【作者】 黄峥；

【导师】 闵航；

【作者基本信息】 浙江大学 ， 微生物学， 2006， 硕士

【摘要】 我国的耕地资源极其匮乏,近年来其数量还在不断减少,成为限制农业可持续发展的重大障碍;另一方面,我国的土壤重金属污染问题日益严峻,不仅危及人类健康,还引起了一系列复杂严重的环境问题,应当引起社会各界的广泛关注。 本研究以稻田土壤为材料,以在我国稻田土壤中污染最为严重的铜、镉为研究对象,采用室内培养、纯培养及统计分析相结合的方法,针对铜离子单一污染及铜镉离子复合污染对土壤中微生物、土壤酶的毒性效应进行比较研究。通过对比分析,研究了铜镉离子对土壤生物学毒性的不同复合效应,并进一步探讨了稻田重金属污染状况与土壤生物学活性之间的相互关系。为重金属复合污染的生物学效应有别于重金属单一污染这一观点提供直观的试验证据,并为确立稻田土壤铜镉污染的早期预警指标提供基础资料。此外,分离筛选到一株高抗铜镉的抗性菌株,为铜镉污染稻田土壤的生物修复提供理论依据和种质资源。主要研究结果如下: 1.以稻田土为材料,在实验室条件下对铜离子单一污染与铜镉离子复合污染对淹水稻田土壤好氧微生物种群数量、区系的多样性影响进行比较研究。结果表明,培养初期（5d）低浓度200mgCu²⁺/kg干土和(200mgCu²⁺+0.5mgCd²⁺)/kg干土的铜镉离子对细菌、放线菌、真菌的生长均有一定的刺激作用。放线菌对铜镉离子极为敏感,数量变化与铜镉离子浓度之间存在显著的剂量效应关系,故可将放线菌作为土壤铜镉污染的早期预警指标。对单一污染与复合污染的抗性次序均为细菌>真菌>放线菌。Cu²⁺和Cd²⁺对细菌和放线菌产生的抑制作用低于Cu²⁺单一污染,毒性作用可能存在拮抗,但拮抗作用并不显著;而对于真菌,铜镉复合污染尤其是高浓度的复合污染产生的抑制作用较Cu²⁺单一污染更强,两者之间可能产生了毒性的协同作用。 2.采用改良的亨格特（Hungate）厌氧技术,通过试管液体培养法及滚管固体培养法系统研究了厌氧水解发酵细菌、厌氧固氮菌和反硝化细菌等几种土壤主要厌氧微生物种群数量在不同污染条件下的动态变化过程。结果表明,厌氧水解发酵细菌与厌氧固氮菌对单一Cu²⁺抗性较强,尤其厌氧固氮菌,12d起各个处理的固氮菌数量呈大幅增长;反硝化细菌对Cu²⁺敏感,可作为Cu²⁺土壤污染的早期预警指标。无论是Cu²⁺单一污染,还是Cu²⁺和Cd²⁺复合污染,对其抗性依次为厌氧固氮菌>水解发酵细菌>反硝化细菌。单一污染与复合污染对反更多还原

【Abstract】 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.更多还原