Non-destructive hydrocarbon extraction from Botryococcus braunii BOT-22 (race B)

There is worldwide interest in developing algal biofuel. One main reason for the lack of success so far in producing a sustainable transport fuel from microalgae is the high cost of biomass processing, especially dewatering and oil extraction. There is also a significant cost involved in the energy content of the nutrient fertilisers required for biomass production. Non-destructive oil extraction or “milking” from algae biomass has the potential to bypass all of these hurdles. Using a “milking” strategy means that there would be no need for (a) biomass dewatering, (b) breaking cells for oil extraction and (c) addition of nutrients to the culture, resulting in a significant reduction in energy and fertiliser cost involved in production of biofuel from algae. We make use of the natural tendency of Botryococcus to produce external hydrocarbon in the extracellular matrix. In current study, we showed that external hydrocarbon from Botryococcus braunii BOT-22 can be non-destructively extracted using n-heptane (optimum contact time with n-heptane?=?20 min). We were able to recover almost the entire de novo-produced external hydrocarbons at 5- and 11-day intervals when the culture was maintained with or without 1 % CO₂ addition, respectively. This repeated non-destructive extraction of external hydrocarbon of B. braunii was possible for up to 70 days when 1 % CO₂ was supplied to the culture. When CO₂ was limited, a 70 % lower external hydrocarbon productivity was achieved using the same process. Although the productivity of external hydrocarbon of 9.33 mg L^?1 day^?1 of the “milked” culture is low in these un-optimised cultures, it was 1.3?±?0.2-fold higher compared with that of a conventional semicontinuous culture, showing the potential of this method.     

Effects of incident light intensity and light path length on cell growth and oil accumulation in Botryococcus braunii (Chlorophyta)

Botryococcus braunii was cultured in different light path length under different incident light intensity to investigate the effect of light on alga growth as well as hydrocarbon and fatty acid accumulation. Results indicated that longer light path length required higher incident light intensity in order to meet the light requirement of algal growth and hydrocarbon accumulation during the course of cultivation. However, hydrocarbon profile was only affected by the incident light intensity and not influenced by the light path length. High incident light intensity enhanced the accumulation of hydrocarbons with longer carbon chains. Besides, the fatty acid content and profiles were significantly influenced by both incident light intensity and light path. Higher fatty acid content and higher percentage of C18 and monounsaturated fatty acid components were achieved at the higher incident light intensity and lower light path length. Taken together, these results are benefit to improve its biomass and oil productivity through the optimization of light and photobioreactor design.     

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.     

Micronutrient Requirements for Growth and Hydrocarbon Production in the Oil Producing Green Alga Botryococcus braunii (Chlorophyta)

The requirements of micronutrients for biomass and hydrocarbon production in Botryococcus braunii UTEX 572 were studied using response surface methodology. The concentrations of four micronutrients (iron, manganese, molybdenum, and nickel) were manipulated to achieve the best performance of B. braunii in laboratory conditions. The responses of algal biomass and hydrocarbon to the concentration variations of the four micronutrients were estimated by a second order quadratic regression model. Genetic algorithm calculations showed that the optimal level of micronutrients for algal biomass were 0.266 μM iron, 0.707 μM manganese, 0.624 μM molybdenum and 3.38 μM nickel. The maximum hydrocarbon content could be achieved when the culture media contained 10.43 μM iron, 6.53 μM manganese, 0.012 μM molybdenum and 1.73 μM nickel. The validation through an independent test in a photobioreactor suggests that the modified media with optimised concentrations of trace elements can increase algal biomass by 34.5% and hydrocarbon by 27.4%. This study indicates that micronutrients play significant roles in regulating algal growth and hydrocarbon production, and the response surface methodology can be used to optimise the composition of culture medium in algal culture.     

Two-phase extraction culture of Botryococcus braunii producing long-chain unsaturated hydrocarbons

Hydrocarbon recovery was 32% and the total production of hydrocarbon in a two-phase extraction culture of Botryococcus braunii was 675 mg l^–1, which was 19% higher than that of single-phase culture. Addition of 60 ml dihexyl ether at mid-growth phase gave the highest yield. Specific growth rate and recovery of hydrocarbon in this case were 0.022 h^–1 and 26%, respectively.     

Hydrocarbon recovery and biocompatibility of solvents for extraction from cultures of Botryococcus braunii

Various water-immiscible solvents were tested for biocompatibility and hydrocarbon recovery under different contact conditions with the hydrocarbon-rich microalga Botryococcus braunii. Eighteen solvents were first selected from a database of 1500 compounds (compiled for solvent selection for ethanol recovery from Saccharomyces cerevisiae fermentation). Nine of these candidate solvents were shown to be biocompatible with B. braunii following short contact times. This biocompatibility tends to be associated with high molecular weights and high boiling points but strongly depends on solvent chemical structure. A low polarity is essential to biocompatibility and calculated octanol-water partition coefficients, or capacity factors determined by reversed-phase high-performance liquid chromatography (HPLC), are suitable predictors of biocompatibility with B. braunii. High recoveries of hydrocarbons directly from the algal culture require relatively polar solvents and are, therefore, inimical with maintenance of cell viability. The inaccessibility of weakly polar solvents to the cell surface appears to protect the algae but also prevents substantial recovery of the hydrocarbons stored in B. braunii outer walls. In order to achieve a high recovery, contact with the solvent must be carried out on algae concentrated by filtration. Then, a large fraction of B. braunii hydrocarbons can be recovered, after a short contact time, without impairing cell viability. Under these conditions, the pertinent solvent property is affinity for the nonpolar hydrocarbons, and the highest recovery yield, approximately 70% after contact for 30 min, is achieved with hexane.     

Mechanism of lipid extraction from Botryococcus braunii FACHB 357 in a biphasic bioreactor

Algal lipid of Botryococcus braunii could be produced continuously and in situ extracted in an aqueous-organic bioreactor. In this study, the cell ultra-structure and cell membrane permeability of B. braunii FACHB 357 were investigated to understand the mechanism of lipid extraction within the biphasic system. The results showed that biocompatible solvent of tetradecane could induce algal lipid accumulation, enable the cell membrane more active and the cell wall much looser. The exocytosis process was observed to be one of the mechanisms for lipid cross-membrane extraction in the presence of organic solvent.     

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...     

Improved algal oil production from Botryococcus braunii by feeding nitrate and phosphate in an airlift bioreactor

To improve biomass and microalgal oil production of Botryococcus braunii, fed‐batch culture was investigated in an airlift photobioreactor. The optimal feeding time of the fed‐batch culture was after 15 days of cultivation, where 1.82 g/L of the microalgal biomass was obtained in the batch culture. Nitrate nutrient was the restrictive factor for the fed‐batch cultivation while phosphate nutrient with high concentration did not affect the microalgal growth. The optimal mole ratio of nitrate to phosphate was 34.7:1, where nitrate concentration reached the initial level and phosphate concentration was one quarter of its initial level. With one feeding, the biomass of B. braunii reached 2.56 g/L after 18 days. Two feedings in 2‐day interval enhanced the biomass production up to 2.87 g/L after 19 days of cultivation. The hydrocarbon content in dry biomass of B. braunii kept at high level of 64.3% w/w. Compared with the batch culture, biomass production and hydrocarbon productivity of B. braunii were greatly improved by the strategic fed‐batch cultivation.     

In situ extraction (milking) of the two promising Botryococcus braunii strains Showa and Bot22 under optimized extraction time

Journal of Applied Phycology - In situ extraction or “milking” of microalgae is a promising approach to reduce downstream costs in order to produce low-value substances such as lipids...     

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.     

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.     

Catalytic conversion of Botryococcus braunii oil to diesel fuel under mild reaction conditions

Although the non-oxygenated triterpenic hydrocarbons (primarily C₃₄H₅₈) produced by the Bot-22 strain of Botryococcus braunii are exceptionally promising candidates for automobile fuel applications, it remains necessary to develop appropriate cracking technologies to convert these oils into a fuel with the required properties. The aim of this research was to develop an on-site process capable of converting the oil extracted from Bot-22 (Bot-oil) to a fuel that satisfies the specifications for diesel fuels, a so-called “drop-in” fuel. One of our primary goals for this on-site conversion was to have it operate as a low-temperature process. In this study, an experimental analysis of Bot-oil catalytic conversion under mild conditions was performed. The results demonstrated that the Bot-oil conversion reaction will proceed at temperatures as low as approximately 200 °C, and that the lowest temperature which resulted in a suitably efficient reaction was found to be 260 °C, generating a yield of 85 %. The physical properties of the converted oil were found to come close to satisfying Japan’s JIS #2 diesel fuel specification. The converted oil’s cetane number (CN), one of the most important quality indexes of diesel fuels, was estimated by measuring its ignition delay in a constant-volume combustion chamber. The estimated CN of the converted oil had a value of 40, which is 5 points below the JIS #2 requirement. It is thought that the Bot-oil can be used to produce a drop-in fuel using a simple on-site conversion process, although further development is required to achieve this aim.     

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.     

能源微藻葡萄藻的研究进展

在石油资源日趋枯竭、能源需求量日益扩大和油品结构性矛盾长期存在的多重压力下,寻找和开发柴油替代能源对保障我国的能源安全、促进国家经济社会可持续发展具有重要战略意义。葡萄藻是一种世界性分布的淡水或微咸水单细胞微藻,富含烃类、油脂和其它化学物质,而且含烃量可高达细胞干重的86%,是一种潜在的能源微藻。论文综述了近年来有关葡萄藻研究的各个方面的进展,主要内容包括葡萄藻的分类学地位、葡萄藻的细胞学特征、烃类的合成途径、葡萄藻产烃的调控因素以及葡萄藻的收集和烃类的回收途径等,期望为葡萄藻的合理开发利用提供参考。     

Simple, rapid and cost-effective method for high quality nucleic acids extraction from different strains of Botryococcus braunii

This study deals with an effective nucleic acids extraction method from various strains of Botryococcus braunii which possesses an extensive extracellular matrix. A method combining freeze/thaw and bead-beating with heterogeneous diameter of silica/zirconia beads was optimized to isolate DNA and RNA from microalgae, especially from B. braunii. Eukaryotic Microalgal Nucleic Acids Extraction (EMNE) method developed in this study showed at least 300 times higher DNA yield in all strains of B. braunii with high integrity and 50 times reduced working volume compared to commercially available DNA extraction kits. High quality RNA was also extracted using this method and more than two times the yield compared to existing methods. Real-time experiments confirmed the quality and quantity of the input DNA and RNA extracted using EMNE method. The method was also applied to other eukaryotic microalgae, such as diatoms, Chlamydomonas sp., Chlorella sp., and Scenedesmus sp. resulting in higher efficiencies. Cost-effectiveness analysis of DNA extraction by various methods revealed that EMNE method was superior to commercial kits and other reported methods by >15%. This method would immensely contribute to area of microalgal genomics.     

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.