Comparative growth and carotenoid production in the green microalga Dunaliella salina from marine and continental ecosystems: harnessing environmental diversity for sustainable industrial applications

N-glycosylation is a major post-translational modification of proteins that has a crucial influence on cell targeting, activity, and half-life. This process starts in the endoplasmic reticulum where an oligosaccharide precursor is added to the newly synthesized protein and continues in the Golgi apparatus where the N-linked carbohydrate sequences are processed. Importantly, the most approved recombinant pharmaceutical proteins (so-called biologics) are glycoproteins mainly currently produced in mammalian cells which is a lengthy, costly, and complex process. Today, several microalgae such as the diatom Phaeodactylum tricornutum, and the green microalgae Chlamydomonas reinhardtii, Chlorella vulgaris, and Dunaliella salina are considered as efficient and eco-friendly alternative platforms for the production of biologics. However, unlike for C. reinhardtii, C. vulgaris, and P. tricornutum, there is to date no data reported regarding the protein N-glycosylation pathway in D. salina. Here, we first investigated the protein N-glycosylation in this green microalga by MALDI-TOF mass spectrometry. These analyses showed that proteins from D. salina are N-glycosylated with Man5GlcNAc2 oligomannoside. Using genome mining approaches, we then identified genes encoding proteins involved in the N-glycosylation pathways in D. salina. Genetic similarities and phylogenetic relationships of the putative sequences with homologues from C. reinhardtii, P. tricornutum, and humans were investigated. These data revealed that in D. salina the biosynthesis of nucleotide sugars and N-glycan biosynthesis share mainly similarities with the GnT I-independent pathway of C. reinhardtii that gives rise to the synthesis of a non-canonical oligomannoside Man5GlcNAc2. Although an α(1,3)-fucosyltransferase is identified in the D. salina genome, impairment of the cytosolic GDP-Fuc biosynthesis prevents the Golgi fucosylation of N-glycans. Taken together, these data demonstrated that proteins from D. salina are homogeneously N-glycosylated with a non-canonical Man5GlcNAc2.

The halotolerant photoautotrophic marine microalga Dunaliella salina is one of the richest sources of natural carotenoids. Here we investigated the effects of high intensity blue, red and white light from light emitting diodes (LED) on the production of carotenoids by strains of D. salina under nutrient sufficiency and strict temperature control favouring growth. Growth in high intensity red light was associated with carotenoid accumulation and a high rate of oxygen uptake. On transfer to blue light, a massive drop in carotenoid content was recorded along with very high rates of photo-oxidation. In high intensity blue light, growth was maintained at the same rate as in red or white light, but without carotenoid accumulation; transfer to red light stimulated a small increase in carotenoid content. The data support chlorophyll absorption of red light photons to reduce plastoquinone in photosystem II, coupled to phytoene desaturation by plastoquinol:oxygen oxidoreductase, with oxygen as electron acceptor. Partitioning of electrons between photosynthesis and carotenoid biosynthesis would depend on both red photon flux intensity and phytoene synthase upregulation by the red light photoreceptor, phytochrome. Red light control of carotenoid biosynthesis and accumulation reduces the rate of formation of reactive oxygen species (ROS) as well as increases the pool size of anti-oxidant.

In this study, carotenoid and glycerol production in two unicellular green algae (Dunaliella salina and D. viridis) isolated from the Gave-Khooni salt marsh grown in media containing five different salt concentrations (0.17, 1, 2, 3, and 4 M NaCl) were evaluated under sterile conditions. Algae growth decreased as the medium salinity increased. Optimum growth of D. salina and D. viridis were obtained at 2 and 1 M NaCl, respectively. As salinity increased, glycerol and carotenoid production were increased in D. salina, whereas lower values for these products were produced in D. viridis under the same conditions. Furthermore, the cell color of D. salina changed from green to orange-red following accumulation of carotenoid, but the color of D. viridis was not changed. Thereby, it seems that the Iranian D. salina may be suitable for carotenoid production (betacarotene) on a large scale. In addition, since carotenoid compounds enhance the efficiency of photosynthesis and glycerol synthesis, it appears that the pathway for glycerol production and mechanisms of salt tolerance in D. viridis are unique from those of D. salina.

There is a particularly high interest to derive carotenoids such as β-carotene and lutein from higher plants and algae for the global market. It is well known that β-carotene can be overproduced in the green microalga Dunaliella salina in response to stressful light conditions. However, little is known about the effects of light quality on carotenoid metabolism, e.g., narrow spectrum red light. In this study, we present UPLC-UV-MS data from D. salina consistent with the pathway proposed for carotenoid metabolism in the green microalga Chlamydomonas reinhardtii. We have studied the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield and identified the optimal photon flux for D. salina growth. We found that the major carotenoids changed in parallel to the chlorophyll b content and that red light photon stress alone at high level was not capable of upregulating carotenoid accumulation presumably due to serious photodamage. We have found that combining red LED (75 %) with blue LED (25 %) allowed growth at a higher total photon flux. Additional blue light instead of red light led to increased β-carotene and lutein accumulation, and the application of long-term iterative stress (adaptive laboratory evolution) yielded strains of D. salina with increased accumulation of carotenoids under combined blue and red light.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-012-4502-5) contains supplementary material, which is available to authorized users.

The effect of salicylic acid (SA), an endogenous plant growth regulator, on adaptation of the green microalga, Dunaliella salina to N starvation was investigated through the study of enzymatic antioxidant system and biochemical changes. Algal cells in the exponential growth phase were exposed to N deficiency with 100 μM of SA. N starvation significantly decreased cell number, chlorophyll a, and hydrogen peroxide and while highly increased levels of fresh weight, soluble sugars, starch, proteins, free amino acids, and the activity of antioxidant enzymes. N-starved cells treated with SA enhanced cell number and hydrogen peroxide content, but accumulated lower amounts of metabolites and enzymatic activities compared to untreated cultures. However, the levels of fresh weight, chlorophyll, β-carotene, and soluble proteins remained roughly unchanged relative to N starvation alone. Proteolytic activity was well correlated with accumulation of amino acids in control and other treatments. The results suggest that exogenous SA treatment can enhance adaptation to N starvation by establishing the enzymatic balance to adjust levels of metabolites and directing them to the growth processes.

Enhanced β-carotene production in Dunaliella salina under relative high flashing light

We recently evaluated the relationship between abiotic environmental stresses and lutein biosynthesis in the green microalga Dunaliella salina and suggested a rational design of stress-driven adaptive evolution experiments for carotenoids production in microalgae. Here, we summarize our recent findings regarding the biotechnological production of carotenoids from microalgae and outline emerging technology in this field. Carotenoid metabolic pathways are characterized in several representative algal species as they pave the way for biotechnology development. The adaptive evolution strategy is highlighted in connection with enhanced growth rate and carotenoid metabolism. In addition, available genetic modification tools are described, with emphasis on model species. A brief discussion on the role of lights as limiting factors in carotenoid production in microalgae is also included. Overall, our analysis suggests that light-driven metabolism and the photosynthetic efficiency of microalgae in photobioreactors are the main bottlenecks in enhancing biotechnological potential of carotenoid production from microalgae.

Dunaliella salina is a high-quality industrial effector for carotenoid production. The mechanism by which red light regulates carotenoid synthesis is still unclear. In this study, a transcription factor of DsGATA1 with a distinct structure was discovered in D. salina. The recognition motif of DsGATA1 was comparable to that of plant and fungal GATA, despite its evolutionary proximity to animal-derived GATA. The expression of DsGATA1 in D. salina was still noticeably decreased when exposed to red light. Analysis of physiological and biochemical transcriptomic data from overexpressed, interfering, and wild-type strains of DsGATA1 revealed that DsGATA1 acts as a global regulator of D. salina carotenoid synthesis. The upregulated genes in the CBP pathway by DsGATA1 were involved in its regulation of the synthesis of carotenoids. DsGATA1 also enhanced carotenoid accumulation under red light by affecting N metabolism. DsGATA1 was found to directly bind to the promoter of nitrate reductase to activate its expression, promoting D. salina nitrate uptake and accelerating biomass accumulation. DsGATA1 affected the expression of the genes encoding GOGAT, GDH, and ammonia transporter proteins. Moreover, our study revealed that the regulation of N metabolism by DsGATA1 led to the production of NO molecules that inhibited carotenoid synthesis. However, DsGATA1 significantly enhanced carotenoid synthesis by NO scavenger removal of NO. The D. salina carotenoid accumulation under red light was elevated by 46% in the presence of overexpression of DsGATA1 and NO scavenger. Nevertheless, our results indicated that DsGATA1 could be an important target for engineering carotenoid production. KEY POINTS: • DsGATA1 with a distinct structure and recognition motif was found in D. salina • DsGATA1 enhanced carotenoid production and biomass in D. salina under red light • DsGATA1 is involved in the regulation of N metabolism and carotenoid synthesis.

Microalgae have become well-known in the last few decades as producers of various compounds of high nutritional value, such as long chain polyunsaturated fatty acids, pigments including carotenoids, proteins, sterols, and vitamins. Among these compounds, carotenoids and phytosterols have been receiving increased attention due to the discoveries that they are capable of preventing various diseases such as cardiovascular problems, certain cancers, and neurological disorders e.g. Alzheimer’s, amyotrophic lateral sclerosis etc. The commercial exploitation of the microalgal strains Dunaliella salina and Haematococcus pluvialis for β-carotene and astaxanthin, respectively, led to the hypothesis for the current study that other microalgal strains may even have a higher potential to produce these compounds, especially when the underlying physiological pathways that lead to elevated levels of these compounds could be stimulated. The aims of this thesis were therefore (1) to screen microalgal strains for their potential to produce carotenoids and phytosterols and (2) to induce the top producer(s) to accumulate higher quantities of carotenoids and phytosterols and (3) to scale up the successful induction method(s) to explore large-scale commercial production of these metabolites. Moreover, the well-known sensitive nature of carotenoids (e.g. astaxanthin and β-carotene) to light, drying and preservation methods led to the idea that freeze-drying rather than the currently popular spray-drying methods could be more suitable for long-term storage of highly valuable carotenoids, such as astaxanthin. As a first step, screening was carried out on twelve microalgal strains collected from brackish and marine waters for carotenoid profiles and contents, antioxidant capacity (total phenolic content and oxygen radical absorbance capacity (ORAC), and phytosterol profiles and contents in order to identify high carotenoid and phytosterol producing strains. Thereafter, the top-ranked strains were treated by plant hormones (salicylic acid (SA) and methyl jasmonate (MJ) for carotenoids), UV-C radiation (for carotenoids and phytosterols), osmotic shock (for phytosterols), nutrient manipulation (for phytosterols), and oxidative stress (H2O2/NaOCl treatments) in order to increase the accumulation of the target compounds. Furthermore, biomass from Haematococcus pluvialis (high astaxanthin producer) was dried either by freeze- or spray-drying, followed by preservation in vacuum or non-vacuum packs at -20oC to 37oC for 20 weeks in order to identify the most suitable drying and packaging methods and preservation temperature for astaxanthin. Among the species screened for carotenoids, the top four producers were Dunaliella salina, Tetraselmis suecica, Isochrysis galbana and Pavlova salina. The total phenolic content was low in these species, with D. salina possessing the highest content. The ORAC values were variable in the twelve strains, with the highest found in D. salina. Based on this ranking, D. salina and T. suecica were selected for further induction studies for carotenoids. These two microalgal strains were treated with SA, MJ, or UV-C radiation, and a combination of UV-C and plant hormones, and the carotenoid levels were measured in the biomass after the treatments. Responses to plant hormones were different in the two strains, with D. salina and T. suecica showing higher carotenoid production compared to mock-treated control cultures, when using MJ and SA, respectively. Both strains also demonstrated increased carotenoid production due to UV-C radiation; however, the dosage and induction time varied in the two strains. D. salina required less dosage but more time than T. suecica to produce more carotenoids than the mock-treated control cultures. The combination of these two treatments showed higher carotenoid production only in T. suecica. The success of the stresses caused by UV-C radiation led to the adoption of treatments with H2O2 or NaOCl in attempts to induce carotenoid production in these two strains as antioxidative defence molecules. However, responses to H2O2 or NaOCl treatments were different in the two strains. Although D. salina demonstrated higher carotenoid production due to H2O2 or NaOCl treatments, T. suecica showed higher carotenoid accumulation only in response to NaOCl. In the carotenoid stability experiment, the sensitivity of astaxanthin to high temperature, and air exposure was confirmed as low storage temperature and vacuum packing showed higher astaxanthin stability. Moreover, freeze- drying yielded higher astaxanthin content in the biomass and lower degradation at the low storage temperatures. Cost-benefit analysis showed freeze-drying followed by vacuum packed storage at -20oC can generate AUD $600 higher profit compared to spray-drying from 100 kg H. pluvialis powder. The screening of twelve microalgal strains for phytosterols identified Pavlova lutheri, Tetraselmis sp. M8, and Nannochloropsis sp. BR2 as the top three producers. Based on this ranking, Pavlova lutheri was selected for further induction studies for phytosterols. As sterols are well known to maintain osmotic balance in the cell membrane, osmotic shock treatment was used in P. lutheri with the hypothesis that the shock would induce higher sterol production. Although no increase in sterol accumulation could be found as a result of osmotic shock, higher sterol contents (up to a 2-fold increase) were found as the cultures grew older. In order to identify whether nutrients contributed to such high sterol accumulation, the P. lutheri cultures were grown in high nitrate or phosphate or nutrient-starved medium. However, no effect of high nitrate or phosphate or nutrient deprivation could be found on sterol accumulation. Moreover, due to evidence of anti-oxidative properties of phytosterols, the P. lutheri cultures were treated in H2O2 or UV-C radiation with the hypothesis that the oxidative stress caused by these treatments would induce higher production of sterols. However, the responses were different with only UV-C causing higher sterol accumulation, while H2O2 showed no effect on sterol production. Taken together, the results from this study identified T. suecica, D. salina, and P. lutheri as high carotenoid and phytosterol producing strains. Moreover, plant hormones such as SA and MJ, H2O2/ NaOCl treatments and UV-C radiation have been identified as useful tools for inducing carotenoid and phytosterol accumulation in these strains. Furthermore, molecular studies are warranted to understand the mechanism of the induction of carotenoids and phytosterols observed in this study. Additionally, freeze-drying has been identified as a mild and more profitable method for ensuring longer shelf life of astaxanthin from the H. pluvialis powder. Due to the scalable nature of the successful induction methods identified in this study, trials should be conducted at larger scale using these tools to explore commercial production of carotenoids and phytosterols from these strains.

Carotenoids are vital for most photosynthetic organisms because of their crucial role in prevention of the damage caused by excess light or stress conditions like heat or nutrient deprivation. Some of them are also valuable for biotechnology with respect to their colorful feature and antioxidant properties. In this study, a natural β-carotene producer, green microalga Dunaliella salina, was evaluated for its potential to produce different valuable carotenoids through the inhibition of cyclization reactions in the carotenoid pathway by the chemical inhibitors 2-methylimidazole (2MI) and 3-amino-1,2,4-triazole (Amitrol). 2MI was shown to be effective on lutein accumulation in D. salina cultures grown at high temperature, without affecting cell viability. Addition of 2MI to the culture at 1 mM concentration resulted a 1.7-fold increase in lutein content in the cell with the highest amount of 3.45 pg/cell, and an associated decrease in β-carotene content suggesting that this inhibitor is more effective on lycopene beta-cyclase activity, thus shifting the pathway from β-carotene arm to the α-carotene direction. The results of this study may give the opportunity to use D. salina for the production of valuable carotenoids other than β-carotene.

Biocompatible extraction emerges recently as a means to reduce costs of biotechnology processing of microalgae. In this frame, this study aimed at determining how specific culture conditions and the associated cell morphology impact the biocompatibility and the extraction yield of β-carotene from the green microalga Dunaliella salina using n-decane. The results highlight the relationship between the cell disruption yield and cell volume, the circularity and the relative abundance of naturally permeabilized cells. The disruption rate increased with both the cell volume and circularity. This was particularly obvious for volume and circularity exceeding 1500 µm3 and 0.7, respectively. The extraction of β-carotene was the most biocompatible with small (600 µm3) and circular cells (0.7) stressed in photobioreactor (30% of carotenoids recovery with 15% cell disruption). The naturally permeabilized cells were disrupted first; the remaining cells seems to follow a gradual permeabilization process: reversibility (up to 20 s) then irreversibility and cell disruption. This opens new carotenoid production schemes based on growing robust β-carotene enriched cells to ensure biocompatible extraction.

Dunaliella salina (Dunal) Teodoresco (1905) is a green unicellular alga able to withstand severe salt, light, and nutrient stress, adaptations necessary to grow in harsh environments such as salt ponds. In response to such growth conditions, this microalga accumulates high amounts of beta-carotene in its single chloroplast. In this study, we show that carotenoid accumulation is consistently inhibited in cells grown in nutrient-supplemented media and exposed either to high-light or medium-low-light conditions. Likewise, carotenogenesis in cells shifted to higher salinity (up to 27% NaCl) under medium-low-light conditions is inhibited by the presence of nutrients. The steady-state levels of transcripts encoding phytoene synthase and phytoene desaturase increased substantially in D. salina cells shifted to high light or high salt under nutrient-limiting conditions, whereas the presence of nutrients inhibited this response. The regulatory effect of nutrient availability on the accumulation of carotenoids and messenger RNA levels of the first two enzymes committed to carotenoid biosynthesis is discussed.

BackgroundRecent years have witnessed a rising trend in exploring microalgae for valuable carotenoid products as the demand for lutein and many other carotenoids in global markets has increased significantly. In green microalgae lutein is a major carotenoid protecting cellular components from damage incurred by reactive oxygen species under stress conditions. In this study, we investigated the effects of abiotic stressors on lutein accumulation in a strain of the marine microalga D. salina which had been selected for growth under stress conditions of combined blue and red lights by adaptive laboratory evolution.ResultsNitrate concentration, salinity and light quality were selected as three representative influencing factors and their impact on lutein production in batch cultures of D. salina was evaluated using response surface analysis. D. salina was found to be more tolerant to hyper-osmotic stress than to hypo-osmotic stress which caused serious cell damage and death in a high proportion of cells while hyper-osmotic stress increased the average cell size of D. salina only slightly. Two models were developed to explain how lutein productivity depends on the stress factors and for predicting the optimal conditions for lutein productivity. Among the three stress variables for lutein production, stronger interactions were found between nitrate concentration and salinity than between light quality and the other two. The predicted optimal conditions for lutein production were close to the original conditions used for adaptive evolution of D. salina. This suggests that the conditions imposed during adaptive evolution may have selected for the growth optima arrived at.ConclusionsThis study shows that systematic evaluation of the relationship between abiotic environmental stresses and lutein biosynthesis can help to decipher the key parameters in obtaining high levels of lutein productivity in D. salina. This study may benefit future stress-driven adaptive laboratory evolution experiments and a strategy of applying stress in a step-wise manner can be suggested for a rational design of experiments.

The microalga Dunaliella salina was studied at the main stages of transition from laboratory to pilot-scale cultivation: strain selection, nutrient medium selection, estimation of influence of physicochemical factors on accumulation of carotenoids and evaluation of the selected strain growing technology at pilot conditions. Dunaliella salina strain IBSS-2 was recognized as promising for commercial cultivation due to the combination of high-production characteristics, environmental stress resistance, and relative easiness of transition to the carotenogenesis stage. The influence of stress factors on the D. salina culture productivity in two nutrient media was estimated. It was shown that light effect combined with nutrients deficiency is the key factor for β-carotene accumulation. The influence of increased irradiance caused the increase of carotenoid content in D. salina cells up to 8%, and increasing irradiance and salinity resulted in carotenoid productivity going up 1.5 times. Testing of D. salina pilot cultivation system demonstrated that productivity at the first cultivation stage was about 6 g m−2 day−1 in both batch and semicontinuous mode. During pilot D. salina cultivation in Crimea, the culture transition to the carotenogenesis stage was achieved both in summer and autumn. The concentration of carotenoids in the ponds was 200 and 600 mg m−2 with a carotenoid/chlorophyll a (Car/Chl a) ratio of 7 and 4.5 in summer and autumn, respectively. The possibility to use natural population of D. salina cells from salterns as inoculum and brine as a nutrient medium base was demonstrated. The study results suggest that the proposed approach can be recommended for D. salina commercial cultivation.

1 - The effect of salinity (9, 14, and 22 % NaCl w/v) and nitrate concentration (882, 435 and 212 imol L-1) on the growth and production of carotenoids was studied in a wild strain of Dunaliella salina Teod. isolated from solar salterns on the Tyrrhenian coast (Tarquinia – Central Italy). 2 - The alga was grown in batch culture at relatively low illumination (40 imol photon m-2s-1). The total content of carotenoids produced from the alga and the amount of all-trans-â-carotene, the isomer showing a relevant commercial value, were determined by UV-Vis spectrophotometry and HPLC analysis. 3 - The highest growth rate in this strain of D. salina was obtained at salinity of 22% NaCl w/v and elevated nitrate concentration (882 imol L-1); a relatively high cell density was also observed at higher salinity and reduced nitrate concentrations. Low nitrate concentration negatively affected growth, but enhanced the carotenoids accumulation in the cells. 4 - The highest concentration of carotenoids (17 pg/cell) was observed in the 28 day old cultures in the late stationary phase at 22% NaCl w/v and 212 imol L-1 nitrate concentration. Also the average of all-trans-â-carotene on total carotenoids was enhanced by low nitrate concentration changing from 5% in 28 day old cultures at 22% salinity and 882 imol L-1 N, to 37% in 28 day old cultures at 22% NaCl w/v and lower nitrate concentration (212 imol L-1).