Breeding, Biosorption Characters And Mechanism Of Heavy Metal Biosorptive Bacteria

Wastewater containing heavy metals is one of the most harmful environmental pollutants to human beings, since heavy metals in wastewater discharged into environment can be enriched by food chain, and finally enter human bodies. On the other hand, heavy metals are important non-renewable resources, the discharge of wastewater containing heavy metals not only leads to environmental pollution, but also results in huge waste of resources. Therefore,wastewater containing heavy metals must be effectively treated before being discharged into environment. Biosorption based on microorganisms and microbial aggregates, one of promising methods, has become well accepted with the advantage of eco-safety, low cost with low concentrations of heavy metals in industrial effluents. With this research background, the selection, breeding and application of heavy metal adsorptive microorganisms were investigated in this thesis. The adsorptive mechanisms were discussed. The results of this study are beneficial for providing excellent microbial resources and powerful technical support for the industrial application of heavy metal wastewater treatment by biosorption method. The main researches and results are summarized as follows.（1） Screening and adsorption test of heavy metal tolerant microbes. The metal-tolerant bacteria strains of BL9, BX and B8 were isolated from polluted environments. The results of 16 S rDNA sequence analysis indicated that thebacteria of BL9, BX and B8 belong to Stenotrophomonas maltophilia,Providencia alcalifaciens and Pseudomonas cedrina, respectively.Experimental results showed that the bacteria BL9, BX are tolerant with wide ranges of metal ions, NaCl, pH and temperature. The bacterium BL9 exhibited a high adsorptive effect towards Ni²⁺, Zn²⁺ and Cu²⁺ with the adsorption capacity of 171.16, 260.13 and 110.53 mg/g（dry bacteria）, respectively. Kinetic study showed that the pseudo-second-order kinetic model fits well with the experimental data, indicating a chemisorptions-controlled process. The isotherm biosorption of BL9 towards Ni²⁺, Zn²⁺ and Cu²⁺ was in accordance with Freundlich model, Sips model and Temkin model, respectively.Thermodynamics study suggested maximum spontaneity at 303 K, 303 K and298K in case of Ni²⁺, Zn²⁺ and Cu²⁺ removal owing to the maximum negative values of Gibbs free energy. The bacterium BX exhibited a high adsorptive effect towards Cu²⁺ and low concentration Cd²⁺, and the adsorption capacity reached 128.74 and 67.16 mg/g, respectively. Kinetic study showed that the pseudo-second-order kinetic model fits well with the experimental data. The isotherm biosorption of BX towards Cu²⁺ and Cd²⁺ was in accordance with Freundlich model and Sips model, respectively. The negative values of GqD for Cu²⁺and Cd²⁺indicated the spontaneous nature of metal biosorption on BX. The bacterium B8 exhibited a maximum adsorptive effect of 94.18% towards 50mg/L Ni²⁺, and the adsorption capacity reached 122.84 mg/g. Kinetic study showed that the pseudo-second-order kinetic model fits well with the experimental data. The isotherm biosorption was in accordance with Sips model. Thermodynamics study showed the spontaneous nature of Ni²⁺biosorption on B8.（2） Mutagenesis of metal tolerant bacterium B8. Microwave and low temperature plasma were used to mutate the strain B8. It was found that low temperature plasma is more suitable for the mutagenesis of B8. The experiments showed that the optimum mutagenic conditions of B8 by plasma are as follows:mutation time 3 min, mutation power of 50?65 W, carrier gas N2 and carrier gas pressure 20 Pa. The mutant Ni12 with high genetic stability was screened under above conditions. The Ni²⁺ adsorption capacity of the mutant Ni12 reached139.83 mg/g（dry bacteria）, 16.13% higher than that of bacterium B8, exhibiting the potential of being used in the treatment of wastewater containing nickel ions.（3） Metal resistance and adsorption mechanisms of BL9. Studies by different methods demonstrated the complex mechanisms for BL9 to sustain the high resistance and biosorption effect of heavy metals. Extra-cellular polymeric substances（EPS）, functional groups of O-H, C-O, C=O and N-H of the cell wall,and the cellular contents were found to be involved in metal resistance and removal. The experiment of iTRAQ（isobaric tags for relative and absolute quantitation） revealed that there are 528 proteins up-expressed and 474down-expressed when stressed for 16?20 hours upon metal ions. DNA sequences of Czc D and Zup T gene were amplified. An up-expressed level in CzcD and a down-expressed level in Zup T gene were found through the method of quantitative PCR in response to Zn²⁺ exposure. Therefore, not only EPS and functional groups on cell wall were explored to resist and remove heavy metals,but also the pump-out proteins to release the metal toxicity.（4） Immobilization of metal tolerant microbes and the continuous adsorption of metal ions on the biofilms. At 15 ± 2℃, the mixed cultures of BL9,BX, Ni12 and FL3 were immobilized on the carrier porous ceramics when ventilated at 32 L/h intermittently. Biofilms formed during 15?60 days after immobilization demonstrated high continuous biosorption of Ni²⁺, Zn²⁺, Cu²⁺and Cd²⁺when wastewater was pulped circularly. HCl（0.1 mol/L） showed effective desorption performance. The experimental results demonstrated the potential application of the biofilm in the treatment of wastewater.（5） Differential expression of ZjMT gene in Z. jujube and the adsorption of metal ions on recombinant E. coli cells. Bioinformatics analysis showed thatZjMT, encoding 74-amino acids homologous to the metallothionein proteins of other plant species, belongs to type 1 metallothionein. Quantitative PCR analysis of plants subjected to cadmium stress showed an enhanced expression of ZjMT in Z. jujube, confirming the important role of ZjMT in metal detoxification. ZjMT gene was integrated into E.coli cells and was expressed as a fusion protein. The recombinant E. coli cells showed high heavy-metal tolerance and binding capacity compared with the control cells. Therefore,ZjMT gene is responsible for the tolerance to, and the accumulation of, heavy metals, in vitro as well in vivo.