University of California, Dept. of Plant and Microbial Biology, Berkeley, CA 94720-3102, USA.
Bioresour Technol. 2010 Apr;101(7):2359-66. doi: 10.1016/j.biortech.2009.11.043. Epub 2009 Dec 14.
Mechanical fractionation and aqueous or aqueous/organic two-phase partition approaches were applied for extraction and separation of extracellular terpenoid hydrocarbons from Botryococcus braunii var. Showa. A direct spectrophotometric method was devised for the quantitation of botryococcene and associated carotenoid hydrocarbons extracted by this method. Separation of extracellular botryococcene hydrocarbons from the Botryococcus was achieved upon vortexing of the micro-colonies with glass beads, either in water followed by buoyant density equilibrium to separate hydrocarbons from biomass, or in the presence of heptane as a solvent, followed by aqueous/organic two-phase separation of the heptane-solubilized hydrocarbons (upper phase) from the biomass (lower aqueous phase). Spectral analysis of the upper heptane phase revealed the presence of two distinct compounds, one absorbing in the UV-C, attributed to botryococcene(s), the other in the blue region of the spectrum, attributed to a carotenoid. Specific extinction coefficients were developed for the absorbance of triterpenes at 190nm (epsilon = 90 +/- 5 mM(-1) cm(-1)) and carotenoids at 450 nm (epsilon=165+/-5mM(-1) cm(-1)) in heptane. This enabled application of a direct spectrophotometric method for the quantitation of water- or heptane-extractable botryococcenes and carotenoids. B. braunii var. Showa constitutively accumulates approximately 30% of the dry biomass as extractable (extracellular) botryococcenes, and approximately 0.2% of the dry biomass in the form of a carotenoid. It was further demonstrated that heat-treatment of the Botryococcus biomass substantially accelerates the rate and yield of the extraction process. Advances in this work serve as foundation for a cyclic Botryococcus growth, non-toxic extraction of extracellular hydrocarbons, and return of the hydrocarbon-depleted biomass to growth conditions for further product generation.
机械分级和水相或水/有机两相分配方法被应用于从 Showa 变种的 Botryococcus braunii 中提取和分离细胞外萜类碳氢化合物。设计了一种直接分光光度法来定量提取的 botryococcene 和相关类胡萝卜素碳氢化合物。通过在水中涡旋微菌落，然后通过浮力密度平衡将碳氢化合物与生物质分离，或者在正庚烷作为溶剂的存在下，将细胞外 botryococcene 碳氢化合物从 Botryococcus 中分离出来，从而实现了细胞外 botryococcene 碳氢化合物的分离。正庚烷可溶解的碳氢化合物（上层）与生物质（下层水相）的水/有机两相分离。上层正庚烷相的光谱分析表明存在两种不同的化合物，一种在 UV-C 区域吸收，归因于 botryococcene(s)，另一种在光谱的蓝色区域吸收，归因于类胡萝卜素。在正庚烷中开发了三萜类物质在 190nm 处的吸光度的特定消光系数（epsilon = 90 +/- 5 mM(-1) cm(-1)）和类胡萝卜素在 450nm 处的吸光度的特定消光系数（epsilon=165+/-5mM(-1) cm(-1)）。这使得可以应用直接分光光度法来定量水相或正庚烷可提取的 botryococcenes 和类胡萝卜素。Showa 变种的 B. braunii 大约以可提取（细胞外）botryococcenes 的形式积累约 30%的干生物质，并且以约 0.2%的干生物质的形式积累类胡萝卜素。进一步证明，Botryococcus 生物质的热处理大大加速了提取过程的速度和产率。这项工作的进展为 Botryococcus 的循环生长、细胞外碳氢化合物的无毒提取以及烃耗尽的生物质返回生长条件以进一步产生产物奠定了基础。
