Breeding And Resistant Mechanisms Of Resistant Bioleaching Bacteria

To screen bioleaching bacteria which are resistant to heavy metals and other toxic ions, and to study the microorganisms' resistance capacities, resistance mechanisms, responsible resistance genes, and the effects of anions. The sulphides bioleaching bacteria were obtained from acid mine drainage (AMD) samples collected from Dexing Copper Mine in Jiangxi Province, Daye Copper Mine in Hubei Province, Dachang Copper Mine in Guangxi Province, China by enrichment, screening, adaptation, and isolation using 9K medium with Fe²⁺ and sulfur as energy sources. Genomic DNA of the isolates was extracted and the 16S rDNA was amplified using PCR technique. The PCR products were compared and analyzed with the known sequences published in GenBank using the BLAST search tool of the National Centre for Biotechnology Information database; the bioleaching bacteria were identificated; the evolutionary relationships were described and the homolog trees were constructed. 9K medium containing several of heavy metal ions (Cu²⁺, Ag⁺, Hg²⁺ , Pb²⁺ , Mg²⁺) was used to culturemetal-resistant bacteria; the ferrous ions concentrations were determined by potassium dichromate titration methods to examine the bacterial oxidation capacities of ferrous ions. The highest toleranceconcentrations to heavy metal ions for metal resistant bacteria were determined and at which the oxidation capacities of ferrous ions of these bacteria were analyzed. The metal resistant bacteria were UV induced mutated to obtain mutants which have higher oxidation capacities of ferrous ions and higher resistance to heavy metal ions. Resistance gene primers were designed according to the data in gene bank. The resistance genes were amplified by PCR (Polymerase Chain Reaction) and the resulting PCR products were cloned and the cloned productswere sequenced. The cloned results were compared and analyzed with the known sequences published in GenBank using the BLAST search tool of the National Centre for Biotechnology Information database. The amino acid sequences of protein were compared with the sequences in the NCBI database, the evolutionary relationships were described and the homolog trees were constructed. The effects of anions were inverstigated when 9K medium containing ZnCl₂, ZnSO₄, Zn(NO₃)₂,respectively, was used to enrich resistant bacteria.The results of 16S rDNA sequences analysis showed that the isolates in this study are all Acidithiobacillus ferrooxidans(At. ferrooxidans). The bacterial resistance capacities to heavy metal ions were determined and the results showed that the tolerate concentration levels of the adaptation bacteria were 32000 mg/L (Cu²⁺ ), 240 mg/L (Ag⁺), 0.9 mg/L (Hg²⁺ ), 3500 mg/L (pb²⁺), 22500 mg/L (Mg²⁺ ), respectively, while the tolerate concentration levels of the wild bacteria were 19000 mg/L (Cu²⁺), 60 mg/L (Ag⁺), 0.1 mg/L (Hg²⁺), 400 mg/L (Pb²⁺), 13500 mg/L (Mg²⁺ ), respectively, which means the adaptation bacteria have higher resistance capacities than that of the wild types. The adapted resistant bacteria were UV induced mutated and the mutants grew well and had higher resistance capacities to heavy metal ions and higher oxidation capacities of ferrous ions than that of the adapted bacteria. Their tolerate capacities to Cu²⁺ and Ag⁺ were 2-3 times higher than that of the wild bacteria and 1.1--1.3 times higher than that of the adapted types, respectively. The resistance mechanisms to Cu²⁺ and Ag⁺ were studied and the results showed that there is copper resistant gene CopD gene but fails to find silver resistant gene silC gene in At. ferrooxidans. The amino acid sequence coded by copper resistant gene was analyzed and two domains were found named CopD and PcoD. The domain CopD coded copper ion resistance protein D and the domainPcoD has a conserved sequence and presumed as a copper ion exporting protein. CopD and PcoD are both responsible to copper resistance. Another gene, arsenic resistance arsH gene of At. ferrooxidans, was amplified by PCR( Polymerase Chain Reaction ) and the resulting PCR products were cloned and sequenced. The cloned results were analyzed and compared by NCBI search tool. The amino acid sequence of arsH protein of At. ferrooxidans was compared with sequences in the NCBI database. The results indicated that there are 14 bp of the arsH gene of the tested strain which are different from those of model At. ferrooxidans ATCC 23270. arsH protein coded by arsH gene was analyzes and a functional domain named FMN _red was found which was proposed to be FMN reductase regarding sulfur as a signal. It is presumed that arsenic-resistance arsH gene is functionally related to redox of arsC reductase.Besides the effects of heavy metal ions, anions also obviously impact the oxidation capacities of ferrous ions of these bacteria and the effect orderly NO₃^-＞Cl^-＞SO₄^2-. And the bacterial tolerance levels to anions had visible differences from different strains.