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.     

Hydrocarbon production from secondarily treated piggery wastewater by the green alga Botryococcus braunii

A laboratory study was conducted on the removal of nitrogen and phosphorus from piggery wastewater during growth of Botryococcus braunii UTEX 572, together with measurements of hydrocarbon formation by the alga. The influence was tested of the initial nitrogen and phosphorus concentration on the optimum concentration range for a culture in secondarily treated piggery wastewater. A high cell density (> 7 g L^–1 d. wt) was obtained with 510 mg L^–1 NO₃-N. Growth increased with nitrogen concentration at the basal phosphorus concentration (14 mg P L^–1). The growth rate was nearly independent ( = 0.027 0.030 h^–1) of the initial phosphate concentration, except under conditions of phosphate deficiency ( = 0.019 h^–1). B. braunii grew well in piggery wastewater pretreated by a membrane bioreactor (MBR) with acidogenic fermentation. A dry cell weight of 8.5 mgL^–1 and hydrocarbon level of 0.95 gL^–1 were obtained, and nitrate was removed at a rate of 620 mg NL^–1. These results indicate that pretreated piggery wastewater provides a good culture medium for the growth and hydrocarbon production by B. braunii.     

The resistant polymer of the walls of the hydrocarbon-rich alga Botryococcus braunii

The outer walls of the green alga Botryococcus braunii (main sites of hydrocarbon production and accumulation) show a complex constitution. They comprise a biopolymer highly resistant to non-oxidative degradation. The resistant polymer accounts for ca 9% of the cell dry wt and appears, along with hydrocarbons, as one of the major constituents of the alga. In addition to chemical resistance, B. braunii polymer exhibits other properties: mode of deposition and fluorescence, often used to identify sporopollenins. (Class of wall components generally regarded as originating from polymerization of carotenoid derivatives.) Nevertheless further studies, using IR spectroscopy and high resolution ¹³C NMR of solids, along with determination of elemental composition and unsaturation levels, indicate that the bulk of the resistant polymer from B. braunii outer walls does not derive from carotenoids; accordingly it cannot be considered, in this respect, as a sporopollenin. In fact the information obtained on the structure of this important constituent of the alga is consistent with its formation via oxidative polymerization of B. braunii dienic hydrocarbons.     

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.     

Botryolins A and B, two tetramethylsqualene triethers from the green microalga Botryococcus braunii

Two new triterpenoid polyethers with a tetramethylsqualene carbon skeleton, botryolins A and B, have been isolated from the green microalga Botryococcus braunii. Their structures were determined by means of spectral analyses including 2D NMR.     

Effect of different media on exopolysaccharide and biomass production by the green microalga Botryococcus braunii

Botryococcus braunii is a colonial green microalga with recognized potential to synthesize lipids and hydrocarbons for biofuel production. Besides this ability, this microalga also produces exopolysaccharides (EPS). Nevertheless, there are few reports about their biotechnological aspects and industrial applications. In this study, the effect of the nutritional conditions was examined by using two different culture media (BG11 and D medium). To our knowledge, the latter has not been reported before for culturing B. braunii. After 49 days of incubation, the final production of EPS was found to be statistically higher (P < 0.05) in the D medium (0.549?±?0.044 g L^?1) than in BG11 (0.336?±?0.009 g L^?1). On the contrary, the biomass production was found to be higher in BG11 (1.019?±?0.051 g L^?1) than in the D medium (0.953?±?0.056 g L^?1). However, this difference was not statistically significant. The difference in salinity and nitrogen concentration between both media is suggested as the main factor involved in the EPS and biomass results. FTIR spectra of B. braunii EPS from both media revealed presence of uronic acids and absence of amino and sulfate groups. Despite the similarity between both spectra, there were some different signals (at 1,921.52 and 720.60 cm^?1) which may mean a difference in glycosyl composition.     

葡萄藻冷冻保藏的研究

为给微藻大规模培养生产生物燃料提供稳定可靠的种质资源,本研究以葡萄藻为研究对象,建立了一套葡萄藻快速高效冷冻保藏的方法.通过对不同冷冻保护剂二甲基亚砜(DMSO)、甲醇(MeOH)、乙二醇(EG)、丙二醇(PG)和甘油(Gly)的毒性测试和冷冻保藏效果的比较,结果表明在以6% MeOH作为冷冻保护剂的条件下葡萄藻的存活...     

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.     

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.     

Improvements in the cultivation of Botryococcus braunii using commercial fertilisers

Journal of Applied Phycology - Agricultural fertilisers (NPKs) have been recognised as an alternative to make microalgae cultivation cheaper as well as simpler in terms of the preparation of the...     

Interactions of Botryococcus braunii Cultures with Bacterial Biofilms

Unicellular microalgae generally grow in the presence of bacteria, particularly when they are farmed massively. This study analyzes the bacteria associated with mass culture of Botryococcus braunii: both the planktonic bacteria in the water column and those forming biofilms adhered to the surface of the microalgal cells (∼10⁷–10⁸ culturable cells per gram microalgae). Furthermore, we identified the culturable bacteria forming a biofilm in the microalgal cells by 16S rDNA sequencing. At least eight different culturable species of bacteria were detected in the biofilm and were evaluated for the presence of quorum-sensing signals in these bacteria. Few studies have considered the implications of this phenomenon as regards the interaction between bacteria and microalgae. Production of C4-AHL and C6-AHL were detected in two species, Pseudomonas sp. and Rhizobium sp., which are present in the bacterial biofilm associated with B. braunii. This type of signal was not detected in the planktonic bacteria isolated from the water. We also noted that the bacterium, Rhizobium sp., acted as a probiotic bacterium and significantly encouraged the growth of B. braunii. A direct application of these beneficial bacteria associated with B. braunii could be, to use them like inoculants for large-scale microalgal cultures. They could optimize biomass production by enhancing growth, particularly in this microalga that has a low growth rate.     

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.     

Characterization of botryococcene synthase enzyme activity, a squalene synthase-like activity from the green microalga Botryococcus braunii, Race B

The extracellular matrix of the alga Botryococcus braunii, Race B, consists mainly of botryococcenes, which have potential as a hydrocarbon fuel. Botryococcenes are structurally similar to squalene raising the possibility of a common enzyme for the biosynthesis of both. While B. braunii squalene synthase (SS) enzyme activity has been documented, botryococcene synthase (BS) enzyme activity has not been. In the current study, an assay for BS activity has been developed and used to show that many of the assay conditions for BS enzyme activity are similar to those of SS. However, SS enzyme activity is stimulated by Tween 80 while BS enzyme activity is inhibited. Moreover, BS enzyme activity was correlated with the accumulation of botryococcenes during a B. braunii culture growth cycle, which was distinctly different from the profile of SS enzyme activity. While the current results indicate a conservation of enzymological features amongst the BS and SS enzymes, raising the possibility of one enzyme capable of catalyzing both activities, they are also consistent with these two activities arising from separate and distinct enzymes.     

Outdoor pilot-scale production of Botryococcus braunii in panel reactors

In this paper, the outdoor production of Botryococcus braunii in pilot-scale panel reactors (0.4?m³) is studied under uncontrolled conditions at a location close to the Atacama Desert (Chile). Discontinuous experiments were performed on different dates to determine the feasibility of the culture and the influence of environmental conditions on the system yield. Data showed that solar radiation is a major parameter in determining system yield, the average irradiance inside the culture determining both the growth rate and biomass productivity. A maximum specific growth rate of 0.09?day^?1 and biomass productivity of 0.02?g?L^?1?day^?1 (dry weight) were measured in discontinuous mode, at an average irradiance of 60?μE?m^?2?s^?1. With respect to lipids, a productivity of 2.5?mg?L^?1?day^?1 was obtained under favourable growth conditions; no accumulation of lipids at the stationary phase was observed. To confirm this behaviour, a semicontinuous culture was performed at 0.04?day^?1 in a larger reactor (1?m³). In this experiment, the biomass concentration and productivity was 0.3?g?L^?1 and 0.015?g?L^?1?day^?1, respectively. The lipid content and productivity was 15.6% and 2.4?mg?L^?1?day^?1, respectively, the mean average irradiance inside the reactor being 60?μmol photons?m^?2?s^?1. The light path of the reactor determines the light availability, thus determining also the biomass concentration and productivity of the reactor once the dilution rate is fixed. Experimentally, biomass productivity of 0.015?g?L^?1?day^?1 was determined for a light path of 0.15?m, but this can be increased by more than three times for a light path of 0.1?m. These data confirm that this alga can be produced outdoors in a secure form, the culture yield improving when optimal conditions are applied, the data reported here establishing the starting point for the development of the process.     

利用烟道气培养产烃葡萄藻的可行性研究

对烟道气培养产烃葡萄藻的可行性进行了研究,重点考察了SO2和NOx的水溶形态对葡萄藻的影响。结果表明,亚硫酸氢盐浓度低于0．8mmol／L时,对葡萄藻生长没有明显抑制作用,可以提供葡萄藻生长的硫源,但高浓度(大于2mmol／L)时抑制细胞生长;当亚硝酸盐浓度小于8mmol／L时,可以作为葡萄藻生长的唯一氮源,亚硝酸盐的去除主要是由微藻利用所致。当起始浓度为2mmol／L和4mmol／L时,亚硝酸盐的去除率分别为100％和99．7％。     

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.     

布朗葡萄藻的培养基选择及其产物代谢规律

通过对布朗葡萄藻分别在Chu13、Chu13×2和BG-11培养基中培养结果的比较,发现在气升式光照生物反应器中Chu13培养基最有利于布朗葡萄藻的生长和烃的合成,培养15d后,其生物量和粗烃质量分数分别为1.82g/L和58.7%;棕榈酸、油酸和亚麻酸是布朗葡萄藻的主要脂肪酸组成,Chu13培养获得的藻体不饱和脂肪酸比例最高。Chu13培养基中布朗葡萄藻代谢规律的研究表明:粗烃含量随着生物量的增加而逐渐增大,15d后粗烃产量达到最大值1.07g/L,不同生长周期烃的组成保持一致,布朗葡萄藻的烃主要由C33H56和C34H58组成;在布朗葡萄藻生长周期中,不饱和脂肪酸的比例显著上升,培养15d达到64%以上。