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Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth
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Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth Jih-Heng Chen a, Chun-Yen Chen b, Jo-Shu Chang a,c,∗ a
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan c Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 701, Taiwan b
a r t i c l e
i n f o
Article history: Received 4 February 2017 Revised 6 April 2017 Accepted 17 April 2017 Available online xxx Keywords: Microalgae Lutein Mixotrophic growth Outdoor cultivation Acetate
a b s t r a c t Lutein is a type of xanthophyll found in the macular region of eye, helps in reducing the risk of agerelated macular degeneration (AMD). In this study, lutein production from the wild type Chlorella sorokiniana MB-1 and the mutants generated by random mutagenesis were tested for their lutein productivity. The wild-type strain of C. sorokiniana MB-1 obtained the highest lutein productivity and lutein content of 2.39 mg/L/day and 5.86 mg/g, respectively, when 6.0 g/L sodium acetate was used as the organic carbon source in mixotrophic cultivation. Under the same conditions, mutant strain MB-1-M12 had better lutein production performance than that of the wild-type strain, exhibiting a lutein content and productivity of 7.52 mg/g and 3.63 mg/L/day, respectively. Outdoor cultivation of mutant strain MB-1-M12 obtained a slightly lower lutein content of 6.85 mg/g but a markedly lower productivity of 1.35 mg/L/day when compared to the indoor culture operated under well-controlled conditions with whole-day illumination. The satisfactory lutein production performance from outdoor culture of MB-1-M12 strain demonstrates the potential in commercial production of lutein using the mutant strain. © 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Carotenoids are accessory photosynthetic pigments seen in microalgae. They have a 40-carbon backbone that contains a bulky conjugated double-bond system and play a key role in photosynthesis and photo-protection [1]. Lutein belongs to xanthophyll family that contains hydroxyl or carbonyl groups; therefore, it has enhanced solubility in animal tissues. Due to its ability to prevent or ameliorate cardiovascular diseases [2], several types of cancer [3,4], and degenerative human diseases [5,6], lutein is frequently used as a food additive, known as E161b, with an annual market size of over $150 million in the U.S.A. [7]. At present, marigold petals are used for the commercial production of lutein. However, extraction of lutein from marigold is limited by the labor-intensive process and the extremely low lutein content of plants (as low as 0.03%) [8]. In recent years, microalgae have been viewed as promising sources to produce lutein due to their high lutein content (up to 7 mg/g) [7]. Some microalgal species are known as potential lutein-producing strains contents, such as Muriellopsis sp. [9], Scenedesmus sp. [10], Chlorella ∗ Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan. E-mail address: [email protected] (J.-S. Chang).
zofingensis [11], and Chlorella protothecoides [12]. The advantages of using microalgae as the lutein source include: (a) excluding the need to remove petals from plants as the whole biomass of the microalgae can be processed, (b) producing microalgae at a high growth rate thereby giving high lutein productivity and (c) yielding relatively high lutein content at high biomass productivity compared to marigold and other terrestrial plants [13]. Altering the growth metabolism of microalgae is one of the most frequently applied strategies used to improve lutein production, as seen in phototrophic [9,14,15], heterotrophic [16], and mixotrophic [14,15] cultivation. Among those, phototrophic cultivation usually shows a higher lutein content but a lower cell growth rate, as the lutein productivity ranged from 0.44 to 4.77 mg/L/day [10,11]. However, the inherent physiological properties and genetic pathways within each strain cannot be completely manipulated by controlling the external parameters during the cultivation processes. Therefore, it is necessary to obtain strains that are genetically capable of higher biomass productivity and higher yield of the desired product (i.e., lutein). Developing microalgal strains with the ability to accumulate lutein at high content with an enhanced production rate appears to be the key to the success of commercializing microalgae-based lutein. In this study, we used random mutagenesis as a strategy for strain improvement to enhance the lutein production of Chlorella sorokiniana MB-1, which
http://dx.doi.org/10.1016/j.jtice.2017.04.022 1876-1070/© 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: J.-H. Chen et al., Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.022
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is an indigenous microalgal strain isolated from southern Taiwan. N-methyl-N -nitro-N-nitrosoguanidine (MNNG) was selected as the mutagen [17] for the random mutagenesis. The mutant strain of C. Sorokiniana MB-1 was cultivated on mixotrophic mode under optimal cultivation conditions using sodium acetate as the organic carbon source [18]. Cultivation of the selected mutant was further scaled up in 1-L photobioreactor to evaluate the feasibility of commercial production of lutein using the mutant strain.
2. Materials and methods 2.1. Microalgal strain, culture condition and photobioreactor operation Chlorella sorokiniana MB-1 was isolated from freshwater in southern Taiwan. The BG-11 medium was used to grow the MB-1 strain under mixotrophic conditions supplemented with 6 g/L sodium acetate, in addition to aeration of 2% (v/v) CO2 . The photobioreactors (PBR) used to cultivate MB-1 strain were 250 mL or 1-L glass vessel equipped with an external light source (14 W TL5 tungsten filament lamps; Philips Co., Taipei, Taiwan), which provides whole-day illumination with a light intensity of 150 μmol/m2 s. A Li-250 Light Meter equipped with a Li-190SA pyranometer sensor (Li-COR Inc., Lincoln, Nebraska, USA) was used to measure the light intensity. The photobioreactors were operated at 25 °C and 300 rpm agitation with continuous aeration of 2% (v/v) CO2 at a flow rate of 0.1 vvm (volume per working volume per minute).
2.2. Random mutagenesis and selection of high lutein yielding mutants of Chlorella sorokiniana C. sorokiniana MB-1 cultures in exponential growth phase were treated with 0.1 mg/mL of 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) for 1 h. After MNNG treatment, the cells were washed twice with sterile water, re-suspended in BG-11 medium and incubated under low light for 25 h. Then, the cells were spread on solid BG-11 medium containing 400 μM nicotine and incubated at 25 °C for 1 week. Nicotine is an inhibitor of the carotenogenesis pathway and nicotine resistant colonies would thus have very powerful carotenoid synthesis [19]. The nicotine-resistant colonies were subcultured several times in nicotine-containing solid medium to confirm their nicotine resistance and the growth on the solid medium. Nicotine-resistant mutants were then cultured in 1-L photobioreactor in order to analyze carotenoids content and growth rate. The algae mutants have been subjected to 10% DMSO storage in liquid nitrogen bucket and their cell growth performance and lutein content are regularly examined every 3 months to monitor their stability in terms of lutein producing activity.
2.3. Effect of outdoor temperature simulation and inoculum size on lutein content and productivity of C. sorokiniana mutant M12 To determine the effect of temperature variation on outdoor cultivation of the wild type and mutant lutein-producing strains, the microalgae were cultivated on BG-11 medium with the following temperature setting: two commonly seen temperature, such as Light/Dark: 35 °C/25 °C and 25 °C/15 °C, representing summer and winter. The cultures were monitored for their growth, lutein content and lutein productivity. The effect of inoculum size on lutein content and productivity of the mutant strain (M12) was also evaluated by using three different inoculum size of 0.03, 0.09 and 0.15 g/L at the two temperature simulation settings.
2.4. Outdoor cultivation For outdoor cultivation of the microalgal strain, the poly (methyl methacrylate) (PMMA)-made tubular photobioreactor (PBR) with a working volume of 60-L (200 cm in height and 20 cm in diameter) were placed outdoors in National Cheng Kung University campus, Tainan City, Taiwan. The medium in the PBR was identical to that used for indoor cultivation. Sunlight was the only light source and the temperature varied naturally depending on the weather situation. The PBR was continuous aerated with 2% (v/v) CO2 at a flow rate of 0.1 vvm. During the microalgal growth period, liquid samples were collected at the desired time period with respect to time to determine microalgal biomass concentration, pH, residual nitrogen concentration, and lutein content. In addition, the water temperature and light intensity were simultaneously monitored by a LI-250 Light Meter with a LI-190SA pyranometer sensor and a temperature sensor (LI-COR, Inc., Lincoln, Nebraska, USA). 2.5. Determination of microalgae cell concentration and biomass productivity The microalgal cell concentration was determined regularly by optical density measurement at a wavelength of 680 nm (i.e., OD680 ) using a spectrophotometer (GENESYS 10S UV–Vis, Thermo Fisher Scientific, MA, USA). The dry cell weight (DCW) of the microalga was obtained by drying certain amount of cultures at 105 °C until the weight was invariant. The OD680 values were converted to biomass concentration via calibration between the OD680 and dry cell weight (i.e., 1.0 OD680 approximately equals to 0.324 ± 0.03 g DCW/L). The centrifuged microalgal biomass was freeze dried by a freeze-dryer (FDU-1100, EYELA, Tokyo, Japan) and collected for subsequent analysis. The biomass productivity (Pbiomass , g DCW/L/day) was calculated from the following equation:
Pbiomass (g DCW/L/day) =
biomass concentration (g DCW/L ) cultivation time (day )
2.6. Measurement of nitrate and acetate concentration The culture samples obtained at regular intervals were filtered with 0.22 μm filters and diluted to an appropriate concentration. The inorganic nitrate and acetate concentration in the samples were measured by ion chromatography (ICS-30 0 0, Thermo Scientific Dionex, CA, USA) equipped with a carbonate eluent anionexchange column (IonPac AS12A, Thermo Scientific Dionex, CA, USA). 2.7. Determination of the lutein content and productivity The lutein content of the microalgae powder was measured using the method described in our recent work [18]. Briefly, 10 mg dry microalgal biomass was added to 1 mL 60% w/v KOH solution to hydrolyze the lipids and disrupt the cells. The suspension was incubated in a hot water bath at 40 °C for 40 min, followed by repeated extraction with diethyl ether, and the supernatant phase was removed and collected until it was clear. Then, the organic solvent was purged with nitrogen gas, and the precipitate was dissolved in acetone. HPLC analysis was performed using the methods described by Kitada et al. [20]. The HPLC instrument was equipped with photodiode array detector (PDA). The separation column (4.6 × 250 mm in size) was packed with YMC Carotenoid RP-30 with a particle size of 5 μm (YMC Schermbeck, Germany). The mobile phase was 1:9 THF: methanol and the flow rate was set at 1.5 mL/min. The lutein standards used for quantification were purchased from Sigma Chemical Co., St. Louis, MO, USA.
Please cite this article as: J.-H. Chen et al., Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.022
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The lutein content (mg/g) was calculated from the following equation:
Lutein content (mg/g DCW ) Lutein production (mg/L ) × Volume of solution (L ) = DCW of microalgal biomass (g ) The lutein productivity (mg/L/day) was calculated from the following equation:
PLutein (mg/L/day ) = Lutein content (mg/g DCW ) × Pbiomass × (g DCW/L/day ) 3. Results and discussion 3.1. Lutein production using mixotrophic cultivation from C. sorokiniana MB-1 The C. sorokiniana MB-1 is an indigenous microalgal strain, isolated from freshwater in southern Taiwan and it produces lutein when cultivated on mixotrophic mode. Mixotrophic cultivation is a mode of microalgal cultivation in which light is used as an energy source and both CO2 and organic carbon are used as the carbon source. The growth rate and biomass concentration is greatly enhanced in mixotrophic cultivation due to the benefits of both photoautotrophic and heterotrophic growth and both organic and inorganic carbon supports the growth of microalgae [14,15]. In this study, sodium acetate was selected as the carbon source and the effect of sodium acetate concentration was evaluated. The microalgal tolerance to acetate might vary from species to species. Thus, the effect of acetate supply on composition of pigment would also be microalgal species dependent [21]. Four different concentrations of sodium acetate were added into the culture; namely, 1.0, 3.0, 6.0 and 9.0 g/L. Nitrogen source was set at 1.0 g/L sodium nitrate and CO2 aeration rate was fixed at 0.1 vvm. The microalgae were grown at 25 °C with 150 μmol/m2 /s light intensity. Fig. 1 shows that maximal biomass concentrations obtained with the four different sodium acetate concentrations were quite similar, but supplementing 6.0 g/L sodium acetate resulted in higher maximal biomass productivity. Maximal lutein content and lutein productivity went along with the maximal biomass concentration, as the highest lutein content (5.86 mg/g) and productivity (2.56 mg/L/day) were reached when 6 g/L sodium acetate was used. Hence, the use of a moderate amount of sodium acetate (i.e., 6 g/L) was optimal for C. sorokiniana MB-1 growth, biomass and lutein production. The carbon source used in mixotrophic cultivation can vary between strains and also mixotrophic growth might not be optimal for all strains. For instance, mixotrophic cultivation of Scenedesmus sp. resulted in lower biomass productivity and lutein content [15]. However, Chlorella protothecoides yielded a high biomass concentration of 22.4 g/L and a high lutein content of 6.48 mg/g under mixotrophic cultivation using glucose as the organic carbon source in the presence of light [22]. In addition, under heterotrophic growth, C. protothecoides could reach a cell concentration of 17.2 g/L and a lutein content of 4.43 mg/g. It is necessary to evaluate the potential of any microbial strain for their performance under a particular metabolic mode. 3.2. Isolation of high lutein yielding mutants of C. sorokiniana MB-1 In order to obtain high lutein producing mutants of C. sorokiniana, cells were subjected to MNNG mutagenesis and mutants were screened on solid BG-11 medium with nicotine. 58 nicotineresistant lines were obtained and cultured under the same conditions in 1-L photobioreactor to analysis the growth and lutein content. The culture conditions were operated at 25 °C, 300 rpm
Fig. 1. Lutein production from mixotrophic cultivation of Chlorella sorokiniana MB-1. (a) Effect of sodium acetate concentration on biomass concentration and productivity, (b) effect of sodium acetate concentration on lutein content and productivity.
agitation supplemented with 2% (v/v) CO2 at an aeration rate of 0.1 vvm with 6 g/L sodium acetate. The biomass production, lutein content and lutein productivity of the mutant strains studied are summarized in Table 1. Four mutants were identified with lutein content exceeding 6.0 mg/g after 6 days of cultivation, with one mutant (M12) having a lutein content of over 7 mg/g (Table 1). The time course profiles of cell growth and lutein production of MB1 and M12 are compared in Fig. 2. The maximum biomass concentration of MB-1-M12 mutant is similar to that of wild type, but the lutein content of MB-1-M12 mutant (7.52 mg/g) was 28.3% higher than that of the wild-type strain (5.86 mg/g). Furthermore, the lutein productivity of mutant MB-1-M12 (3.63 mg/L/day) was 51.9% higher than that of wild-type strain (2.39 mg/L/day). The nitrogen consumption profiles were quite similar in both strains, as the nitrogen source depleted after 2 days of cultivation, while the biomass concentration continued to increase after the depletion of external nitrogen source, which is identical to the observation in our previous study [23]. In a similar study, mutants of C. sorokiniana 211-32 were generated by random mutagenesis and was cultivated under autotrophic and mixotrophic conditions for the evaluation of lutein content and productivity. The authors mentioned that two mutants DMR-5 and DMR-8 attained lutein levels of about 7 mg/g, but the strains were not chosen for further studies. The mutant chosen for further studies MR-12 had a lutein
Please cite this article as: J.-H. Chen et al., Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.022
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J.-H. Chen et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2017) 1–8 Table 1 Cell growth and lutein production performance of Chlorella sorokiniana MB-1 and its mutant strains. Microalgal strain
Max. biomass concentration (g/L)
Max. biomass productivity (g/L/day)
Max. lutein content (mg/g)
Max. lutein productivity (mg/L/day)
MB-1 MB-1-M3 MB-1-M5 MB-1-M9 MB-1-M12
2.26 ± 0.06 1.69 ± 0.05 1.63 ± 0.04 1.99 ± 0.07 2.29 ± 0.03
0.928 ± 0.04 0.643 ± 0.11 0.428 ± 0.08 0.435 ± 0.06 0.932 ± 0.09
5.86 ± 0.11 6.60 ± 0.15 6.32 ± 0.22 6.25 ± 0.17 7.52 ± 0.12
2.39 ± 0.09 2.10 ± 0.17 2.15 ± 0.21 3.60 ± 0.16 3.63 ± 0.15
Fig. 2. The performance of cell growth and lutein production of Chlorella sorokiniana MB-1 and its mutant strain MB-1-M12 under indoor mixotrophic cultivation. (a) Time course profile of biomass concentration and lutein content for MB-1, (b) time course profile of biomass concentration and lutein content for MB-1-M12.
content of 5 mg/g under autotrophic conditions [14]. However, under mixotrophic conditions with the addition of glucose or acetate, biomass concentrations decreased by 40% and the cellular lutein content decreased by 33% [14]. Lutein production performances from various strains reported in the literature are summarized in Table 2 and it can be seen that MB-1-M12 strain has the highest lutein content under mixotrophic mode with the addition of organic carbon source. Thus, the MB-1-M12 strain was used for further experiments because of the higher growth rate and highly enhanced lutein production. 3.3. Outdoor temperature simulated cultivation with C. sorokiniana strain MB-1-M12 Open systems are more advantageous than closed systems for commercial microalgal cultivation, such as smaller capital
investment for construction and operation and easier scale-up [24]. However, open systems are prone to contamination by bacteria, alien microalgae species and protozoa. Very few microalgae like Chlorella sp., Spirulina sp. and Dunaliella sp. with extreme conditions used for cultivation are suited for outdoor open systems. Light intensity and temperature are two important parameters in outdoor cultivation. In this study, the effect of two sets of temperature to simulate summer and winter outdoor conditions was investigated. Light–Dark cycle mode (L/D: 12 h) with two commonly seen temperature, such as Light/Dark: 35 °C/25 °C and 25 °C/15 °C, representing summer and winter were used and the results are shown in Fig. 3. The results show that the microalgal biomass concentration was about 2.3 g/L and the biomass productivity was nearly 0.856 g/L/day under 35 °C/25 °C temperature cycle (Fig. 3(a)), while under 25 °C/15 °C temperature cycle, the biomass concentration increased to nearly 3 g/L (Fig. 3(b)), and the biomass
Please cite this article as: J.-H. Chen et al., Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.022
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Table 2 Comparisons of the operation system, metabolic mode, lutein content and lutein productivity of Chlorella sorokiniana MB-1-M12 with those from related reports. Microalga strain
Operation system
Light supply
Metabolic mode
Lutein content (mg/g)
Lutein productivity (mg/L/day)
Reference
Chlorella zofingiensis Chlorella zofingiensis Chlorella fusca Chlorella proboscideum Scenedesmus obliquus FSP-3 Coccomyxa acidophila Chlorella sorokiniana 211-32 Chlorella sorokiniana MR-16 Chlorella sorokiniana MB-1 Chlorella sorokiniana MB-1-M12
Batch Batch Batch Batch Batch Batch Batch Batch Semi-batch Batch
Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous
Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Mixotrophic Autotrophic Autotrophic Mixotrophic Mixotrophic
2.80 3.50 4.20 3.40 4.52 3.50 3.0 5.0 5.21 7.52 ± 0.12
1.30 1.20 0.80 1.20 4.15 0.90 25 mg/L∗ 42 mg/L∗ 5.67 3.63 ± 0.15
[9] [26] [9] [9] [27] [28] [14] [14] [29] This study
∗
light light light light light light light light light light
Yield depicted as mg/L.
Fig. 3. Effect of temperature simulation outdoor cultivation with mutant strain MB-1-M12 under temperature simulation of outdoor cultivation. (a) Time course profile of biomass concentration and lutein content under 35 °C/25 °C temperature cycle, (b) time course profile of biomass concentration and lutein content under 25 °C/15 °C temperature cycle.
productivity reached 0.740 g/L/day. Although the biomass concentration is higher at the simulated winter temperatures, the biomass productivity was slightly lower when compared with that obtained from using simulated summer temperatures. On the other hand, lutein content and productivity of 5.87 mg/g and 2.7 mg/L/day was obtained under 35 °C/25 °C. In contrast, under the condition of 25 °C/15 °C, the lutein content and productivity markedly increased to 6.19 mg/g and 2.93 mg/L/day, respectively. In summary, for lutein production, the 25 °C/15 °C (known as winter simulation
mode) is better than the 35 °C/25 °C (known as summer simulation mode). Previous reports indicate that high temperature can induce carotenoid accumulation in microalgae, but optimum temperature of 28 °C was found to attain both higher biomass and lutein content. For instance, Muriellopsis sp. obtained a maximal lutein concentration of 30 mg/L at 28 °C under photoautotrophic mode [9]. For Chlorella protothecoides under heterotrophic mode, 28 °C is the optimal temperature for both biomass production (17.2 g/L) and lutein accumulation (74.29 mg/L) [25]. However, it was shown
Please cite this article as: J.-H. Chen et al., Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.022
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Fig. 4. Effect of inoculum size and temperature simulation of outdoor cultivation on cell growth and lutein production of mutant strain MB-1-M12. (a) Time course profile of biomass concentration and productivity with 35 °C/25 °C temperature cycle at an inoculums size of 0.03, 0.09 and 0.15 g/L, (b) time course profile of lutein content and productivity with 35 °C/25 °C temperature cycle at an inoculums size of 0.03, 0.09 and 0.15 g/L, (c) time course profile of biomass concentration and productivity with 25 °C/15 °C temperature cycle at an inoculums size of 0.03, 0.09 and 0.15 g/L, (d) time course profile of lutein content and productivity with 25 °C/15 °C temperature cycle at an inoculums size of 0.03, 0.09 and 0.15 g/L.
that mutant strain MB-1-M12 seems to be able to adapt to the outdoor environment based on the results of the simulated outdoor culture. 3.4. Effect of inoculum size on temperature simulation outdoor cultivation with the mutant strain C. sorokiniana MB-1-M12 As previously mentioned, open systems are susceptible to external environmental influences and can be contaminated by bacteria or other microalgae, and these problems are unavoidable. Therefore, increasing the inoculum size of microalgae is thought to mitigate the influence of contamination during the early stage of microalgal cultivation, thereby increasing the possibility for achieving successful microalgal growth. In this study, three different inoculum sizes were used to examine the effect of inoculum size on microalgal growth, while the same light/dark cycle mode and two sets of temperature cycle (i.e., 35 °C/25 °C and 25 °C/15 °C) were used. As shown Fig. 4, there was no significant change in maximum biomass concentration when using the three inoculum size, but biomass productivity was higher when higher inoculum sizes (i.e., 0.09 and 0.15 g/L) were used. Under temperature cycle
of 35 °C/25 °C, the biomass productivity obtained from using 0.09 and 0.15 g/L inoculum size was 0.704 and 0.692 g/L/day, respectively, which is much higher than that for using 0.03 g/L inoculum size. On the other hand, the highest lutein content (7.57 mg/g) was obtained by using 0.09 g/L inoculum size, while using 0.15 g/L inoculum size achieved the highest lutein productivity of 2.47 mg/L/day (Fig. 4(b)). Although under 35 °C/25 °C temperature cycle, using an inoculum size of 0.15 g/L gave the highest lutein productivity, 0.09 g/L was still considered as the optimal inoculum size when taking all three performances indexes (i.e., biomass productivity, lutein content and lutein productivity). In contrast, under the 25 °C/15 °C temperature cycle, the trends of cell growth are quite similar to those seen for the 35 °C/25 °C temperature cycle (Fig. 4(c)), whereas both lutein content and lutein productivity reached the highest level (7.76 mg/g and 3.45 mg/L/day, respectively) with the inoculum size of 0.09 g/L (Fig. 4(d)), which is different from the trend observed with 35 °C/25 °C temperature cycle (Fig. 4(b)). Overall speaking, using the inoculum size of 0.09 g/L resulted in the highest lutein content and productivity. For both cultivation temperature cycles, the 0.09 g/L inoculum size is considered optimum and was thus used in further experiments.
Please cite this article as: J.-H. Chen et al., Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.022
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Table 3 Comparison of cell growth and lutein production performance of indoor and outdoor cultures of mutant strain Chlorella sorokiniana MB-1-M12. Culture type
Max. biomass concentration. (g/L)
Max. biomass productivity (g/L/day)
Max. lutein concentration (mg/g)
Max. lutein productivity (mg/L/day)
Indoor Outdoor
2.29 ± 0.03 1.18 ± 0.06
0.932 ± 0.09 0.799 ± 0.07
7.52 ± 0.12 6.85 ± 0.60
3.63 ± 0.15 1.35 ± 0.07
content of 4.5 mg/g and a productivity of 290 mg lutein/m2 /day [21]. Although lutein productivity of mutant MB-1-M12 obtained from our outdoor cultivation system is higher or comparable to the reported values from outdoor microalgal cultures [21,24], it is still significantly lower than that obtained in indoor cultures. However, the lutein content of mutant strain MB-1-M12 is similar to that obtained under indoor cultivation, which demonstrates the potential of commercial lutein production with this mutant strain. Nevertheless, better cultivation strategies need to be adopted to attain higher lutein productivity for the outdoor culture. 4. Conclusions In this study, mutants of Chlorella sorokiniana MB-1 with high lutein production ability were successfully generated. Although the biomass production of mutant strains was lower than the wild type, the lutein content and productivity of the selected mutants under indoor cultivation was significantly higher with an increase of 28.3% and 51.9%, respectively. The outdoor culture of the best mutant (MB-1-M12) gave similar lutein content to that obtained in indoor cultivation, whereas a decrease in lutein productivity was observed in the outdoor culture. Further improvement in the lutein productivity using better outdoor cultivation strategies is required for commercialization purposes. Acknowledgments
Fig. 5. Performance of outdoor cultivation with mutant strain Chlorella sorokiniana MB-1-M12 under mixotrophic cultivation. (a) Time course profile of lutein content and productivity for strain MB1-M12, (b) time course profile of light intensity of sunlight and outdoor temperature.
3.5. Performance of outdoor cultivation with mutant strain C. sorokiniana MB-1-M12 The commercial production of lutein can be successful only when outdoor cultivation is possible. Therefore, outdoor cultivation of the mutant strain was conducted to produce lutein with mixotrophic growth under the same conditions used in the indoor tests. As shown in Fig. 5(a), outdoor cultivation of mutant strain MB-1-M12 achieved a lutein content and productivity of 6.85 mg/g and 1.35 mg/L/day (or 202 mg/m2 /day assuming that three 50-L tubular photobioreactors occupy about 1 m2 of land area), respectively (Table 3). The weather condition was not consistent as it can be seen from the differences in light intensity and it is reflected in growth rate and nitrate consumption (Fig. 5(b)). Very few studies report the outdoor cultivation of microalgae for lutein production. Of those, the outdoor cultivation of Muriellopsis sp. in open tanks agitated with a paddle wheel reported a productivity of 100 mg lutein/m2 /day in summer with a dry biomass content of 20 g dry biomass/m2 /day [24]. The biomass obtained was also rich in protein and lipids, with α -linoleic acid amounting to 30% of the total fatty acids [24]. Another report showed that Scenedesmus almeriensis when cultivated in outdoor tubular photobioreactor of 40 0 0 L volume could attain a lutein
The authors gratefully acknowledge financial support received from Taiwan’s Ministry of Science and Technology under grant numbers 106-3113-E-006-011, 106-3113-E-006-004-CC2, and 1052221-E-006-226. This research was also supported by Ministry of Education under the top university Grant number D105-23005. References [1] Demmig-Adams B, Adams WW. The role of xanthophyll cycle carotenoids in the protection of photosynthesis. Trends Plant Sci 1996;1:21–6. [2] Dwyer JH, Navab M, Dwyer KM, Hassan K, Sun P, Shircore A, et al. Oxygenated carotenoid lutein and progression of early atherosclerosis the Los Angeles atherosclerosis study. Circulation 2001;103:2922–7. [3] Astorg P. Food carotenoids and cancer prevention: an overview of current research. Trends Food Sci Technol 1997;8:406–13. [4] Heber D, Lu Q-Y. Overview of mechanisms of action of lycopene. Exp Biol Med 2002;227:920–3. [5] Chiu C-J, Taylor A. Nutritional antioxidants and age-related cataract and maculopathy. Exp Eye Res 2007;84:229–45. [6] Granado F, Olmedilla B, Blanco I. Nutritional and clinical relevance of lutein in human health. Br J Nutr 2003;90:487–502. [7] Cerón MC, Campos I, Sánchez JF, Acién FG, Molina E, Fernández-Sevilla JM. Recovery of Lutein from microalgae biomass: development of a process for Scenedesmus almeriensis biomass. J Agric Food Chem 2008;56:11761–6. [8] Piccaglia R, Marotti M, Grandi S. Lutein and lutein ester content in different types of Tagetes patula and T. erecta. Ind Crops Products 1998;8:45–51. [9] Del Campo JA, Moreno J, Rodriguez H, Vargas MA, Rivas J, Guerrero MG. Carotenoid content of chlorophycean microalgae: factors determining lutein accumulation in Muriellopsis sp. (Chlorophyta). J. Biotechnol. 20 0 0;76:51–9. [10] Sánchez JF, Fernández JM, Acién FG, Rueda A, Pérez-Parra J, Molina E. Influence of culture conditions on the productivity and lutein content of the new strain Scenedesmus almeriensis. Process Biochem 2008;43:398–405. [11] Del Campo JA, Rodriguez H, Moreno J, Vargas MA, Rivas J, Guerrero MG. Accumulation of astaxanthin and lutein in Chlorella zofingiensis (Chlorophyta). Appl Microbiol Biotechnol 2004;64:848–54. [12] Shi X-M, Zhang X-W, Chen F. Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources. Enzyme Microb Technol 20 0 0;27:312–18. [13] Fernandez-Sevilla JM, Fernandez FGA, Grima EM. Biotechnological production of lutein and its applications. Appl. Microbiol. Biotechnol. 2010;86:27–40.
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[14] Cordero BF, Obraztsova I, Couso I, Leon R, Vargas MA, Rodriguez H. Enhancement of lutein production in Chlorella sorokiniana (Chlorophyta) by improvement of culture conditions and random mutagenesis. Mar Drugs 2011;9:1607–24. [15] Yen HW, Sun CH, Ma TW. The comparison of lutein production by Scenedesmus sp. in the autotrophic and the mixotrophic cultivation. Appl Biochem Biotechnol 2011;164:353–61. [16] Wei D, Chen F, Chen G, Zhang X, Liu L, Zhang H. Enhanced production of lutein in heterotrophic Chlorella protothecoides by oxidative stress. Sci China Ser C: Life Sci 2008;51:1088–93. [17] Iskandarov U, Khozin-Goldberg I, Cohen Z. Selection of a DGLA-producing mutant of the microalga Parietochloris incisa: I. Identification of mutation site and expression of VLC-PUFA biosynthesis genes. Appl Microbiol Biotechnol 2011;90:249–56. [18] Chen C-Y, Hsieh C, Lee D-J, Chang C-H, Chang J-S. Production, extraction and stabilization of lutein from microalga Chlorella sorokiniana MB-1. Bioresour Technol 2016;20 0:50 0–5. [19] Cordero BF, Obraztsova I, Couso I, Leon R, Vargas MA, Rodriguez H. Enhancement of lutein production in Chlorella sorokiniana (Chlorophyta) by improvement of culture conditions and random mutagenesis. Mar. Drugs 2011;9:1607–24. [20] Kitada K, Machmudah S, Sasaki M, Goto M, Nakashima Y, Kumamoto S, et al. Supercritical CO2 extraction of pigment components with pharmaceutical importance from Chlorella vulgaris. J Chem Technol Biotechnol 2009;84:657–61.
[21] Del Campo JA, Garcia-Gonzalez M, Guerrero MG. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl Microbiol Biotechnol 2007;74:1163–74. [22] Shi XM, Chen F. Production and rapid extraction of lutein and the other lipid– soluble pigments from Chlorella protothecoides grown under heterotrophic and mixotrophic conditions. Food/Nahrung 1999;43(2):109–13 1 March. [23] Li Y, Han D, Hu G, Sommerfeld M, Hu Q. Inhibition of starch synthesis results in overproduction of lipids in Chlamydomonas reinhardtii. Biotechnol Bioeng 2010;107:258–68. [24] Blanco AM, Moreno J, Del Campo JA, Rivas J, Guerrero MG. Outdoor cultivation of lutein-rich cells of Muriellopsis sp. in open ponds. Appl Microbiol Biotechnol 2007;73:1259–66. [25] Shi X, Wu Z, Chen F. Kinetic modeling of lutein production by heterotrophic Chlorella at various pH and temperatures. Mol Nutr Food Res. 2006;50(8):763–8. [26] Del Campo JA, Rodriguez H, Moreno J, Vargas MA, Rivas J, Guerrero MG. Accumulation of astaxanthin and lutein in Chlorella zofingiensis (Chlorophyta). Appl Microbiol Biotechnol 2004;64:848–54. [27] Ho S-H, Chan M-C, Liu C-C, Chen C-Y, Lee W-L, Lee D-J, et al. Enhancing lutein productivity of an indigenous microalga Scenedesmus obliquus FSP-3 using light-related strategies. Bioresour Technol 2014;152:275–82. [28] Casal C, Cuaresma M, Vega JM, Vilchez C. Enhanced productivity of a lutein-enriched novel acidophile microalga grown on urea. Mar. Drugs 2011;9:29–42. [29] Chen C-Y, Jesisca, Hsieh C, Lee D-J, Chang C-H, Chang J-S. Production, extraction and stabilization of lutein from microalga Chlorella sorokiniana MB-1. Bioresour. Technol. 2016;20 0:50 0–5.
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Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth Jih-Heng Chen a, Chun-Yen Chen b, Jo-Shu Chang a,c,∗ a
Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan c Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 701, Taiwan b
a r t i c l e
i n f o
Article history: Received 4 February 2017 Revised 6 April 2017 Accepted 17 April 2017 Available online xxx Keywords: Microalgae Lutein Mixotrophic growth Outdoor cultivation Acetate
a b s t r a c t Lutein is a type of xanthophyll found in the macular region of eye, helps in reducing the risk of agerelated macular degeneration (AMD). In this study, lutein production from the wild type Chlorella sorokiniana MB-1 and the mutants generated by random mutagenesis were tested for their lutein productivity. The wild-type strain of C. sorokiniana MB-1 obtained the highest lutein productivity and lutein content of 2.39 mg/L/day and 5.86 mg/g, respectively, when 6.0 g/L sodium acetate was used as the organic carbon source in mixotrophic cultivation. Under the same conditions, mutant strain MB-1-M12 had better lutein production performance than that of the wild-type strain, exhibiting a lutein content and productivity of 7.52 mg/g and 3.63 mg/L/day, respectively. Outdoor cultivation of mutant strain MB-1-M12 obtained a slightly lower lutein content of 6.85 mg/g but a markedly lower productivity of 1.35 mg/L/day when compared to the indoor culture operated under well-controlled conditions with whole-day illumination. The satisfactory lutein production performance from outdoor culture of MB-1-M12 strain demonstrates the potential in commercial production of lutein using the mutant strain. © 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Carotenoids are accessory photosynthetic pigments seen in microalgae. They have a 40-carbon backbone that contains a bulky conjugated double-bond system and play a key role in photosynthesis and photo-protection [1]. Lutein belongs to xanthophyll family that contains hydroxyl or carbonyl groups; therefore, it has enhanced solubility in animal tissues. Due to its ability to prevent or ameliorate cardiovascular diseases [2], several types of cancer [3,4], and degenerative human diseases [5,6], lutein is frequently used as a food additive, known as E161b, with an annual market size of over $150 million in the U.S.A. [7]. At present, marigold petals are used for the commercial production of lutein. However, extraction of lutein from marigold is limited by the labor-intensive process and the extremely low lutein content of plants (as low as 0.03%) [8]. In recent years, microalgae have been viewed as promising sources to produce lutein due to their high lutein content (up to 7 mg/g) [7]. Some microalgal species are known as potential lutein-producing strains contents, such as Muriellopsis sp. [9], Scenedesmus sp. [10], Chlorella ∗ Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan. E-mail address: [email protected] (J.-S. Chang).
zofingensis [11], and Chlorella protothecoides [12]. The advantages of using microalgae as the lutein source include: (a) excluding the need to remove petals from plants as the whole biomass of the microalgae can be processed, (b) producing microalgae at a high growth rate thereby giving high lutein productivity and (c) yielding relatively high lutein content at high biomass productivity compared to marigold and other terrestrial plants [13]. Altering the growth metabolism of microalgae is one of the most frequently applied strategies used to improve lutein production, as seen in phototrophic [9,14,15], heterotrophic [16], and mixotrophic [14,15] cultivation. Among those, phototrophic cultivation usually shows a higher lutein content but a lower cell growth rate, as the lutein productivity ranged from 0.44 to 4.77 mg/L/day [10,11]. However, the inherent physiological properties and genetic pathways within each strain cannot be completely manipulated by controlling the external parameters during the cultivation processes. Therefore, it is necessary to obtain strains that are genetically capable of higher biomass productivity and higher yield of the desired product (i.e., lutein). Developing microalgal strains with the ability to accumulate lutein at high content with an enhanced production rate appears to be the key to the success of commercializing microalgae-based lutein. In this study, we used random mutagenesis as a strategy for strain improvement to enhance the lutein production of Chlorella sorokiniana MB-1, which
http://dx.doi.org/10.1016/j.jtice.2017.04.022 1876-1070/© 2017 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: J.-H. Chen et al., Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.022
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is an indigenous microalgal strain isolated from southern Taiwan. N-methyl-N -nitro-N-nitrosoguanidine (MNNG) was selected as the mutagen [17] for the random mutagenesis. The mutant strain of C. Sorokiniana MB-1 was cultivated on mixotrophic mode under optimal cultivation conditions using sodium acetate as the organic carbon source [18]. Cultivation of the selected mutant was further scaled up in 1-L photobioreactor to evaluate the feasibility of commercial production of lutein using the mutant strain.
2. Materials and methods 2.1. Microalgal strain, culture condition and photobioreactor operation Chlorella sorokiniana MB-1 was isolated from freshwater in southern Taiwan. The BG-11 medium was used to grow the MB-1 strain under mixotrophic conditions supplemented with 6 g/L sodium acetate, in addition to aeration of 2% (v/v) CO2 . The photobioreactors (PBR) used to cultivate MB-1 strain were 250 mL or 1-L glass vessel equipped with an external light source (14 W TL5 tungsten filament lamps; Philips Co., Taipei, Taiwan), which provides whole-day illumination with a light intensity of 150 μmol/m2 s. A Li-250 Light Meter equipped with a Li-190SA pyranometer sensor (Li-COR Inc., Lincoln, Nebraska, USA) was used to measure the light intensity. The photobioreactors were operated at 25 °C and 300 rpm agitation with continuous aeration of 2% (v/v) CO2 at a flow rate of 0.1 vvm (volume per working volume per minute).
2.2. Random mutagenesis and selection of high lutein yielding mutants of Chlorella sorokiniana C. sorokiniana MB-1 cultures in exponential growth phase were treated with 0.1 mg/mL of 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) for 1 h. After MNNG treatment, the cells were washed twice with sterile water, re-suspended in BG-11 medium and incubated under low light for 25 h. Then, the cells were spread on solid BG-11 medium containing 400 μM nicotine and incubated at 25 °C for 1 week. Nicotine is an inhibitor of the carotenogenesis pathway and nicotine resistant colonies would thus have very powerful carotenoid synthesis [19]. The nicotine-resistant colonies were subcultured several times in nicotine-containing solid medium to confirm their nicotine resistance and the growth on the solid medium. Nicotine-resistant mutants were then cultured in 1-L photobioreactor in order to analyze carotenoids content and growth rate. The algae mutants have been subjected to 10% DMSO storage in liquid nitrogen bucket and their cell growth performance and lutein content are regularly examined every 3 months to monitor their stability in terms of lutein producing activity.
2.3. Effect of outdoor temperature simulation and inoculum size on lutein content and productivity of C. sorokiniana mutant M12 To determine the effect of temperature variation on outdoor cultivation of the wild type and mutant lutein-producing strains, the microalgae were cultivated on BG-11 medium with the following temperature setting: two commonly seen temperature, such as Light/Dark: 35 °C/25 °C and 25 °C/15 °C, representing summer and winter. The cultures were monitored for their growth, lutein content and lutein productivity. The effect of inoculum size on lutein content and productivity of the mutant strain (M12) was also evaluated by using three different inoculum size of 0.03, 0.09 and 0.15 g/L at the two temperature simulation settings.
2.4. Outdoor cultivation For outdoor cultivation of the microalgal strain, the poly (methyl methacrylate) (PMMA)-made tubular photobioreactor (PBR) with a working volume of 60-L (200 cm in height and 20 cm in diameter) were placed outdoors in National Cheng Kung University campus, Tainan City, Taiwan. The medium in the PBR was identical to that used for indoor cultivation. Sunlight was the only light source and the temperature varied naturally depending on the weather situation. The PBR was continuous aerated with 2% (v/v) CO2 at a flow rate of 0.1 vvm. During the microalgal growth period, liquid samples were collected at the desired time period with respect to time to determine microalgal biomass concentration, pH, residual nitrogen concentration, and lutein content. In addition, the water temperature and light intensity were simultaneously monitored by a LI-250 Light Meter with a LI-190SA pyranometer sensor and a temperature sensor (LI-COR, Inc., Lincoln, Nebraska, USA). 2.5. Determination of microalgae cell concentration and biomass productivity The microalgal cell concentration was determined regularly by optical density measurement at a wavelength of 680 nm (i.e., OD680 ) using a spectrophotometer (GENESYS 10S UV–Vis, Thermo Fisher Scientific, MA, USA). The dry cell weight (DCW) of the microalga was obtained by drying certain amount of cultures at 105 °C until the weight was invariant. The OD680 values were converted to biomass concentration via calibration between the OD680 and dry cell weight (i.e., 1.0 OD680 approximately equals to 0.324 ± 0.03 g DCW/L). The centrifuged microalgal biomass was freeze dried by a freeze-dryer (FDU-1100, EYELA, Tokyo, Japan) and collected for subsequent analysis. The biomass productivity (Pbiomass , g DCW/L/day) was calculated from the following equation:
Pbiomass (g DCW/L/day) =
biomass concentration (g DCW/L ) cultivation time (day )
2.6. Measurement of nitrate and acetate concentration The culture samples obtained at regular intervals were filtered with 0.22 μm filters and diluted to an appropriate concentration. The inorganic nitrate and acetate concentration in the samples were measured by ion chromatography (ICS-30 0 0, Thermo Scientific Dionex, CA, USA) equipped with a carbonate eluent anionexchange column (IonPac AS12A, Thermo Scientific Dionex, CA, USA). 2.7. Determination of the lutein content and productivity The lutein content of the microalgae powder was measured using the method described in our recent work [18]. Briefly, 10 mg dry microalgal biomass was added to 1 mL 60% w/v KOH solution to hydrolyze the lipids and disrupt the cells. The suspension was incubated in a hot water bath at 40 °C for 40 min, followed by repeated extraction with diethyl ether, and the supernatant phase was removed and collected until it was clear. Then, the organic solvent was purged with nitrogen gas, and the precipitate was dissolved in acetone. HPLC analysis was performed using the methods described by Kitada et al. [20]. The HPLC instrument was equipped with photodiode array detector (PDA). The separation column (4.6 × 250 mm in size) was packed with YMC Carotenoid RP-30 with a particle size of 5 μm (YMC Schermbeck, Germany). The mobile phase was 1:9 THF: methanol and the flow rate was set at 1.5 mL/min. The lutein standards used for quantification were purchased from Sigma Chemical Co., St. Louis, MO, USA.
Please cite this article as: J.-H. Chen et al., Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.022
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The lutein content (mg/g) was calculated from the following equation:
Lutein content (mg/g DCW ) Lutein production (mg/L ) × Volume of solution (L ) = DCW of microalgal biomass (g ) The lutein productivity (mg/L/day) was calculated from the following equation:
PLutein (mg/L/day ) = Lutein content (mg/g DCW ) × Pbiomass × (g DCW/L/day ) 3. Results and discussion 3.1. Lutein production using mixotrophic cultivation from C. sorokiniana MB-1 The C. sorokiniana MB-1 is an indigenous microalgal strain, isolated from freshwater in southern Taiwan and it produces lutein when cultivated on mixotrophic mode. Mixotrophic cultivation is a mode of microalgal cultivation in which light is used as an energy source and both CO2 and organic carbon are used as the carbon source. The growth rate and biomass concentration is greatly enhanced in mixotrophic cultivation due to the benefits of both photoautotrophic and heterotrophic growth and both organic and inorganic carbon supports the growth of microalgae [14,15]. In this study, sodium acetate was selected as the carbon source and the effect of sodium acetate concentration was evaluated. The microalgal tolerance to acetate might vary from species to species. Thus, the effect of acetate supply on composition of pigment would also be microalgal species dependent [21]. Four different concentrations of sodium acetate were added into the culture; namely, 1.0, 3.0, 6.0 and 9.0 g/L. Nitrogen source was set at 1.0 g/L sodium nitrate and CO2 aeration rate was fixed at 0.1 vvm. The microalgae were grown at 25 °C with 150 μmol/m2 /s light intensity. Fig. 1 shows that maximal biomass concentrations obtained with the four different sodium acetate concentrations were quite similar, but supplementing 6.0 g/L sodium acetate resulted in higher maximal biomass productivity. Maximal lutein content and lutein productivity went along with the maximal biomass concentration, as the highest lutein content (5.86 mg/g) and productivity (2.56 mg/L/day) were reached when 6 g/L sodium acetate was used. Hence, the use of a moderate amount of sodium acetate (i.e., 6 g/L) was optimal for C. sorokiniana MB-1 growth, biomass and lutein production. The carbon source used in mixotrophic cultivation can vary between strains and also mixotrophic growth might not be optimal for all strains. For instance, mixotrophic cultivation of Scenedesmus sp. resulted in lower biomass productivity and lutein content [15]. However, Chlorella protothecoides yielded a high biomass concentration of 22.4 g/L and a high lutein content of 6.48 mg/g under mixotrophic cultivation using glucose as the organic carbon source in the presence of light [22]. In addition, under heterotrophic growth, C. protothecoides could reach a cell concentration of 17.2 g/L and a lutein content of 4.43 mg/g. It is necessary to evaluate the potential of any microbial strain for their performance under a particular metabolic mode. 3.2. Isolation of high lutein yielding mutants of C. sorokiniana MB-1 In order to obtain high lutein producing mutants of C. sorokiniana, cells were subjected to MNNG mutagenesis and mutants were screened on solid BG-11 medium with nicotine. 58 nicotineresistant lines were obtained and cultured under the same conditions in 1-L photobioreactor to analysis the growth and lutein content. The culture conditions were operated at 25 °C, 300 rpm
Fig. 1. Lutein production from mixotrophic cultivation of Chlorella sorokiniana MB-1. (a) Effect of sodium acetate concentration on biomass concentration and productivity, (b) effect of sodium acetate concentration on lutein content and productivity.
agitation supplemented with 2% (v/v) CO2 at an aeration rate of 0.1 vvm with 6 g/L sodium acetate. The biomass production, lutein content and lutein productivity of the mutant strains studied are summarized in Table 1. Four mutants were identified with lutein content exceeding 6.0 mg/g after 6 days of cultivation, with one mutant (M12) having a lutein content of over 7 mg/g (Table 1). The time course profiles of cell growth and lutein production of MB1 and M12 are compared in Fig. 2. The maximum biomass concentration of MB-1-M12 mutant is similar to that of wild type, but the lutein content of MB-1-M12 mutant (7.52 mg/g) was 28.3% higher than that of the wild-type strain (5.86 mg/g). Furthermore, the lutein productivity of mutant MB-1-M12 (3.63 mg/L/day) was 51.9% higher than that of wild-type strain (2.39 mg/L/day). The nitrogen consumption profiles were quite similar in both strains, as the nitrogen source depleted after 2 days of cultivation, while the biomass concentration continued to increase after the depletion of external nitrogen source, which is identical to the observation in our previous study [23]. In a similar study, mutants of C. sorokiniana 211-32 were generated by random mutagenesis and was cultivated under autotrophic and mixotrophic conditions for the evaluation of lutein content and productivity. The authors mentioned that two mutants DMR-5 and DMR-8 attained lutein levels of about 7 mg/g, but the strains were not chosen for further studies. The mutant chosen for further studies MR-12 had a lutein
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J.-H. Chen et al. / Journal of the Taiwan Institute of Chemical Engineers 000 (2017) 1–8 Table 1 Cell growth and lutein production performance of Chlorella sorokiniana MB-1 and its mutant strains. Microalgal strain
Max. biomass concentration (g/L)
Max. biomass productivity (g/L/day)
Max. lutein content (mg/g)
Max. lutein productivity (mg/L/day)
MB-1 MB-1-M3 MB-1-M5 MB-1-M9 MB-1-M12
2.26 ± 0.06 1.69 ± 0.05 1.63 ± 0.04 1.99 ± 0.07 2.29 ± 0.03
0.928 ± 0.04 0.643 ± 0.11 0.428 ± 0.08 0.435 ± 0.06 0.932 ± 0.09
5.86 ± 0.11 6.60 ± 0.15 6.32 ± 0.22 6.25 ± 0.17 7.52 ± 0.12
2.39 ± 0.09 2.10 ± 0.17 2.15 ± 0.21 3.60 ± 0.16 3.63 ± 0.15
Fig. 2. The performance of cell growth and lutein production of Chlorella sorokiniana MB-1 and its mutant strain MB-1-M12 under indoor mixotrophic cultivation. (a) Time course profile of biomass concentration and lutein content for MB-1, (b) time course profile of biomass concentration and lutein content for MB-1-M12.
content of 5 mg/g under autotrophic conditions [14]. However, under mixotrophic conditions with the addition of glucose or acetate, biomass concentrations decreased by 40% and the cellular lutein content decreased by 33% [14]. Lutein production performances from various strains reported in the literature are summarized in Table 2 and it can be seen that MB-1-M12 strain has the highest lutein content under mixotrophic mode with the addition of organic carbon source. Thus, the MB-1-M12 strain was used for further experiments because of the higher growth rate and highly enhanced lutein production. 3.3. Outdoor temperature simulated cultivation with C. sorokiniana strain MB-1-M12 Open systems are more advantageous than closed systems for commercial microalgal cultivation, such as smaller capital
investment for construction and operation and easier scale-up [24]. However, open systems are prone to contamination by bacteria, alien microalgae species and protozoa. Very few microalgae like Chlorella sp., Spirulina sp. and Dunaliella sp. with extreme conditions used for cultivation are suited for outdoor open systems. Light intensity and temperature are two important parameters in outdoor cultivation. In this study, the effect of two sets of temperature to simulate summer and winter outdoor conditions was investigated. Light–Dark cycle mode (L/D: 12 h) with two commonly seen temperature, such as Light/Dark: 35 °C/25 °C and 25 °C/15 °C, representing summer and winter were used and the results are shown in Fig. 3. The results show that the microalgal biomass concentration was about 2.3 g/L and the biomass productivity was nearly 0.856 g/L/day under 35 °C/25 °C temperature cycle (Fig. 3(a)), while under 25 °C/15 °C temperature cycle, the biomass concentration increased to nearly 3 g/L (Fig. 3(b)), and the biomass
Please cite this article as: J.-H. Chen et al., Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.022
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Table 2 Comparisons of the operation system, metabolic mode, lutein content and lutein productivity of Chlorella sorokiniana MB-1-M12 with those from related reports. Microalga strain
Operation system
Light supply
Metabolic mode
Lutein content (mg/g)
Lutein productivity (mg/L/day)
Reference
Chlorella zofingiensis Chlorella zofingiensis Chlorella fusca Chlorella proboscideum Scenedesmus obliquus FSP-3 Coccomyxa acidophila Chlorella sorokiniana 211-32 Chlorella sorokiniana MR-16 Chlorella sorokiniana MB-1 Chlorella sorokiniana MB-1-M12
Batch Batch Batch Batch Batch Batch Batch Batch Semi-batch Batch
Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous
Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Mixotrophic Autotrophic Autotrophic Mixotrophic Mixotrophic
2.80 3.50 4.20 3.40 4.52 3.50 3.0 5.0 5.21 7.52 ± 0.12
1.30 1.20 0.80 1.20 4.15 0.90 25 mg/L∗ 42 mg/L∗ 5.67 3.63 ± 0.15
[9] [26] [9] [9] [27] [28] [14] [14] [29] This study
∗
light light light light light light light light light light
Yield depicted as mg/L.
Fig. 3. Effect of temperature simulation outdoor cultivation with mutant strain MB-1-M12 under temperature simulation of outdoor cultivation. (a) Time course profile of biomass concentration and lutein content under 35 °C/25 °C temperature cycle, (b) time course profile of biomass concentration and lutein content under 25 °C/15 °C temperature cycle.
productivity reached 0.740 g/L/day. Although the biomass concentration is higher at the simulated winter temperatures, the biomass productivity was slightly lower when compared with that obtained from using simulated summer temperatures. On the other hand, lutein content and productivity of 5.87 mg/g and 2.7 mg/L/day was obtained under 35 °C/25 °C. In contrast, under the condition of 25 °C/15 °C, the lutein content and productivity markedly increased to 6.19 mg/g and 2.93 mg/L/day, respectively. In summary, for lutein production, the 25 °C/15 °C (known as winter simulation
mode) is better than the 35 °C/25 °C (known as summer simulation mode). Previous reports indicate that high temperature can induce carotenoid accumulation in microalgae, but optimum temperature of 28 °C was found to attain both higher biomass and lutein content. For instance, Muriellopsis sp. obtained a maximal lutein concentration of 30 mg/L at 28 °C under photoautotrophic mode [9]. For Chlorella protothecoides under heterotrophic mode, 28 °C is the optimal temperature for both biomass production (17.2 g/L) and lutein accumulation (74.29 mg/L) [25]. However, it was shown
Please cite this article as: J.-H. Chen et al., Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.022
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Fig. 4. Effect of inoculum size and temperature simulation of outdoor cultivation on cell growth and lutein production of mutant strain MB-1-M12. (a) Time course profile of biomass concentration and productivity with 35 °C/25 °C temperature cycle at an inoculums size of 0.03, 0.09 and 0.15 g/L, (b) time course profile of lutein content and productivity with 35 °C/25 °C temperature cycle at an inoculums size of 0.03, 0.09 and 0.15 g/L, (c) time course profile of biomass concentration and productivity with 25 °C/15 °C temperature cycle at an inoculums size of 0.03, 0.09 and 0.15 g/L, (d) time course profile of lutein content and productivity with 25 °C/15 °C temperature cycle at an inoculums size of 0.03, 0.09 and 0.15 g/L.
that mutant strain MB-1-M12 seems to be able to adapt to the outdoor environment based on the results of the simulated outdoor culture. 3.4. Effect of inoculum size on temperature simulation outdoor cultivation with the mutant strain C. sorokiniana MB-1-M12 As previously mentioned, open systems are susceptible to external environmental influences and can be contaminated by bacteria or other microalgae, and these problems are unavoidable. Therefore, increasing the inoculum size of microalgae is thought to mitigate the influence of contamination during the early stage of microalgal cultivation, thereby increasing the possibility for achieving successful microalgal growth. In this study, three different inoculum sizes were used to examine the effect of inoculum size on microalgal growth, while the same light/dark cycle mode and two sets of temperature cycle (i.e., 35 °C/25 °C and 25 °C/15 °C) were used. As shown Fig. 4, there was no significant change in maximum biomass concentration when using the three inoculum size, but biomass productivity was higher when higher inoculum sizes (i.e., 0.09 and 0.15 g/L) were used. Under temperature cycle
of 35 °C/25 °C, the biomass productivity obtained from using 0.09 and 0.15 g/L inoculum size was 0.704 and 0.692 g/L/day, respectively, which is much higher than that for using 0.03 g/L inoculum size. On the other hand, the highest lutein content (7.57 mg/g) was obtained by using 0.09 g/L inoculum size, while using 0.15 g/L inoculum size achieved the highest lutein productivity of 2.47 mg/L/day (Fig. 4(b)). Although under 35 °C/25 °C temperature cycle, using an inoculum size of 0.15 g/L gave the highest lutein productivity, 0.09 g/L was still considered as the optimal inoculum size when taking all three performances indexes (i.e., biomass productivity, lutein content and lutein productivity). In contrast, under the 25 °C/15 °C temperature cycle, the trends of cell growth are quite similar to those seen for the 35 °C/25 °C temperature cycle (Fig. 4(c)), whereas both lutein content and lutein productivity reached the highest level (7.76 mg/g and 3.45 mg/L/day, respectively) with the inoculum size of 0.09 g/L (Fig. 4(d)), which is different from the trend observed with 35 °C/25 °C temperature cycle (Fig. 4(b)). Overall speaking, using the inoculum size of 0.09 g/L resulted in the highest lutein content and productivity. For both cultivation temperature cycles, the 0.09 g/L inoculum size is considered optimum and was thus used in further experiments.
Please cite this article as: J.-H. Chen et al., Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.022
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Table 3 Comparison of cell growth and lutein production performance of indoor and outdoor cultures of mutant strain Chlorella sorokiniana MB-1-M12. Culture type
Max. biomass concentration. (g/L)
Max. biomass productivity (g/L/day)
Max. lutein concentration (mg/g)
Max. lutein productivity (mg/L/day)
Indoor Outdoor
2.29 ± 0.03 1.18 ± 0.06
0.932 ± 0.09 0.799 ± 0.07
7.52 ± 0.12 6.85 ± 0.60
3.63 ± 0.15 1.35 ± 0.07
content of 4.5 mg/g and a productivity of 290 mg lutein/m2 /day [21]. Although lutein productivity of mutant MB-1-M12 obtained from our outdoor cultivation system is higher or comparable to the reported values from outdoor microalgal cultures [21,24], it is still significantly lower than that obtained in indoor cultures. However, the lutein content of mutant strain MB-1-M12 is similar to that obtained under indoor cultivation, which demonstrates the potential of commercial lutein production with this mutant strain. Nevertheless, better cultivation strategies need to be adopted to attain higher lutein productivity for the outdoor culture. 4. Conclusions In this study, mutants of Chlorella sorokiniana MB-1 with high lutein production ability were successfully generated. Although the biomass production of mutant strains was lower than the wild type, the lutein content and productivity of the selected mutants under indoor cultivation was significantly higher with an increase of 28.3% and 51.9%, respectively. The outdoor culture of the best mutant (MB-1-M12) gave similar lutein content to that obtained in indoor cultivation, whereas a decrease in lutein productivity was observed in the outdoor culture. Further improvement in the lutein productivity using better outdoor cultivation strategies is required for commercialization purposes. Acknowledgments
Fig. 5. Performance of outdoor cultivation with mutant strain Chlorella sorokiniana MB-1-M12 under mixotrophic cultivation. (a) Time course profile of lutein content and productivity for strain MB1-M12, (b) time course profile of light intensity of sunlight and outdoor temperature.
3.5. Performance of outdoor cultivation with mutant strain C. sorokiniana MB-1-M12 The commercial production of lutein can be successful only when outdoor cultivation is possible. Therefore, outdoor cultivation of the mutant strain was conducted to produce lutein with mixotrophic growth under the same conditions used in the indoor tests. As shown in Fig. 5(a), outdoor cultivation of mutant strain MB-1-M12 achieved a lutein content and productivity of 6.85 mg/g and 1.35 mg/L/day (or 202 mg/m2 /day assuming that three 50-L tubular photobioreactors occupy about 1 m2 of land area), respectively (Table 3). The weather condition was not consistent as it can be seen from the differences in light intensity and it is reflected in growth rate and nitrate consumption (Fig. 5(b)). Very few studies report the outdoor cultivation of microalgae for lutein production. Of those, the outdoor cultivation of Muriellopsis sp. in open tanks agitated with a paddle wheel reported a productivity of 100 mg lutein/m2 /day in summer with a dry biomass content of 20 g dry biomass/m2 /day [24]. The biomass obtained was also rich in protein and lipids, with α -linoleic acid amounting to 30% of the total fatty acids [24]. Another report showed that Scenedesmus almeriensis when cultivated in outdoor tubular photobioreactor of 40 0 0 L volume could attain a lutein
The authors gratefully acknowledge financial support received from Taiwan’s Ministry of Science and Technology under grant numbers 106-3113-E-006-011, 106-3113-E-006-004-CC2, and 1052221-E-006-226. This research was also supported by Ministry of Education under the top university Grant number D105-23005. References [1] Demmig-Adams B, Adams WW. The role of xanthophyll cycle carotenoids in the protection of photosynthesis. Trends Plant Sci 1996;1:21–6. [2] Dwyer JH, Navab M, Dwyer KM, Hassan K, Sun P, Shircore A, et al. Oxygenated carotenoid lutein and progression of early atherosclerosis the Los Angeles atherosclerosis study. Circulation 2001;103:2922–7. [3] Astorg P. Food carotenoids and cancer prevention: an overview of current research. Trends Food Sci Technol 1997;8:406–13. [4] Heber D, Lu Q-Y. Overview of mechanisms of action of lycopene. Exp Biol Med 2002;227:920–3. [5] Chiu C-J, Taylor A. Nutritional antioxidants and age-related cataract and maculopathy. Exp Eye Res 2007;84:229–45. [6] Granado F, Olmedilla B, Blanco I. Nutritional and clinical relevance of lutein in human health. Br J Nutr 2003;90:487–502. [7] Cerón MC, Campos I, Sánchez JF, Acién FG, Molina E, Fernández-Sevilla JM. Recovery of Lutein from microalgae biomass: development of a process for Scenedesmus almeriensis biomass. J Agric Food Chem 2008;56:11761–6. [8] Piccaglia R, Marotti M, Grandi S. Lutein and lutein ester content in different types of Tagetes patula and T. erecta. Ind Crops Products 1998;8:45–51. [9] Del Campo JA, Moreno J, Rodriguez H, Vargas MA, Rivas J, Guerrero MG. Carotenoid content of chlorophycean microalgae: factors determining lutein accumulation in Muriellopsis sp. (Chlorophyta). J. Biotechnol. 20 0 0;76:51–9. [10] Sánchez JF, Fernández JM, Acién FG, Rueda A, Pérez-Parra J, Molina E. Influence of culture conditions on the productivity and lutein content of the new strain Scenedesmus almeriensis. Process Biochem 2008;43:398–405. [11] Del Campo JA, Rodriguez H, Moreno J, Vargas MA, Rivas J, Guerrero MG. Accumulation of astaxanthin and lutein in Chlorella zofingiensis (Chlorophyta). Appl Microbiol Biotechnol 2004;64:848–54. [12] Shi X-M, Zhang X-W, Chen F. Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources. Enzyme Microb Technol 20 0 0;27:312–18. [13] Fernandez-Sevilla JM, Fernandez FGA, Grima EM. Biotechnological production of lutein and its applications. Appl. Microbiol. Biotechnol. 2010;86:27–40.
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Please cite this article as: J.-H. Chen et al., Lutein production with wild-type and mutant strains of Chlorella sorokiniana MB-1 under mixotrophic growth, Journal of the Taiwan Institute of Chemical Engineers (2017), http://dx.doi.org/10.1016/j.jtice.2017.04.022