【作者】 张芳；

【导师】 王修林；

【作者基本信息】 中国海洋大学 ， 海洋化学， 2008， 博士

【摘要】 中国近海赤潮频发、影响范围扩大、持续时间增长,在一定程度上制约了中国沿海省市海洋经济,甚至损害了公众健康。迫切需要建立一种现场、快速、实时的浮游植物群落组成识别技术,以实现赤潮的常规和应急监测.浮游植物的三维荧光光谱表现出明显的指纹特性,在浮游植物群落组成测定及赤潮发生时对赤潮引发种的识别测定方面具有很大的应用潜力。但是,目前能够应用的浮游植物群落组成荧光识别测定技术主要局限于两大类;a.对某类具有显著荧光特征的浮游植物的测定;b.将浮游植物粗略的分为几大类后进行监测。尚没有针对中国近海具体海区的特定浮游植物群落优势种的识别技术,也没有一种针对赤潮发生时对赤潮引发种进行识别及数量测定的现场监测技术。本文在对中国近海海域赤潮与非赤潮期间浮游植物群落组成特点进行总结的基础上,选取实验室培养的38种东海主要浮游植物（多为赤潮种及优势种,分属7个门10个纲30个属:其中甲藻2纲6属9种,包括塔玛亚历山大藻Alexandrium tamarense、强壮前沟藻Amphidinium carterae、锥状斯比藻Scrippsiella trochoidea、裸甲藻Gymnodinium spp.、简裸甲藻Gymnodinium simplex、东海原甲藻Prorocentrum donghaiense、微小原甲藻Prorocentrum minimum、海洋原甲藻Prococentrum marinum及米氏凯伦藻Kareniamikimotoi,硅藻2纲9属12种,包括尖刺拟菱形藻Pseudo-nitzschia pungens、中肋骨条藻Skeletonema costatuma、圆海链藻Thalassiosira rotula、新月菱形藻Nitzschia closterium、丹麦细柱藻Leptocylindrus danicus、布氏双尾藻Ditylum brightwellii、盒形藻Odontellacf_sinensis、辐射圆筛藻Coscinodiscophyceae Coscinodiscus cf_radiatus、圆筛藻Coscinodiscophyceae Coscinodiscus Jp.、旋链角毛藻Chaetoceros curvisetus、柔弱角毛藻Chaetoceros debilis及双突角毛藻Chaetoceros didymus:金藻2纲5属5种,包括球等鞭金藻Isochrysis galbana、小三毛金藻Prymneium parvum、颗石藻Gephyrocapsa oceanica、球形棕囊藻phaeocystis globosa及六异刺硅鞭藻Distephanus speculum;绿藻1纲4属5种,包括岛国大扁藻Platymonas helgolanidica、亚心型扁藻platymonas subcordiforus、细小微胞藻Micromonas pusilla、盐生杜氏藻Dunaliella salina及小球藻Chlorella pynenoidosa;黄藻1纲2属2种,包括赤潮异湾藻Heterosigma akashiwo及海洋卡盾藻Chattonella marine;隐藻1纲1属2种.包括隐藻Rhodomonas sp.及盐沼隐藻Rhodomonas salina:蓝藻1纲3属3种,包括聚球藻Synechococcus sp.、螺旋藻Spirulina sp.及鱼腥藻Anabaena sp.）进行研究,对活体浮游植物的三维荧光光谱的测量精密度、培养重现性、稳定性（生长期、光照、温度）及不同种类浮游植物的光谱差异性等进行了综合分析,阐述了应用三维荧光光谱识别浮游植物种类的可行性;分别针对已商业化的荧光分光光度计及要自行开发的高灵敏度、低能耗、便携式藻类自动分析仪。基于连续三维荧光光谱及离散三维荧光光谱,利用小波分析等方法建立丁东海浮游植物赤潮种及优势种的识别技术;并初步建立了以叶绿素浓度为指标参数的东海浮游植物（半）定量分析方法,为建立东海赤潮生物种类现场监测技术提供了重要基础。取得的主要成果如下:1.比较系统地总结了中国近海赤潮生物、浮游植物优势种及群落结构特点:a.中国近海海域在非赤潮期间多以1～2个门类的多种浮游植物为优势种,单种优势度一般不会超过75%,而总门类优势度多在90%以上,甚至接近100%;b.中国近海海域以单相型赤潮为主,发生赤潮时绝大多数肇事藻的优势度大于90%,甚至接近100%;双相型赤潮的两种优势种的总优势度也在90%以上,甚至接近100%。2.以相对标准偏差（RSD）及相关性系数（IAC）为标准,对不同三维荧光光谱部分的测量精密度、重现性、稳定性及不同种类差异性等进行分析,阐述了应用三维荧光光谱识别浮游植物种类的可行性:在实验室研究条件下,绝大多数浮游植物的三维荧光光谱具有良好的测量精密度、培养重现性、稳定性（生长期、光照及温度）,大多数不同属（种）的藻间表现出明显的差异性。获得了最佳连续三维荧光光谱部分（Ex=400～600 nm,Em=650～670 nm）。3.以两类小波函数（db7及coif2小波）对浮游植物的最佳连续三维荧光光谱部分进行特征提取,获得了不同层次的尺度分量及小波分量,以Fisher判别法选择了尺度分量及小波分量识别特征谱;对浮游植物在不同条件下的分类识别能力进行了分析,并以系统聚类法提取浮游植物两类小波函数的尺度分量及小波分量识别特征谱的标准谱,分别建立了两级标准谱库,在此基础上,利用非负最小二乘法建立荧光识别技术:对浮游植物在属（种）和门类水平上的正确识别率分别高于86%及96%;可对现场样品中胞密度大于5×10⁶ cell L^-1的赤潮优势种（Pr、Al、诺登海链藻、聚生角毛藻）进行属层次的识别,可对不发生赤潮的浮游植物群落(胞密度为10⁵～10⁶ cell L^-1)优势种进行门类层次的识别。4.选择了12个激发波长点（400 nm,430 nm,450 nm,460 nm,470 nm,490 nm,500nm,510 nm,525 nm,550 nm,570 nm及590 nm）在650～700 nm范围内发射光谱组成离散三维荧光光谱,以两类小波函数（db3及coif2小波）对浮游植物的离散三维荧光光谱进行分解,获得了不同层次的尺度分量及小波分量,以Fisher判别法选择了尺度分量及小波分量识别特征谱;对浮游植物在不同条件下的分类识别能力进行了分析,并以系统聚类法提取浮游植物两类小波函数的尺度分量及小波分量识别特征谱的标准谱,分别建立了两级标准谱库,并利用非负最小二乘法建立荧光识别技术:对浮游植物在属（种）和门类水平上的正确识别率分别高于85%及96%:可对现场样品中胞密度约为10⁷ cell L^-1的赤潮优势种（Pr、诺登海链藻、聚生角毛藻）进行属种层次的识别,可对不发生赤潮的浮游植物群落(胞密度为10⁵-10⁶ cell L^-1)优势种进行门类层次的识别。5.在实验室初步建立了活体浮游植物叶绿素荧光（半）定量分析方法,方法的检测限约为8μgL^-1,叶绿素的最佳线性范围为20～200μg L^-1。本论文的创新之处在于基于小波分析等方法建立了东海主要浮游植物的标准谱库,可对单相型赤潮的引发种进行种属层次的识别,并可对979个浮游植物实验室混合样品中的28种优势种及发生赤潮时的肇事种实现属层次的识别,在属层次上的识别正确率大于85%,门类层次上的识别正确率大于96%。更多还原

【Abstract】 Phytoplankton is the main source of primary production in both marine and freshwater habitats.Some of the species occasionally grow very fast or "bloom" and accumulate into dense,visible patches near the surface of the water,then the harmful algal blooms（HABs） break out.The HABs is very severe in the costal waters of the China Sea.They destroy the zoological balance of the water.Worse still,some species produce potent neurotoxins that can be transferred through the food web,where they affect and even kill the higher forms of life such as zooplankton,shellfish,fish,birds,marine mammals and even humans.This phenomenon becomes very serious.It becomes an urgent need for determining the algae causing HABs in situ,rapidly and reliably.The excitation-emission matrixes（EEMs） has present ’fingerprint feature’ to phytoplankton,which makes it potential to be utilized in the detection of the phytoplankton communities and the identification of HAB causative species when HAB breaks out.However,the utilization of fluormetric methods in discriminating phytoplankton just focus on some divisions that present obvious fluorescent characteristics. There is neither applied fluormetric method that directly aims at the discrimination of the phytoplankton communities in given sea area of the China Sea,nor fluormetric method for the identification of HAB causative species in situ when HABs break out.This paper just focuses on the above problems.Based on the analysis of the characteristics of the phytoplankton communities in coastal water of the China Sea, thirty-eight phytoplankton species,that is Alexandrium tamarense,Amphidinium carterae, Scrippsiella trochoidea,Gymnodinium spp.,Gymnodinium simplex,Prorocentrum donghaiense,Prorocentrum minimum,Prococentrum marinum,Karenia mikimotoi; Pseudo-nitzschia pungens,Skeletonema costatuma,Thalassiosira rotula,Nitzschia closterium, Leptocylindrus danicus,Ditylum brightwellii,Odontella cf_sinensis,Coscinodiscophyceae Coscinodiscus cf_<sub>radiatus,Coscinodiscophyceae Coscinodiscus sp.,Chaetoceros curvisetus, Chaetoceros debilis,Chaetoceros didymus;Isochrysis galbana,Prymneium parvum,Gephyrocapsa oceanic,phaeocystis globosa,Distephanus speculum;Platymonas helgolanidica,platymonas subcordiforus,Micromonas pusilla,Dunaliella salina,Chlorella pynenoidosa;Heterosigrna akashiwo,Chattonella marine;Rhodomonas sp.,Rhodomonas salina;Synechococcus sp.,Spirulina sp.and Anabaena sp.,most of which are HAB causative species and dominant species in the East China Sea,were selected and cultured in the laboratory.They belong to seven divisions and thirty genuses.The EEMs were measured and the scanning precision,culturing recurrence,growth stability（at different growth stages, under different culturing lights and temperatures）,as well as the difference between EEMs of different species are analyzed.Based on this,the feasibility of the utilization of EEMs in discriminating phytoplankton species is expounded.With the utilization of wavelet analysis, the identifying methods of the HAB causative algae and dominant species in the East China Sea are established based on both the continuous and the discrete EEMs,aiming at the commercial fluorescence speetrophotometer and the ones that would be developed by us, respectively.In last section,based on the chlorophyllous eoncentration,the primary semi-quantitative fluormetric method for the in vivo phytoplankton species was established in the lab.All these work can provide important groundwork for the monitoring of the HAB causative phytoplankton species that in the East China Sea.The main conclusions of this paper are as follows:1.On the sum-up of the HAB causative species as well as the domain species in the coastal waters of the China Sea,and the analysis of the composing characteristics of the phytoplankton communities,the following conclusions are gotten:a.during the non-HAB period,there are always more than one phytoplankton dominant species.They usually belong to one or two divisions.The common predominance of one dominant species is less than 75%,while the predominance of one division are usually more than 90%,or even close to 100%.b.The HABs were mostly caused by only one species,the predominance of which are usually more than 90%,or even close to 100%.The HABs caused by two species become more and more.When such HABs take place,the general predominance of the two species are more than 90%,or even close to 100%.2.Two criteria are utilized in the analysis of the feasibility to the discrimination of phytoplankton species by EEMs.The criteria are Relative Standard Deviation（RSD） and Included Angle Cosine（IAC）.The results present that it is possible to identify phytoplankton species based on EEMs.The part with Ex= 400～600 nm,Em= 650～670 nm are selected as the optimal EEMs.Under the culturing conditions in this paper,most phytoplankton species present good scanning precision,culturing recurrence,and growth stability（’at different growth stages,under different culturing irradiances,as well as different culturing temperatures）.Also,there are obvious differences between most phytoplankton species that belonging to different genuses under our experimental conditions.3.Two wavelets（db7 and coif2） are utilized to extract the characteristics of the EEMs. Different scale vectors and time-series vectors are obtained.The discriminating characteristic spectra of both scale and time-series are selected by Fisher discriminant method.Classifying results under different conditions are analized by Bayes discriminant method.Clustering analysis is utilized to extract the reference spectra from both the scale and time-series discriminating characteristic spectra obtained by both wavelets.A two-rank "database of reference spectra" is established for each wavelet.Based on the databases,a fluormetric method for the identification of phytoplankton species is established by nonnegative least squares（NNLS）.This method can identify phytoplankton species at both the genus level as well as the division level.The correct discriminating rates are more than 86%and 96%,respectively.Some HAB causative species （Prorocentrum donghaiense,Alexandrium tamarense,Thalassiosira nordenskioldii and Chaetoceros socialis） can be identified by this fluormetric method when the cellular density is about 10⁷ cell L^-1 in the actual sea waters.The dominant species in the phytoplankton communities can be identified at the division level when the cellular density of the community is 10⁵～10⁶ cell L^-1. 4.Twelve excitation wavelengths,they are,400 nm,430 nm,450 nm,460 nm,470 nm, 490 nm,500 nm,510 nm,525 nm,550 nm,570 nm及590 nm,are selected to compose a discrete EEM.The emission wavelengths are 650～700 nm to the EEM.5.Two wavelets（db3 and coif2） are utilized to extract the characteristics of the discrete EEMs.Classifying results under different conditions are analized by Bayes discriminant method.Different scale vectors and time-series vectors are obtained.The discriminating characteristic spectra of both scale and time-series are selected by Fisher discriminant method.The reference spectra of both the scale and time-series discriminating characteristic spectra obtained by both wavelets were obtained by clustering analysis.A two-rank "data base of reference spectra" is established for both wavelets,respectively. Based on this,a fluormetric method for the identification ofphytoplankton species is established by nonnegative least squares（NNLS）.This method can identify phytoplankton species at both the genus level as well as the division level.The correct discriminating rates are more than 85%and 96%,respectively.Some HAB causative species （Prorocentrum donghaiense,Thalassiosira nordenskioldii and Chaetoceros socialis） can be identified by this fluormetric method when the cellular density is about 10⁷ cell L^-1 in the actual sea waters.The dominant species in the phytoplankton communities can be identified at the division level when the cellular density of the community is 10⁵～10⁶ cell L^-1.6.Based on the chlorophyllous concentration,a primary semi-quantitative fluormetric method for determining the in vivo phytoplankton species was established in the lab.The detecting limit is about 8μg L^-1,and the optimal linear range is 20～200μg L^-1.The innovation in this paper is the establishment of the reference spectra for the main phytoplankton species in the East China Sea.Based on this,28 dominant species in the 979 laboratory mixtures and the HAB causative species during the single type HAB periods can be identified at the genus level.The correct discriminating rates are more than 85%at the genus level,and that are more than 96%at the division level.更多还原