Studies On Variation Of Microbial Activity And Actinomycete Population Under Cadmium Stress As Well As Cadmium Bioadsorption

Soil microbial physiological and ecological characteristics in dry land soil contaminated by heavy metal Cd²⁺ were studied using modern molecule biology methods.Streptomyces sp., designated strain HL-12,capable of inhibiting the growth of Cmm and one,designated strain K33, capable of growing at high Cd²⁺ concentration were isolated and identified.Bioadsorption kinetics and mechanism of the two actinomycete strains in the liquid environment contaminated by Cd²⁺ was also studied in order to set up a useful reference for building up alert index systems in soil polluted by Cd²⁺,environmental quality evaluation.Here are presented the main results of this study:1.Efficient total soil community DNA extraction methods were etablished.Total soil community DNA used for PCR was extracted and the sequence amplified with actinomycetic special primer was got.Variations of actinomycetic gene diversity in soil stressed by Cd²⁺ were observed using the denaturing gradient gel electrophoresis(DGGE) technique.The result showed that there were some significant differences between actinomycetic communities in soils treated with different concentrations of Cd²⁺ and sampled at different cultivation times.Cd²⁺ had obvious toxicity on actinomycetic community during the earlier days after treatment,making gene diversity decreased.There were,however,less differences among variations of actinomycetic genes from soil treated with different concentrations of Cd²⁺ during the later stage after Cd²⁺ treatment than that in the earlier stage,but it could affect the abundances of various kinds of actinomycete.Obviously,it is possible to provide a new way and foundation to illustrated actinomycetic gene diversity in Cd²⁺ contaminated environments.A new band appeared at the lane of extracted DNA from the soil treated with Cd²⁺ of 0.5～1.0 mg·kg^-1 dried soil in the DGGE pattern during the earlier period,but no at that from other treatments.The phenomena mean that there was some actinomycete which could grow in the soil treated with suitable concentrations of Cd²⁺.The sequencing result of the new band showed that there was about 95.9%similarity between the sequences from the isolated strain and Rhodococcus sp.M51-3.3 accessed on the GenBank database.The sequence of another band only appearing in the treatments without receiving any Cd²⁺ showed 98.2%similarity to that of Streptomyces sp.343E07.It is feasible to study as the gene indexes for indicating the contamination degree of soil by Cd²⁺.2.An actinomycete,designated as strain K33,capable of growth in the presence of 300 mg Cd²⁺·l^-1 was isolated from metal contaminated soil and identified as Streptomyces psammoticus and one actinomycete,designated strain HL-12,capable of inhibiting Cmm was isolated from a farmland on Huajiachi compus of Zhejiang University and identified as Streptomyces vinaceus both based on observation,G+C mol%,the 16S rRNA sequence analysis and so on.Unrooted phylogenetic tree was constructed using DNAStar software based on the 16S rRNA sequences of the isolated strains and other strains closed to the isolates.Strain K33 had high resistance to Zn²⁺ and Pb²⁺ but low resistance to Cu²⁺.Strain HL-12 had high inhibition to Cmm and other Gram-positive strains and low inhibition to Gram-negative ones.However HL-12 had little inhibition to fungi,such as Rice Blast Fungus.The diameter of strain HL-12 fermentation broths inhibiting to Cmm was 11mm by filter paper method,while was 13mm by micro-solid agar block method.3.Antimicrobial activity of strain HL-12 both in pure culture and sterile soil stressed by Cd²⁺ was studied.The result showed that the secondary metabolism of strain HL-12 was accelerated with the treatment of suitable Cd²⁺ concentration,making the antimicrobial activity enganced. However,it could cause toxicity to strain HL-12 when the stress of Cd²⁺ exceeded the regulation of itself,leading to the negative effect on the antimicrobial activity.Otherwise,it could reduce the toxicity of Cd²⁺ to strain HL-12 at some extent when adding Ca²⁺.It was propitious to inhition of strain HL-12 to Cmm when used with low concentration of Ca²⁺,while treated with high concentration of Ca²⁺,the inhibition of strain HL-12 to Cmm dropped.Strain HL-12 still had some inhibition to Cmm when Cd²⁺ concentration exceeded the critical point in contaminated soil(1.0 mg·l^-1).As far as the Cd²⁺ concentration was 3～5 times as critical point one,there was a certain extent negative effect on the antimicrobial activity of HL-12 to Cmm.4.Biosorption of Cd²⁺ by the two strains was studied.The result showed that biosorption equilibrium was established earlier by strain K33 than that by HL-12,and K33 had higher adsorption capacity.Langmuir,Freundlich,and Dubinin-Radushkevich(D-R) isotherms were used to describe Cd²⁺ the adsorption processes,Langmuir model fitted the experimental data best. Strain K33 showed greater sorption capacity with 38.49 mg Cd²⁺/g dry cells.Pseudo-first and second-order kinetic models were used to describe the kinetic data,and second-order kinetic model fitted better.About 80%recovery of Cd²⁺ could be obtained at pH≤3 from metal-loaded biomass of strains HL-12 and K33,respectively.5.The biosorption and resistance mechanism of actinomycete to Cd²⁺ was also studied.The result showed that,first,the adsorption of Cd²⁺ by DNA,protein,lipids and amylose in cell had some differences.Of the two strains,Cd²⁺ was combined mainly by protein and amylose,while the composing of strain K33 presented more adsorption ability than that of strain HL-12;second, the investigation of transmission electron microscopy showed that Cd²⁺ exhibited more effect on the changes of strain HL-12 configuration.When HL-12 was repressed by Cd²⁺,the cell volume diminished,bursa wall was changed into amoeboid and largened in the cell.Parts of the cells' surface were destroyed.While taking strain K33 into account,few cells appeared breached,but still remained whole one.In addition,the colour of K33 repressed by Cd²⁺ was darker than that not repressed by Cd²⁺.The surface of strain K33 repressed by high concentration of Cd²⁺ was rougher, and had some crystal granules attached on it.Third,FT-IR was used to characterize the interaction between Cd²⁺ and strain K33 and HL-12,respectivel,revealing that the presence of amino, carboxyl,hydroxyl and carbonyl groups were responsible for biosorption of Cd²⁺.Strain HL-12 had more changes in the functional groups than stain K33.