Screening Of Heavy Metal-Resistant Actinomycetes From Soil Of Tailings And Sewage Irrigation Areas And Research Of Adsorption Mechanism

Actinomycetes are a kind of main microbiology in soil. It has potential application value in the microbial remediation due to its special morphological and physiological metabolism. Excellent heavy metal-resistant actinomycetes were isolated and screened from contaminated soils in heavy metal tailings and sewage irrigation areas. The diversity of these actinomycetes was researched and the potential novel species were identified by polyphasic taxonomy. The adsorption effect was compared between the heavy metal-resisitant strain and the sensitive strain. The adsorption conditions were optimized by orthogonal experiments and adsorption kinetics were researched under efficient and stable adsorption conditions. At the same time, many measures were applied in the adsorption characteristics of the heavy metal-resisitant strain. The mechanism of actinomycetes adsorbing cadmium and zinc were clarified. Major findings and conclusions were summarized in the following:(1) 18 heavy metal-resistant actinomycete strains were screened out from contaminated soils in heavy metal tailings and sewage irrigation aeras. The strain K42(=CCNWNQ0016^T), isolated from lead-zinc tailings, could high resist cadmium and zinc. It could grow in liquid medium of GA contained 5.5 mmol·L^-1 Cd²⁺ and 6.0 mmol·L^-1 Zn²⁺. Strain K42 could strongly resist zinc on solid medium of GA and its maximum resistant levels (MRL) to zinc was 35 mmol·L^-1 and to cadmium was 22 mmol·L^-1.(2) The diversity of 18 strains heavy metal-resistant was researched by classical taxonomy. Results showed that they were Streptomyces and preliminary ranked 8 groups: Griseofuscus (5 strains), Aureus (3 strains), Cyaneus (3 strains), Griseorubroviolaceus (2 strains), Albosporus (2 strains), Lavendulae (1 strains), Flavus ( 1 strain) and Cinerogriseus (1 strain). The evolutionary status of 18 strains was confirmed through 16S rDNA sequence analysis of system development. Some novel species, strain K42, H31, K44 and K30, were found. There were some non- Streptomyces in soil contaminated by heavy metal-resistant through cultural and morphological characteristics. Through 16S rDNA sequence analysis of these non-Streptomyces, 8 strains belonged to 6 genuses that were Micromonospora, Catellatospora, Streptosporangium, Actinoplanes, Nonomuraea and Corynebacterium. Among of them, strains K55, H1 and H6 perhaps were novel species because the similarity between strain K55, H1 and H6 and its closed species was low that were 97.83% 98.65%.(3) The strain K42 and strain H31 were identified by polyphasic taxonomy. Strain K42, that was CCNWNQ 0016^T (= ACCC 41871^T=HAMBI 3107^T) isolated from lead-zinc tailings, denominated Streptomyces zinciresistens. Cells of type strain K42 were Gram-negative, aerobic, resisting zinc and cadmium and the MRL to Cd²⁺ and Zn²⁺ were 35 mmol·L^-1and 22 mmol·L^-1. The DNA G+C content of the type strain K42 is 70.3 mol%. Strain H31, that was CCNWHQ 0031^T(= CCNWTJ 0031^T=JCM 16925^T= ACCC 41873^T)isolated from sewage irrigation aeras, denominated Streptomyces shaanxiensis. It was Gram-negative, aerobic, resisting some heavy metals. It formed light greyish blue aerial mycelium, dark blue substrate mycelium and dark blue diffusible pigment on Gause's synthetic agar. The DNA G+C content of the type strain H31 is 70.7 mol%.(4) Four kinds of heavy metal ions had significant toxicity effect on the strain K42, and the toxicity from the highest to the lowest ranked as Cu²⁺>Ni²⁺>Cd²⁺>Zn²⁺. Strain K42 could grow best under pH 7 or with 4% NaCl. Biosorption capability of strain K42 to cadmium and zinc were effeced by some factors and. Results showed that initial concentration of Cd²⁺ and Zn²⁺, initial pH and inoculation dose had significant effect on biosorption capability of strain K42. However, agitation speed and biosorption temperature had no significant effect. L9(34) orthogonal experiments were designed founded on results of single tests and the optimum conditions for cadmium biosorption by heavy metal-resistant strain K42 were definited to be initial Cd²⁺ concentration 2.0 mmol·L-(1224 mg·L^-1),initial pH4 and 1% inoculation dose. The removal efficiency of Cd²⁺ by K42 reached 91.56%. And the optimum conditions for Zn²⁺ biosorption by K42 were definited to be initial Zn²⁺ concentration 2.0 mmol·L^-1(130 mg·L^-1),initial pH5 and 1% inoculation dose. The removal efficiency of Zn²⁺ by K42 reached 88.14%. However, the optimum conditions for cadmium and zinc biosorption by heavy metal-sensitive strain H7 were definited to be initial Cd²⁺ and Zn²⁺ concentration 2.0 mmol·L^-1, initial pH 5 and 1% inoculation dose. The removal efficiency of Cd²⁺ and Zn²⁺ by strain H7 arrived at 90.33% and 91.49%. The biosorption capacity of Cd²⁺ and Zn²⁺ by mycelia could improve after pretreatment of heating.(5)Compared the biosorption effect of heavy metal-resistant strain K42 and heavy metal-sensitive strain H7 to Cd²⁺ and Zn²⁺. The removal efficiency of strain K42 to Cd²⁺ was more than strain H7, whereas removal efficiency of strain H7 to Zn²⁺ was more than strain K42. Langmuir and Freundlich isotherms were used to describe Cd²⁺ and Zn²⁺ the adsorption processes, and Langmuir model fitted the experimental data best. Pseudo-fiest and second-order kinetic models were used to describe the kinetic data, and second-order kinetic models fitted better. About 98.11% and 88.55% recovery of Cd²⁺ could be obtained at pH=2 from metal-loaed biomass of strain K42 and H7, respectively. About 87.33% and 76.45% recovery of Zn²⁺ could be obtained at pH=1 from metal-loaed biomass of strain K42 and H7, respectively. The biosorption of two strains to Cd²⁺ and Zn²⁺ was restrained due to a variety of heavy metal ions coexisting, especially Cu²⁺ and Cr³⁺.(6) The biosorption and resistance mechanism of strain K42 to Cd²⁺ and Zn²⁺ were also studied. Cellwall of strain K42 was the main part to adsorbing Cd²⁺ and Zn²⁺. Second was intracellular accumulating during logarithmic phase. And the biosorption of cell surface was least when strain was growing. Strain K42 could secrete alkaline substances with or without heavy metal and even produce more alkaline substances with heavy metal making the pH value of medium rising from 6.77 to 8.17. Scanning electron microscopy and transmission electron microscopy results showed that cell morphology and intracellular morphology metal-loaded all changed. Scanning electron microscopy results showed that there was sedimentation floe at surface of cells with lower heavy metal concentration, whereas the cells improved the cadmium and zinc resistance by deformation, fracture and aggregation and there were many crystals and spherical precipitation. Transmission electron microscopy results showed that the cells volume became small and many electron density particles gathered in the intracellular sites due to Cd²⁺ and Zn²⁺ adsorption and accumulation. Based on the results of the energy spectrum analysis, there were many Cd²⁺ and Zn²⁺ on the surface of cells and there were zinc complex in cell. It was speculated that the Cd²⁺ and Zn²⁺ were combined with–OH,–NH2,–COOH,–CO–NH–,–CN, and so on by infrared spectroscopic analysis. Ni²⁺ could improve adsorption sites of cellwall and enhance the adsorption ability of cellwall. However, Cu²⁺ could restrain more groups activity and reduce the biosorption of strain to Cd²⁺ and Zn²⁺.