The Isolation,Identification Of Cd-resistant Bacteria And Their Applications In The Remediation Of Cd-contaminated Environments

Owing to the wide use of cadmium（Cd）and its compounds in human industrial and productive activities,there is a serious environmental problem in both developing and developed countries for the unregulated emissions of the waste water and residues containing this metal,which leads to a series of cadmium contamination.Because of the threats to human health and the environment,problems about Cd-contaminated environments remediation have received great attention in recent years.In this study,a bacterium Achromobacter sp.380 was isolated and evaluated its effect for the in situ remediation of contaminaed agriculture soils.The metal tolerance ability and biosorption characteristics of this bacterium for Cd²⁺were investigated as well.Moreover,another Cd-resistant bacterium Pseudomonas putida X4 isolated in the previous study was evaluated the remediation effect of cadmium contamination by the leaf yield,the Cd content and Cd fractionations of the tobacco in pot experiments.A compound way to remediate the Cd-contaminated（1mg/kg）agriculture soil with bio-organic manure and lime powder was also exploited.The main results were listed as follows:（1）Three bacterial strains 368,375 and 380 with a strong metal resistance were isolated and identified from heavy metal contaminated soils.By phylogenetically analysis,they are named as Brevundimonas sp.368,Pseudomonas sp.375 and Achromobacter sp.380,respectively.Achromobacter sp.380 has higher metal resistance.Its minimum inhibitory concentrations for Cd²⁺,Mn²⁺,Cr⁶⁺,Cu²⁺,Zn²⁺and Co²⁺were 8,12,1,4,12,6and 4,8,0.125,4,8,6 mmol/L in the LB and M9 liquid media,respectively.Moreover,it can resist kinds of antibiotics.（2）The optimum p H was 6.0 without Cd²⁺in the culture,but it was changed to 8.0after added with 6mmol/L of Cd²⁺in the medium.Achromobacter sp.380 could decrease the Cd content by 71.41%when grown in the medium with 6mmol/L of Cd²⁺in 72h.In addtion,the bacterium can growth well in the medium with p H ranged from 6.0 to 10.0.It indicated Achromobacter sp.380 have high Cd-resistance and Cd-biosorption characterias.Fourier transform infrared spectroscopy（FTIR）analysis showed that the strain can produce more functional group especially the group-OH and-COOH for Cd²⁺biosorption under Cd stress.At the same time there are many other dominant binding sites on the 380cell wall for Cd²⁺.-NH,-CH₂,-CN were also involved in binding of Cd²⁺.The results showed that strain 380 can colonize under high Cd stress and has great potential in the in situ remediation of heavy metal-contaminated environments.（3）The inoculation of Achromobacter sp.380 markedly decreased the uptake of Cd by ryegrass and enhanced the ryegrass growth in pot experiments with various levels of Cd contamination（0mg/kg-3mg/kg）.In comparison with control treatment,the application of Achromobacter sp.380 transformed 12.50%-24.86%of soluble/exchangeable Cd to the organic-bound fraction and consequently reduced the ryegrass Cd content of shoots and roots by 35.32%-40.98%and 11.89%-48.38%respectively,improved plant yield by 2.34%-6.77%in Cd-contaminated soils.This result indicated that Achromobacter sp.380 as a metal immobilizing agent is more suitable to remediate Cd-contaminated soils.（4）The metal resistance experiment indicate that P.putida X4 have strong metal resistance,with the minimum inhibitory concentrations for Cd²⁺,Mn²⁺,Pb²⁺,Cr⁶⁺,Cu²⁺,Zn²⁺,Co²⁺were 4,12,12,1,4,10,and 8 mmol/L in liquid LB medium and 2,6,4,0.5,4,5,and 4 mmol/L in M9 medium,respectively.In addition,the strain can resist ampicillin（100mg/L）,chloromycetin（50mg/L）,spectinomycin（25mg/L）and streptomycin（5mg/L）.The biosorption experiment with P.putida X4 live and dead cells indicated that the optimum p H for biosorption of Cd²⁺is 5.0 and the optimum biomass dose is 2g/L for the removal of Cd²⁺from aqueous.The biosorption data obtained by P.putida X4 live and dead cells biomass were quite fitted to Langmuir isotherm model and pseudo-second-order Lagergren kinetic model.According to the Langmuir isotherm,the maximum adsorption of Cd²⁺were 46.10 mg/g and 78.72mg/g by live and dead cells dry biomass,respectively.The results indicate that P.putida X4 biomass is an efficient biosorbent for Cd²⁺and has great potential for the in situ remediation of heavy metal-contaminated environments,especialy for removal Cd²⁺from the aquoues effluent by dead cells.（5）In the tobacco pot experiments,compared with the control treatment,the application for P.putida X4 treatment（X4）can transform 7.47%soluble/exchange Cd to complex Cd,reduced the Cd content of tobacco leaf by 37%and increased the tobacco leaf yield by 13.17%at the harvest periods,which indicated the effect of the bacterium P.putida X4 for the in situ soil remediation is very prominent.When P.putida X4application combined with bio-organic manure or lime powder,the effect for remediation is better than P.putida X4 alone,especially the combined application of bio-organic manure,lime powder and P.putida X4 treatment（B+L+X4）together could increase tobacco yield by 63.08%,decreased the tobacco Cd content by 67.07%,and changed64.14%soluble/exchange Cd to a low toxicity state of complex Cd.In this experiment,all the treatments with P.putida X4 alone or not can enhance plant yields,and decrease Cd content in plants and reduce soil-bioavailable Cd fractions.In addition,the immobilization of Cd can be strengthened in soil.The optimum remediation way is to combine the bio-organic manure,lime powder and P.putida X4,which is a low cost,high efficiency and eco-friendly remediation method for the restoration of Cd-contaminated soils.