【作者】 刘芳明；

【导师】 缪锦来；

【作者基本信息】 国家海洋局第一海洋研究所 ， 海洋生物学， 2008， 硕士

【摘要】 南极具有独特的地理及气候特征,其主要特点是变化极大的光照辐射、季节性的光照时间。南极海洋系统内常年低温(通常在-1.8℃~2.0℃),高盐(海水中的盐度一般为34‰~35‰,海冰中的盐囊和盐通道的盐度可达到150‰),在这样一个低温、强辐射和高盐度的自然环境里,海洋微生物必然具备了相应独特的生理生化和分子机制,因此,南极微生物不仅是研究在低温条件下生物适应性的极好材料,而且也是获得低温生物活性物质(低温酶、PUFA)和其他低温生物工程菌的最佳来源。随着海上油田的开采和海洋运输的日益频繁,海洋的石油烃污染越来越严重,以致被认为人类最后一块净土的南极大陆也未能幸免。近年来,南极考察、探矿和观光旅游的兴起,特别是大型油轮的沉没,导致大量石油烃泄漏,严重威胁了南极生态系统的安全,已经引起世界各国的广泛重视,在南极这样一个非常脆弱的生态系统,任何小的生态环境污染,都会导致严重的生态系统破坏。生物修复是消除海洋石油烃污染的重要途径,本文从南极海水海冰样品中筛选能低温降解石油烃的低温菌,以期为海洋石油烃污染的低温生物修复技术应用奠定基础。本文采用MMC无机盐液体培养和MMC琼脂平板培养结合的方法,从南极海水海冰样品的细菌筛选能够高效低温降解石油烃的细菌,对筛选到的4株南极低温降解菌进行平板生长和降解率试验;采用16S rRNA分子鉴定方法对4株南极低温降解菌进行鉴定,构建系统发育树;通过生物摇床进行单因素试验确定南极低温降解菌NJ49的最适营养和生长条件;通过萘低温降解、柴油低温降解GC-MS图谱、脱氢酶试验、产生物表面活性剂试验、脂肪酸变化试验,对南极低温降解菌NJ49的降解特性和低温适应性进行研究。主要获得以下结果:1.从南极海水海冰样品中筛选到了4株南极低温降解菌,即NJ41、NJ44、NJ49、NJ289。平板可见计数试验表明,培养20d后NJ41、NJ44、NJ49和NJ289均生长良好,南极低温降解菌NJ49菌液细胞可以接近106.8CFU/ml。对4株南极低温降解菌在15℃时降解试验表明,不同菌株对柴油的降解能力不同,其中南极低温降解菌NJ49降解率为最高,可达68%。2.对4株南极低温降解菌的形态特征观察结果表明,南极低温降解菌NJ41是球形,其直径为1.0~1.2μm,其余3株降解菌为杆状,长度范围在0.7~2.8μm之间。4株南极低温降解菌都不形成芽孢,都具有鞭毛可以运动。16S rRNA分子鉴定结果表明:南极低温降解菌NJ41属于动球菌属(Planococcus);南极低温降解菌NJ49属于希瓦氏菌属(Shewanella);南极低温降解菌NJ289是假交替单胞菌属(Pseudoalteromonas);南极低温降解菌NJ44有待进一步鉴定。3.对降解率最高的南极低温降解菌NJ49在各种不同环境条件下的生长以及降解柴油能力的研究结果表明,其生长和降解的最适条件为:初始pH为7.5,温度为15℃,盐度为6%,摇瓶装量为80ml,最佳氮源为硝酸铵,最佳磷源为磷酸二氢钾和磷酸氢二钾的混合物。添加表面活性剂能够促进南极低温降解菌NJ49的生长和降解,尤其是阴离子表面活性剂(SDS)和非离子表面活性剂(鼠李糖脂)合用,提高了菌株的降解率。4.对南极低温降解菌NJ49的低温降解特性的研究结果表明,15℃条件下,它可以在以萘为惟一碳源和能源的无机盐培养基中生长,并对萘进行降解。采用GC-MS测定南极低温降解菌NJ49对柴油的降解,图谱表明,对照柴油中含有C9~C26之间的l8种烷烃,而经南极低温降解菌NJ49低温降解处理后的残油组分中,仅能检测到C15~C217种烷烃,不仅烷烃的种类明显减少,单一烷烃的含量也减少许多。通过对南极低温降解菌NJ49产脱氢酶试验得知,在5℃时脱氢酶酶活力要低于15℃时的酶活力,表明,低温限定了南极低温降解菌NJ49的酶活力;补给柴油的MMC培养基中脱氢酶活力高于2216E培养基中的酶活力,推断测定的脱氢酶是降解柴油的酶。南极低温降解菌NJ49的产表面活性剂试验结果为,在5℃和20℃条件下,该菌均不产生表面活性剂。根据脂肪酸组成的测定数据,发现南极低温降解菌NJ49在低温(5℃)降解时,其脂肪酸组分发生了变化,不饱和脂肪酸的种类和单种脂肪酸含量均有增加。5℃条件下,南极低温降解菌NJ49能产生多种不饱和脂肪酸,其含量高于中温(20℃)条件的。低温时,南极低温降解菌NJ49的脂肪饱和度降低,由1.22降到0.77,推断该低温降解菌通过脂肪酸变化的方式适应低温下的降解。从南极海水海冰中分离到低温降解石油烃的海洋细菌在国内尚属首次,可以认为这些南极低温降解菌为我国低温海洋石油污染的生物修复提供菌源。对南极低温降解菌的石油烃低温降解特性的研究,将为我国低温海域石油烃污染物的清除,提供有效的生物修复技术方法。更多还原

【Abstract】 Antarctica region, due to its specific geographical position and climate, which characterized by strong seasonal changes in light and UV intensity, is considered to be an extreme environment. Antarctic sea environment, which is under low temperature (usually-1.8℃~2.0℃) for all time and high salinity (34‰~35‰in sea water, 150‰in sea ice saline channels). To be colonized in this low-temperature, strong radiation and high-salinity environment, microorganisms form gradually a series of physio- logical, chemical and molecular mechanisms. Therefore, Antarctic microorganisms provide a useful model system for studying cold adaptation, and also are the optimal sources of low temperature bioactive substances ( cold-adapted enzymes , PUFA) and cold-adapted biological engineering bacteria. With the development of sea oil field exploitation and ocean transportation more frenquently, oil hydrocarbon pollution in the sea occurs more seriously. Antarctica, which thought to be the last unpolluted land, were polluted by petroleum oil. Recently, with the development of Antarctic Exploration, mine exploitation and travelling, especially gigantic oil tanker sinking, plenty of pertoleum hydrocarbon leaked out and the security of Antarctic ecosystem had been threatened. The phenomenation had attracted attention of every country. It is so fragile as Antarctic ecosystem that any little pollution could result in serious breakage to ecosystem Bioremidiation is an important method in eliminating oil pollutants from sea, this thesis aims at purpose of which cold-adapted hydrocarbon- degrading bacteria are screened and that will play an fundermental role used for bioremediation technique and method of eliminating sea oil pollution at low tempera- ture.The method of combination MMC liquid culture with MMC agar plate culture was used for screening petroleum hydrocarbon-degrading cold-adapted bacteria from Antarctic seawaters and sea-ice. Tests of bacteria growing and degradation efficiency were carried. Molecular identification of 16S rRNA amplified from four Antarctic cold-adapted degrading bacteria and phylogenetic tree based on 16S rRNA gene was carried. Antarctic cold-adapted degrading bacteria NJ49 was chosen to study the optimization of nutrition and growing condition.Tests of naphthaline degradation, degradable diesel chromatogram, dehydrogenase production, biosurfactant produc- tion, fatty acid change were carried for Antarctic cold-adapted degrading bacteria NJ49 degradation characteristic and cold adaptation study. The results were sum- marized as follows.1. Four petroleum hydrocarbon-degrading cold-adapted bacteria (namely NJ41, NJ44, NJ49, NJ289 ) were screened from Antarctic sea waters and sea-ice samples. Viable counts results showed that four Antarctic cold-adapted degrading bacteria grew well and Antarctic cold-adapted degrading bacteria NJ49 cell number was near to 106.8CFU/ml after twenty days. Degradation datas showed that degradable ablilities of four Antarctic cold-adapted degrading bacteria were different separatetly and degradation efficiency of Antarctic cold-adapted degrading bacteria NJ49, value 68%, were the highest.2. Morphological characteristics results showed that Antarctic hydrocarbon- degrading bacteria NJ41 was spherical,diameter 1.0~1.2μm, and other three bacteria were bacilliform, long 0.7~2.8μm. All Antarctic cold-adapted degrading bacteria did not form spore and all of them had flagellum to move. Results of 16S rRNA identification revealed that Antarctic cold-adapted degrading bacteria NJ41, NJ49 and NJ289 belonged to the described genus Planococcus, Shewanella, Pseudo- alteromonas, respectively. Antarctic cold-adapted degrading bacteria NJ44 need to be studied further.3. Antarctic cold-adapted degrading bacteria NJ49 was chosen to study the optimization of nutrition and growing condition. Results revealed that the optimal combination of nutrient constituents and condition to grow and degrade were determined as NH4NO3, KH2PO4 and K2HPO4, NaCl 6%, pH 7.5, 15℃, 80ml volume. Adding biosurfactant, especially combination negative ion surfactant (SDS) with positive ion surfactant (rhamnolipid) could improve Antarctic cold-adapted degrading bacteria NJ49 growing and degrading.4. Degradation characteristics results showed that Antarctic hydrocarbon- degrading bacteria NJ49 may grow in MMC culture, naphthaline as the sole carbon and energy source and degrade naphthaline at 15℃. Degradable diesel chromatogram revealed that there were eighteen alkane ingredients (C9~C26 ) in control diesel, while only seven alkane ingredients (C15~C21 ) in Antarctic cold-adapted degrading bacteria NJ49 degradable diesel. Furthermore, every alkane ingredients content was decreased. The results of dehydrogenase production revealed that dehydrogenase activity of Antarctic cold-adapted degrading bacteria NJ49 was much lower at 5℃than at 20℃. That indicated low temperature limited enzyme activity. Dehydrogenase activity was higher in 2216E amended diesel than in 2216E culture, indicating that the dehydrogenase may be diesel-degrading enzyme. Results of biosurfactant production showed that Antarctic cold-adapted degrading bacteria NJ49 did not produce bio- surfactant at both 5℃and 20℃. Data of fatty acid change revealed that ouccrred change at low temperature(5℃)condition. Fatty acid components and single fatty acid number were increased induced by low temperature(5℃). Antarctic hydrocarbon- degrading bacteria NJ49 isolate produced kinds of unsaturated fatty acids and the content of them were much higher at 5℃than mesothermal condition(20℃). Saturated degree of Antarctic cold-adapted degrading bacteria NJ49 fatty acid decreased from 1.22 to 0.77 at low temperature(5℃), which indicated that the Antarctic cold-adapted degrading bacteria NJ49 adapt cold condition to degrade by changing fatty acid.Marine bacteria capable of degrading petroleum hydrocarbon at low temperature were isolated from Antarctic seawaters and sea-ice samples, which was the first time in civil country. The Antarctic cold-adapted degrading bacteria provided bacteria sources used for bioremediation of sea oil pollution in our country. The study of degradation characteristics of Antarctic cold-adapted degrading bacteria at low temperature provided efficient bioremediation technique and method of eliminating sea oil pollution at low temperature.更多还原