低温胁迫对三角褐指藻生长和生理生化特性的影响

在温度分别为3、6、9、20℃的条件下培养三角褐指藻,测定其细胞密度、生物量、叶绿素a含量、脯氨酸含量、丙二醛含量在3、6、9、12、15 d和可溶性糖含量随时间的动态变化情况.结果表明:在不同的低温胁迫条件下,三角褐指藻的细胞密度和生物量均明显低于对照组;叶绿素a含量处理前3d有所上升,没有明显低于对照组,处理6d后明显下降;脯氨酸含量、丙二醛含量、可溶性糖含量均有显著上升.该研究结果为进一步揭示三角揭指藻对逆境耐受能力的生理机制提供一定的理论依据.     

猪粪废水用于三角褐指藻培养的研究

研究了以猪粪废水培养一株三角褐指藻(Phaeodactylum tricornutum)的可行性.结果表明:猪粪废水经30g/L盐水适当稀释后可直接用于三角褐指藻的培养,无需添加其他营养物质.在污水稀释率为25倍时,藻细胞可良好地生长,最高密度为合成培养基的1.52倍.同时三角褐指藻有效去除了污水中的氮磷,总氮、氨氮和总磷的去除率分别为23%,93%和93%.与合成培养基养殖的藻相比,猪粪废水养殖的三角褐指藻的二十碳五烯酸和多不饱和脂肪酸的含量略有上升,实现了培养三角褐指藻转化废水为生物量并积累多不饱和脂肪酸的资源化.     

不同起始密度对等鞭金藻和三角褐指藻种间生长的影响

通过共培养方法,研究了不同起始密度对等鞭金藻和三角褐指藻种间生长作用的影响．结果显示：不同起始密度对等鞭金藻和三角褐指藻种间生长竞争具有明显的影响．3种不同的接种比例下,三角褐指藻在与等鞭金藻的竞争中始终占优势,而且随着接种密度的增加,三角褐指藻在种间生长竞争中的优势更加明显．在接种比例为I：Q=4：1时,等鞭金藻对三角褐指藻的生长也会产生一定的抑制作用．     

三角褐指藻的培养条件研究

对三角褐指藻(Phaeodactylum tricornutum)进行了培 养条件研究,结果表明,在20 ℃下,采用去除氮源的No.4基本培养液,起始pH值为8.0,(N H2)2CO为氮源,氮浓度为0.01 mol/ L时,对藻细胞的光合自养生长极为有利,培养10 d,其生物量达441 mg/L.过高的碳酸氢钠 浓度(>0.04 mol/L)易引起培养液pH值的过大改变而导致培养液中出现沉淀,在培养液中加 入葡萄糖有利于藻细胞的生长,光合自养生长生物量浓度较低,混合营养明显促进藻细胞的 生长,且培养液中同时含质量分数0.2%葡萄糖和0.04 mol/L碳酸氢钠后藻生物量达1 630 mg /L,因此光合异养生长明显促进藻细胞的生长.     

快速高通量测定三角褐指藻油脂含量

为了实现三角褐指藻细胞内油脂含量的快速高通量测定,探索了尼罗红荧光染色法检测其油脂含量的检测条件.采用96孔板并配合酶标仪考察三角褐指藻的预处理液的种类及其用量、染色液尼罗红的用量、染色温度、染色时间以及细胞浓度对荧光强度的影响,确立最优染色条件,并研究荧光强度与油脂含量之间的对应关系.结果表明,TritonX-100终浓度为0.05%时,通透效果最佳,每100μL藻悬液中加3μL尼罗红染液(0.1mg/mL),温度40℃,时间10min,细胞浓度低于3×106个/mL时,荧光强度(y)与油脂含量(x)之间呈现良好的线性关系:y=927.37x+15.913,R2=0.9247.方法优化后,90min即内可完成对96个样品的处理及分析,极大提高了分析的通量.     

三角褐指藻对镉的富集机制和转化途径

本论文报道了在典型配体乙二胺四乙酸(EDTA)和半胱氨酸(Cys)的存在下三角褐指藻(Phaeodactylum tricornutum)对镉(Cd)的富集机制和转化途径. 毒性试验表明, 两种配体均可有效降低Cd对三角褐指藻的毒性. ICP-MS分析结果表明, EDTA存在下细胞表面吸附和内部吸收Cd的量随着培养液中EDTA浓度的升高(自由Cd²⁺浓度的降低)而降低, 基本符合自由离子活度模型(Free ion activity model, FIAM)的预测; 而Cys存在时, 细胞表面吸附Cd的量随着Cys浓度的增大呈现先增加后降低的趋势. 在Cys浓度由空白浓度增加至4.45 µmol/L时, 细胞内部吸收Cd的量呈现增加趋势; 而大于4.45 µmol/L时, 又趋于同一水平的现象, 结果偏离FIAM. FTIR和XPS研究确证了细胞壁上的?OH和−NH₂基团对Cd的吸附起主要作用. Cd胁迫后P. tricornutum细胞内诱导合成的植物螯合肽(PCs)的HPLC和ESI-IT-MS分析结果证实造成这两种配体对P. tricornutum积累和转化Cd行为产生不同影响的主要原因是Cys作为Cd²⁺的配体的同时, 又是P. tricornutum细胞内PCs合成的前驱体之一. P. tricornutum细胞内PCs、氧化型PCs以及Cd-PC2的发现证明了Cd诱导P. tricornutum合成的PCs反过来钝化细胞内吸收的Cd, 降低了其对P. tricornutum细胞的氧化毒性.     

Biosynthesis of Cd-bound phytochelatins by <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and their speciation by size-exclusion chromatography and ion-pair chromatography coupled to ICP–MS

Cd-bound phytochelatins (Cd–PCs) have been synthesised by incubation of Phaeodactylum tricornutum cell cultures with Cd and purified by size-exclusion chromatography–UV–Vis. These complexes, which were identified in previous work, have now been used as model substances to develop and optimise ion-pair chromatography (IPC) coupled to inductively coupled plasma–mass spectrometry (ICP–MS) for analysis of Cd–PCs. Subsequent analysis of samples taken from Silene vulgaris plants cultivated under heavy metal stress conditions revealed Cd signals but no Cd–PC signals. By use of isotopically enriched ¹¹⁶Cd–PCs the sample preparation steps were verified to determine the stability of the analytes. We observed species transformation between Cd–PCs and other unidentified Cd complexes. Consequently, the kinetic and thermodynamic lability of Cd–PCs are decisive factors in their detection.     

三角褐指藻和亚心形扁藻对婆罗异剑水蚤繁殖、存活及其脂肪酸的影响

近20年,藻类对桡足类的有害影响已引起越来越多的关注.采用实验生态学方法,探讨不同浓度三角褐指藻(Phaeodactylum tricornutum)和亚心形扁藻(Platymonas subcordi formis)对婆罗异剑水蚤(Apocyclop borneoensis)的繁殖、存活及其脂肪酸的影响.结果表明,单种三角褐指藻对婆罗异剑水蚤世代培养的繁殖和幼体存活具有负面影响,各世代间婆罗异剑水蚤的繁殖率及其幼体存活率差异显著(p＜0.05).而投喂亚心形扁藻的婆罗异剑水蚤其繁殖和幼体存活无显著差异(p＞0.05).且饵料中的脂肪酸组成与含量显著影响桡足类脂肪酸组成与含量,也显著影响桡足类的繁殖性能.     

过表达甘油激酶三角褐指藻藻株富集油脂的研究

为了得到高产并符合生物柴油标准的微藻油脂的藻株,本文研究了在外源甘油存在的情况下野生型（WT）三角褐指藻（Phaeodactylum tricornutum）,甘油激酶过表达藻株（GKOE）以及甘油激酶RNA干扰藻株（GKRNi）在添加浓度为20 mmol/L外源甘油的培养基中的生长情况、油脂产量以及脂肪酸组成.结果显示：（1）甘油兼养的GKOE藻细胞浓度可达到2.23×107个/mL,明显高于甘油兼养的WT的1.79×107个/mL和GKRNi的1.78×107 个/mL;（2）甘油兼养的GKOE油脂含量可达到31.73%,比甘油兼养的WT和GKRNi分别高了27.20%和26.63%;（3）尼罗红染色显示甘油兼养的GKOE荧光强度比甘油兼养的WT和GKRNi分别高16.26%和17.83%;（4）甘油兼养的GKOE总脂肪酸产量可达到0.052 mg/mL,比甘油兼养的WT和GKRNi高了36.84%和10.64%,说明其油脂适合于生物柴油开发.     

Purification and characterization of chlorophyllase from algaPhaeodactylum tricornutum by preparative native electrophoresis

The partially purified chlorophyllase, obtained from the algaPhaeodactylum tricornutum, was further purified by preparative native gel electrophoresis. The purification procedure provided the recovery of large amounts of a single purified chlorophyllase fraction. However, the electrophoretic analyses of the purified enzymatic fraction under denaturing conditions demonstrated the presence of two bands with mol wt of 43 ±3 and 46 ±3 kDa. The purification procedure resulted in 2-and 195-fold increases in chlorophyllase activity compared to that of the partially purified and crude enzymatic extracts, respectively. The optimum pH for chlorophyllase hydrolytic activity was found to be 8.0. The optimum incubation time and temperature for the hydrolytic activity of the purified chlorophyllase were found to be 2 h and 31°C, respectively. The optimum concentrations of magnesium chloride and dithiothreitol, used as activators, were 4 and 5 mM, respectively. The addition of individual plant membrane lipids, including phosphatidylcholine, phosphatidylglycerol, and β-carotene, to the reaction media increased the enzyme activity markedly. The purified enzyme fraction displayed preferential specificity toward selective substrates with an order of activity as follows: purified chlorophyllb > purified chlorophylla > partially purified chlorophyll > crude chlorophyll. Diisopropyl fluorophosphate and phytol, respectively, showed noncompetitive and competitive inhibitory effects on chlorophyllase activity with K_i, values of 0.78 mM and 3.75μM, respectively.