Optimized medium conditions maximize colony regeneration from a single cell of Botryococcus braunii NIES836.

Botryococcus braunii is a colonial alga recognized for its slow growth but high hydrocarbon accumulation. Although using genetic engineering to increase the growth rate and hydrocarbon yield of B. braunii is desirable, the presence of an extracellular matrix (ECM) significantly hinders the emergence of a homogeneous colony from a single DNA-transformed cell. Previously, we developed a method to isolate single cells without ECM from colonies. However, following the isolation of single cells, several months are required to regenerate colonies with a sufficient cell mass for subsequent analysis. To shorten the colony regeneration period, we investigated basal media and medium components, along with growth-promoting additives, in a series of single-factor experiments and optimized the concentrations of the medium constituents via response surface methodology (RSM). The results of the single-factor experiments revealed that the nitrogen source (a mixture of NaNO3 and NH4NO3), 1-naphthylacetic acid (NAA) and Fe(III)-citrate significantly increased the growth of B. braunii single cells into colonies. The optimal medium composition identified by RSM included 151.6 mg/L nitrogen source, 2.419 mg/L NAA and 15.3 mg/L Fe(III)-citrate. Verification experiments showed that the optimized medium resulted in a 1.75-fold increase in colony size compared with that of colonies grown in nonoptimized AF6 medium. This is the first report of the optimal medium composition for the regeneration of B. braunii colonies from single cells.

A bioprocess engineering approach for the production of hydrocarbons and fatty acids from green microalga under high cobalt concentration as the feedstock of high-grade biofuels.

Botryococcus braunii, a colonial green microalga which is well-known for its capacity to synthesize hydrocarbons, has significant promise as a long-term source of feedstock for the generation of biofuels. However, cultivating and scaling up B. braunii using conventional aqua-suspended cultivation systems remains a challenge. In this study, we optimized medium components and light intensity to enhance lipid and hydrocarbon production in a multi-cultivator airlift photobioreactor. BBM 3N medium with 200 µmol/m2/s light intensity and a 16 h light-8 h dark regimen yielded the highest biomass productivity (110.00 ± 2.88 mg/L/day), as well as the highest lipid and hydrocarbon content. Cultivation in a flat-panel bioreactor resulted in significantly higher biomass productivity (129.11 ± 2.74 mg/L/day), lipid productivity (32.21 ± 1.31 mg/L/day), and hydrocarbon productivity (28.98 ± 2.08 mg/L/day) compared to cultivation in Erlenmeyer flasks and open 20-L raceway pond. It also exhibited 20.15 ± 1.03% of protein content including elevated levels of chlorophyll a, chlorophyll b, and carotenoids. This work is noteworthy since it is the first to describe fatty acid and hydrocarbon profiles of B. braunii during cobalt treatment. The study demonstrated that high cobalt concentrations (up to 5 mg/L of cobalt nitrate) during Botryococcus culture affected hydrocarbon synthesis, resulting in high amounts of n-alkadienes and trienes as well as lipids with elevated monounsaturated fatty acids concentration. Furthermore, pyrolysis experiments on microalgal green biomass and de-oiled biomass revealed the lipid and hydrocarbon compounds generated by the thermal degradation of B. braunii that facilitate extra economical value to this system.

A simple method for the preparation of single cells and regeneration of colonies of Botryococcus braunii NIES836.

Botryococcus braunii, a colonial alga, is known for notably slow growth; however, the growth rate and hydrocarbon productivity are expected to be improved using genetic modification techniques. Nevertheless, B. braunii has a hydrocarbon-rich extracellular matrix (ECM), and the ECM is a major barrier to DNA transformation. To analyse and utilize genetically modified B. braunii, it is essential to regenerate genetically homogeneous colonies derived from single cells. In this study, we developed a novel, simple method for harvesting viable single cells of B. braunii by centrifugation of the culture and subsequent filtration alone. The harvest of single cells was made possible by culturing B. braunii colonies in AF6 medium until the depletion of nitrogen and phosphorus sources and then releasing the single cells in colonies into the medium. Twenty-day culture of single cells in a 96-well plate resulted in 96% regeneration of colonies, and the regeneration of colonies was also confirmed on agar medium. This is the first report of colony regeneration from single cells of B. braunii. We believe that our method developed in this study will contribute greatly to the advancement of genetic modification techniques for B. braunii.

Oxidative Stress- and Autophagy-Inducing Effects of PSI-LHCI from Botryococcus braunii in Breast Cancer Cells.

Botryococcus braunii (B. braunii) is a green microalga primarily found in freshwater, reservoirs, and ponds. Photosynthetic pigments from algae have shown many bioactive molecules with therapeutic potential. Herein, we report the purification, characterization, and anticancer properties of photosystem I light-harvesting complex I (PSI-LHCI) from the green microalga B. braunii UTEX2441. The pigment-protein complex was purified by sucrose density gradient and characterized by its distinctive peaks using absorption, low-temperature (77 K) fluorescence, and circular dichroism (CD) spectroscopic analyses. Protein complexes were resolved by blue native-PAGE and two-dimensional SDS-PAGE. Triple-negative breast cancer MDA-MB-231 cells were incubated with PSI-LHCI for all of our experiments. Cell viability was assessed, revealing a significant reduction in a time- and concentration-dependent manner. We confirmed the internalization of PSI-LHCI within the cytoplasm and nucleus after 12 h of incubation. Cell death mechanism by oxidative stress was confirmed by the production of reactive oxygen species (ROS) and specifically superoxide. Furthermore, we monitored autophagic flux, apoptotic and necrotic features after treatment with PSI-LHCI. Treated MDA-MB-231 cells showed positive autophagy signals in the cytoplasm and nucleus, and necrotic morphology by the permeabilization of the cell membrane. Our findings demonstrated for the first time the cytotoxic properties of B. braunii PSI-LHCI by the induction of ROS and autophagy in breast cancer cells.

Exopolysaccharides from the Energy Microalga Strain Botryococcus braunii: Purification, Characterization, and Antioxidant Activity.

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 ß-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.

In Silico and Cellular Differences Related to the Cell Division Process between the A and B Races of the Colonial Microalga Botryococcus braunii.

Botryococcus braunii produce liquid hydrocarbons able to be processed into combustion engine fuels. Depending on the growing conditions, the cell doubling time can be up to 6 days or more, which is a slow growth rate in comparison with other microalgae. Few studies have analyzed the cell cycle of B. braunii. We did a bioinformatic comparison between the protein sequences for retinoblastoma and cyclin-dependent kinases from the A (Yamanaka) and B (Showa) races, with those sequences from other algae and Arabidopsis thaliana. Differences in the number of cyclin-dependent kinases and potential retinoblastoma phosphorylation sites between the A and B races were found. Some cyclin-dependent kinases from both races seemed to be phylogenetically more similar to A. thaliana than to other microalgae. Microscopic observations were done using several staining procedures. Race A colonies, but not race B, showed some multinucleated cells without chlorophyll. An active mitochondrial net was detected in those multinucleated cells, as well as being defined in polyphosphate bodies. These observations suggest differences in the cell division processes between the A and B races of B. braunii.

Exopolysaccharides from microalgae: production, characterization, optimization and techno-economic assessment.

Microalgae cultivation for exopolysaccharide production has getting more attention as a result of their high hydrocarbon biosynthesis skill. The aim of this study is to examine the exopolysaccharide production potential of different species of microalgae. In this context, exopolysaccharides were produced from Chlorella minutissima, Chlorella sorokiniana and Botryococcus braunii microalgae and the effects of carbon and nitrogen content in the growth medium and illumination time on exopolysaccharide production were analyzed statistically using Box-Behnken experimental design. In addition, techno-economic assessment of exopolysaccharide production were also performed by using the most productive microalgae and optimum conditions determined in this study. As a result of the experiments, it was seen that C. minutissima, C. sorokiniana and B. braunii produced 0.245 ± 0.0025 g/L, 0.163 ± 0.0016 g/L and 0.117 ± 0.0007 g/L exopolysaccharide, respectively. Statistically, it was observed that there was an inverse relationship between the exopolysaccharide production and investigated parameters such as illumination period and carbon and nitrogen amounts of culture mediums. The techno-economic assessment comprising microalgal exopolysaccharide (EPS) bioprocess was carried out, and it showed that the system can be considered economically viable, yet can be improved with biorefinery approach.

Channeling of Carbon Flux Towards Carotenogenesis in Botryococcus braunii: A Media Engineering Perspective.

Microalgae, due to their unique properties, gained attention for producing promising feedstocks having high contents of proteins, antioxidants, carotenoids, and terpenoids for applications in nutraceutical and pharmaceutical industries. Optimizing production of the high-value renewables (HVRs) in microalgae requires an in-depth understanding of their functional relationship of the genes involved in these metabolic pathways. In the present study, bioinformatic tools were employed for characterization of the protein-encoding genes of methyl erythritol phosphate (MEP) pathway involved in carotenoid and squalene biosynthesis based upon their conserved motif/domain organization. Our analysis demonstrates nearly 200 putative genes showing a conservation pattern within divergent microalgal lineages. Furthermore, phylogenomic studies confirm the close evolutionary proximity among these microalgal strains in the carotenoid and squalene biosynthetic pathways. Further analysis employing STRING predicts interactions among two rate-limiting genes, i.e., phytoene synthase (PSY) and farnesyl diphosphate farnesyl synthase (FPPS), which are specifically involved in the synthesis of carotenoids and squalene. Experimentally, to understand the carbon flux of these rate-limiting genes involved in carotenogenesis, an industrial potential strain, namely, Botryococcus braunii, was selected in this study for improved biomass productivity (i.e., 100 mg L-1 D-1) along with enhanced carotenoid content [0.18% dry cell weight (DCW)] when subjected to carbon supplementation. In conclusion, our approach of media engineering demonstrates that the channeling of carbon flux favors carotenogenesis rather than squalene synthesis. Henceforth, employing omics perspectives will further provide us with new insights for engineering regulatory networks for enhanced production of high-value carbon biorenewables without compromising growth.

Predicting biomass and hydrocarbon productivities and colony size in continuous cultures of Botryococcus braunii showa.

Mathematical models were developed to predict biomass and hydrocarbon productivities and colony size (ouputs) of Botryococcus braunii showa cultures based on light intensity, temperature and dilution rate (inputs). These models predicted the following maximum values: biomass productivity, 1.3 g L-1 d-1; hydrocarbon productivity, 1.5 mg L-1 d-1; colony size, 320 µm under different culture conditions respectively. These values were confirmed experimentally. Additionally, the combination of inputs that simultaneously maximize all the possible outputs combinations were determined. The prediction for biomass-hydrocarbon-colony size were 1 g L-1 d-1, 12.05 mg L-1 d-1 and 156.8 µm respectively; biomass productivity-hydrocarbon productivities: 1 g L-1 d-1 and 13.94 mg L-1 d-1 respectively; biomass productivity-colony size: 1 g L-1 d-1 and 172.8 µm respectively; hydrocarbon productivity-colony size: 9 mg L-1 d-1 and 240 µm respectively. All these predictions were validated experimentally. These models might be very useful to implement a Botryococcus braunii showa large scale production.

Reducing self-shading effects in Botryococcus braunii cultures: effect of Mg2+ deficiency on optical and biochemical properties, photosynthesis and lipidomic profile.

Microalgae biomass exploitation as a carbon-neutral energy source is currently limited by several factors, productivity being one of the most relevant. Due to the high absorption properties of light-harvesting antenna, photosynthetic cells tend to capture an excessive amount of energy that cannot be entirely channeled through the electron transfer chain that ends up dissipated as heat and fluorescence, reducing the overall light use efficiency. Aiming to minimize this hurdle, in this work we studied the effect of decreasing concentrations of Magnesium (Mg2+) on the chlorophyll a content, photosynthetic performance, biomass and lipid production of autotrophic cultures of Botryococcus braunii LB 572. We also performed, for the first time, a comparative lipidomic analysis to identify the influence of limited Mg2+ supply on the lipid profile of this algae. The results indicated that a level of 0.0037 g L-1 MgSO4 caused a significant decline on chlorophyll a content with a concomitant 2.3-fold reduction in the biomass absorption coefficient. In addition, the Mg2+ limitation caused a decrease in the total carbohydrate content and triggered lipid accumulation, achieving levels of up to 53% DCW, whereas the biomass productivity remained similar for all tested conditions. The lipidome analysis revealed that the lowest Mg2+ concentrations also caused a differential lipid profile distribution, with an enrichment of neutral lipids and an increase of structural lipids. In that sense, we showed that Mg2+ limitation represents an alternative optimization approach that not only enhances accumulation of neutral lipids in B. braunii cells but also may potentially lead to a better areal biomass productivity due to the reduction in the cellular light absorption properties of the cells.

Cobalt enrichment enhances the tolerance of Botryococcus braunii to high concentration of CO2.

This work mainly studied B. braunii adapted to different CO2 concentrations with cobalt enrichment, and developed a process for CO2 capture, hydrocarbon production and cobalt removal. The results showed that B. braunii favored rapid growth at 5.0% (v/v) CO2, and the highest biomass was 1.89 g.L-1 with 4.5 mg.L-1 of cobalt. Hydrocarbon productivity in high concentration CO2 (5.0% and 10.0%) with cobalt enrichment was higher than that in Chu 13 medium. The change in cobalt removal efficiency mainly corresponded to the growth of B. braunii. The LCE of B. braunii in cobalt-rich with high CO2 concentration (5.0% and 10.0%) was 15.7%, and 14.9%, respectively, which was higher than that in normal medium. CO2 fixation rates were also higher in cobalt enrichment coupled with high CO2 concentration. This study not only provides ideas for the removal of toxic metal cobalt, but also has great potential for CO2 biofixation.

Effect of Air Nanobubble Water on the Growth and Metabolism of Haematococcus lacustris and Botryococcus braunii.

Miniature air bubbles with a diameter of less than 200 nm were generated by a nanobubble aerator, and nanobubble water (NBW) was eventually obtained using the gas-liquid mixing system with hydrodynamic function. As the air bubbles have long lifetime and high gas solubility in the liquids, NBW is stable in nature and inside contains sufficient dissolved oxygen. At present, there is no report on the use of NBW to replace ordinary water to cultivate microalgae. In this research, effect of NBW on the growth and metabolism of different microalgae, including Haematococcus lacustris and Botryococcus braunii was investigated. The result demonstrated that the growth of H. lacustris and B. braunii was increased by NBW and the highest promotion ratio was up to 44% and 26%, respectively. For H. lacustris, the astaxanthin content in the NBW treatment group was also improved compared to the control group. As the main product of B. braunii, lipid content in the dry matter was decreased after the treatment of NBW, but total lipid production was significantly higher than that of the control group. In general, NBW could serve as the potentially effective tool to promote the growth of microalgae in the future.

High-value chemicals from Botryococcus braunii and their current applications - A review.

Botryococcus braunii is known for its high yield of extracellular hydrocarbons and polysaccharides. Hydrocarbons, especially botryococcenes and squalene can be used as not only fuels but also alternative feedstock for other fossil-based products. Exopolysaccharides excreted from B. braunii can be used as scaffolds for polyesters production, and have a notable potential for synthesis of nanoparticles. B. braunii is also a rich source of carotenoids, especially the unique secondary carotenoids such as botryoxanthins that have never been found in other microalgae. The morphology, physiology, and outer cell walls of B. braunii are complex. Understanding the colony structure shall provide insights into the mechanism of cell growth and chemicals secretion. It is possible to improve the production economics of the alga with advanced culture systems. Moreover, investigation of metabolic pathways for B. braunii may help us understand their regulation and provide valuable information for strain selection and optimal production of high-value chemicals.

Associated bacteria of Botryococcus braunii (Chlorophyta).

Botryococcus braunii (Chlorophyta) is a green microalga known for producing hydrocarbons and exopolysaccharides (EPS). Improving the biomass productivity of B. braunii and hence, the productivity of the hydrocarbons and of the EPS, will make B. braunii more attractive for industries. Microalgae usually cohabit with bacteria which leads to the formation of species-specific communities with environmental and biological advantages. Bacteria have been found and identified with a few B. braunii strains, but little is known about the bacterial community across the different strains. A better knowledge of the bacterial community of B. braunii will help to optimize the biomass productivity, hydrocarbons, and EPS accumulation. To better understand the bacterial community diversity of B. braunii, we screened 12 strains from culture collections. Using 16S rRNA gene analysis by MiSeq we described the bacterial diversity across 12 B. braunii strains and identified possible shared communities. We found three bacterial families common to all strains: Rhizobiaceae, Bradyrhizobiaceae, and Comamonadaceae. Additionally, the results also suggest that each strain has its own specific bacteria that may be the result of long-term isolated culture.

Type II flavoprotein monooxygenase PsFMO_A from the bacterium Pimelobacter sp. Bb-B catalyzes enantioselective Baeyer-Villiger oxidations with a relaxed cofactor specificity.

Microbial consortia, which degrade branched, long-chain hydrocarbons, can be regarded as a promising source of novel enzymes for the stereo- and regio-selective oxyfunctionalization of hydrocarbons. The hydrocarbon-degrading bacterium Pimelobacter sp. Bb-B was isolated from the consortium associated with the colonial hydrocarbon-excreting microalga Botryococcus braunii. Three new type II flavoprotein monooxygenases (type II FMOs) from this bacterium have been made available in recombinant form through cloning and overexpression in an E. coli host organism. These enzymes (PsFMO_A-C) were characterized in terms of their capability of catalyzing Baeyer-Villiger oxidations with distinct substrates. The highest activity was detected when utilizing camphor and bicyclo[3.2.0]hept-2-en-6-one as substrate in combination with PsFMO_A as the most promising enzyme. Furthermore, synthetic biotransformations with 5 mM of the substrate bicyclo[3.2.0]hept-2-en-6-one, formate and formate dehydrogenase for in situ-cofactor recycling were conducted with this enzyme, leading to a substrate consumption of 85% after 66 h and excellent enantioselectivity of 99% ee for the (1R,5S)-enantiomer. Additionally, an alternative in situ-cofactor recycling based on the use of microalgae for in situ-production of formate from carbon dioxide, water and light together with a formate dehydrogenase was combined successfully with the enzyme PsFMO_A, leading to a substrate consumption of 94% and an enantioselectivity of >99% ee for the so-called "normal lactone"-enantiomer with the absolute configuration 1R,5S.

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.

Comparative transcriptome analyses of oleaginous Botryococcus braunii race A reveal significant differences in gene expression upon cobalt enrichment.

BACKGROUND: Botryococcus braunii is known for its high hydrocarbon content, thus making it a strong candidate feedstock for biofuel production. Previous study has revealed that a high cobalt concentration can promote hydrocarbon synthesis and it has little effect on growth of B. braunii cells. However, mechanisms beyond the cobalt enrichment remain unknown. This study seeks to explore the physiological and transcriptional response and the metabolic pathways involved in cobalt-induced hydrocarbon synthesis in algae cells. RESULTS: Growth curves were similar at either normal or high cobalt concentration (4.5 mg/L), suggesting the absence of obvious deleterious effects on growth introduced by cobalt. Photosynthesis indicators (decline in Fv/Fm ratio and chlorophyll content) and reactive oxygen species parameters revealed an increase in physiological stress in the high cobalt concentration. Moreover, cobalt enrichment treatment resulted in higher crude hydrocarbon content (51.3% on day 8) compared with the control (43.4% on day 8) throughout the experiment (with 18.2% improvement finally). Through the de novo assembly and functional annotation of the B. braunii race A SAG 807-1 transcriptome, we retrieved 196,276 non-redundant unigenes with an average length of 1086 bp. Of the assembled unigenes, 89,654 (45.7%), 42,209 (21.5%), and 32,318 (16.5%) were found to be associated with at least one KOG, GO, or KEGG ortholog function. In the early treatment (day 2), the most strongly upregulated genes were those involved in the fatty acid biosynthesis and metabolism and oxidative phosphorylation, whereas the most downregulated genes were those involved in carbohydrate metabolism and photosynthesis. Genes that produce terpenoid liquid hydrocarbons were also well identified and annotated, and 21 (or 29.2%) were differentially expressed along the cobalt treatment. CONCLUSIONS: Botryococcus braunii SAG 807-1 can tolerate high cobalt concentration and benefit from hydrocarbon accumulation. The time-course expression profiles for fatty acid biosynthesis, metabolism, and TAG assembly were obtained through different approaches but had equally satisfactory results with the redirection of free long-chain fatty acid and VLCFA away from TAG assembly and oxidation. These molecules served as precursors and backbone supply for the fatty acid-derived hydrocarbon accumulation. These findings provide a foundation for exploiting the regulation mechanisms in B. braunii race A for improved photosynthetic production of hydrocarbons.

Effects of limonene, n-decane and n-decanol on growth and membrane fatty acid composition of the microalga Botryococcus braunii.

Botryococcus braunii is a promising microalga for the production of biofuels and other chemicals because of its high content of internal lipids and external hydrocarbons. However, due to the very thick cell wall of B. braunii, traditional chemical/physical downstream processing very often is not as effective as expected and requires high amounts of energy. In this cases, the application of two-phase aqueous-organic solvent systems could be an alternative to cultivate microalgae allowing for a simultaneous extraction of the valuable compounds without significant negative effects on cell growth. Two-phase systems have been applied before, however, there are no studies so far on the mechanisms used by microalgae to survive in contact with solvents present as a second-phase. In this study, the effects of the solvents limonene, n-decane and n-decanol on growth of the microalga B. braunii as well as the adaptive cell response in terms of their phospholipid fatty acid contents were analized. A concentration-dependent negative effect of all three solvents on cell growth was observed. Effects were accompanied by changes of the membrane fatty acid composition of the alga as manifested by a decrease of the unsaturation . In addition, an association was found between the solvent hydrophobicity (given as log octanol-water partition coefficient ([Formula: see text]) values) and their toxic effects, whereby n-decanol and n-decane emerged as the most and least toxic solvent respectively. Among the tested solvents, the latter promises to be the most suitable for a two-phase extraction system.

Bio oil production from microalgae via hydrothermal liquefaction technology under subcritical water conditions.

The upsurge in the concerning issues like global warming, environmental pollution and depletion of fossil fuel resources led to the thrust on third generation biofuels. Algal research has gained a lot of importance in the recent years. Effective utilization of algal biomass in a single step is necessary as it can produce Bio-oil (BO), gases and in addition to a variety of valuable products, along with nutrient recovery. Hydrothermal liquefaction technology does not require the energy intensive drying steps and is an attractive approach for the conversion of algae to liquid fuels. This study investigates direct hydrothermal liquefaction (HTL) of microalgae (Algal biomass) to produce bio-oil using a high-pressure batch reactor under subcritical water conditions. Three different micro algae samples namely, Chlorella vulgaris, Botryococcus braunii and Scenedesmus quadricauda have been examined under hydrothermal liquefaction with different water concentrations (1:6, 1:7, 1:8, 1:9 & 1:10 ratio) at certain temperature range (200-320 °C), pressure (60 bars) and reaction time (30 min). Through liquefaction, the highest BO yield achieved with S. quadricauda was 18 wt% at 1:9 ratio. The chemical components of the obtained bio-oil were analyzed via gas chromatography and the results indicated that the algal BO was composed of furan, phenol, acid, and ester derivatives. Moreover, it was found that by increasing the temperatures, the BO yields increased. This was due to the polymerization reactions that converted the small biomass components into heavier molecules. FTIR spectra showed high percentage of Aliphatic, Phenolic, alcoholic, Carboxylic and Hydroxyl groups for solid residues.

Isolation and characterization of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene from Botryococcus braunii, race B.

The B race of a green microalga Botryococcus braunii Kützing produces triterpene hydrocarbons that is a promising source for biofuel. In this algal race, precursors of triterpene hydrocarbons are provided from the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The terminal enzyme of this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is regarded as one of the key enzymes that affect yields of products in terpene biosynthesis. In order to better understand the MEP pathway of the alga, cDNA and genomic clones of HDR were obtained from B. braunii Showa strain. B. braunii HDR (BbHDR) is encoded on a single copy gene including a 1509-bp open reading frame that was intervened by 6 introns. The exon-intron structure of BbHDR genes did not show clear relation to phylogeny, while its amino acid sequence reflected phyla and classes well. BbHDR sequence was distinctive from that of the HDR protein from Escherichia coli in the residues involved in hydrogen-bond network that surrounds substrate. Introduction of BbHDR cDNA into an E. coli HDR deficient mutant resulted in recovery of its auxotrophy. BbHDR expression level was upregulated from the onset of liquid culture to the 24th day after inoculation with a 2.5-fold increase and retained its level in the subsequent period.