【作者】 董园；

【导师】 杨桂朋； Kam W.Tang；

【作者基本信息】 中国海洋大学 ， 海洋化学， 2013， 博士

【摘要】 β-二甲基巯基丙酸内盐（DMSP）是海水中一种重要的含硫化合物，在微生物食物链中有重要的作用，是海洋细菌、浮游植物生长所需的主要碳源和硫源。DMSP可以经过DMSP裂解酶裂解产生二甲基硫（DMS），DMS对全球气候变化和酸雨的形成有重要影响。DMSP转化为DMS的过程错综复杂，其中生物过程被认为是影响DMSP转化为DMS的主要因素，控制着海水中DMS、DMSP的含量及DMS的海-气通量。研究海洋生物对DMS、DMSP的作用机制，有助于深入了解海洋生源硫的生物地球化学循环。在本论文中，我们以影响DMSP浓度分布的两个生物因素：浮游动物摄食及细菌消耗作为研究重点，旨在深入探究浮游动物摄食和细菌降解对水体中DMS、DMSP产生和转化的影响机制。本文首先以位于青岛近岸的胶州湾海域为调查海区，于2010年7月~11月对胶州湾夏、秋季浮游动物种类和丰度进行现场调查，分析讨论了胶州湾夏、秋季浮游动物丰度的分布与环境因子（水深、温度、盐度、叶绿素a含量）和DMS、溶解态β-二甲基巯基丙酸内盐（DMSPd）、颗粒态β-二甲基巯基丙酸内盐（DMSPp）浓度的相关性。其次，通过实验室培养实验，利用MPN法，研究了摄食不同DMSP含量的无菌浮游植物对桡足类动物体表和体内DMSP消耗细菌（DMSP-Consuming Bacteria，DCB）生物量的影响，并进一步对所分离的DCB进行富集培养，在不同碳源下观测其生长状态，以了解DCB对DMSP的可能裂解机制。最后，在美国切萨皮克湾的约克河流域，定量研究了水体中自由DCB和与该海域的优势种（汤氏纺锤水蚤）相关DCB的分布和季节变化。本论文的主要研究结果如下：1.胶州湾夏、秋季浮游动物丰度与DMS分布之间的关系研究于2010年7月~11月对胶州湾浮游动物丰度进行了现场调查，调查结果显示夏、秋季浮游动物主要优势种有短尾类溞状幼虫、鸟喙尖头溞、长尾类幼体、强壮箭虫、双刺唇角水蚤、背针胸刺水蚤、太平洋纺锤水蚤、五角水母、球型侧腕水母、夜光虫和中华哲水蚤等。（1）胶州湾浮游动物分布受水体的环境因子（水深、温度、表层盐度、叶绿素a含量等）不同程度的影响。胶州湾浮游动物平均丰度分布不均匀，以湾口（E3站）最高，湾内东部沿岸海域（B5站）最低。胶州湾东部海域（B5、C5、D4站）浮游动物平均丰度普遍偏低，均低于100ind/m~3。浮游动物的月际变化中，优势种季节变化控制着水体中浮游动物的丰度，再加上周期性浮游动物幼体和成体的出现对浮游动物丰度的影响较大，胶州湾浮游动物丰度具有明显的季节变化，浮游动物7~11月份平均丰度的变化范围为87.2～246.2ind/m~3，最大丰度出现在夏季8月份（246.2ind/m~3），最低值出现在秋季10月份（87.2ind/m~3）。秋季浮游动物丰度低于夏季浮游动物丰度。（2）胶州湾浮游动物丰度与水深、温度、表层盐度、叶绿素a含量、细菌生物量相关性不明显。2010年10月浮游动物丰度与DMS呈显著正相关（p <0.05），11月浮游动物丰度与DMSPp呈显著正相关（p <0.05），说明浮游动物生物量对DMS、DMSP的释放具有积极的作用。其他月份（7、8、9月）的浮游动物丰度与DMS、DMSPd、DMSPp浓度的相关性均不明显（p>0.05）。由于浮游动物摄食活动对DMS释放的影响受多种因素的制约，因此浮游动物与DMS的相互作用需要进一步研究。（3）桡足类是胶州湾浮游动物的主要类群，调查期间各个月份胶州湾均有桡足类的出现，占总浮游动物总量的3%~60.4%不等，9月桡足类占总浮游动物总量的60%。在胶州湾调查海域，桡足类在各站位呈现分布不均匀的现象，平均丰度相差一个数量级。最大值出现在湾内东北海域的A2站位，桡足类丰度达到80.21ind/m~3，湾口次之，而胶州湾沿岸海域桡足类丰度普遍较低，均低于20ind/m~3，湾内西部沿岸C1站位最低，仅为8.79ind/m~3。（4）桡足类丰度与水深、温度、表层盐度、叶绿素a含量、细菌生物量相关性不明显（p＞0.05）。桡足类丰度只与10月、11月的站位水深呈显著正相关（p＜0.05），在11月与海水温度呈显著负相关（p＜0.05），在其他月份（7、8、9月）与各环境因子均不具有显著相关性（p＞0.05）。桡足类丰度仅与7月的DMS浓度和11月的DMSPp浓度呈显著正相关（p＜0.05），在其他月份（8、9、10月），桡足类丰度与DMS、DMSPd、DMSPp均没有显著相关性（p＞0.05）。2.饵料对汤氏纺锤水蚤体表和体内DMSP消耗细菌的数量及碳利用程度的影响本实验选取了五种不同DMSP含量的无菌浮游植物，即玛氏骨条藻（Skeletonma marinoi）、周氏扁藻（Tetraselmis sp.）、亚历山大藻（Alexandriumtamarense）、盐生红包藻（Rhodomonas salina）、杜氏藻（Dunaliella tertiolecta）作为饵料藻，通过实验室培养实验，利用MPN方法，研究了摄食不同饵料藻后汤氏纺锤水蚤（Acartia tonsa）体表和体内的DCB含量，并研究了DCB在不同结构的碳源有机物上的生长状况。结果表明：（1）在所有的实验组中均发现了DMSP消耗细菌的存在。摄食五种饵料藻的汤氏纺锤水蚤中，摄食玛氏骨条藻的汤氏纺锤水蚤体表和体内DCB密度最大（平均浓度为3.64×10~6/ind），而摄食盐生红包藻的DCB密度最小（平均浓度为1.72×10~5/ind）。几乎检测不到DMSP含量的杜氏藻组却与DMSP高产种亚历山大藻、玛氏骨条藻含有相当数量的DCB，表明桡足体表和体内DCB的含量与饵料中的DMSP含量具有相对独立的关系。（2）在汤氏纺锤水蚤体上分离出的DCB可以在除DMSP外的其他碳源底物上生长，不同的生长速率反映了不同菌种对底物利用能力的差异。在实验中选取的五种底物中，甜菜碱（glycine betaine，GBT）与DMSP具有相似的分子结构，都含有以碳链形式存在的碳，DCB可以在DMSP和GBT中生长，说明DCB具有利用碳链中碳的能力。而甲胺（monomethylamine，MMA）、二甲胺（dimethylamine，DMA）、二甲亚砜（dimethylsulfoxide，DMSO）分子中只含有甲基形式的碳，细菌可以在MMA，DMA，DMSO培养基中生长说明了DCB可以同化还原甲基碳。（3）摄食实验结果中，纺锤水蚤体表和体内DCB的数量不受饵料中DMSP含量的影响，暗示了即使在DMSP含量较少的海洋环境中，浮游动物体内依然可能含有相当数量的DCB。汤氏纺锤水蚤及其体内的DCB在水体中是DMSP迁移去除的一个重要的源，对DMSP的转化有重要作用。3.约克河与桡足类相关的DMSP消耗细菌的昼夜迁移及季节分布DCB广泛存在于海洋环境中，对DMSP的迁移和转化有着重要的作用。本研究于2012年5~10月，在美国弗吉尼亚州约克河流域，对水体中的自由DCB和与汤氏纺锤水蚤相关的DCB做了现场调查。结果发现，DCB普遍存在于水体中，所有的样品中均含有自由DCB和与汤氏纺锤水蚤相关的DCB。对DCB的昼夜迁移研究发现，月初采样时，海水表层自由细菌表现出白天丰度低于夜晚的趋势。相反，月末采样时，夜晚自由细菌丰度低于相应的白天值，具体机制还有待于进一步探讨。自由DCB与温度和盐度呈显著正相关（p<0.05），与浮游动物相关的DCB仅与表层盐度呈显著正相关（p<0.05）。表明在异养微生物生长过程中，底物浓度可能是比温度更为重要的参数。附着在浮游动物营养物质丰富的地方，使细菌更易于接触到可利用的底物有机物，从而减小了细菌丰度对环境因子中温度的依赖程度。更多还原

【Abstract】 Dimethylsulfoniopropionate (DMSP), an important sulfur compound in marinewaters, was considered to play significant roles in microbial food chain and served asprimary carbon/sulfur source for phytoplankton. It was generally known that DMSPcould mainly product dimethylsulfide (DMS) through enzymatic cleavage pathway.DMS has key effects on global climate regulation and the formation of acid rain. Theoccurrence of DMSP and its further turnover to DMS are complex web in whichbiological processes were thought to be the principal factor involved. Biologicalprocesses control the concentration of DMS/DMSP in seawaters and flux of DMSrelease to the atmosphere. Studies on how biological processes were act on the DMSand DMSP contribute to understanding of the biogeochemical cycles of sulfur in theocean.In the present dissertation, we focused on zooplankton grazing and bacteriaconsumption which were the two main factors influence the distribution of DMSP,aimed at exploring the mechanism of how zooplankton grazing and bacteriaconsumption affected the release of DMSP into the water column. At first, we chosethe Qingdao coastal water-Jiaozhou Bay as the study area. Zooplankton compositionand abundance were studied based on the zooplankton in situ samples collected inJiaozhou Bay during the time period from July to November,2010. The distributionand seasonal varied of zooplankton, and their relationships with the environmentalfactors of the seawater (water depth, temperature, salinity and Chl-a concentrations),as well as concentrations of dimethysulfide (DMS), dissolveddimethylsulfoniopropionate (DMSPd), particulate dimethylsulfoniopropionate(DMSPp) were analyzed. Secondly, we used the axenic phytoplankton culture ofdifferent DMSP contents fed copepods. By using MPN (Most Probable Numbers)method, we studied whether the concentration of DMSP-consuming bacteria (DCB)associated with copepod was related to DMSP content in the food under laboratorial experiments. DCB isolated from copepod body were incubated in different carbonsources afterwards. By observing their growth situation, we could get to know thepossible pathway of DCB utilize DMSP. Another objective of this dissertation was toquantitatively study the free-living DCB and Acartia tonsa-associated DCB in YorkRiver, Chesapeake Bay, USA. A. tonsa was the dominant species in this area,furthermore, we monitored the distribution and monthly variation of DCB. The mainconclusions were drawn as follows:1. Study on relationship between zooplankton abundance and DMSdistribution in Jiaozhou Bay in summer and autumn of2010Zooplankton composition and abundance were studied in Jiaozhou Bay during thetime period from July to November,2010. The dominant species in this ara wereBrachyura zoea larva、Penilia avirostris Dana、Macrura larva、 Sagitta crassaTokioka、Labidocera bipinnata Tanaka、Centropages dorsispinatus Thompson etScott、Acartia pacifica Steuer、Muggiaea atlantica Cunningham、Pleurobrachiaglobosa Moser、Noctiluca scientillans Kofoid et Swezy、Calanus sinicus Brodsky, etal.(1) Zooplankton distribution in Jiaozhou Bay were impacted by environmentalfactors of the water column (water depth, temperature, salinity and Chl-aconcentrations, et al) by varying degrees. As it was shown by the results, the averagezooplankton abundance in Jiaozhou Bay was unevenly distributed, with highestzooplankton abundance observed at Bay month (E3station), and the lowest at Easterncoast (B5station). The average zooplankton abundance of eastern stations (B5, C5,D4) were generally low which all below100ind/m~3. For the monthly variation ofzooplankton abundance, on the one hand, because the dominant species of water bodycontroled the total abundance of zooplankton, on the other hand, periodicityzooplankton larvae and adult emergence greatly impacted zooplankton abundance, thezooplankton abundance of Jiaozhou Bay showed significant seasonal variation. Theaverage zooplankton abundance from July to November ranged from87.2~246.2ind/m~3, with highest value found in summer at August (246.2ind/m~3), and lowest one in autumn at October (87.2ind/m~3). Zooplankton abundance in summer was higher thanthat in autumn.(2) Zooplankton abundance didn’t show strong relationships with water depth,temperature, salinity, Chl-a concentration, bacteria number. There was significantlypositive correlation between zooplankton abundance and DMS in October (p<0.05),and zooplankton abundance and DMSPp in Novemeber (p<0.05). Zooplanktonbiomass could accelerate DMSP release. But in other months (July, August,September), the correlations were not significant (p>0.05). Our results showed thatthe influence of zooplankton grazing on the release of DMS from water column wasaffected by complex environmental factors and further research was needed betweenzooplankton and DMS.(3) Copepod was the dominant group of zooplankton in Jiaozhou Bay, it wasobserved in each month during our observation period and could compose about3~60.4%of total zooplankton abundance, with the highest percentage (60%) inSeptember. The copepods were inhomogeneous in their horizon distribution, it couldvaried up to an order of magnitude. The highest copepod abundance, which reach to80.21ind/m~3was found in northeast (A2station), followed by Bay mouth. Generally,copepod abundance of coastal water in Jiaozhou Bay was low, all below20ind/m~3,with the lowest value (8.79ind/m~3) state on the west (C1station).(4) There was no strong correlation between copepod abundance and water depth,temperature, surface salinity or Chl-a concentration (p>0.05). Copepod abundancewas significantly positive related to water depth in October and November (p<0.05),while negatively related to water temperature in November (p<0.05). In other months(July, August, September), the correlations between copepod abundance andenvironemtnal factors were not significant (p>0.05). It only showed significantpositive correlation between copepod abundance and DMS in July, DMSP inNovember (p<0.05), in other months (August, September, October), the correlationsbetween copepod abundance and DMS/DMSPd/DMSPp were not significant(p>0.05).2. Dietary effects on abundance and carbon utilization ability of DMSP-consuming bacteria associated with the copepod Acartia tonsa DanaIn this part, we chose five axenic phytoplankton culture: Skeletonema marinoi、Tetraselmis sp.、Alexandrium tamarense、Rhodomonas salina、Dunaliella tertiolectaas food. Under the laboratorial experiments, we measured DMSP-consuming bacteria（DCB） number associated with A. tonsa by using MPN method. Then studied howDCB growth on different carbon sources. The results showed that:(1) DCB were recovered from all treatments, among which the S. marinoitreatment yielded the highest abundance (3.64×10~6DCB copepod-1) and the R. salinatreatment the lowest (2.40×10~4DCB copepod-1). We found comparable number ofDCB in D. tertiolecta treatment and A. tamarense, while D. tertiolecta had nodetectable amount of DMSP and A. tamarense was a DMSP high producer, DCBabundance associated with A. tonsa was independent from dietary DMSP content.(2) The DCB were able to grow on carbon sources other than DMSP, differentgrowth rates indicated different substrates utilize abilities. Among the five substrates,Glycine betaine and DMSP had similar molecular structure in which all containedcarbon in the carbon chain form, and MMA, DMA, DMSO all contained carbon inmethyl form. The ability of utilize all the substrates indicating their ability toassimilate both carboxyl chain carbon and methyl carbon.(3) The grazing experiment results showed that DCB abundance associated withzooplankton was independence from food DMSP, this would allow DCB to maintaintheir populations among zooplankton even under DMSP-poor conditions, and theywould readily consume DMSP when it became available. This coupling between DCBand zooplankton represented a persistent and potentially important sink of DMSP inthe marine system.3. The diel migration and seasonal distribution of DMSP-consuming bacteriaassociated with copepod in York RiverDCB were ubiquitous in ocean environment and play important roles on DMSPtransportation. In this section, we studied free-living DCB and zooplankton-associatedDCB in York River, Virginia, USA, during May to October,2012. As it was shown inresults, DCB were present in all the samples indicating their universal existence in the seawater. For DCB dial migration, we found it was interestingly that free-living DCBwas lower in the daytime when sample at the beginning of the month, on the contrary,it would be lower in the nighttime when sample at the end of the month.The specificmechanism which controled this process needed to be further studied. Free-livingDCB showed positive correlations with temperature and salinity (p<0.05), whilezooplankton-associated DCB only showed positive correlation with salinity, indicatedthat substrates availability could be as important as or more important thantemperature in regulating heterotrophic microbial process. Association withzooplankton might give attached bacteria access to resources, thereby moderatingtheir responses to environmental temperature.更多还原