丛粒藻形态多样性、遗传多样性分析与培养条件优化
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摘要
丛粒藻(Botry coccus braunii)能够大量合成并积累烃类物质,总烃含量一般可占细胞干重的25%-40%,最高可达76%,其组分和结构与石油十分接近,燃烧后产热值高。因此,丛粒藻是一种具有潜在应用价值的能源微藻。
本文对3株采集自不同地点的丛粒藻(AGB-Bb01、AGB-Bb02和AGB-Bb03)进行了显微结构和亚显微结构观测,研究了不同藻株间的形态变化。PCR扩增了18S rDNA、ITS和rbcL基因序列,并结合GenBank数据库中已登录的丛粒藻相应序列比较分析了16株丛粒藻藻株的遗传距离和序列相似性,重建了系统发生树。结果表明：采集自不同地理位点的丛粒藻藻株在细胞大小、聚落大小和聚落细胞数目方面存在较明显的差异,亚显微结构显示不同藻株的杯状鞘厚度及细胞的包埋程度也存在差异；不同地理株间具有较高的遗传多样性,系统发生树显示所有藻株可分成2个簇群和4个亚群,存在一定程度的地理隔离。研究证明了18S rDNA和ITS区基因序列是进行丛粒藻基因分型和遗传多样性研究的良好位点,而通过分析3株丛粒藻与分类归属地位有争议的不同目的微藻rbcL基因序列发现,采用邻接法、最小进化法、最大简约法和非加权组平均法构建的系统树均显示,该藻与衣藻目的亲缘关系较近且,这与日本学者用18S rDNA研究的结果相一致,为系统了解丛粒藻的遗传多样性和开展优良藻株选育工作奠定了基础。
要实现丛粒藻产烃的工厂规模化生产,优良藻株的选育和培养条件优化必不可少,总烃、总脂含量高和生长速度快的藻株是筛选的首要目标。分别选取丛粒藻藻株AGB-Bb01、AGB-Bb02和AGB-Bb03处于延滞期、对数生长前期、对数生长后期和稳定期的藻细胞,分别采用正己烷法和索氏提取法进行总烃含量及总脂含量的测定,发现3株丛粒藻在不同的生长时期总烃、总脂含量不同,最高值出现在对数生长后期。丛粒藻在Chu10、Chu13、BG11和BBM这4种培养基中的生长适应情况不同,3株实验微藻在不同培养基中的生长速率、生物量、总烃和总脂含量表现出差别。结果表明：AGB-Bb01在Chu13培养基中的生物量最高,AGB-Bb02和AGB-Bb03在BG11培养基中的生物量最高；AGB-Bb01和AGB-Bb02在Chu10培养基中总烃及总脂含量较高,AGB-Bb03在Chul3培养基中总烃及总脂含量最高,为丛粒藻烃类最佳收获期的选择和工业化大规模养殖提供实验基础。
Botryococcus braunii is considered as an energy microalgae having great protential applications because of that it can synthesize and accumulate considerable quantities of hydrocarbons in the metabolic process. The calorific value, composition and structure of these hydrocarbons are very similar to those of oil. Generally the content of total hydrocarbons could reach 25% to 40% of dry biomass and the maximum value that have reported was 76%.
The microscopic structure and sub-microscopic structure of three B. braunii strains (AGB-Bb01,AGB-Bb02 and AGB-Bb03) isolated from different sites were observed by light and electronic microscope observations based on morphological features. AGB-Bb01, AGB-Bb02 and AGB-Bb03 were identified as different genotype and geographical strains of B. braunii by amplifying the 18S-28S rDNA and rbcL gene sequences and blastn analysis, genetic distance and sequence similarities calculation. And it is the first time that the rbcL gene sequences were obtained from this energy microalgae. The phylogenetic trees involved in 16 strains of B. braunii were reconstructed based on the genetic distances and sequence similarities of 18S rDNA, ITS region and rbcL gene sequences at the same time. The results showed that the cell size, colony size and cell number in one colony of the three geographical strains were significantly different. The sub-microscopic structures demonstrated that the thickness of the cup-sheath and extent of cell embedded by the cup-sheath had difference among the three strains as well. There existed high genetic diversity among the different strains living in the different sites and phylogenetic tree showed that all the strains can be divided into 2 groups and 4 sub-groups because of the geographical isolation. It is proved that sequences of 18S rDNA and ITS region can be considered as good tools for gene-type identification and genetic diversity research in B. braunii. Phylogenetic trees were constructed using Neighbor-joining, Minimum evolution, Maximum parsimony and Unweighted pair-group method with arithmetic method, and their reliability was estimated using bootstrap analysis. All the results show the closest relatives are in the Chlamydomonadales with high bootstrap. The study lays the solid basement for the research of the genetic diversity and cultivation in B. braunii.
It is very necessary to breed algae strains and optimize culture conditions in order to achieve large-scale production. The preferred target is to obtain high-yield varieties which can both grow rapidly and have high content of total hydrocarbons and total lipids. Total hydrocarbons and total lipids of B. braunii AGB-Bb01, AGB-Bb02 and AGB-Bb03 was different in different growth phases. The maximum of those appeared in the post-logarithmic phase after algae cells in the lag phase, the early-logarithmic phase, the post-logarithmic phase and the stationary phase were collected and fragmented for determination. The effect of the media on the growth, contents of total hydrocarbons and total lipids of Botryococcus braunii AGB-Bb01, AGB-Bb02 and AGB-Bb03 was studied using different media such as modified Chu10 medium, modified Chul3 medium,BGll and bold basal medium(BBM). Biomass of B. braunii AGB-Bb01 cultured in Chu13 was higher than that cultured in Chu10, BG11 and BBM, while biomass of B. braunii AGB-Bb02 and AGB-Bb03 cultured in BG11 were higher than those cultured in the other medium respectively;Chu10 was found to be the best medium for total hydrocarbon content and total lipid content of B. braunii AGB-Bb01 and AGB-Bb02, whereas Chul3 is the best for total hydrocarbon content and total lipid content of B. braunii AGB-Bb03. This research provides experimental basis for the selection of the best harvest time and industrial large-scale production of B. braunii.
本文对3株采集自不同地点的丛粒藻(AGB-Bb01、AGB-Bb02和AGB-Bb03)进行了显微结构和亚显微结构观测,研究了不同藻株间的形态变化。PCR扩增了18S rDNA、ITS和rbcL基因序列,并结合GenBank数据库中已登录的丛粒藻相应序列比较分析了16株丛粒藻藻株的遗传距离和序列相似性,重建了系统发生树。结果表明：采集自不同地理位点的丛粒藻藻株在细胞大小、聚落大小和聚落细胞数目方面存在较明显的差异,亚显微结构显示不同藻株的杯状鞘厚度及细胞的包埋程度也存在差异；不同地理株间具有较高的遗传多样性,系统发生树显示所有藻株可分成2个簇群和4个亚群,存在一定程度的地理隔离。研究证明了18S rDNA和ITS区基因序列是进行丛粒藻基因分型和遗传多样性研究的良好位点,而通过分析3株丛粒藻与分类归属地位有争议的不同目的微藻rbcL基因序列发现,采用邻接法、最小进化法、最大简约法和非加权组平均法构建的系统树均显示,该藻与衣藻目的亲缘关系较近且,这与日本学者用18S rDNA研究的结果相一致,为系统了解丛粒藻的遗传多样性和开展优良藻株选育工作奠定了基础。
要实现丛粒藻产烃的工厂规模化生产,优良藻株的选育和培养条件优化必不可少,总烃、总脂含量高和生长速度快的藻株是筛选的首要目标。分别选取丛粒藻藻株AGB-Bb01、AGB-Bb02和AGB-Bb03处于延滞期、对数生长前期、对数生长后期和稳定期的藻细胞,分别采用正己烷法和索氏提取法进行总烃含量及总脂含量的测定,发现3株丛粒藻在不同的生长时期总烃、总脂含量不同,最高值出现在对数生长后期。丛粒藻在Chu10、Chu13、BG11和BBM这4种培养基中的生长适应情况不同,3株实验微藻在不同培养基中的生长速率、生物量、总烃和总脂含量表现出差别。结果表明：AGB-Bb01在Chu13培养基中的生物量最高,AGB-Bb02和AGB-Bb03在BG11培养基中的生物量最高；AGB-Bb01和AGB-Bb02在Chu10培养基中总烃及总脂含量较高,AGB-Bb03在Chul3培养基中总烃及总脂含量最高,为丛粒藻烃类最佳收获期的选择和工业化大规模养殖提供实验基础。
Botryococcus braunii is considered as an energy microalgae having great protential applications because of that it can synthesize and accumulate considerable quantities of hydrocarbons in the metabolic process. The calorific value, composition and structure of these hydrocarbons are very similar to those of oil. Generally the content of total hydrocarbons could reach 25% to 40% of dry biomass and the maximum value that have reported was 76%.
The microscopic structure and sub-microscopic structure of three B. braunii strains (AGB-Bb01,AGB-Bb02 and AGB-Bb03) isolated from different sites were observed by light and electronic microscope observations based on morphological features. AGB-Bb01, AGB-Bb02 and AGB-Bb03 were identified as different genotype and geographical strains of B. braunii by amplifying the 18S-28S rDNA and rbcL gene sequences and blastn analysis, genetic distance and sequence similarities calculation. And it is the first time that the rbcL gene sequences were obtained from this energy microalgae. The phylogenetic trees involved in 16 strains of B. braunii were reconstructed based on the genetic distances and sequence similarities of 18S rDNA, ITS region and rbcL gene sequences at the same time. The results showed that the cell size, colony size and cell number in one colony of the three geographical strains were significantly different. The sub-microscopic structures demonstrated that the thickness of the cup-sheath and extent of cell embedded by the cup-sheath had difference among the three strains as well. There existed high genetic diversity among the different strains living in the different sites and phylogenetic tree showed that all the strains can be divided into 2 groups and 4 sub-groups because of the geographical isolation. It is proved that sequences of 18S rDNA and ITS region can be considered as good tools for gene-type identification and genetic diversity research in B. braunii. Phylogenetic trees were constructed using Neighbor-joining, Minimum evolution, Maximum parsimony and Unweighted pair-group method with arithmetic method, and their reliability was estimated using bootstrap analysis. All the results show the closest relatives are in the Chlamydomonadales with high bootstrap. The study lays the solid basement for the research of the genetic diversity and cultivation in B. braunii.
It is very necessary to breed algae strains and optimize culture conditions in order to achieve large-scale production. The preferred target is to obtain high-yield varieties which can both grow rapidly and have high content of total hydrocarbons and total lipids. Total hydrocarbons and total lipids of B. braunii AGB-Bb01, AGB-Bb02 and AGB-Bb03 was different in different growth phases. The maximum of those appeared in the post-logarithmic phase after algae cells in the lag phase, the early-logarithmic phase, the post-logarithmic phase and the stationary phase were collected and fragmented for determination. The effect of the media on the growth, contents of total hydrocarbons and total lipids of Botryococcus braunii AGB-Bb01, AGB-Bb02 and AGB-Bb03 was studied using different media such as modified Chu10 medium, modified Chul3 medium,BGll and bold basal medium(BBM). Biomass of B. braunii AGB-Bb01 cultured in Chu13 was higher than that cultured in Chu10, BG11 and BBM, while biomass of B. braunii AGB-Bb02 and AGB-Bb03 cultured in BG11 were higher than those cultured in the other medium respectively;Chu10 was found to be the best medium for total hydrocarbon content and total lipid content of B. braunii AGB-Bb01 and AGB-Bb02, whereas Chul3 is the best for total hydrocarbon content and total lipid content of B. braunii AGB-Bb03. This research provides experimental basis for the selection of the best harvest time and industrial large-scale production of B. braunii.
引文
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