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Phylogeny tree placement of 16 Botryococcus braunii strains. Strict consensus tree based with overlaid bootstrap values and concensus threshold of 50% obtained by maximum-likelihood analysis on 18-rRNA gene sequences from 19 Botryococcus culture collection strains, 4 reference strains AJ581910 Ayame (B), AJ581911 Songkla (L), AJ581912 (A) and AY197640 Tow (A), 31 Bot strains from Kawachi et al., 2012 and species from other chlorophytes that are used as an outgroup.
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Botryococcus braunii can produce both long-chain hydrocarbons as well as carbohydrates in large quantities, and is therefore a promising industrial organism for the production of biopolymer building blocks. Many studies describe the use of different strains of Botryococcus braunii but differences in handling and cultivation conditions make the comp...
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... assess the relationships between our 16 culture collection strains we used 18S rRNA sequence analysis following the method that was used to characterize 31 BOT strains by Kawachi et al. (2012), who strongly linked the B. braunii chemical races with their phylogenetic placement. Our strains fit well with Kawachi's results (Fig. 1). Strains UTEX 572, UTEX LB572, AC755, CCALA835, CCALA777, CCALA778, K-1489, CCAP807/2 and SAG30.81 are in subclade 5 which refers to race A. Strains AC759, AC760, AC761 and Showa are in subclade 3 which refers to race B, and strains AC765, AC767 and AC768 in subclade 1 and 2 which refers to race L. In Fig. 2, the assigned subclades 1, ...
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... As the Enomoto Alga ( Fig. 2) is capable of proliferating 1000 times faster than the conventional species (doubling time of 2.58 days) within one month, this study aims to use its compositions in the quantitative appraisal that means the production of hydrocarbon oil to the monthly amount of 1800-4200 (g/L). It is done only with the assistance of photosynthesis and even owning the petroleum quality (Alves et al., 2010;Asahi Shimbun, 2022; "Formation of the Joint Venture for Bio-fuel produced by Algae -Aiming for the commercialization in high concentrated fuel producing algae-| Other|2011FY|News|IHI Corporation," n.d.; Gouveia et al., 2017). The optimistic outcome of this research reveals that around 50% dry mass can be used as fuel and it is feasible having a sulfur-free one under particular cultivation (Japan INC, n.d.). ...
Considering algae as a diversified source of various chemicals, the purifying process, and treatment pathways, also deciding on algal strains sound vital rather than coming across. To attain low-emission fuels from Enomoto, fast catalytic pyrolysis reactions could provide anti-knock properties of gasoline blends. The whole issue is under investigation by lifecycle assessment (LCA) and exergy analysis within this scope. There has been no "Mixed Review Study" for these analyses on effective SATBA 1 's goals and Enomoto biogasoline, hitherto. In this study, the consequence of carbon cycle stabilization has been pursued from the beginning of the supply chain, production (electrical energy with the highest contribution), and distribution of Enomoto biogasoline, and the share of total emissions reached 14.14 kgCO 2 /kg fuel. In this research work, LCA outcome was performed with energy balance to optimize the dead states of various exergy losses and compared it with the values of a base reference by the Greenhouse gases, Regulated Emissions, and Energy use in Transportation (GREET) method. For this purpose, for every 838 kJ/L of Enomoto oil, 128834.824 MJ/day of chemical exergy was produced in the system, and the share of physical exergy input at 94355.16 MJ/day, which indicates high efficiency of the process. There was an inextricable correlation between Neat Energy Ratio (NER) 2.27, 80.3% efficiency, and low carbon dioxide (CO 2) emissions with Energy Consumption Ratio (ECR) of approximately 0.001. Also, improving the process and reducing the loss of exergy, i.e. destructive exergy equivalent to 973,879 kJ/day can be significantly improved by removing the heat exchanger and replacing the reactor and condenser type. As a result, it has proved that this process is economically feasible for 1250 L per day of Enomoto biogasoline production with an annual depreciation of about $18,
... For instance, the μ max value of fucose production-capable Botrycoccus braunii was 0.05-0.17 [60,61]. Compared to other microalgae, the growth of Oocystis sp. ...
... To date, there has been limited knowledge about the relationship between Oocystis sp. and fucose. Previously, fucose was found only in the diatoms P. tricornutum [68], Nostoc microscopicum [68], B. braunii (6-463 mg/l) [61], and Graesiella sp. [69] in very low concentrations. ...
Comparative gene identification-58 (CGI-58) is an activating protein of triacylglycerol (TAG) lipase. It has a variety of catalytic activities whereby it may play different roles in diverse organisms. In this study, a homolog of CGI-58 in Phaeodactylum tricornutum (PtCGI-58) was identified. PtCGI-58 was localized in mitochondria by GFP fusion protein analysis, which is different from the reported subcellular localization of CGI-58 in animals and plants. Respectively, PtCGI-58 overexpression resulted in increased neutral lipid content and TAG accumulation by 42-46% and 21-32%. Likewise, it also increased the relative content of eicosapentaenoic acid (EPA), and in particular, the EPA content in TAGs almost doubled. Transcript levels of genes involved in de novo fatty acid synthesis and mitochondrial β-oxidation were significantly upregulated in PtCGI-58 overexpression strains compared with wild-type cells. Our findings suggest that PtCGI-58 may mediate the breakdown of lipids in mitochondria and the recycling of acyl chains derived from mitochondrial β-oxidation into TAG biosynthesis. Moreover, this study potentially illuminates new functions for CGI-58 in lipid homeostasis and provides a strategy to enrich EPA in algal TAGs.
... Biofuel compounds in the range of 25-75% within the biomass have been recorded that can be utilized as feedstock for sustainable bioenergy sources [4][5][6]. However, the growth rate of B. braunii is considerably slower when compared to other oleaginous microalgae [7,8]. Hence, it is not currently feasible to employ this microalga for its biomass and chemical products [9,10]. ...
... The E1.0H15 mutant under control conditions offered biomass productivity at 138.88 ± 1.22 mg L − 1 d − 1 which was considered 184.59% higher in capacity when compared to the wild type. The achieved biomass productivity was also considered larger than other strains such as B. braunii AC755 (60 mg L − 1 d − 1 ) and B. braunii N836 (40 mg L − 1 d − 1 ) in the work carried out by Gouveia et al. [7] and Ranga Rao et al. [81], respectively. Importantly, EMS mutagenesis significantly improved E1.0H15 features involving the accumulation of lipids and hydrocarbons. ...
Green microalga Botryococcus braunii is a eukaryotic photosynthetic microorganism considered as a rich source of biofuel compounds, such as hydrocarbons and lipids, despite its rather slow growth. To improve the biomass, hydrocarbon, and lipid productivity, an ethyl methanesulfonate-induced mutagenesis was carried out on the indigenous microalga, Botryococcus braunii AARL G036. Among those mutants, EMS-mutant E1.0H15 gave the highest biomass and hydrocarbon productivity, which was higher than 1.17, 1.45, and 1.16-fold, respectively, when compared with the wild type (WT). Subsequently, two-stage cultivation with crucial stress factors was successfully exploited to boost the biofuel productivity of EMS-mutant E1.0H15 by increasing the lipid content by 1.22-fold (up to 48.6%) and by 1.20-fold for the hydrocarbon content (up to 71.6%) compared to one-stage cultivation. More importantly, after 24-month maintenance, the high biofuel productivity in EMS-mutant E1.0H15 remains unchanged. In addition, the fatty acid composition (>96% C16–C18) and fuel properties of the mutated microalgal lipid-derived biodiesel indicated good oxidation stability (17.5 h) and high heating values (38.6 MJ kg⁻¹), which were in accordance with those of the international standards, i.e., EN-14214 and ASTM D675140. This strategy could contribute greatly to the further development of the microalgal strain, allowing improvement and cultivation for the desired biofuel properties.
... The BoCAPS allows us to estimate chemical race without the timeconsuming and expensive analysis of hydrocarbons and will be useful for high-throughput screening and ecological studies of wild populations. Because the BoCAPS assumes the consistency of the correspondence of chemical races with the major clades of 18S rRNA phylogenetic tree (Fig. 2), further confirmations of the genetic differentiation between chemical races are important to assure the accuracy of Previous screening studies of appropriate B. braunii strains for biofuel production adopted growth rate and hydrocarbon content as selection criteria [19,[43][44][45]. Recently, Kleinert and Griehl provided a new screening procedure of suitable B. braunii strains for nondestructive in situ hydrocarbon extraction [46]. ...
The colony-forming microalga Botryococcus braunii produces petroleum-like hydrocarbons and is a promising source for biofuel production. B. braunii is classified into three chemical races (A, B, L) plus a tentative class (S), based on the structure of its hydrocarbons. The determination of hydrocarbon structure in a newly isolated strain requires several months for culturing and analysis. As potential for biofuel production differs among races, a rapid method for estimating race is needed for the effective bioprospecting. This study aimed to develop a DNA-based rapid estimation method (BoCAPS) of the chemical races of B. braunii and to test the feasibility of direct PCR from a single colony. A total of 247 wild strains were isolated from 29 ponds in Japan and Indonesia, their 18S rRNA sequences determined, and a phylogenetic tree constructed based on the sequences of standard strains with known chemical races. Polymorphisms between clades of the phylogenetic tree corresponding to chemical races were mapped, and a cleaved amplified polymorphic sequence (CAPS) marker was developed for race discrimination. Direct PCR was tested on a single colony as a template. We demonstrated that HaeIII restriction of the PCR product enables race discrimination on agarose gels, and that direct PCR amplifies the 18S rRNA gene from a single colony without any pre-treatment. With BoCAPS, we investigated race compositions of the isolated strains and determined that the B-race was dominant (74 %). Neither the A-race nor S-race strains were isolated from tropical regions. In addition, we observed a few ponds in which minor races (L or A) were dominant and some ponds with a mixture of different races. The BoCAPS allows us to estimate chemical race without the time-consuming and expensive analysis of hydrocarbons and will be useful for high-throughput screening and ecological studies of wild populations.
... Some very old cultures (more than 1 year old) maintained in our lab exhibited bright red/orange colours. In addition, B. braunii 807/2 has been studied widely in relation to its hydrocarbon production including in outdoor culture (Moheimani et al. 2013b;Zhang 2013;Gouveia et al. 2017;Blifernez-Klassen et al. 2018;Kleinert and Griehl 2021). Hence, we have good understanding about the strain specifically related to its limits to growth factors for hydrocarbon production but not for carotenoid production. ...
Botryococcus braunii CCAP 807/2 has been studied intensively for biofuel production due to its high hydrocarbon content. This strain is also capable of producing high value carotenoids. The aim of the study was to analyse the carotenoid production of B.braunii 807/2 under different growing conditions, first, by using different media and light intensities in indoors, and next, to examine the carotenoid composition between green, intermediately pigmented and red B. braunii grown in indoors and outdoors. The alga was cultured indoors under two different light intensities (100 and 500 μmol photons m⁻² s⁻¹) using three different media: a control with complete modified CHU 13 medium, modified CHU 13 without N and modified CHU13 without N + 2Fe. All cultures were grown at 25 °C with 12:12 h light:dark cycle and were mixed with magnetic stirrers. For the determination of carotenoid composition at different stages, the green, intermediately pigmented and red cells were collected from indoor and outdoor cultures and analysed for their carotenoid composition using HPLC. The cultures grown at high light intensity reached the highest biomass yield at 0.6 g L⁻¹ on day 16, whereas their counterparts at low light intensity took 30 days to reach the same biomass yield. The carotenoid production of B. braunii 807/2 at high light intensity increased up to twofold in 2 days compared to the ones grown at low light. Botryococcus braunii 807/2 accumulates lutein, canthaxanthin and astaxanthin and β,β-carotene as the main carotenoids. Whilst lutein was the major carotenoids of the green/intermediate cells, canthaxanthin and astaxanthin were the predominant carotenoids of the red cells under indoor and outdoor culture, respectively. This study suggests that Botryococcus braunii 807/2 is a potential candidate for the production of lutein and/or astaxanthin. It accumulates a high amount of lutein when grown under optimum conditions and a high amount of astaxanthin when grown under sub-optimum conditions outdoors.
... Based on morphological and molecular identification, it was concluded that the microalga strain SCS-1905 belonged to Chlorophyta, Trebouxiophyceae, Trebouxiales, Botryococcaceae, Botryococcus, Botryococcus braunii, and showed a close phylogenetic relationship with B. braunii AC768. Accordingly, it was classified as race L [33]. Previous studies mostly focused on the excellent hydrocarbon production properties and were committed to its development and application as a source of biofuels, due to the excellent performance in synthesizing and accumulating various lipids and hydrocarbons [34]. ...
... Our results were consistent with previous studies. Metzger and Largeau (2005) analyzed the exopolysaccharides of 16 different strains of B. braunii and found that galactose was the primary monosaccharide in 10 of them, while glucose was the main monosaccharide in the other six [33]. Allard and Casadevall (1990) also identified several uncommon O-methylated sugars, including 3-O-methylrhamnose, 3-O-methylfucose and 6-O-methylhexose [17]. ...
Botryococcus braunii, a prestigious energy microalga, has recently received widespread attention because it can secrete large amounts of exopolysaccharides (EPS) with potential applications in food, cosmetics, and nutraceuticals. Unfortunately, the insufficiency of research on the bioactivity and structure–activity relationship of B. braunii EPS has impeded the downstream applications. In the present study, alcohol precipitation, deproteinization, and DEAE-cellulose column chromatography were used to extract and purify B. braunii SCS-1905 EPS. It was found that B. braunii SCS-1905 EPS were high-molecular-weight heteropolysaccharides containing uronic acid (7.43–8.83%), protein (2.30–4.04%), and sulfate groups (1.52–1.95%). Additionally, the EPS primarily comprised galactose (52.34–54.12%), glucose (34.60–35.53%), arabinose (9.41–10.32%), and minor amounts of fucose (1.80–1.99%), with the presence of a pyranose ring linked by a β-configurational glycosidic bond. Notably, the antioxidant activity of crude exopolysaccharides (CEPS) was stronger, and the half maximal inhibitory concentration (IC50) for ABTS and hydroxyl radicals was significantly lower than that of deproteinized exopolysaccharides (DEPS). Overall, this study indicated a potential application of B. braunii SCS-1905 EPS as a natural antioxidant. In summary, B. braunii EPS could be used as a potential feedstock for the production of antioxidant health foods.
... The B race chemotype has been the most widely studied group, and some strains within it are recognized for producing unsaturated poly-methylated triterpene-like lipids known as botryococcenes (C n H 2n−10 , n = 30-37) which represent 8-35% of total biomass (Okada et al. 1995(Okada et al. , 1997Yoshimura et al. 2013). Other less studied groups are known such as the L race which produces 3-7.5% of a single hydrocarbon known as licopadiene (C 40 H 78 ), and the S race that accumulates an epoxy-n-alkane and a saturated n-alkane with fatty acyl length of C 18 and C 20 , respectively (Gouveia et al. 2017). ...
In this study the metabolic responses of Botryococcus braunii were analyzed upon different inorganic carbon dosages and nutrient limitation conditions in terms of lipid and biomass productivity, as well as photosynthetic performance. The nutritional schemes evaluated included different levels of sodium bicarbonate and nitrogen and phosphorus starvation, which were contrasted against standard cultures fed with CO 2 . Bicarbonate was found to be an advantageous carbon source since high dosages caused a significant increase in biomass and lipid productivity, in addition to an enhanced photosynthetic quantum yield and neutral lipids abundance. This contrasts to the commonly used approach of microalgae nutrient limitation, which leads to high lipid accumulation at the expense of impaired cellular growth, causing a decline in overall lipid productivity. The lipidome analysis served to hypothesize about the influence of the nutritional context on B. braunii structural and storage lipid metabolism, besides the adaptive responses exhibited by cells that underwent nutrient stress.
... Los parámetros más importantes para el crecimiento de microalgas son el carbono y el nitrógeno, y una relación C: N eficiente es crucial para evaluar la productividad de los lípidos y la biomasa de . braunii [103]. así mismo, se han realizado diversos estudios donde se ha demostrado el efecto potenciador de ciertos medios de cultivo como Chu-13, BG-11 [15] y el AF-2 [67]; sin embargo es importante recordar que de acuerdo con los resultados reportados por Largeau et al [104], la síntesis y deposición de hidrocarburos y lípidos son directamente proporcionales al crecimiento del microorganismo. ...
Las microalgas son microorganismos fotosintéticos los cuales pueden ser eucariotas (algas) o procariotas (cianobacterias) y pueden estar presentes en todos los hábitats de la tierra. Este artículo ofrece una descripción bibliográfica sobre análisis técnicos en la producción industrial de los principales metabolitos extraídos de Botryococcus braunii con el objetivo de analizar, determinar e identificar los avances científicos para la utilización de esta alga como un recurso potencial, por medio de una recopilación bibliométrica de 20 años sobre artículos de investigaciones demostrando gran interés en el campo de las microalgas. Se ha realizado este trabajo con el propósito de averiguar los estudios en el área de la microalga Botryococcus braunii y sus aplicaciones industriales, también determinar los principales metabolitos como hidrocarburos, exopolisacáridos, carotenoides, proteínas, en el aprovechamiento de procesos industriales como materiales, alimentos, productos farmacéuticos, nanopartículas y biocombustibles. Debido a sus vías metabólicas particulares, su adaptabilidad fisiológica y su uso en sistemas de cultivo avanzados, existe un gran potencial para producir sustancias químicas de alto valor a partir de cepas de Botryococcus braunii.
... However, few results can be found in the literature, although it is being implemented elsewhere where cultivation at pilot or large scale takes place. Botryococcus braunii is a green colony-forming microalga which has the almost unique capacity to synthesise, accumulate and excrete large amounts of long-chain hydrocarbons and/or interesting groups of polysaccharides, such as exopolysaccharides (EPS) (Gouveia et al. 2017). Such metabolites can be further converted into bio-products, and, accordingly, the attention of researchers has increased in attempts to exploit B. braunii as a renewable source of products (Banerjee et al. 2002;Li and Qin 2005). ...
... Although the use of different commercial low-cost nutrients has been previously reported for B. braunii (Kurinjimalar et al. 2017), to the best of our knowledge, it is the first attempt to cultivate Botryococcus with commercial agricultural NPKs. The algae selection was made based on a previous study that demonstrated their potential as polysaccharide (race A) and hydrocarbon (race B) producers (Gouveia et al. 2017). As a first approach, these microalgae were cultivated with different nitrogen sources in order to identify the preferred one to know which fertilisers could be more suitable to be used. ...
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 medium. Botryococcus braunii, a green microalga, has the almost unique capacity to accumulate and excrete large amounts of long-chain hydrocarbons and/or interesting groups of polysaccharides which can be further converted into bio-products. However, limitations in growth are currently hindering its industrial production. In this work, the use of different agricultural fertilisers (NPKs) was evaluated for the cultivation of two B. braunii races (A and B) in terms of productivity and final media labour and cost. Results corroborated that fertilisers-based media are easier to prepare and their prices are considerably lower compared to common culture media. At the same time, good growth performance and photosynthetic efficiency can be maintained and carbohydrate and hydrocarbon productivities can be further enhanced. However, special attention should be given to each particular strain since different behaviour in growth and metabolite production can be observed depending on the media composition. The significantly higher productivities obtained, together with the important reduction in media price when using commercial fertilisers and the advantages related to the easiness to prepare the culture media based on NPK fertilizers, represent an important achievement for the development of an industry based on these renewable products.
... There should be wild strains that grow faster and are more productive than the Showa strain, but, to the best of our knowledge, there has been no large-scale study seeking these strains, a potential of natural genetic resource. A few wild strains and collections of B. braunii have been investigated [26][27][28][29][30] , but researchers failed to find significantly faster and more productive strains than Showa, probably due to the limited number of strains tested (< 10) and the limited geographic range of locations searched. ...
Algal biofuel research aims to make a renewable, carbon-neutral biofuel by using oil-producing microalgae. The freshwater microalga Botryococcus braunii has received much attention due to its ability to accumulate large amounts of petroleum-like hydrocarbons but suffers from slow growth. We performed a large-scale screening of fast-growing strains with 180 strains isolated from 22 ponds located in a wide geographic range from the tropics to cool-temperate. A fast-growing strain, Showa, which recorded the highest productivities of algal hydrocarbons to date, was used as a benchmark. The initial screening was performed by monitoring optical densities in glass tubes and identified 9 wild strains with faster or equivalent growth rates to Showa. The biomass-based assessments showed that biomass and hydrocarbon productivities of these strains were 12-37% and 11-88% higher than that of Showa, respectively. One strain, OIT-678 established a new record of the fastest growth rate in the race B strains with a doubling time of 1.2 days. The OIT-678 had 36% higher biomass productivity, 34% higher hydrocarbon productivity, and 20% higher biomass density than Showa at the same cultivation conditions, suggesting the potential of the new strain to break the record for the highest productivities of hydrocarbons.
