferior to the case of pH 1.6, suggesting that double stress of low temperature and acidity was detrimental to the bioleaching activity of the evolved strain. Meanwhile, the release of iron ion favored the production of acidity (Fig. 6B). All in all, the evolved L. ferriphilum would be advantageous to leach pyrite for the generation of H2SO4 and Fe3+.
As for PCB bioleaching, no notable difference was observed at pH 1.6 for the cases of the evolved and starting strain (Fig. 6C). The Cu extraction efficiency reached 83% and 88%, respectively. Similar with pyrite bioleaching, low temperature bioleaching environment showed the lesser metal extraction. Notable observation indicated that bioleaching at low pH seriously inhibited the dissolution of Cu. Our previous studies demonstrated that acid catalyst coupling bioleaching could significantly enhance the Cu extraction from PCBs(Liu et al., 2020), together revealed the importance of heavy metal resistance for the application of L. ferriphilum. After adaptation, the acidity adapted L. ferriphilum realized the maximum Cu extraction at pH 1.0 (Fig. 6D), agreeing with previous results that low pH bioleaching eliminated the jarosite barrier and improved the interfacial reaction (Liu et al., 2020). In this regard, it demonstrated that extreme acid tolerance microorganisms would be more promising for the development of bioleaching techniques.