固相萃取法提取典型海水溶解有机质及其红外光谱和固态13C核磁共振表征

由于海水盐度大、有机质含量低,从海水中提取和纯化克级溶解性有机质(dissolved organic matter,DOM)一直十分困难。基于Bond Elut PPL固相萃取小柱法进行海水DOM固体样品提取的放大试验,建立DOM自动化调酸、PPL树脂吸附技术,最后形成提纯、冷冻干燥PPL-DOM(通过PPL树脂提取的DOM)完整工艺。采用元素分析、三维荧光光谱、红外光谱和固态¹³C核磁共振(NMR)波谱法进行样品表征。结果表明:(1)该方法对近海表层海水DOM回收率稳定在50%以上,搭建的提取装置DOM日吸附量可达230mg;(2)鳌山湾四个季节批次PPL-DOM整体分析结果表明,样品的元素组成和光谱特征相似,表明方法适用性和稳定性较好;(3)三种水生态环境的PPL-DOM¹³C NMR谱图显示样品的基本特征相似,以烷基碳、碳水化合物碳、芳香碳和羧基碳/酰胺碳为主,其中偶极去相(dipolar dephasing)谱显示了DOM前体化合物来源的差异。多种结果表明放大后的PPL固相萃取法可稳定提取克级DOM样品。     

海洋环境中多环芳烃的测定与来源解析

从样品采集与保存、样品前处理、多环芳烃的检测3个方面介绍了测定海水和海洋沉积物中多环芳烃的步骤和最新进展.对目前较为常用的几种样品前处理方法,如液-液萃取、固相萃取、固相微萃取、索氏提取、超声提取、微波萃取、超临界流体萃取、加速溶剂萃取;以及检测方法,如气相色谱-质谱法、高效液相色谱法等作了详细介绍,比较了各自的优缺点.对目前较为常用的2种多环芳烃的来源解析方法(分子指纹法和同位素指纹法)的相关研究进展进行了总结.     

海藻的聚酚化合物与腐植质

近海水域中,溶解有机物(DOM)一方面受河流排下的陆源有机物的影响;另一方面也受到潮间带大量生长的海藻,特别是大型褐藻分泌的水溶性胞外产物的影响。海藻的胞外分泌量有时多达其总生产量的37％。如果将活的、死的和腐烂分解的海藻都考虑进去,则进入海水中的DOM总量可超过海藻总生产量的70％。     

分子印迹技术用于海洋微生物生物碱活性成分的发现

以harmine为模板分子,并以甲基丙烯酸(MAA)为功能单体,合成分子印迹聚合物(molecularly imprinted polymer,MIP).将其作为固相萃取材料,对1株海洋共栖细菌Pseudoalteromonas sp. NJ6-3-1次级代谢产物的活性成分进行选择性提取,得到harmine及与harmine结构相似的生物碱.实验结果表明,分子印迹技术是选择性分离提取海洋微生物中活性成分的有效方法.     

基于高效液相色谱法的扇贝毒素-2制备方法研究

本文以渐尖鳍藻(Dinophysis acuminata)为实验原料,建立了基于高效液相色谱法(HPLC)的扇贝毒素-2(Pectenotoxin-2,PTX2)的制备方法。研究重点对鳍藻细胞的破碎、PTX2毒素的提取及HPLC纯化等三个步骤的方法进行筛选和优化。结果表明,反复冻融与超声波细胞破碎相结合可获得最大的细胞破碎率和毒素提取率,分别为99.2%和81.5%。采用四种有机试剂(甲醇、丙酮、乙醚、二氯甲烷)及三种固相萃取小柱(Solid Phase Extraction,SPE)(Oasis HLB SPE,Phenomenex Strata-X SPE,Sep-Pak C18 SPE)对PTX2毒素进行提取,其中乙醚-二氯甲烷的提取效率最高,达78.2%。在HPLC对PTX2毒素的纯化实验中发现,流动相的pH会影响毒素组分的分离,其中在碱性流动相(含6.7mmol/L氨水的水溶液和乙腈溶液,pH=10.94)条件下,PTX2的分离效果最佳,响应值最高。实验使用Phenomenex Kinetex C18(4.8mm×250mm,5μm)色谱柱,在上述碱性流动相条件下,分离得到纯度为93.2%的PTX2毒素。本研究选定的藻细胞破碎、PTX2毒素提取、及HPLC纯化方法简单快捷,高效易操作,为从大规模培养的鳍藻细胞中制备高纯度PTX2毒素提供了技术支持。     

淡水产品中微囊藻毒素固相萃取-高效液相色谱检测方法的建立

研究利用固相萃取-高效液相色谱技术检测淡水产品中的微囊藻毒素MC-LR和MC-RR.样品经80％甲醇水溶液提取后,以固相萃取小柱进行净化,氮吹浓缩后以高效液相色谱进行分析.以65％甲醇水溶液(含0.05％三氟乙酸)为流动相,经C18反相色谱柱分离,以紫外检测器进行定量分析.线性定量范围0.1～2.0μg/g,检出限0....     

海洋水环境中藻毒素的检测技术及分布研究进展

海洋藻毒素是海洋有害藻产生的次级代谢产物,具有种类多、毒性强和生物蓄积性等特点,对海洋生态系统、海产养殖业以及人类健康会造成严重威胁。近年来,近海水环境中的海洋藻毒素已引起人们的广泛关注。通过对近海水环境中海洋藻毒素的富集技术、仪器分析方法和分布特征方面的研究进展进行归纳和总结发现:被动固相吸附和固相萃取技术是海水中海洋藻毒素的主要富集技术,结合液相色谱-质谱联用技术进行检测,是目前近海水环境中各类海洋藻毒素检测的最有效方法;多种海洋藻毒素在全球近海水环境中有分布,且欧洲近海海水中藻毒素种类最丰富、浓度也相对较高;在中国近海水环境中多种脂溶性藻毒素广泛分布。在全球气候变化以及近海污染不断加剧的背景下,海洋藻毒素对近海水环境的影响不容忽视,今后的研究方向将主要包括:重要养殖区海水中藻毒素的长期连续监测;海水中典型藻毒素的现场快速检测技术;近海水环境中海洋藻毒素的来源及其影响因素;典型海洋藻毒素的迁移转化规律及其生物可利用性等。     

海水中颗粒有机碳的测定方法

海水中颗粒有机物(POM)主要来自海洋浮游生物和其他动物的分解碎屑和排泄物。另外,还有经过在海水中,进行的溶解有机物(DOM)和POM之间的平衡而形成的POM。POM在海洋生物食物链中起着重要的作用。因此在研究海域生产力时DOM和POM含量的测定都是很重要的参数。通常以测定颗粒有机碳(POC)的含量作为POM的量度。关于海水POC,     

侧扁软柳珊瑚(Subergorgia suberosa)乙醇提取物抗氧化和抑菌活性研究

对中国南海侧扁软柳珊瑚(Subergorgia suberosa)乙醇提取物及其石油醚相、乙酸乙酯相、正丁醇相进行了抗氧化和抑菌两种生物活性研究。结果表明:中国南海侧扁软柳珊瑚乙醇提取物及其各萃取相对羟自由基的清除效果好,在50mg/L时清除效率均高于同浓度的合成抗氧化剂TBHQ和BHT;石油醚相、乙酸乙酯相对DPPH自由基和超氧阴离子自由基的清除效率优于正丁醇相和乙醇提取物。另外,对5种海洋细菌、4种陆地细菌进行了抑菌活性测定,发现乙醇提取物及其各萃取相对海洋细菌的最低抑菌浓度(MIC)为100g/L左右,对受试陆地细菌未见抑菌作用。     

水环境中的信息有机物与“水域生态讯息学”的提出

信息有机物(或化感物质)系指存在于自然环境,对生物物种或生物类群生存和生长有促进或抑制作用,并能通过调控生物丰度与群落结构来影响生态系统和生态环境的微痕量有机物。目前已知的信息有机物大多是分子质量小于10~4Da的醇、酚、萜、酯、多肽等小分子物质,它们随水体迁移扩散,通过控制酶活性或光合系统等途径影响目标生物生存生长,作用强度受微生物分解和营养水平等多种因素影响。水体中信息有机物一般是通过有机溶剂萃取或固相萃取富集后用色质联用鉴定结构并测定含量。本文系统总结了近年来水环境中信息有机物及其检测方法的主要研究进展,归纳总结了高效灵敏的水体信息有机物检测方法,阐述了水环境中信息有机物种类、结构、作用机制及其对水域生态学的意义,在此基础上,提出了"水域生态讯息学"的新概念,诠释了"水域生态讯息学"的内涵,这对于拓展生态学研究内容,揭示水生生物学发生机制,持续利用水生生物资源具有重要的科学意义和实际价值。     

Effects of seawater acidification on hydrolytic enzyme activities

We have investigated the effects of seawater acidification on the activities of leucine aminopeptidase (LAPase), β-glucosidase (BGase), phosphatase (P-ase), α-glucosidase (AGase), and lipase (L-ase), which are important promoters of degradation of marine organic matter, including proteins, carbohydrates, organic phosphorus compounds, and lipids. Seawater samples were collected from a eutrophic coastal area, from Tokyo Bay, and from oligotrophic pelagic waters outside the Kuroshio Current. Enzyme activities were measured using fluorogenic substrates added to the seawater samples, which were acidified from pH 8.2 to 5.6 by a chemical buffer. Spontaneous hydrolysis of the substrates was shown to be negligible in heat-inactivated control samples, thus validating our results. LAPase was the most sensitive to acidification; enzyme activity rapidly decreased from pH 8.2 to 7.8, corresponding to a realistic scenario of ocean acidification. L-ase activity also decreased with acidification. Activities of P-ase and BGase were relatively constant across the pH levels examined, suggesting that their activity is not appreciably influenced by acidification. The effect of acidification on P-ase activities differed between the coastal and semipelagic samples, and this was likely due to freshwater influence at the nearshore station. Because of the low activity of AGase in the sample, the effect of acidification on this enzyme could not be examined. The effects of acidification on enzyme activity appear to vary depending on enzyme type and location, but we conclude that acidification will cause changes in the cycling of organic matter in marine ecosystems, in particular to proteinous and lipid substances.     

Centropages typicus (Crustacea,Copepoda) reacts to volatile compounds produced by planktonic algae

Volatile organic compounds (VOCs) may play the role of infochemicals and trigger chemotaxis and ecologically relevant responses in freshwater and marine invertebrates. Aquatic grazers use these signals as chemical cues to trace the presence of their food or to detect their predators. However, detailed data are still needed to fully understand the role of these relationships in marine plankton. We investigated the ability of the copepod Centropages typicus to perceive the odour of three planktonic diatoms (Skeletonema marinoi, Pseudonitzschia delicatissima and Chaetoceros affinis) and a dinoflagellate (Prorocentrum minimum). This information is ecologically relevant for orientation, habitat selection, predator avoidance and communication. In addition, as the pH of the medium influences the perception of chemical cues in aquatic environments, we tested the effect of seawater acidification resulting from increasing levels of CO₂, and its influences on the olfactory reactions of copepods. For this reason, our tests were repeated in normal (pH 8.10) and acidified (pH 7.76) seawater in order to simulate future ocean acidification scenarios. Using replicated chemokinetic assays we demonstrated that VOCs produced by Ps. delicatissima and Pr. minimum attract copepods at normal pH, but this effect is lowered in acidified water. By contrast, the odour of S. marinoi mainly induces a reaction of repulsion, but in acidified water and at higher concentrations this toxic diatom becomes attractive for copepods. Our experiments demonstrate, for the first time, that copepods are sensitive to the volatile compounds contained in various microalgae; VOCs prompt chemokinesis according to algal species and odour concentrations. However, seawater acidification induces changes in copepods' perception of odours. These findings highlight the sensitivity of chemically mediated interactions to global changes     

Marine microgels: Optical and proteomic fingerprints

Dissolved organic matter (DOM) is a major carbon reservoir for the global carbon cycle, and its molecules play a key role in the biogeochemistry of the ocean. Colloidal DOM macromolecules assemble to form polymer hydrogels known as marine microgels. Marine microgels represent one of the most dynamic pools of organic carbon in the ocean. However, their optical characteristics and their contribution to ocean optical properties are largely unknown. In this work, we explore the optical and proteomic properties of spontaneously assembled DOM polymer microgels. Microgels from cultures and from Puget Sound seawater were sorted and counted using a dual-laser (365 nm/365 nm) high-speed cell sorter. This sorter has been adapted to interface with a scanning monochromator to measure the fluorescence emission spectrum of the microgels over the range from 300 to 850 nm. Surprisingly, the microgels show a broad fluorescence emission from 420 to 520 nm when excited with UV light. The microgels were classified according to their blue autofluorescence, and by three criteria that are used to define microgels: 1) staining with chlortetracycline 2) the ability to undergo phase transitions at low pH, and 3) dispersion following calcium chelation by EDTA.     

Investigations on dissolved copper—organic substances in Baltic waters

A technique for the pH-controlled isolation and separation of dissolved copper—organic substances from large amounts of seawater is described. The method has been applied in two cruises in the Bothnian and Gotland Seas of the Baltic. In both areas the correlation between the amount of isolated dissolved organic material (DOM) and the concentration of organic copper in seawater has been found to be highly significant. Under the conditions established in our experiments, organic copper fractions detected in Baltic waters were in the range of about 5% of total copper. HPLC chromatograms and UV irradiation experiments from DOM extracts strongly indicate the existence of dissolved organic-copper compounds in Baltic waters.     

Quantitative adsorption of organic matter from seawater on solid matrices

The influence of several operational parameters, including pH, flow rate, total volume of water passed, and choice of eluants, upon the efficiency of extraction of organic matter from seawater by two forms of commercially-available resin, XAD-2 and XAD-8, is considered. The overall efficiency of adsorption is at best 40%. The main factor determining the adsorption efficiency of XAD resin for natural organic matter is pH. The efficiency at normal seawater pH is only half that at pH 2.0. Although the efficiency decreases with increasing flow rate, the decrease is not great up to 3 bed vol./min. With increasing volume of water passed, the adsorption capacity of the resin initially drops, but reaches an approximately constant level after 1000 bed volumes. Alkaline solutions, either NH₄OH or NaOH, followed by methanol, completely recover adsorbed organic material from the resin. There is no significant advantage of XAD-8 over XAD-2 for seawater extractions, and the materials adsorbed by the two forms of resin are not substantially different.A new separation method combining XAD-2 resin and activated carbon as adsorbents in series in a column has been developed. Efficiencies of up to 90% were obtained in the extraction of acidified seawater. When the volume of water passed was less than 500 bed volumes, the adsorption as measured by UV-oxidation was quantitative. Elution of 90–100% of the adsorbed organic carbon was possible when ammonium hydroxide (7 M), methanol and an ammonium hydroxide—methanol mixture were used sequentially.     

Advanced characterization of marine dissolved organic matter by combining reversed-phase liquid chromatography and FT-ICR-MS

Marine dissolved organic matter (DOM) was separated by reversed-phase (RP) liquid chromatography method and analyzed with fluorescence/absorption detection and Fourier transform ion cyclotron mass spectrometry (FT-ICR-MS). The three key characteristics of the RP method are: (a) The C18-RP column chosen provides enhanced separation when the aqueous phase is 100% buffer-free water, and it does not degrade over time; (b) the water eluent adjusted to pH 7 significantly improves the resolution of water soluble compounds; (c) the initial flow maintained at low levels improves the separation of polar compounds. In samples, containing “fresh” DOM, specific peaks were detected, which were absent in “old” DOM samples. The combination with size exclusion chromatography (SEC) also demonstrated the relation between polarity and molecular size of DOM. FT-ICR-MS was applied to evaluate the quality of separation on a molecular scale demonstrating that physico-chemical characteristics of DOM can be related to molecular formulas. Sample extracts were separated into 4 preparative fractions, and a large suite of the identified molecular formulas only occurred in specific fractions. This is an important basis for the application of further analytical techniques in order to perform a more target-oriented analysis aiming at the determination of source and process biomarkers for DOM.     

海水中叶绿素提取方法的研究

本文讨论了用90％丙酮从海藻中提取叶绿素进行测定的三种提取方法(研磨法、浸泡提取法、超声波粉碎法)的最佳条件。并对三种提取方法的提取效率进行了比较。结果表明研磨法的提取效率较高,样品可立即测定,是一种较好的提取方法。 本文还讨论了不同离心力对叶绿素测定的影响。实验表明使用离心力为2000g的[X]—64—01型离心机可基本满足测定要求。     

低共熔溶剂提取海洋活性物质研究进展

海洋活性物质是指对生命活动有影响的海洋生物组分或代谢产物,在医药、化妆品、保健食品等领域具有广泛的应用。低共熔溶剂(deepeutecticsolvent,DES)是基于离子液体所发展而来的一种新型绿色溶剂,它具备可设计、可回收、生物相容性高、合成简易及成本低廉等多重优势,在天然活性物质的提取中具有巨大的应用前景,引起了国内外研究学者的广泛关注。本文综述了DES在海洋活性物质(包括海洋多糖、蛋白质、DNA、生物柴油、酚类和萜烯类化合物等)提取中的应用,以期为海洋活性物质的绿色提取和应用提供新的策略。     

Chromatographic studies of dissolved organic matter and copper-organic complexes isolated from estuarine waters

Dissolved organic matter (DOM) and dissolved copper-organic complexes were isolated from the estuarine waters of Narragansett Bay, RI, using reverse-phase liquid chromatography (RPLC). Different types of reverse-phase BOND ELUT columns (Analytichem International), including C₂, C₁₈ and phenyl-bonded phases, were studied to determine their adsorption efficiency for extracting DOM. Extraction efficiencies followed the order phenyl > C₁₈ > C₂, and phenyl − C₁₈ > C₂ for DOM and organic copper, respectively. However, comparisons of BOND ELUT and C₁₈ SEP-PAK (Waters Associates) columns indicated that SEP-PAK columns were the most efficient when both DOM and organic copper were considered. Chromatographic profiles of the isolated DOM obtained using high-performance liquid chromatography were similar in elution characteristics and resembled chromatograms typical of fulvic acid. The UV-absorption characteristics of the DOM showed small differences and suggested that the different reverse-phase columns isolated material that was qualitatively similar.Copper-organic complexes isolated using C₁₈ RPLC were studied to examine the dissociation of organically bound copper in seawater as the pH is lowered. Only a small amount of the complexed copper was displaced by the H⁺ with about 40% of the copper remaining bound at pH 3. However, the chromatographic elution behavior of the DOM and organic copper was significantly altered under acidic conditions as a result of protonation of acidic functional sites on the organic matter.