The aim of this research was to study the impact of nitrogen starvation on the production of two major secondary metabolites, fatty acids and carbohydrates, in two microalgae: Nannochloropsis sp. and Haematococcus pluvialis. The major response to nitrogen starvation in both algae occurred within the first 2 days, accompanied by a sharp reduction in chlorophyll content. However, the pattern of the response differed between the two microalgae. In H. pluvialis, the first response to nitrogen starvation was intensive production of carbohydrates, accumulating to up to 63% of dry weight by day 1; on day 2, the total carbohydrate content decreased and was partially degraded, possibly to support fatty acid synthesis. Under these conditions, H. pluvialis accumulated up to 35% total fatty acids in the biomass. In Nannochloropsis sp., the immediate and major response, which was maintained throughout the entire period of exposure to stress, was production of fatty acids, accumulating up to 50% of dry weight, while carbohydrate content in the biomass remained stable at 18%. In addition, we tested the effect of the lipid-synthesis inhibitor sesamol, known to inhibit malic enzyme, on the balance between total fatty acid and carbohydrate contents in H. pluvialis and Nannochloropsis sp. In both cultures, sesamol inhibited fatty acid accumulation, but the carbohydrate content was reduced as well, albeit to a lesser extent. These findings demonstrate the complexity of the stress-response and the potential link between fatty acid and carbohydrate synthesis.
A new process evaluation methodology of microalgae biodiesel has been developed. Based on four evaluation criteria, i.e. the net energy ratio (NER), biodiesel production costs, greenhouse gases (GHG) emission rate and water footprint, the model compares various technologies for each step of the process, from cultivation to oil upgrading. An innovative pathway (hybrid raceway/PBR cultivation system, belt filter press for dewatering, wet lipid extraction, oil hydrotreating and anaerobic digestion of residues) shows good results in comparison to a reference pathway (doubled NER, lower GHG emission rate and water footprint). The production costs are still unfavourable (between 1.94 and 3.35€/L of biodiesel). The most influential parameters have been targeted through a global sensitivity analysis and classified: (i) lipid productivity, (ii) the cultivation step, and (iii) the downstream processes. The use of low-carbon energy sources is required to achieve significant reductions of the biodiesel GHG emission rate compared to petroleum diesel.
The effects of exogenous CO(2) on the growth and lipid accumulation of a local screened facultative heterotrophic microalgae strain Auxenochlorella protothecoides (UMN280) as well as nutrient removal from concentrated municipal wastewater stream (centrate) were examined in this study. A 12-day batch experiment was conducted with CO(2) aeration at three levels, namely, 0%, 1%, and 5% (v/v) CO(2) mixed with air, under light intensity of 60 μmol/(m(2) @@s). A two-stage growth pattern was observed. The first stage (first-fifth day) was dominated by heterotrophic growth in which organic carbon was the main carbon source. The second stage (6th-12th day) was dominated by autotrophic growth in which exogenous CO(2) had a positive effect on algal biomass accumulation. The addition of 5% CO(2) was better than that of 1% CO(2) on the biomass and lipid production. The uptakes of nutrients were similar between injection and no injection of CO(2), except on phosphorus removal which was affected by the acidification of CO(2).
Fucoxanthin, one of the main marine carotenoids, is abundant in macro- and microalgae. Here, fucoxanthin was isolated and structurally identified as the major carotenoid in the diatom Phaeodactylum tricornutum through chromatographic and spectroscopic methods, such as liquid chromatography-positive-ion atmospheric pressure chemical ionization/mass spectroscopy and nuclear magnetic resonance. This pigment was quantified by reverse-phase high-performance liquid chromatography, and a number of extraction procedures were assessed to investigate the effect of solvent type, extraction time, temperature, and extraction method (maceration, ultrasound-assisted extraction, Soxhlet extraction, and pressurized liquid extraction). Among the investigated solvents, ethanol provided the best fucoxanthin extraction yield (15.71 mg/g freeze-dried sample weight). Fucoxanthin content in the extracts produced by the different methods was quite constant (15.42-16.51 mg/g freeze-dried sample weight) but increased steeply based on the percentage of ethanol in water, emphasizing the importance of ethanol in the extraction. The results indicate that P. tricornutum is a rich source of fucoxanthin (at least ten times more abundant than that in macroalgae) that is easily extracted with ethanol, suggesting potential applications in human and animal food, health, and cosmetics.
Neochloris oleoabundans is considered one of the most promising oil-rich microalgae because of its ability to store lipids under nitrogen starvation. However, high biomass densities, required for applications on medium to large scale, are not reached in this condition of growth. As previous studies on other microalgae have shown that mixotrophy allows to obtain higher biomass in comparison to autotrophic cultures, we performed morphophysiological analyses in order to test the mixotrophic growth capability of N. oleoabundans. A carbon-rich manure derived from the apple vinegar production (AWP) was added to the medium. Cells were also cultivated under nutrient starvation (tap water), to observe the expected lipids accumulation, and combining AWP to water, to test the potential of this waste in a low-cost culture system. The results highlighted that AWP in the medium allowed to obtain the highest final cell density. Moreover, starch granules were stored inside chloroplast at the beginning of the experiment. The presence of AWP did not induce variations on light harvesting complex II (LHCII)-photosystem II (PSII) assembly, even if an interesting promotion of pigment synthesis in cells was observed. On the other hand, in starved cells, chloroplast degeneration, pigment content decrease, altered LHCII-PSII assembly and accumulation of high amount of lipid globules were observed, irrespective of the presence of AWP. The results suggest that mixotrophy promotes growth in N. oleoabundans and open up the possibility of using waste products from agri-food industries for this purpose. After growth, cells could be transferred under nutrient starvation to induce lipid accumulation.
Linda Nedbalova  travaille depuis longtemps sur les algues des neiges au sein de l'Institut de Botanique  de l'Académie des Sciences . Elle a reçu récemment dans ce cadre un Prix de l'Académie tchèque des Sciences, dans la catégorie des jeunes chercheurs de moins de trente-cinq ans.
Flocculation induced by pH increase for harvesting microalgae and reuse of flocculated medium were evaluated. Increasing the medium pH value induced the highest flocculation efficiency of up to 90% for freshwater microalgae (Chlorella vulgaris, Scenedesmus sp., Chlorococcum sp.) with low/medium biomass concentrations and marine microalgae (Nannochloropsis oculata, Phaeodactylum tricornutum). The mechanism may be explained that Mg(2+) in the growth medium hydrolyzed to form magnesium hydroxide precipitate, which coagulated microalgal cells by sweeping flocculation and charge neutralization. Additionally, this study revealed that the microalgal biomass concentrations and released polysaccharide (RPS) from microalgae could influence the flocculation efficiencies. Furthermore, neutralizing pH and then supplementing nutrients allowed the flocculated medium to maintain an approximate growth yield to that of the fresh medium in algal cultivation. These results suggest that the method presented here is effective, and allows the reuse of the flocculated medium, thereby contributing to the economic production from algae to biodiesel.
Green algae exclusively use the methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of isoprenoids. The first enzyme of this pathway is 1-deoxy-d-xylulose 5-phosphate synthase (DXS, EC 220.127.116.11). Green algae have been thought to possess only a single DXS, in contrast to land plants, which have at least two isoforms that serve different roles in metabolism. The green microalga Botryococcus braunii has an extraordinary isoprenoid metabolism, as it produces large amounts of triterpene hydrocarbons. Here, we did cDNA cloning of DXSs from B. braunii and examined enzyme activities of the heterologously expressed proteins. Three distinct DXS isoforms were identified, all of which were functional and had similar kinetic properties, whereas the temperature dependence of enzyme activity showed considerable differences. Transcription of the genes was examined by real time quantitative RT-PCR. The three DXS genes were simultaneously expressed, and the expression levels were highest on day six after subculturing. B. braunii is the first green microalga demonstrated to have multiple DXS isoforms like land plants. This difference to other microalgae seems to mirror its special needs for extensive triterpene production by increasing the metabolic flow through the MEP pathway.
Considerable research and development is underway to produce fuels from microalgae, one of several options being explored for increasing transportation fuel supplies and mitigating greenhouse gas emissions (GHG). This work models life-cycle GHG and on-site freshwater consumption for algal biofuels over a wide technology space, spanning both near- and long-term options. The environmental performance of algal biofuel production can vary considerably and is influenced by engineering, biological, siting, and land-use considerations. We have examined these considerations for open pond systems, to identify variables that have a strong influence on GHG and freshwater consumption. We conclude that algal biofuels can yield GHG reductions relative to fossil and other biobased fuels with the use of appropriate technology options. Further, freshwater consumption for algal biofuels produced using saline pond systems can be comparable to that of petroleum-derived fuels.
Microalgal based biofuels are discussed as future sustainable energy source because of their higher photosynthetic and water use efficiency to produce biomass. In the context of climate CO2 mitigation strategies, algal mass production is discussed as a potential CO2 sequestration technology which uses CO2 emissions to produce biomass with high-oil content independent on arable land. In this short review, it is presented how complete energy balances from photon to harvestable biomass can help to identify the limiting processes on the cellular level. The results show that high productivity is always correlated with high metabolic costs. The overall efficiency of biomass formation can be improved by a photobioreactor design which is kinetically adapted to the rate-limiting steps in cell physiology. However, taking into account the real photon demand per assimilated carbon and the energy input for biorefinement, it becomes obvious that alternative strategies must be developed to reach the goal of a real CO2 sequestration.
Chlorella vulgaris, a unicellular microalgae, exerts various biological effects; however their effect on proliferation signaling pathways in normal cells has not been studied. We investigated the effect of hot water extracts of Chlorella vulgaris (CVE) on cell proliferation and related signaling pathways in rat intestinal epithelial cells (IEC-6). CVE increased the expression of insulin-like growth factor-I receptor (IGF-IR) and the phosphorylation of focal adhesion kinase (FAK) and Src. In addition, CVE induced activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. We verified the increased phosphorylation of extracellular-signal-related kinase (ERK) and Akt and the increased expression of the PI3K regulatory subunit p85. CVE also influenced the canonical Wnt pathway through increased expression of the nuclear β-catenin, cyclin D1. Tyr-397 of FAK mediates interactions with Src homology 2 (SH2) domains in a number of other signaling proteins, including PI3K, PLC-γ, Shc, Grb7, Src and Nck2. Because CVE induced FAK activation, FAK may affect the Wnt pathway. Addition of a FAK inhibitor decreased the expression of nuclear β-catenin, cyclin D1 and c-myc, and increased the expression of cytosolic β-catenin. We conclude that CVE stimulated proliferation of IEC-6 cells via the MAPK, PI3K/Akt and canonical Wnt pathways, and that this affected the canonical Wnt pathway.
The analysis of the nonpolar extract of the cells of colonies of the green colonial microalgae Botryococcus braunii was performed by gradient HPLC. The growth of B. braunii was stressed by reducing its nitrogen nutrients by 90%, in order to enhance the production of nonpolar compounds. Highly efficient 4.6mm × 100mm columns packed with 2.6μm Kinetex-C(18) core-shell particles (Phenomenex, Torrance, CA, USA) were used. The gradient mobile phase was a mixture of acetonitrile and water (70-97%, v/v). Its initial and final compositions during the gradient elution were chosen so that the retention factors of the last eluted compound at the inlet and outlet of the column were 15 and 1, respectively. The highest peak capacity was obtained by optimizing several experimental parameters, including the injected sample volume, the flow rate, and the column length. The highest resolution was obtained by connecting one 4.6mm × 150mm and three 4.6mm × 100mm columns (total length 45cm). The optimum flow rate was 1.5mL/min, which provided the minimum plate height for the most retained compounds, the optimum inlet pressure was 930bar and the injected volume 2μL. The analysis time was then 14min for a peak capacity of 121. The trends observed for the variation of the experimental peak capacity with the flow rate and the column length are in excellent agreement with theoretical predictions.
There are two major energy and cost constraints to bulk production of single cell microalgae for biofuels or feed: expensive culture systems with high capital costs and high energy requirements for mixing and gas exchange; and the cost of harvesting using high-speed continuous centrifugation for dewatering. This report deals with the latter; harvesting by flocculation where theory states that alkaline flocculants neutralize the repelling surface charge of algal cells, allowing them to coalesce into a floc. It had been assumed that with such electrostatic flocculation, the more cells to be flocculated, the more flocculant needed, in a linear stoichiometric fashion, rendering flocculation overly expensive. Counter to theory of electrostatic flocculation, we find that the amount of alkaline flocculant needed is a function of the logarithm of cell density, with dense cultures requiring an order of magnitude less base than dilute suspensions, with flocculation occurring at a lower pH. Various other theories abound that flocculation can be due to multi-valent cross-linking, or co-precipitation with phosphate or with magnesium and calcium, but are clearly not relevant with the flocculants we used. Monovalent bases that cannot cross-link or precipitate phosphate work with the same log-linear stoichiometry as the divalent bases, obviating those theories, leaving electrostatic flocculation as the only tenable theory of flocculation with the materials used. The cost of flocculation of dense cultures with this procedure should be below $1.00/T algae for mixed calcium:magnesium hydroxides.
Microalgal biomasses have been produced industrially for a long history for application in a variety of different fields. Most recently, microalgae are established as the most promising species for biofuel production and CO(2) bio-sequestration owing to their high photosynthesis efficiency. Nevertheless, design of photobioreactors that maximize solar energy capture and conversion has been one of the major challenges in commercial microalga biomass production. In this review, we systematically survey the recent developments in this field.
As our understanding of the dynamics of lipid droplets (LDs) in animal, plant and fungal cells is rapidly evolving, still little is known about the formation and turnover of these organelles in microalgae. Yet with the growing importance of algal feedstocks for the production of biofuels and high value lipids, there is a need to understand the mechanisms of LD dynamics in microalgae. Thus, we investigated the proteins associated with LDs of the emerging heterokont model alga Nannochloropsis sp. and discovered an abundant hydrophobic Lipid Droplet Surface Protein (LDSP) with unique primary sequence, but structural similarities to other LD proteins. LDSP abundance in Nannochloropsis cells closely tracked the amount triacylglycerols during conditions of oil accumulation and degradation. Functional characterization of LDSP in an Arabidopsis thaliana OLEOSIN 1-deficient mutant allowed a separation of its physical and structural properties in its interaction with LDs from its physiological or biochemical activities. Although LDSP presence in Arabidopsis predictably affected LD size, it could not reverse the physiological impact of OLEOSIN deficiency on triacylglycerol hydrolysis during germination.As our understanding of the dynamics of lipid droplets (LDs) in animal, plant and fungal cells is rapidly evolving, still little is known about the formation and turnover of these organelles in microalgae. Yet with the growing importance of algal feedstocks for the production of biofuels and high value lipids, there is a need to understand the mechanisms of LD dynamics in microalgae. Thus, we investigated the proteins associated with LDs of the emerging heterokont model alga Nannochloropsis sp. and discovered an abundant hydrophobic Lipid Droplet Surface Protein (LDSP) with unique primary sequence, but structural similarities to other LD proteins. LDSP abundance in Nannochloropsis cells closely tracked the amount triacylglycerols during conditions of oil accumulation and degradation. Functional characterization of LDSP in an Arabidopsis thaliana OLEOSIN 1-deficient mutant allowed a separation of its physical and structural properties in its interaction with LDs from its physiological or biochemical activities. Although LDSP presence in Arabidopsis predictably affected LD size, it could not reverse the physiological impact of OLEOSIN deficiency on triacylglycerol hydrolysis during germination.
Microalgal photo-biocatalysis is a green technique for asymmetric synthesis. Asymmetric reduction of nonnatural prochiral ketones to produce chiral alcohols by microalgal photo-biocatalysis was studied in this work. Acetophenone (ACP) and ethyl acetoacetate (EAA) were chosen as model substrates for aromatic ketones and β-ketoesters, respectively. Two prokaryotic cyanophyta and two eukaryotic chlorophyta were selected as photo-biocatalysts. The results proved that nonnatural prochiral ketones can be reduced by microalgal photo-biocatalysis with high enantioselectivity. Illumination is indispensable to the photo-biocatalysis. For aromatic ketone, cyanophyta are eligible biocatalysts. For ACP asymmetric reduction reaction, about 45% yield and 97% e.e. can be achieved by the photo-biocatalysis reaction with Spirulina platensis as biocatalyst. On the contrary, chlorophyta are efficient biocatalysts for β-ketoester asymmetric reduction reaction among the four tested algae. For EAA asymmetric reduction reaction, about 70% yield and 90% e.e. can be achieved with Scenedesmus obliquus as biocatalyst. The microalgae used in this study outperformed other characterized biocatalysts such as microbial and plant cells.