Hydrocarbon productivities in different <Emphasis Type="Italic">Botryococcus</Emphasis> strains: comparative methods in product quantification

Six different strains of the green microalgae Botryococcus belonging to the A-race or B-race, accumulating alkadiene or botryococcene hydrocarbons, respectively, were compared for biomass and hydrocarbon productivities. Biomass productivity was assessed gravimetrically upon strain growth in the laboratory under defined conditions. Hydrocarbon productivities were measured by three different and independent experimental approaches, including density equilibrium of the intact cells and micro-colonies, spectrophotometric analysis of hydrocarbon extracts, and gravimetric quantitation of eluted hydrocarbons. All three hydrocarbon-quantitation methods yielded similar results for each of the strains examined. The B-race microalgae Botryococcus braunii var. Showa and Kawaguchi-1 constitutively accumulated botryococcene hydrocarbons equivalent to 30% and 20%, respectively, of their overall biomass. The A-race microalgae Botryococcus braunii, varieties Yamanaka, UTEX 2441 and UTEX LB572 constitutively accumulated alkadiene hydrocarbons ranging from 14% to 13% and 10% of their overall biomass, respectively. Botryococcus sudeticus (UTEX 2629), a morphologically different green microalga, had the lowest hydrocarbon accumulation, equal to about 3% of its overall biomass. Results validate the density equilibrium and spectrophotometric analysis methods in the quantitation of botryococcene-type hydrocarbons. These analytical advances will serve in the screening and selection of B. braunii and of other microalgae in efforts to identify those having a high hydrocarbon content for use in commercial applications.     

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.     

Sterol profiles of red algae

Twelve species of red algae belonging to the Orders Gelidiales, Cryptonemiales and Gigartinales were examined for sterols. Four species contained cholestan-3β-ol as the major sterol, accompanied by C₂₆, C₂₈ and C₂₉ stanols. Sterols not previously reported in algae were 24-dimethyl-5α-chol-22-en-3β-ol, cholest-22-en-3β-ol, cholest-7-en-3β-ol, 24ξ-methylcholest-22-en-3β-ol, 24-methylenecholestan-3β-ol, 24ξ-ethylcholestan-3β-ol and isofucostanol.     

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.     

Six novel tetraterpenoid ethers, lycopanerols B-G, and some other constituents from the green microalga Botryococcus braunii

Six novel tetraterpenoid ethers, lycopanerols B-G, were isolated from lipidic extracts of the green microalga Botryococcus braunii (L race), along with a series of phytyl esters and alpha- and beta-tocopherols. The structures of the compounds were determined by means of spectral analyses including 2D NMR techniques. A biogenetic relationship is proposed between lycopanerols and lycopadiene, the acyclic diunsaturated tetraterpenoid hydrocarbon synthesized by the alga.     

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。     

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.     

STEROLS OF 14 SPECIES OF MARINE DIATOMS (BACILLARIOPHYTA)1

The sterol compositions of 14 species of marine diatoms were determined by gas chromatography and gas chromatography-mass spectrometry. A variety of sterol profiles were found. The sterols 24-methylcholesta-5,22E-dien-3β-ol, cholest-5-en-3β-ol, and 24-methylcholesta-5,24(28)-dien-3β-ol, previously described as the most common sterols found in diatoms, were major sterols in only a few of the species. In light of this and other recent data, it is clear that these three sterols are not typical constituents of many diatom species. Most of the centric species examined had 24-methylcholesta-5,24(28)-dien-3β-ol and 24-methylcholest-5-en-3β-ol as two of their major sterols. The exception was Rhizosolenia setigera, which possessed cholesta-5,24-dien-3β-ol as its single major sterol. In contrast to the centric species, the pennate diatoms examined did not have any particular sterols common to most species. Minor levels ofΔ⁷-sterols, rarely found in large amounts in diatoms, were found in four species. C₂₉sterols were found in many species; seven contained 24-ethylcholest-5-en-3β-ol and three contained 24-ethylcholesta-5,22E-dien-3β-ol, reinforcing previous suggestions that C₂₉ sterols are not restricted to higher plants and macroalgae. 24-Ethylcholesta-5,22E-dien-3β-ol may prove to be useful for taxonomy of the genus Amphora and the order Thalassiophysales. A major sterol of Fragilaria pinnata was the uncommon algal sterol 23,24-dimethylcholesta-5,22E-dien-3β-ol. Cholesta-5,24-dien-3β-ol was the only sterol found in the culture of Nitzschia closterium. This differed from previous reports of 24-methylcholesta-5,22E-dien-3β-ol as the single major sterol in N. closterium. Two C₂₈ sterols possessing an unusual side chain were found in Thalassi-onema nitzschioides, a C_28:2 sterol (16%) and a C_28:1 sterol in lower abundance (2.5%), which may be 23-methylcholesta-5,22E-dien-3β-ol and 23-methyl-5α-cholest-22E-en-3β-ol, respectively. The species Cylindrotheca fusiformis, T. nitzschioides, and Skeletonema sp. may be useful as direct sources of cholesterol in mariculture feeds due to their moderate to high content of this sterol.     

PIGMENTS,FATTY ACIDS,AND STEROLS OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM1

Cultures and field samples of the toxic dinoflagellate Gymnodinium catenatum Graham from Tasmania, Australia, were analyzed for pigment, fatty acid, and sterol composition. Gymnodinium catenatum contained the characteristic pigments of photosynthetic dinoflagellates, including chlorophyll a, chlorophyll c₂, and the carotenoids peridinin, dinoxanthin, diadinoxanthin, diatoxanthin, and β,β-carotene. In midlogarithmic and early stationary phase cultures, the chlorophyll a content ranged 50–72 pg · cell^?1, total lipids 956–2084 pg · cell^?1, total fatty acids 426–804 pg · cell^?1, and total sterols 8–20 pg · cell^?1. The major fatty acids (in order of decreasing abundance) were 16:0, 22:6(n-3), and 20:5(n-3) (collectively 65–70% of the total fatty acids), followed by 16:1(n-7), 18:2(n-6), and 14:0. This distribution is characteristic of most dinoflagellates, except for the low abundance (<3%) of the fatty acid 18:5(n-3), considered by some authors to be a marker for dinoflagellates. The three major sterols were 4α-methyl-5α-cholest-7-en-3β-ol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (the dinoflagellate sterol, dinosterol), and 4α,23,24-trimethyl-5α-cholest-7-en-3β-ol. These three sterols comprised about 75% of the total sterols in both logarithmic and early stationary phase cultures, and they were also found in high proportions (22–25%) in natural dinoflagellate bloom samples. 4-Desmethyl sterols, which are common in most microalgae, were only present in trace amounts in G. catenatum. The chemotaxonomic affinities of G. catenatum and the potential for using specific signature lipids for monitoring toxic dinoflagellate blooms are discussed.     

The fatty acid and sterol composition of two marine dinoflagellates

The fatty acid, sterol and chlorophyll pigment compositions of the marine dinoflagellates Gymnodinium wilczeki and Prorocentrum cordatum are reported. The fatty acids of both algae show a typical dinoflagellate distribution pattern with a predominance of C₁₈, C₂₀ and C₂₂ unsaturated components. The acid 18:5ω3 is present at high concentration in these two dinoflagellates. G. wilczeki contains a high proportion (93.4%) of 4-methyl-5α-stanols including 4,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol), dinostanol and 4,23,24-trimethyl-5α-cholest-7-en-3β-ol reported for the first time in dinoflagellates. The role of this sterol in the biosynthesis of 5α-stanols in dinoflagellates is discussed. P. cordatum contains high concentrations of a number of δ ²⁴⁽²⁸⁾-sterols with dinosterol, 24-methylcholesta-5,24(28)-dien-3β-ol, 23,24-dimethylcholesta-5,22E-dien-3β-ol, 4,24-dimethyl-5α-cholest-24(28)-en-3β-ol and a sterol identified as either 4,23,24-trimethyl- or 4-methyl-24-ethyl-5α-cholest-24(28)-en-3β-ol present as the five major components. The role of marine dinoflagellates in the input of both 4-methyl- and 4-desmethyl-5α-stanols to marine sediments is discussed.     

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.     

Engineering linear,branched‐chain triterpene metabolism in monocots

Triterpenes are thirty‐carbon compounds derived from the universal five‐carbon prenyl precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Normally, triterpenes are synthesized via the mevalonate (MVA) pathway operating in the cytoplasm of eukaryotes where DMAPP is condensed with two IPPs to yield farnesyl diphosphate (FPP), catalyzed by FPP synthase (FPS). Squalene synthase (SQS) condenses two molecules of FPP to generate the symmetrical product squalene, the first committed precursor to sterols and most other triterpenes. In the green algae Botryococcus braunii, two FPP molecules can also be condensed in an asymmetric manner yielding the more highly branched triterpene, botryococcene. Botryococcene is an attractive molecule because of its potential as a biofuel and petrochemical feedstock. Because B. braunii, the only native host for botryococcene biosynthesis, is difficult to grow, there have been efforts to move botryococcene biosynthesis into organisms more amenable to large‐scale production. Here, we report the genetic engineering of the model monocot, Brachypodium distachyon, for botryococcene biosynthesis and accumulation. A subcellular targeting strategy was used, directing the enzymes (botryococcene synthase [BS] and FPS) to either the cytosol or the plastid. High titres of botryococcene (>1 mg/g FW in T₀ mature plants) were obtained using the cytosolic‐targeting strategy. Plastid‐targeted BS + FPS lines accumulated botryococcene (albeit in lesser amounts than the cytosolic BS + FPS lines), but they showed a detrimental phenotype dependent on plastid‐targeted FPS, and could not proliferate and survive to set seed under phototrophic conditions. These results highlight intriguing differences in isoprenoid metabolism between dicots and monocots.     

Analysis of sterols in selected bloom-forming algae in China

Sterols, a group of stable lipid compounds, are often used as biomarkers in marine biogeochemical studies to indicate sources of organic matter. In this study, sterols in 13 species of major bloom-forming algae in China, which belong to Dinophyceae, Bacillariophyceae, Ulvophyceae, and Pelagophyceae, were analyzed with gas chromatography-mass spectrometry (GC–MS) to test their feasibility in representing different types of harmful algal blooms (HABs). It was found that (24Z)-stigmasta-5,24-dien-3β-ol (28-isofucosterol) was a major sterol component in green-tide forming macroalga Ulva prolifera. In bloom-forming dinoflagellates Alexandrium spp., Prorocentrum micans and Scrippsiella trochoidea, (22E)-4α,23-dimethyl-5α-ergost-22-en-3β-ol (dinosterol) was detected in addition to cholest-5-en-3β-ol (cholesterol), (22E)-ergosta-5,22-dien-3β-ol, (22E)-stigmasta-5,22-dien-3β-ol and other minor sterol components. In brown-tide forming pelagophyte Aureococcus anophagefferens, (24E)-24-propylcholesta-5,24-dien-3β-ol ((24E)-24-propylidenecholesterol) and (24Z)-24-propylcholesta-5,24-dien-3β-ol ((24Z)-24-propylidenecholesterol) were detected together with cholesterol, (22E)-stigmasta-5,22-dien-3β-ol, stigmast-5-en-3β-ol and campest-5-en-3β-ol. Among the selected bloom-forming diatoms, Chaetoceros sp. and Pseudo-nitzschia spp. only produced cholesterol, while Cylindrotheca closterium produced solely (22E)-ergosta-5,22-dien-3β-ol. Sterol content in four bloom-forming algal species correlates well with their biomass or abundance. It's proposed that 28-isofucosterol could serve as a promising biomarker for green algae in green-tide studies. Dinosterol and (24Z)-24-propylidenecholesterol can be used as potential biomarkers to represent bloom-forming dinoflagellates and pelagophytes, while (22E)-ergosta-5,22-dien-3β-ol is not a good indicator for diatoms.     

STEROL DISTRIBUTION IN THE GENUS HETEROCAPSA (PYRRHOPHYTA)1

The sterol composition of seven strains of marine peridinioid dinoflagellates comprising the four known species of Heterocapsa Stein was examined by gas chromatography-mass spectrometry to determine the utility of these compounds in systematics. Cholest-5-en-3β-ol (cholesterol), 24-methyl-cholest-5-en-3β-ol (24-methylcholesterol), 4α,24(S)-dimethyl-5α-cholestan-3β-ol (4,24-dimethylcholestanol), 4α,23,24(R)-trimethyl-5α-cholest-22-en-3β-ol (dinosterol), 4α,23ξ,24ξ-trimethyl-5α-cholestan-3β-ol (dihydrodinosterol), and an unknown sterol were detected. Sterol composition does not vary significantly from species to species within the genus Heterocapsa and thus cannot be used for species differentiation. Sterols may, however, have value in defining the properties of dinoflagellate taxa above the family level. Over the course of the growth curve for Heterocapsa niei (Loeblich) Morrill & Loeblich 4,24-dimethylcholestanol and dinosterol covaried, suggesting that 4,24-dimethylcholestanol is converted into dinosterol by a previously proposed bioalkylation scheme.     

LIPIDS IN BLUE-GREEN ALGAL MATS (MODERN STROMATOLITES) FROM ANTARCTIC OASIS LAKES1

Lipids comprising the stenols, stanols, polar lipid fatty acids, alkanes and alkenes of blue-green algal-(diatomaceous)-microbial mats and cores (modern cold water stromatolites) collected from three Antarctic lakes were identified and compared with those of other algae. The major stenols were: (cholesta-5, 22-dien-3β-ol, cholest-5-en-3β-ol, 24-methylcholesta-5, 22-dien-3β-ol, 24-methyl-cholest-5-en-3β-ol, 24-ethylcholesta-5, 22-dien-3β-ol, and 24-ethylcholest-5-en-3β-ol). The presence of C28 Δ^{3, 22} stenols, as well as other C28 stenols, was suggestive of diatom input. C29 stenols may have originated from blue-grern algae. However, the high concentrations of stenols present and the lack of Δ⁷ stenols was atypical for known stenol components of several blue-green algal species previously reported. The occurrence of these stenols and other lipid markers strongls implicate diatoms as well as blue-green algae as important biogenetic sources of lipids and has established the potential for studies of lipid diagenesis in these unique cold, freshwater stromatolites .     

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.     

INVESTIGATION OF 4-METHYL STEROLS FROM CULTURED DINOFLAGELLATE ALGAL STRAINS

The marine dinoflagellates Prorocentrum micans, Gonyaulax polyedra, Gymnodinium sp., and Alexandrium tamarense, collected from the Adriatic Sea during red-tide blooms, were cultured to investigate the 4-methyl sterol constituents. To ascertain a possible influence of cell age on the 4-methyl sterol content, for one strain (Gymnodinium sp.)we investigated the composition of these constituents at exponential and stationary growing phases. The lipid material extracted with acetone from the lyophilized algal samples was fractionated by thin-layer chromatography. The 4-methyl sterols recovered from the layer were converted into the corresponding OTMS derivatives. Nine of 11 constituents were identified by gas chromatography and gas chromatography-mass spectrometry; only two minor constituents were characterized by their gas chromatographic parameters. All free methyl sterols identified in the algal samples had been detected previously in various dinoflagellates. The 4-methyl sterol fractions generally contained very few constituents. Except for the Gymnodinium sp. sample, collected at the exponential growing phase (GyD2 exp), which contains 4,24-dimethylcholestan-3-ol as a unique constituent, dinosterol was the major component. Moreover, 4,24-ethylcholestan-3-ol was also an important constituent of both Prorocentrum and Gonyaulax strains, whereas considerable amounts of dinostanol characterized all the Gymnodinium sp. strains. In addition, the latter contained several minor constituents such as 4-methylcholestan-3-ol, 4,24-dimethylcholesta-22-en-3-ol, and 4-methyl-24-ethylcholestan-3-ol. 4-Methyl-24-methylene-cholestan-3-ol was a constituent of the Gymnodinium sp. sample, collected at the stationary growing phase (GyD2 stat)only, whereas 4-methylgorgostanol was identified only in the Alexandrium tamarense Gt4 strain. Except for 4-methyl-24-ethylcholesta-8(14)-en-3-ol, all the methyl sterol constituents from our algae show a saturated polynuclear system. The pathways by which side-chain modifications occur in dinoflagellate 4-methyl sterols are considered, and a map of the fragmentation pattern of the trimethylsilyl-4-methyl sterols under electronic impact is also reported.