Isolation and Phylogenetic Analysis of Chromium ( VI ) Reducing Bacteria of a Magnetite Mine Drainage from Hebei China

A magnetite mine drainage sample collected from Han-Xing Iron mine area in Hebei Province was domesticated with potassium dichromate. Four kinds of media supplemented with 300mg/L chromium(VI) were used to isolate chromium(VI) tolerant bacteria. 10 Cr(VI) reducing strains which can tolerant at least 350 mg/L Cr(VI) and reduce the Cr(VI) efficently are screened by observing the growth station and measuring the remaining chromium (VI) concentration in media. 16S rDNA sequencing and phylogenetic analysis showed that Brevundimonas spp. are the main group of chromium(VI) tolerant bacteria in the magnetite mine drainage water sample.


Source of the sample
The magnetite mine drainage used for bacteria isolation was collected from the Qianshan Mining of Han-Xing iron mine area in Shahe, Hebei Province, China, on November 4, 2009.The depth of the mine is about 300 meters; the pH value of the water is 6.5 by field testing with a portable acidometer.The mine drainage sample was collected by a sterilized plastic bottle and used for domestication immediately after taken back to the laboratory.

Domestication and isolation
The chromium tolerant bacteria in the sample were domesticated by the following procedure: the mine drainage sample was inoculated by 5%(v/v) to the domestication medium (yeast extracts 1.0 g, glucose 1.0 g, deionized water 1000 mL, natural pH, autoclaving at 121 for 20 min) supplemented with 200 mg/L of potassium ℃ dichromate, cultured for 3 days at 28 by shaking at 200r/min; then the domesticated sample was transferred ℃ by 5%(v/v) to a new domestication medium supplemented with 400 mg/L of potassium dichromate , culturing for 3 days by the same condition; and the like, until the potassium dichromate concentration up to 1000 mg/L for domestication culture.
After the final domestication, the Cr(VI) tolerant bacteria were isolated by dilution plating method on four kinds of media according to the reported methods and modified by this experiment.9K medium (Silverman et al., 1959) and9K' medium (Wan et al., 2008) were used to isolate acidophilus bacteria from other acidic metal minerals, the modified media at pH 5.5 were used in this study for the pH of the sample is near the neutral.M5' medium (Lee et al., 2000) was reported for the isolation of actinomycete from gold-cave and used for isolation in this trail.PCA is the most commonly used medium for counting the "total" bacteria of various water medium (Greenberg et al., 1992;Marshall., 1992), in this study, we chose to use 1/5 PCA for bacteria isolation for the oligotrophy fact of the sample.
All of the media above were supplemented with 300mg/L of Cr(VI) (equals to 847.8 mg/L potassium dichromate) and 1.25g/L of sodium pyruvate for isolation.The sodium pyruvate can promote the growth of the difficult-to-culture bacteria (Lee, 1996).
The sequences obtained were initially estimated by the BLAST facility of NCBI (www.ncbi.nlm.nih.gov/BLAST) and then aligned with all related sequences obtained from GenBank by Cluastal W (Thompson et al., 1994).Evolutionary distance matrices were calculated by using the method of Jukes & Cantor (Jukes et al., 1969), and a phylogenetic tree was reconstructed by the neighbour-joining method (Saitou et al.,1987).

Biodiversity analysis
The alpha biodiversity of Cr(VI) tolerant bacteria of the sample was estimated basing on Shannon-wiener index and Simpson index.

Isolation
The isolation results (Table 1) were analyzed by SPSS11.5 software.It showed that the isolated bacteria quantity of 1/5 PCA and M5' reach significant difference at 5% level comparing with modified 9K and modified 9K'media.Although the isolation effect of 1/5 PCA and M5' did not reach significant difference at 5% level, the bacteria number on 1/5 PCA is a little more than that of M5' media, and they grow faster and stronger .While the bacteria on M5' medium grow slower and relatively weaker.The results showed that Cr(VI) tolerant bacteria of the magnetite mine drainage are neutrophilic bacteria, 1/5 PCA medium is suitable for the isolation, but the modified 9K and modified 9K' media are not.

Screening result of chromium (VI) reducing strains
All the 83 Cr(VI) tolerant isolates were screenned, and 10 strains with good ability to reduce Cr(VI) at the concentraion of 350 mg/L were obtained.The 10 Cr(VI) reducing strains can tolerate to Cr(VI) up to a concentration of 450 mg/L and remove the Cr(VI) by efficently after cultured for 3 days(Table 2).All the strains showed good ability to reduce Cr(VI) at the initial concentration of 350 mg/L, of which strain cp03 has the highest reduction rate of 80%.The reduction rate of the 10 strains descend at different ratios as the Cr(VI) increase.

Phylogenetic and Biodiversity analysis
All the 16S rDNA sequences of the 10 Cr(VI) reducing bacteria are submmited to GenBank with the accession numbers HQ830175 to HQ830184.Phylogenetic analysis (Figure 1) indicates that the10 strains belong to 5 OTUs, and they all share highly homologous to α-Proteobacteria with the highest sequence similarities from 96.5 % to 99.9%.

Conclusion
The bacteria isolated from the magnetite mine drainage sample have strong toleration and reduction power to the heavy metal Cr(VI), and they are hopeful adopted for microbial remediation of chromium (VI) pollutted environments.
Neutrophilic bacteria are the main chromium (VI) toleration groups in the analyzed magnetite mine drainage mine sample, and 1/5 PCA medium is suitable for bacteria isolation.
The alpha diversity of Cr(VI) reducing bacteria on genus level of the magnetitle mine drainage sample is poor.
Strains cf02, cf01, cp2, cp04, cp07 and cf04 are closest to Brevundimonas intermedia ATCC 15262 T with the similarity of 99.9%; Strains cp03 and cp05 are closest to Brevundimonas mediterranea V4.BO.10 T with the sequence similarities of 99.6 % and 99.8%, respectively; Strain cp06 shares the same similarity of 97.8% with its nearest neighbors Sphingomonas desiccabili CP1D T and Sphingomonas koreensis JSS-26; Strain cf03 shares highest homologous to Azospirillum rugosum IMMIB AFH-6 with the similarity of 96.5 %.The alpha biodiversity of Cr(VI) reducing bacteria on genus level of the sample evaluated by Shannon-wiener index and Simpson index are 0.64 and 0.34, respectively, which indicate that the magnetite mine drainage sample have low richness and evenness on genus level, and the diversity is not rich.

Table 1 .
Brevundimonas spp.are the dominant Cr(VI) reducing group of the sample, accompanied with little amount of Sphingomonas spp.and Azospirillum spp.. Higgins, D G, and Gibson, T J. (1994).Clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighing, position-specifc gap penalties and weight matrix choice.Nucleic Acids Res 22, 4673-4680.Jukes, T H, and Cantor, C R. (1969).Evolution of protein molecules.In Mammalian Protein Metabolism, Edited by H. N. Munro.New York: Academic Press, pp.21-132.Saitou, N, and Nei, M. (1987).The neighbor-joining method: a new method for constructing phylogenetic trees.Molecular Biology and Evolution, 4: 406-425.Isolation result of Cr(VI) tolerant bacteria from magnitite mine drainage sample on four different media

Table 2 .
The reduction rate of chromium reducing bacterium after cultured for 3 days