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A highly efficient two-stage cultivation strategy for lutein production using heterotrophic culture of Chlorella sorokiniana MB-1-M12
Bioresource Technology 253 (2018) 141–147
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A highly efficient two-stage cultivation strategy for lutein production using heterotrophic culture of Chlorella sorokiniana MB-1-M12
T
Chun-Yen Chena, I-Chia Lub, Dillirani Nagarajanb,c, Chien-Hsiang Changb, I-Son Ngb, ⁎ Duu-Jong Leec, Jo-Shu Changb,d, a
University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan c Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan d 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
A B S T R A C T
Keywords: Lutein Microalgae Chlorella sorokiniana Heterotrophic growth Two-stage cultivation
A heterotrophic mutant of Chlorella sorokiniana MB-1-M12 was evaluated for its ability to produce lutein using organic carbon and nitrogen sources and without light irradiation. In batch fermentation, the maximal lutein content (3.67 mg lutein/g biomass) and productivity (2.84 mg/L/d) could be obtained when cultivated in BG-11 medium with 7.5 g/L glucose, 0.75 g/L urea, pH 7.5 and a C/N ratio of 10. A novel two-stage cultivation strategy that integrates fed-batch and semi-batch operations was applied to enhance the lutein production performance. When growing MB-1-M12 strain in a 5L fermenter using the optimal operation strategies, the maximum biomass concentration, biomass productivity, lutein content and lutein productivity could reach 25 g/L, 4.88 mg/L/d, 5.88 mg/g and 16.2 mg/L/d, respectively. This high lutein productivity could significantly reduce the cultivation time and the associated costs, indicating the potential of using MB-1-M12 strain for heterotrophic lutein production in commercial scale.
1. Introduction Lutein is a primary xanthophyll carotenoid, serving as light-harvesting antenna pigment and antioxidant in microalgae, plants and other photosynthetic organisms (Pascal et al., 2005). Other than light harvesting, the major function of lutein is to protect the photosystems from oxidative damage at high light intensities, especially blue light (Roberts et al., 2009). The structure of lutein is composed of a long conjugated double bond backbone chain with aromatic rings at either end. Lutein is lipo-soluble and is capable of scavenging free radicals and singlet oxygen, acting as a natural antioxidant (Britton, 1995). In humans, lutein along with zeaxanthin, accumulates in the macula of the eye, as the macular pigment and protects the retina from blue light and aids in improving visual acuity (Krinsky et al., 2003). Lutein has been implicated in human health mainly because of its ocular-protective activity and antioxidant property against neurodegenerative diseases, cardio-vascular diseases, diabetic retinopathy (Zhang et al., 2014) and respiratory health (Melo van Lent et al., 2016). However, humans are incapable of de novo synthesis of lutein and are strictly dependent on dietary intake to fulfil their lutein requirement. A daily dose of 5 mg is recommended for patients with Age related Macular Degeneration
⁎
(AMD), but the status of lutein as an essential nutrient has not been approved yet by pharmaceutical authorities (Fernandez-Sevilla et al., 2010). Other than this, lutein has also been used as colorant and food additive for human consumption (known as colorant E161b in the European Union) and also as a feed additive to deepen the yellow color of egg yolks (Lin et al., 2015). With all these applications, market demand for lutein is increasing and in 2015 the global lutein market was estimated at US$ 135 million and will continue to rise (Global Market Insights Lutein Market Report, 2016). Currently, the market demand for lutein is met by the extraction of lutein from the bright yellow petals of the marigold flowers of the genus Tagetes (Fernandez-Sevilla et al., 2010). However, lutein extraction from Tagetes petals are challenged by the seasonal availability of the flowers, large requirement of land for the cultivation of the plants, requirement of skilled labor and the very low lutein content present in the flower petals (Lin et al., 2015). Lutein primarily exists in mono- and diesterified form in marigold flowers (Del Campo et al., 2007), and chemical saponification is required to extract lutein from them. Microalgae are a potential source of lutein and the advantages are: i) high cellular lutein content at 0.5–1.2% by weight, ii) unaffected by seasonal variation as in the case of Tagetus, iii) very low land and water
Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan. E-mail address: [email protected] (J.-S. Chang).
https://doi.org/10.1016/j.biortech.2018.01.027 Received 9 December 2017; Received in revised form 30 December 2017; Accepted 5 January 2018
Available online 06 January 2018 0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
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2.2. Determination of microalgal biomass dry cell weight, biomass productivity and specific growth rate
requirements, and iv) the possibility to generate other high value products from the microalgal biomass (Lin et al., 2015). However, the major bottleneck in the mass production of microalgae is the supply of optimal light intensity, which is the prime criteria to stimulate pigment production in photoautotrophic cultures. As a result, effective new photobioreactor designs are being implemented to cope with this issue; however, this usually causes a significant increase in the product costs. Therefore, in contrast to phototrophic culture systems, heterotrophic cultivation of microalgae for the mass production of high-value products has been considered an economically feasible and commercially favorable option since the heterotrophic growth could attain much higher microalgal biomass productivity without the need of light supply and CO2 aeration (Hu et al., 2017). It is easier to control the product quality in a heterotrophic culture due to being operated in a wellcontrol closed system. Also, since the growth and metabolism of microalgae occurs in dark conditions for heterotrophic cultures, conventional bacterial fermenters can be used for microalgal cultures without much modifications (Bumbak et al., 2011). Although microalgae are genetically capable of utilizing organic carbon sources and are endowed with a central carbon catabolic pathway, the predisposition for heterotrophy is mainly strain dependent (Perez-Garcia et al., 2011). The other potential problem associated with producing pigments (e.g., lutein) under heterotrophic conditions would be the absence of light, as light illumination is usually required for promoting the production of pigments (Hu et al., 2017). Therefore, selecting a suitable heterotrophic strain for efficient lutein production under optimal operating conditions would be a pivotal step to realize the idea of producing microalgal lutein heterotrophically. In this study, a heterotrophic Chlorella sorokiniana MB-1-M12, was examined for its effectiveness in lutein production and the culture conditions were optimized to achieve the maximal lutein producing performance. In particular, a novel two-stage cultivation strategy was devised to further improve the lutein productivities to make it suitable for industrial scale applications.
A 5 mL of cell suspension was collected from the bioreactor, and was centrifuged at 10,000 rpm for 4 min to remove the culture medium. Then, the sample was washed twice with 5 mL of deionized water and transferred to an aluminum pan. The aluminum pan was placed in an infrared moisture determination balance (FD-720, KETT, Japan) at 100 °C until all the water was removed. After drying, the dry cell weight (DW) was determined as the weight difference between the pan with and without the microalgal cells. The biomass productivity (Pbiomass, mg DCW/L/d) and specific growth rate (μ) were calculated from the following equation (Bailey and Ollis, 1986):
Biomass productivity (mg DCW/L/d) = Pbiomass =
ΔX X −X = t initial Δt t (1)
d lnX ⎞ Specific growth rate (d−1) = ⎛ ⎝ dt ⎠max
(2)
where X is dry cell weight (g/L), ΔX (mg/L) is the variation of dry cell weight and Δt is the cultured time (d). 2.3. Determination of nutrient concentration The nitrate concentration was determined using the spectrophotometric method described earlier (Collos et al., 1999). In general, the liquid samples were collected from the bioreactor and filtered via a 0.22 μm pore size filter. After filtration, samples were diluted with deionized water as determined by measuring the optical density (OD) of the samples at a wavelength of 220 nm (denoted as OD220) using a UV/ Vis spectrophotometer (model U-2001, Hitachi, Tokyo, Japan). A standard curve was generated using pure sodium nitrate (obtained from Sigma Chemical Co.), and the concentrations of nitrate in the samples were calculated. The ammonium concentration of the culture was analyzed by Berthelot’s reaction (Patton and Crouch, 1977). A 250 μL deionized water, 500 μL phenol nitroprusside solution (SIGMA) and 500 μL 0.2% alkaline hypochlorite solution (SIGMA) were added into 50 μL diluted liquid sample obtained from the bioreactor and the mixture was incubated at room temperature for 30 min. Then, the optical density of the sample was measured at a wavelength of 630 nm by a UV/Vis spectrophotometer (model U-2001, Hitachi, Tokyo, Japan). A standard curve was generated using pure ammonium chloride (obtained from Sigma Chemical Co.), and the concentrations of ammonium in the samples were calculated. The urea concentration of the medium was analyzed by hydrolysis of urea with urease, followed by estimation of the ammonia released by the Berthelot’s reaction. Briefly, 50 μL filtered and appropriately diluted liquid sample from the bioreactor was added to 250 μL 1% urease solution (SIGMA) and the mixture was incubated at 37 °C for 10 min. Next, 500 μL phenol nitroprusside solution (SIGMA) and 500 μL 0.2% alkaline hypochlorite solution (SIGMA) was added and the mixture was incubated at room temperature for 30 min. the optical density of the sample was measured at a wavelength of 630 nm by a UV/Vis spectrophotometer (model U-2001, Hitachi, Tokyo, Japan) for measuring ammonia concentration as described previously. Urea concentration was calculated from the calibration curve obtained using standard urea solution. The concentrations of glucose, acetate and glycerol were determined by high-performance liquid chromatography (HPLC) equipped with a refraction index detector (RID-10A, Shimadzu, Japan) and an ICSep ICE-COREGEL 87H3 column (Transgenomic, USA). The mobile phase was 0.008 N H2SO4, which was eluted at 0.4 mL/min at room temperature.
2. Materials and methods 2.1. Microalga used and its culture conditions Chlorella sorokiniana MB-1-M12, is a glucose tolerant mutant of C. sorokiniana MB-1, which is originally a photoautotrophic lutein-rich strain isolated in southern Taiwan. C. sorokiniana MB-1-M12 possesses a high lutein content and productivity under heterotrophic conditions, making it a potential candidate for mass production of lutein. The MB1-M12 strain was cultivated on BG-11 medium consisting of (mg/L): NaNO3, 1.0; Na2CO3, 20; CaCl2·2H2O, 36; Citric acid, 6.0; MgSO4·7H2O, 75; K2HPO4, 40; Ferric ammonium citrate, 6.0; EDTA·2Na, 1.0; H3BO3, 2.86; MnCl2·4H2O, 1.81; CuSO4·5H2O, 0.079; ZnSO4·7H2O, 0.222; Na2MoO4·2H2O, 0.39; Co(NO3)2·6H2O, 0.049. All experiments were conducted in serum bottle cultures containing 1000 mL sterilized medium (120 °C, 20 min) at 27 ± 1 °C with continuous stirring (300 rpm of agitation rate) and the said reactors were covered with aluminum foil to avoid light penetration. Glucose, acetate and glycerol were evaluated as carbon source (at a concentration of 0.08 mol/L) for growth and the metabolism of MB-1M12 in dark conditions. Four nitrogen sources, namely, sodium nitrate (NaNO3), potassium nitrate (KNO3), ammonium chloride (NH4Cl) and urea (CO(NH2)2), at a concentration of 0.01 mol/L, were evaluated for their effect on lutein content and productivity of MB-1-M12. The effect of C/N ratio on lutein content and productivity of MB-1-M12 was also determined by testing five different C/N ratios (3, 5, 10, 15, 20 for glucose versus urea). The optimal pH for growth and lutein production was determined after culturing the microalgal strain at pH 6.5, 7, 7.5, 8, 8.5.
2.4. Determination of lutein content The lutein content of microalgae was estimated as described 142
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Table 1 The effect of carbon sources on biomass, lutein content, yield, and productivity of C. sorokiniana MB-1-M12 under heterotrophic cultivation. Carbon source
Dry weight (g/L)
Biomass productivity (mg/L/d)
Yield (g DW/g substrate)
Lutein content (mg/g)
Lutein productivity (mg/L/d)
Glucose Acetate Glycerol
1.20 ± 0.09 0.68 ± 0.06 N.A.
392 ± 14.04 190 ± 12.16 N.A.
0.45 0.19 N.A.
2.88 ± 0.25 3.06 ± 0.28 N.A.
0.85 ± 0.13 0.47 ± 0.18 N.A.
designated intervals.
previously (Chen et al., 2017). First, 10 mg of lyophilized microalgal biomass and 0.5 g glass beads were added to 1 mL of 60% w/w KOH solution to promote cell disruption by bead-beating. After cell disruption, the mixture was placed in a water bath at 40 °C for 40 min for converting esterified form of lutein to free form completely and lutein was extracted with diethyl ether. The supernatant phase was separated by centrifugation and collected until it was clear. Lutein in solvent was purged with nitrogen gas, the precipitate was re-dissolved in acetone, and lutein content was measured by HPLC equipped with a photodiode array detector (PDA) and a YMC Carotenoid RP-30 column with a particle size of 5 μm and a column size of 4.6 × 250 mm (YMC Schermbeck, Germany). The temperatures of column and tray were set at 30 °C and 4 °C, respectively. The mobile phase contained 10% (v/v) tetrahydrofuran (THF) and 90% (v/v) methanol, and the extracts were eluted at a flow rate of 1.5 mL/min. Lutein was detected by measuring the absorbance at the wavelength range of 220–750 nm, and the maximal absorbance at 450 nm was used for quantification of lutein content. The lutein standards for quantification of lutein were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The lutein content (mg/ g) was obtained from the following equation:
Lutein content (mg/g DCW) Lutein concentration (mg/L) × Volume of solution (L) = Microalgal biomass (g)
3. Results and discussion Chlorella sorokiniana MB-1-M12 is capable of heterotrophic growth under dark conditions using glucose as the carbon source. Preliminary experiments showed that optimal growth of the MB-1-M12 strain was obtained with BG-11 medium, while other frequently used media (such as Basal medium and Bold’s basal medium) did not provide optimal growth (data not shown). Therefore, BG-11 medium was used for the experiments investigating the optimal carbon source, nitrogen source, pH and the C/N ratio as well as the determination of optimal culture conditions. The results are presented in the following sections. 3.1. Effect of different carbon sources on growth and lutein production of MB-1-M12 Glucose is the preferred carbon source in most heterotrophic microalgae, while acetate and glycerol have also been variably used depending on the tolerance and metabolic capacity of the cultivated strains. The effect of glucose, glycerol and acetate on biomass growth and lutein production from MB-1 M12 has been evaluated with 1 g/L sodium nitrate as the nitrogen source and the tested carbon source at 80 mM. The results are summarized in Table 1. C. sorokiniana MB-1M12 is incapable of utilizing glycerol as the sole carbon source for growth. Using glucose as the carbon source exhibited much better maximum biomass concentration (1.20 g/L), biomass productivity (392 mg/L/d) and cell growth yield coefficient of (0.45 g biomass/ g substrate) when compared with using acetate, whereas cultivating MB-1-M12 on acetate resulted in a slightly higher lutein content than that obtained with glucose (3.0 mg/g lutein from acetate vs. 2.88 mg/g lutein from glucose). Nevertheless, using glucose still showed a higher lutein productivity due mainly to a higher biomass productivity when compared to using acetate. It is also known that glucose has higher energy content compared to acetate (2.8 kJ/mol vs. 0.8 kJ/mol). Considering lutein productivity as the most important parameter from practical application aspect, glucose was chosen as the optimal carbon source for lutein production with MB-1-M12. Previous work on lutein production with heterotrophic Chlorella sp. also showed that using glucose as the carbon source to grow C. protothecoides in large scale cultures could obtain a lutein content of as high as 5 mg/g (Shi et al., 2002).
(3)
The lutein productivity (Plutein, mg/L/d) was obtained from the following equation:
Plutein (mg/L/d) = Lutein content (mg/g DCW) × Pbiomass (g DCW/L/d) (4) 2.5. Operation of two-stage cultivation strategy for enhanced lutein production from MB-1-M12 under heterotrophic conditions All experiments were performed in 1-L serum bottles with BG-11 medium containing 7.5 g/L initial glucose concentration, 0.75 g/L initial urea concentration, pH 7.5, 0.3 vvm aeration rate under dark heterotrophic conditions. An integrated fed-batch/semi-batch strategy was applied for enhancing lutein production. The experiment was started in a fed-batch mode to accumulate maximum biomass. A concentrated feed solution containing 500 g/L glucose and 50 g/L urea was prepared and when the residual glucose concentration was below 2 g/L, the feed solution was fed into the bioreactor to keep the glucose concentration in the range of 2.0–7.5 g/L and maintain sufficient nitrogen source for growth. After maximal biomass was accumulated, the fermentation was switched to semi-batch mode and when the glucose in medium was completely depleted by MB-1M12, 50% of the culture broth (volume of 1 L) was removed and 500 mL of fresh medium was added into the cultivation in order to restart a new cycle of growth. The scale-up fermentation of the fed-batch/semi-batch integrated two-stage operation strategy for lutein production from MB-1-M12 was carried out in a 5L stirred tank fermenter with a working volume of 3 L. The fermentation conditions were maintained at 25 ± 1 °C, 100 rpm of agitation rate, 0.3 vvm of air aeration rate (giving a mass transfer coefficient (kLa) of 12 ± 1.5 h−1) and pH 7.5 ± 0.1. The pH of culture was maintained by periodic addition of 1 N HCl or 1 N NaOH with a pH controller. The samples were collected to measure microalgal cell growth, glucose concentration, urea concentration and lutein content at
3.2. Effect of culture pH on growth and lutein production of MB-1-M12 The pH of culture medium is a significant parameter for cell growth and lutein accumulation of microalgal strains, and the optimal condition of pH for the algal growth and lutein production is highly strain dependent. C. sorokiniana species could grow in the pH range from 5.0 to 9.0 (Qiu et al., 2017). In heterotrophic microalgal cultures where pH is uncontrolled or the medium pH is towards the acidic range, the culture becomes more acidic due to the oxidative assimilation of glucose and the release of metabolic acids such as citric acid, pyruvic acid and malic acid (Wu et al., 2005). Hence, MB-1-M12 was grown in medium with different pH values of 6.5, 7, 7.5, 8, and 8.5, while pH uncontrolled cultures served as a control. The results are summarized in Table 2. The results show that there was no significant difference in 143
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reduced energy requirement for its uptake and metabolism (Grobbelaar, 2007; Shi et al., 2000). In contrast, nitrate requires more energy for transport into cells and further conversion to ammonia (Perez-Garcia et al., 2011). Our lutein production results show no significant difference in lutein content for the cultures grown with nitrate and ammonium, but a relatively lower level of lutein content were obtained when urea was used as a nitrogen source. However, when ammonia is used as a sole nitrogen source in pH uncontrolled cultures, the medium pH would drop significantly during algal growth due to the release of H+ ions during the metabolism of ammonium (Grobbelaar, 2007), and the sudden pH drift is often lethal to cells. It was observed that after cultivation for 2 days, the culture pH dropped to below 3.5 when using ammonium as the nitrogen source. With respect to cost, for an equivalent nitrogen concentration, the order of nitrogen cost for lutein production with various nitrogen sources is as follows: in declining order, urea < ammonium < nitrate. When urea replaces nitrate as nitrogen source, the cost of nitrogen source significantly decreased from 445.2 to 70.9 NTD/g lutein (NTD denotes new Taiwan dollar), achieving a cost reduction of 84%. It is clear that urea is an economical and effective nitrogen source for MB-1-M12, compared to ammonium and nitrate for lutein production. Therefore, with respect to biomass concentration, productivity and cost, urea was chosen as the suitable nitrogen source for C. sorokiniana MB-1-M12. Similar results were obtained for Chlorella protothecoides CS-41, where urea proved to be a better nitrogen source, compared to nitrate and ammonium. They found that even though all the above-mentioned three nitrogen sources yielded similar biomass, lutein content was considerably higher when urea was used as a nitrogen source (2 mg lutein/g glucose) (Shi et al., 2000).
Table 2 The effect of culture medium pH on biomass, lutein content, yield, and productivity of C. sorokiniana MB-1-M12 under heterotrophic cultivation. pH
Dry weight (g/L)
Biomass productivity (mg/L/d)
Lutein content (mg/g)
Lutein productivity (mg/L/d)
6.5 7.0 7.5 8.0 8.5 Control
2.35 2.32 2.39 2.32 2.32 2.27
423 584 651 641 599 583
2.55 2.79 3.38 3.34 2.98 2.98
0.88 1.06 1.27 1.25 1.16 1.14
± ± ± ± ± ±
0.11 0.11 0.13 0.12 0.14 0.13
± ± ± ± ± ±
19 22 23 26 25 24
± ± ± ± ± ±
0.12 0.11 0.15 0.14 0.13 0.11
± ± ± ± ± ±
0.03 0.04 0.05 0.05 0.03 0.03
biomass concentration with different pH, but lutein content and productivity differed considerably. The highest biomass productivity (650.58 mg/L/d) and lutein content (3.42 mg/g) were obtained at a pH of 7.5. In addition, there was a dramatic decrease in biomass productivity and lutein content when the pH decreased to 6.5. The slightly acidic pH 6.5 severely inhibited biomass growth (423.01 mg/L/d) and lutein accumulation (2.55 mg/g). The pH drop in microalgal cultures is generally accompanied by the destruction of the carotenoids and chlorophylls (Theriault, 1965). The alkaline pH of 8.5 slightly inhibited microalgal growth with a biomass productivity and lutein content of 599.0 mg/L/d and 2.98 mg/g, respectively. Therefore, pH 7.5 was chosen as the optimal pH for lutein production from MB-1-M12. In a related study, (Shi et al., 2006) reported that C. protothecoides CS-41 was capable of heterotrophic growth and lutein production, and pH 6.6 yielded the highest lutein content and biomass productivity of 4.75 mg/ g cells and 77.9 mg/L, respectively. Alkaline pH of 8 inhibited biomass growth of C. protothecoides CS-41, which is similar to our observation.
3.4. Effect of urea concentration and C/N ratio on growth and lutein production of MB-1-M12
3.3. Effect of different nitrogen sources on growth and lutein production of MB-1-M12
The effect of urea concentration (along with an initial glucose concentration of 5 g/L) on the growth and lutein production from MB-1M12 was investigated in batch fermentation. Nitrogen is an essential macro nutrient for growth and biomass yield in microalgae and optimal nitrogen source concentration is crucial as lower nitrogen levels might decrease the lutein yield. Five different C/N ratios (3, 5, 10, 15, 20 for glucose versus urea) in the culture medium were examined for their effects on the lutein content and productivity. As shown in Table 3, there was no significant difference in biomass concentration and biomass productivity when different urea concentrations were used, while a marked decrease in lutein content (below 3 mg/g) was observed when the C/N ratio was greater than 10 (Table 3). Lutein content markedly decreased when nitrogen is insufficient (or when the C/N ratio is high). At C/N ratios of 15 and 20 with the same glucose concentration of 5 g/ L, nitrogen limitation occurs resulting in a lower lutein content. A lower C/N ratio and sufficient nitrogen supply could ensure higher lutein content, but when the C/N ratio is too low (e.g., lower than 10), lutein formation in the microalgae also slightly decreases. Thus, there is an optimal C/N ratio of 5, at which the lutein content and lutein productivity could reach the highest level of 3.76 mg/g of 2.12 mg/L/d, respectively. However, considering the cost for nitrogen in large scale cultures, the C/N ratio of 10 that gives the second best lutein producing performance was selected for lutein production with MB-1-M12 in the following experiments.
Nitrogen is an important macronutrient for the growth and metabolism of microalgal cells. Microalgae are able to utilize a variety of nitrogen sources, such as ammonia, nitrate, urea, and amino acids (Perez-Garcia et al., 2011). The effect of four various nitrogen sources, sodium nitrate (NaNO3), potassium nitrate (KNO3), ammonium chloride (NH4Cl) and urea (CO(NH2)2) on the production of lutein by MB-1-M12 under heterotrophic conditions was investigated. The concentration of nitrogen source in the medium was maintained at 10 mM, along with 5 g/L glucose, pH 7.5. The results are shown in Table 3. Nitrate has been used as a common nitrogen source for the cultivation of Chlorella spp. in BG-11 or BM medium. However, Table 3 shows that higher maximum biomass concentration was obtained with ammonium chloride and urea at 2.78 and 2.83 g/L dry cells, respectively. The efficacy of the various nitrogen sources with respect to biomass concentration and productivity were, in declining order: urea > ammonium > nitrate. C. sorokiniana MB-1-M12 preferentially absorbed ammonium and urea, and the highest lutein productivity was achieved with ammonia (2.11 mg/L/d). Ammonium is considered as the most preferred and suitable nitrogen source for algae growth due to Table 3 The effect of C/N ratio on biomass, lutein content, yield, and productivity of C. sorokiniana MB-1-M12 under heterotrophic cultivation. C/N ratio*
Urea (g/L)
Biomass productivity (mg/ L/d)
Lutein content (mg/g)
Lutein productivity (mg/L/d)
3.5. Effect of glucose concentration on the growth and lutein production of MB-1-M12
20 15 10 5 3
0.25 0.35 0.50 1.00 1.66
957 936 935 928 942
1.73 2.97 3.64 3.76 3.13
1.31 1.91 2.10 2.12 1.57
In order to achieve high biomass production in batch culture, it is required to use high initial carbon source concentration (i.e., glucose), and hence it is essential to determine the optimal glucose concentration without generating substrate inhibition. The effect of initial glucose and urea concentration on biomass growth and lutein production of MB-1-
± ± ± ± ±
43 38 37 42 41
± ± ± ± ±
0.09 0.12 0.13 0.13 0.11
± ± ± ± ±
0.08 0.07 0.11 0.12 0.07
144
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at pH 7.5 ± 0.1, 27 ± 1 °C and 0.3 vvm aeration rate under continuous stirring (300 rpm of agitation rate) in the dark. The remains of the first stage served as inoculum and fresh BG-11 medium, concentrated glucose solution and concentrated urea solution (500 g/L glucose and 50 g/L urea, respectively) were added for the new semibatch cycle. Based on our previous results, it was shown that the culture reached a biomass concentration of 10 g/L with two cycles of fed-batch cultivation and after that glucose consumption and biomass accumulation decreases. Therefore, one replacement of culture was conducted every two cycles of fed-batch culture (in 1-L serum bottles) in the twostage strategy and the results are shown in Fig. 1. The two stage strategy was conducted with three different media replacement ratios (25%, 50%, 75%) was conducted and best results were shown for 75% medium replacement in the second stage semi-batch mode. The twostage strategy with 75% medium replacement ratio significantly increased the growth rate decreasing the operation time, allowing to increase the number of cycles. Also, increasing the number of cycles leads to an increase in the total lutein content as lutein content considerably increased in the latter cycles. A biomass productivity of around 2700 mg/L/d and lutein productivity of 7 mg/L/d were achieved after 4 or 5 cycles of integrated semi-batch operations in the two-stage strategy (Table 4). The productivity achieved in this study is the highest achieved for heterotrophic lutein production from microalgae reported so far, as per our earlier review on heterotrophic pigment production from microalgae (Table 1 in Hu et al., 2017).
M12 was investigated in batch culture. The C/N ratio was maintained at 10, while different glucose concentrations (5, 7.5, 10, 15, and 20 g/L) were used. Biomass yield of MB-1-M12 increased as the initial glucose concentration was increased from 5 to 10 g/L, while the highest glucose consumption rate and specific growth rate was obtained at a glucose concentration of 10 g/L. Further increase in the glucose concentration (above 10 g/L) did not lead to obvious improvement in biomass yield but caused an obvious lag phase along with a decreased specific growth rate and glucose consumption efficiency. Lutein content and productivity reached their maximal value of 3.79 mg/g and 2.07 mg/L/d at 5 and 7.5 g/L glucose, respectively, while they also decreased when the glucose concentration was above 10 g/L. Furthermore, when the glucose concentration was greater than 20 g/L, biomass productivity and yield rapidly dropped to 570 mg/L/d and 0.34 g biomass/g glucose, respectively, probably due to substrate inhibition. Substrate inhibition not only brings about slower growth rate but also results in a decrease in biomass concentration, yield and the production of target product (i.e., lutein in this case). The optimal substrate concentration varies with species and for the heterotrophic Chlorella protothecoides CS-41, 40 g/L glucose yielded higher biomass production and cellular lutein content of 18.4 g/L and 4.4 mg lutein/g cells, respectively, as indicated in a previous work (Shi et al., 1999). In this study, maximum biomass productivity (1113 mg/L/d) and maximum lutein productivity (2.07 mg/L/d) were obtained with 7.5 g/L of initial glucose concentration. The optimal initial glucose and urea concentrations for the heterotrophic lutein production with MB-1-M12 were found to be 7.5 and 0.75 g/L respectively.
3.7. Scale up of integrated fed-batch/semi-batch two-stage operations in a 5 L bioreactor
3.6. Integrated fed-batch/semi-batch two-stage operations for high efficient lutein production with MB-1-M12
After the successful employing the two-stage cultivation strategy for heterotrophic lutein production from MB-1-M12 in small-scale serum bottles (working volume = 1 L), the two-stage cultivation was performed in a 5 L bioreactor with a 3 L working volume to check the feasibility of scale-up fermentation. With better control of the operational conditions in fermenters, such as efficient oxygen transfer and mixing for homogeneous culture through mechanical agitation, the performance of heterotrophic culture might be further improved. Timecourse cell growth profile and fermentation characteristics of the 4 cycles in the two-stage process operated in bench-top fermenter are illustrated in Fig. 2. It can be seen that glucose consumption efficiency was higher in the fermenter compared to serum bottles, thus increasing amounts of glucose could be fed, leading to a maximal biomass accumulation of 25 g/L and after that the cell growth rate decreased. In addition, maximal biomass concentration could be reached in about 6 days in the first cycle, and then it decreased to about 4 days in the following cycles with the two-stage strategy, which dramatically enhanced biomass productivity from 2982 to 4805 mg/L/d. The lutein content reached about 5.5 mg/g biomass in each cycle, and high lutein productivity of 16.2 mg/L/d was obtained with the combination of high biomass production and lutein content in the fermenter. The results shown above clearly indicate that the two-stage cultivation strategy proposed in this study can be applied in fermenters for better performance of heterotrophic cultivation of MB-1-M12 for lutein production. The lutein content and lutein productivity achieved in this study are much higher than most reported values in the literature (Chan, 2012; Casal et al., 2011; Shi et al., 2000, 2002) and we also report for the first time a novel and effective two-stage cultivation integrating fed-batch/ semi-batch operation strategies suitable for heterotrophic lutein production with microalgae. In addition to lutein productivity, the integrated fed-batch/semi-batch two-stage strategy also had the lowest cost for lutein production, as the estimated costs per gram of lutein with different operation modes decrease in the order of batch ≫ fed-batch alone > semi-batch alone > two-stage strategies. Therefore, the twostage process proposed in this work is an excellent and affordable engineering strategy for lutein production by C. sorokiniana MB-1-M12. Experiments are under way for further scaling up of the cultivation
Previous studies show that heterotrophic Chlorella sp. could grow in up to 100 g/L glucose with compromised biomass levels, while 40 g/L seems to the optimal concentration in many cases reported. Since C. sorokiniana MB-1-M12 is a mutant of a photoautotrophic native strain, the tolerance for glucose was quite low, as growth inhibition started to occur when the glucose concentration was higher than 10 g/L. Hence, appropriate bioprocess engineering strategies, such as fed-batch and semi-batch fermentation modes, should be applied to enhance lutein production from MB-1-M12. A fed-batch fermentation experiment was conducted with glucose and nitrogen feeding with a fixed glucose concentration of 2 g/L but this operation did not significantly promote biomass growth when the experiment was performed in 1L serum bottles. A similar fed-batch strategy with the feed containing trace elements could significantly improve biomass content by 81% and improve the lutein concentration to 39.8 mg/L. However, a low biomass and lutein productivity was encountered. Since mineral nutrients was necessary in addition to carbon and nitrogen, a semi-batch strategy with the partial replacement of fresh medium (50%) was applied to increase lutein content and productivity. Higher biomass (7.91 g/L) and lutein productivity (4.56 mg/L/d) were obtained by using the semibatch strategy when compared to those obtained with batch and fedbatch fermentation modes (Table 4). The harvest time (medium replacement time) is a glucose depleted culture and since lutein accumulation also occurs at this stage, it resulted in lower lutein content (1.95 mg/g biomass) at each cycle. To combine the advantages of fed-batch and semi-batch operations and to avoid their drawbacks, a novel two-stage fed-batch/semi-batch operation strategy was devised to enhance both biomass and lutein productivity of MB-1-M12. In the first stage, the fed-batch fermentation (as described in the methods) was carried out to significantly increase biomass production until cell growth rate decreases. However, owing to lower lutein content at complete glucose exhaustion, the semi-batch system was applied as the second stage to the culture with maximal dry cell weight and glucose depletion to trigger lutein accumulation by supplying fresh medium and glucose. The second stage was carried out 145
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Table 4 Performance obtained from using different engineering strategies for enhanced lutein production from C. sorokiniana MB-1-M12. Cultivation mode
Batch Semi-batch (75% replacement ratio)
Fed-batch Fed-batch/semi-batch integrated with two-stage strategy (75% replacement ratio)
1st cycle 2nd cycle 3rd cycle 4th cycle 1st cycle 2nd cycle 3rd cycle 4th cycle 5th cycle
Dry weight (g/L)
Overall biomass productivity (mg/L/d)
Lutein content (mg/ g)
Lutein production of harvest (mg)
Lutein productivity (mg/L/d)
Total lutein accumulation (mg)
4.46 4.43 6.44 7.28 7.91 11.82 9.40 10.1 10.14 10.34 10.80
1497 1484 2365 2294 2219 980 1754 2581 2548 2726 2767
3.67 1.93 1.95 1.97 1.95 3.79 3.87 3.73 3.71 4.90 4.84
14.28 4.32 6.22 6.45 6.84 39.80 21.95 22.85 22.12 28.91 28.86
2.84 2.92 4.56 4.06 3.84 2.77 3.7 4.87 4.84 7.14 6.96
14.28 4.32 10.54 16.99 23.83 39.80 21.95 44.80 66.92 95.83 124.69
Fig. 1. Time course profile of (A) dry cell weight in the first stage, (B) residual glucose in first stage and (C) lutein content in the second stage in the cultivation of C. sorokiniana MB-1-M12 with two-stage strategy at 75% medium replacement ratio in 1 L serum bottles.
Fig. 2. Time course profile of (A) dry cell weight in the first stage, (B) residual glucose in first stage and (C) lutein content in the second stage in the cultivation of C. sorokiniana MB-1-M12 with two-stage strategy at 75% medium replacement ratio in 5 L fermenters.
volume to assess the feasibility of commercial production of microalgae-based lutein by using this approach.
integrating fed-batch and semi-batch modes. High lutein content (5.88 mg/g biomass) and lutein productivity (16.2 mg/L/d) were achieved with the two-stage strategy, which seems to be economically feasible for large-scale heterotrophic lutein production with the microalgal mutant used in this study.
4. Conclusions Chlorella sorokiniana MB-1-M12, a heterotrophic high lutein accumulating microalga, could grow well and accumulate lutein using glucose and urea as the carbon and nitrogen sources with maximal lutein content obtained at pH 7.5 and C/N ratio of 10. The lutein content and productivity were much improved when the fermentation mode was changed from batch mode to a novel two-stage process
Acknowledgements This work was supported by Taiwan’s Ministry of Science and Technology (MOST) under grant numbers of MOST 106-3113-E-006146
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011, 106-3113-E-006-004-CC2, 106-2621-M-006-007, 106-3114-E006-008, 104-2221-E-006-227-MY3, and 103-2221-E-006-190-MY3.
of microalgae for pigment production: a review. Biotechnol. Adv. Patton, J.C., Crouch, S., 1977. Spectrophotometric and Kinetics Investigation of the Berthelot Reaction for the Determination of Ammonia. [More sensitive, Precise Procedure Presented]. Krinsky, N.I., Landrum, J.T., Bone, R.A., 2003. Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye. Annu. Rev. Nutr. 23, 171–201. Lin, J.-H., Lee, D.-J., Chang, J.-S., 2015. Lutein production from biomass: Marigold flowers versus microalgae. Bioresour. Technol. 184 (Suppl. C), 421–428. Melo van Lent, D., Leermakers, E.T.M., Darweesh, S.K.L., Moreira, E.M., Tielemans, M.J., Muka, T., Vitezova, A., Chowdhury, R., Bramer, W.M., Brusselle, G.G., Felix, J.F., Kiefte-de Jong, J.C., Franco, O.H., 2016. The effects of lutein on respiratory health across the life course: a systematic review. Clin. Nutr. ESPEN 13, e1–e7. Pascal, A.A., Liu, Z., Broess, K., van Oort, B., van Amerongen, H., Wang, C., Horton, P., Robert, B., Chang, W., Ruban, A., 2005. Molecular basis of photoprotection and control of photosynthetic light-harvesting. Nature 436 (7047), 134–137. Perez-Garcia, O., Escalante, F.M.E., de-Bashan, L.E., Bashan, Y., 2011. Heterotrophic cultures of microalgae: metabolism and potential products. Water Res. 45 (1), 11–36. Qiu, R., Gao, S., Lopez, P.A., Ogden, K.L., 2017. Effects of pH on cell growth, lipid production and CO2 addition of microalgae Chlorella sorokiniana. Algal Res. 28 (Suppl. C), 192–199. Roberts, R.L., Green, J., Lewis, B., 2009. Lutein and zeaxanthin in eye and skin health. Clin. Dermatol. 27 (2), 195–201. Shi, X.-M., Liu, H.-J., Zhang, X.-W., Chen, F., 1999. Production of biomass and lutein by Chlorella protothecoides at various glucose concentrations in heterotrophic cultures. Process Biochem. 34 (4), 341–347. Shi, X., Wu, Z., Chen, F., 2006. Kinetic modeling of lutein production by heterotrophic Chlorella at various pH and temperatures. Mol. Nutr. Food Res. 50 (8), 763–768. Shi, X., Zhang, X., Chen, F., 2000. Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources. Enzyme Microb. Technol. 27 (3–5), 312–318. Shi, X.M., Jiang, Y., Chen, F., 2002. High-yield production of lutein by the green microalga Chlorella protothecoides in heterotrophic fed-batch culture. Biotechnol. Prog. 18 (4), 723–727. Theriault, R.J., 1965. Heterotrophic growth and production of xanthophylls by Chlorella pyrenoidosa. Appl. Microbiol. 13, 402–416. Wu, S.-T., Yu, S.-T., Lin, L.-P., 2005. Effect of culture conditions on docosahexaenoic acid production by Schizochytrium sp. S31. Process Biochem. 40 (9), 3103–3108. Zhang, J., Sun, Z., Sun, P., Chen, T., Chen, F., 2014. Microalgal carotenoids: beneficial effects and potential in human health. Food Funct. 5 (3), 413–425.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2018.01.027. References Bailey, J.E., Ollis, D.F., 1986. Biochemical Engineering Fundamentals, second ed. McGraw-Hill Publishers, New York, U.S.A. Britton, G., 1995. Structure and properties of carotenoids in relation to function. FASEB J. 9 (15), 1551–1558. Bumbak, F., Cook, S., Zachleder, V., Hauser, S., Kovar, K., 2011. Best practices in heterotrophic high-cell-density microalgal processes: achievements, potential and possible limitations. Appl. Microbiol. Biotechnol. 91 (1), 31–46. Casal, C., Cuaresma, M., Vega, J.M., Vilchez, C., 2011. Enhanced productivity of a luteinenriched novel acidophile microalga grown on urea. Mar. Drugs 9 (1), 29–42. Chan, M.C., 2012. Producing lutein from indigenous microalgae – optimization of microalgae cultivation extraction protocols storage conditions and mass production processes. In: Chemical Engineering, vol. MS. National Cheng Kung University, pp. 122. Chen, C.-Y., Ho, S.-H., Liu, C.-C., Chang, J.-S., 2017. Enhancing lutein production with Chlorella sorokiniana Mb-1 by optimizing acetate and nitrate concentrations under mixotrophic growth. J. Taiwan Inst. Chem. Eng. 79 (Suppl. C), 88–96. Collos, Y., Mornet, F., Sciandra, A., Waser, N., Larson, A., Harrison, P.J., 1999. An optical method for the rapid measurement of micromolar concentrations of nitrate in marine phytoplankton cultures. J. Appl. Phycol. 11 (2), 179–184. Del Campo, J.A., Garcia-Gonzalez, M., Guerrero, M.G., 2007. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl. Microbiol. Biotechnol. 74 (6), 1163–1174. Fernandez-Sevilla, J.M., Acien Fernandez, F.G., Molina Grima, E., 2010. Biotechnological production of lutein and its applications. Appl. Microbiol. Biotechnol. 86 (1), 27–40. Grobbelaar, J.U., 2007. Algal nutrition – mineral nutrition. In: Handbook of Microalgal Culture. , Blackwell Publishing Ltd, pp. 95–115. Hu, J., Nagarajan, D., Zhang, Q., Chang, J.-S., Lee, D.-J., 2017. Heterotrophic cultivation
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A highly efficient two-stage cultivation strategy for lutein production using heterotrophic culture of Chlorella sorokiniana MB-1-M12
T
Chun-Yen Chena, I-Chia Lub, Dillirani Nagarajanb,c, Chien-Hsiang Changb, I-Son Ngb, ⁎ Duu-Jong Leec, Jo-Shu Changb,d, a
University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan c Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan d 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
A B S T R A C T
Keywords: Lutein Microalgae Chlorella sorokiniana Heterotrophic growth Two-stage cultivation
A heterotrophic mutant of Chlorella sorokiniana MB-1-M12 was evaluated for its ability to produce lutein using organic carbon and nitrogen sources and without light irradiation. In batch fermentation, the maximal lutein content (3.67 mg lutein/g biomass) and productivity (2.84 mg/L/d) could be obtained when cultivated in BG-11 medium with 7.5 g/L glucose, 0.75 g/L urea, pH 7.5 and a C/N ratio of 10. A novel two-stage cultivation strategy that integrates fed-batch and semi-batch operations was applied to enhance the lutein production performance. When growing MB-1-M12 strain in a 5L fermenter using the optimal operation strategies, the maximum biomass concentration, biomass productivity, lutein content and lutein productivity could reach 25 g/L, 4.88 mg/L/d, 5.88 mg/g and 16.2 mg/L/d, respectively. This high lutein productivity could significantly reduce the cultivation time and the associated costs, indicating the potential of using MB-1-M12 strain for heterotrophic lutein production in commercial scale.
1. Introduction Lutein is a primary xanthophyll carotenoid, serving as light-harvesting antenna pigment and antioxidant in microalgae, plants and other photosynthetic organisms (Pascal et al., 2005). Other than light harvesting, the major function of lutein is to protect the photosystems from oxidative damage at high light intensities, especially blue light (Roberts et al., 2009). The structure of lutein is composed of a long conjugated double bond backbone chain with aromatic rings at either end. Lutein is lipo-soluble and is capable of scavenging free radicals and singlet oxygen, acting as a natural antioxidant (Britton, 1995). In humans, lutein along with zeaxanthin, accumulates in the macula of the eye, as the macular pigment and protects the retina from blue light and aids in improving visual acuity (Krinsky et al., 2003). Lutein has been implicated in human health mainly because of its ocular-protective activity and antioxidant property against neurodegenerative diseases, cardio-vascular diseases, diabetic retinopathy (Zhang et al., 2014) and respiratory health (Melo van Lent et al., 2016). However, humans are incapable of de novo synthesis of lutein and are strictly dependent on dietary intake to fulfil their lutein requirement. A daily dose of 5 mg is recommended for patients with Age related Macular Degeneration
⁎
(AMD), but the status of lutein as an essential nutrient has not been approved yet by pharmaceutical authorities (Fernandez-Sevilla et al., 2010). Other than this, lutein has also been used as colorant and food additive for human consumption (known as colorant E161b in the European Union) and also as a feed additive to deepen the yellow color of egg yolks (Lin et al., 2015). With all these applications, market demand for lutein is increasing and in 2015 the global lutein market was estimated at US$ 135 million and will continue to rise (Global Market Insights Lutein Market Report, 2016). Currently, the market demand for lutein is met by the extraction of lutein from the bright yellow petals of the marigold flowers of the genus Tagetes (Fernandez-Sevilla et al., 2010). However, lutein extraction from Tagetes petals are challenged by the seasonal availability of the flowers, large requirement of land for the cultivation of the plants, requirement of skilled labor and the very low lutein content present in the flower petals (Lin et al., 2015). Lutein primarily exists in mono- and diesterified form in marigold flowers (Del Campo et al., 2007), and chemical saponification is required to extract lutein from them. Microalgae are a potential source of lutein and the advantages are: i) high cellular lutein content at 0.5–1.2% by weight, ii) unaffected by seasonal variation as in the case of Tagetus, iii) very low land and water
Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan. E-mail address: [email protected] (J.-S. Chang).
https://doi.org/10.1016/j.biortech.2018.01.027 Received 9 December 2017; Received in revised form 30 December 2017; Accepted 5 January 2018
Available online 06 January 2018 0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
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2.2. Determination of microalgal biomass dry cell weight, biomass productivity and specific growth rate
requirements, and iv) the possibility to generate other high value products from the microalgal biomass (Lin et al., 2015). However, the major bottleneck in the mass production of microalgae is the supply of optimal light intensity, which is the prime criteria to stimulate pigment production in photoautotrophic cultures. As a result, effective new photobioreactor designs are being implemented to cope with this issue; however, this usually causes a significant increase in the product costs. Therefore, in contrast to phototrophic culture systems, heterotrophic cultivation of microalgae for the mass production of high-value products has been considered an economically feasible and commercially favorable option since the heterotrophic growth could attain much higher microalgal biomass productivity without the need of light supply and CO2 aeration (Hu et al., 2017). It is easier to control the product quality in a heterotrophic culture due to being operated in a wellcontrol closed system. Also, since the growth and metabolism of microalgae occurs in dark conditions for heterotrophic cultures, conventional bacterial fermenters can be used for microalgal cultures without much modifications (Bumbak et al., 2011). Although microalgae are genetically capable of utilizing organic carbon sources and are endowed with a central carbon catabolic pathway, the predisposition for heterotrophy is mainly strain dependent (Perez-Garcia et al., 2011). The other potential problem associated with producing pigments (e.g., lutein) under heterotrophic conditions would be the absence of light, as light illumination is usually required for promoting the production of pigments (Hu et al., 2017). Therefore, selecting a suitable heterotrophic strain for efficient lutein production under optimal operating conditions would be a pivotal step to realize the idea of producing microalgal lutein heterotrophically. In this study, a heterotrophic Chlorella sorokiniana MB-1-M12, was examined for its effectiveness in lutein production and the culture conditions were optimized to achieve the maximal lutein producing performance. In particular, a novel two-stage cultivation strategy was devised to further improve the lutein productivities to make it suitable for industrial scale applications.
A 5 mL of cell suspension was collected from the bioreactor, and was centrifuged at 10,000 rpm for 4 min to remove the culture medium. Then, the sample was washed twice with 5 mL of deionized water and transferred to an aluminum pan. The aluminum pan was placed in an infrared moisture determination balance (FD-720, KETT, Japan) at 100 °C until all the water was removed. After drying, the dry cell weight (DW) was determined as the weight difference between the pan with and without the microalgal cells. The biomass productivity (Pbiomass, mg DCW/L/d) and specific growth rate (μ) were calculated from the following equation (Bailey and Ollis, 1986):
Biomass productivity (mg DCW/L/d) = Pbiomass =
ΔX X −X = t initial Δt t (1)
d lnX ⎞ Specific growth rate (d−1) = ⎛ ⎝ dt ⎠max
(2)
where X is dry cell weight (g/L), ΔX (mg/L) is the variation of dry cell weight and Δt is the cultured time (d). 2.3. Determination of nutrient concentration The nitrate concentration was determined using the spectrophotometric method described earlier (Collos et al., 1999). In general, the liquid samples were collected from the bioreactor and filtered via a 0.22 μm pore size filter. After filtration, samples were diluted with deionized water as determined by measuring the optical density (OD) of the samples at a wavelength of 220 nm (denoted as OD220) using a UV/ Vis spectrophotometer (model U-2001, Hitachi, Tokyo, Japan). A standard curve was generated using pure sodium nitrate (obtained from Sigma Chemical Co.), and the concentrations of nitrate in the samples were calculated. The ammonium concentration of the culture was analyzed by Berthelot’s reaction (Patton and Crouch, 1977). A 250 μL deionized water, 500 μL phenol nitroprusside solution (SIGMA) and 500 μL 0.2% alkaline hypochlorite solution (SIGMA) were added into 50 μL diluted liquid sample obtained from the bioreactor and the mixture was incubated at room temperature for 30 min. Then, the optical density of the sample was measured at a wavelength of 630 nm by a UV/Vis spectrophotometer (model U-2001, Hitachi, Tokyo, Japan). A standard curve was generated using pure ammonium chloride (obtained from Sigma Chemical Co.), and the concentrations of ammonium in the samples were calculated. The urea concentration of the medium was analyzed by hydrolysis of urea with urease, followed by estimation of the ammonia released by the Berthelot’s reaction. Briefly, 50 μL filtered and appropriately diluted liquid sample from the bioreactor was added to 250 μL 1% urease solution (SIGMA) and the mixture was incubated at 37 °C for 10 min. Next, 500 μL phenol nitroprusside solution (SIGMA) and 500 μL 0.2% alkaline hypochlorite solution (SIGMA) was added and the mixture was incubated at room temperature for 30 min. the optical density of the sample was measured at a wavelength of 630 nm by a UV/Vis spectrophotometer (model U-2001, Hitachi, Tokyo, Japan) for measuring ammonia concentration as described previously. Urea concentration was calculated from the calibration curve obtained using standard urea solution. The concentrations of glucose, acetate and glycerol were determined by high-performance liquid chromatography (HPLC) equipped with a refraction index detector (RID-10A, Shimadzu, Japan) and an ICSep ICE-COREGEL 87H3 column (Transgenomic, USA). The mobile phase was 0.008 N H2SO4, which was eluted at 0.4 mL/min at room temperature.
2. Materials and methods 2.1. Microalga used and its culture conditions Chlorella sorokiniana MB-1-M12, is a glucose tolerant mutant of C. sorokiniana MB-1, which is originally a photoautotrophic lutein-rich strain isolated in southern Taiwan. C. sorokiniana MB-1-M12 possesses a high lutein content and productivity under heterotrophic conditions, making it a potential candidate for mass production of lutein. The MB1-M12 strain was cultivated on BG-11 medium consisting of (mg/L): NaNO3, 1.0; Na2CO3, 20; CaCl2·2H2O, 36; Citric acid, 6.0; MgSO4·7H2O, 75; K2HPO4, 40; Ferric ammonium citrate, 6.0; EDTA·2Na, 1.0; H3BO3, 2.86; MnCl2·4H2O, 1.81; CuSO4·5H2O, 0.079; ZnSO4·7H2O, 0.222; Na2MoO4·2H2O, 0.39; Co(NO3)2·6H2O, 0.049. All experiments were conducted in serum bottle cultures containing 1000 mL sterilized medium (120 °C, 20 min) at 27 ± 1 °C with continuous stirring (300 rpm of agitation rate) and the said reactors were covered with aluminum foil to avoid light penetration. Glucose, acetate and glycerol were evaluated as carbon source (at a concentration of 0.08 mol/L) for growth and the metabolism of MB-1M12 in dark conditions. Four nitrogen sources, namely, sodium nitrate (NaNO3), potassium nitrate (KNO3), ammonium chloride (NH4Cl) and urea (CO(NH2)2), at a concentration of 0.01 mol/L, were evaluated for their effect on lutein content and productivity of MB-1-M12. The effect of C/N ratio on lutein content and productivity of MB-1-M12 was also determined by testing five different C/N ratios (3, 5, 10, 15, 20 for glucose versus urea). The optimal pH for growth and lutein production was determined after culturing the microalgal strain at pH 6.5, 7, 7.5, 8, 8.5.
2.4. Determination of lutein content The lutein content of microalgae was estimated as described 142
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Table 1 The effect of carbon sources on biomass, lutein content, yield, and productivity of C. sorokiniana MB-1-M12 under heterotrophic cultivation. Carbon source
Dry weight (g/L)
Biomass productivity (mg/L/d)
Yield (g DW/g substrate)
Lutein content (mg/g)
Lutein productivity (mg/L/d)
Glucose Acetate Glycerol
1.20 ± 0.09 0.68 ± 0.06 N.A.
392 ± 14.04 190 ± 12.16 N.A.
0.45 0.19 N.A.
2.88 ± 0.25 3.06 ± 0.28 N.A.
0.85 ± 0.13 0.47 ± 0.18 N.A.
designated intervals.
previously (Chen et al., 2017). First, 10 mg of lyophilized microalgal biomass and 0.5 g glass beads were added to 1 mL of 60% w/w KOH solution to promote cell disruption by bead-beating. After cell disruption, the mixture was placed in a water bath at 40 °C for 40 min for converting esterified form of lutein to free form completely and lutein was extracted with diethyl ether. The supernatant phase was separated by centrifugation and collected until it was clear. Lutein in solvent was purged with nitrogen gas, the precipitate was re-dissolved in acetone, and lutein content was measured by HPLC equipped with a photodiode array detector (PDA) and a YMC Carotenoid RP-30 column with a particle size of 5 μm and a column size of 4.6 × 250 mm (YMC Schermbeck, Germany). The temperatures of column and tray were set at 30 °C and 4 °C, respectively. The mobile phase contained 10% (v/v) tetrahydrofuran (THF) and 90% (v/v) methanol, and the extracts were eluted at a flow rate of 1.5 mL/min. Lutein was detected by measuring the absorbance at the wavelength range of 220–750 nm, and the maximal absorbance at 450 nm was used for quantification of lutein content. The lutein standards for quantification of lutein were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The lutein content (mg/ g) was obtained from the following equation:
Lutein content (mg/g DCW) Lutein concentration (mg/L) × Volume of solution (L) = Microalgal biomass (g)
3. Results and discussion Chlorella sorokiniana MB-1-M12 is capable of heterotrophic growth under dark conditions using glucose as the carbon source. Preliminary experiments showed that optimal growth of the MB-1-M12 strain was obtained with BG-11 medium, while other frequently used media (such as Basal medium and Bold’s basal medium) did not provide optimal growth (data not shown). Therefore, BG-11 medium was used for the experiments investigating the optimal carbon source, nitrogen source, pH and the C/N ratio as well as the determination of optimal culture conditions. The results are presented in the following sections. 3.1. Effect of different carbon sources on growth and lutein production of MB-1-M12 Glucose is the preferred carbon source in most heterotrophic microalgae, while acetate and glycerol have also been variably used depending on the tolerance and metabolic capacity of the cultivated strains. The effect of glucose, glycerol and acetate on biomass growth and lutein production from MB-1 M12 has been evaluated with 1 g/L sodium nitrate as the nitrogen source and the tested carbon source at 80 mM. The results are summarized in Table 1. C. sorokiniana MB-1M12 is incapable of utilizing glycerol as the sole carbon source for growth. Using glucose as the carbon source exhibited much better maximum biomass concentration (1.20 g/L), biomass productivity (392 mg/L/d) and cell growth yield coefficient of (0.45 g biomass/ g substrate) when compared with using acetate, whereas cultivating MB-1-M12 on acetate resulted in a slightly higher lutein content than that obtained with glucose (3.0 mg/g lutein from acetate vs. 2.88 mg/g lutein from glucose). Nevertheless, using glucose still showed a higher lutein productivity due mainly to a higher biomass productivity when compared to using acetate. It is also known that glucose has higher energy content compared to acetate (2.8 kJ/mol vs. 0.8 kJ/mol). Considering lutein productivity as the most important parameter from practical application aspect, glucose was chosen as the optimal carbon source for lutein production with MB-1-M12. Previous work on lutein production with heterotrophic Chlorella sp. also showed that using glucose as the carbon source to grow C. protothecoides in large scale cultures could obtain a lutein content of as high as 5 mg/g (Shi et al., 2002).
(3)
The lutein productivity (Plutein, mg/L/d) was obtained from the following equation:
Plutein (mg/L/d) = Lutein content (mg/g DCW) × Pbiomass (g DCW/L/d) (4) 2.5. Operation of two-stage cultivation strategy for enhanced lutein production from MB-1-M12 under heterotrophic conditions All experiments were performed in 1-L serum bottles with BG-11 medium containing 7.5 g/L initial glucose concentration, 0.75 g/L initial urea concentration, pH 7.5, 0.3 vvm aeration rate under dark heterotrophic conditions. An integrated fed-batch/semi-batch strategy was applied for enhancing lutein production. The experiment was started in a fed-batch mode to accumulate maximum biomass. A concentrated feed solution containing 500 g/L glucose and 50 g/L urea was prepared and when the residual glucose concentration was below 2 g/L, the feed solution was fed into the bioreactor to keep the glucose concentration in the range of 2.0–7.5 g/L and maintain sufficient nitrogen source for growth. After maximal biomass was accumulated, the fermentation was switched to semi-batch mode and when the glucose in medium was completely depleted by MB-1M12, 50% of the culture broth (volume of 1 L) was removed and 500 mL of fresh medium was added into the cultivation in order to restart a new cycle of growth. The scale-up fermentation of the fed-batch/semi-batch integrated two-stage operation strategy for lutein production from MB-1-M12 was carried out in a 5L stirred tank fermenter with a working volume of 3 L. The fermentation conditions were maintained at 25 ± 1 °C, 100 rpm of agitation rate, 0.3 vvm of air aeration rate (giving a mass transfer coefficient (kLa) of 12 ± 1.5 h−1) and pH 7.5 ± 0.1. The pH of culture was maintained by periodic addition of 1 N HCl or 1 N NaOH with a pH controller. The samples were collected to measure microalgal cell growth, glucose concentration, urea concentration and lutein content at
3.2. Effect of culture pH on growth and lutein production of MB-1-M12 The pH of culture medium is a significant parameter for cell growth and lutein accumulation of microalgal strains, and the optimal condition of pH for the algal growth and lutein production is highly strain dependent. C. sorokiniana species could grow in the pH range from 5.0 to 9.0 (Qiu et al., 2017). In heterotrophic microalgal cultures where pH is uncontrolled or the medium pH is towards the acidic range, the culture becomes more acidic due to the oxidative assimilation of glucose and the release of metabolic acids such as citric acid, pyruvic acid and malic acid (Wu et al., 2005). Hence, MB-1-M12 was grown in medium with different pH values of 6.5, 7, 7.5, 8, and 8.5, while pH uncontrolled cultures served as a control. The results are summarized in Table 2. The results show that there was no significant difference in 143
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reduced energy requirement for its uptake and metabolism (Grobbelaar, 2007; Shi et al., 2000). In contrast, nitrate requires more energy for transport into cells and further conversion to ammonia (Perez-Garcia et al., 2011). Our lutein production results show no significant difference in lutein content for the cultures grown with nitrate and ammonium, but a relatively lower level of lutein content were obtained when urea was used as a nitrogen source. However, when ammonia is used as a sole nitrogen source in pH uncontrolled cultures, the medium pH would drop significantly during algal growth due to the release of H+ ions during the metabolism of ammonium (Grobbelaar, 2007), and the sudden pH drift is often lethal to cells. It was observed that after cultivation for 2 days, the culture pH dropped to below 3.5 when using ammonium as the nitrogen source. With respect to cost, for an equivalent nitrogen concentration, the order of nitrogen cost for lutein production with various nitrogen sources is as follows: in declining order, urea < ammonium < nitrate. When urea replaces nitrate as nitrogen source, the cost of nitrogen source significantly decreased from 445.2 to 70.9 NTD/g lutein (NTD denotes new Taiwan dollar), achieving a cost reduction of 84%. It is clear that urea is an economical and effective nitrogen source for MB-1-M12, compared to ammonium and nitrate for lutein production. Therefore, with respect to biomass concentration, productivity and cost, urea was chosen as the suitable nitrogen source for C. sorokiniana MB-1-M12. Similar results were obtained for Chlorella protothecoides CS-41, where urea proved to be a better nitrogen source, compared to nitrate and ammonium. They found that even though all the above-mentioned three nitrogen sources yielded similar biomass, lutein content was considerably higher when urea was used as a nitrogen source (2 mg lutein/g glucose) (Shi et al., 2000).
Table 2 The effect of culture medium pH on biomass, lutein content, yield, and productivity of C. sorokiniana MB-1-M12 under heterotrophic cultivation. pH
Dry weight (g/L)
Biomass productivity (mg/L/d)
Lutein content (mg/g)
Lutein productivity (mg/L/d)
6.5 7.0 7.5 8.0 8.5 Control
2.35 2.32 2.39 2.32 2.32 2.27
423 584 651 641 599 583
2.55 2.79 3.38 3.34 2.98 2.98
0.88 1.06 1.27 1.25 1.16 1.14
± ± ± ± ± ±
0.11 0.11 0.13 0.12 0.14 0.13
± ± ± ± ± ±
19 22 23 26 25 24
± ± ± ± ± ±
0.12 0.11 0.15 0.14 0.13 0.11
± ± ± ± ± ±
0.03 0.04 0.05 0.05 0.03 0.03
biomass concentration with different pH, but lutein content and productivity differed considerably. The highest biomass productivity (650.58 mg/L/d) and lutein content (3.42 mg/g) were obtained at a pH of 7.5. In addition, there was a dramatic decrease in biomass productivity and lutein content when the pH decreased to 6.5. The slightly acidic pH 6.5 severely inhibited biomass growth (423.01 mg/L/d) and lutein accumulation (2.55 mg/g). The pH drop in microalgal cultures is generally accompanied by the destruction of the carotenoids and chlorophylls (Theriault, 1965). The alkaline pH of 8.5 slightly inhibited microalgal growth with a biomass productivity and lutein content of 599.0 mg/L/d and 2.98 mg/g, respectively. Therefore, pH 7.5 was chosen as the optimal pH for lutein production from MB-1-M12. In a related study, (Shi et al., 2006) reported that C. protothecoides CS-41 was capable of heterotrophic growth and lutein production, and pH 6.6 yielded the highest lutein content and biomass productivity of 4.75 mg/ g cells and 77.9 mg/L, respectively. Alkaline pH of 8 inhibited biomass growth of C. protothecoides CS-41, which is similar to our observation.
3.4. Effect of urea concentration and C/N ratio on growth and lutein production of MB-1-M12
3.3. Effect of different nitrogen sources on growth and lutein production of MB-1-M12
The effect of urea concentration (along with an initial glucose concentration of 5 g/L) on the growth and lutein production from MB-1M12 was investigated in batch fermentation. Nitrogen is an essential macro nutrient for growth and biomass yield in microalgae and optimal nitrogen source concentration is crucial as lower nitrogen levels might decrease the lutein yield. Five different C/N ratios (3, 5, 10, 15, 20 for glucose versus urea) in the culture medium were examined for their effects on the lutein content and productivity. As shown in Table 3, there was no significant difference in biomass concentration and biomass productivity when different urea concentrations were used, while a marked decrease in lutein content (below 3 mg/g) was observed when the C/N ratio was greater than 10 (Table 3). Lutein content markedly decreased when nitrogen is insufficient (or when the C/N ratio is high). At C/N ratios of 15 and 20 with the same glucose concentration of 5 g/ L, nitrogen limitation occurs resulting in a lower lutein content. A lower C/N ratio and sufficient nitrogen supply could ensure higher lutein content, but when the C/N ratio is too low (e.g., lower than 10), lutein formation in the microalgae also slightly decreases. Thus, there is an optimal C/N ratio of 5, at which the lutein content and lutein productivity could reach the highest level of 3.76 mg/g of 2.12 mg/L/d, respectively. However, considering the cost for nitrogen in large scale cultures, the C/N ratio of 10 that gives the second best lutein producing performance was selected for lutein production with MB-1-M12 in the following experiments.
Nitrogen is an important macronutrient for the growth and metabolism of microalgal cells. Microalgae are able to utilize a variety of nitrogen sources, such as ammonia, nitrate, urea, and amino acids (Perez-Garcia et al., 2011). The effect of four various nitrogen sources, sodium nitrate (NaNO3), potassium nitrate (KNO3), ammonium chloride (NH4Cl) and urea (CO(NH2)2) on the production of lutein by MB-1-M12 under heterotrophic conditions was investigated. The concentration of nitrogen source in the medium was maintained at 10 mM, along with 5 g/L glucose, pH 7.5. The results are shown in Table 3. Nitrate has been used as a common nitrogen source for the cultivation of Chlorella spp. in BG-11 or BM medium. However, Table 3 shows that higher maximum biomass concentration was obtained with ammonium chloride and urea at 2.78 and 2.83 g/L dry cells, respectively. The efficacy of the various nitrogen sources with respect to biomass concentration and productivity were, in declining order: urea > ammonium > nitrate. C. sorokiniana MB-1-M12 preferentially absorbed ammonium and urea, and the highest lutein productivity was achieved with ammonia (2.11 mg/L/d). Ammonium is considered as the most preferred and suitable nitrogen source for algae growth due to Table 3 The effect of C/N ratio on biomass, lutein content, yield, and productivity of C. sorokiniana MB-1-M12 under heterotrophic cultivation. C/N ratio*
Urea (g/L)
Biomass productivity (mg/ L/d)
Lutein content (mg/g)
Lutein productivity (mg/L/d)
3.5. Effect of glucose concentration on the growth and lutein production of MB-1-M12
20 15 10 5 3
0.25 0.35 0.50 1.00 1.66
957 936 935 928 942
1.73 2.97 3.64 3.76 3.13
1.31 1.91 2.10 2.12 1.57
In order to achieve high biomass production in batch culture, it is required to use high initial carbon source concentration (i.e., glucose), and hence it is essential to determine the optimal glucose concentration without generating substrate inhibition. The effect of initial glucose and urea concentration on biomass growth and lutein production of MB-1-
± ± ± ± ±
43 38 37 42 41
± ± ± ± ±
0.09 0.12 0.13 0.13 0.11
± ± ± ± ±
0.08 0.07 0.11 0.12 0.07
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at pH 7.5 ± 0.1, 27 ± 1 °C and 0.3 vvm aeration rate under continuous stirring (300 rpm of agitation rate) in the dark. The remains of the first stage served as inoculum and fresh BG-11 medium, concentrated glucose solution and concentrated urea solution (500 g/L glucose and 50 g/L urea, respectively) were added for the new semibatch cycle. Based on our previous results, it was shown that the culture reached a biomass concentration of 10 g/L with two cycles of fed-batch cultivation and after that glucose consumption and biomass accumulation decreases. Therefore, one replacement of culture was conducted every two cycles of fed-batch culture (in 1-L serum bottles) in the twostage strategy and the results are shown in Fig. 1. The two stage strategy was conducted with three different media replacement ratios (25%, 50%, 75%) was conducted and best results were shown for 75% medium replacement in the second stage semi-batch mode. The twostage strategy with 75% medium replacement ratio significantly increased the growth rate decreasing the operation time, allowing to increase the number of cycles. Also, increasing the number of cycles leads to an increase in the total lutein content as lutein content considerably increased in the latter cycles. A biomass productivity of around 2700 mg/L/d and lutein productivity of 7 mg/L/d were achieved after 4 or 5 cycles of integrated semi-batch operations in the two-stage strategy (Table 4). The productivity achieved in this study is the highest achieved for heterotrophic lutein production from microalgae reported so far, as per our earlier review on heterotrophic pigment production from microalgae (Table 1 in Hu et al., 2017).
M12 was investigated in batch culture. The C/N ratio was maintained at 10, while different glucose concentrations (5, 7.5, 10, 15, and 20 g/L) were used. Biomass yield of MB-1-M12 increased as the initial glucose concentration was increased from 5 to 10 g/L, while the highest glucose consumption rate and specific growth rate was obtained at a glucose concentration of 10 g/L. Further increase in the glucose concentration (above 10 g/L) did not lead to obvious improvement in biomass yield but caused an obvious lag phase along with a decreased specific growth rate and glucose consumption efficiency. Lutein content and productivity reached their maximal value of 3.79 mg/g and 2.07 mg/L/d at 5 and 7.5 g/L glucose, respectively, while they also decreased when the glucose concentration was above 10 g/L. Furthermore, when the glucose concentration was greater than 20 g/L, biomass productivity and yield rapidly dropped to 570 mg/L/d and 0.34 g biomass/g glucose, respectively, probably due to substrate inhibition. Substrate inhibition not only brings about slower growth rate but also results in a decrease in biomass concentration, yield and the production of target product (i.e., lutein in this case). The optimal substrate concentration varies with species and for the heterotrophic Chlorella protothecoides CS-41, 40 g/L glucose yielded higher biomass production and cellular lutein content of 18.4 g/L and 4.4 mg lutein/g cells, respectively, as indicated in a previous work (Shi et al., 1999). In this study, maximum biomass productivity (1113 mg/L/d) and maximum lutein productivity (2.07 mg/L/d) were obtained with 7.5 g/L of initial glucose concentration. The optimal initial glucose and urea concentrations for the heterotrophic lutein production with MB-1-M12 were found to be 7.5 and 0.75 g/L respectively.
3.7. Scale up of integrated fed-batch/semi-batch two-stage operations in a 5 L bioreactor
3.6. Integrated fed-batch/semi-batch two-stage operations for high efficient lutein production with MB-1-M12
After the successful employing the two-stage cultivation strategy for heterotrophic lutein production from MB-1-M12 in small-scale serum bottles (working volume = 1 L), the two-stage cultivation was performed in a 5 L bioreactor with a 3 L working volume to check the feasibility of scale-up fermentation. With better control of the operational conditions in fermenters, such as efficient oxygen transfer and mixing for homogeneous culture through mechanical agitation, the performance of heterotrophic culture might be further improved. Timecourse cell growth profile and fermentation characteristics of the 4 cycles in the two-stage process operated in bench-top fermenter are illustrated in Fig. 2. It can be seen that glucose consumption efficiency was higher in the fermenter compared to serum bottles, thus increasing amounts of glucose could be fed, leading to a maximal biomass accumulation of 25 g/L and after that the cell growth rate decreased. In addition, maximal biomass concentration could be reached in about 6 days in the first cycle, and then it decreased to about 4 days in the following cycles with the two-stage strategy, which dramatically enhanced biomass productivity from 2982 to 4805 mg/L/d. The lutein content reached about 5.5 mg/g biomass in each cycle, and high lutein productivity of 16.2 mg/L/d was obtained with the combination of high biomass production and lutein content in the fermenter. The results shown above clearly indicate that the two-stage cultivation strategy proposed in this study can be applied in fermenters for better performance of heterotrophic cultivation of MB-1-M12 for lutein production. The lutein content and lutein productivity achieved in this study are much higher than most reported values in the literature (Chan, 2012; Casal et al., 2011; Shi et al., 2000, 2002) and we also report for the first time a novel and effective two-stage cultivation integrating fed-batch/ semi-batch operation strategies suitable for heterotrophic lutein production with microalgae. In addition to lutein productivity, the integrated fed-batch/semi-batch two-stage strategy also had the lowest cost for lutein production, as the estimated costs per gram of lutein with different operation modes decrease in the order of batch ≫ fed-batch alone > semi-batch alone > two-stage strategies. Therefore, the twostage process proposed in this work is an excellent and affordable engineering strategy for lutein production by C. sorokiniana MB-1-M12. Experiments are under way for further scaling up of the cultivation
Previous studies show that heterotrophic Chlorella sp. could grow in up to 100 g/L glucose with compromised biomass levels, while 40 g/L seems to the optimal concentration in many cases reported. Since C. sorokiniana MB-1-M12 is a mutant of a photoautotrophic native strain, the tolerance for glucose was quite low, as growth inhibition started to occur when the glucose concentration was higher than 10 g/L. Hence, appropriate bioprocess engineering strategies, such as fed-batch and semi-batch fermentation modes, should be applied to enhance lutein production from MB-1-M12. A fed-batch fermentation experiment was conducted with glucose and nitrogen feeding with a fixed glucose concentration of 2 g/L but this operation did not significantly promote biomass growth when the experiment was performed in 1L serum bottles. A similar fed-batch strategy with the feed containing trace elements could significantly improve biomass content by 81% and improve the lutein concentration to 39.8 mg/L. However, a low biomass and lutein productivity was encountered. Since mineral nutrients was necessary in addition to carbon and nitrogen, a semi-batch strategy with the partial replacement of fresh medium (50%) was applied to increase lutein content and productivity. Higher biomass (7.91 g/L) and lutein productivity (4.56 mg/L/d) were obtained by using the semibatch strategy when compared to those obtained with batch and fedbatch fermentation modes (Table 4). The harvest time (medium replacement time) is a glucose depleted culture and since lutein accumulation also occurs at this stage, it resulted in lower lutein content (1.95 mg/g biomass) at each cycle. To combine the advantages of fed-batch and semi-batch operations and to avoid their drawbacks, a novel two-stage fed-batch/semi-batch operation strategy was devised to enhance both biomass and lutein productivity of MB-1-M12. In the first stage, the fed-batch fermentation (as described in the methods) was carried out to significantly increase biomass production until cell growth rate decreases. However, owing to lower lutein content at complete glucose exhaustion, the semi-batch system was applied as the second stage to the culture with maximal dry cell weight and glucose depletion to trigger lutein accumulation by supplying fresh medium and glucose. The second stage was carried out 145
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Table 4 Performance obtained from using different engineering strategies for enhanced lutein production from C. sorokiniana MB-1-M12. Cultivation mode
Batch Semi-batch (75% replacement ratio)
Fed-batch Fed-batch/semi-batch integrated with two-stage strategy (75% replacement ratio)
1st cycle 2nd cycle 3rd cycle 4th cycle 1st cycle 2nd cycle 3rd cycle 4th cycle 5th cycle
Dry weight (g/L)
Overall biomass productivity (mg/L/d)
Lutein content (mg/ g)
Lutein production of harvest (mg)
Lutein productivity (mg/L/d)
Total lutein accumulation (mg)
4.46 4.43 6.44 7.28 7.91 11.82 9.40 10.1 10.14 10.34 10.80
1497 1484 2365 2294 2219 980 1754 2581 2548 2726 2767
3.67 1.93 1.95 1.97 1.95 3.79 3.87 3.73 3.71 4.90 4.84
14.28 4.32 6.22 6.45 6.84 39.80 21.95 22.85 22.12 28.91 28.86
2.84 2.92 4.56 4.06 3.84 2.77 3.7 4.87 4.84 7.14 6.96
14.28 4.32 10.54 16.99 23.83 39.80 21.95 44.80 66.92 95.83 124.69
Fig. 1. Time course profile of (A) dry cell weight in the first stage, (B) residual glucose in first stage and (C) lutein content in the second stage in the cultivation of C. sorokiniana MB-1-M12 with two-stage strategy at 75% medium replacement ratio in 1 L serum bottles.
Fig. 2. Time course profile of (A) dry cell weight in the first stage, (B) residual glucose in first stage and (C) lutein content in the second stage in the cultivation of C. sorokiniana MB-1-M12 with two-stage strategy at 75% medium replacement ratio in 5 L fermenters.
volume to assess the feasibility of commercial production of microalgae-based lutein by using this approach.
integrating fed-batch and semi-batch modes. High lutein content (5.88 mg/g biomass) and lutein productivity (16.2 mg/L/d) were achieved with the two-stage strategy, which seems to be economically feasible for large-scale heterotrophic lutein production with the microalgal mutant used in this study.
4. Conclusions Chlorella sorokiniana MB-1-M12, a heterotrophic high lutein accumulating microalga, could grow well and accumulate lutein using glucose and urea as the carbon and nitrogen sources with maximal lutein content obtained at pH 7.5 and C/N ratio of 10. The lutein content and productivity were much improved when the fermentation mode was changed from batch mode to a novel two-stage process
Acknowledgements This work was supported by Taiwan’s Ministry of Science and Technology (MOST) under grant numbers of MOST 106-3113-E-006146
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011, 106-3113-E-006-004-CC2, 106-2621-M-006-007, 106-3114-E006-008, 104-2221-E-006-227-MY3, and 103-2221-E-006-190-MY3.
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