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Enhancing production of lutein by a mixotrophic cultivation system using microalga Scenedesmus obliquus CWL-1
Bioresource Technology 291 (2019) 121891
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Enhancing production of lutein by a mixotrophic cultivation system using microalga Scenedesmus obliquus CWL-1 Wei-Chuan Chena, Yin-Che Hsua, Jo-Shu Changb,c, Shih-Hsin Hod, Li-Fen Wange, Yu-Hong Weia,
T ⁎
a
Graduate School of Biotechnology and Bioengineering, Yuan Ze University, No. 135, Yuan-Tung Road, Chung-Li Dist, Taoyuan City 32003, Taiwan, ROC Department of Chemical Engineering, National Cheng Kung University, Tainan City 701, Taiwan, ROC c Department of Chemical and Materials Engineering, College of Engineering, Tunghai University, Taichung 407, Taiwan, ROC d Department of Environmental Science and Technology, Harbin Institute of Technology, China e Department of Applied Chemistry and Materials Science, Fooyin University, 151 Jinxue Rd, Daliao Dist., Kaohsiung City 83102, Taiwan, ROC b
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Lutein Scenedesmus obliquus Light-related strategies Nitrate concentration Fed-batch fermentation
This work studies a series of strategies in the production of lutein by Scenedesmus obliquus CWL-1 under mixotrophic cultivation. Our experimental results revealed that the optimal conditions associated with light-related strategies were 12 h light period followed by a 12 h dark period and blue to red light under mixotrophic cultivation. Under such conditions, the biomass, lutein content and lutein productivity were maximized to 9.88 (g/ L), 1.78 (mg/g) and 1.43 (mg/L/day), respectively. Moreover, the assimilation of 4.5 g/L of calcium nitrate into S. obliquus CWL-1 increased the maximal biomass (12.73 g/L) and the highest maximal lutein productivity (3.06 mg/L/day), while the assimilation of 1.5 g/L of calcium nitrate yielded the highest maximal lutein content of 2.45 mg/g. The highest maximal lutein productivity of 4.96 (mg/L/day) was obtained when fed-batch fermentation was conducted, and this value was approximately 11-folds that obtained using the batch system.
1. Introduction Lutein ((3R, 3′R, 6′R)-β, ε-carotene-3, 3′-diol), a photosynthetic pigment, is a type of xanthophyll and one of 600 well-known carotenoids that are present in the natural world (Humphries and Khachik, 2003). Lutein is simply synthesized by green plants and is found in large
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amounts in green leafy vegetables. In the human retina, lutein is absorbed from blood specifically into the macula lutea (Del Campo et al., 2007), although its precise function in the body is unknown. Lutein is also a natural colorant in poultry feathers, drugs, and cosmetics and is extensively used in the food industry. Its aforementioned important properties make lutein a highly valuable chemical. Lutein production is
Corresponding author. E-mail address: [email protected] (Y.-H. Wei).
https://doi.org/10.1016/j.biortech.2019.121891 Received 31 May 2019; Received in revised form 22 July 2019; Accepted 23 July 2019 Available online 26 July 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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expected to grow rapidly at 3.6% annually (Sun et al., 2016). Most of the current commercial production of lutein is from marigold petals. However, its high production cost, the low lutein content (0.03%) of the marigold petals, and their harvesting and separation, pose serious challenges (Piccaglia et al., 1998). Microalgae, a group of photosynthetic microorganisms, are commonly found in aquatic environments. Many specifies of microalgae accumulate large amounts of lutein in their biomass. Recently, the production of lutein from microalgae, including Chlorella protothecoides (Shi et al., 2000), Dunaliella salina (Fu et al., 2014), Scenedesmus almeriensis (Sánchez et al., 2008), and Galdieria sulphuraria (Marquardt, 1998), has been widely studied. This production has been one of the most successful applications of microalgal biotechnology. Lutein production using microalgae as a source has many merits, including the following (Ho et al., 2014); (a) the biomass of the microalgae can be utilized without the need to process petals from plants; (b) a high growth rate in the biomass of the microalgae can support high lutein productivity and (c) a larger amount of lutein is obtained than can be obtained using traditional methods, reflecting high biomass productivity. However, the technology for lutein production using microalgae is not mature. Accordingly, to improve the feasibility of the commercial production of lutein, the cultivation of microalgae must be further studied, focusing especially on cell growth, lutein content, and overall lutein productivity. The photo-autotrophic cultivation of microalgae is frequently used in the production of lutein, because of the cells’ physiological response to high light stress in photosynthetic pigment synthesis (Chen and Liu, 2018). Recently, mixotrophic cultivation combines the advantages of both photoautotrophy and heterotrophy. Mixotrophic cultivation involves the uptake of organic and inorganic nutrient sources in the presence of light by microalgae under environments of aerobic respiration and photosynthesis (Velea et al., 2017; Yen et al., 2011). Reports have established that mixotrophic the cultivation of microalgae has many merits, including reduction of the photo inhibitory effect of photosynthetic capacity, nighttime loss of biomass, and photo-oxidative damage during the cultivation period (Chen et al., 2018; Chen and Liu, 2018; Velea et al., 2017). Mixotrophic cultivation has been become a feasible strategy for the production of lutein from microalgae with high biomass, high lutein content and high productivity. Several value-added metabolites are produced by the genus Scenedesmus in autotrophic culture (Sánchez et al., 2008), which grows heterotrophically (Ren et al., 2013; Flórez-Mirandaa et al., 2017). S. obliquus is a freshwater chlorophycean microalga, which has been isolated from freshwater in southern Taiwan (Ho et al., 2014). It has recently attracted attention as a commercially valuable source of bioactive compounds, including antimicrobial and anticancer compounds (Marrez et al., 2019). However, the productivity of lutein using mixotrophic cultures of S. obliquus has so far been relatively low. This study concerns the optimization of mixotrophic cultivation conditions for the production of lutein by microalga, and studies the effects thereon of various light-related strategies, involving various types of light, light/dark periods and two-stage light source operations. Then, the mixotrophic cultivation of microalga is studied by investigating the effect of nitrate concentration on the mixotrophic cultivation of microalga is studied. Finally, mixotrophic cultivation was performed in a bioreactor in a manner determined by the obtained experimental results.
0.25 g/L of KCl (Sigma), 0.25 g/L of MgSO4·7H2O, 0.26 g/L of KH2PO4, 0.02 g/L of FeSO4·7H2O, 0.2 g/L of EDTA·2Na, 2.9 × 10−3 g/L of H3BO3, 1.8 × 10−3 g/L of MnCl2, 1.1 × 10−4 g/L of ZnCl, 0.8 × 10−3 g/L of CuSO4·5H2O and 1.8 × 10−5 g/L of (NH4)6Mo7O24·4H2O. To study the effects of different light-related strategies on the production of lutein by microalga, light of four wavelengths from an LED (T8 lamp) with a light intensity of 150 μmol/m2/s was used. The four wavelengths corresponded to red, blue, green and white light: the wavelengths were 600–690, 435–515, and 480–580 nm, respectively. To study the effect of the light period/dark period, two conditions (a 24 h light period and then a 12 h light period followed by a 12 h dark period (12L:12D)) were used to optimize the illumination to enhance the production of microalgal biomass and its lutein content. This study was performed to evaluate the microalgal biomass production, lutein concentration, lutein content and lutein productivity. In this experiment, 2% of glucose and 1 g/L of Ca(NO3)2·4H2O in 200 mL of DM medium in a flask was used. A 0.2 g mass of dry microalgae was inoculated for four days of cultivation under blue light (or red light) (first stage). The pellet of microalgae was then moved to fresh DM medium under red light (or blue light) for four days (second stage). The microalgal biomass production, lutein concentration, lutein content and lutein productivity were determined. To study the effect of the assimilation of a nitrogen source into S. obliquus CWL-1, various concentrations of Ca(NO3)2·4H2O from 0.5–5 g/L (in steps of 0.5 g/L) were used and the corresponding microalgal biomass production, lutein concentration, lutein content and lutein productivity were monitored.
2. Methods
The residual nitrate concentration was modified based on the work of Ho et al. (2014). In brief, samples were taken from the microalgal culture and filtered through a filter with 0.22 μm pores. The samples were properly diluted to ensure that the absorbance (optical density at a wavelength of 220 nm, OD220), measured using a UV/VIS spectrophotometer (model U-2001, Hitachi, Tokyo, Japan), was linearly correlated with the nitrate concentration. Calcium nitrate was purchased from Sigma for use as the standard.
2.2. Operation of stirred tank reactor The stirred tank reactor (STR) was used in the cultivation of S. obliquus CWL-1, respectively. The STR with a 7 L glass vessel was illuminated by sources of white light (150 μmol/m2/s, 12 h light period followed by a 12 h dark period) that were mounted around the reactor. The pre-cultured S. obliquus CWL-1 was inoculated into STR with a 400 mg/L inoculum. The STR was operated at 30℃ with an agitation rate of 150 rpm. Two percent glucose in 3 L of DM medium with 2% CO2 was continuously fed into the culture for nine days of cultivation. Liquid samples were collected from the sealed glass vessel to measure microalgae concentration, residual nitrate concentrations, and lutein concentration, lutein content and lutein productivity.
2.3. Determination of microalgal biomass concentration The biomass concentrations in the microalgal cultures in flasks and reactors were determined by measuring the optical density at a wavelength of 680 nm (OD680) using a UV/VIS spectrophotometer. The samples were properly diluted to ensure that the absorbance was linearly correlated with the biomass concentration. The OD680 values were converted to dry cell weight (DCW) using the following calibration formula: DCW = 0.4695 × OD680 − 0.2634. The calibration between the DCW and OD680 was performed repeatedly to ensure the accuracy of the conversion factor.
2.4. Determination of residual nitrate concentration
2.1. Microalgal strain, culture medium and culture conditions Professor Jo-Shu Chang from National Cheng Kung University kindly provided S. obliquus CWL-1 (Ho et al., 2014). Detmer’s medium (DM) was used to grow a pure culture of S. obliquus CWL-1, it comprised 1 g/L of Ca(NO3)2·4H2O (Sigma-Aldrich, St. Louis, Missouri, USA), 2
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Under 24 h of illumination, the highest maximal lutein content and lutein productivity of S. obliquus CWL-1 were 1.20 (mg/g) and 1.17 (mg/L/day), respectively. White LEDs provide higher lutein production efficiency than the three monochromatic LEDs (red, blue, and green) (Ho et al., 2014). Other works have also proved that the pigment content of coastal plankton diatoms increases under illumination by white light (Sanchez-Saavedra and Voltolina, 2002), possibly because of a complementary chromatic adaptation by which white light not only participates in the rearrangement of chloroplasts but also stimulates the synthesis of chlorophyll. As already mentioned, illumination for 24 hr facilitates an increase in not only the biomass of S. obliquus CWL-1, but also the lutein content and lutein productivity of S. obliquus CWL-1. These results suggest that the right light source is critical to the accumulation of microalgal lutein.
2.5. Determination of microalgal lutein content The method used for lutein extraction from microalgal biomass was a modified protocol of that presented by Yen et al. (2011). Briefly, 10 mL of thoroughly mixed culture fluid from the fermenter was transferred to a 15 mL conical tube. The culture was then centrifuged at 8000 rpm for 15 min, and the supernatant was discarded. Ten milliliters of methanol were subsequently added to the mixture for the ultrasonic extraction of lutein. The mixture was then centrifuged at 8000 rpm for 15 min at 4 °C. Pigments were analyzed using an HPLC system with an RP-18e (250 × 4.68 mm) column under ambient temperature (27 ± 1 °C). The mobile phase used initially consisted of 60% solvent A (acetonitrile/methanol, 20/80 v/v) and 40% solvent B (methanol/ acetone, 80/20 v/v) and was then modified to 30% solvent A and 70% solvent B in a period of 15 min. The column was subsequently returned to its original solvent composition (i.e., 60% solvent A and 40% solvent B) for the next 6 min before the injection of a new sample. The flow rate used was 1.0 mL/min and the UV detector (Hitachi) was operated at 450 nm.
3.1.2. Effect of dark/light period on cell growth, lutein content and lutein productivity of S. obliquus CWL-1 As displayed in Table 1, light provides the energy that is used by microalgae, owing to the photosynthetic process. Accordingly, the dark/light period affects algal growth and biomass production. The light regimen in the natural environment is not constant and changes in the period of illumination (such as to 12L:12D) may increase productivity and photosynthetic efficiency, improving cell growth, lutein content and lutein productivity. Table 2 presents the effects of the dark/light period on the maximal biomass of S. obliquus CWL-1. The maximal biomass of S. obliquus CWL1 (4.48 g/L) was reached when the blue light source was used for six days cultivation. Clearly, the maximal biomass under 12L:12D was higher than that under 24 h of light (Table 1 vs. Table 2). These results reveal that the light period greatly influenced the growth of microalgal biomass. Changing the light/dark periods may change the biochemical composition of the microalgae (Krzemińska et al., 2014). Another work has established that cyanobacterium Aphanothece microscopica Nägeli exhibits higher productivity and maximal cell density under a 12:12 (night:day) photoperiod than with other photoperiods (Jacob-Lopes et al., 2009). The highest maximal lutein productivity of S. obliquus CWL-1 was 1.36 (mg/L/day), which was reached under 12L:12D using white light (Table 2). Increasing the frequency of the light/dark period greatly increases the productivity of metabolites and photosynthetic efficiency (Fábregas et al., 2002). Grobbelaar (2009) showed that pulsing light at frequencies equivalent to those of electron turnover in the electron transport chains associated with photosynthesis increases the yields of metabolites and photosynthetic efficiency. Additionally, the dissipation of excess heat as a result of light-induced damage to photosynthetic organisms is the primary function of lutein (Chen et al., 2019). Therefore, more lutein accumulates under 12L:12D than under 24 h of light.
3. Results and discussion 3.1. Effects of light-related strategies on cell growth, lutein content and lutein productivity of S. obliquus CWL-1 3.1.1. Effect of various wavelength of light source on cell growth, lutein content and lutein productivity of S. obliquus CWL-1 The photoautotrophy of microalgae cultivation for the production of bioactive compounds such as lutein may require exposure to sunlight. The sunlight is captured and photosynthesis is the main source of energy for the survival and growth of the microalgae. Reports have established that light of a specific wavelength not only favors microalgal growth but also facilitates the accumulation of valuable-added bioactive compounds, such as natural pigments and fatty acids (Kim et al., 2013). Therefore, a series of mixotrophic light-related strategies were used with S. obliquus CWL-1 to examine its cell growth, lutein content and lutein productivity. They involved illuminating the microalgae for 24 h, and separately for 12 h followed by a 12 h dark period (12L:12D). Four light sources with different wavelengths, corresponding to the colors red, blue, green and white (T8 lamp), were used. For microalgae cultivation, the intensity of each light source was fixed at 150 μmol/ m2/s. As indicated in Table 1, the effects of wavelength on S. obliquus CWL-1 under 24 h of illumination differed significantly from maximal biomass. The maximal biomass of S. obliquus CWL-1 after six days cultivation increased in the order of red, green, blue and white. Therefore, the maximal biomass decreased as the light wavelength increased. The effect of the wavelength of light on the growth of microalgae herein was consistent with the literature (Ho et al., 2014), white light is the most favorable light for the growth of biomass of S. obliquus. Indeed, white light (400–700 nm) is typically used for microalgal growth (Carvalho et al., 2011). Table 1 Effects of wavelength of light source on cell growth and lutein production of S. obliquus CWL-1 under 24 h illumination: maximal biomass, maximal lutein content and maximal lutein productivity. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp).
Table 2 Effects of wavelength of light source on cell growth and lutein production of S. obliquus CWL-1 under 12L:12D condition: maximal biomass, maximal lutein content and maximal lutein productivity. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp).
Types of light
Maximal biomass (g/L)
Maximal lutein content (mg/g)
Maximal lutein productivity (mg/L/Day)
Types of light
Maximal biomass (g/L)
Maximal lutein content (mg/g)
Maximal lutein productivity (mg/L/Day)
White Red Blue Green
4.26 2.17 2.97 2.53
1.20 1.14 0.99 1.51
1.17 0.44 0.63 0.53
White Red Blue Green
4.34 4.37 4.48 3.96
1.48 1.68 1.26 1.54
1.36 1.09 1.28 1.07
± ± ± ±
0.30 0.06 0.07 0.31
± ± ± ±
0.08 0.25 0.17 0.13
± ± ± ±
0.03 0.02 0.10 0.04
3
± ± ± ±
0.02 0.41 0.15 0.19
± ± ± ±
0.20 0.36 0.45 0.21
± ± ± ±
0.07 0.08 0.09 0.07
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fresh DM medium was exchanged, and then declined with increasing cultivation time to the end of the experiment (Fig. 1A and 1B). These results may be explained by the fact that the biomass increased with the cultivation time, rapidly consuming the fresh medium, reducing lutein productivity. The maximal lutein content was obviously higher under the two-stage light source operation than under the 24 h light period or 12L:12D. However, the lutein content was not effectively increased, suggesting that the high biomass of microalgae did not adsorb light evenly. An increase in the area of the photon-rich zone increased the proportion of cells that received enough light to perform photosynthesis (Carvalho et al., 2011).
3.2. Effect of nitrate concentration on cell growth, lutein content and lutein productivity of S. obliquus CWL-1 Nitrogen has an important role of microalgal cells, which is a significant fraction, one to ten percent, of the dry weight of microalgal cells (Martin-Jézéquel et al., 2000). Therefore, nitrogen is vital for the growth and metabolism of microalgal cells (Perez-Garcia et al., 2011). Microalgal cells can use various nitrogen sources, mainly ammonia (NH4+), nitrate (NO3–), and urea, as well as yeast extract and amino acids can be used by Liu and Chen (2016). With respect to lutein accumulation, the availability of a nitrogen source strongly affects microalgae under phototrophic cultivation (Xie et al., 2013). Therefore, the effects of the concentration of nitrate on cell growth, lutein content and lutein productivity were studied to determine the optimal calcium nitrate concentration for mixotrophic cultivation. S. obliquus CWL-1 was cultivated under 12L:12D with a light intensity of 150 μmol/m2/s for six days. The experimental results in Fig. 2 reveal that the use of calcium nitrate was related to lutein content and lutein productivity. The lutein content increased with the biomass for the first three days and then decreased as the system became deficient in calcium nitrate. Both the maximal lutein content (1.37 mg/g) and the maximal lutein productivity (0.95 mg/L/day) were obtained at the beginning of the period of deficient calcium nitrate. This finding is similar to an earlier finding of maximal lutein in the early stationary phase in Muriellopsis sp., approximately when all available nitrogen was consumed (Del Campo et al., 2000). Ten concentrations of calcium nitrate 0.5–5 g/L (in steps of 0.5 g/L) were used and the microalgal biomass production, lutein content and lutein productivity observed. As shown in Fig. 3, the maximal biomass dramatically increased with the concentration of calcium nitrate from 0.5 g/L to 3.5 g/L. As the concentration increased above 3.5 g/L, the amount of biomass increased more slowly. The most maximal biomass (12.73 g/L) was obtained when 4.5 g/L of calcium nitrate was used. The maximal lutein productivity and maximal lutein productivity (3.06 mg/ L/day) were also highest when 4.5 g/L of calcium nitrate was used, because the higher cell density that was obtained using a higher nitrate concentration caused stronger light shading (Ho et al., 2015). The experimental results in Fig. 3 reveal that the maximal lutein content increased from 1.37 mg/g to 2.45 mg/g as the nitrate concentration increased from 0.5 g/L to 1.5 g/L. These experimental results are in good agreement with similar results concerning other microalgal strains, indicating an improvement in lutein content with increasing nitrogen concentration (Cordero et al., 2011; Xie et al., 2013; Ho et al., 2015). However, the maximal lutein content decreased from 1.87 mg/g
Fig. 1. Effects of two-stage light operation, red light to blue light (A) and blue light to red light (B)) on cell growth and lutein production of S. obliquus CWL-1: biomass, lutein concentration, lutein content and lutein productivity. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp).
3.1.3. Effect of two-stage light operation on cell growth, lutein content and lutein productivity of S. obliquus CWL-1 As depicted in Table 2, the experimental results reveal that the wavelength of light significantly affected cell growth, lutein content and lutein productivity sources. The maximal lutein content (1.68 mg/ g) was highest when red light was used under the 12L:12D condition and the maximal biomass was highest (4.48 g/L) when blue light was used under the 12L:12D condition (Table 2). To study further the effect of a two-stage light source operation on lutein production under 12L:12D, the microalgal biomass production, lutein concentration, lutein content and lutein productivity were studied. The experimental results in Fig. 1 and Table 3 reveal that the maximal biomass and maximal lutein productivity of S. obliquus CWL-1 markedly increased to approximately 10 g/L and 8.2 (mg/L) from the cultivation of beginning under blue light and then red light or red light and then blue light. Wavelengths between 400 and 700 nm are typically used for microalgal growth (Carvalho et al., 2011). Additionally, blue light (420–450 nm) and red light (660–700 nm) are equally efficient for photosynthesis (Carvalho et al., 2011). The maximal lutein productivity in Fig. 1 and Table 3 was 1.32 (mg/ L/day) under red light to blue light and 1.43 (mg/L/day) under blue light to red light. Lutein productivity remained stable for two days after
Table 3 Effects of two-stage light operation on cell growth and lutein production of S. obliquus CWL-1: maximal biomass, maximal lutein content and maximal lutein productivity. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp). Types of light
Maximal biomass (g/L)
Maximal lutein content (mg/g)
Maximal lutein productivity (mg/L/Day)
Red light to Blue light Blue light to Red light
10.12 ± 0.30 9.88 ± 0.98
1.67 ± 0.08 1.78 ± 0.10
1.32 ± 0.02 1.43 ± 0.18
4
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Fig. 2. Profiles of cell growth, residual nitrate concentration, lutein content, and productivity of S. obliquus CWL-1. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp).
Fig. 4. Effects of fed-batch fermentation on cell growth and lutein production of S. obliquus CWL-1: (A) biomass, residual nitrate concentration, lutein content and maximum lutein productivity with 1.0 g/L of calcium nitrate as a feeding substrate and (B) biomass, residual nitrate concentration, lutein content and maximum lutein productivity with 4.5 g/L of calcium nitrate as a feeding substrate. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp). Fig. 3. Effects of concentration of calcium nitrate on cell growth and lutein production of S. obliquus CWL-1 under two-stage light operation: maximal biomass, maximal lutein content and maximum lutein productivity. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp).
nitrate was fed into the STR. The highest biomass (12.93 g/L) was achieved after the second feeding of calcium nitrate on the fifth day of cultivation. Then the biomass slightly decreased, even though calcium nitrate was repeatedly fed into the STR until the end of cultivation. Similar results are observed in Fig. 4B, which reveals that the highest biomass (12.80 g/L) was reached on the fifth day of cultivation, when the initial calcium nitrate concentration of 4.5 g/L declined for the first time, and the biomass then slightly declined until the end of the cultivation. A report has also found that the biomass concentration increased with the amount of added urea under the fed-batch cultivation of Chlorella sp. (Hsieh and Wu, 2009). The lutein productivity varied similarly with both initial concentrations of calcium nitrate, 1.0 g/L or 4.5 g/L (Fig. 4A and B). The highest lutein productivity that was reached with 1.0 g/L or 4.5 g/L calcium nitrate was 4.44 (mg/L/day) or 4.96 (mg/L/day), respectively. These results are attributed to the light shading effect. The light only slightly penetrated the cell broth, and the specific light intensity decreased sharply with penetration, because the biomass concentration and lutein productivity increased with the cultivation time at a high nitrate concentration (Cheirsilp and Torpee, 2012). The lutein contents in Fig. 4A and 4B are correlated with calcium nitrate concentration. The highest lutein contents were quite close, being 2.58 mg/g at 1.0 g/L and 2.55 mg/g at 4.5 g/L of calcium nitrate. These results suggest that the lutein content was raised when the calcium nitrate had not completely depleted. Under the fed-batch strategy, the calcium concentration must be kept sufficiently high. However, the concentrations of other nutrients, such as the carbon source (glucose) and trace elements, decreased as the cultivation time increased. This variation facilitated the production and storage of to the metabolites such as lutein, by the microalgae, providing energy for survival. Xie
to 1.29 mg/g as the concentrations of nitrogen increased above 1.5 g/L (to 2 g/L and 5 g/L). Plant cells must reduce assimilated nitrate to ammonia by membrane transportation, consuming large amounts of energy, carbon, and protons (Crawford and Forde, 2002). The maximal lutein content herein agrees closely with that finding. Also, an excess of calcium nitrate might have led to a low pH value when nitrate reductase is fully expressed in most microalgae and the sole nitrogen source is nitrate, reducing the lutein content (Perez-Garcia et al., 2011). 3.3. Effect of fed-batch fermentation on cell growth, lutein content and lutein productivity of S. obliquus CWL-1 under mixotrophic cultivation The results in Fig. 4 reveal that an initial concentration of calcium nitrate of 4.5 g/L with yielded the highest lutein productivity (3.06 mg/ L/day) and the highest biomass (12.73 g/L), with the complete consumption of the calcium nitrate on the fifth day of cultivation. Accordingly, to measure the cell growth, lutein content and lutein productivity of S. obliquus CWL-1, fed-batch fermentation using an STR was conducted. A constant calcium nitrate concentration of 4.5 g/L is manipulated in the STR. In the control, the concentration was 1.0 g/L. The cell growth, lutein content and lutein productivity of S. obliquus CWL-1 were studied to observe how calcium nitrate affected by fed-batch fermentation. Fig. 4A shows that the initial concentration of calcium nitrate, 1.0 g/L, declined on the third day of cultivation, when 1.0 g/L calcium 5
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et al. (2017) presented similar results, which revealed that nitrate pulse feeding promoted β-carotene biosynthesis and the bioconversion of αcarotene to lutein. Based on these results, maintaining the nitrogen concentration at an appropriate level facilitated the accumulation of lutein. By this fed-batch strategy, the highest lutein productivity of 4.96 mg/L/day was achieved, which is about 11-fold that obtained using the batch system. This result demonstrates the feasibility of commercializing fed-batch fermentation with calcium nitrate supplementation.
production in the green microalga Dunaliella salina. Microb. Cell Fact. 13, 3. Grobbelaar, J.U., 2009. Upper limits of photosynthetic productivity and problems of scaling. J. Appl. Phycol. 21, 519–522. Ho, S.H., Chan, M.C., Liu, C.C., Chen, C.Y., Lee, W.L., Lee, D.J., Chang, J.S., 2014. Enhancing lutein productivity of an indigenous microalga Scenedesmus obliquus FSP-3 using light-related strategies. Bioresour. Technol. 152, 275–282. Ho, S.H., Xie, Y., Chan, M.C., Liu, C.C., Chen, C.Y., Lee, D.J., Huang, C.C., Chang, J.S., 2015. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. 184, 131–138. Hsieh, C.H., Wu, W.T., 2009. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresour. Technol. 100, 3921–3926. Humphries, J.M., Khachik, F., 2003. Distribution of lutein, zeaxanthin, and related geometrical isomers in fruit, vegetables, wheat, and pasta products. J. Agric. Food Chem. 51, 1322–1327. Flórez-Mirandaa, L., Cañizares-Villanuevaa, R.O., Melchy-Antonioa, O., MartínezJerónimob, F., Flores- Ortízc, C.M., 2017. Two stage heterotrophy/photoinduction culture of Scenedesmus incrassatulus: potential for lutein production. J. Biotechnol. 262, 67–74. Jacob-Lopes, E., Scoparo, C., Lacerda, L., Franco, T., 2009. Effect of light cycles (night/ day) on CO2 fixation and biomass production by microalgae in photobioreactors. Chem. Eng. Process. 48, 306–310. Liu, J., Chen, F., 2016. Biology and industrial applications of chlorella: advances and prospects. Adv. Biochem. Eng. Biotechnol. 153, 1–35. Kim, T.H., Lee, Y., Han, S.H., Hwang, S.J., 2013. The effects of wavelength and wavelength mixing ratios on microalgae growth and nitrogen, phosphorus removal using Scenedesmus sp. for wastewater treatment. Bioresour. Technol. 130, 75–80. Krzemińska, I., Pawlik-Skowrońska, B., Trzcińska, M., Tys, J., 2014. Influence of photoperiods on the growth rate and biomass productivity of green microalgae. Bioprocess Biosyst. Eng. 37, 735–741. Marrez, D.A., Naguib, M.M., Sultan, Y.Y., Higazy, A.M., 2019. Antimicrobial and anticancer activities of Scenedesmus obliquus metabolites. Heliyon 5, e01404. Marquardt, J., 1998. Effects of carotenoid-depletion on the photosynthetic apparatus of a Galdieria sulphuraria (rhodophyta) strain that retains its photosynthetic apparatus in the dark. J. Plant Physiol. 152, 372–380. Martin-Jézéquel, V., Hildebrand, M., Brzezinski, M.A., 2000. Silicon metabolism in diatoms: implications for growth. J. Phycol. 36, 821–840. Perez-Garcia, O., Escalante, F.M., de-Bashan, L.E., Bashan, Y., 2011. Heterotrophic cultures of microalgae: metabolism and potential products. Water Res. 45, 11–36. Piccaglia, R., Marotti, M., Grandi, S., 1998. Lutein and lutein ester content in different types of Tagetes patula and T. erecta. Ind. Crops Prod. 8, 45–51. Ren, H.Y., Liu, B.F., Ma, C., Zhao, L., Ren, N.Q., 2013. A new lipid-rich microalga Scenedesmus sp: strain R-16 isolated using Nile red staining: effects of carbon and nitrogen sources and initial pH on the biomass and lipid production. Biotechnol. Biofuel. 6, 143. Sánchez, J.F., Fernández-Sevilla, J.M., Acién, F.G., Cerón, M.C., Pérez-Parra, J., MolinaGrima, E., 2008. Biomass and lutein productivity of Scenedesmus almeriensis: influence of irradiance, dilution rate and temperature. Appl. Microbiol. Biotechnol. 79, 719–729. Sanchez-Saavedra, M.P., Voltolina, D., 2002. Effect of photon fluence rates of white and blue–green light on growth efficiency and pigment content of three diatom species in batch cultures. Cien. Marinas 28, 273–279. Shi, X., Zhang, X., Chen, F., 2000. Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources. Enzyme Microb. Technol. 27, 312–318. Sun, Z., Li, T., Zhou, Z.G., Jiang, Y., 2016. Microalgae as a source of lutein: chemistry, biosynthesis, and carotenogenesis. Adv. Biochem. Eng. Biotechnol. 153, 37–58. Xie, Y., Ho, S.H., Chen, C.N., Chen, C.Y., Ng, I.S., Jing, K.J., Chang, J.S., Lu, Y., 2013. Phototrophic cultivation of a thermo-tolerant Desmodesmus sp. for lutein production: effects of nitrate concentration, light intensity and fed-batch operation. Bioresour. Technol. 144, 435–444. Xie, Y., Zhao, X., Yang, X., Lu, Y., Zheng, X., Chen, J., 2017. Effect of nitrate pulse-feeding cultivation on cell growth and cell composition of thermo-tolerant Desmodesmus sp. F51. Food Sci. 38, 64–70. Velea, S., Oancea, F., Fischer, F., Muñoz, R., 2017. 2 - Heterotrophic and mixotrophic microalgae cultivation A2 - Gonzalez-Fernandez, Cristina2 - Heterotrophic and mixotrophic microalgae cultivation A2 - Gonzalez-Fernandez, Cristina. In: Microalgae-Based Biofuels and Bioproducts. Woodhead Publishing, pp. 45–65. Yen, H.W., Sun, C.H., Ma, T.W., 2011. The comparison of lutein production by Scenesdesmus sp. in the autotrophic and the mixotrophic cultivation. Appl. Biochem. Biotechnol. 164, 353–361.
4. Conclusions This study utilized a series of light-related strategies for the production of lutein by S. obliquus CWL-1 under mixotrophic cultivation using fed-batch operation and various nitrate concentrations. The highest lutein production (9.88 g/L) was achieved in a 12L:12D blue to red light regime with mixotrophic cultivation. The lutein productivity increased to 3.06 (mg/L/day) upon the assimilation of 4.5 g/L of calcium nitrate. The highest lutein productivity of 4.96 (mg/L/day) was achieved using fed-batch operation. The strategies used in this work seem to demonstrate the potential for commercial production of microalgal lutein. Acknowledgements The authors gratefully acknowledge the financial support provided by the Ministry of Science and Technology of the Republic of China under grant number MOST 107-2221-E-155-022-MY3. References Carvalho, A.P., Silva, S.O., Baptista, J.M., Malcata, F.X., 2011. Light requirements in microalgal photobioreactors: an overview of biophotonic aspects. Appl. Microbiol. Biotechnol. 89, 1275–1288. Cheirsilp, B., Torpee, S., 2012. Enhanced growth and lipid production of microalgae under mixotrophic culture condition: effect of light intensity, glucose concentration and fed-batch cultivation. Bioresour. Technol. 110, 510–516. Chen, C.Y., Liu, C.C., 2018. Optimization of lutein production with a two-stage mixotrophic cultivation system with Chlorella sorokiniana MB-1. Bioresour. Technol. 262, 74–79. Chen, C.-Y., Lu, I.C., Nagarajan, D., Chang, C.-H., Ng, I.S., Lee, D.-J., Chang, J.-S., 2018. A highly efficient two-stage cultivation strategy for lutein production using heterotrophic culture of Chlorella sorokiniana MB-1-M12. Bioresour. Technol. 253, 141–147. Chen, J.H., Chen, C.Y., Hasunuma, T., Kondo, A., Chang, C.H., Ng, I.S., Chang, J.S., 2019. Enhancing lutein production with mixotrophic cultivation of Chlorella sorokiniana MB-1-M12 using different bioprocess operation strategies. Bioresour. Technol. 278, 17–25. Cordero, B.F., Obraztsova, I., Couso, I., Leon, R., Vargas, M.A., Rodriguez, H., 2011. Enhancement of lutein production in Chlorella sorokiniana (Chorophyta) by improvement of culture conditions and random mutagenesis. Mar. Drugs 9, 1607–1624. Crawford, N.M., Forde, B.G., 2002. Molecular and developmental biology of inorganic nitrogen nutrition. Arabidopsis Book. 1, e0011. Del Campo, J.A., Moreno, J., Rodríguez, H., Vargas, M.A., Rivas, J., Guerrero, M.G., 2000. Carotenoid content of chlorophycean microalgae: factors determining lutein accumulation in Muriellopsis sp. (Chlorophyta). J. Biotechnol. 76, 51–59. Del Campo, J.A., García-González, M., Guerrero, M.G., 2007. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl. Microbiol. Biotechnol. 74, 1163–1174. Fábregas, J., Maseda, A., Domínguez, A., Ferreira, M., Otero, A., 2002. Changes in the cell composition of the marine microalga, Nannochloropsis gaditana, during a light:dark cycle. Biotechnol. Lett. 24, 1699–1703. Fu, W., Paglia, G., Magnúsdóttir, M., Steinarsdóttir, E.A., Gudmundsson, S., Palsson, B.Ø., Andrésson, Ó.S., Brynjólfsson, S., 2014. Effects of abiotic stressors on lutein
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Enhancing production of lutein by a mixotrophic cultivation system using microalga Scenedesmus obliquus CWL-1 Wei-Chuan Chena, Yin-Che Hsua, Jo-Shu Changb,c, Shih-Hsin Hod, Li-Fen Wange, Yu-Hong Weia,
T ⁎
a
Graduate School of Biotechnology and Bioengineering, Yuan Ze University, No. 135, Yuan-Tung Road, Chung-Li Dist, Taoyuan City 32003, Taiwan, ROC Department of Chemical Engineering, National Cheng Kung University, Tainan City 701, Taiwan, ROC c Department of Chemical and Materials Engineering, College of Engineering, Tunghai University, Taichung 407, Taiwan, ROC d Department of Environmental Science and Technology, Harbin Institute of Technology, China e Department of Applied Chemistry and Materials Science, Fooyin University, 151 Jinxue Rd, Daliao Dist., Kaohsiung City 83102, Taiwan, ROC b
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Lutein Scenedesmus obliquus Light-related strategies Nitrate concentration Fed-batch fermentation
This work studies a series of strategies in the production of lutein by Scenedesmus obliquus CWL-1 under mixotrophic cultivation. Our experimental results revealed that the optimal conditions associated with light-related strategies were 12 h light period followed by a 12 h dark period and blue to red light under mixotrophic cultivation. Under such conditions, the biomass, lutein content and lutein productivity were maximized to 9.88 (g/ L), 1.78 (mg/g) and 1.43 (mg/L/day), respectively. Moreover, the assimilation of 4.5 g/L of calcium nitrate into S. obliquus CWL-1 increased the maximal biomass (12.73 g/L) and the highest maximal lutein productivity (3.06 mg/L/day), while the assimilation of 1.5 g/L of calcium nitrate yielded the highest maximal lutein content of 2.45 mg/g. The highest maximal lutein productivity of 4.96 (mg/L/day) was obtained when fed-batch fermentation was conducted, and this value was approximately 11-folds that obtained using the batch system.
1. Introduction Lutein ((3R, 3′R, 6′R)-β, ε-carotene-3, 3′-diol), a photosynthetic pigment, is a type of xanthophyll and one of 600 well-known carotenoids that are present in the natural world (Humphries and Khachik, 2003). Lutein is simply synthesized by green plants and is found in large
⁎
amounts in green leafy vegetables. In the human retina, lutein is absorbed from blood specifically into the macula lutea (Del Campo et al., 2007), although its precise function in the body is unknown. Lutein is also a natural colorant in poultry feathers, drugs, and cosmetics and is extensively used in the food industry. Its aforementioned important properties make lutein a highly valuable chemical. Lutein production is
Corresponding author. E-mail address: [email protected] (Y.-H. Wei).
https://doi.org/10.1016/j.biortech.2019.121891 Received 31 May 2019; Received in revised form 22 July 2019; Accepted 23 July 2019 Available online 26 July 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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expected to grow rapidly at 3.6% annually (Sun et al., 2016). Most of the current commercial production of lutein is from marigold petals. However, its high production cost, the low lutein content (0.03%) of the marigold petals, and their harvesting and separation, pose serious challenges (Piccaglia et al., 1998). Microalgae, a group of photosynthetic microorganisms, are commonly found in aquatic environments. Many specifies of microalgae accumulate large amounts of lutein in their biomass. Recently, the production of lutein from microalgae, including Chlorella protothecoides (Shi et al., 2000), Dunaliella salina (Fu et al., 2014), Scenedesmus almeriensis (Sánchez et al., 2008), and Galdieria sulphuraria (Marquardt, 1998), has been widely studied. This production has been one of the most successful applications of microalgal biotechnology. Lutein production using microalgae as a source has many merits, including the following (Ho et al., 2014); (a) the biomass of the microalgae can be utilized without the need to process petals from plants; (b) a high growth rate in the biomass of the microalgae can support high lutein productivity and (c) a larger amount of lutein is obtained than can be obtained using traditional methods, reflecting high biomass productivity. However, the technology for lutein production using microalgae is not mature. Accordingly, to improve the feasibility of the commercial production of lutein, the cultivation of microalgae must be further studied, focusing especially on cell growth, lutein content, and overall lutein productivity. The photo-autotrophic cultivation of microalgae is frequently used in the production of lutein, because of the cells’ physiological response to high light stress in photosynthetic pigment synthesis (Chen and Liu, 2018). Recently, mixotrophic cultivation combines the advantages of both photoautotrophy and heterotrophy. Mixotrophic cultivation involves the uptake of organic and inorganic nutrient sources in the presence of light by microalgae under environments of aerobic respiration and photosynthesis (Velea et al., 2017; Yen et al., 2011). Reports have established that mixotrophic the cultivation of microalgae has many merits, including reduction of the photo inhibitory effect of photosynthetic capacity, nighttime loss of biomass, and photo-oxidative damage during the cultivation period (Chen et al., 2018; Chen and Liu, 2018; Velea et al., 2017). Mixotrophic cultivation has been become a feasible strategy for the production of lutein from microalgae with high biomass, high lutein content and high productivity. Several value-added metabolites are produced by the genus Scenedesmus in autotrophic culture (Sánchez et al., 2008), which grows heterotrophically (Ren et al., 2013; Flórez-Mirandaa et al., 2017). S. obliquus is a freshwater chlorophycean microalga, which has been isolated from freshwater in southern Taiwan (Ho et al., 2014). It has recently attracted attention as a commercially valuable source of bioactive compounds, including antimicrobial and anticancer compounds (Marrez et al., 2019). However, the productivity of lutein using mixotrophic cultures of S. obliquus has so far been relatively low. This study concerns the optimization of mixotrophic cultivation conditions for the production of lutein by microalga, and studies the effects thereon of various light-related strategies, involving various types of light, light/dark periods and two-stage light source operations. Then, the mixotrophic cultivation of microalga is studied by investigating the effect of nitrate concentration on the mixotrophic cultivation of microalga is studied. Finally, mixotrophic cultivation was performed in a bioreactor in a manner determined by the obtained experimental results.
0.25 g/L of KCl (Sigma), 0.25 g/L of MgSO4·7H2O, 0.26 g/L of KH2PO4, 0.02 g/L of FeSO4·7H2O, 0.2 g/L of EDTA·2Na, 2.9 × 10−3 g/L of H3BO3, 1.8 × 10−3 g/L of MnCl2, 1.1 × 10−4 g/L of ZnCl, 0.8 × 10−3 g/L of CuSO4·5H2O and 1.8 × 10−5 g/L of (NH4)6Mo7O24·4H2O. To study the effects of different light-related strategies on the production of lutein by microalga, light of four wavelengths from an LED (T8 lamp) with a light intensity of 150 μmol/m2/s was used. The four wavelengths corresponded to red, blue, green and white light: the wavelengths were 600–690, 435–515, and 480–580 nm, respectively. To study the effect of the light period/dark period, two conditions (a 24 h light period and then a 12 h light period followed by a 12 h dark period (12L:12D)) were used to optimize the illumination to enhance the production of microalgal biomass and its lutein content. This study was performed to evaluate the microalgal biomass production, lutein concentration, lutein content and lutein productivity. In this experiment, 2% of glucose and 1 g/L of Ca(NO3)2·4H2O in 200 mL of DM medium in a flask was used. A 0.2 g mass of dry microalgae was inoculated for four days of cultivation under blue light (or red light) (first stage). The pellet of microalgae was then moved to fresh DM medium under red light (or blue light) for four days (second stage). The microalgal biomass production, lutein concentration, lutein content and lutein productivity were determined. To study the effect of the assimilation of a nitrogen source into S. obliquus CWL-1, various concentrations of Ca(NO3)2·4H2O from 0.5–5 g/L (in steps of 0.5 g/L) were used and the corresponding microalgal biomass production, lutein concentration, lutein content and lutein productivity were monitored.
2. Methods
The residual nitrate concentration was modified based on the work of Ho et al. (2014). In brief, samples were taken from the microalgal culture and filtered through a filter with 0.22 μm pores. The samples were properly diluted to ensure that the absorbance (optical density at a wavelength of 220 nm, OD220), measured using a UV/VIS spectrophotometer (model U-2001, Hitachi, Tokyo, Japan), was linearly correlated with the nitrate concentration. Calcium nitrate was purchased from Sigma for use as the standard.
2.2. Operation of stirred tank reactor The stirred tank reactor (STR) was used in the cultivation of S. obliquus CWL-1, respectively. The STR with a 7 L glass vessel was illuminated by sources of white light (150 μmol/m2/s, 12 h light period followed by a 12 h dark period) that were mounted around the reactor. The pre-cultured S. obliquus CWL-1 was inoculated into STR with a 400 mg/L inoculum. The STR was operated at 30℃ with an agitation rate of 150 rpm. Two percent glucose in 3 L of DM medium with 2% CO2 was continuously fed into the culture for nine days of cultivation. Liquid samples were collected from the sealed glass vessel to measure microalgae concentration, residual nitrate concentrations, and lutein concentration, lutein content and lutein productivity.
2.3. Determination of microalgal biomass concentration The biomass concentrations in the microalgal cultures in flasks and reactors were determined by measuring the optical density at a wavelength of 680 nm (OD680) using a UV/VIS spectrophotometer. The samples were properly diluted to ensure that the absorbance was linearly correlated with the biomass concentration. The OD680 values were converted to dry cell weight (DCW) using the following calibration formula: DCW = 0.4695 × OD680 − 0.2634. The calibration between the DCW and OD680 was performed repeatedly to ensure the accuracy of the conversion factor.
2.4. Determination of residual nitrate concentration
2.1. Microalgal strain, culture medium and culture conditions Professor Jo-Shu Chang from National Cheng Kung University kindly provided S. obliquus CWL-1 (Ho et al., 2014). Detmer’s medium (DM) was used to grow a pure culture of S. obliquus CWL-1, it comprised 1 g/L of Ca(NO3)2·4H2O (Sigma-Aldrich, St. Louis, Missouri, USA), 2
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Under 24 h of illumination, the highest maximal lutein content and lutein productivity of S. obliquus CWL-1 were 1.20 (mg/g) and 1.17 (mg/L/day), respectively. White LEDs provide higher lutein production efficiency than the three monochromatic LEDs (red, blue, and green) (Ho et al., 2014). Other works have also proved that the pigment content of coastal plankton diatoms increases under illumination by white light (Sanchez-Saavedra and Voltolina, 2002), possibly because of a complementary chromatic adaptation by which white light not only participates in the rearrangement of chloroplasts but also stimulates the synthesis of chlorophyll. As already mentioned, illumination for 24 hr facilitates an increase in not only the biomass of S. obliquus CWL-1, but also the lutein content and lutein productivity of S. obliquus CWL-1. These results suggest that the right light source is critical to the accumulation of microalgal lutein.
2.5. Determination of microalgal lutein content The method used for lutein extraction from microalgal biomass was a modified protocol of that presented by Yen et al. (2011). Briefly, 10 mL of thoroughly mixed culture fluid from the fermenter was transferred to a 15 mL conical tube. The culture was then centrifuged at 8000 rpm for 15 min, and the supernatant was discarded. Ten milliliters of methanol were subsequently added to the mixture for the ultrasonic extraction of lutein. The mixture was then centrifuged at 8000 rpm for 15 min at 4 °C. Pigments were analyzed using an HPLC system with an RP-18e (250 × 4.68 mm) column under ambient temperature (27 ± 1 °C). The mobile phase used initially consisted of 60% solvent A (acetonitrile/methanol, 20/80 v/v) and 40% solvent B (methanol/ acetone, 80/20 v/v) and was then modified to 30% solvent A and 70% solvent B in a period of 15 min. The column was subsequently returned to its original solvent composition (i.e., 60% solvent A and 40% solvent B) for the next 6 min before the injection of a new sample. The flow rate used was 1.0 mL/min and the UV detector (Hitachi) was operated at 450 nm.
3.1.2. Effect of dark/light period on cell growth, lutein content and lutein productivity of S. obliquus CWL-1 As displayed in Table 1, light provides the energy that is used by microalgae, owing to the photosynthetic process. Accordingly, the dark/light period affects algal growth and biomass production. The light regimen in the natural environment is not constant and changes in the period of illumination (such as to 12L:12D) may increase productivity and photosynthetic efficiency, improving cell growth, lutein content and lutein productivity. Table 2 presents the effects of the dark/light period on the maximal biomass of S. obliquus CWL-1. The maximal biomass of S. obliquus CWL1 (4.48 g/L) was reached when the blue light source was used for six days cultivation. Clearly, the maximal biomass under 12L:12D was higher than that under 24 h of light (Table 1 vs. Table 2). These results reveal that the light period greatly influenced the growth of microalgal biomass. Changing the light/dark periods may change the biochemical composition of the microalgae (Krzemińska et al., 2014). Another work has established that cyanobacterium Aphanothece microscopica Nägeli exhibits higher productivity and maximal cell density under a 12:12 (night:day) photoperiod than with other photoperiods (Jacob-Lopes et al., 2009). The highest maximal lutein productivity of S. obliquus CWL-1 was 1.36 (mg/L/day), which was reached under 12L:12D using white light (Table 2). Increasing the frequency of the light/dark period greatly increases the productivity of metabolites and photosynthetic efficiency (Fábregas et al., 2002). Grobbelaar (2009) showed that pulsing light at frequencies equivalent to those of electron turnover in the electron transport chains associated with photosynthesis increases the yields of metabolites and photosynthetic efficiency. Additionally, the dissipation of excess heat as a result of light-induced damage to photosynthetic organisms is the primary function of lutein (Chen et al., 2019). Therefore, more lutein accumulates under 12L:12D than under 24 h of light.
3. Results and discussion 3.1. Effects of light-related strategies on cell growth, lutein content and lutein productivity of S. obliquus CWL-1 3.1.1. Effect of various wavelength of light source on cell growth, lutein content and lutein productivity of S. obliquus CWL-1 The photoautotrophy of microalgae cultivation for the production of bioactive compounds such as lutein may require exposure to sunlight. The sunlight is captured and photosynthesis is the main source of energy for the survival and growth of the microalgae. Reports have established that light of a specific wavelength not only favors microalgal growth but also facilitates the accumulation of valuable-added bioactive compounds, such as natural pigments and fatty acids (Kim et al., 2013). Therefore, a series of mixotrophic light-related strategies were used with S. obliquus CWL-1 to examine its cell growth, lutein content and lutein productivity. They involved illuminating the microalgae for 24 h, and separately for 12 h followed by a 12 h dark period (12L:12D). Four light sources with different wavelengths, corresponding to the colors red, blue, green and white (T8 lamp), were used. For microalgae cultivation, the intensity of each light source was fixed at 150 μmol/ m2/s. As indicated in Table 1, the effects of wavelength on S. obliquus CWL-1 under 24 h of illumination differed significantly from maximal biomass. The maximal biomass of S. obliquus CWL-1 after six days cultivation increased in the order of red, green, blue and white. Therefore, the maximal biomass decreased as the light wavelength increased. The effect of the wavelength of light on the growth of microalgae herein was consistent with the literature (Ho et al., 2014), white light is the most favorable light for the growth of biomass of S. obliquus. Indeed, white light (400–700 nm) is typically used for microalgal growth (Carvalho et al., 2011). Table 1 Effects of wavelength of light source on cell growth and lutein production of S. obliquus CWL-1 under 24 h illumination: maximal biomass, maximal lutein content and maximal lutein productivity. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp).
Table 2 Effects of wavelength of light source on cell growth and lutein production of S. obliquus CWL-1 under 12L:12D condition: maximal biomass, maximal lutein content and maximal lutein productivity. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp).
Types of light
Maximal biomass (g/L)
Maximal lutein content (mg/g)
Maximal lutein productivity (mg/L/Day)
Types of light
Maximal biomass (g/L)
Maximal lutein content (mg/g)
Maximal lutein productivity (mg/L/Day)
White Red Blue Green
4.26 2.17 2.97 2.53
1.20 1.14 0.99 1.51
1.17 0.44 0.63 0.53
White Red Blue Green
4.34 4.37 4.48 3.96
1.48 1.68 1.26 1.54
1.36 1.09 1.28 1.07
± ± ± ±
0.30 0.06 0.07 0.31
± ± ± ±
0.08 0.25 0.17 0.13
± ± ± ±
0.03 0.02 0.10 0.04
3
± ± ± ±
0.02 0.41 0.15 0.19
± ± ± ±
0.20 0.36 0.45 0.21
± ± ± ±
0.07 0.08 0.09 0.07
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fresh DM medium was exchanged, and then declined with increasing cultivation time to the end of the experiment (Fig. 1A and 1B). These results may be explained by the fact that the biomass increased with the cultivation time, rapidly consuming the fresh medium, reducing lutein productivity. The maximal lutein content was obviously higher under the two-stage light source operation than under the 24 h light period or 12L:12D. However, the lutein content was not effectively increased, suggesting that the high biomass of microalgae did not adsorb light evenly. An increase in the area of the photon-rich zone increased the proportion of cells that received enough light to perform photosynthesis (Carvalho et al., 2011).
3.2. Effect of nitrate concentration on cell growth, lutein content and lutein productivity of S. obliquus CWL-1 Nitrogen has an important role of microalgal cells, which is a significant fraction, one to ten percent, of the dry weight of microalgal cells (Martin-Jézéquel et al., 2000). Therefore, nitrogen is vital for the growth and metabolism of microalgal cells (Perez-Garcia et al., 2011). Microalgal cells can use various nitrogen sources, mainly ammonia (NH4+), nitrate (NO3–), and urea, as well as yeast extract and amino acids can be used by Liu and Chen (2016). With respect to lutein accumulation, the availability of a nitrogen source strongly affects microalgae under phototrophic cultivation (Xie et al., 2013). Therefore, the effects of the concentration of nitrate on cell growth, lutein content and lutein productivity were studied to determine the optimal calcium nitrate concentration for mixotrophic cultivation. S. obliquus CWL-1 was cultivated under 12L:12D with a light intensity of 150 μmol/m2/s for six days. The experimental results in Fig. 2 reveal that the use of calcium nitrate was related to lutein content and lutein productivity. The lutein content increased with the biomass for the first three days and then decreased as the system became deficient in calcium nitrate. Both the maximal lutein content (1.37 mg/g) and the maximal lutein productivity (0.95 mg/L/day) were obtained at the beginning of the period of deficient calcium nitrate. This finding is similar to an earlier finding of maximal lutein in the early stationary phase in Muriellopsis sp., approximately when all available nitrogen was consumed (Del Campo et al., 2000). Ten concentrations of calcium nitrate 0.5–5 g/L (in steps of 0.5 g/L) were used and the microalgal biomass production, lutein content and lutein productivity observed. As shown in Fig. 3, the maximal biomass dramatically increased with the concentration of calcium nitrate from 0.5 g/L to 3.5 g/L. As the concentration increased above 3.5 g/L, the amount of biomass increased more slowly. The most maximal biomass (12.73 g/L) was obtained when 4.5 g/L of calcium nitrate was used. The maximal lutein productivity and maximal lutein productivity (3.06 mg/ L/day) were also highest when 4.5 g/L of calcium nitrate was used, because the higher cell density that was obtained using a higher nitrate concentration caused stronger light shading (Ho et al., 2015). The experimental results in Fig. 3 reveal that the maximal lutein content increased from 1.37 mg/g to 2.45 mg/g as the nitrate concentration increased from 0.5 g/L to 1.5 g/L. These experimental results are in good agreement with similar results concerning other microalgal strains, indicating an improvement in lutein content with increasing nitrogen concentration (Cordero et al., 2011; Xie et al., 2013; Ho et al., 2015). However, the maximal lutein content decreased from 1.87 mg/g
Fig. 1. Effects of two-stage light operation, red light to blue light (A) and blue light to red light (B)) on cell growth and lutein production of S. obliquus CWL-1: biomass, lutein concentration, lutein content and lutein productivity. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp).
3.1.3. Effect of two-stage light operation on cell growth, lutein content and lutein productivity of S. obliquus CWL-1 As depicted in Table 2, the experimental results reveal that the wavelength of light significantly affected cell growth, lutein content and lutein productivity sources. The maximal lutein content (1.68 mg/ g) was highest when red light was used under the 12L:12D condition and the maximal biomass was highest (4.48 g/L) when blue light was used under the 12L:12D condition (Table 2). To study further the effect of a two-stage light source operation on lutein production under 12L:12D, the microalgal biomass production, lutein concentration, lutein content and lutein productivity were studied. The experimental results in Fig. 1 and Table 3 reveal that the maximal biomass and maximal lutein productivity of S. obliquus CWL-1 markedly increased to approximately 10 g/L and 8.2 (mg/L) from the cultivation of beginning under blue light and then red light or red light and then blue light. Wavelengths between 400 and 700 nm are typically used for microalgal growth (Carvalho et al., 2011). Additionally, blue light (420–450 nm) and red light (660–700 nm) are equally efficient for photosynthesis (Carvalho et al., 2011). The maximal lutein productivity in Fig. 1 and Table 3 was 1.32 (mg/ L/day) under red light to blue light and 1.43 (mg/L/day) under blue light to red light. Lutein productivity remained stable for two days after
Table 3 Effects of two-stage light operation on cell growth and lutein production of S. obliquus CWL-1: maximal biomass, maximal lutein content and maximal lutein productivity. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp). Types of light
Maximal biomass (g/L)
Maximal lutein content (mg/g)
Maximal lutein productivity (mg/L/Day)
Red light to Blue light Blue light to Red light
10.12 ± 0.30 9.88 ± 0.98
1.67 ± 0.08 1.78 ± 0.10
1.32 ± 0.02 1.43 ± 0.18
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Fig. 2. Profiles of cell growth, residual nitrate concentration, lutein content, and productivity of S. obliquus CWL-1. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp).
Fig. 4. Effects of fed-batch fermentation on cell growth and lutein production of S. obliquus CWL-1: (A) biomass, residual nitrate concentration, lutein content and maximum lutein productivity with 1.0 g/L of calcium nitrate as a feeding substrate and (B) biomass, residual nitrate concentration, lutein content and maximum lutein productivity with 4.5 g/L of calcium nitrate as a feeding substrate. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp). Fig. 3. Effects of concentration of calcium nitrate on cell growth and lutein production of S. obliquus CWL-1 under two-stage light operation: maximal biomass, maximal lutein content and maximum lutein productivity. (Operating conditions: light intensity, 150 μmol/m2/s; temperature, 30 °C; agitation speed, 150 rpm; light source, T8 LED lamp).
nitrate was fed into the STR. The highest biomass (12.93 g/L) was achieved after the second feeding of calcium nitrate on the fifth day of cultivation. Then the biomass slightly decreased, even though calcium nitrate was repeatedly fed into the STR until the end of cultivation. Similar results are observed in Fig. 4B, which reveals that the highest biomass (12.80 g/L) was reached on the fifth day of cultivation, when the initial calcium nitrate concentration of 4.5 g/L declined for the first time, and the biomass then slightly declined until the end of the cultivation. A report has also found that the biomass concentration increased with the amount of added urea under the fed-batch cultivation of Chlorella sp. (Hsieh and Wu, 2009). The lutein productivity varied similarly with both initial concentrations of calcium nitrate, 1.0 g/L or 4.5 g/L (Fig. 4A and B). The highest lutein productivity that was reached with 1.0 g/L or 4.5 g/L calcium nitrate was 4.44 (mg/L/day) or 4.96 (mg/L/day), respectively. These results are attributed to the light shading effect. The light only slightly penetrated the cell broth, and the specific light intensity decreased sharply with penetration, because the biomass concentration and lutein productivity increased with the cultivation time at a high nitrate concentration (Cheirsilp and Torpee, 2012). The lutein contents in Fig. 4A and 4B are correlated with calcium nitrate concentration. The highest lutein contents were quite close, being 2.58 mg/g at 1.0 g/L and 2.55 mg/g at 4.5 g/L of calcium nitrate. These results suggest that the lutein content was raised when the calcium nitrate had not completely depleted. Under the fed-batch strategy, the calcium concentration must be kept sufficiently high. However, the concentrations of other nutrients, such as the carbon source (glucose) and trace elements, decreased as the cultivation time increased. This variation facilitated the production and storage of to the metabolites such as lutein, by the microalgae, providing energy for survival. Xie
to 1.29 mg/g as the concentrations of nitrogen increased above 1.5 g/L (to 2 g/L and 5 g/L). Plant cells must reduce assimilated nitrate to ammonia by membrane transportation, consuming large amounts of energy, carbon, and protons (Crawford and Forde, 2002). The maximal lutein content herein agrees closely with that finding. Also, an excess of calcium nitrate might have led to a low pH value when nitrate reductase is fully expressed in most microalgae and the sole nitrogen source is nitrate, reducing the lutein content (Perez-Garcia et al., 2011). 3.3. Effect of fed-batch fermentation on cell growth, lutein content and lutein productivity of S. obliquus CWL-1 under mixotrophic cultivation The results in Fig. 4 reveal that an initial concentration of calcium nitrate of 4.5 g/L with yielded the highest lutein productivity (3.06 mg/ L/day) and the highest biomass (12.73 g/L), with the complete consumption of the calcium nitrate on the fifth day of cultivation. Accordingly, to measure the cell growth, lutein content and lutein productivity of S. obliquus CWL-1, fed-batch fermentation using an STR was conducted. A constant calcium nitrate concentration of 4.5 g/L is manipulated in the STR. In the control, the concentration was 1.0 g/L. The cell growth, lutein content and lutein productivity of S. obliquus CWL-1 were studied to observe how calcium nitrate affected by fed-batch fermentation. Fig. 4A shows that the initial concentration of calcium nitrate, 1.0 g/L, declined on the third day of cultivation, when 1.0 g/L calcium 5
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et al. (2017) presented similar results, which revealed that nitrate pulse feeding promoted β-carotene biosynthesis and the bioconversion of αcarotene to lutein. Based on these results, maintaining the nitrogen concentration at an appropriate level facilitated the accumulation of lutein. By this fed-batch strategy, the highest lutein productivity of 4.96 mg/L/day was achieved, which is about 11-fold that obtained using the batch system. This result demonstrates the feasibility of commercializing fed-batch fermentation with calcium nitrate supplementation.
production in the green microalga Dunaliella salina. Microb. Cell Fact. 13, 3. Grobbelaar, J.U., 2009. Upper limits of photosynthetic productivity and problems of scaling. J. Appl. Phycol. 21, 519–522. Ho, S.H., Chan, M.C., Liu, C.C., Chen, C.Y., Lee, W.L., Lee, D.J., Chang, J.S., 2014. Enhancing lutein productivity of an indigenous microalga Scenedesmus obliquus FSP-3 using light-related strategies. Bioresour. Technol. 152, 275–282. Ho, S.H., Xie, Y., Chan, M.C., Liu, C.C., Chen, C.Y., Lee, D.J., Huang, C.C., Chang, J.S., 2015. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. 184, 131–138. Hsieh, C.H., Wu, W.T., 2009. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresour. Technol. 100, 3921–3926. Humphries, J.M., Khachik, F., 2003. Distribution of lutein, zeaxanthin, and related geometrical isomers in fruit, vegetables, wheat, and pasta products. J. Agric. Food Chem. 51, 1322–1327. Flórez-Mirandaa, L., Cañizares-Villanuevaa, R.O., Melchy-Antonioa, O., MartínezJerónimob, F., Flores- Ortízc, C.M., 2017. Two stage heterotrophy/photoinduction culture of Scenedesmus incrassatulus: potential for lutein production. J. Biotechnol. 262, 67–74. Jacob-Lopes, E., Scoparo, C., Lacerda, L., Franco, T., 2009. Effect of light cycles (night/ day) on CO2 fixation and biomass production by microalgae in photobioreactors. Chem. Eng. Process. 48, 306–310. Liu, J., Chen, F., 2016. Biology and industrial applications of chlorella: advances and prospects. Adv. Biochem. Eng. Biotechnol. 153, 1–35. Kim, T.H., Lee, Y., Han, S.H., Hwang, S.J., 2013. The effects of wavelength and wavelength mixing ratios on microalgae growth and nitrogen, phosphorus removal using Scenedesmus sp. for wastewater treatment. Bioresour. Technol. 130, 75–80. Krzemińska, I., Pawlik-Skowrońska, B., Trzcińska, M., Tys, J., 2014. Influence of photoperiods on the growth rate and biomass productivity of green microalgae. Bioprocess Biosyst. Eng. 37, 735–741. Marrez, D.A., Naguib, M.M., Sultan, Y.Y., Higazy, A.M., 2019. Antimicrobial and anticancer activities of Scenedesmus obliquus metabolites. Heliyon 5, e01404. Marquardt, J., 1998. Effects of carotenoid-depletion on the photosynthetic apparatus of a Galdieria sulphuraria (rhodophyta) strain that retains its photosynthetic apparatus in the dark. J. Plant Physiol. 152, 372–380. Martin-Jézéquel, V., Hildebrand, M., Brzezinski, M.A., 2000. Silicon metabolism in diatoms: implications for growth. J. Phycol. 36, 821–840. Perez-Garcia, O., Escalante, F.M., de-Bashan, L.E., Bashan, Y., 2011. Heterotrophic cultures of microalgae: metabolism and potential products. Water Res. 45, 11–36. Piccaglia, R., Marotti, M., Grandi, S., 1998. Lutein and lutein ester content in different types of Tagetes patula and T. erecta. Ind. Crops Prod. 8, 45–51. Ren, H.Y., Liu, B.F., Ma, C., Zhao, L., Ren, N.Q., 2013. A new lipid-rich microalga Scenedesmus sp: strain R-16 isolated using Nile red staining: effects of carbon and nitrogen sources and initial pH on the biomass and lipid production. Biotechnol. Biofuel. 6, 143. Sánchez, J.F., Fernández-Sevilla, J.M., Acién, F.G., Cerón, M.C., Pérez-Parra, J., MolinaGrima, E., 2008. Biomass and lutein productivity of Scenedesmus almeriensis: influence of irradiance, dilution rate and temperature. Appl. Microbiol. Biotechnol. 79, 719–729. Sanchez-Saavedra, M.P., Voltolina, D., 2002. Effect of photon fluence rates of white and blue–green light on growth efficiency and pigment content of three diatom species in batch cultures. Cien. Marinas 28, 273–279. Shi, X., Zhang, X., Chen, F., 2000. Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources. Enzyme Microb. Technol. 27, 312–318. Sun, Z., Li, T., Zhou, Z.G., Jiang, Y., 2016. Microalgae as a source of lutein: chemistry, biosynthesis, and carotenogenesis. Adv. Biochem. Eng. Biotechnol. 153, 37–58. Xie, Y., Ho, S.H., Chen, C.N., Chen, C.Y., Ng, I.S., Jing, K.J., Chang, J.S., Lu, Y., 2013. Phototrophic cultivation of a thermo-tolerant Desmodesmus sp. for lutein production: effects of nitrate concentration, light intensity and fed-batch operation. Bioresour. Technol. 144, 435–444. Xie, Y., Zhao, X., Yang, X., Lu, Y., Zheng, X., Chen, J., 2017. Effect of nitrate pulse-feeding cultivation on cell growth and cell composition of thermo-tolerant Desmodesmus sp. F51. Food Sci. 38, 64–70. Velea, S., Oancea, F., Fischer, F., Muñoz, R., 2017. 2 - Heterotrophic and mixotrophic microalgae cultivation A2 - Gonzalez-Fernandez, Cristina2 - Heterotrophic and mixotrophic microalgae cultivation A2 - Gonzalez-Fernandez, Cristina. In: Microalgae-Based Biofuels and Bioproducts. Woodhead Publishing, pp. 45–65. Yen, H.W., Sun, C.H., Ma, T.W., 2011. The comparison of lutein production by Scenesdesmus sp. in the autotrophic and the mixotrophic cultivation. Appl. Biochem. Biotechnol. 164, 353–361.
4. Conclusions This study utilized a series of light-related strategies for the production of lutein by S. obliquus CWL-1 under mixotrophic cultivation using fed-batch operation and various nitrate concentrations. The highest lutein production (9.88 g/L) was achieved in a 12L:12D blue to red light regime with mixotrophic cultivation. The lutein productivity increased to 3.06 (mg/L/day) upon the assimilation of 4.5 g/L of calcium nitrate. The highest lutein productivity of 4.96 (mg/L/day) was achieved using fed-batch operation. The strategies used in this work seem to demonstrate the potential for commercial production of microalgal lutein. Acknowledgements The authors gratefully acknowledge the financial support provided by the Ministry of Science and Technology of the Republic of China under grant number MOST 107-2221-E-155-022-MY3. References Carvalho, A.P., Silva, S.O., Baptista, J.M., Malcata, F.X., 2011. Light requirements in microalgal photobioreactors: an overview of biophotonic aspects. Appl. Microbiol. Biotechnol. 89, 1275–1288. Cheirsilp, B., Torpee, S., 2012. Enhanced growth and lipid production of microalgae under mixotrophic culture condition: effect of light intensity, glucose concentration and fed-batch cultivation. Bioresour. Technol. 110, 510–516. Chen, C.Y., Liu, C.C., 2018. Optimization of lutein production with a two-stage mixotrophic cultivation system with Chlorella sorokiniana MB-1. Bioresour. Technol. 262, 74–79. Chen, C.-Y., Lu, I.C., Nagarajan, D., Chang, C.-H., Ng, I.S., Lee, D.-J., Chang, J.-S., 2018. A highly efficient two-stage cultivation strategy for lutein production using heterotrophic culture of Chlorella sorokiniana MB-1-M12. Bioresour. Technol. 253, 141–147. Chen, J.H., Chen, C.Y., Hasunuma, T., Kondo, A., Chang, C.H., Ng, I.S., Chang, J.S., 2019. Enhancing lutein production with mixotrophic cultivation of Chlorella sorokiniana MB-1-M12 using different bioprocess operation strategies. Bioresour. Technol. 278, 17–25. Cordero, B.F., Obraztsova, I., Couso, I., Leon, R., Vargas, M.A., Rodriguez, H., 2011. Enhancement of lutein production in Chlorella sorokiniana (Chorophyta) by improvement of culture conditions and random mutagenesis. Mar. Drugs 9, 1607–1624. Crawford, N.M., Forde, B.G., 2002. Molecular and developmental biology of inorganic nitrogen nutrition. Arabidopsis Book. 1, e0011. Del Campo, J.A., Moreno, J., Rodríguez, H., Vargas, M.A., Rivas, J., Guerrero, M.G., 2000. Carotenoid content of chlorophycean microalgae: factors determining lutein accumulation in Muriellopsis sp. (Chlorophyta). J. Biotechnol. 76, 51–59. Del Campo, J.A., García-González, M., Guerrero, M.G., 2007. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl. Microbiol. Biotechnol. 74, 1163–1174. Fábregas, J., Maseda, A., Domínguez, A., Ferreira, M., Otero, A., 2002. Changes in the cell composition of the marine microalga, Nannochloropsis gaditana, during a light:dark cycle. Biotechnol. Lett. 24, 1699–1703. Fu, W., Paglia, G., Magnúsdóttir, M., Steinarsdóttir, E.A., Gudmundsson, S., Palsson, B.Ø., Andrésson, Ó.S., Brynjólfsson, S., 2014. Effects of abiotic stressors on lutein
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