一株葡萄藻的分离、鉴定及产烃性能评价

研究从我国海南博鳌海边的淡水池塘水样分离获得一株绿藻ENN41。显微形态观察表明,ENN41的形态特征属于葡萄藻。进一步克隆ENN41的核糖体小亚基18S rRNA片段,利用分子生物学软件进行比对分析,结果表明ENN41的18S rRNA基因序列与布朗葡萄藻（Botryococcus braunii）同源性最高,说明ENN41为一株布朗葡萄藻（B.braunii）。ENN41在柱式反应器中培养16d,单位体积产率为0.483 g/（Ld）,粗烃占干重的含量为56.6%;主要脂肪酸为油酸（C18:1）、十八碳四烯酸（C18:4）和棕榈酸（C16:0）,三者之和占总脂肪酸的72.6%;利用尼罗红染色,清晰可见大量的油脂分布在细胞内和胞外基质中。ENN41在板式反应器中培养16d,单位体积产率为0.234 g/（Ld）,粗烃占干重的含量为20.0%。上述研究表明,ENN41是具有较高的生长速度和粗烃积累能力的布朗葡萄藻（B.braunii）藻株,具有产业化应用潜力。     

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.     

串珠藻目分子系统学研究进展

串珠藻目(Batrachospermales)是淡水红藻中最主要的类群。近年来, 应用DNA序列分析探讨串珠藻目的系统发育, 并与传统的形态学和生态学特征相结合, 为串珠藻目系统学研究拓展了新的思路。本文回顾了串珠藻目的建立及其所含类群的研究历史, 归纳了目前在串珠藻目系统发育与进化研究中常用的分子标记方法, 其中包括核基因组的18S rDNA、26S rDNA和ITS序列, 叶绿体基因组的rbcL序列, 线粒体基因组的cox2-3序列, 以及新兴的ISSR技术, 并对各种分子标记的特点及适用范围做了评述。结果表明, ITS序列多适用于种群分化及相近种间遗传分析, ISSR标记适用于种下分类群间及同一种群不同个体间基因多态性分析, cox2-3序列在一定程度上也可用于同一种群不同个体间的基因多态性分析, 而18S rDNA 与rbcL序列既可用于种间关系分析, 又可用于更高水平分析的构建系统树。这些分子标记已被证明在研究串珠藻目系统地理、物种起源和散布机制方面有着广泛的应用前景。同时, 本文对串珠藻目分子系统学研究的最新进展也进行了概述,并对今后的研究方向做了展望。     

布朗葡萄藻转录组研究进展

布朗葡萄藻是一种能够在细胞内积累多种油脂和碳氢化合物的绿色微藻,根据其积累的不同碳氢化合物,可以将其分为A、B和L型3个主要化学种,最近也有发现其存在S亚型。之前对葡萄藻的研究大多都围绕其培养优化、产物分离及品系筛选等方面展开。伴随着高通量测序的发展,通过转录组测序可以一次获得个体内大量未知基因并对其进行注释及定量分析,完备的转录组数据可以为后续的基因分析提供大量的可用信息。本综述主要介绍了布朗葡萄藻各化学种的碳氢化合物及其合成途径、已报道的A、B和L型转录组研究,并且对未来基于基因组的葡萄藻转录组分析进行了探讨。     

串珠藻目分子系统学研究进展

串珠藻目（Batrachospermales）是淡水红藻中最主要的类群。近年来,应用DNA序列分析探讨串珠藻目的系统发育,并与传统的形态学和生态学特征相结合,为串珠藻目系统学研究拓展了新的思路。本文回顾了串珠藻目的建立及其所含类群的研究历史,归纳了目前在串珠藻目系统发育与进化研究中常用的分子标记方法,其中包括核基因组的18SrDNA、26SrDNA和ITS序列,叶绿体基因组的rbcL序列,线粒体基因组的cox9．-3序列,以及新兴的ISSR技术,并对各种分子标记的特点及适用范围做了评述。结果表明,ITS序列多适用于种群分化及相近种间遗传分析,ISSR标记适用于种下分类群间及同一种群不同个体间基因多态性分析,cox2-3序列在一定程度上也可用于同一种群不同个体间的基因多态性分析,而18SrDNA与rbcLFF列既可用于种问关系分析,又可用于更高水平分析的构建系统树。这些分子枥对己已被证明在研究串珠藻目系统地理、物种起源和散布机制方面有着广泛的应用前景。同时,本文对串珠藻目分子系统学研究的最新进展也进行了概述,并对今后的研究方向做了展望。     

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。     

紫芝基因组rDNA序列特征及ITS2二级结构比较

紫芝栽培品种‘紫芝S2’(武芝2号)的ITS序列与NCBI数据库中5个紫芝菌株/分离株相似度高达99.79%-100%,在系统进化树上相聚成一类。本研究预测‘紫芝S2’基因组与参考基因组中的rRNA基因簇,分析rDNA结构及各构件序列间的多态性。从高质量‘紫芝S2’基因组中挖掘得到完整rDNA,序列全长40.377 kb,由4组串联重复的(18S、5.8S、28S、5S) rRNA基因簇组成,并含有完整的基因内间隔区(ITS1、ITS2)和基因间间隔区(IGS1、IGS2)。在紫芝S2的rDNA中,高度保守的28S rRNA基因间出现3个SNP和2个插入(1 bp,10 bp)位点;虽然第4条ITS2中有1个SNP位点,但紫芝S2的4条ITS2在二级结构上的分子形态高度一致,与ITS2数据库中其他紫芝菌株仅存在螺旋区间夹角的微小差异。由‘紫芝S2’基因组rDNA的ITS2生成的DNA条形码与二维码,可以作为该栽培品种鉴定与同源物种其他菌株鉴别的分子标记。     

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.     

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。     

基于核及叶绿体基因序列探讨中国绿水螅共生单细胞绿藻系统发生地位

研究对中国绿水螅共生绿藻的核18S rRNA基因全长序列及其叶绿体9个基因(atpA、chlB、chlN、petA、psaB、psbA、psbC、psbD及rbcL)片段序列进行了克隆和测序, 并基于18S rRNA基因序列及叶绿体9个基因序列的整合数据分别通过最大似然法(Maximum-likelihood)和贝叶斯分析(Bayesian inference)对中国绿水螅(Hydra sinensis)共生单细胞绿藻的系统发生地位进行了探讨。系统发生表明: (1)中国绿水螅共生绿藻属于共球藻纲(Trebouxiophyceae)小球藻目(Chlorellales), 但不属于其中的小球藻属(Chlorella); (2)来源于草履虫、水螅、地衣及银杏的共生绿藻均在共球藻纲支系, 而来源于蛙类和蝾螈的共生绿藻属于绿藻纲(Chlorophyceae)支系。无论在共球藻纲支系还是在绿藻纲支系, 不同来源的共生藻并没有排他性地聚为单系群而在系统树中与其他自由生活的绿藻混杂排列, 来自不同宿主的共生绿藻没有共同起源。     

Genome size and phylogenetic analysis of the A and L races of <Emphasis Type="Italic">Botryococcus braunii</Emphasis>

Botryococcus braunii (Chlorophyta, Botryococcaceae) is a colony-forming green microalga that produces large amounts of liquid hydrocarbons, which can be converted into transportation fuels. There are three different races of B. braunii, A, B, and L, that are distinguished based on the type of hydrocarbon each produces. Each race also has many strains that are distinguished by the location from which they were collected. While B. braunii has been well studied for the chemistry of the hydrocarbon production, very little is known about the molecular biology of B. braunii. To begin to address this problem, we determined the genome size of the A race, Yamanaka strain, and the L race, Songkla Nakarin strain, of B. braunii. Flow cytometry analysis indicates that the A race of B. braunii has a genome size of 166.0 ± 0.4 Mb, while the L race has a substantially larger genome size at 211.3 ± 1.7 Mb. We also used phylogenetic analysis with the nuclear small subunit (18S) rRNA gene to classify strains of the A and B races that have not yet been compared evolutionarily to previously published B. braunii phylogenetics. The analysis suggests that the evolutionary relationship between B. braunii races is correlated with the type of liquid hydrocarbon they produce.     

PHYLOGENETIC PLACEMENT,GENOME SIZE,AND GC CONTENT OF THE LIQUID‐HYDROCARBON‐PRODUCING GREEN MICROALGA BOTRYOCOCCUS BRAUNII STRAIN BERKELEY (SHOWA) (CHLOROPHYTA)1

We report the genome size and the GC content, and perform a phylogenetic analysis on Botryococcus braunii Kütz., a green, colony‐forming, hydrocarbon‐rich alga that is an attractive source for biopetroleum. While the chemistry of the hydrocarbons produced by the B race of B. braunii has been studied for many years, there is a deficiency of information concerning the molecular biology of this alga. In addition, there has been some discrepancy as to the phylogenetic placement of the Berkeley (or Showa) strain of the B race. To clarify its classification, we isolated the Berkeley strain nuclear SSU (18S) rRNA gene and β‐actin cDNA and used these sequences for phylogenetic analysis to determine that the Berkeley strain belongs to the Trebouxiophyceae class. This finding is in agreement with other B races of B. braunii, indicating the Berkeley strain is a true B race of B. braunii. To better understand molecular aspects of B. braunii, we obtained the Berkeley strain genome size as a first step in genome sequencing. Using flow cytometry, we determined the B. braunii Berkeley genome size to be 166.2 ± 2.2 Mb. We also estimated the GC content of the Berkeley strain as 54.4 ± 1.2% for expressed gene sequences.     

Alkadiene- and botryococcene-producing races of wild strains of Botryococcus braunii

Samples of the green colonial alga Botryococcus braunii, collected from various localities, were grown in the laboratory and examined for their hydrocarbon content and morphology. Although few differences appeared between the ultrastructures of the samples, the nature of their hydrocarbons, which remains unchanged at any stage of growth, allows the distinction of two physiological races viz algae producing odd-numbered unbranched alkadienes and trienes (C₂₅C₃₁) (the A race) and those producing polymethylated triterpenes C_nH_2n-₁₀ (C₃₀C₃₇), the botryococcenes (the B race). In laboratory culture, the hydrocarbon content of these new strains is very high, from 30 to 60% of the dry biomass. For the two races the greatest hydrocarbon productivity takes place during the active growth phase. The important variability observed in botryococcene distribution could originate both from genetic and environmental factors.     

STRUCTURE AND CHEMISTRY OF A NEW CHEMICAL RACE OF BOTRYOCOCCUS BRAUNII (CHLOROPHYCEAE) THAT PRODUCES LYCOPADIENE,A TETRATERPENOID HYDROCARBON1

New strains of the hydrocarbon rich alga Botryococcus braunii Kützing were isolated from water samples collected in three tropical freshwater lakes. These strains synthesize lycopadiene, a tetraterpenoid metabolite, as their sole hydrocarbon. The morphological and ultrastructural characteristics of these algae are similar to those reported for previously described strains which produce either alkadienes or botryococcenes. The pyriform shaped cells are embedded in a colonial matrix formed by layers of closely appressed external walls: this dense matrix is impregnated by the hydrocarbon and some other lipids. We believe the new strains synthesizing lycopadiene form a third chemical race in B. braunii, besides the alkadiene and botryococcene races, rather than a different species. Like the other two types of hydrocarbons, lycopadiene was produced primarily during the exponential and linear growth phases. The major fatty acid in the three races was oleic acid. This fatty acid was predominant in the alkadiene race; palmitic and octacosenoic acid also were present in appreciable amounts in the three races. Cholest-5-en-3β-ol, 24-methylcholest-5-en-3β-ol and 24-ethylcho-lest-5-en-3β-ol occurred in the three races; three unidentified sterols also were detected in the lycopadiene race. Moreover, the presence of very long chain alkenyl-phenols in the lipids of algae of the alkadiene race was not observed in the botryococcene and lycopadiene races. Of the polysaccharides released in the medium, galactose appeared as a primary component: it predominated in the botryococcene race. The other major constituents were fucose for the alkadiene race and glucose and fucose for the lycopadiene race. Although morphologically similar, some important chemical differences exist among algae classified as B. braunii.     

Culture of the green microalga Botryococcus braunii Showa with LED irradiation eliminating violet light enhances hydrocarbon production and recovery

The green microalga Botryococcus braunii (B. braunii), race B, was cultured under light-emitting diode (LED) irradiation with and without violet light. This study examined the effect of violet light on hydrocarbon recovery and production in B. braunii. C34 botryococcene hydrocarbons were efficiently extracted by thermal pretreatments at lower temperatures when the alga was cultured without violet light. The hydrocarbon content was also higher (approximately 3%) in samples cultured without violet light. To elucidate the mechanism of effective hydrocarbon recovery and production, we examined structural components of the extracellular matrix (ECM). The amounts of extracellular carotenoids and water-soluble polymers extracted by thermal pretreatment from the ECM were decreased when the alga was cultured without violet light. These results indicate that LED irradiation without violet light is more effective for hydrocarbon recovery and production in B. braunii. Furthermore, structural ECM components are closely involved in hydrocarbon recovery and production in B. braunii.     

PHYLOGENETIC PLACEMENT OF BOTRYOCOCCUS BRAUNII (TREBOUXIOPHYCEAE) AND BOTRYOCOCCUS SUDETICUS ISOLATE UTEX 2629 (CHLOROPHYCEAE)1

The phylogenetic placement of four isolates of Botryococcus braunii Kützing and of Botryococcus sudeticus Lemmermann isolate UTEX 2629 was investigated using sequences of the nuclear small subunit (18S) rRNA gene. The B. braunii isolates represent the A (two isolates), B, and L chemical races. One isolate of B. braunii (CCAP 807/1; A race) has a group I intron at Escherichia coli position 1046 and isolate UTEX 2629 has group I introns at E. coli positions 516 and 1512. The rRNA sequences were aligned with 53 previously reported rRNA sequences from members of the Chlorophyta, including one reported for B. braunii (Berkeley strain). Phylogenetic trees were constructed using distance, weighted maximum parsimony, and maximum likelihood, and their reliability was estimated using bootstrap analysis for distance and parsimony and Bayesian inference for likelihood. All methods showed, with high bootstrap or credibility support, that the four isolates of B. braunii form a monophyletic group whose closest relatives are in the genus Choricystis in the Trebouxiophyceae, whereas the previously reported B. braunii sequence is from a member of the Chlamydomonadales in the Chlorophyceae and isolate UTEX 2629 is a member of the Sphaeropleales in the Chlorophyceae. Polyphyly of these sequences was confirmed by Kishino‐Hasegawa tests on artificial trees in which sequences were moved to a single lineage.     

Hydrocarbon composition of newly isolated strains of the green microalga Botryococcus braunii

Four new strains of Botryococcus braunii were isolated from Japanese waters and cultured under defined conditions. Their hydrocarbon content and composition were analyzed and compared with those of the Darwin and Berkeley strains. The Yamanaka strain produced only alkadienes characteristic of the A race, whereas the others, the Yayoi, Kawaguchi-1 and -2 strains as well as the Darwin and Berkeley strains, produced botryococcenes peculiar to the B race. The hydrocarbon content of the Yamanaka strain was 16.1 % dry weight and that of the B race strains ranged from 9.7 to 37.9%. Botryococcene composition of the Japanese strains differed from each other as well as from the Darwin and Berkeley strains. More than 50% of the hydrocarbons in the Yayoi, Darwin, and Berkeley strains were composed of C₃₄H₅₈, but the main components were different from one another as isomers. The Kawaguchi-1 and -2 strains did not have a high level of C₃₄ botryococcenes, C₃₂ ones being the main components. In these strains significant amounts of squalene-related compounds were detected.     

Transformation of Lipid Bodies Related to Hydrocarbon Accumulation in a Green Alga,Botryococcus braunii (Race B)

The colonial microalga Botryococcus braunii accumulates large quantities of hydrocarbons mainly in the extracellular space; most other oleaginous microalgae store lipids in the cytoplasm. Botryococcus braunii is classified into three principal races (A, B, and L) based on the types of hydrocarbons. Race B has attracted the most attention as an alternative to petroleum by its higher hydrocarbon contents than the other races and its hydrocarbon components, botryococcenes and methylsqualenes, both can be readily converted into biofuels. We studied race B using fluorescence and electron microscopy, and clarify the stage when extracellular hydrocarbon accumulation occurs during the cell cycle, in a correlation with the behavior and structural changes of the lipid bodies and discussed development of the algal colony. New accumulation of lipids on the cell surface occurred after cell division in the basolateral region of daughter cells. While lipid bodies were observed throughout the cell cycle, their size and inclusions were dynamically changing. When cells began dividing, the lipid bodies increased in size and inclusions until the extracellular accumulation of lipids started. Most of the lipids disappeared from the cytoplasm concomitant with the extracellular accumulation, and then reformed. We therefore hypothesize that lipid bodies produced during the growth of B. braunii are related to lipid secretion. New lipids secreted at the cell surface formed layers of oil droplets, to a maximum depth of six layers, and fused to form flattened, continuous sheets. The sheets that combined a pair of daughter cells remained during successive cellular divisions and the colony increased in size with increasing number of cells.     

Botryococcus braunii: A Renewable Source of Hydrocarbons and Other Chemicals

ABSTRACT:?

Botryococcus braunii, a green colonial microalga, is an unusually rich renewable source of hydrocarbons and other chemicals. Hydrocarbons can constitute up to 75% of the dry mass of B. braunii. This review details the various facets of biotechnology of B. braunii, including its microbiology and physiology; production of hydrocarbons and other compounds by the alga; methods of culture; downstream recovery and processing of algal hydrocarbons; and cloning of the algal genes into other microorganisms. B. braunii converts simple inorganic compounds and sunlight to potential hydrocarbon fuels and feedstocks for the chemical industry. Microorganisms such as B. braunii can, in the long run, reduce our dependence on fossil fuels and because of this B. braunii continues to attract much attention.     

BACTERIAL INFLUENCE UPON GROWTH AND HYDROCARBON PRODUCTION OF THE GREEN ALGA BOTRYOCOCCUS BRAUNII1

Batch cultures of the hydrocarbon-rich alga Botryococcus braunii, Kütz. (axenic strains, non-axenic strains, associations with selected microorganisms) were examined with regard to total biomass and hydrocarbons at the onset of the stationary phase. Pronounced variations, related to the origin of the strains and to growth conditions, were observed with axenic cultures. It also appeared that the presence of microorganisms is not essential for high hydrocarbon production. Nevertheless, numerous bacteria were shown to exert considerable influence, antagonistic or beneficial, on B. braunii growth yield and hydrocarbon production. Such effects were strongly dependent on the species involved and on culture conditions. The presence of various microorganisms can influence not only the quantity of hydrocarbons produced, but also their level in the algal biomass and their relative abundance. However, their chemical structure is not affected. Intricate relationships were observed in B. braunii-bacteria systems and numerous factors (including, in some cultures, large positive effects due to bacterially produced CO₂) were implicated. Accordingly, specific associations should provide appropriate conditions for renewable hydrocarbon production via B. braunii large scale cultures.