【作者】 宣瑛；

【导师】 闵航；

【作者基本信息】 浙江大学 ， 微生物学， 2006， 硕士

【摘要】 本文以华家池菜园土为研究材料,采用室内培养、纯培养与数理统计分析相结合的方法,探讨了重金属镉和除草剂乙草胺对旱地土壤中自生固氮菌数量及酶活变化的影响,并筛选、分离和鉴定了一株高效自生固氮菌,对污染物胁迫下纯菌的生长和固氮酶活及氧化应激反应进行了研究,对C4菌株抗污染的机理进行了初步的探讨,为建立有效的镉和乙草胺复合污染预警指标体系、环境质量评价提供了有益的参考。本研究所获得主要结论如下: 1.采用传统方法研究了镉和乙草胺单一及复合污染对旱地土壤自生固氮菌生长及对土壤固氮强度的影响。研究结果表明,土壤中好氧固氮菌对Cd²⁺非常敏感。低浓度的Cd²⁺可以刺激土壤中好氧固氮菌的数量,但高浓度对其有强烈的抑制作用。而乙草胺对好氧固氮菌数量没有实质上的影响。Cd²⁺对前8d土壤中的厌氧固氮菌数量表现为强烈的刺激作用,随后固氮菌数量下降。最终除最高浓度组(5.0mg·kg^-1干土)受明显的抑制作用,其余组与对照相当。从长期来看,施用乙草胺后厌氧固氮菌数量变化不大,但施用高浓度时（2.0mg/kg干土）可显著抑制固氮菌的数量。Cd²⁺与乙草胺复合污染对土壤好氧和厌氧固氮菌均表现为协同毒性效应。Cd²⁺对培养末期土壤固氮强度的影响表现为负相关作用,呈良好的浓度—剂量效应。而一定浓度范围内的乙草胺可以在短期内刺激固氮酶活性,但最终则表现为抑制作用。两者复合污染下的固氮酶活前期表现为协同刺激作用,而末期则为协同毒性效应。 2.从菜园中分离得到一株高效自生固氮菌株C4。C4菌株16S rDNA与GenBank数据库中拜式固氮菌（Azotobacter beijerinckii）的16S rDNA序列具有99%同源性,结合形态、生理生化特性判断,该菌为拜式固氮菌。对该菌固氮酶活的测定发现,菌株在培养24h时固氮酶活性最高,乙烯生成量达1203.779nmol/h.ml菌液,随后酶活逐步下降,至90h时的酶活仅75.66nmol/h.ml菌液。菌株的抗药性的检测结果表明,菌株C4对庆大霉素、氨苄青霉素、丁胺卡那霉素、红霉素敏感,而对青霉素钠、头孢霉素和氯霉素不敏感。 3.以菜园里分离得到的固氮菌株C4为实验材料,通过对其生长和固氮酶活两方面的影响,研究了Cd²⁺、乙草胺两者复合污染对C4菌株的毒害作用,初步获得以下结论:Cd²⁺对刚培养或已培养12h后的纯培养物菌株C4的生长和酶活都有较强的抑制作用,其中Cd²⁺对已培养12h菌株的危害更大。0.5mg L^-1 Cd²⁺浓度即可显著地抑制菌株的生长和酶活水平。而C4菌株对乙草胺有较强的耐受能力,特别是对已培养12h的菌株,乙草胺浓度高达1000mg/L时,其酶活仍可与对照水平相近。在Cd²⁺和乙草胺复合污染中,也观察到已培养更多还原

【Abstract】 In this thesis, the effects of Cd²⁺, acetochlor and their association on the number and nitrogenase activity of nitrogen-fixing bacteria were studied in upland soil samples. Moreover, a bacterium capable of fixing nitrogen efficiently was isolated, identified and phylogenetically analyzed. The growth and nitrogenase activity of Azotobacter beijerinckii C4 （G^-） under the pollution was studied. The activity response of the antioxidant enzymes superoxide dismutase, catalase, ATP enzyme activities of A.beijerinckii C4 following exposure to the pollution was also investigated. The results will be valuable to discuss the influence mechanism of strain C4 with pollution and to build up alert index systems and environmental quality evaluation in upland soil polluted by Cd²⁺ and atecholor. Here the main results of this study are presented as the follows:1. The influences of Cd²⁺, acetochlor and their association on the cultural nitrogen-fixing bacteria in upland soil and the intensity of N₂-fixation were investigated using traditional methods. The results showed that the aerobic nitrogen-fixing bacteria were sensitive to Cd²⁺。The low concentration of Cd²⁺ could increase the numbers of aerobic nitrogen-fixing bacteria, while the CFU of N₂-fixing bacteria were strongly inhibited by higher concentration of Cd²⁺. Acetochlor had no essential effect on aerobic nitrogen-fixing bacteria. Anaerobic nitrogen-fixing bacteria were stimulated by Cd²⁺ at the first 8 days, and then restrained. Finally, its numbers were remarkably restrained by the treatment of 5.0 mg kg^-1 dry soil, while others were nearly equal to that of the control. Acetochlor had no evident influence on the numbers of anaerobic nitrogen-fixing bacteria during the whole experiment phase except the group of 2.0 mg kg^-1 dry soil. Association of Cd²⁺ and acetochlor demonstrated joint inhibitory effects on both aerobic and anaerobic nitrogen-fixing bacteria. The inhibition of nitrogenase activity became stronger with the increase of Cd²⁺ concentration, showing a striking dose-response relationship. The concentrations of acetochlor used in this experiment had a relatively mild effect on the intensity of N₂-fixation in a short term. However, the inhibition to that was found at the end of experiments. The nitrogenase activity under the combined pollution accumulated originally, but declined ultimately.2. A bacterium, named strain C4, capable of fixing nitrogen efficiently, was isolated from a kailyard and identified as Azotobacter beijerinckii based on morphology, physio-biochemical properties and a partial sequencing of 16S rDNA. Strain C4 was sensitive to gentamicin, ampicillin, amikacin and erythromycin, but insensitive to penicillin, cephalothin and chloramphenicol. The nitrogenase activity of strain C4 was highest after growth for 24h, with the acetylene reduction activity （ARA） of 1203.779 nmol C2H4 per h per ml culture. The nitrogenase activity declined subsequently, and was only 75.66 nmol C2H4 per h per ml culture grown for 90h.3. The impacts of growth and nitrogenase activity of strain C4 isolated from garden was studied to illuminate the toxic effect of compound pollution on strain C4. Results obtained indicated that the growth and nitrogenase activity of strain C4, especially after growing for 12h, was strongly restrained by Cd2+, and that treated with 0.5 mg L"1 was inhibited evidentIy（p<0.05）. Strain C4 demonstrated good tolerance to acetochlor, especially the nitrogenase activity of strain C4 growing for 12h could reach the level of the control even exposed to concentration of 1000 mg/L. The higher sensitivity of strain C4 growing for 12h to the association of Cd2+ and acetochlor was also observed. The growth and nitrogenase activity of strain C4 could only retain at a low level under the pollution stress of the two compounds. It indicated that Cd2+ is the key factor while acetochlor has no marked influence on strain C4 in the associated pollution of compounds. However, notable interaction between Cd2+and acetochlor was not detected.4. The activity response of the antioxidant enzymes of Azotobacter beijerinckii C4 （G） following exposure to Cd2+, acetochlor and their association was investigated. Results showed that activities of SOD, CAT and ATPase in the bacterium induced by Cd2+ or acetochlor were positive and obvious, especially in shorter time after treatments. The associated pollution of Cd2+ and acetochlor had different impacts on the activity of the antioxidant enzymes with different concentrations and treatment times. SOD and ATPase activities of all association pollution appeared stronger than those of a single factor, but the activities recovered finally to that of the control. It demonstrated that various concentration association of the two pollutants caused the inducing or inhibiting actions primarily, but inhibited effects ultimately on CAT activity.更多还原