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.

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。

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.

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.

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℃. The SiO2 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 SiO2 morphologies of both biosilica are typical amorphous silica. Besides, IV. 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, 1 125, 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, 16:1n7 and 20:5n3 ( EPA ) , B13 also has high percentage -of 20:4n6 ( 6.0 ~ 9.1% ).

Purification of a Diatom and Its Identification to Cylindrotheca closterium

A diatom was purified with colony selection and continuous dilution methods. It was identified to Cylindrotheca closterium according to its morphological characteristics and rbc L and 18 s r RNA gene sequences. The alga was not sensitive to ampicillin and neomycin, but sensitive to chloramphenicol which inhibited its growth at concentrations ranging from 50 to 150 μg m L-1. The purified alga was easy to culture and its specific growth rate was 0.207 ± 0.002(d-1). It was resistant to pollution and could be harvested in an easy way. It was relatively high in lipid content(20.08% ± 0.67% of dry weight) and the combined amount of its 16:0 and 16:1(n-7), the most suitable resource of biodiesel, was as high as 64% of the total fatty acids, while the amount of polyunsaturated fatty acids reached 19.96%–20% of the total fatty acids. Thus the purified C. closterium can be cultured as a biodiesel producer or a nutrition supplement producer.

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

The maximum growth rate ofNitzschia elosterium 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 experi- mental results showed that the growth rate of Nitzschia elosterium 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 distribu- tion were observed during the growth period ofNitzsehia closterium. The Gaussian function could give a clear description of the cell size distribution ofNitzsehia 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.

Cylindrotheca closterium Is a Species Complex as Was Evidenced by the Variations of rbcL Gene and SSU rDNA

The genus Cylindrotheca consists of a small group of marine diatoms with a few species described. Eleven isolates of diatoms identified as Cylindrotheca closterium morphologically were obtained from Jiaozhou Bay with their nuclear-encoded small-subunit ribosomal RNA （SSU rDNA） and chloroplast-encoded rbcL gene sequences determined in this study. Interestingly, very high sequence divergences of SSU rDNA and rbcL gene were found among these isolates, and numerous nucleotide variation of rbcL gene caused relatively few variation of deduced amino acid sequence. Phylogenetic analyses based on SSU rDNA and rbcL gene, respectively, grouped the isolates into 6 clades. Phylogenetic tree of SSU rDNA placed all the Cylindrotheca isolates together, separating them into two lineages clearly. LineageⅠ was composed of the eleven C. closterium isolates obtained in this study together with another C. closterium isolate, but some clades were not well supported. LineageⅠwas contained two C. closterium isolates and one C. fusiformis isolate. Phylogenetic analysis of rbcL gene also separated the Cylindrotheca isolates into two well-defined lineages. The eleven C. closterium isolates formed a lineage and all clades were supported strongly. Statistical comparisons of SSU rDNA indicated that the average distance within lineageⅠwas significantly higher than that of other microalgae species （P 〈0.01）. These results suggested the existence of cryptic species within C. closterium.

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。

用包埋脱水法冰冻保存小新月菱形藻

采用包埋脱水法研究了小新月菱形藻(Nitzschia closterium minutissima)的超低温保存,探讨了影响存活率的内、外因素:藻细胞年龄、脱水速率、胶球含水量和化冻后的恢复方法等。结果表明,将处于静止初期的藻细胞包埋在含有0.5 mol/L蔗糖的3%褐藻酸钙胶球中,以含水量每小时减少0.9%的平均速率脱水至胶球含水量为40%,化冻后在冻存管中室温下暗恢复12 h的条件下冰冻存活率较高,可达到18.3%左右。与常规的两步法冰冻保存相比,包埋脱水法可大大简化操作程序,是保存小新月菱形藻的一种有潜力的方法。

不同氮磷浓度对莱布新月藻生长与脂溶性物质含量的影响

以莱布新月藻Closterium leibleinii为试材,利用单因子分析研究不同氮、磷浓度培养的莱布新月藻生长及脂溶性物质含量变化,为其作为生物柴油开发提供基础资料。结果表明,莱布新月藻生长的最适氮浓度为5.30 mg·L^(-1),其最大生长速率为3.675×10^(6)个·L^(-1)·d^(–1);最适磷浓度为0.02 mg·L^(-1),最大生长速率为4.05×10^(5)个·L^(-1)·d^(–1)。在最适氮磷浓度BG11培养下粗脂约占藻体干重的88.04%。莱布新月藻在适宜的氮磷浓度培养基中生长速率较快,脂溶性物质含量高,可作为一种较有潜力的植物开发生物柴油。

甲基磺酸乙酯对小新月菱形藻的生物学效应

为提高小新月菱形藻(Nitzschia closterium)的产油脂能力、获得油脂含量高的突变株,利用甲基磺酸乙酯(EMS)对该藻株进行了化学诱变处理,并对其生物学作用进行了考察。结果显示,EMS对小新月菱形藻具有较好的诱变作用,经0.1 mol/L和0.05 mol/L EMS处理后分别得到油脂生产能力较强的突变藻株YA和YB,在同等培养条件下两株突变藻单位体积细胞的内叶绿素a、类胡萝卜素、蛋白质、可溶性糖和油脂相对含量分别比对照组提高了30.05%和25.77%、22.15%和19.26%、9.06%和8.27%、48.25%和45.48%、44.06%和12.86%。

铜、锌和锰抑制月形藻生长的毒性效应

运用评价化学品毒性藻类测试的标准实验方法 ,得到铜、锌和锰对月形藻生长的最小无显著差异浓度 (LNOEC)分别为31.8μg L、16 .4μg L和 2 .7mg L ,抑制月形藻生长的 96h半效应浓度 (96h EC50 )分别为 199.5 μg L、38.0 μg L和 12 .6mg L .实验结果表明无论从LNOEC还是从 96h EC50 考虑 ,都证明抑制月形藻生长的毒性由大到小的顺序是锌 >铜 >锰 .此结果对铜抑制藻类生长的毒性远远大于锌的毒性这一人们普遍认同的观点构成挑战 .

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。

铜抑制单细胞绿藻生长的毒性效应

运用评价化学品对藻类毒性的标准实验方法,根据实验结果采用机率单位法进行数据处理,分别得出铜抑制蛋白核小球藻(Chlorella pyrenoidosa),斜生栅藻(Scenedesmus obliquus),月形藻(Closterium lunula)生长的96h半效应浓度(96h-EC50)分别为67,51,202(g/L,并对各剂量反应方程进行X2检验,结果表明均符合精度要求,计算出的96h-EC50真实可靠.研究结果表明, 斜生栅藻对铜最敏感,而月形藻对铜有很强的抵抗能力.这3种藻类对铜敏感度由大到小的顺序为斜生栅藻、蛋白核小球藻、月形藻.不同藻种细胞的大小不同和藻细胞壁表面的含硫基团不同是导致藻类对铜敏感性不同的主要原因.

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％ ．

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。

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。