Effects Of Mono-contamination Of Cu～(2+) And Associated-contamination Of Cu～(2+) And Cd～(2+) On Microecosystem In Paddy Soil And Isolation, Characterization Of A Cu～(2+)-Cd～(2+)-resisting Bacterium

The infield resource in china, which is destitute and reducing continually years-by-year, has badly impeded the Sustainable development of agriculture. Meanwhile, pollution of heavy metals, which should be paid more attention to, has not only brought complicated environmental problems from bad to worse, but also disserved people's health.In this thesis, biological community assessments including cuturable bacteria and soil enzyme activities were carried out under laboratory condition to investigate comparatively the effects of mono-contamination of Cu²⁺ and associated-contamination of Cu²⁺ and Cd²⁺ at different concentrations on soil micro ecosystem. A bacterial strain which resists highly to Cu²⁺ and Cd²⁺ was also isolated and identified. These results will be useful reference to building up alert index systems in Cu-Cd-polluted paddy soil, environmental quality evaluation and bioremediation of Cu-Cd-polluted paddy soil. The main results are reported as follows.1. Changes of the amounts of three aerobic microbes in soil polluted with mono-contamination of Cu²⁺ and associated-contamination of Cu²⁺ and Cd²⁺ were simultaneously observed during 40-day period. Results showed that there was a slight stimulation to the culturable bacteria, actinomycetes, fungi during the initial incubation stage (5 days) of the soil samples with low concentrations of 200mgCu²⁺/kg dried soil and (200mgCu²⁺+0.5mgCd²⁺)/kg dried soil. Actinomycetes appeared sensitive to heavy metals. There existed an obvious negative relationship between does of heavy metals and microbial population. The resistance order was bacteria > fungi > actinomycetes for aerobic bacteria to no matter Cu²⁺ or associated-contamination of Cu²⁺ andCd2+. It is likely to be seen that there was an antagonistic effect between Cu2+ and Cd2+ to aerobic bacteria and actinomycetes when they were applied together, the association inhibition of Cu2+ and Cd2+ seemed not so intense as that of mono-pollution of Cu2+ applied but enhanced inhibition to fungi was investigated here, it could be conjectured that there was a synergistic effect of association of Cu2+ and Cd2+.2. The influence of Cu2+ and mixture of Cu2+ with Cd2+ on four anaerobic microbes in paddy soil was assessed using the improved Hungate anaerobic technique, including anaerobic MPN (most probable number) and roll tube methods. Results indicated that hydrolytic-fermentative bacteria and anaerobic nitrogen-fixing bacteria resist mightily to Cu2+, especially the anaerobic nitrogen-fixing bacteria, it had a definite increase after 12 days of the soil samples with each concentration of Cu2+ applied;denitrifying bacteria was sensitive to Cu2+, its population could be used as indexes to indicate the contamination degree of paddy soil by Cu2+. The resistant order of anaerobic bacteria to no matter the pollution of Cu2+ or Cu2+ and Cd2+ mixture from strong to weak was anaerobic nitrogen-fixing bacteria > hydrolytic-fermentative bacteria > denitrifying bacteria. The inhibition of Cd2+ and Cu + has been enhanced applied together to hydrolytic-fermentative and anaerobic nitrogen-fixing bacteria, it can be estimated that there was synergistic effect between Cu2+ and Cd2+.3. The activities of three types of enzymes, including catalase, phosphorylase and urease were measured to evaluate the different biological effect of Cu2+ and associated-contamination of Cu2+ and Cd24 to soil environment. Results pointed out that the pollution of association-contamination of heavy metals was not only different from simple element, but also the distinct effects on soil enzyme activities. In paddy soil, Cu2+ inhibited the enzyme activities in this orderof urease > phosphatase > hydrogen peroxidase, differed from the inhibition order of phosphatase > urease > hydrogen peroxidase for Cu2+-Cd2+ associated-contamination. Urease and phosphatase activities decreased significantly with increasing contamination and the multivariate regression analysis indicated that urease and phosphatase activities were very significantly related to both mono-contamination of Cu2+ and Cu2+-Cd2+ associated-contamination. The characteristics of Cu2+-Cd2+ associated pollution for urease exhibited synergistic effect, however, antagonistic effect for phosphatase and hydrogen peroxidase at different levels. It suggested that it is feasible to use urease and phosphatase activities as a primary biochemical parameter to evaluate and assess heavymetals compound pollution in paddy soil.4. A bacterial strain HLC2, resisting highly to Cu2+ and Cd2+, was isolated from soil sample polluted by chemical wastewater for half century. The strain was identified as Brevundimonas diminuta based on morphological, physio-biochemical characteristics, Biolog-GN and the 16S rDNA sequence analysis. The cells of the strain were non-motile, non-spore, short-rod and Gram-negative;the colonies formed on agar medium were pinpoint, hard picking up and configuration of straw hat.Strain HLC2 is more sensitive to pencicillinum and benzylpenicillinum than erythromycin and cefuroxime within the tested concentrations but it has a stronger resistance to gentamicin, amikacin and chloramphenicol. The optimal temperature ranges from 30 °C to 37°Cand the optimal pH of 8.5 for its growth. Cu2\ Cd2\ Zn2\ Pb2\ Cr24 and Hg2+ should be essential element for strain HLC2 in basal medium and the optimal concentration for its growth respectively was4900mg/L 5900mg/L, 600mg/L, lOOOmg/L, 500mg/L, 600mg/L and 5mg/L, and Cd2+ 600mg/L + Cu2+ 2000mg/L for associated treated by Cu2+ and Cd2+. The adsorption amount of strain HLC2 to Cu2+ was equal to Cd2+ when grown separately on medium containing Cu + or Cd +, meanwhile, nearly 5 times more when containing Cu2+ and Cd2+ together. It seemed that strain HLC2 has the higher adaptability and could decrease more amount of Cu2+ and Cd2+ within same time when both of Cu2+ and Cd2+ existed. Further studies are necessary to determine the potential use of the isolate in the disposal of Cu and Cd in agriculture and industry processes.