Liquid Nitrogen Preservation And Study Of Molecular Polymorphisms Of Acidithiobacillus Ferrooxidans Strains

Abstract:The Acidithiobacillus ferrooxidans is an important species of bioleaching. The preservation of Acidithiobacillus ferrooxidans is vital when the strains with good properties are acquired. Research showed that the strains were diverse in physiological and biochemical and genetic trait. Study of these traits is in favor of the preservation of Acidithiobacillus ferrooxidans, the data combing of the culture collection and the preservation of gene information.Liquid nitrogen cryoprotectants polyethylene glycol (PEG) and glycerin were used as protective agents for exploration of molecular weight and concentration of effects. There are three kinds of molecular weight of PEG, PEG2000, PEG12000and PEG20000. Strains which were preserved by20%PEG2000showed highest survival rate and shortest active recovery cycle time after preservation, so its preservation effect was the best.20%PEG2000and30%glycerin were selected for long-term preservation of A. ferrooxidans. Survival rate and ferrous iron oxidation active recovery cycle were determined after preservation of specific time, namely two months, six months and one year. With the extension of storage time, the survival rate reduced, ferrous iron oxidation active recovery cycle extended. The preservation effect of20%PEG is superior to the traditional liquid nitrogen protectant, glycerin. It was characterized by shorter activation cycle and higher survival rate.20%PEG is a good protective agent for liquid nitrogen preservation of A. ferrooxidans. The preservation of20%PEG can obtain good preservation effect, namely a higher survival rate, shorter period of activation cycle compared with30%glycerin.In order to study the phylogenetic relationship and genetic features of17Acidithiobacillus ferrooxidans strains isolated from different areas, a Acidithiobacillus thiooxidans strain(A. thiooxidans A01) and three Acidithiobacillus caldus strains (A. caldus, D-1, S-1, S-2) were regarded as exogenous strains.16S rDNA and16S-23S intergenic sequences of all Acidithiobacillus strains in this study were amplified and sequenced. Similar sequences searched from NCBI database and the sequencing results of house keeping genes were used to build the complete phylogenetic trees. All Acidithiobacillus strains can be divided into four or five major groups by phylogenetic analysis of each gene. Cluster analysis of REP-PCR (repetitive element PCR) and AP-PCR (arbitrarily primed PCR) DNA fingerprinting revealed that21isolates can be divided into four genomes, and the division was in conformity with the phylogenetic grouping. A. ferrooxidans showed as two distinct groups by phylogenetic evolution and genomic grouping.In order to study the differences and similarities in iron and sulfur oxidation mechanisms between two groups of A. ferrooxidans, four genes (rusA, rusB, iro and hip) which were closely related to the sulfur and iron oxidation were amplified and sequenced. Seven A. ferrooxidans strains contain rusA gene, which encodes RUS protein. The RUS is a very important protein. It functions well in iron metabolism respiratory chain. A gene encodes a homologous protein (RusB) were amplified from other A. ferrooxidans strains, but the function of rusB is not clear at present. Allocation of hip and iro in A. ferrooxidans was almost the same as rus. All the Acidithiobacillus strains were seperated into three groups according to the different capacity of ferrous iron and sulfur oxidation. Grouping based on iron sulfur oxidation ability and grouping based on molecular characterization did not showed significant correlation.The above experimental results showed that phylogenetic heterogeneity of A. ferrooxidans was correlated with their genetic polymorphisms. Two kinds of A. ferrooxidans may have different pathways of iron metabolism. In terms of ferrous iron and sulfur oxidation, and manifestation after liquid nitrogen preservation, the different phylogenetic groups of strains showed no big difference.20%PEG is the ideal protective agent of A. ferrooxidans, it functions well in the long-term preservation.