CHEMICAL ENGINEERINGTRANSACTIONS 
 

VOL. 62, 2017 

A publication of 

 
The Italian Association 

of Chemical Engineering 
Online at www.aidic.it/cet 

Guest Editors: Fei Song,Haibo Wang, Fang He 
Copyright © 2017, AIDIC Servizi S.r.l. 
ISBN978-88-95608- 60-0; ISSN 2283-9216 

Heavy Metal Transport Genes and Bioinformatics Analysis of 
At. Ferrooxidans  

Fei Mi*, Zhixing Xiang 
City College of Dongguan University of Technology, Dongguan 523419, China 
18900820211@163.com 

This paper analyzes the resistance mechanism of At. Ferrooxidans to heavy metal ions, and compares the 
tolerance of different strains to Cu2+ and the expression of At. Ferrooxidans strains under different 
concentrations of Mn2+, Zn2+ and Cd2+. The results show that: the three selected At. Ferrooxidans strains, 
DY1, DY2 and DY3, differ in terms of Cu2+ toleration and S and Fe2+ oxidizing abilities. The maximum 
tolerable Cu2+ concentrations of DY1, DY2 and DY3 are 0.41mol/L, 0.20mol/L and 0.04mol/L, respectively. 
DY1 has the strongest oxidizing ability to S element, but the weakest oxidizing ability to Fe2+ ions among the 
three strains; DY3 has the strongest oxidizing ability to Fe2+ ions. There are differences in the adaptability of 
different strains to the leaching system. The copper resistance gene of the strains are stimulated to a certain 
degree by the Cu2+ in the culture solutions. the Cu2+ resistance of the strains is proportional to the resistance 
gene of the strain itself. The stronger the resistance of the strain to Cu2+, the more unlikely for the relevant 
resistance gene to be expressed in large quantities at low Cu2+ concentration in the culture solution. The 
maximum tolerated concentrations of Mn2+, Zn2+ and Cd2+ are 0.38 mol/L, 0.18 mol/L and 0.08 mol/L for the 
selected At. Ferrooxidans strains. The three heavy metal elements on the strains are ranked as Mn2+ <Zn2+ 
<Cd2+ by toxic effect in descending order. The relative expression of the four genes of the strains is 
proportional to the concentration of heavy metal elements. The proteins encoded in the four genes are 
involved in the transport of heavy metals. 

1. Introduction 
Acidithiobacillus Ferrooxidans (At. Ferrooxidans) is a leaching bacterium proven to be an efficient reducing 
agent for metals like gold, copper, manganese and zinc (Sasaki et al., 2009). It can also oxidize Fe2+, S2+ 
and other cations and absorbs energy (Paulino et al., 2002; Luo et al., 2008). Living in high concentrations of 
heavy metals, At. Ferrooxidans boasts a strong tolerance to heavy metals. Recent years has seen more and 
more attention been paid to the strong heavy metal tolerance of At. Ferrooxidans, and the gradual application 
of the bacterium in bio-metallurgy and heavy metal pollution control (Rawlings, 2005; Dopson et al., 2014). 
The DNA sequence of the standard strain was detected in 2007, but the principle of heavy metal transport 
genes in the strain has not been scientifically explained. Therefore, it is of great theoretical and practical 
significance to study the relative expression of metal transport genes in At. Ferrooxidans strains, and to 
analyze the mechanism of At. Ferrooxidans to withstand high concentrations of heavy metal ions (Qin et al., 
2011; Pittman, 2005; Silver and Phung, 2005). 
This paper analyzes the resistance mechanism of At. Ferrooxidans to heavy metal ions, and compares the 
tolerance of different strains to Cu2+ and the expression of At. Ferrooxidans strains under different 
concentrations of Mn2+, Zn2+ and Cd2+, laying the theoretical basis for relevant research. 

2. Test materials and methods  
2.1 Test materials  

The At. Ferrooxidans strains were taken from the acidic abandoned pits in several large mines. The strains 
were cultured in the following conditions: temperature 26°C; shaking speed 165r/min; concentration 
0.48×107cell/ml. The 9k medium was divided into 4 groups. The solutions in Group 1 was added 0.1-0.4mol/L 

                                

 
 

 

 
   

                                                  
DOI: 10.3303/CET1762222 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Please cite this article as: Fei Mi,  Zhixing Xiang, 2017, Heavy metal transport genes and bioinformatics analysis of at. ferrooxidans, Chemical 
Engineering Transactions, 62, 1327-1332  DOI:10.3303/CET1762222   

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Mn2+; the solutions in Group 2 was supplemented with 0.04—0.12 mol/L Zn2+; the solutions in Group 3 was 
treated with 0.04—0.06 mol/L Cd2+; the solutions in Group 4 was provided with 0.02—0.1 mol/L Cu2+. The 
original strains were put into the medium for short culture, and centrifuged to extract ENA. The test adopted 
Tiangen DNA extraction kit, Tiangen DNA recovery kit, TRIzol RNA extraction reagent and Qiagen RNA 
purification reagent. Fe2+ was extracted from the strains by potassium dichromate. 

2.2 Measuring the tolerance of at. Ferrooxidans to metal elements  

Different concentrations of Mn2+, Zn2+, Cd2+ and Cu2+ were used to create stress environments. At. 
Ferrooxidans strains were inoculated into the culture solutions. In the control group, the culture solution is 
replaced with clean water. The pH value of the culture medium was tested every 12 hours. The extracted RNA 
was purified by Qiagen, some of the RNA was subjected to reverse transcription, and the cDNA obtained by 
transcription was measured by NanoDrop. After measurement, the solutions were diluted to 180 mg/L and 
stored. 

2.3 RT-PCR and bioinformatics analysis  

The general PCR was amplified and purified based on cDNA, and the purified PCR was diluted in 12 folds. 
The general PCR was produced in the following conditions: pre-denaturation (4min) and denaturation (30s) at 
95°C; extension (40s) at 70°C (40 cycles); annealing at 50°C-90°C (the temperature increased by 1°C in each 
cycle). Five parallel tests were performed for each heavy metal test, and 16SrRNA was selected as the 
reference gene. 
The bioinformatics method was employed for target gene analysis. Specifically, the search for target similarity 
was based on BLAST; the ORF Finder was used to search the related gene reading frame; the protein 
isoelectric points, relative molecular weight and protein cell structure were calculated and evaluated by 
ExPASy and PSORTb; Tmhmm and Ncbi were the transmembrane structure and conserved region of protein. 

3. Test results and analysis 
3.1 Leaching of copper pyrites using At. Ferrooxidans 

Three types of At. Ferrooxidans strains were selected from different sources and denoted as DY1, DY2 and 
DY3, respectively. The strains were separately placed in Cu2+ solutions for Cu2+ resistance test. It was found 
that the highest Cu2+ resistances of the three strains were 0.41mol/L, 0.20mol/L and 0.04mol/L, respectively, 
signifying significant difference in Cu2+ resistance of the three At. Ferrooxidans strains. In descending order, 
the ranking of Cu2+ resistance is DY1> DY2> DY3. 

0 1 2 3 4 5 6
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 DY1
 DY2
 DY3

0 5 10 15 20
1.0

1.2

1.4

1.6

1.8

2.0

2.2

pH

Time/d

 DY1
 DY2
 DY3
 Blank

 

Figure 1: Growth curves of 3 types of strains                        Figure 2: pH variation curves of 3 types of strains 

Figure 1 illustrates the growth curves of the three strains when the culture mediums were added with the S 
element. Figure 2 shows the pH variation curves of the culture solutions of the three strains, where “Blank” 
stands for the control group that replaced the culture solution with clean water. It can be seen from Figure 1 
that DY1 and DY2 increased linearly, and the cell densities of the culture solutions after 6 days were 
1.08×108cell/ml and 9.7×108cell/ml, respectively. DY3, however, increased before the ultimate decrease; 
After 6 days, the cell density was merely 0.9×107cell/ml, up by 0.4×107cell/ml. This means the growth of DY3 
strain was inhibited. As shown in Figure 2, DY1 suffered the most significant pH variation as its pH increased 
before eventually decreasing to 1.07; the pH value plunged deeply on the 12th day. In contrast, the pH values 

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of DY2 and DY3 were in a gradual decline. The final pH values of DY2 and DY3 were 1.18 and 1.39, 
respectively. Therefore, the three strains should be ranked as DY1> DY2 >DY3 by the sulfur oxidizing ability. 
When the substance of energy source of the culture solutions was changed to Fe2+, the cell density growth 
curves and Fe2+ oxidizing abilities of the three strains were as shown in Figure 3. According to the illustration, 
the results were different from those in the S element culture solutions. After 42h, the three strains were 
ranked as DY2> DY3 >DY1 by the cell density in Fe2+ culture solution. The cell density in DY3 grew by a 
wider margin than in DY1 and DY2. In the latter two strains, the growth of cell density was slower than that of 
S element culture solutions. After 40h, the Fe2+ oxidation rates of DY2 and DY3 both reached 100%, but that 
of DY1 was only 81%. Comparing Figures 1-3, it is clear that DY1 has the strongest oxidizing ability to S 
element, but the weakest oxidizing ability to Fe2+ ions among the three strains; on the contrary, DY3 has the 
strongest oxidizing ability to Fe2+ ions. From the above analysis, it is concluded that the appropriate At. 
Ferrooxidans strains should be selected for different heavy metal pollution sources to maximize the survival 
rate of the strains and the leaching effect of metal minerals.  

0 10 20 30 40
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0.05mol/L

Time/d  

Figure 3: Cell densities and oxidation rates with the       Figure 4: Cell density with different initial Cu2+ energy 
resource of Fe2+                                                              concentrations 

Figure 4 shows the strain growth when the solutions were treated with different concentrations of Cu2+ ions. 
The concentrations of Cu2+ ions in the solutions were set to 0.001mol/L and 0.05mol/L, respectively. The initial 
cell density was set as 1.0×107cell/mL. After 30 days of soaking, the results show the decline of cell density 
across the three strains in the first 6 days. In particular, the cell density decreased by 60% at the solution 
concentration of 60%. The phenomenon reflects that the three strains died in large numbers as they were not 
adapted to the new culture environment. Since the 6thday, the three strains have gradually adapted to the Cu2+ 
environment, resulting in a gradual increase in the cell density. However, the growth differs from strain to 
strain, which again reveals the different adaptability of different strains to the leaching system. 
It can also be inferred that the strains of DY1, DY2 and DY3 maintained different growth rates when the 
Cu2+concentration was 0.001mol/L; nevertheless, when the Cu2+concentration was 0.05mol/L, the growth of 
the three strains was significantly inhibited, leading to continuous decline in cell density. The cell density of 
DY3 even suffered negative growth. After 30 days, the cell densities of DY1 and DY2 were 6.1×107cell/ml and 
5.8×107cell/ml, respectively. This also demonstrates the advantage of DY1 in actual leaching, that is, the DY1 
strain can extract Cu2+ at high concentration of the ion by extending the reaction time, thus saving the cost of 
frequent replacement of extracted ores and improving the economic efficiency. 
Figure 5 displays the leaching of copper ore concentrates after the three types of At. Ferrooxidans strains had 
been immersed for 30 days in culture solutions of different Cu2+ concentrations (0.001mol/L and 0.05mol/L). 
According to the figure, the leaching amount of copper was very limited in the first 6 days and the cell 
densities were very low because none of the three strains had adapted to the heavy metal environment; After 
6 days, the strains started to grow rapidly, accompanied by the gradual increase in copper leaching amount 
and leaching efficiency. At the Cu2+ concentration of 0.001mol/L, the inhibition of the growth and bioactivity of 
the strains was small. After 30 days, the leaching amounts of the three strains were DY1-2.958g, DY2-2.633g 
and DY3-2.392g. When the concentration of Cu2+ reached 0.05mol/L, the bioactivities of the three strains 
were obviously inhibited, and the leaching efficiencies were suppressed. After 30 days, the leaching amounts 
of the three strains was DY1-1.825g, DY2-1.603g and DY3-0.326g. Hence, high Cu2+ concentration has a 
negative effect on the growth and decomposition of the strains. 
Table 1 presents the up-regulated folds of copper resistance gene in the three strains DY1-DY3 under 
different Cu2+ concentrations. According to previous studies, when the concentration of Cu2+ in the culture 

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solution is lower than the maximum tolerable Cu2+ concentration of At. Ferrooxidans strain, the gene Afe0022 
in the strain will be up-regulated. It can be seen from the table that, under the maximum tolerable Cu2+ 
concentration, the gene were up-regulated by 18.26, 54.56 and 185.58 folds in DY1-DY3, respectively.The 
copper resistance gene of the strains were stimulated to a certain degree by the Cu2+ in the culture solutions. 
Featuring stronger Cu2+ resistance than the other two strains, DY1 has the highest gene up regulated fold, 
followed by DY2 and DY3 in descending order. This shows that the Cu2+ resistance of the strains is 
proportional to the resistance gene of the strain itself. The stronger the resistance of the strain to Cu2+, the 
more unlikely for the relevant resistance gene to be expressed in large quantities at low Cu2+ concentration in 
the culture solution. 

0 5 10 15 20 25 30
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 DY1
 DY2
 DY3

C
u2

+  
co

nc
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io
ns

0.05mol/L

Time/d  

Figure 5: Cu2+ leaching curves with different initial Cu2+ concentrations 

Table 1: Differentially expression of 3 types of strains under different Cu2+ concentrations 

Strain  Cu2+ concentration (mol/L) Up-regulated fold 

DY3 
0.02 1.35 
0.04 18.26 

DY2 
0.02 1.40 
0.04 11.42 
0.20 54.56 

DY1 
0.04 1.21 
0.20 72.18 
0.41 185.58 

3.2 Analysis of the resistance of At. Ferrooxidans strains to Mn2+, Zn2+ and Cd2+ 

Figure 6 shows the pH variations in each culture solution after 10 days of culture of At. Ferrooxidans strains at 
different concentrations of Mn2+, Zn2+ and Cd2+. The substance of energy source in the solutions is S element. 
0mol/L stands for the control group in which the culture solution did not contain any heavy metal ion. As 
shown in the figure, the pH of the control group fell from 2 to 1.20 after 10 days of culture. In comparison, the 
final pH values of the culture solutions were increased in different degrees after 10 days of culture in solutions 
added with Mn2+, Zn2+ and Cd2+. In other words, the strain growth and decomposition were suppressed. The 
less the variation in pH, the more inhibited the activity of the strains. As can be seen from Figure 6(a), the final 
pH values of the culture solutions stood at 1.41, 1.56 and 1.90 when the Mn2+ concentrations were 0.3mol/L, 
0.34mol/L and 0.38mol/L, respectively. As the strain activity was almost completely inhibited at the Mn2+ 
concentration of 0.38mol/L, the maximum tolerable Mn2+ concentration of the strains is 0.38mol/L; similarly, 
the maximum tolerable Zn2+ concentration of the strains is 0.18mol/L for the final pH values of the culture 
solutions were 1.32, 1.57 and 1.93 when the Zn2+ concentrations were 0.06mol/L, 0.12mol/L and 0.18mol/L; 
the maximum tolerable Cd2+ concentration (0.08mol/L) is obtained in similar manner. 
The difference in the maximum tolerable concentration of Mn2+, Zn2+ and Cd2+ is an evidence to the varied 
degrees of toxic effect of the three heavy metal ions on the bacterium. As two of the essential elements for 
biological growth, suitable amounts of Mn2+ and Zn2+ are conducive to strain growth while excessive amounts 
of the two ions will hinder the growth. Cd2+, however, is not a biologically essential element. Any trace of Cd2+ 
will harm the bacterium in the surrounding environment.  
Figure 7 describes the relative expression levels of the genes in the strains stimulated by Mn2+ and Zn2+. The 
abscissa 1-4 in the figure represent genes (Afe-671, Afe-674, Afe-1143 and Afe-1144) in the strains, 
respectively. As shown in Figure 7(a), the expression levels of Afe-671, Afe-1143 and Afe-1144 were 
increased by 2 times, 3.5 times and 2.7 times, respectively, when Mn2+ was 0.1 mol/L in the culture solutions. 

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When the concentration of Mn2+ was increased to 0.4mol/L, the expression levels of the four genes were 
increased by 428.2 times, 688.1 times, 289 times and 620.6 times, respectively. According to Figure 7(b), the 
expression levels of Afe-671, Afe-674, Afe-1143 and Afe-1144 in the culture solutions were increased by 1.35 
times, 1.47 times, 5.23 times and 6.2 times, respectively, when Zn2+ concentration was 0.04mol/L; when Zn2+ 
concentration was raised to 0.16mol/L, the expression levels of the four genes were increased by 229 times, 
86.4 times, 216.6 times and 153.7 times, respectively. To sum up, Figure 7 demonstrates that the expression 
levels of the genes in the strains experienced different degrees of upregulation with the increase in the 
concentrations of heavy metal ions in the culture solutions. Comparatively speaking, the four genes in the At. 
Ferrooxidans strain were more sensitive to Mn2+ and Zn2+. 
The genes in the above four genes were then analyzed by bioinformatics. The reading frame of Afe-671 is 
3114bp, which encodes the protein COG3696. Known as the silver efflux pump, it is mainly responsible for the 
transport of inorganic ions. The open reading frame lengths of Afe-671, Afe-1143 and Afe-1144 are 363bp, 
1320bp and 3111bp, respectively. In the test, the proteins encoded by the four genes participated in the 
transport of heavy metals. The proteins encoded in Afe-671 and Afe-1144 are multiple transmembrane 
proteins, while the protein of Afe-1143 is a metal ion transport protein. 

0 2 4 6 8 10
1.0

1.2

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Mn2+

pH

Time/d

 0 mol/L
 0.3mol/L
 0.34mol/L
 0.38mol/L

0 2 4 6 8 10
1.0

1.2

1.4

1.6

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Zn2+

 0 mol/L
 0.06mol/L
 0.12mol/L
 0.18mol/L

pH

Time/d  
(a) Mn2+                                                                (b) Zn2+ 

0 2 4 6 8 10
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1.4

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Cd2+

 0 mol/L
 0.02mol/L
 0.05mol/L
 0.08mol/L

pH

Time/d  

(c) Cd2+ 

Figure 6: pH variation curves at different concentrations of metal ions 

1 2 3 4
-100

0

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Mn2+

R
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 0 mol/L
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 0 mol/L
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 0.18 mol/L

 

(a) Mn2+                                                                     (b) Zn2+ 

Figure 7: Differentially expressed of metal genes under different metal ions concentrations 

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4. Conclusion 
This paper analyzes the resistance mechanism of At. Ferrooxidans to heavy metal ions, and compares the 
tolerance of different strains to Cu2+ and the expression of At. Ferrooxidans strains under different 
concentrations of Mn2+, Zn2+ and Cd2+. The conclusions are as follows: 
(1) The three selected At. Ferrooxidans strains, DY1, DY2 and DY3, differ in terms of Cu2+ toleration and S 
and Fe2+ oxidizing abilities. The maximum tolerable Cu2+ concentrations of DY1, DY2 and DY3 are 
0.41mol/L, 0.20mol/L and 0.04mol/L, respectively. DY1 has the strongest oxidizing ability to S element, but the 
weakest oxidizing ability to Fe2+ ions among the three strains; DY3 has the strongest oxidizing ability to Fe2+ 
ions. There are differences in the adaptability of different strains to the leaching system. The copper 
resistance gene of the strains are stimulated to a certain degree by the Cu2+ in the culture solutions. The 
Cu2+ resistance of the strains is proportional to the resistance gene of the strain itself. The stronger the 
resistance of the strain to Cu2+, the more unlikely for the relevant resistance gene to be expressed in large 
quantities at low Cu2+ concentration in the culture solution.  
(2) The maximum tolerated concentrations of Mn2+, Zn2+ and Cd2+ are 0.38 mol/L, 0.18 mol/L and 0.08 
mol/L for the selected At. Ferrooxidans strains. The three heavy metal elements on the strains are ranked as 
Mn2+<Zn2+<Cd2+ by toxic effect in descending order. The relative expression of the four genes of the strains 
is proportional to the concentration of heavy metal elements. The proteins encoded in the four genes are 
involved in the transport of heavy metals. 

Acknowledgment 

Project supported by the Young teacher growth fund of Dongguan university of technology (Study on the 
relationship between social medical insurance and commercial medical insurance in Dongguan). 

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