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.     

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.     

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.     

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.     

Seawater-Cultured Botryococcus braunii for Efficient Hydrocarbon Extraction

As a potential source of biofuel, the green colonial microalga Botryococcus braunii produces large amounts of hydrocarbons that are accumulated in the extracellular matrix. Generally, pretreatment such as drying or heating of wet algae is needed for sufficient recoveries of hydrocarbons from B. braunii using organic solvents. In this study, the Showa strain of B. braunii was cultured in media derived from the modified Chu13 medium by supplying artificial seawater, natural seawater, or NaCl. After a certain period of culture in the media with an osmotic pressure corresponding to 1/4-seawater, hydrocarbon recovery rates exceeding 90% were obtained by simply mixing intact wet algae with n-hexane without any pretreatments and the results using the present culture conditions indicate the potential for hydrocarbon milking.

Highlights

Seawater was used for efficient hydrocarbon extraction from Botryococcus braunii. The alga was cultured in media prepared with seawater or NaCl. Hydrocarbon recovery rate exceeding 90% was obtained without any pretreatment.     

Active Hydrocarbon Biosynthesis and Accumulation in a Green Alga,Botryococcus braunii (Race A)

Among oleaginous microalgae, the colonial green alga Botryococcus braunii accumulates especially large quantities of hydrocarbons. This accumulation may be achieved more by storage of lipids in the extracellular space rather than in the cytoplasm, as is the case for all other examined oleaginous microalgae. The stage of hydrocarbon synthesis during the cell cycle was determined by autoradiography. The cell cycle of B. braunii race A was synchronized by aminouracil treatment, and cells were taken at various stages in the cell cycle and cultured in a medium containing [¹⁴C]acetate. Incorporation of ¹⁴C into hydrocarbons was detected. The highest labeling occurred just after septum formation, when it was about 2.6 times the rate during interphase. Fluorescent and electron microscopy revealed that new lipid accumulation on the cell surface occurred during at least two different growth stages and sites of cells. Lipid bodies in the cytoplasm were not prominent in interphase cells. These lipid bodies then increased in number, size, and inclusions, reaching maximum values just before the first lipid accumulation on the cell surface at the cell apex. Most of them disappeared from the cytoplasm concomitant with the second new accumulation at the basolateral region, where extracellular lipids continuously accumulated. The rough endoplasmic reticulum near the plasma membrane is prominent in B. braunii, and the endoplasmic reticulum was often in contact with both a chloroplast and lipid bodies in cells with increasing numbers of lipid bodies. We discuss the transport pathway of precursors of extracellular hydrocarbons in race A.     

Botryococcus braunii: A Renewable Source of Hydrocarbons and Other Chemicals

ABSTRACT:?

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.     

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.     

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.     

Echinenone production of a dark red-coloured strain of Botryococcus braunii

Echinenone has been used as an edible orange pigment, antioxidant and provitamin A. An echinenone-accumulating strain, BOT-20, of Botryococcus braunii was isolated from freshwater environments in Japan. The B. braunii BOT-20 strain is different from other strains of B. braunii, as it appeared dark red during its growth in the laboratory culture as opposed to green. The biomass of the strain was 1.9?g?L^?1 at 1?month after cultivation. The n-hexane/acetone (3:1, v/v) extract of the strain was 45.5% of the dry biomass weight and consisted of carotenoids (92%, of which 73% was echinenone) and hydrocarbons (8%). The echinenone content was 30.5% of the dry biomass weight, and production was 630?mg?L^?1. Hydrocarbons comprised only 3.7% of the total dry biomass weight. The main component of hydrocarbon was an analogue of botryococene by ¹H and ¹³C NMR. With high values of echinenone content and production, the B. braunii strain BOT-20 is expected to be a new bioresource for the commercial production of echinenone.     

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。     

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.     

Growth and photosynthetic activity of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> biofilms

Botryococcus braunii is a green microalga capable of producing large amounts of external long-chain hydrocarbons suitable as a source of biofuel. There have been several studies indicating that cultures of B. braunii can reduce the energy and water requirement for mass biofuel production, especially if non-destructive extraction methods for milking hydrocarbons are used. Growing microalgae as a raw material for biofuel using conventional liquid-based cultivation (i.e., raceway ponds) has yet to be shown to be economically successful. An alternative solid growth (biofilm) cultivation method can markedly reduce the energy requirements and costs associated with the harvesting and dewatering processes. We evaluated the growth of biofilms of several strains of B. braunii (from races A, B, L and S) and found that three of the four tested races successfully grew to stationary phase in 10 weeks with no contamination. Among all races, B. braunii BOT22 (race B) reached the highest biomass and lipid yields (3.80 mg dry weight cm^?2 day^?1 and 1.11 mg dry weight cm^?2). Irrespective of the race, almost all photosynthetic parameters (F _V /F ₀ , PI_ABS and the OJIP curve) showed that the biofilm cultures were more stressed during lag and stationary phases than in logarithmic phase. We also studied the Botryococcus biofilm profiles using confocal microscopy and found that this method is suitable for estimating the overall biomass yield when compared with gravimetric measurement. In conclusion, the growth characteristics (biomass and lipid) and photosynthetic performance of all races indicated that B. braunii BOT22 is the most promising strain for biofilm cultivation.     

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.     

Non-destructive oil extraction from Botryococcus braunii (Chlorophyta)

Some of the key reasons for why the production of biofuels from microalgae have not yet succeeded as a source of sustainable transport fuel are the costs involved and the amount of energy needed to obtain the oils compared to the energy contained in the final fuel. The key energy costs are in the dewatering of biomass followed by extraction of the oil, disposal of biomass, and the energy content of the nutrient fertiliser needed for regrowing the algae. In this study, we bypass all of these barriers by using a different approach towards cutting energy and fertiliser costs in the production of biofuels from microalgae—rather than growing the algae in the presence of fertilisers such as N and P, followed by harvesting the whole algae cells, and the energetically costly drying of cells and extraction of the fuel from the cells, this process makes use of the natural tendency of the green alga, Botryococcus braunii to release oils from the cell into the extracellular matrix during and after growth. Here, we non-destructively and repeatedly harvest the external oil (hydrocarbons) from B. braunii CCAP 807/2. Extraction with several solvents showed that hexane was not compatible with B. braunii, but that heptane in contact with B. braunii for less than 20 min did not negatively affect this alga. As an alternative, solvent-free method, we tested physical methods of extracting the extracellular oil. Light and temperature did not affect the extraction of the external oil from Botryococcus, but gentle pressure (i.e. ‘blotting’) was an effective method for external oil recovery. Less than 1 h of blotting also did not affect the physiology of Botryococcus. Both the heptane extraction and the non-destructive ‘blotting’ methods had no significant effect on growth and photosynthesis (F _v/F _m, ETR_max) of B. braunii. Our results indicate that over a period of 6 days, we can repeatedly extract over 35 % (using heptane) and 1 % (using ‘blotting’) of the total oil, mainly in the form of external hydrocarbon in stationary phase cells without damage to the cells.     

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.     

Colony Organization in the Green Alga Botryococcus braunii (Race B) Is Specified by a Complex Extracellular Matrix

Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous β-1, 4- and/or β-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form “drapes” between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.     

Measurement of individual cell strength of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> in cell culture

Botryococcus braunii is a microalga considered for biofuel production and may require physical disruption of cells/colonies for efficient hydrocarbon extraction. In this study, the strength of individual cells of B. braunii was measured using a nanoindenter. From the load and cell size, the pressure for bursting the cell was calculated to be 56.9 MPa. This value is 2.3–10 times those of Saccharomyces cerevisiae and Chlorella vulgaris found in another research, because B. braunii has two types of cell walls with different thicknesses. The energy required to disrupt 1 g of dry B. braunii cells, estimated by load-displacement curves, is 3.19 J g^?1 which is 0.19–1.2 times higher than those of S. cerevisiae and C. vulgaris. When using a high-pressure homogenizer for disrupting B. braunii cells, the cell disruption degree increased with the treatment pressure at above 30 MPa, and 70% of cells were disrupted at 80 MPa.     

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.