海洋环境中多环芳烃污染的微生物修复作用研究
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摘要
本研究针对环境科学研究领域的热点和难点，以厦门西港为主要研究区域，将生物修复的新思路引入海洋环境中多环芳烃(PAHs)污染的治理，对厦门西港PAHs污染水平、PAHs降解菌数量分布进行现场跟踪调查；采用富集培养的方法，从自然海区分离筛选高效降解四环以上PAHs的微生物，对海洋环境中PAHs生物降解过程及机制进行研究。取得了目前国内关于海洋环境中PAHs生物降解研究最为丰富、系统的研究成果。
主要研究结果归纳如下：
1、首次对厦门西港表层海水和沉积物中16种优先监控的PAHs的污染状况进行不同季节的连续调查，结果表明：表层海水中PAHs的含量与组成具有明显的时间差异。在4月和10月的PAHs含量较7月高；PAHs的组成在4月以2环的萘为优势组分，在7月以3环的苊烯为优势组分，10月以5环的屈为优势组分。分析三个航次的调查结果发现：厦门西港表层海水中的PAHs主要来源于石油类物质的输入。厦门西港表层海水各种PAHs的含量与国际生物学组织或国家制定的评价水生生物暴露于水体的安全食用标准相比虽未超标，但个别组分如蒽、苯并[a]芘的含量已达到生态毒理评价标准。
2、厦门西港沉积物中检出的PAHs均以4-6环的为主，其中荧蒽和芘在不同航次及不同站位均为优势组分。PAHs的总含量变化不明显，但高分子量的PAHs(5～6环)的含量却有升高的趋势。厦门西港沉积物中的PAHs主要来源于矿物的不完全燃烧，与1993年的调查结果相比厦门西港沉积物中PAHs的含量有所减少，与国内外其他相似地区相比属中等水平。
3、首次对厦门西港表层海水和沉积物中PAHs降解菌的数量分布进行连续两个航次的调查，并借鉴现行国际上测定PAHs降解微生物的方法，建立了适合样品量大、现场调查快速准确的细菌计数方法——单层平板方格法。研究结果表明：厦门西海域表层海水及沉积物中PAHs降解菌丰富可得，在夏季表层海水中PAHs降解菌的数量可达10~3CFU／mL以上，沉积物中PAHs降解菌的数量可达10~4CFU／g以上；在秋季表层海水中PAHs降解菌的数量可达10~2CFU／mL以上，沉积物中PAHs降解菌的数量可达10~3CFU／g以上。
4、表层水中低分子量PAHs降解菌的数量在一定程度上可以指示低分子量PAHs的污染程度，而高分子量PAHs的含量与其降解菌数量之间没有表现出相关性。沉积物中PAHs降解菌的数量分布对PAHs污染有一定的指示作用，即沉积物中PAHs降解菌的数量与PAHs含量呈正相关。但特殊区域(养殖海区)环境因素对降解菌的影响不可忽视。
5、对表层海水及沉积物样品进行驯化，富集培养，从中分离出芘-降解菌3株，编号为PYSP1和PYSP22；荧蒽-降解菌株3株，编号为FLSP2、FLSP6和FLSP8；菲-降解菌株2株，编号为PHSP2和PHSP6。3株芘-降解菌经分子生物学鉴定分别为：PYSP1为Ochrobactrum sp., PYSP6为Pandoraea sp., PYSP22为Ochrobactrum sp．。
6、以芘富集培养的单菌株PYSP22能很好地利用不同分子量的PAHs-芘、荧蒽、菲作为其生长的碳源和能源，经过约3个星期的培养，单菌株PYSP22可分别将培养液中的芘、荧蒽、菲降解70.3％、85.4％和85.8％。另外，单菌株PYSP22可以同时利用这三种PAHs，
摘 要
3个星期后，将混合液中的花、荧葱、菲降解了80．6％、90．6％和78．3％。
7、以茁富集培养的混合微生物SS6由四种菌落特征不同的细菌组成，可以很好地利用不同
分子量的PAHs－茂、荧葱、菲作为其生长的碳源和能源，经过约3个星期的培养，混合
培养菌能6可分别将培养液中的蓖、荧葱、菲降解gi．2o、89．35％、92．sl％；SS6可以
同时利用花、荧蓉、菲，经过3个星期可将混合液中的蓖、荧葱、菲降解89．5％、90．2％、
90．7％O
8、环境因子对于PAHS的代谢和微生物的生长有着重要的影响。在海水培养液中，混合培
养菌SS6生长的最适pH值为8．0，在pH6－8的范围内可以良好的生长。培养液中pH值
超过9刀会抑制微生物的生长：混合培养菌％6在盐度为二0－30的培养液中可以良好地
生长，在盐度为 40－50的培养液中生长受到明显的抑制。海水培养液中外加 N源（KNO3）
可以提高混合培养菌 SS6的生长水平，促进培养液中 PAH－花的降解，而外加 P源
（KZHPO4）的影响却不明显，表明厦门西港的海水中，N是PAHS生物降解的限制因素，
而P则不是。
9、对海水环境中4环PAH－花的共代谢机制开展研究，结果表明共代谢促进PAHs的降解。
混合培养菌在对四环PAH－茁和荧葱的降解过程中，培养液中优势微生物的种类和数量
发生明显变化，SPI在花和荧惫降解的启动过程中起关键作用；分别以茁的代谢中间产
物一水杨酸和邻苯二甲酸、同系化合物一菲、容易
The investigation of the pollution level of polycyclic aromatic hydrocarbons (PAHs) and the distribution characteristics of PAHs-degrading bacteria in surface water and sediments has been conducted at six locations in Xiamen harbor (24?9' N, 118?4' E) on spring, summer and autumn cruises in 2001. The enrichment culture method was used to isolate the high molecular weight PAHs-degrading bacteria from samples contaminated with PAHs (pyrene, phenanthrene and fluoranthene) in order to have a better understanding of biodegradation mechanism of PAHs in marine environment.
In this study, the level of sixteen individual polycyclic aromatic hydrocarbon compounds has been identified as priority pollutants in different seasons in Xiamen harbor. The results showed that the concentration and composition of PAHs in the surface water were unstable. The pollution level of PAHs in April and October were higher than that in July. PAHs in the surface water were mainly dominated by naphthalene (2 rings) in April, acenaphthene (3 rings) in July and chrysene (4 rings) in the October. The concentration of anthracene and benzo(a)pyrene in surface water was higher than the toxicity guidelines. The origin of PAH pollution in surface water of Xiamen harbor was mostly petrogenic.
PAHs in sediments were mainly dominated by high molecular weight PAH components (4-6 rings). Fluoranthene and pyrene were the most dominant at different seasons and stations. The change of total PAHs concentration was insignificant in different seasons, but the level of high molecular weight PAH compounds showed an increasing tendency. Comparing with the results obtained from Xiamen harbor in 1993, the pollution level of PAH in sediments has decreased and the origin of PAH was the same as that found for the surface water.
The distribution characteristics of PAH-degrading bacteria were realized for the first time in Xiamen harbor during the two cruises in surface water and sediments of Xiamen Western harbor. For this purpose, the spray-plate technique with modification was used to numerate PAH-degrading bacteria. The results showed that, PAH-degrading bacteria were abundant in both of surface water and sediments of Xiamen Western harbor. The numbers of PAH-degrading bacteria were more than 103CFU/mL in surface water and 104 CFU/g in sediments during summer season. However, during the autumnthe numbers of PAH-degrading bacteria were more than 102CFU/mL in surface water and 103 CFU/g in sediments.
In the surface water, the concentration of high molecular weight PAH compounds fluoranthene and pyrene was very low during the two cruises. No correlation was found between the concentrations of high molecular weight PAH compounds and their degrading bacteria numbers. The positive correlation was found between the concentrations of low molecular weight PAH compounds (fluorene and
phenanthrene) and their degrading bacteria numbers. This may imply that the inducement of high molecular weight PAH compounds to bacterium was limited by their low concentration in the surface water, high chemical stability and hydrophobicity and short exposure time. Consequently, the numbers of high molecular weight PAH compounds degrading bacteria might not indicate the extent of PAHs contamination in the surface water.
In the sediments, positive correlation was significantly found between PAH-degrading bacteria numbers and PAHs concentration, expect the station 6. Those suggest that the relationship between the concentrations of high molecular weight PAH compounds and their degrading bacteria numbers is only significant for highly contaminated sites and long-term exposure. The PAHs-degrading bacteria number may indicate the PAHs pollution at some extent in the relative stable sediment environment, but it would not be omitted to well consider other influential factors.
The enrichment of three pure cultures (PYSP1, PYSP6 and PYSP22) has been done successfully by using a spray plate method with pyrene as the sole carbon source. Other three pure cultures (FLSP2, FLSP6 and FLSP8) were is
主要研究结果归纳如下：
1、首次对厦门西港表层海水和沉积物中16种优先监控的PAHs的污染状况进行不同季节的连续调查，结果表明：表层海水中PAHs的含量与组成具有明显的时间差异。在4月和10月的PAHs含量较7月高；PAHs的组成在4月以2环的萘为优势组分，在7月以3环的苊烯为优势组分，10月以5环的屈为优势组分。分析三个航次的调查结果发现：厦门西港表层海水中的PAHs主要来源于石油类物质的输入。厦门西港表层海水各种PAHs的含量与国际生物学组织或国家制定的评价水生生物暴露于水体的安全食用标准相比虽未超标，但个别组分如蒽、苯并[a]芘的含量已达到生态毒理评价标准。
2、厦门西港沉积物中检出的PAHs均以4-6环的为主，其中荧蒽和芘在不同航次及不同站位均为优势组分。PAHs的总含量变化不明显，但高分子量的PAHs(5～6环)的含量却有升高的趋势。厦门西港沉积物中的PAHs主要来源于矿物的不完全燃烧，与1993年的调查结果相比厦门西港沉积物中PAHs的含量有所减少，与国内外其他相似地区相比属中等水平。
3、首次对厦门西港表层海水和沉积物中PAHs降解菌的数量分布进行连续两个航次的调查，并借鉴现行国际上测定PAHs降解微生物的方法，建立了适合样品量大、现场调查快速准确的细菌计数方法——单层平板方格法。研究结果表明：厦门西海域表层海水及沉积物中PAHs降解菌丰富可得，在夏季表层海水中PAHs降解菌的数量可达10~3CFU／mL以上，沉积物中PAHs降解菌的数量可达10~4CFU／g以上；在秋季表层海水中PAHs降解菌的数量可达10~2CFU／mL以上，沉积物中PAHs降解菌的数量可达10~3CFU／g以上。
4、表层水中低分子量PAHs降解菌的数量在一定程度上可以指示低分子量PAHs的污染程度，而高分子量PAHs的含量与其降解菌数量之间没有表现出相关性。沉积物中PAHs降解菌的数量分布对PAHs污染有一定的指示作用，即沉积物中PAHs降解菌的数量与PAHs含量呈正相关。但特殊区域(养殖海区)环境因素对降解菌的影响不可忽视。
5、对表层海水及沉积物样品进行驯化，富集培养，从中分离出芘-降解菌3株，编号为PYSP1和PYSP22；荧蒽-降解菌株3株，编号为FLSP2、FLSP6和FLSP8；菲-降解菌株2株，编号为PHSP2和PHSP6。3株芘-降解菌经分子生物学鉴定分别为：PYSP1为Ochrobactrum sp., PYSP6为Pandoraea sp., PYSP22为Ochrobactrum sp．。
6、以芘富集培养的单菌株PYSP22能很好地利用不同分子量的PAHs-芘、荧蒽、菲作为其生长的碳源和能源，经过约3个星期的培养，单菌株PYSP22可分别将培养液中的芘、荧蒽、菲降解70.3％、85.4％和85.8％。另外，单菌株PYSP22可以同时利用这三种PAHs，
摘 要
3个星期后，将混合液中的花、荧葱、菲降解了80．6％、90．6％和78．3％。
7、以茁富集培养的混合微生物SS6由四种菌落特征不同的细菌组成，可以很好地利用不同
分子量的PAHs－茂、荧葱、菲作为其生长的碳源和能源，经过约3个星期的培养，混合
培养菌能6可分别将培养液中的蓖、荧葱、菲降解gi．2o、89．35％、92．sl％；SS6可以
同时利用花、荧蓉、菲，经过3个星期可将混合液中的蓖、荧葱、菲降解89．5％、90．2％、
90．7％O
8、环境因子对于PAHS的代谢和微生物的生长有着重要的影响。在海水培养液中，混合培
养菌SS6生长的最适pH值为8．0，在pH6－8的范围内可以良好的生长。培养液中pH值
超过9刀会抑制微生物的生长：混合培养菌％6在盐度为二0－30的培养液中可以良好地
生长，在盐度为 40－50的培养液中生长受到明显的抑制。海水培养液中外加 N源（KNO3）
可以提高混合培养菌 SS6的生长水平，促进培养液中 PAH－花的降解，而外加 P源
（KZHPO4）的影响却不明显，表明厦门西港的海水中，N是PAHS生物降解的限制因素，
而P则不是。
9、对海水环境中4环PAH－花的共代谢机制开展研究，结果表明共代谢促进PAHs的降解。
混合培养菌在对四环PAH－茁和荧葱的降解过程中，培养液中优势微生物的种类和数量
发生明显变化，SPI在花和荧惫降解的启动过程中起关键作用；分别以茁的代谢中间产
物一水杨酸和邻苯二甲酸、同系化合物一菲、容易
The investigation of the pollution level of polycyclic aromatic hydrocarbons (PAHs) and the distribution characteristics of PAHs-degrading bacteria in surface water and sediments has been conducted at six locations in Xiamen harbor (24?9' N, 118?4' E) on spring, summer and autumn cruises in 2001. The enrichment culture method was used to isolate the high molecular weight PAHs-degrading bacteria from samples contaminated with PAHs (pyrene, phenanthrene and fluoranthene) in order to have a better understanding of biodegradation mechanism of PAHs in marine environment.
In this study, the level of sixteen individual polycyclic aromatic hydrocarbon compounds has been identified as priority pollutants in different seasons in Xiamen harbor. The results showed that the concentration and composition of PAHs in the surface water were unstable. The pollution level of PAHs in April and October were higher than that in July. PAHs in the surface water were mainly dominated by naphthalene (2 rings) in April, acenaphthene (3 rings) in July and chrysene (4 rings) in the October. The concentration of anthracene and benzo(a)pyrene in surface water was higher than the toxicity guidelines. The origin of PAH pollution in surface water of Xiamen harbor was mostly petrogenic.
PAHs in sediments were mainly dominated by high molecular weight PAH components (4-6 rings). Fluoranthene and pyrene were the most dominant at different seasons and stations. The change of total PAHs concentration was insignificant in different seasons, but the level of high molecular weight PAH compounds showed an increasing tendency. Comparing with the results obtained from Xiamen harbor in 1993, the pollution level of PAH in sediments has decreased and the origin of PAH was the same as that found for the surface water.
The distribution characteristics of PAH-degrading bacteria were realized for the first time in Xiamen harbor during the two cruises in surface water and sediments of Xiamen Western harbor. For this purpose, the spray-plate technique with modification was used to numerate PAH-degrading bacteria. The results showed that, PAH-degrading bacteria were abundant in both of surface water and sediments of Xiamen Western harbor. The numbers of PAH-degrading bacteria were more than 103CFU/mL in surface water and 104 CFU/g in sediments during summer season. However, during the autumnthe numbers of PAH-degrading bacteria were more than 102CFU/mL in surface water and 103 CFU/g in sediments.
In the surface water, the concentration of high molecular weight PAH compounds fluoranthene and pyrene was very low during the two cruises. No correlation was found between the concentrations of high molecular weight PAH compounds and their degrading bacteria numbers. The positive correlation was found between the concentrations of low molecular weight PAH compounds (fluorene and
phenanthrene) and their degrading bacteria numbers. This may imply that the inducement of high molecular weight PAH compounds to bacterium was limited by their low concentration in the surface water, high chemical stability and hydrophobicity and short exposure time. Consequently, the numbers of high molecular weight PAH compounds degrading bacteria might not indicate the extent of PAHs contamination in the surface water.
In the sediments, positive correlation was significantly found between PAH-degrading bacteria numbers and PAHs concentration, expect the station 6. Those suggest that the relationship between the concentrations of high molecular weight PAH compounds and their degrading bacteria numbers is only significant for highly contaminated sites and long-term exposure. The PAHs-degrading bacteria number may indicate the PAHs pollution at some extent in the relative stable sediment environment, but it would not be omitted to well consider other influential factors.
The enrichment of three pure cultures (PYSP1, PYSP6 and PYSP22) has been done successfully by using a spray plate method with pyrene as the sole carbon source. Other three pure cultures (FLSP2, FLSP6 and FLSP8) were is
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