光发酵强化三角褐指藻生产岩藻黄素

本研究旨在建立光发酵培养三角褐指藻高效生产岩藻黄素的技术体系。在5 L光发酵罐中,系统研究了兼养条件下初始光强、氮源种类和浓度以及光质对于三角褐指藻生物量浓度和岩藻黄素积累的效果。结果表明,在初始光强为100μmol/(m²·s)红蓝(R:B=6:1)混合光、含氮量为0.02 mol/L的胰蛋白胨和尿素混合氮源(1:1, N mol/N mol)优化条件下,三角褐指藻生物量浓度、岩藻黄素含量和产率分别达到了最大值3.80 g/L、13.44 mg/g和4.70 mg/(L·d),比优化前分别提高了1.41、1.33和2.05倍。本研究开发了强化三角褐指藻光发酵生产岩藻黄素的关键技术,促进了海洋天然产物开发。     

三角褐指藻dxs基因克隆与诱导表达

为研究6种外源因素处理对三角褐指藻(Phaeodactylum tricornutum)1-脱氧-D-木酮糖-5-磷酸合成酶(DXS)基因表达的影响, 通过高通量转录组测序技术获得三角褐指藻dxs基因cDNA全长序列, 并对其进行生物信息学分析。研究结果表明, 三角褐指藻dxs基因cDNA全长2476 bp, ORF全长2193 bp, 编码730个氨基酸, 具有高度保守的ThDP结合位点和转酮醇酶结构域。三角褐指藻DXS蛋白为亲水性稳定蛋白, 相对分子质量(M_w)为79.31 kD, 理论等电点为6.65, 具有信号肽、跨膜区域、卷曲螺旋和TM-螺旋等。系统进化树分析结果表明, DXS蛋白进化树分为高等植物和藻类2个分支, 高等植物DXS蛋白进一步分为DXS1和DXS2两支, 藻类DXS蛋白聚类为3个分支, 分别为硅藻门、红藻门和绿藻门分支, 三角褐指藻聚类在硅藻门分支上。诱导表达调控结果表明, 三角褐指藻dxs基因受到茉莉酸甲酯(MeJA)、花生四烯酸(AA)、硫酸铈铵(ACS)、光合诱导素(PIF)、光合抑制剂(DCMU)和光质等6种外源因素的诱导调控。在100 μmol/L MeJA、62.5 mg/L AA、1.6 mg/L ACS、1.00 μg/L PIF、0.2 mg/L DCMU和紫光处理下, 三角褐指藻dxs基因表达量最高。在MeJA和光质处理下, 三角褐指藻dxs基因表达量与岩藻黄素含量变化趋势一致, 说明DXS是MeJA和光质等诱导子促进三角褐指藻岩藻黄素积累的关键酶之一。研究为进一步利用代谢工程手段提高藻细胞内岩藻黄素含量提供了良好的基因资源, 为深入探索三角褐指藻岩藻黄素合成的分子调控机制提供了一定的理论依据。     

长山群岛海域真核微藻粒级结构及扇贝摄食选择性

采用高通量测序-分子鉴定分级技术于2019年对长山群岛全海域真核微藻粒级结构进行了研究。结果发现,春季以中（47%）、小粒级（41%）为主,夏季以小（39%）、大粒级（38%）为主,秋季以大粒级（60%）为主,春、夏、秋季小、中、大粒级微藻比例为42：47：11、39：23：38、22：18：60。小粒级微藻优势种为细小微胞藻（Micromonas pusilla）、融合微胞藻（Micromonas commoda）和金牛微球藻（Ostreococcus tauri）,中粒级微藻优势种为剧毒卡尔藻（Karlodinium veneficum）、大粒级微藻优势种为柔弱几内亚藻（Guinardia delicatula）、平野亚历山大藻（Alexandrium hiranoi）、多纹膝沟藻（Gonyaulax polygramma）,综合整个真核微藻群落,春季由中粒径的剧毒卡尔藻占据优势（23.9%）,夏季由大粒径的平野亚历山大藻占据优势（29.4%）,秋季由大粒径的多纹膝沟藻占据优势（66.8%）,有毒甲藻在该海域中占有绝对优势,贝毒累积风险较高,小粒径微藻中金牛微球藻和抑食金球藻曾在渤海引发褐潮,潜在威胁该海域贝类养殖业。虾夷扇贝对小粒级和大粒级微藻的选择性较低,对中粒级微藻的选择性较高,尤其对水体中优势种剧毒卡尔藻一直表现出主动选择。光学需氧量、无机氮、溶解氧、石油类及部分重金属Cd、As、Hg影响着整个长山群岛海域真核微藻粒级结构时空演变。     

三角褐指藻岩藻黄素-叶绿素蛋白复合体的分离纯化和功能研究

通过改进硅藻主要捕光天线(FCP)的分离和提取方法, 得到高纯度、高均一性的三角褐指藻FCP蛋白,并通过电泳、液相色谱、质谱和吸收荧光光谱学等手段研究三角褐指藻FCP的氨基酸序列、色素组成和捕光特点等, 初步预测三角褐指藻的结构和功能特点。结果表明三角褐指藻FCP含有198个氨基酸, 与高等植物LHCII的序列Identity约为24%。三维结构预测显示FCP具有与LHCII相似的三次跨膜螺旋框架结构, 但跨膜螺旋较短, 且无膜表面螺旋结构。FCP中主要结合了叶绿素a、叶绿素c、岩藻黄素, 不含叶绿素b, Chl. a/c为3.0。光谱学分析表明岩藻黄素可以在水下弱光环境中有效地捕获绿光, 并高效地传递至叶绿素。而岩藻黄素在400-500 nm区域吸收的光能, 向叶绿素传递效率较低, 预示着岩藻黄素在强光下也有一定的光保护功能。FCP中有4个叶绿素结合的保守氨基酸位点, 可能是其叶绿素结合位置, 但岩藻黄素的结合位置因其结构和结合位点的变化而无法预测。研究为进一步探索FCP的结构和功能特性奠定了基础。     

13种微藻的脂肪酸组成分析

分析了13种微藻(包括7种绿藻,5种杂色藻和1种红藻)的总脂含量和脂肪酸组成,结果表明,不同门类微藻的脂肪酸组成差异较大:绿藻的脂肪酸组成以C16和C18为主;杂色藻类的脂肪酸组成相近,金藻门含有14:0、16:0、18:1、18:4等特征脂肪酸,三角褐指藻主要的脂肪酸为14:0、16:0、16:1、16:3和20:5,而粉核油球藻的脂肪酸以14:0、16:0、20:5为主;紫球藻的脂肪酸组成以16:0、20:4和20:5为主。在测试的13种微藻中,杜氏盐藻的亚麻酸含量最高,占总脂肪酸的60.9%;等鞭金藻的十八碳四烯酸含量最高,占总脂肪酸的19.6%;紫球藻和粉核油球藻中花生四烯酸与二十碳五烯酸(EPA)含量分别占总脂肪酸的17.1%和20.9%。     

云南石蕊共生藻形态及分子系统学研究

该研究以2016年采自云南凤凰山的云南石蕊(Cladonia yunnan)为材料,在无菌条件下,采用微量微管吸法和直接法分离纯化培养云南石蕊共生藻,利用光学显微镜观察共生藻细胞的形态结构特征,并对该共生藻进行分子系统学分析。结果表明：(1)从云南石蕊中仅分离到1种共生藻,该藻细胞大小约2.5~6 μm,球形或椭球形,单细胞,不运动,色素体片状,无性繁殖,含有中央蛋白核和淀粉粒,且3~5个细胞包裹在粘液状的胶壳中,细胞核不可见。(2)该共生藻主要形态学特征与自由生活的胶球藻高度相似,根据采集地及形态特征将其命名为云南胶球藻(Coccomyxa sp. YN)。(3)基于对云南石蕊分子数据(rbcL, ITS)构建的云南石蕊共生藻的系统发育关系显示,云南胶球藻与胶球藻聚为一枝; ITS2 rRNA二级结构分析发现,云南胶球藻与胶球藻具有几乎一致的ITS2 rRNA二级结构。该研究首次揭示了胶球藻属(Coccomyxa)藻细胞与石蕊科地衣共生,且云南胶球藻与胶球藻具有高度一致的亲缘关系,而“地衣化”使两者细胞形态特征有明显差异,推测共生藻细胞可能是由自由生活的藻细胞进化而来。     

蚯蚓两种抗菌肽的分离纯化及部分性质

经硫酸铵沉淀、超滤、阳离子交换分离和反相快速蛋白质液相色谱（FPLC）分析,得到了两种新的蚯蚓抗菌肽F-1与F-2,经电喷雾离子源质谱（ESI-MS）测定,其相对分子质量为535.27和519.27.串联质谱（MS/MS）数据表明F-1的肽序列为Ac-Ala-Met-Val-Ser-Ser,F-2的肽序列为Ac-Ala-Met-Val-Gly-Thr.最小抑菌浓度（MIC）实验表明,F-1与F-2对鹑鸡肠球菌(Enterococcus gallinarum)、绿脓杆菌(Pseudomonas pyocyanea)、鲍氏不动杆菌(Acinetobacter baumanii)、土生克雷伯氏菌（Klebsiella terrigena）的最小抑菌浓度分别为11.4 mg/L和12.85 mg/L,对粪肠球菌(Enterococcus faecalis)的最小抑菌浓度分别为22.8 mg/L和25.68 mg/L.对真菌白色念株菌（Candida albicans）没有表现为完全的抑制作用.     

短头熊蜂肠道拮抗菌株的分离筛选鉴定及其生物特性评价

【背景】近年来,由于栖息地减少、农药的大量使用及病原菌侵染等综合因素,导致全世界的熊蜂种类与数量逐年减少,病原菌的侵染可通过微生物在自身生长过程中会产生的抑菌物质进行有效抑制或杀灭。【目的】短头熊蜂(Bombus breviceps)长期生存在野外环境中,其肠道内存在着大量微生物资源。从短头熊蜂肠道内筛选拮抗菌株,并对其抑菌特性进行研究。【方法】采用牛津杯双层法筛选拮抗菌株,测定抑菌活性最佳菌株发酵液的抑菌物质稳定性与抑菌广谱性等抑菌特性,并借助细胞膜通透性、流式细胞仪检测等试验探究其抑菌机制。【结果】得到了5株具有明显抑菌作用的拮抗菌株,其中果杆菌(Fructobacillus tropaeoli)CZ01对金黄色葡萄球菌(Staphylococcus aureus)、沙门氏菌(Salmonella choleraesuis)、大肠杆菌(Escherichia coli)、福氏志贺氏菌(Shigella flexneri)和无乳链球菌(Streptococcus agalactiae)这5种病原指示菌都具有高度抑菌效果。菌株CZ01对金黄色葡萄球菌的抑菌效果最佳,抑菌圈直径可达到(21.21±0.25) mm,在121 ℃处理后仍具有67.36%以上的抑菌活性,调整pH值为10.0时仍具有78.16%的抑菌活性。【结论】短头熊蜂肠道微生物资源较丰富,尤其是果杆菌(F.tropaeoli)CZ01具有抑菌活性高、稳定性好、抑菌谱广等特性,对金黄色葡萄球菌具有良好的杀灭效果,显示出良好的应用潜能。     

通过同源重组在聚球藻7002中表达小鼠金属硫蛋白-Ⅰ的初步研究

用PCR方法从海洋单细胞蓝藻聚球藻7002（Synechococcus sp. PCC 7002）基因组DNA中扩增得到藻蓝蛋白β亚基基因（cpcβ）的上游序列（Pcpcβ）,及编码谷氨酰胺合成酶的glnA基因片段．以Pcpcβ作为启动子、以glnA基因片段作为整合平台,构建含有小鼠金属硫蛋白-Ⅰ（mMT-Ⅰ）cDNA的同源整合表达载体pKGC-MT.通过自然转化法将整合表达载体导入聚球藻7002中,经氨苄青霉素筛选,得到遗传性状稳定的转基因藻.PCR检测证明mMT-Ⅰ基因已整合到蓝藻基因组DNA上;蛋白质印迹表明mMT-Ⅰ已在蓝藻中表达;ELISA结果显示mMT-Ⅰ在蓝藻中的表达量约为800 μg/g.     

新疆粉衣科地衣分类学研究

该研究以采自新疆的100余份粉衣科地衣标本为研究材料,通过形态解剖学、地衣化学以及分子生物学的方法鉴定出6个种和1个变种,分别为中央黑瘤衣 (Buellia centralis)、丽黑瘤衣 (B. elegans)、蒙古黑瘤衣 (B. mongolica)、鳞饼衣(Dimelaena oreina)、鳞饼衣白磷变种(D. oreina var. exalbescens)、海登氏多瘤胞 (Diplotomma hedinii)和绿色四孢黑瘤衣(Tetramelas chloroleucus),其中丽黑瘤衣、蒙古黑瘤衣和绿色四孢黑瘤衣为新疆新增粉衣科地衣新记录,至此新疆粉衣科地衣共有6属13种1变种;并提供了新疆粉衣科地衣的分种检索表、物种描述、系统发育分析以及形态解剖结构照片。     

The influence of different flocculants on the physiological activity and fucoxanthin production of Phaeodactylum tricornutum

Phaeodactylum tricornutum is an economically important species of microalgae that is widely used in aquaculture, and it is rich in bioactive substances including eicosapentaenoic acid and fucoxanthin. The major bottleneck for industrialization of this species is harvesting. Flocculation is used to harvest microalgae, thus the selection of flocculants is of great importance. In this study, we compared the flocculation effect of four different chemicals (ferric chloride, aluminum sulphate, polyaluminum chloride, and aluminum potassium sulphate) on P. tricornutum. Microexamination showed that ferric and aluminum salts had similar flocculation effects on the algae. Growth and chlorophyll fluorescence measurements showed that P. tricornutum can be re-cultured after flocculation. Pigment analysis showed that flocculation did not result in degradation of fucoxanthin, which suggests that the four flocculants tested may be useful for industrial applications. The results also showed that ferric chloride was the best flocculant for harvesting P. tricornutum when the target product was fucoxanthin, as it had the least influence on the physiological activity of P. tricornutum and it did not lead to degradation of cell components. In contrast, aluminum is poisonous to the nervous system of animals and humans. In addition, the culture medium can be recycled after flocculation by ferric chloride.     

Xanthophyll synthesis in diatoms: quantification of putative intermediates and comparison of pigment conversion kinetics with rate constants derived from a model

Recently, we reported the presence of the violaxanthin-antheraxanthin-zeaxanthin cycle in diatoms, and showed that violaxanthin is the putative precursor of both diadinoxanthin and fucoxanthin in the diatom Phaeodactylum tricornutum Bohlin (M. Lohr and C. Wilhelm, 1999, Proc. Natl. Acad. Sci. USA 96: 8784–8789). In the present study, two possible intermediates in the synthesis of violaxanthin from β-carotene were identified in P. tricornutum, namely β-cryptoxanthin and β-cryptoxanthin epoxide. In low light, the latter pigment prevails, but in high light β-cryptoxanthin accumulates, probably as the result of an increased activity of the xantophyll-cycle de-epoxidase. The apparent kinetics of several xanthophyll conversion steps were determined for P. tricornutum and Cyclotella meneghiniana Kützing. The experimentally determined conversion rates were used to evaluate the hypothetical pathway of xanthophyll synthesis in diatoms. For this purpose a mathematical model was developed which allows the calculation of theoretical rates of pigment conversion for microalgae under steady-state growth conditions. A comparison between measured and calculated conversion rates agreed well with the proposal of a sequential synthesis of fucoxanthin via violaxanthin and diadinoxanthin. The postulation of zeaxanthin as an obligatory intermediate in the synthesis of violaxanthin, however, resulted in large discrepancies between the measured and calculated rates of its epoxidation. Instead of zeaxanthin, β-cryptoxanthin epoxide may be involved in the biosynthesis of violaxanthin in diatoms. Received: 16 March 2000 / Accepted: 30 June 2000     

Potentiation of concurrent expression of lipogenic genes by novel strong promoters in the oleaginous microalga Phaeodactylum tricornutum

There has been growing interest in using microalgae as production hosts for a wide range of value-added compounds. However, microalgal genetic improvement is impeded by lack of genetic tools to concurrently control multiple genes. Here, we identified two novel strong promoters, designated Pt202 and Pt667, and delineated their potential role on simultaneously driving the expression of key lipogenic genes in Phaeodactylum tricornutum. In silico analyses of the identified promoter sequences predicted the presence of essential core cis elements such as TATA and CAAT boxes. Regulatory role of the promoters was preliminarily assessed by using GUS reporter which demonstrated strong GUS expression. Thereafter, two key lipogenic genes including malic enzyme (PtME) and 5-desaturase (PtD5b), were overexpressed by the two promoters Pt202 and Pt667, respectively, in P. tricornutum. Combinatorial gene overexpression did not impair general physiological performance, meanwhile neutral lipid content was remarkably increased by 2.4-fold. GC-MS analysis of fatty acid methyl esters revealed that eicosapentaenoic acid (EPA; C20:5) was increased significantly. The findings augment a crucial kit to microalgal genetic tools that could facilitate the multiple-gene expression driven by various promoters, and promote microalgae for industrial bioproduction.     

Growth kinetics and fucoxanthin production of <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and <Emphasis Type="Italic">Isochrysis galbana</Emphasis> cultures at different light and agitation conditions

Fucoxanthin is a carotenoid that exerts multiple beneficial effects on human health. However, reports comparing microalgae culture conditions and their effect on growth and fucoxanthin production are still limited. Isochrysis galbana and Phaeodactylum tricornutum cultures in different light (62.0, 25.9, 13.5, or 9.1 μmol photons m^-2 s^-1), mixing conditions (1 vvm aeration or 130 rpm agitation), and media compositions (F/2 and Conway medium) were studied for comparison of cellular growth and fucoxanthin production on F/2 medium. I. galbana showed a better adaptation to tested culture conditions in comparison with P. tricornutum, reaching 2.15?×?10⁷?±?4.07?×?10⁶ cells mL^-1 and a specific growth rate (μ) of 1.12?±?0.05 day^-1 under aerated conditions and 62.0 μmol photons m^-2 s^-1 light intensity. Fucoxanthin concentration was about 25 % higher in P. tricornutum cultures under 13.5 μmol photons m^-2 s^-1 light intensity and aerated conditions, but the highest fucoxanthin total production was higher in I. galbana, where 3.32 mg can be obtained from 1 L batch cultures at the 16th day under these conditions. Moreover, higher cell densities (~32.41 %), fucoxanthin concentration (~42.46 %), and total production (~50.68 %) were observed in I. galbana cultures grown in Conway medium, if compared with cultures grown in F/2 medium. The results show that the best growth conditions did not result in the best fucoxanthin production for either microalgae, implying that there is not a direct relationship between cellular growth and fucoxanthin production. Moreover, the results suggest that I. galbana cultures on Conway medium are strong candidates for fucoxanthin production, where 1.2 to 15 times higher fucoxanthin concentration are observed in comparison to macroalgal sources.     

Light-harvesting proteins of diatoms: Their relationship to the chlorophyll a/b binding proteins of higher plants and their mode of transport into plastids

Summary We have cloned and characterized members of a gene family encoding polypeptide constituents of the fucoxanthin, chlorophyll a/c protein complex, a light-harvesting complex associated with photosystem II of diatoms and brown algae. Three cDNA clones encoding proteins associated with this complex in the diatom Phaeodactylum tricornutum have been isolated. As deduced from the nucleotide sequences, these light-harvesting proteins show homology to the chlorophyll a/b binding polypeptides of higher plants. Specifically, the N-terminal regions of the fucoxanthin, chlorophyll a/c-binding proteins are homologous to the chlorophyll a/b binding proteins in both the third membrane-spanning domain and the stroma-exposed region between membrane-spanning domains 2 and 3. Like the chlorophyll a/b-binding proteins, the mature fucoxanthin, chlorophyll a/c polypeptides have three hydrophobic -helical domains which could span the membrane bilayer. The similarities between the two light-harvesting proteins might reflect the fact that both bind chlorophyll molecules and/or might be important for maintaining certain structural features of the complex. There is little similarity between the N-terminal sequences of the primary translation products of the fucoxanthin, chlorophyll a/c proteins and any transit sequences that have been characterized. Instead, the N-terminal sequences have features resembling those of signal sequences. Thus either transit peptides used in P. tricornutum show little resemblance to those of higher plants and green algae or the nuclear-encoded plastid proteins enter the organelle via a mechanism different from that used in higher plants.     

Test for allelopathic interactions between two marine microalgal species grown in intensive cultures

Both high pH and cell senescence are believed to lead to the production of toxic extracellular metabolites in freshwater microalgae. However, there was no evidence for allelopathic suppression of photosynthesis when filtrates of either of two marine microalgae,Phaeodactylum tricornutum andDunaliella tertiolecta, were mixed with whole cultures of the other species. This was true even when filtrate ofP. tricornutum sample was derived from a culture at high pH or from one in various stages of senescence. It is believed that the major factor leading to the dominance ofP. tricornutum in intensive outdoor cultures is the unique ability of this alga to tolerate pH levels above 9.5, not the allelopathic inhibition of competing species.     

Pressurized extraction of unsaturated fatty acids and carotenoids from wet Chlorella vulgaris and Phaeodactylum tricornutum biomass using subcritical liquids

The objective of this study was to investigate the extraction of lipids, for example, mono‐ and polyunsaturated fatty acids (PUFA) as well as carotenoids, from wet microalgae biomass using pressurized subcritical extraction solvents, which meet the requirements of food and feed applications. To demonstrate the effect of the solvent and temperature on the lipid yield, we chose two microalgae species, viz. Chlorella vulgaris and Phaeodactylum tricornutum, differing in their biochemical composition fundamentally. In case of P. tricornutum, ethanol showed the highest fatty acid yield of 85.9% w/w. In addition to eicosapentaenoic acid (EPA), the ethanolic extracts contained exceptional amounts of fucoxanthin (up to 26.1 mg/g d. w.), which can be beneficial to protect unsaturated fatty acids from oxidation processes and in terms of human nutrition. For C. vulgaris, a fatty acid yield of 76.5% w/w was achieved from wet biomass using ethyl acetate at 150°C. In general, an increase in the extraction temperature up to 150°C was found to be important in terms of fatty acid yield when extracting wet microalgae biomass. The results suggest that it is possible to efficiently extract both fatty acids and carotenoids from wet microalgae by selecting suitable solvents and thus circumvent energy‐intensive drying of the biomass.     

Integration of consumer-targeted microalgal production with marine fish effluent biofiltration – a strategy for mariculture sustainability

EU regulations recommend effluent treatment and nutrient recycling for aquaculture sustainability, so a study was undertaken to provide base-line data for the integration of commercial fish-farm effluents with the production of microalgae. The project relates to a specific bivalve consumer (Tapes decussatus) and biofiltration. Effluent inorganic nutrient composition was assessed and evaluated as culture media for Phaeodactylum tricornutum, Tetraselmis suecica and Tetraselmis sp. Optimization of the microalgal compartment included studies on preparation of a simple medium, nutrient or dilution rate manipulation and nutrient removal. Cell harvest was increased chiefly by N correction (6-fold for Tetraselmis sp.) and semi-continuous or continuous operation (by a factor of 3 to 11). Nutrient removal efficiency was high for ammonium and nitrite-nitrogen (80–100%), depending on species, nutrient ratio (Si correction for P. tricornutum) and culture regime for nitrate (41–100%) or phosphorus (21–99%). Data obtained under cyclostat cultivation (yields of 1.38 and 0.50×10⁶ P. tricornutum or Tetraselmis sp. cells mL⁻¹ d⁻¹ and nutrient uptake rates of 2.32 mg N L⁻¹ d⁻¹ and 0.96 mg P L⁻¹ d⁻¹) were used to show clam production and simultaneous wastewater treatment possibilities through the proposed fish-microalgae-clam integrated aquaculture system.