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Background: Traditional methods of obtaining arsenic have disadvantages such as high cost and high energy consumption. Realgar is one of the most abundant arsenic sulphide minerals and usually treated as waste in industry. The aim of the present study was to screen an arsenic tolerant bacterium used for bioleaching arsenic from realgar. Results: An acidophilic iron-oxidizing bacterium BYQ-12 was isolated from Wudalianchi volcanic lake in northeast China. BYQ-12 was a motile, rod-shaped gram-negative bacterium with an optimum growth at 30°C and pH 2.5. 16S rDNA phylogeny showed that BYQ-12 was a new strain of Acidithiobacillus ferrooxidans. The inhibitory concentrations (ICs) of arsenite and arsenate were 32 and 64 mM, respectively. A significant second-order model was established using a BoxBehnken design of response surface methodology (BBD-RSM) and it estimated that a maximum arsenic bioleaching rate (73.97%) could be obtained when the pulp concentration, pH and initial ferrous ion concentration were set at optimized values of 0.95% w/v, 1.74 and 3.68 g/L, respectively. SEM, EDS and XRD analyses also revealed that there was direct bioleaching besides indirect electrochemical leaching in the arsenic bioleaching system. Conclusion: From this work we were successful in isolating an acidophilic, arsenic tolerant ferrous iron-oxidizing bacterium. The BBD-RSM analysis showed that maximum arsenic bioleaching rate obtained under optimum conditions, and the most effective factor for arsenic leaching was initial ferrous ion concentration. These revealed that BYQ-12 could be used for bioleaching of arsenic from arsenical minerals.
Subject(s)
Arsenic/metabolism , Arsenic/chemistry , Acidithiobacillus/isolation & purification , Oxidation-Reduction , Hydrogen-Ion Concentration , Iron/metabolismABSTRACT
OBJECTIVE: To detect the key arsenic-resistance domain in the second transmembrane domain (TMD2) of ABCA1 protein. METHODS: These mutant genes were amplified by gene splicing by overlap extension, then transfected into HeLa cells. The location and arsenic-resistant ability of the mutant were investigated by confocal microscopy, CCK-8 assay and atomic fluorescence spectrophotometry (AFS). RESULTS: These mutants were all located at the plasma membrane. HeLa cells were exposed to arsenic for 48 h, then CCK-8 assay showed that the survival rate of these cells in ABCA1 mutant groups were higher than that of the control group at all concentrations of arsenic as the wild-type ABCA1 (WT), except the sixth extracellular loop. The difference of the survival rates between the two groups had statistical significance (P < 0.01). The IC50 of the WT and ABCA1 mutant groups were 33.18, 32.84, 33.45 and 34.29 μmol · L-1, respectively, which were higher than the 19.01 μmol · L-1 of the control group and 19.95 μmol · L-1 of the sixth extracellular loop. At the same time, the AFS determination showed similar result as the CCK-8 assay. CONCLUSION: ABCA1 mutants were successfully constructed, and the expressed protein was correctly located at the plasma membrane with some arsenic-resistant ability, which was not different with wild-type ABCA1, except the sixth extracellular loop. Therefore, it is possible that the sixth extracellular loop may be a key arsenic-resistance domain in ABCA1 gene.
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Objective To examine the expression of ATP-binding cassette transporter A1(ABCA1)in eukaryotie cells and the effect of arsenic resistance after the transfection of eukaryotic expression vector containing ABCA1 gene.Methods HeLa cells were transfected with the recombinant plasmid by lipofectaonmine 2000 (recombinant plasmid group),empty plasmid and untransfected HeLa cell as the control group.The level of the mRNA was examined by real-time PCR,and the expression of ABCA1 protein wag examined by Western blot,the change of cell survival rate was examined by methyl thiazolyl tetrazolium(MTT)after exposure in a series of arsenic [0(contro1),4,8,16,32,64,128 μmol/L]for 48 hours.Results Expression level of ABCA1 mRNA in recombinant plasmid,empty plasmid and untransfeeted groups was(2.09±0.08)×10-4,(0.09±0.02)×10-4,(0.08±0.02)×10-4,there was a significant difference between the groups(F=1499.23,P<0.01).The level of ABCA1 mRNA in recombinant plasmid group was higher than empty plasmid and untransfected group(all P<0.01).Western blot showed that specific protein straps existed at 254×103 in all the three groups,with a similar size to the ABCA1 protein.The amount of the recombinant plasmid group was higher than the other two groups.MTT shows that arsenic concentration at 4,8,16,32,64,128 μmol/L,the survival rates of recombinant plasmid group was(94.8±0.9)%,(86.5 ± 2.6)%, (77.8 ± 2.0)%, (56.0 ± 2.0)%, (23.8 ± 1.7)%, (18.6 ± 0.6)%, higher than that of empty plasmid group[ (85.3 ± 1.1)%, (78.7 ± 0.6)%, (67.8 ± 2.4)%, (43.2 ± 1.5)%, (14.5 ± 1.3)%, (8.0 ± 0.4)%], the difference of survival rate had a statistical signifieance(t = 18.985,6.689,5.922,9.504,9.481,32.634, all P < 0.01). Conclusions ABCA1 protein is over expressed in HeLa cells after transfect ABCA1 gene. ABCA1 protein increases resistance of arsenic in HeLa cells.
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Objective To obtain stable arsenic-resistance cells,the fetal bone marrow mesenchymal stem cells(BMSCs)were exposed to low-level arsenite for 18 weeks. Methods Cells from 4 months fetal bone marrow were cultured in ?-MEM medium to obtain BMSCs.After 24h cytotoxicity test of the fetal BMSCs,we chosed 1?mol/L NaAsO_2 to be the dose under which the cell death-rate was 5%-10%.The fetal BMSCs were exposed to low-level arsenite.MTT was used to detect the survival rate and IC_(50) of arsenic-exposed cells and the control cells,which can reflect the change of arsenic tolerance.A hydride generationatomic fluorescence spectrometry method was used to detect arsenic in the arsenic-resistance cells and the control cells.In order to study the mechanism of arsenic-resistance,we also examined the intracellular GSH and GST content in the arsenic-resistance cells and the control cells. Results The fetal BMSCs were continuously exposed to 1?mol/L NaAsO_2.Parallel cells were cultured in medium without arsenic provided passage-matched control.After the fetal BMSCs were continuously exposed to low level NaAsO_2 for 18 weeks,cells exhibited dramatic resistance to acute arsenite toxicity.Compared to control cells,arsenic-resistance cells showed reduction in arsenic accumulation in cells.The GSH levels and GST activity of arsenic-resistance cells were higher than that of control cells.Conclusion Acquisition of stable arsenic-resistance in BMSCs was available by using cell culture and adding low-level arsenic.Our research established a solid basis for further study about the mechanisms of arsenic-resistance in human being.