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.     

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.     

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.     

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.     

Botryococcus braunii: a rich source for hydrocarbons and related ether lipids

This paper presents a review on Botryococcus braunii, a cosmopolitan green colonial microalga characterised by a considerable production of lipids, notably hydrocarbons. Strains like wild populations of this alga differ in the type of hydrocarbons they synthesise and accumulate: (1) n-alkadienes and trienes, (2) triterpenoid botryococcenes and methylated squalenes, or (3) a tetraterpenoid, lycopadiene. In addition to hydrocarbons and some classic lipids, these algae produce numerous series of characteristic ether lipids closely related to hydrocarbons. This review covers the algal biodiversity, the chemical structures and biosynthesis of hydrocarbons and ether lipids and the biotechnological studies related to hydrocarbon production.     

Growth of the hydrocarbon-rich microalga Botryococcus braunii in secondarily treated sewage

Summary A hydrocarbon-rich green microalga, Botryococcus braunii, was able to grow well in secondarily treated sewage (STS) from domestic waste-water in a batch system. The growth in STS from domestic waste-water was as good as in the common artificial medium of modified Chu 13 and its hydrocarbon contents were high enough at 53% and 40% compared with 58% in the case of the modified Chu 13 medium. B. braunii utilized nitrate from 7.67 or 4.48 mg/l to a level below detection of < 0.01 mg/l in STS. After this consumption of nitrate, nitrite was consumed, and ammonium was not. Phosphate, even at an extremely low concentration, was also consumed by B. braunii. These results show the possibility of using STS as a medium to grow B. braunii and for removal of nitrogen and phosphorus by algal consumption in STS.Correspondence to: S. Yokoyama     

Effects of carbon source on growth and morphology of <Emphasis Type="Italic">Botryococcus braunii</Emphasis>

The green colonial alga Botryococcus braunii is characterized by the ability to produce and accumulate large amounts of hydrocarbons. We isolated and established an axenic clonal strain of B. braunii B70 and investigated the effects of organic carbon sources, including glucose, mannose, fructose, galactose, or acetate, on growth under light and dark conditions. This algal strain had the capacity to grow photo-, mixo-, or heterotrophically. Growth was promoted substantially following exposure of the algae to glucose or mannose under light exposure. Cells could grow under continuous darkness with glucose or mannose. In the presence of glucose under light or dark conditions, cell and colony size, and the intracellular granules containing oil, were markedly larger than those cultured without glucose.     

Sites of accumulation and composition of hydrocarbons in Botryococcus braunii

Raman spectrometry and electron microscopy show that, in the hydrocarbon-rich alga Botryococcus braunii, hydrocarbons accumulate in two distinct sites; internally in cytoplasmic inclusions and externally in successive outer walls and derived globules. No other classes of lipid are present in noticeable amounts in the cytoplasmic inclusions and in the external globules. The same hydrocarbons are observed in the internal and external pools but with different relative abundances, the shorter hydrocarbons being more abundant in the internal pool. The bulk of B. braunii hydrocarbons (ca 95%) is located in the external pool. Such an extracellular location allows this species to exhibit both an unusually high hydrocarbon content (15% of dry wt) and a normal level (0.75%) within the cells. The hydrocarbon pattern and location of B. braunii were compared with that of other organisms; a close relation appears between higher plant epidermal cells and this green alga. The trilaminar outer walls of B. braunii, at whose contact external hydrocarbon globules accumulate, contain a sporopollenin-like compound.     

Mechanism of non-isoprenoid hydrocarbon biosynthesis in Botryococcus braunii

The green unicellular alga Botryococcus braunii shows unusually high concentrations of non-isoprenoid very long chain hydrocarbons. The structure of such hydrocarbons, the relative efficiency of various long chain fatty acids as precursors, the relationship between fatty acid and hydrocarbon concentrations (over the different physiological stages of the alga achieved during batch cultures) and the preferential localization of fatty acids lead to the conclusion that all the major non-isoprenoid hydrocarbons of B. braunii derive from the same direct precursor, oleic acid. Feeding experiments, using doubly labelled oleic acid, show that the whole carbon chain of the latter is incorporated into final hydrocarbons; accordingly such compounds do not originate from a head-to-head condensation mechanism with oleic acid acting as donor. Various features (regarding chiefly the systematic occurrence of a terminal double bond in B. braunii hydrocarbon, their close specific activities after feeding and the large inhibition in their production achieved using dithioerythritol) show that the biosynthesis of B. braunii hydrocarbons probably takes place via an elongation-decarboxylation mechanism related to that operating in some higher plants.     

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.     

Desiccation tolerance of Botryococcus braunii (Trebouxiophyceae,Chlorophyta) and extreme temperature tolerance of dehydrated cells

Botryococcus braunii Kützing, a green colonial microalga, occurs worldwide in both freshwater and brackish water environments. Despite considerable attention to B. braunii as a potential source of renewable fuel, many ecophysiological properties of this alga remain unknown. Here, we examined the desiccation and temperature tolerances of B. braunii using two newly isolated strains BOD-NG17 and BOD-GJ2. Both strains survived through 6- and 8-month desiccation treatments but not through a 12-month treatment. Interestingly, the desiccation-treated cells of B. braunii gained tolerance to extreme temperature shifts, i.e., high temperature (40 °C) and freezing (?20 °C). Both strains survived for at least 4 and 10 days at 40 and ?20 °C, respectively, while the untreated cells barely survived at these temperatures. These traits would enable long-distance dispersal of B. braunii cells and may account for the worldwide distribution of this algal species. Extracellular substances such as polysaccharides and hydrocarbons seem to confer the desiccation tolerance.     

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.     

Isolation and Characterization of Two Squalene Epoxidase Genes from Botryococcus braunii,Race B

The B race of the green microalga Botryococcus braunii produces triterpene hydrocarbons, botryococcenes and methylsqualenes that can be processed into jet fuels with high heating values. In this alga, squalene is also converted into membrane sterols after 2,3-epoxidation. In the present study, cDNA clones of two distinct squalene epoxidases (BbSQE-I and -II) were isolated. Predicted amino acid sequences encoded on these genes are 45% identical with each other. Introduction of BbSQE-I or -II into Saccharomyces cerevisie erg1 mutants resulted in the complementation of ergosterol auxotrophy. The relative expression level of SQE-II increased 3.5-fold from the early stage to the middle phase of a culture period of 42 days, while that of SQE-I was almost constant throughout the culture period. Southern blot analyses suggested that these genes are single-copied genes. This is the first report on the isolation of functional SQEs that are encoded in duplicated loci in the algal genome.     

Herbicide-resistant mutants of Botryococcus braunii race B (strain BOT-22)

The freshwater green alga Botryococcus braunii produces long-chain hydrocarbon oil in large quantities and secretes these from the cells. To exploit B. braunii as a source of next-generation biofuel, development of cost-effective cultivation systems are required. One of the most cost-effective methods for large-scale production is to simply grow B. braunii in open ponds. However, trials to cultivate B. braunii in open ponds often fail due to thriving of other green algal and cyanobacterial species because of the relatively slow growth of B. braunii. We previously demonstrated that herbicides are effective in control of contaminating algae. In order to use herbicide-assisted cultivation systems, we generated herbicide-resistant mutants of an oil-rich strain of B. braunii race B (strain BOT-22) by ethyl methanesulfonate mutagenesis. We isolated 44 glufosinate-resistant and 21 methyl viologen-resistant mutant lines. Some of these mutant lines exhibited vigorous growth and oil production in herbicide-containing liquid media, suggesting that they can be directly used in herbicide-containing cultivation systems.     

Growth and hydrocarbon production ofBotryococcus braunii immobilized in calcium alginate gel

Summary The colonial microalgaB. braunii, immobilized in calcium alginate beads, shows active photoautotrophic growth. Nevertheless, the rates of increase in cell number and, to a lesser extent, in biomass are substantially lower when compared to free cultures. Such features are related to steric contraints which occasion also the formation of large spherical colonies in the gel, showing an unsual mulberry organization. Some cracks due to the development of underlying colonies appear at the surface of the beads. Alga release remains low, however, during the cultures. EntrappedB. braunii retain the ability to produce extracellular hydrocarbons; the structure of the latter is not affected by immobilization but their relative abundances can undergo some variations. Entrapment leads to marked improvements in hydrocarbon production; decrease in growth rates is therefore associated, in alginate gel, with a still more pronounced diversion ofB. braunii metabolic activity towards hydrocarbon generation. It appears also that the improvements in hydrocarbon production, due to strain selection and to culture condition adjustment, obtained in free cultures, can be directly applied toB. braunii immobilized in alginate beads.     

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.     

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。     

ALLELOCHEMICALS OF BOTRYOCOCCUS BRAUNII (CHLOROPHYCEAE)1

The alga Botryococcus braunii Kützing (Chlorophyceae) present in Liyu Lake (Huanlien County, Taiwan) has toxic effects on a variety of aquatic organisms. Blooms of this alga, which typically occur in autumn, are associated with fish deaths in this lake. Experiments using 15 phytoplankton and 5 zooplankton isolated from Liyu Lake indicate that these plankton exhibit various susceptibilities to B. braunii. A close correlation between the degree of susceptibility tested in the laboratory and the absence of certain phytoplankton during B. braunii blooms in the lake was observed, suggesting allelopathic effects. Isolation, identification, and verification with authentic compounds indicated that allelochemicals were a mixture of free fatty acids, including α‐linolenic, oleic, linolic, and palmitic acids. Compared with other phytoplankton isolates, B. braunii produced significantly higher amounts of free fatty acids, particularly of oleic and α‐linolenic acids. The role of these fatty acids in favoring dominance of B. braunii in the natural environment was elucidated.     

Continuous culture of hydrocarbon-rich microalga Botryococcus braunii in secondarily treated sewage

The hydrocarbon-rich green microalga, Botryococcus braunii, was grown on secondarily treated sewage (STS) in a continuous bioreactor system. The algal biomass increased at a sustained rate of 196 mg dry weight/l per week for 1 month. The hydrocarbon content of algae grown on STS (49%) compared well with that of algae grown on an artificial medium. The concentrations of nitrate and phosphate ions in STS decreased from 5.5 to 4.0 mg nitrogen/l and 0.08 to 0.03 mg phosphorus/l, respectively, by algal consumption. STS would thus appear to suffice as an appropriate medium for continuously sustainable growth of B. braunii and the algal consumption of nitrate and phosphate should help in removing these ions from STS.     

The biosynthesis of long-chain hydrocarbons in the green alga Botryococcus braunii

The green colonial alga Botryococcus braunii has unusually high levels of hydrocarbons. Two distinct sites of hydrocarbon accumulation are present in the species: an internal pool present in cytoplasmic inclusions and an external pool in the trilaminar outer walls and associated globules. It is generally assumed that the hydrocarbons are produced within the cells and then excreted into the external pool to maintain the intracellular content at a normal value. Various feeding experiments showed, however, that the radioactivity of the external pool is much higher than the internal one. On the other hand, there was no decrease in the labelling of internal hydrocarbons in chase experiments. Therefore, an excretory process apparently does not take place in B. braunii. The outer wall, therefore, is the main site of hydrocarbon accumulation and also the place where the bulk of B. braunii hydrocarbons are produced. The outer wall also is involved in the matrix of colony formation and the above findings account for the sharp decrease of hydrocarbon production which is associated with the loss of colonial habit. The cultures were also shown to be unable, under usual growth conditions, to catabolize their own hydrocarbons. Such a feature, along with the extracellular location of the main site of production, may account for the abnormally high content of hydrocarbons typical of B. braunii.