Biosynthesis of triterpenoid hydrocarbons in the B-race of the green alga Botryococcus braunii. Sites of production and nature of the methylating agent

The B race of the green alga Botryococcus braunii is characterized by the production of large amounts of botryococcenes, i.e. triterpenoid hydrocarbons of general formula C_πH_2π-109 n= 30–37. The axenic strain used in this work produces botryococcenes ranging from C₃₀ to C₃₄ when fast growth is promoted by air-lift. Sequential extraction of hydrocarbons with solvents showed that botryococcenes accumulate in two distinct sites: externally in the successive outer walls forming a dense matrix and internally, probably in cyctoplasmic inclusions. Moreover, chase experiments after feeding the algae with sodium [1,2-¹⁴C]acetate, and feeding experiments with L-[Me-¹⁴C]methionine established the existence of an excretory process from the cells towards the matrix. The results of the radio GC analyses of the botryococcenes synthesized during the feeding experiments provided good evidence to show that the C₃₀ botryococcene is the precursor of all the higher hydrocarbons, and that each intermediate botryococcene C₃₁-_C33 is the precursor of its next highest homologue. L-Methionine acts as the methyl donor in the methylation process, leading from the C₃₀ precursor to the botryococcene family. The ¹³C NMR spectra of the botryococcenes produced when the algae were fed with L-[Me-¹³C]methionine indicate that the methylation takes place on the C₃₀ backbone in positions 37, 16 and 20.     

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.     

Raman Spectroscopy Analysis of Botryococcene Hydrocarbons from the Green Microalga Botryococcus braunii

Botryococcus braunii, B race is a unique green microalga that produces large amounts of liquid hydrocarbons known as botryococcenes that can be used as a fuel for internal combustion engines. The simplest botryococcene (C₃₀) is metabolized by methylation to give intermediates of C₃₁, C₃₂, C₃₃, and C₃₄, with C₃₄ being the predominant botryococcene in some strains. In the present work we have used Raman spectroscopy to characterize the structure of botryococcenes in an attempt to identify and localize botryococcenes within B. braunii cells. The spectral region from 1600–1700 cm⁻¹ showed ν(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm⁻¹ were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm⁻¹ was assigned to the backbone C=C bond stretching. Density function theory calculations were used to determine the Raman spectra of all botryococcenes to compare computed theoretical values with those observed. The analysis showed that the ν(C=C) stretching bands at 1647 and 1670 cm⁻¹ are actually composed of several closely spaced bands arising from the six individual C=C bonds in the molecule. We also used confocal Raman microspectroscopy to map the presence and location of methylated botryococcenes within a colony of B. braunii cells based on the methylation-specific 1647 cm⁻¹ botryococcene Raman shift.     

Biosynthesis of unusual acyclic isoprenoids in the Alga Botryococcus braunii

A ‘resting state’ isolate of the hydrocarbon-producing alga Botryococcus braunii photoassimilated sodium [¹⁴C]bicarbonate at rates comparable to fast growing algae, such as Chlorella (> 1.50 μg atoms ¹⁴C/mg chlorophyll·hr). Early in the reaction (up to several min), most of the radioactivity was associated with water-soluble metabolites. However, labelling of hexane-soluble compounds steadily from ca 3% at 15 sec to over 50% of the total incorporated ¹⁴C at 60 min. The purified hexane fraction, which consisted of a series of botryococcenes and squalene, constituted a relatively constant proportion (40–45%) of the total hexane-soluble radioactivity at all but the earliest time points (< 60 sec). This fraction initially consisted almost exclusively of a C₃₀ botryococcene (ca 91%) and squalene (ca 8%); however, small amounts of radioactivity sequentially appeared in the C₃₁, C₃₂ and C₃₄ botryococcenes. The results of pulse-chase experiments implicated the C₃₀ botryococcene as the precursor of the higher homologues; during the chase, loss of radioactivity from the C₃₀ compound was accompanied by a concomitant increase in the labelling of the C₃₁ and C₃₂ compounds. This study provides further evidence that the relatively slow growth of Botryococcus in culture may result, in part, from the diversion of a large proportion of reduced carbon into energetically expensive compounds and that the slower growth rate in the ‘resting state’ cannot be totally attributed to an impaired or intrinsically slow metabolism.     

Structures of some botryococcenes: branched hydrocarbons from the b-race of the green alga Botryococcus braunii

Nine branched hydrocarbons of the botryococcene type (C_nH_2n-10 30 ? n ? 37) have been isolated from the green alga Botryococcus braunii. Hydrocarbon mixtures were recovered from wild algae collected in fresh water lakes or from the same strains growing in laboratory; they were further separated by reversed-phase, and in some cases by normal phase, HPLC. From chemical investigations, GC/MS analyses, ¹H and ¹³C NMR spectroscopy, the structures of four new botryococcenes (one C₃₃H₅₆, two C₃₄H₅₈ and one C₃₇H₆₄) were elucidated.     

Hydrocarbon composition of newly isolated strains of the green microalga Botryococcus braunii

Four new strains of Botryococcus braunii were isolated from Japanese waters and cultured under defined conditions. Their hydrocarbon content and composition were analyzed and compared with those of the Darwin and Berkeley strains. The Yamanaka strain produced only alkadienes characteristic of the A race, whereas the others, the Yayoi, Kawaguchi-1 and -2 strains as well as the Darwin and Berkeley strains, produced botryococcenes peculiar to the B race. The hydrocarbon content of the Yamanaka strain was 16.1 % dry weight and that of the B race strains ranged from 9.7 to 37.9%. Botryococcene composition of the Japanese strains differed from each other as well as from the Darwin and Berkeley strains. More than 50% of the hydrocarbons in the Yayoi, Darwin, and Berkeley strains were composed of C₃₄H₅₈, but the main components were different from one another as isomers. The Kawaguchi-1 and -2 strains did not have a high level of C₃₄ botryococcenes, C₃₂ ones being the main components. In these strains significant amounts of squalene-related compounds were detected.     

Presence of methyl branched fatty acids and saturated hydrocarbons in botryococcene producing strain of Botryococcus braunii

Botryococcus braunii (N-836) produced 60 – 73% hydrocarbons on dry weight basis, of which C₃₄ botryococcene was found to be the major hydrocarbon, constituting about 50 – 76 % of total content throughout the experimental studies. Major fatty acids present in this organism were C18:1 and C16:0. Saturated hydrocarbons like docosane, hexacosane and heptacosane were also found to be produced by the organism. Methyl branched fatty acids, were identified as 16-methyl heptadecanoic and 5, 9, 13 - trimethyl tetradecanoic acids by GC-MS. Maximum hydrocarbon accumulation was observed during third week of its growth.     

STRUCTURE AND CHEMISTRY OF A NEW CHEMICAL RACE OF BOTRYOCOCCUS BRAUNII (CHLOROPHYCEAE) THAT PRODUCES LYCOPADIENE,A TETRATERPENOID HYDROCARBON1

New strains of the hydrocarbon rich alga Botryococcus braunii Kützing were isolated from water samples collected in three tropical freshwater lakes. These strains synthesize lycopadiene, a tetraterpenoid metabolite, as their sole hydrocarbon. The morphological and ultrastructural characteristics of these algae are similar to those reported for previously described strains which produce either alkadienes or botryococcenes. The pyriform shaped cells are embedded in a colonial matrix formed by layers of closely appressed external walls: this dense matrix is impregnated by the hydrocarbon and some other lipids. We believe the new strains synthesizing lycopadiene form a third chemical race in B. braunii, besides the alkadiene and botryococcene races, rather than a different species. Like the other two types of hydrocarbons, lycopadiene was produced primarily during the exponential and linear growth phases. The major fatty acid in the three races was oleic acid. This fatty acid was predominant in the alkadiene race; palmitic and octacosenoic acid also were present in appreciable amounts in the three races. Cholest-5-en-3β-ol, 24-methylcholest-5-en-3β-ol and 24-ethylcho-lest-5-en-3β-ol occurred in the three races; three unidentified sterols also were detected in the lycopadiene race. Moreover, the presence of very long chain alkenyl-phenols in the lipids of algae of the alkadiene race was not observed in the botryococcene and lycopadiene races. Of the polysaccharides released in the medium, galactose appeared as a primary component: it predominated in the botryococcene race. The other major constituents were fucose for the alkadiene race and glucose and fucose for the lycopadiene race. Although morphologically similar, some important chemical differences exist among algae classified as B. braunii.     

Hydrocarbon production in Anacystis montana and Botryococcus braunii

The production of labelled aliphatic hydrocarbons in Anacystis montana and Botryococcus braunii has been studied using Na₂CO₃ [¹⁴C] as a carbon source. The major hydrocarbon produced by A. montana is pentadecane (ca 93%) accompanied by a pentadecene (ca 4%) and other hydrocarbons in the range C₁₃-C₁₇. Long chain (C₂₁-C ₃₃) hydrocarbons could not be detected in this organism. The variety of unsaturated hydrocarbons (C₂₅-C₃₁) previously reported in Botryococcus braunii is confirmed and contrasts with the synthesis of unsaturated C₁₇ hydrocarbons only, in axenic cultures prepared from single cell isolates of this colonial alga.     

Photosynthetic acclimation of maize to growth under elevated levels of carbon dioxide

The effects of elevated CO₂ concentrations on the photochemistry, biochemistry and physiology of C₄ photosynthesis were studied in maize (Zea mays L.). Plants were grown at ambient (350 μL L⁻¹) or ca. 3 times ambient (1100 μL L⁻¹) CO₂ levels under high light conditions in a greenhouse for 30 d. Relative to plants grown at ambient CO₂ levels, plants grown under elevated CO₂ accumulated ca. 20% more biomass and 23% more leaf area. When measured at the CO₂ concentration of growth, mature leaves of high-CO₂-grown plants had higher light-saturated rates of photosynthesis (ca. 15%), lower stomatal conductance (71%), higher water-use efficiency (225%) and higher dark respiration rates (100%). High-CO₂-grown plants had lower carboxylation efficiencies (23%), measured under limiting CO₂, and lower leaf protein contents (22%). Activities of a number of C₃ and C₄ cycle enzymes decreased on a leaf-area basis in the high-CO₂-grown plants by 5–30%, with NADP-malate dehydrogenase exhibiting the greatest decrease. In contrast, activities of fructose 1,6-bisphosphatase and ADP-glucose pyrophosphorylase increased significantly under elevated CO₂ condition (8% and 36%, respectively). These data show that the C₄ plant maize may benefit from elevated CO₂ through acclimation in the capacities of certain photosynthetic enzymes. The increased capacity to synthesize sucrose and starch, and to utilize these end-products of photosynthesis to produce extra energy by respiration, may contribute to the enhanced growth of maize under elevated CO₂. Received: 30 April 1999 / Accepted: 17 June 1999     

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.     

Characteristics of Five New Photoautotrophic Suspension Cultures Including Two Amaranthus Species and a Cotton Strain Growing on Ambient CO(2) Levels

Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura innoxia, and a Nicotiana tabacum-N. glutinosa fusion hybrid were adapted to grow photoautotrophically under continuous light. The cotton strain grew with an atmosphere of ambient CO₂ (about 0.06 to 0.07% in the culture room) while the other strains required elevated CO₂ levels (5%). Photoautotrophy was indicated by the requirement for CO₂ and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate carboxylase (RuBPcase) activity levels were well below those of mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C₄Amaranthus species. The cells showed high dark respiration rates and had lower net CO₂ fixation under high O₂ conditions. Dark CO₂ fixation rates ranged from near 10 to 30% of that in light. Fluorescence emission spectra measurements show that the cell antenna pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO₂ conditions showed the unique properties of a high RuBPcase activation level in ambient CO₂ and a stable ability to show net CO₂ fixation in 21% O₂ conditions.     

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.     

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.     

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.     

Growth and senescence in plant communities exposed to elevated CO2 concentrations on an estuarine marsh

Summary Three high marsh communities on the Chesapeake Bay were exposed to a doubling in ambient CO₂ concentration for one growing season. Open-top chambers were used to raise CO₂ concentrations ca. 340 ppm above ambient over monospecific communities of Scirpus olneyi (C₃) and Spartina patens (C₄), and a mixed community of S. olneyi, S. patens, and Distichlis spicata (C₄). Plant growth and senescence were monitored by serial, nondestructive censuses. Elevated CO₂ resulted in increased shoot densities and delayed sensecence in the C₃ species. This resulted in an increase in primary productivity in S. olneyi growing in both the pure and mixed communities. There was no effect of CO₂ on growth in the C₄ species. These results demonstrate that elevated atmospheric CO₂ can cause increased aboveground production in a mature, unmanaged ecosystem.     

Importance of Different Volatile Petroleum Hydrocarbon Fractions in Human Health Risk Assessment

Soil vapor data for benzene and four aliphatic and aromatic hydrocarbon fractions from five volatile petroleum hydrocarbon (VPH)-contaminated sites in western Canada were used together with the Canadian Council of Ministers of the Environment (CCME) Canada-Wide Standard for petroleum hydrocarbons to investigate the relative importance of benzene and the different fractions in human health risk assessment. VPH concentrations in soil vapor samples ranged from 4.0 to 4200?mg/m³, of which 0 to 4.6% was BTEX and 90 to 95% was hydrocarbons of the C_5–8 aliphatic fraction. VPH inhalation exposure by an adult receptor in a hypothetical, commercial building was modelled deterministically assuming 16- and 70 year occupational tenures. The magnitude of hazard quotients varied widely between sites, but their hydrocarbon fraction signatures were consistent, and characterized by higher hazard quotients in the C_5–8 and C_9–10 aliphatic and C_9–10 aromatic fractions relative to benzene and the TEX aromatic fraction. This work has shown that the C_5– and C_9–10 aliphatic fractions yield greater relative risk than the commonlyregulated TEX compounds, and that benzene becomes the primary chemical of potential concern only when an occupational tenure approaching 70 years is assumed.     

Nitrogen and carbon dynamics in C3 and C4 estuarine marsh plants grown under elevated CO2 in situ

Summary Carbon dioxide concentrations were elevated in three estuarine communities for an entire growing season. Open top chambers were used to raise CO₂ concentrations ca. 336 ppm above ambient in monospecific communities of Scirpus olneyi (C₃) and Spartina patens (C₄), and a mixed community of S. olneyi, S. patens and Distichlis spicata (C₄). Nitrogen and carbon concentration (% wt) of aboveground tissue was followed throughout growth and senescence. Green shoot %N was reduced and %C was unchanged under elevated CO₂ in S. olneyi. This resulted in a 20%–40% increase in tissue C/N ratio. There was no effect of CO₂ on either C₄ species. Maximum aboveground N (g/m²) was unchanged in S. olneyi, indicating that increased productivity under elevated CO₂ was dependent on reallocation of stored N. There was no change in the N recovery efficiency of S. olneyi in pure stand and a decrease in the mixed community. Litter C/N ratio was not affected by elevated CO₂ suggesting that decomposition and N mineralization rates will also remain unchanged. Continued growth responses to elevated CO₂ could, however, be limited by the ability of S. olneyi to increase the total aboveground N pool.     

Acclimation of photosynthesis in relation to Rubisco and non-structural carbohydrate contents and in situ carboxylase activity in Scirpus olneyi grown at elevated CO2 in the field

Stands of Scirpus olneyi, a native saltmarsh sedge with C₃ photosynthesis, had been exposed to normal ambient and elevated atmospheric CO₂ concentrations (C_a) in their native habitat since 1987. The objective of this investigation was to characterize the acclimation of photosynthesis of Scirpus olneyi stems, the photosynthesizing organs of this species, to long-term elevated C_a treatment in relation to the concentrations of Rubisco and non-structural carbohydrates. Measurements were made on intact stems in the Held under existing natural conditions and in the laboratory under controlled conditions on stems excised in the field early in the morning. Plants grown at elevated C_a had a significantly higher (30–59%) net CO₂ assimilation rate (A) than those grown at ambient C_a when measurements were performed on excised stems at the respective growth C_a. However, when measurements were made at normal ambient C_a, A was smaller (45–53%) in plants grown at elevated C_a than in those grown at ambient C_a. The reductions in A at normal ambient C_a, carboxylation efficiency and in situ carboxylase activity were caused by a decreased Rubisco concentration (30–58%) in plants grown at elevated C_a; these plants also contained less soluble protein (39–52%). The Rubisco content was 43 to 58% of soluble protein, and this relationship was not significantly altered by the growth CO₂ concentrations. The Rubisco activation state increased slightly, but the in situ carboxylase activity decreased substantially in plants grown at elevated C_a. When measurements were made on intact stems in the field, the elevated C_a treatment caused a greater stimulation of,A (100%) and a smaller reduction in carboxylation efficiency (which was not statistically significant) than when measurements were made on excised stems in the laboratory. The possible reasons for this arc discussed. Plants grown at elevated C_a contained more non-structural carbohydrates (25–53%) than those grown at ambient C_a. Plants grown at elevated C_a appear to have sufficient sink capacity to utilize the additional carbohydrates formed during photosynthesis. Overall, our results are in agreement with the hypothesis that elevated C_a leads to an increased carbohydrate concentration and the ensuing acclimation of the photo-synthetic apparatus in C₃ plants results in a reduction in the protein complement, especially Rubisco, which reduces the photosynthetic capacity in plants grown at elevated C_a, relative to plants grown at normal ambient C_a. Nevertheless, when compared at their respective growth C_a, Scirpus olneyi plants grown at elevated C_a in their native habitat maintained a substantially higher rate of photosynthesis than those grown at normal ambient C_a even after 8 years of growth at elevated C_a.     

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。