Optimisation of biomass and lipid production by adjusting the interspecific competition mode of Dunaliella salina and Nannochloropsis gaditana in mixed culture

The high cost of algal cultivation has been a barrier associated with the commercialisation of algal biodiesel. Therefore, this study aimed to enhance lipid production by optimising the nutrient supply to benefit the coexistence of Dunaliella salina and Nannochloropsis gaditana. The effects on biomass and lipid production of using different proportions of D. salina and N. gaditana, urea and NaHCO₃ were optimised by response surface method with a 17-run Box–Behnken design. The optimal conditions for the algal growth are 58 % of D. salina in the mixture at OD₆₈₀, 150 μL day^?1 urea (0.0044 g day^?1) and no addition of NaHCO₃. The biomass concentration and lipid production reached 1.00 and 0.383 g L^?1, respectively, which are exceeded by the amount before optimisation, indicating the efficiency of the model obtained by response surface method.     

Lipid composition of the plasma-membrane of the halotolerant alga,Dunaliella salina

The major lipids of the isolated plasma-membrane of the halotolerant alga Dunaliella salina are diacylglyceroltrimethylhomoserine (DGTS, 23.5%), sterol peroxides (7-dehydroporiferasterol peroxide and ergosterol peroxide, 22%), phosphatidylcholine (13%) and phosphatidylethanolamine (11%). Free sterols comprised 5% of the lipids and contained predominantly 7-dehydroporiferasterol and ergosterol. The major fatty acids of the plasma-membrane were palmitic (31%), oleic (13%), linoleic (20%) and γ-linolenic (17%) acids. In constrast to the whole cells, the plasma-membrane contained less (11%) α-linolenic acid and no 16-carbon unsaturated fatty acids. Sterol peroxides were identified by ¹H-NMR and ¹³C-NMR spectroscopy, mass spectrometry, and by comparison on thin-layer chromatography to the product of ergosterol photooxygenation. We believe that this is the first report on the occurrence of sterol peroxides as major constituents of a biological membrane. It is suggested that they may play a role in the unusual membrane-permeability properties of the plasma-membrane of Dunaliella.     

Milking microalga Dunaliella salina for beta-carotene production in two-phase bioreactors

A new method was developed for production of beta-carotene from Dunaliella salina. Cells were grown in low light intensity and then transferred to a production bioreactor illuminated at a higher light intensity. It was a two-phase bioreactor consisting of an aqueous and a biocompatible organic phase. Mixing of the cells and extraction were performed by recirculation of the organic phase. Two experiments were performed. In the first experiment, bioreactors were operated at two different solvent recirculation rates of 150 and 200 mL min(-1). The beta-carotene extraction rate increased significantly at the higher recirculation rate, without exerting any influence on cell number and viability. A second experiment was carried out at a recirculation rate (200 mL min(-1)) appropriate for the study of long-term production of beta-carotene. The results show that D. salina at high light intensity remained viable for a long period (>47 days) in the presence of a biocompatible organic phase; however, cell growth was very slow. beta-Carotene could be continuously extracted to the organic phase; the cells continued to produce beta-carotene and the extracted molecules were continuously reproduced. As a result, beta-carotene was continuously removed ("milked") from the cells. beta-Carotene extraction efficiency in this system was >55%, and productivity was 2.45 mg m(-2) day(-1), much higher than that of commercial plants.     

Bio-diesel production directly from the microalgae biomass of Nannochloropsis by microwave and ultrasound radiation

This work offers an optimized method for the direct conversion of harvested Nannochloropsis algae into bio-diesel using two novel techniques. The first is a unique bio-technology-based environmental system utilizing flue gas from coal burning power stations for microalgae cultivation. This method reduces considerably the cost of algae production. The second technique is the direct transesterification (a one-stage method) of the Nannochloropsis biomass to bio-diesel production using microwave and ultrasound radiation with the aid of a SrO catalyst. These two techniques were tested and compared to identify the most effective bio-diesel production method. Based on our results, it is concluded that the microwave oven method appears to be the most simple and efficient method for the one-stage direct transesterification of the as-harvested Nannochloropsis algae.     

Growth aspects of the marine microalga Nannochloropsis gaditana

Nannochloropsis is well appreciated in aquaculture due to its nutritional value and the ability to produce valuable chemical compounds, such as pigments (zeaxanthin, astaxanthin...) and polyunsaturated fatty acids (EPA). Commercial exploitation needs high cell densities but the low growth rate and the small size of cells are practical difficulties. To increase biomass concentration the positive effect of several factors was evident: (i) pH approximately 8 control (with dilute Tris-HCl buffer); (ii) the continuous illumination (no evidence of photo-inhibition was observed); (iii) a quite large temperature range (25+/-5 degrees C); (iv) the presence of organic carbon source (with the danger of contamination); (v) the presence of urea as an additional nitrogen source (10 mM); (vi) a small air flow rate with large bubbles can be more efficient for CO(2) mass transfer (associated to reduced shearing).     

Effect of different media on exopolysaccharide and biomass production by the green microalga Botryococcus braunii

Botryococcus braunii is a colonial green microalga with recognized potential to synthesize lipids and hydrocarbons for biofuel production. Besides this ability, this microalga also produces exopolysaccharides (EPS). Nevertheless, there are few reports about their biotechnological aspects and industrial applications. In this study, the effect of the nutritional conditions was examined by using two different culture media (BG11 and D medium). To our knowledge, the latter has not been reported before for culturing B. braunii. After 49 days of incubation, the final production of EPS was found to be statistically higher (P < 0.05) in the D medium (0.549?±?0.044 g L^?1) than in BG11 (0.336?±?0.009 g L^?1). On the contrary, the biomass production was found to be higher in BG11 (1.019?±?0.051 g L^?1) than in the D medium (0.953?±?0.056 g L^?1). However, this difference was not statistically significant. The difference in salinity and nitrogen concentration between both media is suggested as the main factor involved in the EPS and biomass results. FTIR spectra of B. braunii EPS from both media revealed presence of uronic acids and absence of amino and sulfate groups. Despite the similarity between both spectra, there were some different signals (at 1,921.52 and 720.60 cm^?1) which may mean a difference in glycosyl composition.     

Antifouling potential of the marine microalga Dunaliella salina

Marine organisms have usually been viewed as sources of environmentally friendly compounds with antifouling activity. We performed a series of operations to investigate the antifouling potential of the marine microalga Dunaliella salina. For the ethyl acetate crude extract, the antialgal activity was significant, and the EC₅₀ value against Skeletonema costatum was 58.9 μg ml^?1. The isolated purified extract was tested for antifouling activity, the EC ₅₀ value against S. costatum was 21.2 μg ml^?1, and the LC₅₀ against Balanus amphitrite larvae was 18.8 μg ml^?1. Subsequently, both UHR–TOF–MS and GC–MS were used for the structural elucidation of the compounds, and a series of unsaturated and saturated 16- and 18-carbon fatty acids were detected. The data suggested that the fatty acid extracts from D. salina possess high antifouling activity, and could be used as substitutes for potent, toxic antifouling compounds.     

The utilization of bicarbonate ions by the marine microalga Nannochloropsis oculata (Droop) Hibberd

HCO₃^? utilization by the marine microalga Nannochloropsis oculata was investigated using a pH drift technique in a closed system. Light-dependent alkalization of the medium resulted in a final pH of 10.5, confirming substantial HCO₃^? use by this alga. Alkalinity remained constant throughout the pH drift. Measurement of dissolved inorganic carbon (DIC) or the uptake of H¹⁴CO₃^? showed that nearly 50% of the total DIC remained external to the plasma membrane on completion of a pH drift. The rate of light-driven alkalization was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and thus was dependent on photosynthesis. Light-driven alkalization was not inhibited by a membrane-impermeable inhibitor of carbonic anhydrase (CA), dcxtran-bound sulphonamide (DBS), indicating that external CA was not involved in HCO₃^? utilization. The anion-cxchangc inhibitor 4′,4′-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) completely inhibited light-driven alkalization of the medium and H¹⁴CO₃^? uptake, providing unequivocal support for a direct uptake of H¹⁴CO₃^?. Chloride ions were essential for DIC-dependent photosynthetic oxygen evolution, suggesting that bicarbonate transport occurs by HCO₃^?/CI^? exchange.     

Enhanced production of biomass and lipids by supplying CO2 in marine microalga Dunaliella sp.

Non-food-based biofuel feedstocks are in high demand worldwide. Among the various feedstocks, microalgae are the most promising feedstock for mitigating atmospheric CO₂ and producing biodiesel. In this study, various concentrations of CO₂, from 0.03 to 12%, were used to investigate their effect on the cell growth, biomass and lipid production and fatty acid composition of Dunaliella sp. in a closed photobioreactor. The results showed that the highest biomass and total lipids, 521 mg/L/d and 40 mg/L/d, respectively, were produced with 5% CO₂ aeration during the logarithmic growth phase. The oleic acid (18:1n9c) and elaidic acid (18:1n9t) contents were increased approximately two fold. The physiological responses of Dunaliella sp. at 10% CO₂ were similar to those at 5% CO₂. Therefore, the present results suggest that 5–10% is a suitable CO₂ concentration range for Dunaliella sp. growth to mitigate atmospheric CO₂ and increase biofuel production.     

Serial optimization of biomass production using microalga Nannochloris oculata and corresponding lipid biosynthesis

As energy and environment have become urgent issues, there has been increasing needs to develop alternative energy source, such as microalgal bio-fuel. In this study, we investigated the growth and lipid contents of microalgae Nannochloris oculata under various environmental conditions for biodiesel production. Our results indicated that biomass productivities of N. oculata were correlated with increasing nitrogen concentrations up to 37.5 ppm. High irradiance using 230-250 μmol/m(2) led to higher biomass yields than low irradiance of 160-180 μmol/m(2). Biomass productivities increased further by manipulating surface to volume ratio (S/V), which in turn enhanced light penetration. Finally, optimal biomass productivities (1.04 g/l day) could be achieved by the supplementation of yeast extract. Lipid contents and fatty acid profiles of N. oculata were affected by the different growth conditions. Lipid contents of N. oculata decreased as nitrogen concentration increased. Lower temperature (15 °C) resulted in higher lipid content than higher temperature (25 °C). Fatty acid profiles of N. oculata indicated that palmitic acid (C16:0) and linoleic acid (C18:2) were the two most abundant fatty acids, but the supplementation of yeast extract increased linolenic acid (C18:3) content. Our results suggested the feasibility of N. oculata for the biodiesel production.     

Light-dependent bicarbonate uptake and CO2 efflux in the marine microalga Nannochloropsis gaditana

 Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO₂ fluxes in cell suspensions using mass spectrometry. Addition of H¹³CO₃ ⁻ to cell suspensions in the dark caused a transient increase in the CO₂ concentration in the medium far in excess of the equilibrium CO₂ concentration. The magnitude of this release was dependent on the length of time the cells had been kept in the dark. Once equilibrium between the Ci species had been achieved, a CO₂ efflux was observed after saturating light intensity was applied to the cells. External carbonic anhydrase (CA) was not detected nor does this species demonstrate a capacity to take up CO₂ by active transport. Photosynthetic O₂ evolution and the release CO₂ in the dark depend on HCO₃ ⁻ uptake since both were inhibited by the anion exchange inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark. Ethoxyzolamide, a CA inhibitor, markedly inhibited CO₂ efflux in the dark, indicating that CO₂ efflux was dependent upon the intracellular dehydration of HCO₃ ⁻. These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which maintains the equilibrium between CO₂ and HCO₃ ⁻ and thus causes a subsequent release of CO₂ to the external medium. Received: 20 September 1999 / Accepted: 25 October 1999     

Functional Organization of Chlorophyll a and Carotenoids in the Alga, Nannochloropsis salina

Chlorophyll-protein complexes were isolated from a yellow-green alga, Nannochloropsis salina after mild detergent treatment and gel electrophoresis. Three different complexes were obtained which correspond to the three major kinds of chlorophyll-proteins isolated from spinach chloroplasts by the same procedure and previously identified as reaction center complexes for photosystems I and II and a light-harvesting complex. The analogy between the algal complexes and those from spinach was drawn from their absorption and fluorescence spectra and relative pigment content. The identities and amounts of the major carotenoids associated with each isolated complex were determined by HPLC. Although the reaction center complexes accounted for only 14% of the total chlorophyll, they were highly enriched in β-carotene, whereas the light-harvesting complex contained a high proportion of xanthophylls (mainly violaxanthin and vaucheriaxanthin-ester). Fluorescence excitation spectra of the algal membranes showed that one or both of the major xanthophylls may act as antenna pigment for photosynthesis.     

Mono- and digalactosyldiacylglycerols: potent nitric oxide inhibitors from the marine microalga Nannochloropsis granulata

Chemical investigation of a marine microalga, Nannochloropsis granulata, led to the isolation of four digalactosyldiacylglycerols namely, (2S)-1-O-eicosapentaenoyl-2-O-palmitoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (1), (2S)-1-O-eicosapentaenoyl-2-O-palmitoleoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (2), (2S)-1-O-eicosapentaenoyl-2-O-myristoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (3), and (2S)-1,2-bis-O-eicosapentaenoyl-3-O-(β-d-galactopyranosyl-6-1α-d-galactopyranosyl)-glycerol (4), together with their monogalactosyl analogs (5–8). Among the isolated galactolipids 2 and 3 were new natural products. Complete stereochemistry of 1, 4, 5, 7, and 8 was determined for the first time by both spectroscopic techniques and classical degradation methods. Both mono- and digalactosyldiacylglycerols isolated from N. granulata possessed strong nitric oxide (NO) inhibitory activity against lipopolysaccharide-induced NO production in RAW264.7 macrophage cells through downregulation of inducible nitric oxide synthase expression indicating the possible use as anti-inflammatory agents.     

New diacylglyceryltrimethylhomoserines from the marine microalga Nannochloropsis granulata and their nitric oxide inhibitory activity

Chemical investigation of polar lipids from the marine eustigmatophyte microalga Nannochloropsis granulata led to the isolation of six betaine lipid diacylglyceryltrimethylhomoserine (DGTS), namely, (2S)-1,2-bis-O-eicosapentaenoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (1), (2S)-1-O-eicosapentaenoyl-2-O-arachidonoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (2), (2S)-1-O-eicosapentaenoyl-2-O-myristoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (3), (2S)-1-O-eicosapentaenoyl-2-O-palmitoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (4), (2S)-1-O-eicosapentaenoyl-2-O-palmitoleoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (5), and (2S)-1-O-eicosapentaenoyl-2-O-linoleoylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (6). Structures of the isolated DGTSs were elucidated based on both spectroscopic technique and degradation methods. This is the first report of isolation of 1 in pure state, and 2–6 are all new compounds. The isolated betaine lipids showed dose-dependent nitric oxide (NO) inhibitory activity against lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophage cells. Further study suggested that these betaine lipids (1–6) inhibit NO production in RAW264.7 macrophage cells through downregulation of inducible nitric oxide synthase expression, indicating the possible use as an anti-inflammatory agent. This is the first report of DGTS with anti-inflammatory activity.     

Inorganic carbon transport across cell compartments of the halotolerant alga Dunaliella salina

The inorganic carbon (C_i) accumulation and the intracellular location of carbonic anhydrase (CA, EC 4.2.1.1) in the halotolerant unicellular alga Dunaliella salina have been investigated. The rate of HCO₃ -dependent O₂ evolution was determined by growth conditions. Algae grown under high CO₂ conditions (5% CO₂ in air, v/v; high C_i cells) had a very low affinity for HCO₃^? at pH 7.0 and 8.2, whereas algae grown under low CO₂ conditions (0.03% CO₂ in air; low C_i cells) showed a high affinity for HCO₃^? at both pH values and were sensitive to Dextran-bound sulfonamide (DBS), an inhibitor of extracellular CA. The photosynthetic rate or HCO₄^? dependent O₂ evolution was always higher at pH 7.0 than at pH 8.2. Ethoxyzolamide (EZ), an inhibitor of total (extacellular plus intracellular) CA activity, strongly inhibited photosynthesis at both pH values. During adaptation from high to low CO₂ conditions CA activity increased in chloroplasts in a process dependent on the novo protein synthesis. Carbonic anhydrase activity was found in the supernatant and pellet fractions of chloroplast homogenates. The rate of photosynthesis of chloroplasts from low C_i cells was higher at pH 7.0 than at pH 8.2. The alkalinization of the growth medium, which took place only in the presence of C_i, was partially inhibited by DBS and completely by EZ. We suggest that in D. salina CO₂ is the general form of C_i transported across the plasma membrane and the chloroplast envelope and that bicarbonate enters the cell mainly, although not entirely, by an ‘indirect’ mechanism after dehydration to CO₂.     

Effects of nitrogen concentration and media replacement on cell growth and lipid production of oleaginous marine microalga Nannochloropsis oceanica DUT01

Cell growth and lipid production of a marine microalga Nannochloropsis oceanica DUT01 were investigated, and fresh medium replacement with different ratios to promote long term cell growth and lipid accumulation was also tested. The highest lipid content reached 64% in nitrogen deplete f/2 medium containing 37.5 mg/L NaNO₃ combined with 1/5 fresh medium replacement, however, the highest lipid titer (0.6 g/L) and lipid productivity (31 mg/L/d) were achieved using BG11 medium containing 1.5 g/L NaNO₃, taking advantage of 1/5 fresh medium replacement as well, which corresponded to the maximum biomass production of 1.4 g/L, highlighting the importance of high biomass accumulation for efficient lipid production. When biomass compositions were monitored throughout the culture, decreased protein content was found to be coupled with increased lipid production, whereas relatively stable carbohydrate content was observed. The fatty acids in the lipid of N. oceanica DUT01 comprise over 65% saturated fatty acids and monounsaturated acids (i.e. palmitic acid (C16:0) and oleic acid (C18:1)), suggesting that N. oceanica DUT01 is a promising candidate for biodiesel production. Interestingly, very high content of hexadecadienoic acid (C16:2, about 26–33%) was produced by DUT01, which distinguished this microalga with other microalgae strains reported so far.     

Cold-acclimation protects photosystem II against freezing damage in the halotolerant alga Dunaliella salina

Cold-acclimation (CA) of the halotolerant alga Dunaliella was inhibited by light and by high salt. CA was associated with enhanced resistance to freezing in saline growth solutions, as manifested by protection of photosynthetic oxygen evolution and by reduced permeabilisation of the plasma membrane. Oxygen evolution activity in isolated chloroplasts was not affected by freezing, but was inhibited by high salt and the inhibition could be reversed or protected by glycerol. The activity of chloroplasts from cold-acclimated cells was more resistant to salt than of non-acclimated cells. Electron transport measurements in chloroplasts indicated that high salt inhibited PS-II, but not PS-I electron transport. High salt also inhibited PS-II thermoluminescence (TL) activity in chloroplasts. Similar inhibition of PS-II TL was observed by freezing intact cells in saline solutions. Chloroplasts from cold-acclimated cells had enhanced resistance to inhibition of PS-II electron transport and of PS-II TL by high salt. These results suggest that inhibition of oxygen evolution upon freezing Dunaliella cells may result from inactivation of PS-II due to massive influx of salt and loss of glycerol. The enhanced freeze-resistance of cold-acclimated cells to inhibition of oxygen evolution can be accounted for partly by protection of PS-II against high salt.     

Temperature-regulated expression of a glycine-rich RNA-binding protein in the halotolerant alga Dunaliella salina

Acclimation of the halotolerant alga Dunaliella salina to low temperature induced the accumulation of a 12.4 kDa protein (DsGRP-1) and reduction of a 13.1 kDa protein (DsGRP-2). DsGRP-1 and DsGRP-2 are boiling-stable proteins that are localised in the cytoplasm, as revealed by sub-cellular fractionation and by immuno-localisation. The proteins were partially purified and their corresponding genes were cloned. The predicted sequences are homologous to Glycine-Rich RNA-binding Proteins (GRPs) from plants and cyanobacteria. The nucleotide sequences of grp1 and grp2 differ in a short insert encoding 9 amino acids in the glycine-rich domain of DsGRP-2. grp2 contains a single intron at position 179 indicating that DsGRP-1 and DsGRP-2 are not derived from alternative splicing of a common gene. The level of grp mRNA increased at 7 degrees C and was rapidly depressed at 24 degrees C. Analysis of binding to ribonucleotide homopolymers revealed that DsGRP-1 and DsGRP-2 bind preferentially to poly-G and to poly-U indicating that they are RNA-binding proteins. It is proposed that DsGRP-1 and DsGRP-2 are encoded by distinct genes which are differentially regulated by temperature.