Effects of L-carnitine on Population Growth of Nannochloropsis oculata and Tetrahymena sp.

[Objective] The aim was to study the effects of L-carnitine on population growth of Nannochloropsis oculata and Tetrahymena sp..[Method] When the concentration of L-carnitine was 0,50,100 and 1 000 mg/L,population densities of Nannochloropsis oculata and Tetrahymena were determined respectively.[Result] Adding high-dose L-carnitine had a significant inhibition effect on the population growth of Nannochloropsis oculata （P0.05）.Adding L-carnitine had a significant proliferation promoting effect on the population growth of Tetrahymena （P0.05）.[Conclusion] The research provides theoretical basis for the application of L-carnitine as feed additive in aquaculture.

Potency of Nannochloropsis oculata as Antibacterial, Antioxidant and Antiviral on Humpback Grouper Infected by Vibrio alginolyticus and Viral Nervous Necrotic

The aim of this study was to explore the potency of N. oculata extracts as antibacterial, antioxidant and antiviral on grouper Cromileptes altivelis （C. altivelis） infected by Vibrio alginolyticus （V. alginolyticus） and Viral Nervous Necrotic （VNN）. Dilution test was used to measure antibacterial activity of N. oculata extracts. Antioxidant activity of extracts was expressed by levels of MDA （Malondialdehyde） and SOD （superoxide dismutase） in the brain and kidneys of fish. Antiviral capability of N. oculata was determined by the expression of cellular immune cells Major Histocompatibility Complex （MHC） Class I for proliferation and inhibition of VNN in blood cells with immunocytochemistry. The results showed that N. oculata extract was able to suppress the growth of V. alginolyticus at a concentration of 40%. Increasing levels of SOD and reducing level of MDA indicated that N. oculata extracts may serve as an antibacterial and antioxidant. Providing cellular response of MHC class I cell expressed on C. altive/is blood cells, N. oculata demonstrated its antiviral activity.

一种提取眼点拟微球藻(Nannochloropisis oculata)DNA的简便方法

用氯化锂法从眼点拟微球藻 (Nannochloropisisoculata)中提取DNA并对其提取条件进行了优化。新鲜藻细胞在提取液 (0.6mol/LLiCl,0.8%Sakosyl,20mmol/LEDTA(pH8.0) ,0.4%PVP,10mmol/LTris HCl(pH8.0) ,5% β 巯基乙醇 )中经过55℃温育30min,每mg鲜藻可提取20～40ngDNA ,提取的DNA分子量在23kb左右 ,可用于PCR、限制性酶切等。

海绵Haliclona oculata化学成分的研究

目的研究海绵Haliclona oculata中的化学成分。方法运用正反相硅胶、凝胶(Sephadex LH-20)等多种柱色谱和HPLC进行分离纯化,根据理化性质和波谱数据进行结构鉴定。结果从海绵Haliclona oculata的二氯甲烷萃取部分分离得到4个化合物,分别鉴定为胆甾醇(Ⅰ),胆甾-4,22-二烯-6α-羟基-3-酮(Ⅱ),胆甾-4-烯-6α-羟基-3-酮(Ⅲ),麦角甾-4,24(28)-二烯-6-羟基-3-酮(Ⅳ)。结论化合物Ⅱ,Ⅲ,Ⅳ均为首次从该种海绵中分离得到。

硝态氮对Nannochloropsis oculata生长及合成胞内组分的影响

研究了硝态氮对Nannochloropsis oculata C170细胞生长及胞内组分合成的影响。以硝酸钠为氮源考察不同氮浓度下藻细胞生长、氮消耗及油脂含量变化,并在此基础上分析氮限制处理时油脂、总糖及蛋白等胞内组分合成差异。结果表明:N.oculata C170在42.5mg/L氮浓度时生长最佳,其生物量、比生长速率及产率最高,分别为0.59g/L、0.275d-1和50.98mg/(L·d)。油脂含量与氮浓度呈明显负相关,氮浓度为12.5mg/L时,油脂含量和产率最高,分别为29.17%和11.40mg/(L·d)。而胞内多不饱和脂肪酸比例随着氮浓度的升高显著增加,其中EPA含量由8.06%升高到18.76%。此外藻细胞在限氮培养时,其油脂含量增加至23.07%,为最初含量的2.8倍,同时总糖含量升高了76%左右,叶绿素a和蛋白含量则分别下降了大约29%和24%。高浓度硝态氮有利于N.oculata C170细胞生长及胞内EPA积累,低浓度硝态氮诱导藻细胞油脂积累并与糖类合成、色素和蛋白降解相关联。

Cloning and characterization of a delta-6 desaturase encoding gene from Nannochloropsis oculata

A gene （NANOC-D6D） encoding a desaturase that removes two hydrogen atoms from fatty acids at delta 6 position was isolated from a cDNA library of Nannochloropsis oculata （Droop） D. J. Hibberd （Eustigmatophyceae）. The unicellular marine microalga N. oculata synthesizes rich long chain polyunsaturated fatty acids （LCPUFAs）, including eicosapentaenoic acid （20：5n-3, EPA）. The deduced protein contains 474 amino acids that fold into 4 trans-membrane domains. The neighbor-joining phylogenetic tree indicates that NANOC-D6D is phylogenetically close to the delta-6 fatty acid desaturase of marine microalgae such as Glossomastix chrysoplasta, Thalassiosira pseudonana, and Phaeodactylum tricornutum. The gene was expressed in Saccharomyces cerevisiae INVScl to verify the substrate specificity of NAN OC-D6D. Our results suggest that the recombinant NANOC-D6D simultaneously desaturates linoleic acid （LA） and a-linolenic acid （ALA）.

微绿球藻(Nannochloropsis oculata)培养基的优化

【目的】微绿球藻(Nannochloropsis oculata)是一种优良的单细胞饵料微藻,广泛应用于水产养殖。优化微绿球藻培养基,以期提高其生长速率,降低生产成本。【方法】以宁波大学3#配方为基础微藻培养液,以乙酸钠为碳源,硝酸钾、尿素和氯化铵为氮源,磷酸二氢钾和磷酸二氢钠为磷源,硫酸亚铁和柠檬酸铁为铁源,通过单因子和正交试验,研究了碳、氮、磷、铁、维生素B_1(VB_1)和B_(12)(VB_(12))等主要营养元素对微绿球藻生长、繁殖的影响。【结果】获得了以天然海水为基础的微绿球藻优化培养基:3 g/L CHCOONa、20 mg/L NH_4CL-N、2 mg/L KH_2PO_4-P、3 mg/L FeSO_4-Fe、0.05 mg/L VB_1和 0.005mg/L VB_(12);采用优化培养基与宁波大学3#培养基对比培养微绿球藻,结果表明,培养 2～6 d,优化培养基收获微绿球藻的生物量( 细胞密度)比宁波大学 3#培养基的分别提高了2.21、2.55、2.30、1.97、1.7倍;培养6 d,优化培养基中微绿球藻收获的生物量(细胞密度)达到1.74×10~7个/mL,是宁波大学3#培养基的1.7倍。【结论】优化培养基极显著地提高了微绿球藻的生物量,是微绿球藻的良好培养基。

Cloning and Phylogenetic Analysis of a Fatty Acid Elongase Gene from Nannochloropsis oculata CS179

Nannochloropsis oculata CS179, a unicellular marine microalga, is rich in long-chain polyunsaturated fatty acids （LCPUFAs）. Elongase and desaturase play a key role in the biosynthesis of PUFAs. A new elongase gene, which encodes 322 amino acids, was identified via RT-PCR and 5＇ and 3＇ RACE. The sequence of the elongase gene was blast-searched in the NCBI GenBank and showed a similarity to those of the coptosporidium. But the NJ-tree revealed that the N. oculata CS 179 elongase clustered with those of the microalgae Phaeodac^lum tricornutum, Ostreocoecus tauri and Thalassiosira pseudonana.

Construction and characterization of a normalized cDNA library of Nannochloropsis oculata(Eustigmatophyceae)

We constructed and characterized a normalized cDNA library of Nannochloropsis oculata CS-179,and obtained 905 nonredundant sequences(NRSs) ranging from 431-1 756 bp in length.Among them,496 were very similar to nonredundant ones in the GenBank(E ≤1.0e-05),and 349 ESTs had significant hits with the clusters of eukaryotic orthologous groups(KOG).Bases G and/or C at the third position of codons of 14 amino acid residues suggested a strong bias in the conserved domain of 362 NRSs(>60%).We also identified the unigenes encoding phosphorus and nitrogen transporters,suggesting that N.oculata could efficiently transport and metabolize phosphorus and nitrogen,and recognized the unigenes that involved in biosynthesis and storage of both fatty acids and polyunsaturated fatty acids(PUFAs),which will facilitate the demonstration of eicosapentaenoic acid(EPA) biosynthesis pathway of N.oculata.In comparison with the original cDNA library,the normalized library significantly increased the efficiencies of random sequencing and rarely expressed genes discovering,and decreased the frequency of abundant gene sequences.

The Response of Bio-Component Production of Nannochloris oculata to the Combinations of Monochromatic Light

Illuminating conditions are crucial factors affecting the microalgal growth and biosynthesis.The effects of illuminating spectral quality on the growth and bio-component production of Nannochloris oculata were investigated.The results indicated that a high proportion of Red-light enhanced the pigments and carbohydrate production but reduced those of the biomass and lipid.Mono-chromatic Blue-light has advantageous effects on lipid production compared with Red-and White-light.The optimal light spectrum for the protein production was the combination of Red-and Blue-light at a ratio of 4:3 or 5:2.Among the seven fatty acids identified from N.oculata,the contents of C16:0,C18:0,and C18:3(n-3)in the lipid were inhibited by the increased proportion of Red-light while those of C18:2(n-9),C16:2(n-6),and C20:0 were inhibited by Blue-light.Monochromatic Red-and Blue-light and their com-binations were proposed as a promising illuminating strategy for the large-scale cultivation aiming for various bio-components.

Low-Temperature Affected LC-PUFA Conversion and Associated Gene Transcript Level in Nannochloropsis oculata CS-179

Nannochloropsis oculata CS-179, a marine eukaryotic unicellular microalga, is rich in long-chain polyunsaturated fatty acids (LC-PUFAs). Culture temperature affected cell growth and the composition of LC-PUFAs. At an initial cell density of 1.5 × 106 cell mL-1, the highest growth was observed at 25℃ and the cell density reached 3 × 107 cell mL-1 at the beginning of loga-rithmic phase. The content of LC-PUFAs varied with culture temperature.The highest content of LC-PUFAs (43.96%) and EPA (36.6%) was gained at 20℃. Real-time PCR showed that the abundance of Δ6-desaturase gene transcripts was significantly different among 5 culture temperatures and the highest transcript level (15℃) of Nanoc-D6D took off at cycle 21.45. The gene transcript of C20-elongase gene was higher at lower temperatures (10, 15, and 20℃), and the highest transcript level (20℃) of Nanoc-E took off at cycle 21.18. The highest conversion rate (39.3%) of Δ6-desaturase was also gained at 20℃.But the conversion rate of Nanoc-E was not detected. The higher content of LC-PUFAs was a result of higher gene transcript level and higher enzyme activity. Compared with C20-elongase gene, Δ6-desaturase gene transcript and enzyme activity varied significantly with temperature. It will be useful to study the mechanism of how the content of LC-PUFAs is affected by temperature.

Temperature effects on lipid properties of microalgae Tetraselmis subcordiformis and Nannochloropsis oculata as biofuel resources

Microalgae Tetraselmis subcordiformis and Nannochloropsis oculata were cultured at 15,20,25,30,and 35℃ and their properties as potential biofuel resources were examined.The results indicate that T.subcordiformis and N.oculata grew best at 20℃ and 25℃ and yielded the highest total lipids at 20℃and 30℃,respectively.With increased temperature,neutral lipid and polyunsaturated fatty acids(FAs)decreased while saturated FAs increased,accompanied by increased monounsaturated FAs(MUFAs) in T.subcordiformis and decreased MUFAs in N.oculata;meanwhile,the predicted cetane number of FA methyl esters increased from 45.3 to 47.6 in T.subcordiformis and from 52.3 to 60.3 in N.oculata.Therefore,optimizing culture temperatures is important for improving microalgal biodiesel production.

氮源对Nannochloropsis oculata生化组成、超微结构及生产的影响

为了提高眼点微拟球藻生物量及生产强度,比较了氯化铵、乙酸铵及硝酸钠三种氮源对其生理生化及生产的影响。结果显示:铵盐组胞内蛋白质质量分数高于硝酸钠组,叶绿素a质量分数结果相反;乙酸铵组总油质量分数最高,为13.5%;硝酸钠组总碳水化合物质量分数最高,为16.5%。硝酸钠组极性油/中性油比值及EPA相对含量最高,分别为5.9和26.6%。铵盐组藻细胞尺寸大于硝酸钠组;硝酸钠组叶绿体数量、相对体积、类囊体密度高于铵盐组;仅乙酸铵组胞内含有油体。乙酸铵组生物量及生产强度最大,分别为873 mg/L和25.3 mg/(L·d)。

Genetic Transformation of Nannochloropsis oculata with a Bacterial Phleomycin Resistance Gene as Dominant Selective Marker

The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2× 10^7 cells mL-1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS 188. The construct was transferred into the protoplasts through electroporation （1 kV, 15 μS）. The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL-t zeoein. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.

Hydroesterification of <i>Nannochloropsis oculata</i>microalga’s biomass to biodiesel on Al₂O₃supported Nb₂O₅catalyst

Hydroesterification process has been presented biodiesel production from oil the green microalga Nannochloropsis oculata raw materials. Biodiesel studied in this work is the main product got from the hydroesterification of biomass the Nannochloropsis oculata and was obtained from esterification of fatty acid (product of a hydrolysis reaction) with methanol. It was used as catalyst the niobic acid pure and supported on δ-aluminum. The product was evaluated by gas chromatography and other analyses. The optimum conditions found in the conversion (%) for the hydrolysis reactions of the biomass (92.3%). Better results were observed in the algae concentration 20%, lead at 300?C with 20% of catalyst. For esterification of fatty acids of Nannochloropsis oculata (92.24%), were observed the molar ratio methanol: fat acid 3, lead at 200°C with 20% of catalyst supported.

Fatty Acid Composition Analyses of the DCMU Resistant Mutants of Nannochloropsis oculata(Eustigmatophyceae)

Ultraviolet mutagenesis was applied to Nannochloropsis oculata and three mutants resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were isolated. The cellular chlorophyll a and total lipid content of the wild are higher in the medium supplemented with DCMU than in the control without DCMU. Without DCMU, the growth rates and chlorophyll a contents of the mutants are similar to those of the wild. Significant changes of fatty acid content and composition have occurred in DCMU-resistant mutants growing in the medium supplemented with DCMU. The total lipid, palmitic acid (16∶0), palmitoleic acid (16∶1ω9) and oleic (18∶1ω9) contents decrease significantly, while the vaccenic acid (18∶1ω11) increases significantly and the EPA content of dried powder increases slightly in the mutants. The study may provide a basis to improve EPA content in Nannochloropsis oculata in the future.

光周期对微绿球藻生长和物质生产的影响

为研究光周期对微绿球藻(Nannochloris oculata)生长及物质生产的影响,该文设置了4种长时光周期(光∶暗(h∶h)=24∶0、16∶8、12∶12或8∶16),5种频率光周期(按照光∶暗=16∶8,24 h内分别设置1、4、9、16或25次光暗交替)。结果发现,长时光周期对微绿球藻的生物量干重无显著性影响,而频率光周期对生物量干重影响显著。2种光周期对于微绿球藻的叶绿素a含量有极显著影响,生长速度最快的处理组叶绿素a含量最低。2类光周期对微绿球藻脂质生产均有显著影响,24∶0及16∶8处理组获得较高的脂肪产量,24 h内光暗交替9次处理比交替1次的脂肪产量提高了55.65%。微绿球藻的蛋白质生产受光周期影响不大,但多糖生产受光周期影响显著。微绿球藻的脂肪酸中含量最丰富的是C18∶3(n-3)、C16∶0和C18∶2(n-9),频率光周期对其脂肪酸组成和生物柴油性能影响不显著。

氮源和N/P对眼点拟微球藻的生长、总脂含量和脂肪酸组成的影响

报道了NaNO3,NH4Cl和NH4NO3 3种氮源及N/P对海洋微藻眼点拟微球藻(Nannochloropsisoculata)的生长、总脂含量和脂肪酸组成的影响。结果表明 ,N/P从10增加到90 ,对细胞生长的影响不大 ,总脂含量最终都稳定在干重的20 %左右。16∶0、16∶1n 9和20∶5n 3 (EPA)是脂肪酸的主要组分 ,三者之和占总脂肪酸的70 %～80 %。在培养基N/P>20时 ,20∶5n 3和PUFA占总脂肪酸的比例接近30 %和35 %。在3种氮源中 ,使用NaNO3 可获得更高的生长速率 ,并在相对较低的N/P下 (N/P=54)时获得约30 %的EPA ,故NaNO3 是眼点拟微球藻生长、合成和积累EPA的最佳氮源。将培养基中所有营养盐增加4倍 ,EPA含量可提高到35 % ,说明富含氮、磷的培养基可使细胞始终保持旺盛生长、富含EPA的状态 ,这也是该种微藻大规模培养中高产EPA的关键措施。

两种微藻改善虾池环境增强凡纳对虾抗病力的研究

人工引入波吉卵囊藻和微绿球藻于凡纳对虾养殖环境中 ,检测与凡纳对虾抗病力有关因子变化和测定主要水质因子 ,研究微藻生态调控对凡纳对虾抗病力的影响。结果表明 ,引入波吉卵囊藻和微绿球藻能改善养殖水体的水质 ,凡纳对虾的血细胞数目 ,血清蛋白的含量以及酚氧化酶、超氧化物歧化酶 ,溶菌酶 ,抗菌酶的活性都较对照组显著提高。因此 。