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。

Growth performance of Chlorella ellipsoidea as biomicrocapsule using supernatant of digested sugar mill waste effluent

Biomicrocapsules mean microscopic living organisms which carry important nutrients, very essential for the growth and development of aquatic organisms as well as other animals. Among these biomicrocapsules, Chlorella ellipsoidea, an important green microalga (Chlorophyceae) which contains 40-45% crude protein, 12-16% crude lipid, 14-15% minerals, colour pigments, vitamins and carotene. The microalga, C. ellipsoidea was cultured in four different dilutions of supernatant of digested sugar mill effluent (DSME) i.e. 25, 50, 75 and 100% DSME and Bold basal medium (BBM) as control in laboratory condition. Maximum cell growth and chlorophyll a content of C. ellipsoidea were obtained on l0th day of culture in supernatant of 50% diluted DSME followed by those of this biomicrocapsule grown in BBM, and 75, 25 and 100% DSME at stationary phase. Cell number had highly (p<0.01) direct correlation with chlorophyll a (r = 0.889) of C. ellipsoidea, and optical density (r = 0.926) of media. Chlorophyll a was also highly (p<0.01) and directly correlated with optical density (r= 0.877) of media. The specific growth rates (µ/day) of cell and chlorophyll a of C. ellipsoidea grown in supernatant of 50% DSME were significantly (p<0.01) varied from those of C. ellipsoidea cultured in BBM followed by other DSME. Total biomass of C. ellipsoidea cultured in supernatant of 50% DSME was found significantly (p<0.01) higher than that of this microalga cultured in BBM, and supernatant of 25, 75 and 100% DSME. Similar trend was also observed in the case of optical density. The physico-chemical properties of media were varied with the growth of cell of this microalga. It was recorded that cell number, chlorophyll a of biomicrocapsule, and optical density of media were highly (p<0.01) and directly correlated with pH, hardness and alkalinity, and inversely correlated with nitrate-N. Crude protein and crude lipid of C. ellipsoidea grown in supernatant of 50% DSME were significantly (p<0.01) higher than those of C. ellipsoidea cultured in other DSME and BBM. Due to best growth performance exhibited by this microalga grown in supernatant of 50% DSME, it may be used to grow in supernatant of 50% DSME to get more essential nutrients than that cultured in supernatant of other DSME media.

Culture of green algae Chlorella ellipsoidea in inexpensive media

The culture of the of green alga Chlorella ellipsoidea was conducted under natural conditions at the same place simultaneously in five different media, viz., medium-I (inorganic medium), medium-II (powdered whole-pulse medium), medium-III (medium of pulse bran), medium-IV (mixed medium = 50% inorganic medium + 50% whole-pulse powder medium), medium-V (mixed medium = 50% inorganic medium + 50% pulse bran medium). The culture was done in 500 ml conical flask. Growth rates of C. ellipsoidea in five different media were different and reached maximum cell densities of 0.63 x 10^6 cells per ml in 8 days in medium-I, 4.02 x 10^6 cells per ml in 10 days in medium-II, 3.62 x 10^6 cells per ml in 9 days in medium-III, 4.38 x 10^6 cells per ml in 11 days in medium-IV and 4.36 x 10^6 cells per ml in 11 days in medium-V. The range of air temperature was 20 to 33°C and that of culture media was 24 to 32°C and light intensity was 2000 to 7000 lux during the culture period. The inexpensive culture media were found to be significantly useful for algal culture.

Effect of two green algae species (Chlorella sp. & Scenedesmus obliquus) enriched with B group vitamins on food chain of Rutilus frisii kutum (Kamensky, 1901) fry

The Effect of two freshwater green algae species Chlorella sp. & Scenedesmus obliquus enriched (from the beginning of culture and after 96 hours) with different dosages of B group vitamins (0, 0.5, 1, and 2 ml of enriching solution per each liter of algae medium) on fecundity of Daphnia magna and growth of Rutilus frisii kutum fry were investigated in a research from spring, 2008 to autumn, 2009. First, each of the green algae species were cultured purely and massively in the Zander (Z-8+N) medium and then the nutritional value (the amount of protein, lipid, and carbohydrate) of enriched algae were meausered. In this study, enriching of Chlorella sp. & S. obliquus with a suitable mix of B group vitamins significantly improved their nutritive value. So the highest amount of nutritional value of Chlorella sp. was obtained because of enriching with dosage 0.5 ml.l-1 (366.654Kcal) and for Scenedesmus obliquus with dosage of 1 ml.l-1 (376.95Kcal). The acquired amount from control group showed an increase of respectively 42% and 11%. According to the results, increased dosages of enriching solution caused Daphnia fecundity to increase (at both stages : enrichment from the beginning of culture and after 96 hours). So the highest average of D. magna reproduction rate was obtained through being fed with Chlorella sp. and S. obliquus enriched with dosage of 2 ml enriching solution per liter of algae medium. The average fecundity of D. magna fed with Chlorella sp. enriched with dosage of 2 ml.l-1 enriching solution from the beginning of culture and after 96 hours was obtained respectively 2.128 ± 0.375 and 2.1 ± 0.69 and the average fecundity of D. magna fed with S. obliquus enriched with dosage of 2 ml enriching solution from the beginning of culture and after 96 hours was obtained respectively 2.128 ± 0.375 and 2.1 ± 0.69 which showed respectively an increase of 61 ٪, 91٪, 77 ٪, and 83٪ in proportion to the acquired amount from control group. When enriching solution was added to either algae culture medium from the beginning of culture, showed statistically significant differences (P<0.05) between dosages of 0 and 2 ml.l-1, 1 and 2 ml.l-1, and 0.5 and 2 ml enriching solution per each liter of Chlorella sp. culture medium and between dosages of 0 and 1 ml.l-1, and 0 and 2 ml enriching solution per each liter of S. obliquus culture medium. The highest average of body weight gain percentage and specific growth rate of kutum fry was obtained respectively 21.19%, 26.63%, 1.92, and 2.34 from the beginning of culture and after 96 hours with dosage of 1 ml B group vitamins per each liter of Chlorella sp. culture medium, which showed respectively an increase of 50%, 70%, 46%, and 62% in proportion to the acquired amount from control group. In the cases which Chlorella sp. were grown in the medium containing vitamin, from point of view of the average percentage of weight and specific growth rate of kutum fry significant differences were observed on the basis of the result of One-way ANOVA between dosages of 0 and 1, 1 and 2 , 0.5 and 1 ml B group vitamins per each liter. The highest average of body weight gain percentage and specific growth rate of kutum fry was obtained respectively 32.02%, 29.42%, 2.78, and 2.34 from the beginning of culture and after 96 hours with dosage of 2 ml B group vitamins per each liter of S. obliquus culture medium, which showed respectively an increase of 32%, 19%, 28%, and 17% in proportion to the acquired amount from control group. In the cases which S. obliquus were grown in the medium containing vitamin, from point of view of the average percentage of weight and specific growth rate of kutum fry significant differences were observed on the basis of the result of One-way ANOVA between dosages of 0 and 1, 0 and 2. According to the results of the present research we can say that considerable enhancement in the quality of the food of D. magna can be made by manipulation of the nutritional value of fresh water unicellular green algae with suitable mixture of B group vitamins, so that both the fecundity of D. magna will increase and the nutritional requirements of the kutum fry will be filled in this way.

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.

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.

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.

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 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.

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.