Advanced Materials Research Vol.

402 (2012) pp 3-6

© (2012) Trans Tech Publications, Switzerland
doi:10.4028/www.scientific.net/AMR.402.3

Molecular Identification of a Strain Acidithiobacillus ferrooxidans and Its

Biological Characteristics

Chengxun DENG 1, a, Mei YUE 1, 2, b, Ling WANG 1, c, Yuhui LI 1,d

1
Department of Biology and Environment Engineering, Hefei University, Hefei 230022, China
2
State Key Laboratory of Coal Resources and Safe Mining, China University of Mining and
Technology, Beijing 100083, China
a
dcx@hfuu.edu.cn, byuemei8027@163.com, cyuhuili112@163.com, dwangling@hfuu.edu.cn

Keywords: Acidithiobacillus ferrooxidans; strain isolation; oxidation; phylogenetic tree; 16S rDNA

Abstract. A strain of Acidithiobacillus ferrooxidans designated S t1 was isolated and identified from
the acid mine drainage of Tianmashan Coal Mine, Tongling county, Anhui province. Its
morphological, physiological and biochemical characters, growth conditions of pH value and
temperature, 16S rDNA sequence and the phylogenetic tree was studied. Results showed that the
bacteria strains from different sites have divergence at the curve of pH and Eh values during the
enriching culture. The isolated strain S t1 grew in a pH rang from 2.0 to 3.0 and temperature 25 to
35˚C with over 98％similarity to Acidithiobacillus ferrooxidans in 16S rDNA sequence.

Introduction
In the mining industry, dissolution of sulfide ores exposed to oxygen, water, and microorganisms
results in acid production and environmentally detrimental acid mine drainage (AMD). The damage
of AMD with low pH, high concentration sulfate, heavy metal ions such as copper , zinc, nickel,
chromium , lead , iron and cyanide is serious to the water ecosystem, biodiversity and biomass, even
humanity survival[1]. Microbial community plays a leading role in the information of AMD though the
catalytic oxidation of sulfide [2, 3, 4]. The Acidithiobacillus ferrooxidans is the dominant bacteria in the
acid mine drainage. They promote and accelerate the degradation of sulfide. Therefore, it is important
to study the microbiology of AMD. Therefore, a strain of Acidithiobacillus ferrooxidans designated
S t1 was isolated and identified from the AMD of Tianmashan Coal Mine, Tongling County, Anhui
Province .Its morphological, physiological and biochemical, phylogenetic tree of 16S rDNA was
studied in order to provide the theoretical foundation for inhibiting or slowing down the process of
sulfate oxidation.
Copyright © 2012. Trans Tech Publications, Limited. All rights reserved.

Materials and Methods

Isolation of Microorganisms. The water samples were taken from an extreme acid mine drainage
site with pH 2.7 of Tianmashan Coal Mine in Tongling of Anhui Province, China. The water samples
were first enriched at pH 2.7 in 250 ml Erlenmeyer flasks containing 100 ml of Silverman and
Lundgren liquid 9K medium[5] on a shaker at 180rpm at a temperature of 28˚C. Subsequently , the
enrichment cultures above were cultivated at pH 2.0 in a modified liquid 9K medium[6] supplemented
with FeSO4.7H2O（80 g/L） as bacteria’s energy resource under shaking at 180rpm, 28˚C. After the
second procedure was repeated three times, cell grown in a modified liquid 9K medium were serially
diluted to 10-9. All serial dilutions were spread to solid medium, incubated at 28˚C for ten days until
single colonies appeared. The bacteria from single colony was spread to solid medium for isolation
and incubated to liquid medium for enrichment. Pure strain designated strain S t1 was gotten after
three times of repeat of isolation.
Morphological Characteristics. Cells of strain S t1 were collected from a three-day-old culture
by centrifugation, then washed three times with 0.02 mol/L H 2 SO 4 , and observed under
optical microscope. Bacteria in the exponential growth phase were performed grams staining.

Advances in Metallurgical and Mining Engineering : Advances in Metallurgical and Mining Engineering, edited by Hong Xi Zhu, and Lin Jiang Wang, Trans Tech Publications,
Limited, 2012. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/canterbury/detail.action?docID=1872756.
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 4 Advances in Metallurgical and Mining Engineering

Physiological and Biochemical Analysis. During the period of culture and isolation, the pH and
Eh values were measured. The influence of temperature and pH value on its living activity was
studied in terms the variation of Fe2+ concentrate in the liquid medium for cultivation. Concentration
of Fe2+ was assayed by the method of potassium dichromate titration [7].
DNA extraction and 16S rDNA Sequencing. The bacteria DNA was extracted with UNIP-10 kit
(Shanghai Sangon Biology Engineering Technology & Service Co., Ltd) according the manual. A
partial DNA sequence for the 16S rDNA gene was amplified by PCR with primers [8] FC27
(5’-AGAGTTTGATCCTGGCTCAG-3’) and RC1492( 5’-TACGGCTACCTTGTTACTT-3’) .
The amplification was performed using the ABI 2720 with the following cycling parameter: 94˚C for
5 min, followed by 35 cycles of 30 s at 94˚C, 30 s at 52˚C and 1 min at 72˚C with final extension for
5 min. The purification and sequence analysis of the 16S rDNA PCR products was performed by
DNA Analyser.
Phylogenetic Analysis. The 16S rDNA gene sequences were submitted to the GenBank database
using the BLAST algorithm. Sequences having ≥98％ similarity and matching the same GenBank
sequence were assigned to the same phylotype. The sequences were aligned with their closest
relatives and the phylogenetic trees (neighbour-joining algorithm with Jukes-Cantor corrections)
were constructed using Mega4.0 [9].

Results
Morphologic Characters. After inoculation the bacteria to solid medium for isolation, a serial of
single colonies designated S t1 appeared. The color of colonies was white (Fig. 1) at the beginning
and turned to yellow brown at the end. The shape of colonies was round and glossy, the border was
smooth (Fig. 2). Cells of strain S t1 are Gram-negative with moving activity at the result of
morphological identification.
Copyright © 2012. Trans Tech Publications, Limited. All rights reserved.

Fig. 1 The colonies of S t1 on the solid medium Fig. 2 The T.f under optical microscope

Physiological and Biochemical Characters. The profiles of pH and Eh values were measured
during the enriching culture of bacteria from four sample sites (3#, 4#, 7#, 9#) .The real-time curves
were made with origin 6.0 software (Fig. 3). The Fig. 3(a) showed that the pH value of culture
medium increased in the first day, and then decreased from the second day. The Fig 3(b) indicated that
the value of Eh increased quickly at the beginning, then kept stable. This indicated that the activity of
acidophil strains is closely related to their growth environment.
The effect of pH value and temperature on oxidation activity of strain S t1 could be seen from Fig.
4, Fig5. Strain S t1 grew in a pH rang from 2.0 to 3.0; The temperature for the growth of strain S t1
ranged from 25˚C to 35˚C.

Advances in Metallurgical and Mining Engineering : Advances in Metallurgical and Mining Engineering, edited by Hong Xi Zhu, and Lin Jiang Wang, Trans Tech Publications,
Limited, 2012. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/canterbury/detail.action?docID=1872756.
Created from canterbury on 2020-10-11 12:05:47.
 Advanced Materials Research Vol. 402 5

(a) (b)
600
2.4
3# 550
2.3 4#
2.2 7# 500
9# 3#
450

Eh/mV
2.1
4#
pH

2.0 400 7#
1.9 9#
350
1.8
300
1.7
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
Time(d) Time(d)
Fig. 3 The profiles of pH and Eh value of acidophil strains during the enriching culture

Fe 2 + oxidised percentage(％ )
Fe oxidised percentage(％ )

100
100 20℃
pH1.5 80
80 25℃
pH2.0 60
60 30℃
pH2.5 40
40 35℃
pH3.0 20
20
pH3.5 0 40℃
0
2+

20 30 40 50 60
20 30 40 50 60
Time(h)
Time(h)
Fig. 4 Effect of pH value on Fig. 5 Effect of temperature on
oxidation activity of T.f S t1 oxidation activity of T.f S t1
Phylogenetic Analysis. A 1,439-bp region of the S t1 16S rDNA gene was amplified using
universal bacterial primers and the sequence was compared to those residing in the GenBank
database. The similarity rank analysis of 16S rDNA showed that it is closely related to
Acidithiobacillus ferrooxidans (Fig. 6) . The relationship between St1 and related taxa is illustrated in
Fig. 6. we only get one type of acidophil strains in this study.
Copyright © 2012. Trans Tech Publications, Limited. All rights reserved.

Fig. 6 Phylogenetic tree of the 16S rDNA sequence of T.f S t1

Advances in Metallurgical and Mining Engineering : Advances in Metallurgical and Mining Engineering, edited by Hong Xi Zhu, and Lin Jiang Wang, Trans Tech Publications,
Limited, 2012. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/canterbury/detail.action?docID=1872756.
Created from canterbury on 2020-10-11 12:05:47.
 6 Advances in Metallurgical and Mining Engineering

Conclusions
The strain St1 was identified as a strain of Acidithiobacillus ferrooxidans based on phenotypic,
physiological and biochemical characteristics, as well as 16S rDNA phylogenetic analysis. Strain S t1
grew in a pH rang from 2.0 to 3.0 and temperature 25 to 35˚C.
Owing to the limitations of bacterial culture and isolation, Only one type of acidophil strains was
gotten in this study. Other symbiotic microbes, especially uncultivable microorganisms can’t be
identified in this experiment. More works are needed for this aspect in the future.
The acidophil strains from different sites have divergence at pH and Eh values during the enriching
culture, and the oxidant activity of them to mine metals and sulphide are different. Therefore,
different interfering and modifying ways are needed for different acid mine drainage sites to inhibit or
slow down the process of sulphate oxidation.

Acknowledgements
This work was financially supported by the State Key Laboratory of Coal Resources and Safe Mining
(No.SKLCRSM09KFB03), Anhui Province Outstanding Talented University Youth Fund Project
(2010AQRL157) and Nature Science Research and Development funds of Hefei University
(10KY10ZR).

References
[1] Feng Y, Kang Y Z, Fan F Z, Kong Q, Action of miroorganism on generation and treatment of acid
mine drainage, Nonferrous Metals. 57(2005)103-108 (in Chinese)
[2] Southam G, Beveridge TJ , Enumeration of thiobacilli within Ph-neutral and acidic mine tailings
and their role in the development of secondary mineral soil, Appl Environ Microbiol .
58(1992)1904-1912.
[3] Wulf-Durand DP, Bryant LJ, Sly LI , PCR mediated detection of acidophilic bioleaching
associated bacteria, Appl Environ Mirobiol .63 (1997) 2944-2948.
[4] Wielinga B, Lucy JK, Moore JN, Seastone OF, Gannon JE, Microbiological and geochemical
characterization of fluvially deposited sulfidic mine tailings, Appl Environ Mirobiol. 65(1999)
[5] Silverman M P, Lundgren D C, Study on the chemoautotrophic iron bacterium Ferrobacillus
ferrooxidans (Ⅰ): an improved medium and harvesting procedure for securing high cell yield,
Journal of Bacteriology. 77(1959) 642-647.
Copyright © 2012. Trans Tech Publications, Limited. All rights reserved.

[6] Zhang Z H, Qiu G Z, Hu Y H, The investigation of the colony isolation of Thiobacillus

ferrooxidans, Multipurpose Utilization of Mineral Resource(2002)19-23.
[7] Peng H, Yang Y, Li X, et al, Struture analysis of 16S rDNA sequence from strains of
Acidithiobacillus ferrooxidans, Biochem. Mol. Bio. 39(2006) 178-182.
[8] Yang Y, Peng H, Wan M X, et al, Identification of new subspecies Acidithiobacillus ferrooxidans
strain from complexsulfide mines, The Chinese Journal of Nonferrous Metals.
16(2006):1094-1099.
[9] Information on http://www.megasoftware.net

Advances in Metallurgical and Mining Engineering : Advances in Metallurgical and Mining Engineering, edited by Hong Xi Zhu, and Lin Jiang Wang, Trans Tech Publications,
Limited, 2012. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/canterbury/detail.action?docID=1872756.
Created from canterbury on 2020-10-11 12:05:47.
 Advanced Materials Research Vol. 402 (2012) pp 7-11
© (2012) Trans Tech Publications, Switzerland
doi:10.4028/www.scientific.net/AMR.402.7


  

   
  


 







  

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Advances in Metallurgical and Mining Engineering : Advances in Metallurgical and Mining Engineering, edited by Hong Xi Zhu, and Lin Jiang Wang, Trans Tech Publications,
Limited, 2012. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/canterbury/detail.action?docID=1872756.
Created from canterbury on 2020-10-11 12:05:47.