Effects of Two Potential Allelochemicals on the Photosystem II of Nitzschia closterium and Monostroma nitidum

In aquaculture,high-density seaweed farming brings higher economic benefits but also increases outbreaks of diatom felt.The effective control of diatom felt in high-density seaweed farming has always been a research hotspot.This study selected two potential allelochemicals 2-hydroxycinnamic acid and quinic acid to explore their effects on a diatom Nitzschia closterium and an economic seaweed Monostroma nitidum.The results showed that 2-hydroxycinnamic acid had better inhibitory effects than quinic acid on the growth,pigment content and photosynthetic efficiency of N.closterium.Their half-maximal inhibitory concentrations at 120 h(IC_(50–120 h))were 0.9000 and 1.278 mM,respectively.Additionally,these allelochemicals had limited inhibitory effects on the growth,pigment content and photosynthetic efficiency of M.nitidum before 24 h.To further explore the allelopathic effect of these chemicals,this study focused on the photosystem II energy fluxes of N.closterium.It was found that 3 mM 2-hydroxycinnamic acid could destroy the whole photosynthetic system by devastating the PSII reaction centre(RC)before 24 h;however,the same concentration of quinic acid could only down-regulate the electron transport efficiency by changing the effective antenna size of an active RC and downregulating the PSII reaction centre density.These experimental results are expected to provide a new strategy to control diatom felt blooms on the high-density seaweed farming areas.

新月菱形藻(Nitzschia closterium)制备参数对藻球性质及去氮磷效果的影响

为了制备出高效的固定化藻球,完善固定化藻球的制备工艺,以新月菱形藻为藻种,采用褐藻胶包埋技术和单因子试验,研究了不同褐藻酸钠质量分数(2.0%、2.5%、3.0%)和不同溶剂(海水、蒸馏水、NaCl溶液)对藻球形状、强度、传质性、去除氮磷效果及藻细胞生长的影响.结果表明:不同褐藻酸钠质量分数对藻球形状影响显著(P<0.05),褐藻酸钠质量分数为2.0%和2.5%时藻球呈球形,为3.0%时藻球呈不规则的水滴形;不同溶剂种类对藻球形状则无影响.不同褐藻酸钠质量分数对藻球强度影响显著(P<0.05),随着褐藻酸钠浓度的增加,藻球强度逐渐增强;不同溶剂对藻球强度的影响显著(P<0.05),影响大小的排序为海水>NaCl>蒸馏水.不同褐藻酸钠质量分数和不同溶剂对藻球传质性影响显著(P<0.05),随着褐藻酸钠浓度的增加,藻球传质性也逐渐增强;溶剂对传质性影响的强弱排序为海水>NaCl>蒸馏水;不同褐藻酸钠质量分数和不同溶剂对固定化藻球藻细胞生长及去氮磷效率影响显著(P<0.05),其中以3.0%褐藻酸钠+海水的藻细胞生长最快,去除氮磷效率最佳.

Analysis of phospholipids in microalga Nitzschia closterium by UPLC-Q-TOF-MS

Precise structural identification of phospholipids in the microalga Nitzschia closterium has been established using ultra performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS) for direct analysis of total lipid extracts.Mass spectrometry was performed in reflective time-of-flight using electron spraying ionization in negative mode.Phospholipid molecular species identification was based on the characteristic product ions and neutral loss yielded by different phospholipids under ESI-MS/MS mode.The molecular species were confirmed by the carboxylate anions produced by phospholipids in negative mode;the regiospecificity of the two acyl chains was determined from the ratio of sn-1 to sn-2 carboxylate anion abundances.As a result,18 lipid molecular species were identified for the first time in this microalga,comprising seven phosphatidylcholines (PC),two phosphatidylethanolamines (PE),two phosphatidylinositols (PI),and seven phosphatidylglycerols (PG).Lipid standards of PC,PE,PI,and PG were added to the total lipids as internal standards for semiquantitative analysis,revealing concentrations of phospholipids in this species between 0.09 and 3.37 nmol/mg.This method can produce a full structural profile of intact phospholipid molecular species and can be used for study of the physiological and ecological functions of lipids by monitoring their individual changes over time.

Preparation and characteristics of biosilica derived from marine diatom biomass of Nitzschia closterium and Thalassiosira

In this study, biosilica of high purity was successfully prepared from marine diatom( Nitzschia closterium and Thalassiosira) biomass using an optimized novel method with acid washing treatment followed by thermal treatment of the biomass. The optimal condition of the method was 2% diluted HCl washing and baking at 600°C. The SiO_2 contents of N. closterium biosilica and Thalassiosira biosilica were 92.23% and 91.52%, respectively, which were both higher than that of diatomite biosilica. The SiO_2 morphologies of both biosilica are typical amorphous silica. Besides, N. closterium biosilica possessed micropores and fibers with a surface area of 59.81 m^2/g. And Thalassiosira biosilica possessed a mesoporous hierarchical skeleton with a surface area of 9.91 m^2/g. The results suggest that the biosilica samples obtained in this study present highly porous structures. The prepared porous biosilica material possesses great potential to be used as drug delivery carrier, biosensor, biocatalyst as well as adsorbent in the future.

Effect of NaNO_3 Concentrations on the Growth and Fatty Acid Compositions of Nitzschia closterium and Chaetoceros gracilis*

Fatty acid compositions of two strains of marine diatoms Nitzschia closterium ( MACC B222 ) and Chaetoceros gracilis ( MACC B13 ) have been examined. The microalgae have been grown at different initial NaNO3 concentrations (75,375, 750, 1125,1 500, 1 875 mg/L ) and harvested at the late exponential phase. The results by one factor analysis of variance show that the NaNO3 concentrations have no significant influence on the relative growth rate of two strains of marine diatoms; the influence of NaNO3 concentrations on the fatty acids differs from species to species. The major fatty acids of two diatom strains are 14:0, 16:0, 16:ln- 7 and 20:5n- 3 ( EPA ) , B13 also has high percentage of20:4n-6 (6.0～9.1%).

Size-Dependent Growth Rate of Nitzschia closterium at Different Concentrations of Petroleum Hydrocarbon

The maximum growth rate of Nitzschia closterium at various concentrations of petroleum hydrocarbon was studied. The influence of petroleum hydrocarbon on cell size as a function of concentration was discussed. The relationship between maximum growth rate of Nitzschia closterium and cell median equivalent spherical diameter (MESD) was also carefully studied. The experimental results showed that the growth rate of Nitzschia closterium was generally suppressed by petroleum hydrocarbon, which had greater effects at both low and high concentrations than at intermediate concentrations. No significant changes in cell size distribution were observed during the growth period of Nitzschia closterium. The Gaussian function could give a clear description of the cell size distribution of Nitzschia closterium, and the MESD value ranged from 2.71 to 6.82 μm. The MESD decreased when the cell was exposed to petroleum hydrocarbon, and the reduction of cell MESD was much more significant at both relatively high and low hydrocarbon concentrations. The presence of petroleum hydrocarbon changed the relationship between μmax and MESD from an allometric function to a U-shaped curve. When the MESD was below 5.07 μm, μmax decreased along with increased MESD, whereas when MESD was above 5.07 μm, μmax increased along with MESD, which deviated from the allometric model.

长菱形藻(Nitzschia longissima)和新月细柱藻(Cylindrotheca closterium)分子分类研究

本研究自胶州湾分离了两种底栖硅藻,形态学初步鉴定为长菱形藻和新月细柱藻。对其18S rDNA和rbcL基因进行了测序并用邻接法构建了系统树。结果表明,两藻在18S rD-NA和rbcL基因序列上均存在较大的差异,基于DNA序列的分类结果与形态学分类结果一致。在依据形态指标难以确定藻类分类地位的情况下,18S rDNA和rbcL基因序列是有用的鉴定工具。

Hierarchical recognition on the taxonomy of Nitzschia closterium f. minutissima

Nitzschia closterium f. minutissima, a marine eukaryotic unicellular diatom, originally classified as Bacillariophyta/Bacillariophyceae/Bacillariales/Bacillariaceae/Nitzschia, is one of the most important feed sources in mariculture. In this study, its morphological features were examined under DIC Microscopy (differential interference contrast microscope); its pigments and fatty acids composition were analyzed by using High Performance Liquid Chromatography (HPLC) and gas chromatography (GC); the complete Actin cDNA, part 18S rDNA, complete ITS1 and ITS2 sequences, part 28S rDNA se- quences, and a putatively encoding ?5 fatty acid desaturase gene were cloned respectively and further functioned in transgenic yeast. The sequence alignments were separately conducted using the related sequences from Nitzschia closterium f. minutissima, Cylindrotheca closterium (Bacillariophyta/Baci- llariales/Bacillariaceae/Cylindrotheca) and Phaeodactylum tricornutum (Naviculales) with ClustalX 1.83. No distinct difference was discovered between N. closterium f. minutissima and P. tricornutum in both biochemical and molecular level. Their identity was more than 99.6% among 18S rDNA, 5.8S rDNA and actin-gene sequences, and is up to 98.6% even among ITS1 and ITS2 sequences. Their ?5 desaturase similarity was 99.4%. However, the lower similarity was disclosured between N. closterium f. minutis- sima and Cylindrotheca closterium, which shared less than 40% identity in the ITS1 and ITS2 se- quences. So, N. closterium f. minutissima should not be placed in Bacillariales, Bacillariaceae, Nitzschia, but in Naviculales, Phaeodactylaceae, Phaeodactylum, and it was actually a strain of P. tri- cornutum.

光质对新月菱形藻生长、总脂含量和脂肪酸组成的影响

【目的】研究不同光质对新月菱形藻(Nitzschia closterium)生长、总脂含量和脂肪酸组成的影响,确定新月菱形藻生长及油脂产量的最适光质。【方法】在智能光质培养箱的静置培养条件下,采用单因子试验分别研究不同光质(LED白光、LED红光、LED蓝光、LED绿光和LED黄光)条件下新月菱形藻的生长、总脂含量和脂肪酸组成。【结果】光质对新月菱形藻的生长、总脂累积和脂肪酸合成有显著影响(P<0.05):LED绿光和黄光培养下新月菱形藻比生长速率显著高于白光、蓝光和红光组;LED白光、绿光和黄光培养下新月菱形藻的叶绿素a含量显著高于红光和蓝光组;LED白光、绿光和黄光培养下新月菱形藻的总脂含量显著高于红光和蓝光组;LED蓝光、绿光和黄光培养下新月菱形藻的多不饱和脂肪酸(PUFAs)含量没有显著差异(P>0.05),显著高于白光和红光组;LED蓝光培养下新月菱形藻的二十碳五烯酸(EPA)含量最高,显著高于其他组;LED红光培养下新月菱形藻的二十二碳六烯酸(DHA)含量最高,显著高于其他组。【结论】LED绿光和黄光能提高新月菱形藻的生长速率,LED蓝光有利于提高新月菱形藻的PUFAs和EPA的含量,LED红光有利于提高新月菱形藻的DHA含量。

快速测定藻类生物量的方法探讨

对新月菱形藻(Nitzschiaclosterium)藻细胞进行超声波破碎并离心,用分光光度计在672nm处测定其吸光度。这种方法与细胞计数法、叶绿素a含量测定法相比较,既快速又准确,且获得十分理想的线性相关性,其测定的吸光度A值可以作为衡量藻类生物量的指标。

6种苯系物对球等鞭金藻和新月菱形藻的生长抑制

海洋环境中苯系物污染主蜃来源于海洋溢油事故以及沿海石油化工企业的废水排放。为探究苯系物对海洋微藻的毒性作用，选择球等鞭金藻和新月菱形藻作为受试生物，分别考察了苯、甲苯、乙基苯、邻-二甲苯、间-二甲苯和对-二甲苯6种苯系物对2种海洋微藻生长的影响，结果表明，在0．25～64．0 mg·L-1暴露浓度下，6种苯系物对2种微藻生长具有显著的抑制作用，随着暴露浓度的升高，抑制作用明显增强。苯、甲苯、乙基苯、邻-二甲苯、间-二甲苯、对-二甲苯对球等鞭金藻的24 h的半数效应浓度（24 h-EC50）分别为：17 07、12．88、7．58、0．55、0．36、0．27 mg·L-1；对新月菱形藻的24 h-EC50值分别为：1．03、0．68、0．46、0．40、0．42、0．38 mg·L-1。上述研究结果勾确定苯系物海洋环境质量标准、保护海洋生态环境提供了基础数据。

两种海洋饵料硅藻的超低温保存

用两步冷冻法对牟氏角剌藻和新月菱形藻进行了超低温保存，研究了影响存活率的内外因素．结果表明，对这两种藻分别选用１０％ 和１５％ ＤＭＳＯ为抗冻保护剂，预冻至－ ４０℃并保持３０ ｍ ｉｎ 后投入液氮，样品化冻后分别在室温及０℃采用较慢的稀释速率去除ＤＭＳＯ时可获得较高的存活率．此外，处于静止期或经过低温驯化的藻细胞也表现出较大的抗冻性．通过优化内外条件，两种藻的存活率分别达到９１．５％ 和６３．１％ ．

温度对小新月菱形藻叶绿素荧光特性及生长的影响

以小新月菱形藻为试验材料,研究了其在一次性培养过程中,不同温度（5~30℃）对其叶绿素荧光参数[光系统Ⅱ的最大光能转化效率（Fv/Fm）、光系统Ⅱ的潜在活性（Fv/Fo）、光系统Ⅱ的实际光能转化效率（ΦPSⅡ）、相对光合电子传递效率（rETR）、光化学淬灭（qP）和非光化学淬灭（NPQ）]、叶绿素相对含量以及细胞密度的影响。单因子方差分析结果表明,在整个培养周期中,温度对小新月菱形藻各叶绿素荧光参数、细胞密度和叶绿素相对含量均有显著影响（P〈0.05）。多重比较结果表明,接种后1~2 d,20℃处理组的主要荧光参数（Fv/Fm、Fv/Fo、rETR、ΦPSⅡ）显著高于其他处理组。30℃的处理组的上述荧光参数从第1 d开始均显著低于其他处理组。20℃处理组的细胞密度和叶绿素相对含量均显著高于其他处理组。在本试验条件下,适宜小新月菱形藻生长的温度为10~25℃,最适温度为20℃。相关性分析结果表明,在整个培养周期中,小新月菱形藻的叶绿素相对含量和细胞密度之间存在显著的正相关。

不同强化剂及其剂量对褶皱臂尾轮虫成活率和脂肪酸组成的影响

以小新月菱形藻强化效果为对照，研究了3种油脂型强化剂（墨鱼油、精制鱼油、混合鱼油）在不同剂量（0．25g／L、0．35g／L和0．45g／L）下强化12h后褶皱臂尾轮虫的脂肪酸组成和成活率。结果表明，不同强化剂和强化剂量下褶皱臂尾轮虫的成活率存在显著差异。用小新月菱形藻强化轮虫的成活率最高（96．82％），随着油脂型强化剂剂量的增大，轮虫的成活率显著下降。当强化剂量为0．45g／L时，墨鱼油型强化剂强化过程中轮虫的成活率仅为72．54％。不同强化剂强化后轮虫的脂肪酸组成有显著差异，强化前轮虫的EPA、ARA和DHA的量分别为2．56％、0．95％和7．73％，PUFA含量仅为16．65％。小新月菱形藻强化轮虫的EPA、ARA和DHA的量分别为12．27％、1．80％和8．61％，PUFA含量为26．00％。墨鱼油型强化剂强化的轮虫EPA、ARA和DHA的量分别为12．73％～13．47％、1．41％～1．46％和17．04％～17．96％，PUFA含量为39．36％～40．82％。精制油型强化剂强化的轮虫EPA、ARA和DHA的量分别为14．58％～17．00％、1．48％～2．78％和30．24％～31．93％，PUFA含量为55．53％～59．32％。混合鱼油型强化剂强化的轮虫EPA、ARA和DHA的量分别为13．34％～14．13％、1．38％～1．42％和24．88％～26．60％，PUFA含量为48．87％～50．70％。综合考虑轮虫成活率及脂肪酸组成，采用0．35g／L精制鱼油或0．25g／L混合鱼油强化银鲳仔鱼的饵料轮虫是合适的。

NaHCO_3浓度对塔胞藻、小球藻和新月菱形藻生长的影响

在温度为 ( 2 2± 1 )℃ ,盐度为 2 8的条件下 ,用不同浓度的NaHCO3( 0 ,1 0 0 ,2 0 0 ,40 0 ,80 0 ,1 2 0 0和 1 60 0mg/L)对青岛海洋大学微藻种质库保存的塔胞藻 (Pyramidomonassp.)、小球藻 (Chlorellaspp .)和新月菱形藻 (Nitzschiaclosterium )进行培养。实验结果表明 ,NaHCO3浓度对 3种海洋微藻生长的影响差异显著 ,经过 6d的培养 ,塔胞藻和小球藻的细胞浓度都在NaHCO3浓度为 1 2 0 0mg/L时达到最大值 ;新月菱形藻的细胞浓度在NaHCO3浓度为 40 0mg/L时达到最大值。

共培养系统中4种微藻生态因子的研究

通过二次回归通用旋转组合设计安排实验,研究光强、温度和盐度对分离自对虾池塘水环境中的啮蚀隐藻、新月菱形藻、微绿球藻和蛋白核小球藻的增长率的影响。获得各微藻的最适生态因子及其影响度。啮蚀隐藻的最适生态因子为:光强5750～7944lx,温度21.3～28.3℃,盐度13.3～23.0;影响度依次为:盐度>温度>光强。新月菱形藻的最适生态因子为:光强5761～8697lx,温度23.4～29.6℃,盐度11.9～25.7;影响度依次为:盐度>光强>温度。蛋白核小球藻的最适生态因子为:光强6754～8775lx,温度17.1～20.7℃,盐度19.6～26.4;影响度依次为:温度>盐度>光强。微绿球藻的最适生态因子为:光强7128～9012lx,温度18.7～26.7℃,盐度17.9～24.3;影响度为光强>温度>盐度。啮蚀隐藻和新月菱形藻的增长率受到以盐度为基础、以光强和温度为协同因子的显著影响。

悬浮物对浮游植物和浮游动物的急性毒性效应

通过悬浮物对浮游植物和浮游动物的急性毒性实验,研究了不同浓度悬浮物对浮游植物和浮游动物生长的影响。浮游植物选取中肋骨条藻Skeletonema costatum和新月菱形藻Nitzschia closterium为受试生物,浮游动物为胶州湾近岸拖网所得,主要优势种类包括藤壶无节幼体、太平洋哲水蚤、腹剑水蚤和小拟哲水蚤。实验结果表明,不同浓度的悬浮物对两种浮游植物的生长都具有非常显著的影响(P<0.01);悬浮物浓度与中肋骨条藻的生长抑制率呈线性关系,半抑制浓度48 h-EC50为1.58 g/L,R值为0.977,96 h-EC50为2.82 g/L,R值为0.993;悬浮物浓度和新月菱形藻的生长抑制率也呈线性关系,其半抑制浓度48 h-EC50为4.27 g/L,R值为0.933,96 h-EC50为4.47 g/L,R值为0.939。悬浮物对浮游动物的生长也有显著影响,其死亡率随着悬浮物浓度的增加而升高,其半致死浓度96 h-LC50为60.172 mg/L。

Cd(Ⅱ)、Zn(Ⅱ)对新月菱形藻生长及生化成分的影响

研究了不同浓度的Cd(Ⅱ)、Zn(Ⅱ)对新月菱形藻Nitzschia closterium生长及细胞内蛋白质、多糖、叶绿素等生化成分含量的影响。结果表明:新月菱形藻对两种重金属均有一定的耐受力;Cd(Ⅱ)、Zn(Ⅱ)浓度分别低于3.0、1.0 mg/L时对新月菱形藻的生长没有显著影响(P>0.05),高于此浓度则产生明显的抑制作用(P<0.05);Zn(Ⅱ)对新月菱形藻胞内蛋白质含量的影响不明显(P>0.05),Cd(Ⅱ)能抑制新月菱形藻蛋白质的合成;低于3.0 mg/L的Cd(Ⅱ)、Zn(Ⅱ)能促进新月菱形藻胞内多糖和叶绿素的合成。

新月菱形藻胞外多糖的成分及其硫酸酯的制备

目的阐明新月菱形藻胞外多糖(exopolysaccharides of Nitzschia closterium,EPN)的成分、单糖组成及其硫酸酯化条件。方法新月菱形藻胞外多糖以醇沉法从该藻的培养液中制备;DEAE-Sephedex25柱分离,HPLC法测定其质量分数,HPLC测定其相对分子质量;以氯磺酸-吡啶法探索新月菱形藻硫酸酯化反应条件。结果新月菱形藻向胞外分泌3种多糖,相对分子质量分别为EPN11.886×105,EPN21.41×105,EPN31.134×105。3种多糖的单糖组成均为甘露糖、鼠李糖、葡萄糖醛酸、葡萄糖和岩藻糖。甘露糖、鼠李糖、葡萄糖醛酸、葡萄糖和岩藻糖的摩尔比在EPN1为4.3∶1∶5.1∶8.9∶5.6,EPN2为4.2∶7.3∶3.7∶12.8∶3.4,EPN3为3.5∶2.1∶4.3∶4.9∶4.7。3种多糖的硫酸酯化条件基本一致,提高氯磺酸比例和延长反应时间均有利于增加酯化产物的硫酸根、提高取代度和酸酯化反应的产率。结论新月菱形藻胞外多糖为酸性多糖,其硫酸酯化产物多糖硫酸酯可能具有潜在的药理活性。

高浓度小新月菱形藻保存方法的研究

对小新月菱形藻（Ｎｉｔｚｃｈｉａｃｌｏｓｔｅｒｉｕｍｆ．ｍｉｎｕｔｉｓｓｉｍａ）的保存方法进行了研究，试验证明，小新月菱形藻用不同的方法保存３个月，其最高存活率分别为：（１）在２０℃下，１５．０％的ＳＡＭ８组为７６．４％，１．５％的抗生素组为５４．６％，０．１％的防腐剂组为４５．３％，对照组仅为３５．４％。（２）在０℃下，１０．０％的ＳＡＭ８组为９８．４％，１．５％的抗生素组为７８．８％，０．１％的防腐剂组为６３．１％，低于对照组（６５．５％）．（３）－３０℃下，１０．０％的ＤＭＳＯ组为８４．６％，１０．０％的甘油组为８３．１％，对照组为４６．０％。为微藻的浓缩保存提供了新的方法。