应用发光细菌Acinetobacter sp.Tox2和Acinetobacter sp.RecA快速检测海水生物毒性的

发布时间：2018-07-04 13:24

本文选题：发光细菌 + 急性毒性　；参考：《国家海洋局第一海洋研究所》2015年硕士论文

【摘要】：发光菌检测法(luminescent bacteria test,简称LBT)是以天然或人工改造的发光细菌为受试生物进行毒性检测的技术。发光细菌能够表达细菌荧光素酶,从而产生生物发光现象。当细菌暴露于毒性环境中时,外界有毒物质会使发光细菌的发光强度发生变化,借助于高灵敏度的生物发光检测仪器,可以定性或定量的表征有毒物质的毒性大小。然而,现今报道的多数发光细菌多用于检测淡水的急性毒性,而对海洋污染遗传毒性的相应检测技术或标准更是尚未起步,其次,鱼类等普通生物检测生物毒性有耗时耗力的缺陷。本文以两株发光细菌Acinetobacter sp.Tox2和Acinetobacter sp.RecA为研究对象,以发光抑制率和发光诱导率表示急性毒性和遗传毒性的大小,优化了生物毒性快速检测方法,并对实际海水样品的生物毒性进行评价,在此基础上考察了这两株发光细菌可用于快速检测海水生物毒性的可行性。该研究首先对两种发光细菌的培养条件、检测条件进行了优化。研究表明,Tox2菌株的次代培养时间(B液)为8 h,RecA菌株的次代培养时间为16 h。两株细菌可以检测盐度为30左右的样品,在检测过程中需要保持室温30℃。检测过程中急性毒性最短可以在30 min内得到实验结果,而遗传毒性最短可以在3h内得到实验结果。其次利用这两株发光细菌检测了模拟污染海水中总石油烃(TPH)、4种重金属离子(Hg2+、Zn2+、Cu2+、Cd2+)、4种多环芳烃(菲、芘、荧蒽、苯并[a]芘)的急性毒性和遗传毒性。研究发现,菌株Tox2和RecA对总石油烃的检出限达到了0.001 mg/L;菌株Tox2对四种重金属离子表现出不同的敏感度,并可以计算样品浓度与发光抑制率的回归方程,四种重金属对Tox2的EC50分别为:HgCl2 0.108 mg/L、ZnSO4 46.0 mg/L、CuSO4 0.816 mg/L、CdCl2 14.2 mg/L;菌株Tox2对多环芳烃中的菲和芘比较敏感,EC50分别是1.46 mg/L和0.266 mg/L。在现场实验中,采用发光细菌法快速评价了环渤海排污口12份污水样品的生物毒性。12个排污口的污水样品均表现出不同水平的急性毒性和遗传毒性,所有排污口样品的急性毒性整体偏低,只有BH3站位污水的急性毒性达到了剧毒级别,当量HgCl2浓度达到0.193 mg/L,多数站位样品的当量HgCl2浓度小于0.02 mg/L。遗传毒性整体偏高,多数站位的当量MMC浓度高于0.2 mg/L而BH4站位样品的当量MMC浓度高达2.41 mg/L。生物毒性同时也呈现出一定的地理分布特征。其中,高毒水质集中出现在山东半岛污水样品中,中毒水质集中出现在辽东半岛地区,而低毒水质主要集中于京津冀地区。同时,针对黄岛“11.22”溢油事故进行了海水遗传毒性的快速检测。选取溢油后90 d内距离溢油点1~15km的5个站点,结合化学荧光分析,将发光细菌RecA应用于溢油污染海水样品的遗传毒性评价。研究发现,溢油1d后距离泄漏原油入海口1 km以内的S1、S2、S3站点海水样品的石油烃含量为0.55 mg/L~1.04 mg/L,其遗传毒性相当于0.60 mg/L~1.78 mg/L的丝裂霉素C(MMC);海水中的石油烃含量随时间逐渐降低至0.02 mg/L,其遗传毒性也随之逐渐降低。而距离泄漏原油入海口5km的S4、S5站点海水样品的石油烃含量一直较低(0.02 mg/L),表现为低毒或无毒。综上所述,使用基因工程改造的两株发光细菌可以用于对受污染的海水进行快速、简便、高灵敏度的生物毒性检测。使用该发光细菌在后期有关于海水生物毒性快速检测的行业标准的制定中打下基础,并且在应对突发海洋污染事件的海水水质快速检测评价中具有很好的应用前景。
[Abstract]:Luminescent bacteria test (LBT) is a technique to detect the toxicity of the natural or artificial luminescent bacteria. The luminescent bacteria can express the bacterial luciferase, thus producing bioluminescence. When the bacteria are exposed to the toxic environment, the external toxic substances will make the luminescent bacteria strong. However, most of the reported bacteria are used to detect the acute toxicity of fresh water, and the related detection techniques or standards for the genetic toxicity of marine pollution are not yet to be started. Second, fish and other universal methods. The biological toxicity of two strains of luminescent bacteria, Acinetobacter sp.Tox2 and Acinetobacter sp.RecA, was studied in this paper. The acute toxicity and the size of genotoxicity were expressed by the luminescence inhibition rate and the luminescence induction rate. The rapid detection method of biological toxicity and the biological toxicity of the actual seawater samples were optimized. On the basis of the evaluation, the feasibility of the two luminescent bacteria can be used to detect the biological toxicity of the sea water quickly. First, the culture conditions and the conditions of the two kinds of luminescent bacteria were optimized. The study showed that the secondary culture time of the Tox2 strain (B liquid) was 8 h, and the secondary culture time of the RecA strain was 16 h. Two Strains of bacteria. The sample with a salinity of about 30 can be detected in the process of testing at 30 degrees C. The shortest acute toxicity in the detection process can be obtained in 30 min, and the shortest genetic toxicity can be obtained in 3H. Secondly, the total petroleum hydrocarbons (TPH) and 4 kinds of heavy metals in the simulated polluted sea water are detected by the two strains of luminescent bacteria. The acute toxicity and genetic toxicity of 4 polycyclic aromatic hydrocarbons (phenanthrene, pyrene, fluoranthene, benzo [a] pyrene) of 4 kinds of polycyclic aromatic hydrocarbons (phenanthrene, pyrene, fluoranthene, benzo [a] pyrene). The study found that the detection limit of Tox2 and RecA on total petroleum hydrocarbons reached 0.001 mg/L, and the strain Tox2 showed different sensitivity to four kinds of heavy metal ions and could calculate the regression of sample concentration and luminescence inhibition rate. The EC50 of four kinds of heavy metals to Tox2 are HgCl2 0.108 mg/L, ZnSO4 46 mg/L, CuSO4 0.816 mg/L, CdCl2 14.2 mg/L. Strain Tox2 is sensitive to phenanthrene and pyrene in polycyclic aromatic hydrocarbons, EC50 is 1.46 and 0.266 respectively in the field experiment, and the production of 12 sewage samples in the ring Bohai sewage outlet is quickly evaluated by the light emitting bacteria method. The acute toxicity and genotoxicity of the sewage samples at the.12 sewage outlet were all shown at different levels. The acute toxicity of all the effluent samples was low. Only the acute toxicity of the sewage at the BH3 station reached the toxic level, the equivalent HgCl2 concentration reached 0.193 mg/L, and the equivalent HgCl2 concentration of most of the station samples was less than 0.02 mg/L. genetic poison. The equivalent MMC concentration of most stations is higher than 0.2 mg/L, while the equivalent MMC concentration of the BH4 station samples is up to 2.41 mg/L. and also presents a certain geographical distribution. In the area of Beijing, Tianjin and Hebei Province, the rapid detection of genetic toxicity of sea water was carried out in Huangdao "11.22" oil spill accident. 5 sites of the oil spill point 1~15km within 90 d of the spilled oil were selected and combined with chemical fluorescence analysis to evaluate the genotoxicity of the luminescent bacteria RecA in the samples of the spilled oil contaminated seawater. The petroleum hydrocarbon content of the sea water samples of S1, S2 and S3 sites within 1 km of Haikou is 0.55 mg/L~1.04 mg/L, and its genetic toxicity is equivalent to 0.60 mg/L~1.78 mg/L of mitomycin C (MMC), and the petroleum hydrocarbon content in the sea water decreases with time to 0.02 mg/L, and its genetic toxicity decreases gradually. S5 site sea water samples have low petroleum hydrocarbons (0.02 mg/L), showing low toxicity or non-toxic. In summary, the use of two strains of genetically engineered luminescent bacteria can be used for rapid, simple, highly sensitive biotoxicity test for polluted sea water. It lays a solid foundation for the formulation of the industry standard, and has a good application prospect in the rapid detection and evaluation of marine water quality in response to sudden marine pollution incidents.
【学位授予单位】：国家海洋局第一海洋研究所
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X834

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