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.     

Selective extraction of carotenoids from the microalga Dunaliella salina with retention of viability

Simultaneous production and selective extraction of beta-carotene from living cells of Dunaliella salina in a two-phase system of aqueous and organic phases has been investigated. Solvents with values of log P(octanol), which denotes hydrophobicity of a compound, ranging from 3 to 9 were used as organic phase. Viability and activity of Dunaliella salina in the presence of organic solvents were checked by microscopic observation and photosynthetic oxygen-production-rate measurements, respectively. Extraction ability of different solvents for both beta-carotene and chlorophyll was determined spectrophotometrically. In addition, beta-carotene contents of the cells growing in the aqueous phase and extracted beta-carotene by the different organic phases were quantified by the same method. Results showed that solvents having log P(octanol) > 6 can be considered biocompatible for this alga. Moreover, pigment extraction ability of a solvent is inversely dependent on its log P(octanol) value. By increasing the degenerative hydrophobicity the extraction ability for both chlorophyll and beta-carotene, decreases. However, this decrease is more profound for chlorophyll. Therefore, selective extraction of beta-carotene becomes feasible. Comparison of the total beta-carotene produced in the presence and in the absence of solvents shows that the presence of a second phase of biocompatible solvents in the culture media may induce the beta-carotene production pathway. The beta-carotene productivity per cell in a two-phase system with dodecane was the highest observed. Extraction ability of the biocompatible solvents dodecane, tetradecan, and hexadecane was similar.     

Microalgae mediated photoproduction of beta-carotene in aqueous-organic two phase systems

Improving productivity is a usual requirement for most biotechnological processes, and the utilisation of two-phase aqueous organic systems has proved to be an effective way to improve the productivity of poorly water-soluble or toxic compounds. The high hydrophobicity of beta-carotene, which is highly demanded by the pharma and agrofood industry, makes it a good candidate for aqueous/organic biphasic photoproduction. In the present work we have investigated the viability of a two-phase system for the production of beta-carotene by the marine microalgae Dunaliella salina using decane as organic phase. Decane, with a logP(octanol) value of 5.6, showed no toxicity to Dunaliella cells for more than 72 h, and its ability for beta-carotene extraction is acceptable. Transferring Dunaliella cells from standard to carotenogenic conditions caused inhibition of chlorophyll production and induced a strong synthesis of beta-carotene. The two-phase aqueous/decane system was stable and beta-carotene content of the cells was increasing during 4-days. About 8% of the total carotenoids produced were excreted and extracted into the decane phase.     

Physiological characterization of Dunaliella sp. (Chlorophyta, Volvocales) from Yucatan, Mexico

Physiological responses of Dunaliella salina and Dunaliella viridis, isolated from solar saltworks on the Yucatan Peninsula, were studied. Optimal growth temperature for D. salina was 22 degrees C (3.06 x 10(6) cells mL(-1)) and 26 degrees C for D. viridis (4.04 x 10(6)cells mL(-1)). Total carotenoid content in D. salina increased with temperature to a maximum of 35.14 pg cell(-1) at 38 degrees C. Dunaliella salina alpha-carotene and beta-carotene content was 0.083+/-0.003 and 0.598+/-0.020 mg 100g dry wt(-1) respectively, whereas lower values were found in D. viridis cultured under same experimental conditions (0.018+/-0.002 and 0.136+/-0.012 mg 100g dry wt(-1) respectively). The highest specific growth rate in D. salina was obtained at 10% NaCl (0.28 d(-1)), while its cell volume increased from 524 to 2066.93 microm(3) when cultured from 10% to 35% NaCl. Maximum photosynthetic rates were attained when increasing from optimal growing temperature to 30 degrees C for D. viridis (108 n mol O(2)microg chl alpha h(-1)) and D. salina (139 n mol O(2)microg chl alpha h(-1)). Photosynthetic responses to temperature variations indicated physiological adjustments in both species, with higher acclimation in D. salina. Evaluation of physiological attributes of these species will be used for to carry out mass cultivation.     

Effect of light intensity on beta-carotene production and extraction by Dunaliella salina in two-phase bioreactors

Application of two-phase bioreactors is a useful technique for improvement of the productivity of fermentations. Fermentative extraction of the products in situ is performed in this technique. The effect of light intensity on the extraction of beta-carotene from Dunaliella salina, in the fermentative extraction, has been investigated. Three different average light exposures were applied: 1.5 x 10(-8) (low), 2.7 x 10(-8) (intermediate) and 4.5 x 10(-8) (high) micromol s(-1) per cell. Results show that beta-carotene content of the cells increases by increasing the light exposure. Increase in the beta-carotene content of the cells is not necessarily coupled with an increase in the volumetric production of beta-carotene. Final volumetric production is about the same for the three bioreactors. beta-Carotene extraction rate is enhanced by the increase in the light exposure. The results suggest that extraction rate is related to beta-carotene content of the cells and is not essentially related to the volumetric production of beta-carotene. Although the effectiveness of extraction with respect to the light input is comparable for all light intensities applied, increasing the light input per cell leads to a higher volumetric extraction rate. Moreover, extracted beta-carotene stays very pure even so the extraction increased by the increase of light intensity.     

Enhancement of innate immunity in rainbow trout (Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products

The effects of orally administered carotenoids from natural sources on the non-specific defense mechanisms of rainbow trout were evaluated in a nine-week feeding trial. Fish were fed four diets containing either beta-carotene or astaxanthin at 100 and 200 mg kg-1 from the marine algae Dunaliella salina and red yeast Phaffia rhodozyma, respectively, and a control diet containing no supplemented carotenoids. Specific growth rate and feed:gain ratio were not affected by dietary carotenoid supplementation. Among the humoral factors, serum alternative complement activity increased significantly in all carotenoid supplemented groups when compared to the control. On the other hand, serum lysozyme activity increased in the Dunaliella group but not in the Phaffia group, whereas plasma total immunoglobulin levels were not altered by the feeding treatments. As for the cellular responses, the superoxide anion production from the head kidney remained unchanged while the phagocytic rate and index in all supplemented groups were significantly higher than those of the control. These findings demonstrate that dietary carotenoids from both D. salina and P. rhodozyma can modulate some of the innate defense mechanisms in rainbow trout.     

Mechanism of extraction of beta-carotene from microalga Dunaliellea salina in two-phase bioreactors

We show that it is possible to extract beta-carotene selectively from Dunaliella salina in two-phase bioreactors. The cells continue to produce beta-carotene and the extracted part is substituted by newly produced molecules. This process is called "milking." We performed several experiments to understand the exact mechanism of the extraction process. The results show that direct contact between the cells and the biocompatible organic solvent was not a requirement for the extraction but it accelerated the extraction. Electron microscopy photographs showed an undulated shape of the cell membrane and a space between the cell and the chloroplast membranes in the cells growing in the presence of dodecane (a biocompatible solvent). Extra-chloroplast beta-carotene globules located in the space between the cell and the chloroplast membranes were observed in these cells as well. It was shown that dodecane was taken up by the cells. The concentration of dodecane in the cells was about 13 pg.cll(-1). It can be concluded that dodecane uptake by the cells is responsible for the morphological changes in the cells and leads to more activity in the cell membrane. The results suggest two possible modes of extraction. One of the mechanisms is transport of the globules from the chloroplast to the space between the cell and the chloroplast membranes and subsequently from there to the outside by exocytosis. Another possible mode for the extraction could be release of beta-carotene from the globules as a result of alterations in the membrane in response to the uptake of dodecane. beta-Carotene molecules diffuse from the chloroplast to the space between the cell and the chloroplast membranes and from there to the medium either by diffusion or by exocytosis after accumulation in the vesicles.     

Production of Dunaliella salina biomass rich in 9-cis-beta-carotene and lutein in a closed tubular photobioreactor

Performance of Dunaliella salina cultures outdoors in a closed tubular photobioreactor has been assessed. Optimization of conditions involved verification of the effect of several determining factors on the yield of both biomass and carotenoids. Maximal biomass productivity (over 2g (dry weight) m(-2) d(-1) or 80 gm(-3) d(-1)) was achieved at 38 cm s(-1), flow rate; 2 x 10(9) cells l(-1), initial population density; 25 degrees C, temperature; semi-continuous regime, keeping a cell density interval between 2 x 10(9) and over 4 x 10(9) cells l(-1). Coverage of the tubular loop with a sunshade screen to avoid light-induced damage of cells was essential to maintain growth performance. The cellular beta-carotene level increased significantly during the light period, as also did that of lutein. The rise in the beta-carotene level could be accounted by the 9-cis-isomer, with all-trans-beta-carotene remaining steady during the light period. By sunset, the ratio between 9-cis- and all-trans-isomers of beta-carotene amounted to 1.5, with over 60% of total beta-carotene corresponding to the 9-cis-isomer. Removal of sunshade enhanced carotenoid accumulation by cells to reach up to 10% of dry biomass. Cultivation of Dunaliella in closed tubular photobioreactor, thus represents a suitable approach for the production of a high-quality microalgal biomass enriched in the valuable 9-cis-isomer of beta-carotene and lutein.     

Effect of nitrate concentration on growth and pigment synthesis of Dunaliella salina cultivated under low illumination and preadapted to different salinities

A wild strain of Dunaliella salina was isolated from a solar evaporation salt-pond in Araya (Estado Sucre, Venezuela) and grown in batch culture using relatively low illumination (80 μmol photon m-2 s-1). After the alga had been adapted to various salinities (9, 14, 21% w/v NaCl), the influence of nitrate concentration (882, 435, 212 μmol L-1 N) on growth rate and chlorophyll a and total carotenoid concentrations was measured. Low nitrate concentration negatively affected growth, but enhanced carotenoid accumulation. A slight increase in carotenogenesis was also observed in alga grown at the highest salinity. There were no significant additive or synergistic effects of salinity and nutrient concentrations on the concentrations of chlorophyll a or total carotenoid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Continuous production of carotenoids from Dunaliella salina

During the in situ extraction of β-carotene from Dunaliella salina, the causal relationship between carotenoid extraction and cell death indicated that cell growth and cell death should be at equilibrium for a continuous in situ extraction process. In a flat-panel photobioreactor that was operated as a turbidostat cell numbers of stressed cells were kept constant while attaining a continuous well-defined light-stress. In this way it was possible to study the balance between cell growth and cell death and determine whether both could be increased to reach higher volumetric productivities of carotenoids. In the two-phase system a volumetric productivity of 8.3 mg β-carotene L(RV)(-1)d(-1) was obtained. In situ extraction contributed only partly to this productivity. The major part came from net production of carotenoid-rich biomass, due to a high growth rate of the cells and subsequent dilution of the reactor. To reach equilibrium between cell growth and cell death, sparging rates of dodecane could have been increased. However, already at the applied sparging rate of 286 L(dod)L(RV)(-1)min(-1) emulsion formation of the dodecane in the aqueous phase appeared. In a turbidostat without in situ extraction a volumetric productivity of 13.5 mg β-caroteneL(RV)(-1)d(-1) was reached, solely based on the continuous production of carotenoid-rich biomass.     

Selective separation of cis-trans geometrical isomers of beta-carotene via CO2 supercritical fluid extraction

We investigated a novel method for the selective separation of beta-carotene isomers from a freeze-dried powder of the algae Dunaliella bardawil using supercritical fluid extraction. The separation method relies on the different dissolution rate of the 9Z and all-E isomers of beta-carotene in SC-CO(2). At first, the equilibrium solubility of the two isomers in SC-CO(2) was determined at the extraction conditions of 44.8 MPa and 40 degrees C. The solubility of the 9Z isomer was found to be nearly 4 times higher than that of the all-E isomer (1.92 x 10(-5) g all-E isomer/g CO(2) compared to 7.64 x 10(-5) g 9Z isomer/g CO(2)). When supercritical fluid extraction was applied to a carotenoid concentrate from the algae (29 wt% beta-carotene) or a freeze-dried powder of the algae (3.1% beta-carotene), a selective separation of the 9Z/all-E isomers of beta-carotene was obtained. Thirty-nine percent recovery of beta-carotene with 80% purity of 9Z isomer was achieved at the initial stages of extraction (40 mL CO(2)). The extraction rate of beta-carotene from the freeze-dried algae powder was slower than that from the carotenoid concentrate, resulting in a reduction in the recovery and purity of the 9Z isomer. This indicates that even at the initial stage of the extraction the internal mass resistance is significant. Isomer purity and recovery could be enhanced upon grinding of the algae powder.     

Identification and characterization of a new strain of the unicellular green alga Dunaliella salina (Teod.) from Korea

The unicellular green alga Dunaliella salina is a halotolerant eukaryotic organism. Its halophytic properties provide an important advantage for open pond mass cultivation, since D. salina can be grown selectively. D. salina was originally described by E. C. Teodoresco in 1905. Since that time, numerous isolates of D. salina have been identified from hypersaline environments on different continents. The new Dunaliella strain used for this study was isolated from the salt farm area of the west coastal side of South Korea. Cells of the new strain were approximately oval- or pear-shaped (approximately 16-24 microm long and 10-15 microm wide), and contained one pyrenoid, cytoplasmatic granules, and no visible eyespot. Although levels of beta-carotene per cell were relatively low in cells grown at salinities between 0.5 to 2.5 M NaCl, cells grown at 4.5 M NaCl contained about a ten-fold increase in cellular levels of beta-carotene, which demonstrated that cells of the new Korean strain of Dunaliella can overaccumulate beta- carotene in response to salt stress. Analysis of the ITS1 and ITS2 regions of the new Korean isolate showed that it is in the same clade as D. salina. Consequently, based on comparative cell morphology, biochemistry, and molecular phylogeny, the new Dunaliella isolate from South Korea was classified as D. salina KCTC10654BP.     

Effect of salinity on the quantity and quality of carotenoids accumulated by Dunaliella salina (strain CONC-007) and Dunaliella bardawil (strain ATCC 30861) Chlorophyta

Dunaliella salina and D. bardawil are well-known microalgae accumulating high levels of beta-carotene under growth-limiting conditions. In both taxa, this pigment is primarily composed of the isomers 9-cis and all-trans. The 9-cis beta-carotene occurs only in natural sources and is the most attractive from a commercial point of view. The conditions that enhance the preferred accumulation of 9-cis beta-carotene in D. salina are controversial and they have not been well established yet. This study examined the effect of salinity on the quantity and quality of total carotenoids and beta-carotene isomers accumulated by D. salina (strain CONC-007) and D. bardawil (strain ATCC 30861) grown in two media with different nutritional compositions (PES and ART) and at salt concentrations of 1M, 2M and 3M NaCl. Total carotenoids were determined by spectrophotometry and beta-carotene isomers, by HPLC. The highest carotenoid contents per cell were obtained at 2M NaCl in both taxa. In both media, an increase of the 9-cis/all-trans beta-carotene ratio was observed in D. bardawil when the salt concentration increased, with a maximum value of 2.6 (in ART medium at 3M NaCl). In D. salina this ratio did not exhibit the same pattern, and the salt concentrations for maximal ratios were different in both media. The highest ratio obtained for this strain was 4.3 (in ART medium at 2M NaCl).     

高浓度钾抑制杜氏盐藻生长的生理机制

在含１ｍｏｌ／ＬＮａＣｌ的杜氏盐藻（Ｄｕｎａｌｉｅｌａｓａｌｉｎａ（Ｄｕｎａｌ）Ｔｅｏｄ．）培养液中加入５０ｍｍｏｌ／Ｌ以上的ＫＣｌ可观察到Ｋ＋对杜氏盐藻生长有明显的抑制作用,而当ＫＣｌ达１００ｍｍｏｌ／Ｌ时,杜氏盐藻的生长被完全抑制。另一方面,当培养液中缺乏Ｋ＋时,杜氏盐藻的生长也被显著抑制。在正常培养条件下,伴随着杜氏盐藻的生长,培养液的ｐＨ由８左右升高至１０左右,而高浓度Ｋ＋则显著抑制杜氏盐藻培养液ｐＨ的升高;而在培养液ｐＨ为７．０至１０．０的范围内,不同ｐＨ对杜氏盐藻的生长无明显影响。将杜氏盐藻在高浓度Ｋ＋条件下预处理１２ｈ以上,杜氏盐藻的光合放氧速率显著下降,光合速率下降的程度与Ｋ＋浓度的高低和预处理的时间长短呈正相关。高浓度Ｋ＋处理也引起杜氏盐藻叶绿素含量的显著下降。对经高浓度Ｋ＋预处理的杜氏盐藻的光合放氧速率与培养液中ｐＨ变化同时进行测定的结果表明,Ｋ＋抑制杜氏盐藻光合速率的同时也显著抑制了光照条件引起的培养液ｐＨ的上升。实验结果表明,Ｋ＋抑制杜氏盐藻光合作用以及抑制杜氏盐藻生长与Ｋ＋影响跨盐藻质膜的质子运输之间可能存在一定关系。     

Exposure to low irradiances favors the synthesis of 9-cis beta, beta-carotene in Dunaliella salina (Teod.)

We examined the effect of irradiance on the synthesis of beta-carotene and its isomers by Dunaliella salina. Growth irradiance had a marked effect both on growth of the alga (which was suppressed at both low and high irradiances) and on the accumulation of beta-carotene. The accumulation of beta-carotene but not alpha-carotene was closely linked to an increase in irradiance. Growth at low irradiances (20-50 micromol m(-2) s(-1)) promoted a high ratio of 9-cis to all-trans beta-carotene (>2:1), while exposure to high irradiances (200-1,250 micromol m(-2) s(-1)) resulted in a large reduction in this ratio (to <0.45:1). A similar pattern was seen for the geometric isomers of alpha-carotene, with exposure to low irradiance favoring the accumulation of the 9-cis form. The carotenoid biosynthesis inhibitors 4-chloro-5(methylamino)-2-(alpha-alpha-alpha-trifluoro-m-tolyl)-3-(sH )-pyridazinone and 2-(4-chlorophenylthio)triethylamine caused the accumulation of the precursors phytoene and lycopene, respectively, in D. salina. High-performance liquid chromatography and infrared analysis showed that phytoene adopted the 15-cis and all-trans forms (as in higher plants), and that lycopene primarily adopted the all-trans form. This indicates that isomerization of beta-carotene takes place during or after cyclization.     

Physiological Mechanisms of Growth inhibition by Concentrated Potassium in Dunaliella Salina

It was observed in our preliminary experiments that the growth of Dunaliella salina (Dunal) Teed. cultured in medium containing 1 mol/L NaC1 was almost completely inhibited by the addition of 100 mmol/L KC1. This study was focused on elucidating the physiological mechanisms by which high K+ inhibits D. salina growth. Under the control conditions, the pH of the medium rapidly increased from 8 to 10 along with the growth of D. salina. However, addition of 100 mmol/L KCI significantly inhibited the increase of medium pH. The results suggested that the growth of D. salina may require a low environmental of H + concentration or be related to the H + transport into the D. salina cells. Interestingly, D. salina cultured in various media with different pH buffered with 40 mmol/L Tris-HC1 (or Tris-NaOH) did not show any significant difference in the growth rates. This result brings up the question on the previous hypothesis that Na+/H+ antiport plays a major role in Na + extrusion in D. salina. The photosynthetic rate of D. salina was not inhibited immediately following the addition of high K+ in the reaction medium, but it was significantly inhibited after the pretreatment of algae with high K+ for 12 hours or longer. The results from simultaneous measurements of O2 evolution of D. salina and the changes in pH of the medium revealed that the inhibitory effect of high K + on D. salina growth was somehow associated with K+ effect on pH changes in the medium. The high K+ treatment of D. salina also resulted in significant decrease of chlorophyll contents in the algae. It is concluded that the inhibitory effects of high K + on photosynthesis of D. salina may, at least partially, account for the growth-inhibition of D. salina growth by high K+; the inhibitory effects of high K+ on D. salina photosynthesis may result from the influence of K + on H + transport across the plasma membranes of D. salina cells.     

Effect of nutrient depletion on beta-carotene and glycerol accumulation in two strains of Dunaliella sp

The response of two strains of Dunaliella, a beta-carotene accumulating halotolerant alga, was evaluated under sulphate, nitrate and phosphate limitation. All these factors decreased the growth rate and chlorophyll content but, increased the beta-carotene content of the two isolates of Dunaliella, D1, obtained from GTCC and D2 an indigenous strain isolated from Sambhar salt lake, India. Both the strains exhibited accumulation of beta-carotene and glycerol under the different nutrient limiting conditions. A maximum accumulation of 3.99 pg/cell of beta-carotene was observed under phosphate depletion. However, nutrient depletion did not significantly affect the glycerol accumulation in these cells. D2, the indigenous isolate, was found to be a better accumulator of beta-carotene than D1.