ISOLATION AND CHARACTERIZATION OF CHLORELLA SOROKINIANA GXNN01 (CHLOROPHYTA) WITH THE PROPERTIES OF HETEROTROPHIC AND MICROAEROBIC GROWTH

Heterotrophic and anaerobic microalgae are of significance in both basic research and industrial application. A microalga strain was isolated from a wastewater treatment pond and identified as Chlorella sorokiniana Shihira et W. R. Krauss GXNN01 in terms of morphology, physiology, and phylogeny. The strain grows rapidly in heterotrophic or mixotrophic conditions with addition of various carbon sources, and even in anaerobic conditions. The maximum growth rate reached 0.28 d(-1) when using D,L-malate as the carbon source, and the protein content of the microalgae was 75.32% in cell dry weight. The strain was shown to be capable of (1) utilizing D, L-malate only with light, (2) inhibiting photosynthesis in mixotrophic growth, and (3) growing in anaerobic conditions with regular photosynthesis and producing oxygen internally. This study demonstrates the influence of oxygen (aerobic vs. anaerobic) and metabolic regime (autotrophy, mixotrophy, heterotrophy) on the physiological state of the cell.

Effect of carbon source on growth and lipid accumulation in Chlorella sorokiniana GXNN01

Heterotrophic culture of microalgae to develop methods of increasing biomass productivity and storage lipids has brought new insight to commercial biodiesel production. To understand the relationship between heterotrophy and lipid production, the effects of carbon sources on the growth and lipid accumulation of Chlorella sorokiniana GXNN01 was studied. The alga exhibited an increased growth rate in response to the addition of carbon sources, which reached the stationary phase after 48 h at 30A degrees C. In addition, glucose and NaAc had a significant effect on the lipid accumulation during the early-stationary phase. Specifically, the lipid content was 0.237 +/- 0.026 g g(-1) cell dry weight and 0.272 +/- 0.041 g L-1 when glucose was used as the carbon source, whereas the lipid content reached 0.287 +/- 0.018 g g(-1) cell dry weight and 0.288 +/- 0.008 g L-1 when NaAc was used as the carbon source. The neutral lipid content was found to first decrease and then increase over time during the growth phase. A glucose concentration of 20 mmol L-1 gave the maximal lipid yield and the optimum harvest time was the early-stationary phase.

海洋小球藻Chlorella sp.甘油脂及脂肪酸、光合色素特征的研究

海洋微藻是海洋生态系统中最主要的初级生产者，也是海洋生物资源的重要来源。许多海洋微藻富含对人体具有重要的生理作用与保健功能的长链多不饱和脂肪酸，因此，筛选富含EPA、DHA等长链多不饱和脂肪酸的微藻和利用人工培养方法提高这些脂肪酸的产量是当前海洋生物学研究领域的热点之一。在本研究中，我们对被中科院海洋所定名为“Chlorella sp”（编号为1061）的一种海洋微藻的化学分类、甘油脂及其脂肪酸组成和外源葡萄糖和抗氧化剂（硫代硫酸钠）对这种微藻的脂肪酸含量的影响进行了研究，取得了以下主要结果。 海洋微藻是我固海水养殖中广泛使用的优良饵料藻。脂类物质是微藻最重要的营养指标之一，在本研究中，我们首先分析了被中科院海洋所定名为“Chlorellasp”的海洋微藻中的甘油脂及其脂肪酸种类和组成特点。结果表明，Chlorella sp.中的非极性脂主要为三脂酰甘油，极性甘油脂有10种。其中，一般培养条件下（温度23℃：光照，周期L/D14:10，强度60umolm-2-S-l）三脂酰甘油约占总脂的31 mol%，极性甘油脂约占总脂的69 rriol%。10种极性甘油脂是单半乳糖甘油二脂( monogalactosyldiacylglycerol． MGDG)、 双半乳糖甘油二脂( diagalactosyldiacylglycerol ， DGDG)、 硫代异鼠李糖甘油二脂( sulfoquinovosyldiacylglycerol，SQDG)、磷脂酰甘油(phosphatatidylglycerol，PG)、磷脂酰乙醇胺(phosphatidylethanolamine，PE)、磷脂酰胆碱( phosphatidylcholine，PC)、磷脂酰肌醇(phosphatidylinositol，PI)、磷脂酰丝氨醴(phosphatidylserine，Ps)、l，2-二酰基甘油-0-4，．（ⅣMⅣ-三甲基）高丝氨酸（diacylglyceryltrimethylhomoserine，DGTS）以及一种未能完全肯定，但可能是一中氯硫脂( chlorosulfolipid，CSL)。其中MGDG、DGDG、SQDG和PG是构成光合膜的主要成分，也是Chlorella sp中的主要极性脂。甜菜碱脂DGTS和磷脂PC是构成非光合膜的主要组分。Chlorella sp.中的主要脂肪酸为C16:0、C16:1耜C20：5（EPA)，后者主要存在于MGDG、DGDG和DGTS中，而三脂酰甘油也含有接近7%的EPA。 海洋微藻Chlorella sp.1061虽然被归属到绿藻纲绿藻目小球藻属，但是我们的研究表明，其色素、极性脂皮其脂肪酸组成与其它小球藻属藻类存在这很大差异Chl b是绿藻纲藻类中最主要的光合色素之一，1 6：4(n-3)和l 8：3(n-3)是绿藻微藻的主要脂肪酸，然而所有这些绿藻的特征化合物均未在Chlorella sp. 1061中检测到。DGTS和20:5(n-3)存在于很多的海洋微藻中，我们从Chlorella sp. 1061 中分离到占总极性甘油脂8 mo1%的DGTS，并从MGDG、DGDG和DGTS等极性甘油脂中检测到大量的20:5(n-3)。但是一般认为，小球藻属藻类中不舍这两种化合物。根据Chlorella sp. 1061的以上特点，这种藻不应该被归到小球藻属中。另外，由于Chlorella sp. 1061在色素、膜脂和脂肪酸组成特征方面大眼藻纲( Eustigmatophyceae)中的微绿球藻（Nannochloropsis）非常相似，因此，我们认为ChloreHa sp. 1061可能是Nannochloropsis中的一个种。但是未得到更进一步的证明和权威的认可之前，本文中我们仍然沿用ChloreHa sp,这一名称。 许多藻类中DGTS和PC -般不会同时存在，或者说一个存在时另外的一个的含量非常低。由此有人认为DGTS和PC之间存在着相互替代的关系。然而本研究中发现正常培养条件下Chlorella sp.中的DGTS和PC含量均较高（约10%）。磷处理实验结果表明，磷缺乏时Chlore Ha sp,中DGTS舍量大幅升高，而同时PC含量相应下降许多：但高浓度的磷并不能提高PC含量和降低DGTS含量，说明Chlorella sp,中DGTS仍可起替代PC的作用，然而PC可能并不能替代DGTS。Chlorella sp.中MGDG和DGTS脂肪酸组成及其位置分布结果显示，它们的组成和分布相似;在老化培养过程中MGDG和DGTS表现出周期性的相反的含量升高、降低的趋势，这进一步说明MGDG和DGTS之间存在着特殊的关系，MGDG可能合成自DGTS。 海洋微藻富含有利于人体健康的长链不饱和脂肪酸，如何提高微藻脂肪酸特别是多不饱和脂肪酸产量是目前研究的热点之一。本文首次报道了同时加入葡萄糖和硫代硫酸钠对Chlorelta sp,的生长、脂类组成和脂肪酸总产量的影响，结果显示葡萄糖和硫代硫酸钠存在明显而且强烈的互作，二者在培养液中的同时存在显著刺激了脂肪酸总产量的积累，在培养液中分别加入2.5 mM的葡萄糖和5mM的硫代硫酸钠，脂肪酸的产量可以比对照提高78%。而低浓度的葡萄糖和硫代硫酸钠对Chlorella sp.脂肪酸组成影响变化不明显，甚至在硫代硫酸钠存在下令人感兴趣的EPA含量还略有升高。显然，在Chlorella sp.培养中同时加入低浓度的葡萄糖和硫代硫酸钠是极具潜力的提高脂产量的方法，也可作为提高培养微藻其它活性物质产量借鉴的方法。在不久的将来，这种培养方法很可能发展成为生产实践中提高Chlore sp．乃至其它微藻脂肪酸、EPA和其它活性物质产量的经济有效的新途径。

Enhanced expression of antifreeze protein genes drives the development of freeze tolerance in an Antarctica isolate of Chlorella vulgaris

In adaptation to new environments, organisms may accumulate mutations within encoding sequences to modify protein characteristics or acquire mutations within regulatory sequences to alter gene expression levels. With the development of antifreeze capability as the example, this study presents the evidence that change in gene expression level is probably the most important mechanism for adaptive evolution in a green alga Chlorella vulgaris. C. vulgaris NJ-7, an isolate from Antarctica, possesses an 18S rRNA sequence identical to that of a temperate isolate, SAG211-11b/UTEX259, but shows much higher freeze tolerance than the later isolate. The chromosomal DNA/cDNA of four antifreeze genes, namely hiC6, hiC12, rpl10a and hsp70, from the two isolates of C. vulgaris were cloned and sequenced, and very few variations of deduced amino acid sequences were found. In contrast, the transcription of hiC6, hiC12 and rpl10a was greatly intensified in NJ-7 compared to that in UTEX259, which is correlated to the significantly enhanced freeze tolerance of the Antarctica isolate. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Alternative cold response modes in Chlorella (Chlorophyta, Trebouxiophyceae) from Antarctica

Chlorella was known to show enhanced antifreeze capability after cold hardening. We isolated Chlorella strains NJ-7 and NJ-18, which display alternative cold response modes from rock surfaces in Antarctica. On the basis of 18S ribosomal (rRNA) sequences, NJ-7 is an Antarctic type of Chlorella vulgaris; NJ-18 is also a 'true' Chlorella species but differs from any previously reported species in structure. NJ-7 partially retained the enhancing effects of low temperature cultivation on freeze tolerance, which correlates with an increase of C18:3-fatty acid content and up-regulation of two antifreeze protein genes. NJ-18, however, showed stable freeze tolerance regardless of the precultivation temperature. We propose that cold response modes vary widely in Chlorella and that the adaptation of C. vulgaris to Antarctica may serve as a model system for the evolution of antifreeze mechanisms in a single species of photosynthetic microorganism.

Effects of CO2 concentrations on the freshwater microalgae, Chlamydomonas reinhardtii, Chlorella pyrenoidosa and Scenedesmus obliquus (Chlorophyta)

In order to investigate the possible impacts of increased atmospheric CO2 levels on algal growth and photosynthesis, the influence of CO2 concentration was tested on three planktonic algae (Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Scenedesmus obliquus). Increased CO2 concentration enhanced significantly the growth rate of all three species. Specific growth rates reached maximal values at 30, 100, and 60 muM CO2 in C. reinhardtii, C pyrenoidosa, and S. obliquus, respectively. Such significant enhancement of growth rate with enriched CO2 was also confirmed at different levels of inorganic N and P, being more profound at limiting levels of N in C pyrenoidosa and P in S. obliquus. The maximal rates of net photosynthesis, photosynthetic efficiency and light-saturating point increased significantly (p<0.05) in high-CO2-grown cells. Elevation of the CO2 levels in cultures enhanced the photoinhibition of C. reinhardtii, but reduced that of C pyrenoidosa and S. obliquus when exposed to high photon flux density. The photo-inhibited cells recovered to some extent (from 71% to 99%) when placed under dim light or in darkness, with better recovery in high-CO2-grown C. pyrenoidosa and S. obliquus. Although pH and pCO(2) effects cannot be distinguished from this study, it can be concluded that increased CO2 concentrations with decreased pH could affect the growth rate and photosynthetic physiology of C. reinhardtii, C. pyrenoidosa, and S. obliquus.

Effects of doubled atmospheric CO2 concentration on the photosynthesis and growth of Chlorella pyrenoidosa cultured at varied levels of light

Chlorella pyrenoidosa was cultured with 350 and 700 p.p.m.v. CO2 at varied levels of light to see the impacts of doubled atmospheric CO2 concentration on its growth and photosynthesis. The CO2 enrichment did not affect the growth rate (mu), but significantly increased the cell density when light was sufficiently supplied. The CO2 enrichment significantly depressed light-saturated photosynthesis and dark respiration in the cells grown under a high-light regime, but not those under a low-light regime. The light-saturating point for photosynthesis and photosynthetic efficiency was not affected by the CO2 enrichment under either the high-light or low-light conditions.

Growth of Chlorella vulgaris in cultures with low concentration dimethoate as source of phosphorus

The growth response of Chlorella vulgaris to low concentration of dimethoate, an organophosphorus pesticide, was studied. Results show that cell density, protein content, chlorophyll pigment and alkaline phosphatase activity were all increased, which indicates that low concentration dimethoate can accelerate growth of Chlorella vulgaris. (C) 1997 Elsevier Science Ltd.

Anti-inflammatory and immunomodulatory activities of polysaccharide from Chlorella stigmatophora and Phaeodactylum tricornutum

Crude polysaccharide extracts were obtained from aqueous extracts of the microalgae Chlorella stigmatophora and Phaeodactylum tricornutum. The crude extracts were fractionated by ion-exchange chromatography on DEAE-cellulose columns. The molecular weights of the polysaccharides in each fraction were estimated by gel filtration on Sephacryl columns. The crude polysaccharide extracts of both microalgae showed anti-inflammatory activity in the carrageenan-induced paw edema test. In assays of effects on the delayed hyper-sensitivity response, and on phagocytic activity assayed in vivo and in vitro, the C. stigmatophora extract showed immunosuppressant effects, while the P. tricornutum extract showed immunostimulatory effects. Copyright © 2003 John Wiley & Sons, Ltd.

Molecular cloning and stress-dependent expression of a gene encoding Delta(12)-fatty acid desaturase in the Antarctic microalga Chlorella vulgaris NJ-7

The psychrotrophic Antarctic alga, Chlorella vulgaris NJ-7, grows under an extreme environment of low temperature and high salinity. In an effort to better understand the correlation between fatty acid metabolism and acclimation to Antarctic environment, we analyzed its fatty acid compositions. An extremely high amount of Delta(12) unsaturated fatty acids was identified which prompted us to speculate about the involvement of Delta(12) fatty acid desaturase in the process of acclimation. A full-length cDNA sequence, designated CvFAD2, was isolated from C. vulgaris NJ-7 via reverse transcription polymerase chain reaction (RT-PCR) and RACE methods. Sequence alignment and phylogenetic analysis showed that the gene was homologous to known microsomal Delta(12)-FADs with the conserved histidine motifs. Heterologous expression in yeast was used to confirm the regioselectivity and the function of CvFAD2. Linoleic acid (18:2), normally not present in wild-type yeast cells, was detected in transformants of CvFAD2. The induction of CvFAD2 at an mRNA level under cold stress and high salinity is detected by real-time PCR. The results showed that both temperature and salinity motivated the upregulation of CvFAD2 expression. The accumulation of CvFAD2 increased 2.2-fold at 15A degrees C and 3.9-fold at 4A degrees C compared to the alga at 25A degrees C. Meanwhile a 1.7- and 8.5-fold increase at 3 and 6% NaCl was detected. These data suggest that CvFAD2 is the enzyme responsible for the Delta(12) fatty acids desaturation involved in the adaption to cold and high salinity for Antarctic C. vugaris NJ-7.

Effect of iron on growth and lipid accumulation in Chlorella vulgaris

The economic feasibility of algal mass culture for biodiesel production is enhanced by the increase in biomass productivity and storage lipids. Effect of iron on growth and lipid accumulation in marine microalgae Chlorella vulgaris were investigated. In experiment I, supplementing the growth media with chelated FeCl3 in the late growth phase increased the final cell density but did not induce lipid accumulation in cells. In experiment II, cells in the late-exponential growth phase were collected by centrifugation and re-inoculated into new media supplemented with five levels of Fe3+ concentration. Total lipid content in cultures supplemented with 1.2 x 10(-5) mol L-1 FeCl3 was up to 56.6% biomass by dry weight and was 3-7-fold that in other media supplemented with lower iron concentration. Moreover, a simple and rapid method determining the lipid accumulation in C. vulgaris with spectrofluorimetry was developed. (c) 2007 Elsevier Ltd. All rights reserved.

两种真核微藻逆境响应关键基因的调控机理研究

非生物逆境胁迫严重影响植物的生长发育和作物的产量。解析逆境下植物体基 因表达及信号传导网络调控机制对于阐明植物的适应性反应及培育耐逆性作物具有 重要理论和现实意义。 雨生红球藻（Haematoccoccus pluvialis）通过累积虾青素来适应胁迫环境，茉莉 酮酸甲酯（methyl jasmonate，MJ）和赤霉素（gibberellins，GA）对植物胁迫适应过 程中相关基因的诱导表达起到重要的调控作用。本文选取 H. pluvialis 作为出发株， 研究了 MJ 和 GA 对虾青素合成途径关键酶 β-胡萝卜素酮化酶基因的调控作用。 在其中一种 H. pluvialis 中克隆获得了三种不同的 β-胡萝卜素酮化酶基因。通过基 因组步移，获得了三种 β-胡萝卜素酮化酶的5’ 侧翼序列（5’-flanking region），其 中存在着多样的顺式作用元件，包括 MJ 和 GA 的顺式作用位点。进一步的实验 表明，MJ 和 GA 的诱导处理，可以促进 H. pluvialis 中虾青素的累积；同时，MJ 和 GA 调控了三种 β-胡萝卜素酮化酶基因的转录水平。 南极小球藻（Chlorella vulgaris NJ-7）可以在较大温度和盐度变化范围内存活， 是研究生物胁迫适应机理的理想模式生物。对数期的 C. vulgaris NJ-7 主要的脂肪酸

复合藻-菌净化系统处理麦秆造纸废液初步研究

在室内模拟条件下,采用正交设计法建立了一种由蛋白核小球藻(Chlorella pyrenoidosa)、鱼腥藻7120(Anabaena sp.PCC7120)、硝化细菌(Nitrate bacteria)和荚膜红假单胞菌(Rhodopseudomonas capsulata)组成的复合藻-菌净化系统去除造纸废液中有机质和氨态氮的最优化模型,确定了藻类与细菌的最优化数量配比关系为蛋白核小球藻∶鱼腥藻7120∶硝化细菌∶荚膜红假单胞菌=1∶2.17∶2.83∶5.09.比较了复合藻-菌净化系统最优化模型与单

焦酚对蓝藻和绿藻生长、光合色素及微量元素的作用

研究了焦酚对4种常见蓝藻和绿藻的生长影响.结果表明,焦酚对几种藻的生长具有抑制作用.相同的处理浓度,4种藻生长率从小到大依次是:铜绿微囊藻(Microcystic aeruginosa)、羊角月牙藻(Selenastrum capri-cornutum)、蛋白核小球藻(Chlorella pyrenoidosa)、水华鱼腥藻(Anabaena flos-aquae).焦酚作用后铜绿微囊藻和羊角月牙藻的叶绿素a和胡萝卜素的含量有显著的变化,随着焦酚浓度的升高,铜绿微囊藻和羊角月牙藻叶绿素a和胡萝卜素含量减少