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。     

Studies on batch and continuous cultures of Botryococcus braunii: hydrocarbon production in relation to physiological state, cell ultrastructure, and phosphate nutrition

The growth of the hydrocarbon-rich alga Botryococcus braunii was studied under "air-lift" conditions using batch and continuous cultures. Large variations in the physiological state of B. braunii were achieved in batch cultures and in continuous cultures with various dilution rates. The possible effects of these variations upon hydrocarbons (nature, relative abundance, location, level, productivity) and also on the production of exocellular polysaccharides were examined. The relationships between the physiological state of B. braunii and its hydrocarbon and polysaccharide production were discussed and compared with those generally observed in unicellular algae. The factors giving rise to the transition from high to low productivity stages were considered. To this end we examined, at first, the variations in cell ultrastructure and the resulting degeneration occurring during batch cultures. Afterward the parallel changes in some parameters of the medium (pH, phosphate level) were determined and their possible relationships with B. braunii growth and hydrocarbon production were discussed. The main features of phosphate nutrition in B. braunii and its effects on hydrocarbons were finally examined.     

6 种无机絮凝剂对布朗葡萄藻的絮凝效应

探讨了6种无机絮凝剂对布朗葡萄藻的絮凝效应。采用光密度法测定布朗葡萄藻的生物量,分别研究了硫酸铝、硫酸铝铵、硫酸铝钾、硫酸镁、硫酸铁、氯化铁对布朗葡萄藻的絮凝效应。结果表明,光密度法可应用于布朗葡萄藻生物量的测定,可间接测定絮凝效应,且最佳测定波长为680nm;6种无机絮凝刺的絮凝时间为60min,同时6种无机试剂对布朗葡萄藻均有明显的絮凝效应,硫酸铝、硫酸铝钾、氯化铁对布朗葡萄藻的絮凝效应较硫酸铝铵、硫酸镁、硫酸铁明显（P〈0．05）。硫酸铝浓度控制在1．1g／L,硫酸铝钾浓度控制在0．45g／L以内,氯化铁浓度控制在0．9g／L。     

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。     

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。       

葡萄藻冷冻保藏的研究

为给微藻大规模培养生产生物燃料提供稳定可靠的种质资源,本研究以葡萄藻为研究对象,建立了一套葡萄藻快速高效冷冻保藏的方法.通过对不同冷冻保护剂二甲基亚砜(DMSO)、甲醇(MeOH)、乙二醇(EG)、丙二醇(PG)和甘油(Gly)的毒性测试和冷冻保藏效果的比较,结果表明在以6% MeOH作为冷冻保护剂的条件下葡萄藻的存活...     

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.     

Localization of human gene loci using spontaneous chromosome rearrangements in human-Chinese hamster somatic cell hybrids.

Analysis of human-Chinese hamster somatic cell hybrids with spontaneously derived chromosome structural changes has provided data for the regional and subregional localization of gene loci which have previously been assigned to human chromosomes 2, 12, and X. Correlation of the expression of human gene loci with the human chromosome complements present in somatic cell hybrids indicates that the cytoplasmic malate dehydrogenase (MDH1) locus is in the 2p23yields2pter region, and red cell acid phosphatase (AcP1) is at or adjacent to 2p23. The cytoplasmic isocitrate dehydrogenase (IDH1) locus is at or adjacent to 2q11, peptidase B (Pep B) is at or adjacent to 12q21, lactate dehydrogenase B (LDH B) is in the 12q21yiedls12pter region, glucose-6-phosphate dehydrogenase (G6PD) is in the Xq24yieldsXqter region, and the gene loci for phosphoglycerate kinase (PGK), alpha-galactosidase (alpha-gal), and hypoxanthine guanine phosphoribosyltransferase (GPRT) are in the Xp21yieldsXq24 region.     

Botryococcus braunii: a renewable source of hydrocarbons and other chemicals

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.     

Simple, rapid and cost-effective method for high quality nucleic acids extraction from different strains of Botryococcus braunii

This study deals with an effective nucleic acids extraction method from various strains of Botryococcus braunii which possesses an extensive extracellular matrix. A method combining freeze/thaw and bead-beating with heterogeneous diameter of silica/zirconia beads was optimized to isolate DNA and RNA from microalgae, especially from B. braunii. Eukaryotic Microalgal Nucleic Acids Extraction (EMNE) method developed in this study showed at least 300 times higher DNA yield in all strains of B. braunii with high integrity and 50 times reduced working volume compared to commercially available DNA extraction kits. High quality RNA was also extracted using this method and more than two times the yield compared to existing methods. Real-time experiments confirmed the quality and quantity of the input DNA and RNA extracted using EMNE method. The method was also applied to other eukaryotic microalgae, such as diatoms, Chlamydomonas sp., Chlorella sp., and Scenedesmus sp. resulting in higher efficiencies. Cost-effectiveness analysis of DNA extraction by various methods revealed that EMNE method was superior to commercial kits and other reported methods by >15%. This method would immensely contribute to area of microalgal genomics.     

PHYLOGENETIC POSITION OF BOTRYOCOCCUS BRAUNII (CHLOROPHYCEAE) BASED ON SMALL SUBUNIT RIBOSOMAL RNA SEQUENCE DATA1

A small subunit ribosomal RNA (16S-like rRNA) in the hydrocarbon-rich microalga Botryococcus braunii Kützing (Chlorophyceae) was amplified using RNA polymerase chain reaction, and its sequence was determined. The sequence data of B. braunii were analyzed with those of several other algae in order to determine phylogenetic relationships among these algae. Phylogenetic analysis indicated B. braunii to be a member of the Chlorophyta and possibly related to Characium vacuolatum and Dunaliella parva.     

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。     

Botryolins A and B, two tetramethylsqualene triethers from the green microalga Botryococcus braunii

Two new triterpenoid polyethers with a tetramethylsqualene carbon skeleton, botryolins A and B, have been isolated from the green microalga Botryococcus braunii. Their structures were determined by means of spectral analyses including 2D NMR.     

Rapid method for the determination of lipid from the green alga Botryococcus braunii

Of various methods for lipid recovery in Botryococcus braunii UTEX 572, the most effective method was disruption of the cells with a bead-beater followed by extraction with chloroform/methanol (2:1, v/v). This gave a lipid content of 28.6% of dry wt. There was a significant relationship between in vivo fluorescence of cells stained with Nile Red and lipid content in B. braunii determined gravimetrically (r2 = 0.997). This suggested that the Nile Red staining as a rapid method was as good as the gravimetric method commonly used for lipid determination which requires toxic solvents and considerable time-consuming manipulations. © Rapid Science Ltd. 1998     

Hydrocracking of the oils of Botryococcus braunii to transport fuels

Hydrocarbon oils of the alga Botryococcus braunii, extracted from a natural "bloom" of the plant, have been hydrocracked to produce a distillate comprising 67% gasoline fraction, 15% aviation turbine fuel fraction, 15% diesel fuel fraction, and 3% residual oil. The distillate was examined by a number of standard petroleum industry test methods. This preliminary investigation indicates that the oils of B. braunii are suitable as a feedstock material for hydrocracking to transport fuels.     

Isolation and expression of the gene encoding yeast mitochondrial malate dehydrogenase.

The mitochondrial tricarboxylic acid cycle enzyme malate dehydrogenase was purified from Saccharomyces cerevisiae, and an antibody to the purified enzyme was obtained in rabbits. Immunoscreening of a yeast genomic DNA library cloned into a lambda gt11 expression vector with anti-malate dehydrogenase immunoglobulin G resulted in identification of a lambda recombinant encoding an immunoreactive beta-galactosidase fusion protein. The yeast DNA portion of the coding region for the fusion protein translates into an amino acid sequence which is very similar to carboxy-terminal sequences of malate dehydrogenases from other organisms. In s. cerevisiae transformed with a multicopy plasmid carrying the complete malate dehydrogenase gene, the specific activity and immunoreactivity of the mitochondrial isozyme are increased by eightfold. Expression of both the chromosomal and plasmid-borne genes is repressed by growth on glucose. Disruption of the chromosomal malate dehydrogenase gene in haploid S. cerevisiae produces mutants unable to grow on acetate and impaired in growth on glycerol plus lactate as carbon sources.     

利用烟道气培养产烃葡萄藻的可行性研究

对烟道气培养产烃葡萄藻的可行性进行了研究,重点考察了SO2和NOx的水溶形态对葡萄藻的影响。结果表明,亚硫酸氢盐浓度低于0．8mmol／L时,对葡萄藻生长没有明显抑制作用,可以提供葡萄藻生长的硫源,但高浓度(大于2mmol／L)时抑制细胞生长;当亚硝酸盐浓度小于8mmol／L时,可以作为葡萄藻生长的唯一氮源,亚硝酸盐的去除主要是由微藻利用所致。当起始浓度为2mmol／L和4mmol／L时,亚硝酸盐的去除率分别为100％和99．7％。     

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.