布朗葡萄藻的培养基选择及其产物代谢规律

通过对布朗葡萄藻分别在Chu13、Chu13×2和BG-11培养基中培养结果的比较,发现在气升式光照生物反应器中Chu13培养基最有利于布朗葡萄藻的生长和烃的合成,培养15d后,其生物量和粗烃质量分数分别为1.82g/L和58.7%;棕榈酸、油酸和亚麻酸是布朗葡萄藻的主要脂肪酸组成,Chu13培养获得的藻体不饱和脂肪酸比例最高。Chu13培养基中布朗葡萄藻代谢规律的研究表明:粗烃含量随着生物量的增加而逐渐增大,15d后粗烃产量达到最大值1.07g/L,不同生长周期烃的组成保持一致,布朗葡萄藻的烃主要由C33H56和C34H58组成;在布朗葡萄藻生长周期中,不饱和脂肪酸的比例显著上升,培养15d达到64%以上。     

共培养对布朗葡萄藻764、765株生长的影响

以布朗葡萄藻(Botryococcus braunii )764 株(B764 )和765 株(B765 )为实验材料,研究了株间共培养(接种比例为纯B764 、1 :9 、3 :7 、5 :5 、7 :3 、9 :1 、纯B765 )对藻细胞生物量、总烃含量、总脂肪酸含量的影响,以及添加另一株的藻滤液对其生长的影响.结果表明:两株布朗葡萄藻的混合培养能够增加藻细胞群体的生物量,且布朗葡萄藻764 株占主体,同时存在一定量765 的混合培养群体生物量明显高于纯培养的布朗葡萄藻(P＜0.05);布朗葡萄藻株间共培养可以发生互利促进效应, 低浓度的布朗葡萄藻765 藻滤液对布朗葡萄藻764 的生长具有显著促进作用(P＜0.05).布朗葡萄藻能够向胞外培养液中释放化感物质,藻滤液中的低浓度的化感物质可以对受体微藻产生一定促进作用.     

N、P营养盐胁迫对两株布朗葡萄藻生长的影响

采用批次培养方法,在光照强度60、110mol/m2s下分别设置了7个不同的氮、磷浓度(N:0-3500g/L,P:15-775g/L),研究两株布朗葡萄藻(Botryococcus braunii)对氮、磷胁迫的敏感性差异,筛选高营养利用效率的优良藻株。结果表明:两株藻对氮磷营养胁迫的耐受性存在差异,B.braunii764株对氮胁迫具有较高耐受性,而B.braunii765株对磷胁迫具有较高耐受性。光照强度110mol/m2s,不同氮浓度下B.braunii764株其平均生长速率均显著高于其他各处理组;不同磷浓度下B.braunii765株其平均生长速率显著高于B.braunii764株。在试验设定的光照强度条件下,适当增加光照强度能够显著降低氮胁迫对布朗葡萄藻生长的抑制效应。在光照强度110mol/m2s下,氮浓度3500g/L时两株布朗葡萄藻平均生长速率与在正常Chu-10培养基条件下无显著差异。磷浓度775g/L时两株布朗葡萄藻的平均生长速率均显著低于正常Chu-10培养基条件,增加光照强度对磷胁迫下藻细胞的生长无显著作用。两株布朗葡萄藻在第2天时磷吸收与初始磷浓度呈正相关关系,氮吸收在3500g/L时出现饱和现象。布朗葡萄藻的生长更容易受到培养基中磷营养胁迫的影响。       

富油微藻布朗葡萄藻分子生态学研究进展

分子生态学是研究生命系统与环境系统相互作用机理及其分子机制的科学,可以从宏观和微观结合的角度真实反映生态现象的本质。简述产烃布朗葡萄藻形态与化学种等生理生态特征的基础上,综述了近年来国内外布朗葡萄藻分子生态学研究的新进展,主要包括分子系统发育学及其与化学种、基因组、地理来源等之间的关系。经典分类学上,关于布朗葡萄藻属于绿藻门(Chlorophyta)还是黄藻门(Xanthophyta)存在争议,而基于18S核糖体核糖核酸(18S ribosomal ribonucleic acid,18S rRNA)序列的分子系统发育学研究结果将布朗葡萄藻界定为绿藻门、共球藻纲(Trebouxiophyceae)。依据藻株的产烃种类和化学结构特征,可将布朗葡萄藻划分为A、B和L 3个化学种,而布朗葡萄藻的分子系统学进化关系与化学种间高度统一。在基因组大小上,位于同一大亚聚群中的化学种B与L间却存在明显差异,而进化关系较远的化学种B与A间则更相近。不同地理来源布朗葡萄藻的18S rRNA序列和内部转录间隔区(internal transcribed spacer,ITS)多态性较高,提示不同地缘藻株间存有较高的遗传多样性。探讨了布朗葡萄藻分子生态学研究尚待解决的问题,并对今后相关研究做了展望。     

不同氮源对布朗葡萄藻生长、总脂和总烃含量的影响

以能源微藻布朗葡萄藻Botryococcus braunii 764和Botryococcus braunii 765为实验材料,采用实验室一次性培养的方法,研究了不同氮源及浓度对其生长、总脂和总烃含量的影响.结果表明,B. braunii 764和B. braunii 765的最适氮源均为硝态氮,且均能够利用硝态氮、亚硝态氮、铵态氮和尿素进行生长,但是不同氮源及其浓度对这两株藻的生长、总脂和总烃含量的影响不同.B. braunii 764生长速度较B. braunii 765缓慢,但是B. braunii 764的总脂和总烃含量均高于B. braunii 765,最高分别达27.61%和34.21%.以硝态氮为氮源时,B. braunii 764的细胞OD值、生物量、总脂和总烃含量分别为1.38、1.81 g/L、27.61%和34.21%,均显著高于其它试验组,而以尿素为氮源时,B. braunii 765的最大OD值为1.87,以硝态氮为氮源时,其生物量(2.15 g/L)和总烃含量(27.89%)最高,而铵态氮对二者生长的促进作用以及总脂和总烃含量的影响不明显.综合考虑,硝态氮是两株葡萄藻较为理想的氮源,而B. braunii 764可以作为一种较有潜力的能源微藻进行开发利用.     

微拟球藻富油藻株筛选及柱状光生物反应器培养评价研究

研究以亲脂性荧光染料BODIPY505/515和流式细胞仪为基础, 从多株诱变海洋微拟球藻(Nannochloropsis oceanica)中筛选到4株候选富油藻株(MT-1,2,3,4), 并利用柱状光生物反应器对诱变株的产油能力进行了综合评价。结果表明, 藻株筛选时最佳BODIPY505/515使用浓度为0.87 μg/mL, 染色时间为10min; 4株诱变株产油性能较野生株有较大提高, 其中MT-4油脂积累达到了干重的66%, 油脂产率比野生型藻株提高了45%, 达到了27.32 mg/(L·d)。4株诱变株的脂肪酸组成合适, 其中C16和C18之和占78%以上, 且主要以饱和脂肪酸和单不饱和脂肪酸为主; 多不饱和脂肪酸只占总脂肪酸的6%—8%, 非常适合生物柴油生产。研究提供了一种针对海洋微拟球藻富油藻株快速、有效的筛选方法, 并以此为基础筛选得到4株极具生物柴油生产潜力的候选藻株, 有望用于规模化生产。     

一株产油微藻的筛选及分子鉴定

利用通气培养系统, 对10株经过初步生长筛选的微藻进行培养, 以总脂单位体积产率为主要指标, 筛选具有产油潜力的优良藻种。结果表明, 10株微藻的生物质干重、总脂含量分别为0.81.6 g/L、14.8%39.7%, 总脂单位体积产率大于30 mg/(Ld)的有6株, 其中藻株HY-6总脂单位体积产率达到最高的50.8 mg/(Ld), 是一株具有潜力的产油微藻。运用形态学特征和18S rDNA及ITS系统学分析相结合的方法对藻种HY-6进行分类鉴定。依据形态学特征, HY-6为球状单细胞, 具有1个明显的蛋白核, 杯状色素体周生, 从而初步判断该藻株可能属于小球藻属(Chlorella)或拟小球藻属(Parachlorella); 18S rDNA及ITS系统学分析表明HY-6与小球藻属分为两个不同的进化支, 但与凯氏拟小球藻(Parachlorella kessleri)的亲缘关系较近, 且具有较高的自展支持率, 因此将其鉴定为凯氏拟小球藻(P. kessleri)。研究结果将为产油微藻资源的收集、筛选及后续研究提供基础。       

富营养废水中碳氮磷浓度对布朗葡萄藻总脂和总烃的影响

以经过二次过滤的富营养化鱼塘养殖污水为培养液,添加外源的碳、氮、磷元索,研究了污水中不同的外源无机碳、总氮和总磷浓度对布朗葡萄藻(Botryococcus braunii)生物量、总脂和总烃含量的影响.结果表明:(1)以NaHCO3作为碳源,布朗葡萄藻的生物量和总脂含量在外源无机碳浓度为5～10 mg/L时最高,总烃含量在外源无机碳浓度为15mg/L时最高.(2)以KNO3作为氮源,布朗葡萄藻的生物量在总氮浓度为15mg/L时最高,总脂含量在总氮浓度为2mg/L时最高,总烃含量在总氮浓度为20mg/L时最高.(3)以KH2 PO4作为磷源,布朗葡萄藻生物量在总磷浓度为2mg/L时最高,总脂含量和总烃含量在总磷浓度为1.5 mng/L时最高.     

富碳培养对海洋富油微藻油脂积累特性的影响

实验针对三株海洋富油微藻:球等鞭金藻（Isochrysis galbana CCMM5001）、一种等鞭金藻（Isochrysis sp. CCMM5002）和一种微拟球藻（Nannochloropsis sp. CCMM7001）,研究了它们在通入0.03%（空气）、5%、10%三个浓度CO2培养条件下的生长特性,同时考察了其总油脂及中性脂的累积情况。结果显示,富碳培养有利于这三株海洋微藻的生长,但最适生长的CO2浓度不同。球等鞭金藻（Isochrysis galbana CCMM5001）和等鞭金藻（Isochrysis sp. CCMM5002）在通入10% CO2时具有最大产率,分别达到（182.287.07） mg/（Ld）和（164.227.10） mg/（Ld）,而微拟球藻在通入5%时具有最大产率,达到（122.251.17） mg/（Ld）,随着CO2浓度的增加,三株海洋微藻的总脂含量和中性脂含量有明显提高。在通入10% CO2条件下,球等鞭金藻（Isochrysis galbana CCMM5001）、等鞭金藻（Isochrysis sp. CCMM5002）和微拟球藻（Nannochloropsis sp. CCMM7001）的总脂含量分别达到（45.154.03）%、（47.151.20）%和（41.201.69）%;从中性脂的累积规律来看,三株藻均在平台期的累积达到最大,脂肪酸分析结果表明三株藻种适合制备生物柴油的C14-C18系脂肪酸相对含量在不同CO2条件下基本保持不变,维持在90%左右。实验结果显示,研究的藻株作为富油高固碳优良藻株,具备用于海洋生物质能耦合CO2减排开发的潜力。       

布朗葡萄藻：从二氧化碳固定到烷烃、烯烃与萜烯的生物合成

高效生物生产碳氢化合物是解决石油等液体燃料短缺的有效手段之一,而微藻油是生产可持续生物燃料的可靠选择。布朗葡萄藻(Botryococcus braunii)是一种由单细胞组成的不定形群体绿藻,能够积累大量碳氢化合物,最高含量可达其干重的75%,因而受到广泛关注。近年来随着对葡萄藻生物学特性和生长生理的不断深入研究,提高了其规模化培养及其碳氢化合物工业化生产的可行性。从生物学特性、碳氢化合物合成途径及调控因子、多组学研究和规模化培养技术几方面简单叙述了布朗葡萄藻作为新型产油微藻生产碳氢化合物的潜力,为探索利用布朗葡萄藻大规模工业化生产生物燃料提供参考,从而加速该微藻资源的开发利用。     

Specificity of Lipid Composition in Two Botryococcus Strains,the Producers of Liquid Hydrocarbons

The structure of liquid hydrocarbons and fatty acids produced by the green alga Botryococcus was identified. Two representatives of this alga, Botryococcus braunii Kütz, strain IPPAS H-252, introduced into culture earlier and an organism isolated for the first time from the Shira Lake, were used for this identification. Fatty acid composition of B. braunii, strain H-252, lipids was characterized by a high content of trienoic acids of C16–C18 series. The hydrocarbon composition of this strain was represented by straight-chain and branched-chain C14–C28 components; long-chain linear aliphatic C20–C27 hydrocarbons (54.4%) and 2,6,10,14-tetramethylhexadecane (20.5%) predominated among them. The strain H-252 differed in its fatty acid and hydrocarbon composition from the strains described earlier as Botryococcus braunii. The fatty acid composition of the Botryococcus isolate was represented mainly by C12–C32 saturated and monoenoic acids. The hydrocarbons formed by this isolate were represented by dienoic and trienoic components. C29 (48.9–56.3%) and C31 (11.1–16.3%) hydrocarbons predominated among the C23–C31 dienoic hydrocarbons, and C27, C29, and C31 trienoic hydrocarbons comprised 2.5–2.6% of total hydrocarbons. This type of hydrocarbons and the lipid fatty acid composition were characteristic for the race A of B. braunii.     

几株丛粒藻烃类的气相色谱—质谱分析

用气相色谱-质谱分析定性鉴定了三株不同来源的丛粒藻(Botryococcus braunii)产生的烃类组分。丛粒藻株A(USA)和B(Germany)产生以C_nH_(2n-2)和C_nH_(2n-4)为主的烯基直链烃,丛粒藻C得自我国云南省抚仙湖,产生以丛粒藻烯和异丛粒藻烯为主的多分枝烃。讨论了不同丛粒藻产生不同烃类的原因。     

Hydrocarbon productivities in different <Emphasis Type="Italic">Botryococcus</Emphasis> strains: comparative methods in product quantification

Six different strains of the green microalgae Botryococcus belonging to the A-race or B-race, accumulating alkadiene or botryococcene hydrocarbons, respectively, were compared for biomass and hydrocarbon productivities. Biomass productivity was assessed gravimetrically upon strain growth in the laboratory under defined conditions. Hydrocarbon productivities were measured by three different and independent experimental approaches, including density equilibrium of the intact cells and micro-colonies, spectrophotometric analysis of hydrocarbon extracts, and gravimetric quantitation of eluted hydrocarbons. All three hydrocarbon-quantitation methods yielded similar results for each of the strains examined. The B-race microalgae Botryococcus braunii var. Showa and Kawaguchi-1 constitutively accumulated botryococcene hydrocarbons equivalent to 30% and 20%, respectively, of their overall biomass. The A-race microalgae Botryococcus braunii, varieties Yamanaka, UTEX 2441 and UTEX LB572 constitutively accumulated alkadiene hydrocarbons ranging from 14% to 13% and 10% of their overall biomass, respectively. Botryococcus sudeticus (UTEX 2629), a morphologically different green microalga, had the lowest hydrocarbon accumulation, equal to about 3% of its overall biomass. Results validate the density equilibrium and spectrophotometric analysis methods in the quantitation of botryococcene-type hydrocarbons. These analytical advances will serve in the screening and selection of B. braunii and of other microalgae in efforts to identify those having a high hydrocarbon content for use in commercial applications.     

Effect of salinity on growth of green alga Botryococcus braunii and its constituents

Growth of Botryococcus braunii (race 'A') and production of its constituents viz, hydrocarbon, carbohydrate, fatty acid, and carotenoids were influenced by different levels of salinity. Under salinity at 34 mM and 85 mM, 1.7-2.25-fold increase in the relative proportion of palmitic acid and two fold increase in oleic acid were observed. A twofold increase in carotenoid content was noticed at 85 mM salinity with lutein (75% of total carotenoid) as the major carotenoid followed by beta-carotene. The increase in biomass yields and changes in other constituents indicated the influence of salinity and the organism's adaptability to the tested levels of salinity (17 mM to 85 mM).     

Influence of CO2 on growth and hydrocarbon production in Botryococcus braunii

Botryococcus braunii is a green colonial fresh water microalga and it is recognized as one of the renewable resources for production of liquid hydrocarbons. CFTRI-Bb-1 and CFTRI-Bb-2 have been reported for the first time and their performance with regard to growth and biochemical profile is presented here. The present study focused on effect of carbon dioxide (CO2) on biomass, hydrocarbon, carbohydrate production, fatty acid profile, and carotenoid content in various species of B. braunii (LB-572, SAG 30.81, MCRC-Bb, N-836, CFTRI-Bb-1, and CFTRI-Bb-2) at 0.5, 1.0, and 2.0% (v/v) levels using a two-tier flask. CO2 at 2.0% (v/v) level enhanced growth of the organism, and a two-fold increase in biomass and carotenoid contents was observed in all the B. braunii strains studied compared with control culture (without CO2 supplementation). At 1% and 2% (v/v) CO2 concentrations, palmitic acid and oleic acid levels increased by 2.5 to 3 folds in one of the strains of B. braunii (LB-572). Hydrocarbon content was found to be above 20% at 2% CO2 level in the B. braunii LB-572, CFTRI-Bb-2, CFTRI-Bb-1, and N-836 strains, whereas it was less than 20% in the SAG 30.81 and MCRC-Bb strains compared with control culture. This culture methodology will provide information on CO2 requirement for growth of algae and metabolite production. B. braunii spp. can be grown at the tested levels of CO2 concentration without much influence on culture pH.     

Effects of incident light intensity and light path length on cell growth and oil accumulation in Botryococcus braunii (Chlorophyta)

Botryococcus braunii was cultured in different light path length under different incident light intensity to investigate the effect of light on alga growth as well as hydrocarbon and fatty acid accumulation. Results indicated that longer light path length required higher incident light intensity in order to meet the light requirement of algal growth and hydrocarbon accumulation during the course of cultivation. However, hydrocarbon profile was only affected by the incident light intensity and not influenced by the light path length. High incident light intensity enhanced the accumulation of hydrocarbons with longer carbon chains. Besides, the fatty acid content and profiles were significantly influenced by both incident light intensity and light path. Higher fatty acid content and higher percentage of C18 and monounsaturated fatty acid components were achieved at the higher incident light intensity and lower light path length. Taken together, these results are benefit to improve its biomass and oil productivity through the optimization of light and photobioreactor design.     

CHEMICAL PROFILE OF SELECTED SPECIES OF MICROALGAE WITH EMPHASIS ON LIPIDS1

The lipid profile of seven species of unicellular eukaryotic microalgae grown under controlled conditions was studied with emphasis on the hydrocarbons and the fatty acids as part of a search for oil-producing algae. Green, slow-growing colonies of Botryococcus braunii Kutz contained the highest lipid content of 45% based on the organic weight, with an increase to 55% under nitrogen deficiency and with no effect of sodium chloride stress. Ankistrodesmus sp. Thomas, Dunaliella spp., Isochrysis sp., Nannochloris sp. Thomas, and Nitzschia sp. Chapman contained an average of 25% lipids under nitrogen sufficient conditions. Nitrogen deficiency resulted in significant increase in the lipid content in all species but Dunaliella spp., which produced a higher content of carbohydrates. Significant low amounts of acyclic hydrocarbons were detected only in Botryococcus braunii Kutz and not in the other algae. The major hydrocarbon fractions in nitrogen deficient Botryococcus braunii Kutz, Dunaliella salina Thomas, Isochrysis spp. and Nannochloris sp. Thomas were cyclic and branched polyunsaturated components which were identified as various isoprenoid derivatives. The polar lipid composition of glycolipids and phospholipids of all species investigated was fairly typical of photosynthetic eukaryotic algae. Fatty acid composition was species specific, with changes occurring in the relative amounts of individual acids of cells cultivated under different conditions and growth phases. All species synthesized C14:0, C16.0, C18:1, C18:2 and C18:3 fatty acids; C 16:4 in Ankistrodesmus sp. Thomas; C18:4 and C 22.6 in Isochrysis sp.; C16:2, C16:3 and C20:5 in Nannochloris sp. Thomas; C16:2, C16:3 and C20:5 in Nitzschia sp. Chapman. Nitrogen deficiency and salt stress induced accumulation of C18:1 in all treated species and to a lesser extent in Botryococcus braunii Kutz. The low production of hydrocarbons under optimal growth conditions and the high production of hydrocarbons under limited growth conditions cannot support the notion that microalgae can be utilized as biosolar energy converters for the production of liquid fuel, but point to the availability of a variety of neutral and polar lipid products.