Studies on batch and continuous cultures of Botryococcus braunii: hydrocarbon production in relation to physiological state, cell ultrastructure, and phosphate nutrition

The growth of the hydrocarbon-rich alga Botryococcus braunii was studied under "air-lift" conditions using batch and continuous cultures. Large variations in the physiological state of B. braunii were achieved in batch cultures and in continuous cultures with various dilution rates. The possible effects of these variations upon hydrocarbons (nature, relative abundance, location, level, productivity) and also on the production of exocellular polysaccharides were examined. The relationships between the physiological state of B. braunii and its hydrocarbon and polysaccharide production were discussed and compared with those generally observed in unicellular algae. The factors giving rise to the transition from high to low productivity stages were considered. To this end we examined, at first, the variations in cell ultrastructure and the resulting degeneration occurring during batch cultures. Afterward the parallel changes in some parameters of the medium (pH, phosphate level) were determined and their possible relationships with B. braunii growth and hydrocarbon production were discussed. The main features of phosphate nutrition in B. braunii and its effects on hydrocarbons were finally examined.     

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.     

Improvement of hydrocarbon recovery by spouting solvent into culture of Botryococcus braunii

Botryococcus braunii, a green microalga, is known to produce plentiful liquid hydrocarbons as promising biodiesel resources. However, the hydrocarbon extraction methods that have so far achieved have several problems such as low efficiency and high cost. In our study, a solvent-spouted extraction process integrated with photo-bioculture was designed for simultaneous realization of hydrocarbon extraction and cell culture in two phases. The n-octane was selected as the best solvent among several solvents because its biocompatibility was highest for B. braunii. As a result, high level of biomass and hydrocarbon, 4.17 and 893.79 mg/L, respectively, was attained at 100 mL/min of solvent recycling rate through three times of processes for 66 days. Moreover, formation of cell clump was suppressed in solvent extraction, cells were regenerated after it, and thus cell viability was maintained even after repeated cycles of it. Finally, this solvent-spouted culture process required the smaller cost due to reuse of the less solvent and regenerated cells, compared with the other conventional methods. Accordingly, this technique would be applicable to exploit the continuous extraction of hydrocarbon from the algal biomass, especially for application on a large scale.     

Influence of CO2 on growth and hydrocarbon production in Botryococcus braunii

Botryococcus braunii is a green colonial fresh water microalga and it is recognized as one of the renewable resources for production of liquid hydrocarbons. CFTRI-Bb-1 and CFTRI-Bb-2 have been reported for the first time and their performance with regard to growth and biochemical profile is presented here. The present study focused on effect of carbon dioxide (CO2) on biomass, hydrocarbon, carbohydrate production, fatty acid profile, and carotenoid content in various species of B. braunii (LB-572, SAG 30.81, MCRC-Bb, N-836, CFTRI-Bb-1, and CFTRI-Bb-2) at 0.5, 1.0, and 2.0% (v/v) levels using a two-tier flask. CO2 at 2.0% (v/v) level enhanced growth of the organism, and a two-fold increase in biomass and carotenoid contents was observed in all the B. braunii strains studied compared with control culture (without CO2 supplementation). At 1% and 2% (v/v) CO2 concentrations, palmitic acid and oleic acid levels increased by 2.5 to 3 folds in one of the strains of B. braunii (LB-572). Hydrocarbon content was found to be above 20% at 2% CO2 level in the B. braunii LB-572, CFTRI-Bb-2, CFTRI-Bb-1, and N-836 strains, whereas it was less than 20% in the SAG 30.81 and MCRC-Bb strains compared with control culture. This culture methodology will provide information on CO2 requirement for growth of algae and metabolite production. B. braunii spp. can be grown at the tested levels of CO2 concentration without much influence on culture pH.     

布朗葡萄藻在不同培养基中的生长效应

研究了布朗葡萄藻(Botryococcus braunii)764株和765株在3种培养基Chu10、Chu13×2和SE中的生长效应.结果表明:(1)B.braunii764在Chu10中的细胞密度、生物量、总脂含量和总烃含量均高于Chu13×2和SE;B.braunii765在Chu10中的细胞密度、生物量和总脂含量高于Chu13×2和SE,而总烃含量在Chu13×2中较高.(2)B.braunii764的总脂和总烃含量分别为19.4%、23.4%,显著高于B.braunii765.     

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...     

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.     

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.     

Rapid method for the determination of lipid from the green alga Botryococcus braunii

Of various methods for lipid recovery in Botryococcus braunii UTEX 572, the most effective method was disruption of the cells with a bead-beater followed by extraction with chloroform/methanol (2:1, v/v). This gave a lipid content of 28.6% of dry wt. There was a significant relationship between in vivo fluorescence of cells stained with Nile Red and lipid content in B. braunii determined gravimetrically (r2 = 0.997). This suggested that the Nile Red staining as a rapid method was as good as the gravimetric method commonly used for lipid determination which requires toxic solvents and considerable time-consuming manipulations. © Rapid Science Ltd. 1998     

Toxicity effects of compressed and supercritical solvents on thermophilic microbial metabolism

Selection of biocompatible solvents is critical when designing bioprocessing applications for the in situ biphasic extraction of metabolic end-products. The prediction of the biocompatibility of supercritical and compressed solvents is more complicated than for liquid solvents, because their properties can change significantly with pressure and temperature. The activity of the anaerobic thermophilic bacterium, Clostridium thermocellum, was studied when the organism was incubated in the presence of compressed nitrogen, ethane, and propane at 333 K and multiple pressures. The metabolic activity of the organisms in contact with compressed solvents was analyzed using traditional indicators of solvent biocompatibility, such as log P, interfacial tension, and solvent density. The toxicity of the compressed solvents was compared with the phase and molecular toxicity effects measured in liquid alkanes at atmospheric pressure. Inactivation increased with time in the presence of the compressed solvents, but was constant in the presence of atmospheric liquid solvents. Knowledge of molecular and phase toxicity provides a framework for the interpretation of C. thermocellum metabolism in contact with atmospheric and compressed solvents.     

Dielectric Spectroscopy: a Rapid Method for the Determination of Solvent Biocompatibility During Biotransformations

Dielectric spectroscopy provides a convenient means of determining the degree of intactness of biological cells. 4-terminal dielectric measurements of suspensions of Saccharomyces cerevisiae at 0.4 MHz show that, as with all other biological cells, these organisms possess a substantial β-dispersion. The additional of octanol to such suspensions causes a rapid decrease in the electrical capacitance of the suspension, which parallels the cellular viability as determined by methylene blue staining. The kinetics of cell death are determined in part by the rate of dissolution of the organic solvent in the aqueous phase. The toxicity of several organic solvents to S. cerevisiae is studied using this technique, and is found to be dependent upon the polarity of the solvent. The present method provides a simple and rapid means for assessing the biocompatibility of solvents used in biotransformations.     

Purification and concentration of alkaline phosphatase by selective permeabilization of Escherichia coli using reverse micellar solutions

Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface-active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single-step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible.     

Solvation properties of ubiquinone-10 in solvents of different polarity

The solvation properties of ubiquinone-10 and ubiquinol-10 in a wide variety of solvents of polarity varying from alkanes to water are reported. Greatest solubility is observed in solvents of intermediate polarity and particularly where low polarity is combined with a pronounced tendency to interact with the benzoquinone substituent of the ubiquinone molecule. This includes solvents like chloroform and benzene. Ubiquinone-10 is somewhat less polar than ubiquinol-10 as judged by comparative solubilities of the two molecules. Proton-NMR chemical shift measurements and aggregation studies in selected solvents indicate that in ubiquinone-10 in the liquid phase and in solution in hydrocarbons like dodecane the molecules have a preferred association possibly involving stacking of the benzoquinone rings. Surface balance studies indicated that the surface-active character of ubiquinone-10 is relatively weak and only in a comparatively polar and highly structured solvent, formamide, was there evidence of an effect on surface tension of the solvent. The critical micelle concentratiom in this solvent was estimated to be about 5 M on the basis of surface tension measurements. Ubiquinone-10 is well known to form virtually insoluble monolayers at the air/water interface. Studies of the partition of ubiquinone-10 in binary mixtures of solvents suggest that the interaction of the benzoquinone ring substituent with structured polar solvents is considerably weaker than the internal cohesion between molecules of the solvent. No evidence on the basis of wide-angle X-ray diffraction measurements was obtained to indicate that solvent molecules were a component of the crystal lattice of ubiquinone-10 that had precipitated from solvent mixtures.     

Acyl Group Migrations in 2-Monoolein

Acyl migration in 2-monoolein dissolved in solvents under conditions common in lipid modification reactions has been studied. The effects on acyl migration of solvent, incubation temperature, water activity, polar additives and solid additives have been investigated. Extensive acyl migration occured in aliphatic hydrocarbons and water-miscible alcohols under dry conditions. The acyl migration rate could be decreased in several nonpolar solvents by adding a small amount of water or an alcohol. Increasing water activity had no effect in isooctane, but decreased the acyl migration rate dramatically in methyl tert-butyl ether and methyl isobutyl ketone. Several commonly used enzyme supports catalysed acyl migration, showing that supports with surface charges could catalyse acyl migration.     

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。     

Solvent selection strategies for extractive biocatalysis.

This report follows the development of systematic solvent screening strategies for the identification of superior pure solvents and introduces techniques for the identification of effective coextractants. Specifically, methods to predict the biocompatibility and extractant capability of solvents are discussed. Biocompatibility is predicted by using heuristic data or the correlations between bioactivity and the logarithm of the partition coefficient of the solvent or the concentration of solvent in the cell membrane. A computer program, known as the extractant screening program or ESP, has been developed to effectively predict the behavior of virtually any product in any solvent/aqueous system. It is demonstrated that a biocompatible yet poor solvent can be mixed with a toxic solvent that has better extractant properties to yield a mixture with improved solvent characteristics that is still biocompatible. The fact that solvents do not mix in an ideal manner is exploited by using ESP to identify solvent mixtures that are still biocompatible at relatively high concentrations of toxic solvent.     

几株丛粒藻烃类的气相色谱—质谱分析

用气相色谱-质谱分析定性鉴定了三株不同来源的丛粒藻(Botryococcus braunii)产生的烃类组分。丛粒藻株A(USA)和B(Germany)产生以C_nH_(2n-2)和C_nH_(2n-4)为主的烯基直链烃,丛粒藻C得自我国云南省抚仙湖,产生以丛粒藻烯和异丛粒藻烯为主的多分枝烃。讨论了不同丛粒藻产生不同烃类的原因。     

Formation of planar bilayer membranes from lipid monolayers. A critique.

The formation of planar bilayer membranes from lipid monolayers as described by Montal and Mueller (Proc. Natl. Acad. Sci. 1972. 69:3561) is analyzed. Bilayers absolutely free of alkane solvents or other nonpolar hydrocarbons can be formed on polytetrafluoroethylene (PTFE) (e.g. Teflon) septa only if certain boundary conditions are satisfied. Measurements have been made of the contact angles between monolayer-coated water and PTFE in the presence and absence of alkane solvents. The measurement suggest that the boundary conditions for formation of stable bilayers can be satisfied only when a nonpolar solvent is present. We conclude that the bilayer must be surrounded by a torus of alkane solvent, petroleum jelly, or silicone grease depending upon the details of technique used to form the bilayer. The non-polar solvent used in the formation of the bilayer may or may not be present in the bilayer depending upon the water solubility and size of the solvent molecule relative to the size of the alkyl chain of the lipid. Detailed sketches describing the formation of bilayers from monolayers are presented.     

Use of non-conventional media inMorinda citrifolia cell cultures

Non-conventional media, containing organic solvents as supplement, were exploited to obtain the non-lethal product release of plant cell secondary metabolites, using suspension cultures ofMorinda citrifolia as model system. The results of our preliminary studies about solvent biocompatibility show that the discrimination between biocompatible and toxic solvents can be achieved by means of two parameters: log P and critical solvent concentration. The last one seems to be a better indicator of solvent toxicity for living cells.Abbreviations log P logarithm of P - P solvent partition coefficient in a standard system n-octanol/water     

Evaluation of silicone tubing toxicity using tobacco BY2 culture

Summary Silicone tubing is frequently used for gas exchange in cell culture systems, due to its biocompatibility and high permeability to CO₂ and O₂. In cell culture chambers, medium pH and oxygen levels are often maintained by gas exchange through a coil of silicone tubing. Culture medium is recirculated between the gas exchanger and the culture chamber which contains a suspension of cells. We report that the type of agent used for silicone vulcanization (peroxide or platinum) can markedly affect its biocompatibility, and that tobacco cell culture represents a particularly sensitive indicator of tubing cytotoxicity. Under the conditions studied (cell suspension maintained with forward-reverse flow and stirring), peroxide-cured silicone tubing was toxic to the tobacco BY2 cell culture, in contrast to the platinum-cured silicone tubing that was completely biocompatible. Upon further investigation by mass spectrometry, it was determined that a component with a molecular mass of 288 Da, possibly a tetrachlorinated biphenyl, was present in culture medium in contact with peroxide-cured tubing but not in medium in contact with platinum-cured tubing. Additional curing of peroxide-cured tubing resulted in cell morphology and viability comparable to controls. These data suggest that improperly cured silicone tubing can release catalytic byproducts which can be toxic to plant cells, and that the BY2 tobacco cells represent a suitable model system for studies of materials biocompatibility.