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Isolation, Cadmium-resistance Mechanism and Cadmiums Biosorption of Heavy-metal-resistant Microbes

Author: ZengXiaoXi
Tutor: LiuXueDuan;TangJianXin
School: Central South University
Course: Biological Engineering
Keywords: heavy-metal-resistant microbe cadmium-resistant mechanism cadmium biosorption mercury-resistant gene
CLC: X172
Type: PhD thesis
Year: 2010
Downloads: 1197
Quote: 4
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Abstract


Heavy metal pollution is increasing with metals exploitation and application. Bioremediation plays a role in pollution treatment. Using the microbe isolated from indigenous soils can improve the efficiency. So the study of the indigenous heavy-metal-resistant strains is significant for bioremediation.Some strains resisted to high concentration of cadmium were isolated by acclimation of concentration gradient from the polluted soils of the smeltery in Hunan province, China. Strains marked E1 and M1 with higher resistance were selected in the following experiments. Strain E1 could survive in 18 mmol/L Cd2+ solid medium and 10 mmol/L liquid medium. M1 could survive in 80 mmol/L Cd2+solid medium and 40 mmol/L liquid medium. E1 presents Gram-negative, rod-shaped with polar flagellum, and the size is (0.5-1.0)μm×(2.0-3.0)μm. It produced green or blue pigment, and the pigment disappeared in the absence of cadmium. Based on the characterizations of the physiological and biochemical characteristics 16S rDNA sequence analysis, strain E1 was identified as Pseudomonas aeruginosa.On Czapek and PDA solid medium, strain M1 appeared white density mycelia and the bottom of plates showed yellow at the first 3-5 day. But after then the mycelia gradually became yellow or brown. Scanning electron microscope observation showed, the aerial mycelia had septate and branches, slender conidiophores grew out from aerial mycelia and conidia present oval. The strain M1 was identified as Paecilomyces lilacinus based on the morphologic characteristics and the sequences analysis of 18s rRNA, ITS region andβ-tubulin.The strains could resist not only to cadmium but also to other metals including Cu, Co, Mn, Pb and Zn. P.aeruginosa E1 and P.lilacinus M1 have broad-spectrum resistance to heavy metals and antibiotics. The growth conditions of two strains were similar. The suitable of conditions were 20-35℃, pH 6-8,80-100 mL medium in 250 mL shaker. But the optimum initial pH in medium was 7 and 6 for E1 and M1.50 mg/L Zn and Mn had some help to the growth. While 50 mg/L Pb and Co would restrain the growths of two strains in cadmium and no cadmium mediums.The plasmid did not be extracted from P.aeruginosa E1 by the methods of boiling, SDS and plasmid kit. SDS, acridine and sodium benzoate were singlely added into medium to eliminate plasmid. The resistance ability to cadmium of the cultures performed plasmid eliminations were tested and did not present obviously difference. It was demonstrated that the strain has not plasmid and the resistant gene maybe locate on chromosome. The response of strain E1 to cadmium ion was investigated based on the genes expression profiling examined by using Real-time PCR. Exposure to cadmium for 1 h, the gene czcA,czcB and czcC expression reached the highest level, and then declined to a stable range. czcD gene expression was up-regulated 15.67-fold after exposure to cadmium for 10 min. With the fast and high up-regulation expression, czcD gene was thought as more important in the response of strain E1 to cadmium stress than czcABC. Exposure to cadmium cysM gene expression was present more up-regulation than cysK. It showed that o-acetylserine sulphydrylase-B coded by the cysM played a role in cysteine biosynthesis to resist cadmium. The response of mgt gene to cadmium press was fast and the gene expression up regulated mainly in earlier stage, while the up-regulation of znu was gently and lasting.Both living and non-living cells of P.aeruginosa E1 can remove Cd from solution. After being treated for 24 h, the cadmium removal rates of the living cell and non-living cell reached 43.3% and 22.6%, respectively. It suggested that the biosorption ability of the living cell was better than the non-living cell. During the log phase, the cadmium removal by P.lilacinus M1 displayed a rising from 8.09%to 68.8%and reached a plateau phase on day 4. The biosorption amount also presented a increasing trend, from 17.98 mg/g on day 1 to 24.23 mg/g on day 3, and subsequently following a minimal decreasing. The result showed that the better cadmium adsorption by the fungus occurred on after culturing for 3-4 day.P.aeruginosa E1 and P.lilacinus M1 were single immobilized on bio-ceramic to adsorb cadmium ion from solution. The biosorptions both of batch and reactor confirmed that immobilized microbes could remove cadmium. The removal rates were 42.36% and 64.9% for P.aeruginosa E1 and M1. The EDX analysis confirmed the above result. Moreover,extracellular polysaccharide was presumed as the main role in M1 biosorption based on EDX analysis. FTIR spectrum suggested that biosorption of cadmium ion by biomass of strain E1 mainly related to C=O, C-O, C-H, O-H, C-N, N-H groups and phosphate functional groups, M1 related to C=O、C-H、N-H. The biosorption reactions occurred between cadmium ion and the above groups of biomass.Strain D2 resistance to high concentration mercury ion was isolated. Based on analysis of 16S rDNA the strain was identified as P. aeruginosa. It can grow in the soil medium containing 60 mg/L Hg2+. But when the concentration of mercuric ion in liquid medium reaches 60 mg/L, the strain hardly grows. Besides mercury, strain D2 can tolerant to other heavy metals Cu, Co, Mn, Zn, Cd, and Pb. Antibiotics disc tests demonstrated it could resistant to the 12 antibiotics. The resistance to mercuric ion of strain D2 was related to chromosome. The merRT and merA gene sequences were obtained. Sequence alignment demonstrated that the mer gene was high similar to R. Metallidurans CH34, Tn501 and so on.

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