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Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3
Bioresource Technology xxx (2014) xxx–xxx
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Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3 Shih-Hsin Ho a, Youping Xie b, Ming-Chang Chan c, Chen-Chun Liu d, Chun-Yen Chen d, Duu-Jong Lee e, Chieh-Chen Huang f, Jo-Shu Chang c,d,g,⇑ a
Organization of Advanced Science and Technology, Kobe University, Kobe 657-8501, Japan College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, PR China Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan d Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan e Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan f Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan g Research Center for Energy Technology and Strategy Center, National Cheng Kung University, Tainan 701, Taiwan b c
h i g h l i g h t s N source/concentration were optimized for better lutein production from microalgae. The light intensity shift strategy enhanced lutein content of S. obliquus FSP-3. Semi-continuous operation improved the productivity of lutein from microalgae. The highest lutein productivity of 6.01 mg/L/day is high than most reported values.
a r t i c l e
i n f o
Article history: Received 9 September 2014 Received in revised form 12 October 2014 Accepted 13 October 2014 Available online xxxx Keywords: Microalgae Scenedesmus obliquus Lutein Light intensity shift Bioreactor strategy
a b s t r a c t In this study, the effects of the type and concentration of nitrogen sources on the cell growth and lutein content of an isolated microalga Scenedesmus obliquus FSP-3 were investigated. With batch culture, the highest lutein content (4.61 mg/g) and lutein productivity (4.35 mg/L/day) were obtained when using 8.0 mM calcium nitrate as the nitrogen source. With this best nitrogen source condition, the microalgae cultivation was performed using two bioreactor strategies (namely, semi-continuous and two-stage operations) to further enhance the lutein content and productivity. Using semi-continuous operation with a 10% medium replacement ratio could obtain the highest biomass productivity (1304.8 mg/L/day) and lutein productivity (6.01 mg/L/day). This performance is better than most related studies. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Lutein, an important photosynthetic pigment present in plants and phototrophic microorganisms, belongs to the xanthophyllic pigment groups (Fernandez-Sevilla et al., 2010). Lutein is extremely necessary to support the life of photoautotrophs owing to its role in assisting photosynthesis and photo-protection (Demmig-Adams and Adams, 1996). Lutein has many functions for the protection of the human’s health. It can prevent cardiovascular diseases
⇑ Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan. Tel.: +886 6 2757575x62651; fax: +886 6 2357146. E-mail address: [email protected] (J.-S. Chang).
(Dwyer et al., 2001), age-related macular degeneration (Chiu and Taylor, 2007; Granado et al., 2003), and some types of cancers (Astorg, 1997). For the multiple curing effects of human life, lutein is largely consumed and listed as E161b for food additives with the market size of nearly $150 million in the US and its market size is still expanding (Ceron et al., 2008). Nowadays, the main lutein feedstock for commercial lutein production focuses on the marigold petals. However, the major hurdle of using marigold petals for lutein production is their low lutein content (ca. 0.03%) and low biomass productivity. In addition, the harvesting and separation of the marigold flowers is labor intensive. Thus, there is still a demand to look for more promising alternatives for lutein production. In recent years, microalgae are emerged as promising sources for lutein production. Some
http://dx.doi.org/10.1016/j.biortech.2014.10.062 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062
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microalgal species are known to produce lutein effectively, such as Chlorella zofingiensis (Del Campo et al., 2004), Chlorella protothecoides (Shi et al., 2000), Scenedesmus sp. (Ho et al., 2014a), Desmodesmus sp. (Xie et al., 2013) and Muriellopsis sp. (Del Campo et al., 2000). The advantages of using microalgae for lutein production include (1) microalgae have higher growth rate with the relatively higher lutein content (typically 0.2–0.7%) than marigold and other terrestrial plants (Fraser and Bramley, 2004; Ho et al., 2014a; Kandlakunta et al., 2008; Xie et al., 2013); (2) Microalgae could be harvested daily under optimal operating condition; (3) there is no need to separate the petals like marigold, while the whole microalgal biomass can be used to extract lutein; and (4) valuable byproducts could also be obtained, such as protein, carbohydrates and lipids (Fernandez-Sevilla et al., 2010). However, since the microalgal lutein is tightly bound with the cell wall of microalgae, it might be more difficult to extract lutein from microalgal biomass when compared with marigold. Moreover, due to the small cell size and the dilute concentration in the culture, it may require more energy consumption to harvest the microalgal biomass from the culture. As a result, the cost of microalgae-based lutein production is still too high to be economically feasible in an industrial scale. Therefore, the lutein productivity needs to be significantly improved to make the microalgae-based lutein more commercially viable. In order to achieve a higher lutein productivity, both lutein content and biomass productivity should be improved. The combination of these two parameters gives the lutein productivity, which is considered the most important indicator in the commercial process. Our recent studies (Ho et al., 2014a) and some related studies showed that the irradiance and nitrogen availability could significantly affect the accumulation of lutein or the biomass productivity of microalgae (Borowitzka et al., 1991; Del Campo et al., 2000; Xie et al., 2013). Therefore, acquiring the suitable light intensity, nitrogen sources, and nitrogen concentration is extremely vital in microalgae cultivation, aiming to enhance the lutein content and overall lutein productivity (Del Campo et al., 2000). In this study, the initial nitrogen sources and nitrogen concentration of the medium (i.e., Detmer’s medium) for the lutein production of Scenedesmus obliquus FSP-3 were optimized under phototrophic conditions. Moreover, the feasibility of using bioreactor operating strategies (i.e., two-stage process and semi-continuous operation) to improve the production of lutein was also assessed. The results obtained in this work could provide useful information for the assessment of full-scale lutein production using microalgae as the feedstock.
2. Methods 2.1. Microalgae culture conditions The microalga used in this study was S. obliquus FSP-3, which was isolated from freshwater located in southern Taiwan (Ho et al., 2010). A modified version of Detmer’s Medium (DM) was used to grow the pure culture of S. obliquus FSP-3. The medium consisted of (g/L): Ca(NO3)24H2O, 1.00; KH2PO4, 0.26; MgSO47H2O, 0.55; KCl, 0.25; FeSO47H2O, 0.02; EDTA.2Na, 0.2; H3BO3, 2.9 103; ZnCl2 1.1 104; MnCl24H2O, 1.81 103; (NH4)6Mo7O244H2O, 1.8 105; CuSO45H2O, 0.8 103. The FSP-3 strain was grown at 28 °C for 6–10 days under an optimal light intensity of 300 lmol/m2/s (illuminated by TL5 tungsten filament lamp). The light intensity was measured by a LI-250 Light Meter with a LI-200SA pyranometer sensor (LI-COR, Inc., Lincoln, Nebraska, USA). The centrifuged microalgal biomass was lyophilized using a freeze-dryer (FDU-1100, EYELA, Tokyo, Japan) for storage and lutein content analysis.
2.2. Operation of photobioreactor The photobioreactor (PBR) used for cultivating S. obliquus FSP-3 was a 1-liter glass-made vessel illuminated with an external light source mounted on both sides. The S. obliquus FSP-3 was precultured and inoculated into the PBR with a inoculum size of 40 mg/L. The PBR was operated at 28 °C, pH 6.0, and an agitation rate of 300 rpm. Serving as the sole carbon source, 2.5% CO2 was fed into the culture continuously during 6–10 day cultivation. The liquid samples were collected from the sealed glass vessel to determine microalgae cell concentration, culture pH, residual nitrate concentration, and lutein content. 2.3. Determination of microalgal biomass concentration The biomass concentration of S. obliquus FSP-3 in PBR was determined regularly using UV/VIS spectrophotometer via the measurement of optical density at a wavelength 685 nm (denoted as OD685). The absorbance values were converted to dry cell weight (denoted as DCW) concentration via the appropriate calibration (i.e., 1.0 OD685 equals to 0.2–0.3 g DCW/L). Intermittent measurements of DCW were performed to confirm the calibration relationship between the values of OD685 and DCW concentration. 2.4. Determination of growth kinetics parameters The specific growth rate (l, day1) of microalgal culture was calculated with the equation shown below:
l¼
d ln X dt
ði:e:; maximum slope from lynx versus plotÞ
max
where X and t indicate the biomass concentration (g/L) and cultivation time (day), respectively. The overall biomass productivity (Poverall, g DCW/L/d) was calculated as follows:
Poverall ¼
DX Dt
where DX is the variation of biomass concentration (g DCW/L) within a cultivation time of DtðdÞ. 2.5. Determination of residual nitrogen concentration The residual nitrate concentration was determined by a modified method reported in our previous study (Ho et al., 2013b). In general, microalgae samples were taken from the cultures and filtered through a 0.22 lm pore size filter and then diluted 30-fold with DI water for each sample. The absorbance (at 220 nm) of the filtered solution was measured with an UV/VIS spectrophotometer (model U-2001, Hitachi, Tokyo, Japan). The NH+4 concentration of the culture medium was determined with an ammonia photometer (model HI 93715, Hanna Instruments, Italy). A liquid sample collected from the PBR was filtered using a 0.22 lm pore size filter and then 10 mL was taken with 4 drops of specific reagents for each sample. After setting for 3.5 min, the sample was examined with an ammonia photometer. The urea concentration of the culture medium was analyzed with the manual urease procedure proposed by Butler et al. (1996). Samples (1.0 mL each) were taken from the microalgal culture, and then centrifuged at 10,000 rpm for 5 min. The supernatant (100 lL) was taken and then mixed with 0.5 mL urease solution, which was incubated in a 37 °C water bath for 10 min. After that, the reaction mixture was removed from the water bath, and the reaction was stopped by the addition of 1.0 mL phenol nitroprusside (enzyme terminator) and 1.0 mL alkaline hypochlorite. After setting at room temperature for 30 min, the absorbance
Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062
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The lutein was extracted from microalgae by the modified protocols reported in our previous study (Chan et al., 2013). In general, 10–15 mg dry microalgal biomass was treated with 1 mL KOH solution (at a concentration of 60% w/w) to hydrolyze the lipids and assist cell disruption. After the alkaline treatment, diethyl ether was added into the mixture placed in 40 °C hot-water bath to extract lutein for 40 min. When the color of microalgal biomass turned clear, the supernatant phase was collected for the analysis. The lutein content of the solvent extracts was measured by purging the organic solvent with a N2 flow and then dissolving the precipitate in acetone for the analysis by high-performance liquid chromatography (HPLC) (Taylor et al., 2006). The separation column used in HPLC was YMC Carotenoid RP-30 (particle size 5 lm) column (4.6 mm 250 mm) (YMC Europe, Schermbeck, Germany). The binary mobile phase consisted of (A) 3% ddH2O in methanol containing 0.05 M ammonium acetate, and (B) 100% TBME (tert-butyl methyl ether). Both mobile phases contained 0.1% (w/v) BHT and 0.05% triethylamine (TEA). The extracts were eluted at a flow rate of 1 mL/min and the lutein content was detected by measuring absorbance at the wavelength range of 220–750 nm. Three absorbance wavelengths were used (i.e., 450, 652, and 478 nm) and the maximal absorbance (450 nm) was chosen for quantification of lutein extracts. The lutein standards used for quantification purpose were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3. Results and discussion
4
3
2
1
5
3.1. Effect of nitrogen sources on cell growth and the lutein content of S. obliquus FSP-3 Literature shows that selecting suitable nitrogen source is of importance in improving biomass productivity of microalgal species (Hsieh and Wu, 2009). However, the effect of nitrogen source on lutein accumulation in microalgae is still not clear. To investigate an optimal nitrogen source for cell growth and lutein accumulation of S. obliquus FSP-3, three different nitrogen sources (Ca(NO3)2, (NH4)2SO4 and urea) at an identical concentration of 8.0 mM were examined, respectively. As shown in Fig. 1, under the same culture conditions, nitrate appeared to support better growth of S. obliquus FSP-3 than NH4 and urea. The biomass production using nitrate as nitrogen source was around 2–3-fold higher than that obtained from using NH4 and urea, indicating that nitrate is the favorable nitrogen source for the growth of S. obliquus FSP-3. This result was consistent with results obtained from the cultivation of other microalgal species. For example, the growth of Neochloris oleoabundans was better when using nitrate as the nitrogen source when compared with using NH4 and urea (Li et al., 2008). However, the other microalgal species, Ellipsoidion
Nitrogen concentration (mM)
0 8
4 6 3 4
2
2
1
0
Lutein content (mg/g DCW)
2.6. Determination of the lutein content of microalgal biomass
sp., had a higher growth rate with ammonia (Xu et al., 2001). Hence, the suitable nitrogen source for the microalgal growth is largely species-dependent (Hsieh and Wu, 2009). Ammonium is usually favorable over nitrate as a nitrogen source by microalgae due to no redox reaction and less energy required for its assimilation (Ramanna et al., 2014). However, the lowest biomass production obtained from S. obliquus FSP-3 with NH4 may be attributed to a sharp decrease in pH from 7.0 to around 5.0 during the assimilation of ammonium ions (data not shown). Wu et al. (2013) also reported similar results, showing that ammonium consumption by Monoraphidium sp. SB2 resulted in a pH decrease from 8.0 to 4.4 during the cultivation. On the other hand, urea dissociates to form CO2 and ammonium in solution via the urea amino hydrolase pathway (urease enzyme) (Ramanna et al., 2014). The lower biomass production with urea-N might be due to a relatively low urease activity of this strain. Moreover, Fig. 1 shows that the lutein content increased gradually along with the consumption of NH4 and urea, while the maximum lutein content was obtained at the beginning of nitrate depletion, while the lutein content started to decrease under the
Biomass concentration (g/L)
of the resulting mixture was measured at a wavelength of 570 nm and the value of absorbance was converted to urea concentration via appropriate calibration.
0 0
1
2
3
Time (d)
4
5
Lutein content (Nitrate) Lutein content (Ammonia) Lutein content (Urea) Biomass and Nitrogen (Nitrate) Biomass and Nitrogen (Ammonia) Biomass and Nitrogen (Urea)
Fig. 1. Time-course profiles of biomass concentration, nitrogen source concentration, lutein content and lutein productivity during the growth of S. obliquus FSP-3 using different nitrogen sources. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s).
Table 1 Effect of different nitrogen sources on biomass production, biomass productivity, lutein content and lutein productivity of S. obliquus FSP-3. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s). Nitrogen source
Biomass production (g/L)a
Biomass productivity (mg/L/day)a
Lutein content (mg/g)a
Lutein productivity (mg/L/day)a
Ca(NO3)2 Urea (NH4)2SO4
1.84 ± 0.08 0.96 ± 0.05 0.60 ± 0.04
942.0 ± 14.1 477.8 ± 8.4 286.3 ± 6.1
4.61 ± 0.11 2.18 ± 0.13 1.58 ± 0.08
4.35 ± 0.09 1.04 ± 0.07 0.45 ± 0.03
Data shown in this table are average values of triplicates ± SD (standard deviation). a The values were calculated at the time when the maximal lutein productivity was reached.
Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062
S.-H. Ho et al. / Bioresource Technology xxx (2014) xxx–xxx
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3 2
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Biomass concentration (g/L)
0
0 25
16mM Nitrate
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15
3 2
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Biomass concentration (g/L)
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0 25
24mM Nitrate
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0
0 0
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3
Time (d)
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Lutein content & productivity (mg/g, mg/L/d)
25
8mM Nitrate
5
0 5 4 3 2 1
Lutein contnt & productivity (mg/g, mg/L/d)
Biomass concentration (g/L)
Our previous study showed that nitrogen availability is a vital factor affecting the lutein accumulation of microalgae during
0 5 4 3 2 1
Lutein content & productvity (mg/g, mg/L/d)
3.2. Effect of nitrate concentration on cell growth and lutein content of S. obliquus FSP-3
Nitrate concentration (mM)
cultivation (Ho et al., 2014a). However, the nitrogen concentration in media used for producing lutein from S. obliquus FSP-3 was not optimized. Thus, in this study, modified Detmer’s Medium containing 8, 16 and 24 mM calcium nitrate, respectively, were examined. The time-course profiles of cell growth and lutein production obtained with different nitrate concentrations are shown in Fig. 2. As indicated in Fig. 2 and Table 2, the biomass concentration of S. obliquus FSP-3 improved significantly when the nitrate concentration increased from 8 to 24 mM. However, the biomass productivity was slightly lower when using higher nitrate concentration. This could be attributed to more severe light shading effects arising from the higher cell density obtained from using higher nitrate concentration. Moreover, Fig. 2 shows that in all different nitrate concentrations groups, the maximum lutein content occurred at the beginning of nitrate depletion, and increased slightly from 4.61 to 4.95 mg/g as nitrate concentration increased from 8 to 24 mM (Table 2). Similar result was also reported in other studies with other microalgal strains, showing an improvement in lutein content when increasing nitrogen concentration (Cordero et al.,
Nitrate concentration (mM)
nitrate depletion condition. It was suggested that nitrogen-sufficient condition was required for lutein accumulation (Xie et al., 2013). As shown in Table 1, the lutein content of S. obliquus FSP3 obtained from using nitrate as the nitrogen source was nearly 1–2 folds higher than that obtained from using NH4 and urea. In contrast, Del Campo et al. (2000) reported that the nitrogen sources (NaNO3, NH4Cl and NH4NO3) did not influence the lutein level in Muriellopsis sp. This means that the correlation of nitrogen source and lutein content may also be species-specific. Further, the highest lutein productivity (4.35 mg/L/day) was also obtained when using nitrate as nitrogen source. Thus, it is clear that among the three nitrogen sources examined, nitrate is the most favorable nitrogen source for both cell growth and lutein accumulation of S. obliquus FSP-3 under the culture conditions.
Nitrate concentration (mM)
4
0
6 Lutein content Lutein productivity Biomass concentration Nitrate concentration
Fig. 2. Time-course profiles of biomass concentration, nitrate concentration, lutein content and lutein productivity during the growth of S. obliquus FSP-3 using different nitrate concentrations. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s).
Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062
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Table 2 Effect of different nitrate concentrations on biomass production, biomass productivity, lutein content and lutein productivity of S. obliquus FSP-3. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s). Nitrate concentration (mM)
Culture time (day)a
Biomass production (g/L)a
Biomass productivity (mg/L/day)a
Lutein content (mg/g)a
Lutein productivity (mg/L/day)a
8 16 24
1.9 3.8 4.9
1.84 ± 0.08 3.38 ± 0.14 4.30 ± 0.21
942.0 ± 14.1 872.1 ± 10.9 859.1 ± 11.3
4.61 ± 0.11 4.87 ± 0.16 4.95 ± 0.22
4.35 ± 0.09 4.25 ± 0.12 4.25 ± 0.14
Data shown in this table are average values of triplicates ± SD (standard deviation). a The values were calculated at the time when the maximal lutein productivity was reached.
2011; Xie et al., 2013). As the biomass concentration increased with the cultivation time under high nitrate concentration, the light may hardly penetrated to cell broth and the specific light intensity (i.e., the amount of light intensity received per cell) decreased sharply (Cheirsilp and Torpee, 2012). Thus, the size of the light-harvesting antenna complex should be increased to absorb more light energy, resulting in higher lutein content (Solovchenko et al., 2008). As shown in Table 2, the highest lutein productivity of S. obliquus FSP-3 (4.35 mg/L/day) was obtained with 8 mM nitrate concentration, which is slightly higher than that obtained with the other nitrate concentrations. This result might be due to the highest biomass productivity with 8 mM nitrate concentration. In addition, when using a nitrate concentration of 8 mM, the culture time required to reach the highest lutein content was significantly shorter than that required for other nitrate concentrations examined. This would benefit the economic feasibility of lutein production. When the nitrate concentration was decreased to 4 mM or 2 mM, the overall biomass productivity slightly dropped due to the presence of a longer lag phase in the early stage of the cultivation (data not shown). Moreover, low nitrate concentration is also not conducive to the accumulation of lutein because of the excess light exposure. Therefore, based on the results shown above, 8 mM seems to be the best nitrate concentration for lutein production of S. obliquus FSP-3. 3.3. Improvement of the lutein content of S. obliquus FSP-3 by twostage process with light-intensity shift Many environmental stresses can lead to changes in the biochemical composition and cell growth of microalgae, such as nitrogen depletion (Ho et al., 2013a), light intensity (Xie et al., 2013), and high salinity (Ho et al., 2014b). Among them, light intensity seems to be the most direct factor influencing the production of photosynthesis-associated pigments, such as lutein, while the effects of light intensity on lutein production of microalgae, as reported in the literature, have been very diverse. Sanchez et al. (2008) reported that elevating the light intensity could increase
the lutein content of Scenedesmus almeriensis. In contrast, Solovchenko et al. (2008) found that the lutein content increases upon the decrease of light supply. This difference might be associated with the function of lutein, which could play a role in either light protection or light energy capture (Demmig-Adams and Adams, 1996). Our recent work also investigated the relationship between the lutein content and irradiance of the S. obliquus FSP3 strain, and observed a higher lutein content when using lower light intensity (Ho et al., 2014a), while on the other hand higher light intensity is favorable for cell growth. Accordingly, in this study, a two-stage process with a switch in light intensity was attempted to enhance the lutein productivity of S. obliquus FSP-3. In the first stage, microalgae were cultivated under a higher light intensity of 300 lmol/m2/s to facilitate cell growth. At difference nitrogen consumption stages (i.e., 70%, 80%, 90% and 95% of nitrogen source consumption), the microalgae culture was shifted to lower irradiance (75 lmol/m2/s) in the second stage to stimulate the lutein accumulation until nitrogen starvation occurred (i.e., 100% nitrogen source consumption). Single-stage cultivation at a fixed light intensity of 300 lmol/m2/s was performed as the control. As indicated in Table 3, switching the light intensity on microalgae culture to a lower level of 75 lmol/m2/s could indeed lead to an significant increase in lutein content. In particular, the light intensity shift at 70% nitrogen consumption resulted in a lutein content increase from 4.20 to 5.04 mg/g. The light intensity shift activated at 80%, 90%, and 95% nitrogen consumption also showed a slight increase in the lutein content when compared to the control group (Table 3). However, because of the lower biomass productivity in the second stage due to lower irradiance on the culture, the overall lutein productivity in this two-stage operation is still markedly lower when compared to the control, in which the microalga was grown in a single-stage process with a constant light intensity of 300 lmol/m2/s. In summary, using the two-stage operation strategy, the lutein productivity was not improved when compared to the single-stage operation, whereas the lutein content could be promoted to a higher level when applying the light intensity shift strategy. The
Table 3 Effect of different light-intensity (from 300 lmol/m2/s to 75 lmol/m2/s) and switching timing (at 70%, 80%, 90% and 95% of nitrogen consumption) on biomass productivity, lutein content and lutein productivity of S. obliquus FSP-3. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s; nitrogen source, calcium nitrate at 8.0 mM). Percentage of nitrogen consumptiona (%)
Lutein accumulation timec (hr)
Biomass productivity (mg/L/day)
Lutein content (mg/g)
70% 80% 90% 95% Controlb
44.0 ± 2.1 16.5 ± 1.3 10.5 ± 1.1 3.0 ± 0.5 –
572.4 ± 19.1 765.3 ± 28.8 836.8 ± 24.7 901.2 ± 29.6 942.0 ± 14.1
4.20 ± 0.09 4.25 ± 0.13 4.41 ± 0.06 4.57 ± 0.16
Before light intensity shift
After light intensity shift
5.04 ± 0.15 4.89 ± 0.11 4.75 ± 0.09 4.69 ± 0.12 4.61 ± 0.11
Lutein productivity (mg/L/day)
2.88 ± 0.07 3.74 ± 0.10 3.97 ± 0.07 4.23 ± 0.14 4.35 ± 0.09
a
The switch point of light-intensity was based on the percentage of nitrogen consumption (%). Single-stage operation at a fixed light intensity of 300 lmol/m2/s without light intensity shift. The time interval starting from the shift of light intensity from 300 to 75 lmol/m2/s till the time when nitrogen starvation was reached (i.e., the time when 100% nitrate consumption occurred). b
c
Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062
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Table 4 Performance of semi-batch culture with different broth replacement ratios in terms of biomass productivity, lutein content and lutein productivity of S. obliquus FSP-3. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s; nitrogen source, calcium nitrate at 8.0 mM). Replacement ratio (%)
Time interval (hr)
Biomass productivity (mg/L/day)
Lutein content (mg/g)
Lutein productivity (mg/L/day)
10% 30% 50% 70% Batch
4±0 13.3 ± 1.2 22.5 ± 0.8 31.9 ± 1.2 48(2 days)
1304.8 ± 33.7 1159.8 ± 102.5 1118.2 ± 37.7 1087.2 ± 30.6 942.0 ± 14.1
4.61 ± 0.16 4.53 ± 0.21 4.42 ± 0.14 4.54 ± 0.17 4.61 ± 0.11
6.01 ± 0.24 5.25 ± 0.44 4.95 ± 0.27 4.94 ± 0.27 4.35 ± 0.09
2.2 10% replacement
3
2.0
2 1.9
1 0 0.6
0.8 30% replacement
5
2.0
4
1.8
3
1.6
2
1.4
1
0.5
1.0
1.5
2.0
2.4
Lutein content (mg/g DCW)
0.4
0 3.0
2.5 50% replacement
2.2
5
2.0
4
1.8 3
1.6 1.4
2
1.2
Lutein content (mg/g DCW)
Biomass concnetration (g/L)
0.2
2.2
0.0
1
1.0 0.8
0 0
Biomass concentration (g/L)
Lutein conent (mg/g DCW)
4
0.0
Biomass concentration (g/L)
5
2.1
1
2
3
4
5 6
70% replacement 5
2.0
4 1.5
3 2
1.0
Lutein content (mg/g DCW)
Biomass concentration (g/L)
Data shown in this table are average values of five replicates ± SD (standard deviation).
1 0.5 0 0
2
4
6
Time(d) Fig. 3. Profiles of cell growth and lutein content of S. obliquus FSP-3 cultivated with semi-continuous operation with intermittent replacement of culture broth with various replacement ratios (10%, 30%, 50%, 70%). (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s; nitrogen source, calcium nitrate at 8.0 mM).
Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062
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S.-H. Ho et al. / Bioresource Technology xxx (2014) xxx–xxx Table 5 Comparison of the performance of lutein content and lutein productivity in this study and in other related studies. Microalga source
Operation mode
Chlorella protothecoides Chlorella protothecoides Chlorella zofingiensis Chlorella zofingiensis Chlorella fusca Chlorella proboscideum Muriella aurantiaca Murielladecolor Muriellopsis sp. Neospongiococcus gelatinosum Desmodesmus sp. F2 Muriellopsis sp. Scenedesmus almeriensis Coccomyxa acidophila Scenedesmus obliquus FSP-3 Scenedesmus obliquus FSP-3 Scenedesmus obliquus FSP-3
Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Pulse-feeding Continuous Continuous Batch Batch Two-stage Semi-batch
Cultivating conditions Light supply
Metabolic mode
Absence of light Absence of light Continuous light Continuous light Continuous light Continuous light Continuous light Continuous light Continuous light Continuous light Continuous light Sunlight period Sunlight period Continuous light Continuous light Continuous light Continuous light
Heterotrophic Heterotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Mixotrophic Autotrophic Autotrophic Autotrophic
increase in lutein content per unit cell may lead to a decrease in the cost for product separation and purification, especially when the process is scaled up. Therefore, the proposed two-stage process may still have the niche. To further enhance the lutein content and productivity of the two-stage process, the light intensity employed in each stage and the duration time of the second stage may need to be optimized to achieve the best performance. 3.4. Improvement of lutein productivity of S. obliquus FSP-3 by using semi-batch operation From the results shown above, using two-stage operation could improve the lutein content of the FSP-3 strain but with a side-effect of lower biomass productivity. Therefore, another engineering strategy must be developed to further enhance the lutein productivity. As depicted in Fig. 2, in batch culture, using calcium nitrate dehydrate at a concentration of 8 mM as the nitrogen source resulted in the highest lutein productivity of 4.35 mg/L/ day. Therefore, this nitrogen source condition was adapted for conducting semi-continuous operations, aiming to enhance the biomass productivity, while maintaining the lutein content at a relatively high level (Chiu et al., 2009; Ho et al., 2013b). Before the semi-continuous operation, S. obliquus FSP-3 was cultivated for two days until the biomass concentration reached 1.9–2.1 g/L, at which the microalgal strain had the maximal lutein content and maximal lutein productivity, which has the same trend as earlier results shown in Fig. 2. The operation mode was then changed to semi-continuous cultivation by intermittent replacement of culture broth with fresh medium at different replacement ratios. As shown in Table 4, the replacement ratios included 10% replacement (at intervals of 4 h), 30% replacement (at intervals of 12–15 h), 50% replacement (at intervals of 21–24 h) and 70% replacement (at intervals of 30–34 h),. The new cycle was started when the biomass concentration again reached 1.9–2.1 g/L again (Fig. 3). As shown in Fig. 3, the results of semi-continuous operation, such as biomass productivity and lutein content, were consistent during the five cycles with four replacement ratios, suggesting that the operation of semi-continuous is stable and feasible for industrial production of lutein from S. obliquus FSP-3. Table 4 summarizes the biomass and lutein production performance of semibatch operation of S. obliquus FSP-3 under the four medium replacement ratios. There is no significant difference in lutein content for the semi-continuous cultures at all the four replacement ratios used, as the values fall within the range of 4.40–4.60 mg/g.
Lutein content (mg/g)
Lutein productivity (mg/L/day)
References
1.9 4.6 2.8 3.5 4.2 3.4 2.6 0.5 5.5 4.4 5.1 4.3 5.5 3.5 4.6 4.7 4.6
3.6 12.0 1.3 1.2 0.8 1.2 3.1 1.3 4.3 6.0 3.6 7.2 4.9 0.9 4.4 4.2 6.0
Wei et al. (2008) Shi et al. (2000) Del Campo et al. (2000) Del Campo et al. (2004) Del Campo et al. (2000) Del Campo et al. (2000) Del Campo et al. (2000) Del Campo et al. (2000) Del Campo et al. (2000) Del Campo et al. (2000) Xie et al. (2013) Sanchez et al. (2008) This study This study This study
However, semi-continuous operation with 10% medium replacement could achieve the highest biomass productivity and lutein productivity of 1304.8 and 6.01 mg/L/day, respectively. As shown in Table 5, the biomass productivity and lutein productivity are much higher than those obtained from batch culture (942.0 and 4.35 mg/L/day, respectively) and are also higher than most of the reported values in related studies (Del Campo et al., 2000; Hodaifa et al., 2009; Mandal and Mallick, 2009; Sanchez et al., 2008; Wei et al., 2008). To the best of our knowledge, this is the first study reporting the use of semi-continuous cultivation to improve lutein production from microalgae. By using this strategy, the highest lutein productivity of 6.01 ± 0.24 mg/L/day was achieved, which is approximately 40% higher than that obtained with the batch system. This demonstrates the feasibility of using semi-continuous process with 10% medium replacement ratio for lutein production from the lutein-rich S. obliquus FSP-3 strain. 4. Conclusions Calcium nitrate at a concentration of 8 mM was the favorable nitrogen source condition for both cell growth and lutein production of S. obliquus FSP-3. The two-stage operation with light intensity shift elevated the lutein content to 5.04 mg/g, but obtained lower lutein productivity due to a marked decrease in biomass productivity. The semi-continuous operation with 10% medium replacement ratio effectively enhanced microalgal biomass (1304.8 mg/L/day) and lutein productivity (6.01 mg/L/day), which are higher than most reported values. This semi-continuous strategy should be further assessed in outdoor scale-up cultivation to evaluate its feasibility in commercial production of microalgal lutein. Acknowledgements The authors gratefully acknowledge the support from the Top University Project of NCKU, Energy Technology Program for Academia of Bureau of Energy of Ministry of Economic Affairs, and from Taiwan’s Ministry of Science and Technology (MOST) under grant numbers 103-3113-P-006-006, 102-3113-P-110-011 and 1012221-E-006-209-MY3. References Astorg, P., 1997. Food carotenoids and cancer prevention: an overview of current research. Trends Food Sci. Technol. 8 (12), 406–413.
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obliquus CNW-N used for bioethanol fermentation. Bioresour. Technol. 143, 163–171. Ho, S.-H., Nakanishi, A., Ye, X., Chang, J.-S., Hara, K., Hasunuma, T., Kondo, A., 2014b. Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy. Biotechnol. Biofuels 7 (1), 97. Ho, S.H., Chen, C.Y., Yeh, K.L., Chen, W.M., Lin, C.Y., Chang, J.S., 2010. Characterization of photosynthetic carbon dioxide fixation ability of indigenous Scenedesmus obliquus isolates. Biochem. Eng. J. 53 (1), 57–62. Hodaifa, G., Martinez, M.E., Sanchez, S., 2009. Daily doses of light in relation to the growth of Scenedesmus obliquus in diluted three-phase olive mill wastewater. J. Chem. Technol. Biotechnol. 84 (10), 1550–1558. Hsieh, C.H., Wu, W.T., 2009. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresour. Technol. 100 (17), 3921–3926. Kandlakunta, B., Rajendran, A., Thingnganing, L., 2008. Carotene content of some common (cereals, pulses, vegetables, spices and condiments) and unconventional sources of plant origin. Food Chem. 106 (1), 85–89. Li, Y., Horsman, M., Wang, B., Wu, N., Lan, C.Q., 2008. Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Appl. Microbiol. Biotechnol. 81 (4), 629–636. Mandal, S., Mallick, N., 2009. Microalga Scenedesmus obliquus as a potential source for biodiesel production. Appl. Microbiol. Biotechnol. 84 (2), 281–291. Ramanna, L., Guldhe, A., Rawat, I., Bux, F., 2014. The optimization of biomass and lipid yields of Chlorella sorokiniana when using wastewater supplemented with different nitrogen sources. Bioresour. Technol. 168, 127–135. Sanchez, J.F., Fernandez-Sevilla, J.M., Acien, F.G., Ceron, M.C., Perez-Parra, J., MolinaGrima, E., 2008. Biomass and lutein productivity of Scenedesmus almeriensis: influence of irradiance, dilution rate and temperature. Appl. Microbiol. Biotechnol. 79 (5), 719–729. Shi, X.M., Zhang, X.W., Chen, F., 2000. Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources. Enzyme Microb. Technol. 27 (3–5), 312–318. Solovchenko, A.E., Khozin-Goldberg, I., Didi-Cohen, S., Cohen, Z., Merzlyak, M.N., 2008. Effects of light and nitrogen starvation on the content and composition of carotenoids of the green microalga Parietochloris incisa. Russ. J. Plant Physiol. 55 (4), 455–462. Taylor, K.L., Brackenridge, A.E., Vivier, M.A., Oberholster, A., 2006. High-performance liquid chromatography profiling of the major carotenoids in Arabidopsis thaliana leaf tissue. J. Chromatogr. A 1121 (1), 83–91. Wei, D., Chen, F., Chen, G., Zhang, X.W., Liu, L.J., Zhang, H., 2008. Enhanced production of lutein in heterotrophic Chlorella protothecoides by oxidative stress. Sci. Chin. C 51 (12), 1088–1093. Wu, L.F., Chen, P.C., Lee, C.M., 2013. The effects of nitrogen sources and temperature on cell growth and lipid accumulation of microalgae. Int. Biodeterior. Biodegrad. 85, 506–510. Xie, Y., Ho, S.-H., Chen, C.-N.N., Chen, C.-Y., Ng, I.S., Jing, K.-J., Chang, J.-S., Lu, Y., 2013. Phototrophic cultivation of a thermo-tolerant Desmodesmus sp. for lutein production: effects of nitrate concentration, light intensity and fed-batch operation. Bioresour. Technol. 144, 435–444. Xu, N., Zhang, X., Fan, X., Han, L., Zeng, C., 2001. Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta). J. Appl. Phycol. 13 (6), 463–469.
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Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3 Shih-Hsin Ho a, Youping Xie b, Ming-Chang Chan c, Chen-Chun Liu d, Chun-Yen Chen d, Duu-Jong Lee e, Chieh-Chen Huang f, Jo-Shu Chang c,d,g,⇑ a
Organization of Advanced Science and Technology, Kobe University, Kobe 657-8501, Japan College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, PR China Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan d Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan e Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan f Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan g Research Center for Energy Technology and Strategy Center, National Cheng Kung University, Tainan 701, Taiwan b c
h i g h l i g h t s N source/concentration were optimized for better lutein production from microalgae. The light intensity shift strategy enhanced lutein content of S. obliquus FSP-3. Semi-continuous operation improved the productivity of lutein from microalgae. The highest lutein productivity of 6.01 mg/L/day is high than most reported values.
a r t i c l e
i n f o
Article history: Received 9 September 2014 Received in revised form 12 October 2014 Accepted 13 October 2014 Available online xxxx Keywords: Microalgae Scenedesmus obliquus Lutein Light intensity shift Bioreactor strategy
a b s t r a c t In this study, the effects of the type and concentration of nitrogen sources on the cell growth and lutein content of an isolated microalga Scenedesmus obliquus FSP-3 were investigated. With batch culture, the highest lutein content (4.61 mg/g) and lutein productivity (4.35 mg/L/day) were obtained when using 8.0 mM calcium nitrate as the nitrogen source. With this best nitrogen source condition, the microalgae cultivation was performed using two bioreactor strategies (namely, semi-continuous and two-stage operations) to further enhance the lutein content and productivity. Using semi-continuous operation with a 10% medium replacement ratio could obtain the highest biomass productivity (1304.8 mg/L/day) and lutein productivity (6.01 mg/L/day). This performance is better than most related studies. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Lutein, an important photosynthetic pigment present in plants and phototrophic microorganisms, belongs to the xanthophyllic pigment groups (Fernandez-Sevilla et al., 2010). Lutein is extremely necessary to support the life of photoautotrophs owing to its role in assisting photosynthesis and photo-protection (Demmig-Adams and Adams, 1996). Lutein has many functions for the protection of the human’s health. It can prevent cardiovascular diseases
⇑ Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan. Tel.: +886 6 2757575x62651; fax: +886 6 2357146. E-mail address: [email protected] (J.-S. Chang).
(Dwyer et al., 2001), age-related macular degeneration (Chiu and Taylor, 2007; Granado et al., 2003), and some types of cancers (Astorg, 1997). For the multiple curing effects of human life, lutein is largely consumed and listed as E161b for food additives with the market size of nearly $150 million in the US and its market size is still expanding (Ceron et al., 2008). Nowadays, the main lutein feedstock for commercial lutein production focuses on the marigold petals. However, the major hurdle of using marigold petals for lutein production is their low lutein content (ca. 0.03%) and low biomass productivity. In addition, the harvesting and separation of the marigold flowers is labor intensive. Thus, there is still a demand to look for more promising alternatives for lutein production. In recent years, microalgae are emerged as promising sources for lutein production. Some
http://dx.doi.org/10.1016/j.biortech.2014.10.062 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062
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microalgal species are known to produce lutein effectively, such as Chlorella zofingiensis (Del Campo et al., 2004), Chlorella protothecoides (Shi et al., 2000), Scenedesmus sp. (Ho et al., 2014a), Desmodesmus sp. (Xie et al., 2013) and Muriellopsis sp. (Del Campo et al., 2000). The advantages of using microalgae for lutein production include (1) microalgae have higher growth rate with the relatively higher lutein content (typically 0.2–0.7%) than marigold and other terrestrial plants (Fraser and Bramley, 2004; Ho et al., 2014a; Kandlakunta et al., 2008; Xie et al., 2013); (2) Microalgae could be harvested daily under optimal operating condition; (3) there is no need to separate the petals like marigold, while the whole microalgal biomass can be used to extract lutein; and (4) valuable byproducts could also be obtained, such as protein, carbohydrates and lipids (Fernandez-Sevilla et al., 2010). However, since the microalgal lutein is tightly bound with the cell wall of microalgae, it might be more difficult to extract lutein from microalgal biomass when compared with marigold. Moreover, due to the small cell size and the dilute concentration in the culture, it may require more energy consumption to harvest the microalgal biomass from the culture. As a result, the cost of microalgae-based lutein production is still too high to be economically feasible in an industrial scale. Therefore, the lutein productivity needs to be significantly improved to make the microalgae-based lutein more commercially viable. In order to achieve a higher lutein productivity, both lutein content and biomass productivity should be improved. The combination of these two parameters gives the lutein productivity, which is considered the most important indicator in the commercial process. Our recent studies (Ho et al., 2014a) and some related studies showed that the irradiance and nitrogen availability could significantly affect the accumulation of lutein or the biomass productivity of microalgae (Borowitzka et al., 1991; Del Campo et al., 2000; Xie et al., 2013). Therefore, acquiring the suitable light intensity, nitrogen sources, and nitrogen concentration is extremely vital in microalgae cultivation, aiming to enhance the lutein content and overall lutein productivity (Del Campo et al., 2000). In this study, the initial nitrogen sources and nitrogen concentration of the medium (i.e., Detmer’s medium) for the lutein production of Scenedesmus obliquus FSP-3 were optimized under phototrophic conditions. Moreover, the feasibility of using bioreactor operating strategies (i.e., two-stage process and semi-continuous operation) to improve the production of lutein was also assessed. The results obtained in this work could provide useful information for the assessment of full-scale lutein production using microalgae as the feedstock.
2. Methods 2.1. Microalgae culture conditions The microalga used in this study was S. obliquus FSP-3, which was isolated from freshwater located in southern Taiwan (Ho et al., 2010). A modified version of Detmer’s Medium (DM) was used to grow the pure culture of S. obliquus FSP-3. The medium consisted of (g/L): Ca(NO3)24H2O, 1.00; KH2PO4, 0.26; MgSO47H2O, 0.55; KCl, 0.25; FeSO47H2O, 0.02; EDTA.2Na, 0.2; H3BO3, 2.9 103; ZnCl2 1.1 104; MnCl24H2O, 1.81 103; (NH4)6Mo7O244H2O, 1.8 105; CuSO45H2O, 0.8 103. The FSP-3 strain was grown at 28 °C for 6–10 days under an optimal light intensity of 300 lmol/m2/s (illuminated by TL5 tungsten filament lamp). The light intensity was measured by a LI-250 Light Meter with a LI-200SA pyranometer sensor (LI-COR, Inc., Lincoln, Nebraska, USA). The centrifuged microalgal biomass was lyophilized using a freeze-dryer (FDU-1100, EYELA, Tokyo, Japan) for storage and lutein content analysis.
2.2. Operation of photobioreactor The photobioreactor (PBR) used for cultivating S. obliquus FSP-3 was a 1-liter glass-made vessel illuminated with an external light source mounted on both sides. The S. obliquus FSP-3 was precultured and inoculated into the PBR with a inoculum size of 40 mg/L. The PBR was operated at 28 °C, pH 6.0, and an agitation rate of 300 rpm. Serving as the sole carbon source, 2.5% CO2 was fed into the culture continuously during 6–10 day cultivation. The liquid samples were collected from the sealed glass vessel to determine microalgae cell concentration, culture pH, residual nitrate concentration, and lutein content. 2.3. Determination of microalgal biomass concentration The biomass concentration of S. obliquus FSP-3 in PBR was determined regularly using UV/VIS spectrophotometer via the measurement of optical density at a wavelength 685 nm (denoted as OD685). The absorbance values were converted to dry cell weight (denoted as DCW) concentration via the appropriate calibration (i.e., 1.0 OD685 equals to 0.2–0.3 g DCW/L). Intermittent measurements of DCW were performed to confirm the calibration relationship between the values of OD685 and DCW concentration. 2.4. Determination of growth kinetics parameters The specific growth rate (l, day1) of microalgal culture was calculated with the equation shown below:
l¼
d ln X dt
ði:e:; maximum slope from lynx versus plotÞ
max
where X and t indicate the biomass concentration (g/L) and cultivation time (day), respectively. The overall biomass productivity (Poverall, g DCW/L/d) was calculated as follows:
Poverall ¼
DX Dt
where DX is the variation of biomass concentration (g DCW/L) within a cultivation time of DtðdÞ. 2.5. Determination of residual nitrogen concentration The residual nitrate concentration was determined by a modified method reported in our previous study (Ho et al., 2013b). In general, microalgae samples were taken from the cultures and filtered through a 0.22 lm pore size filter and then diluted 30-fold with DI water for each sample. The absorbance (at 220 nm) of the filtered solution was measured with an UV/VIS spectrophotometer (model U-2001, Hitachi, Tokyo, Japan). The NH+4 concentration of the culture medium was determined with an ammonia photometer (model HI 93715, Hanna Instruments, Italy). A liquid sample collected from the PBR was filtered using a 0.22 lm pore size filter and then 10 mL was taken with 4 drops of specific reagents for each sample. After setting for 3.5 min, the sample was examined with an ammonia photometer. The urea concentration of the culture medium was analyzed with the manual urease procedure proposed by Butler et al. (1996). Samples (1.0 mL each) were taken from the microalgal culture, and then centrifuged at 10,000 rpm for 5 min. The supernatant (100 lL) was taken and then mixed with 0.5 mL urease solution, which was incubated in a 37 °C water bath for 10 min. After that, the reaction mixture was removed from the water bath, and the reaction was stopped by the addition of 1.0 mL phenol nitroprusside (enzyme terminator) and 1.0 mL alkaline hypochlorite. After setting at room temperature for 30 min, the absorbance
Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062
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The lutein was extracted from microalgae by the modified protocols reported in our previous study (Chan et al., 2013). In general, 10–15 mg dry microalgal biomass was treated with 1 mL KOH solution (at a concentration of 60% w/w) to hydrolyze the lipids and assist cell disruption. After the alkaline treatment, diethyl ether was added into the mixture placed in 40 °C hot-water bath to extract lutein for 40 min. When the color of microalgal biomass turned clear, the supernatant phase was collected for the analysis. The lutein content of the solvent extracts was measured by purging the organic solvent with a N2 flow and then dissolving the precipitate in acetone for the analysis by high-performance liquid chromatography (HPLC) (Taylor et al., 2006). The separation column used in HPLC was YMC Carotenoid RP-30 (particle size 5 lm) column (4.6 mm 250 mm) (YMC Europe, Schermbeck, Germany). The binary mobile phase consisted of (A) 3% ddH2O in methanol containing 0.05 M ammonium acetate, and (B) 100% TBME (tert-butyl methyl ether). Both mobile phases contained 0.1% (w/v) BHT and 0.05% triethylamine (TEA). The extracts were eluted at a flow rate of 1 mL/min and the lutein content was detected by measuring absorbance at the wavelength range of 220–750 nm. Three absorbance wavelengths were used (i.e., 450, 652, and 478 nm) and the maximal absorbance (450 nm) was chosen for quantification of lutein extracts. The lutein standards used for quantification purpose were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3. Results and discussion
4
3
2
1
5
3.1. Effect of nitrogen sources on cell growth and the lutein content of S. obliquus FSP-3 Literature shows that selecting suitable nitrogen source is of importance in improving biomass productivity of microalgal species (Hsieh and Wu, 2009). However, the effect of nitrogen source on lutein accumulation in microalgae is still not clear. To investigate an optimal nitrogen source for cell growth and lutein accumulation of S. obliquus FSP-3, three different nitrogen sources (Ca(NO3)2, (NH4)2SO4 and urea) at an identical concentration of 8.0 mM were examined, respectively. As shown in Fig. 1, under the same culture conditions, nitrate appeared to support better growth of S. obliquus FSP-3 than NH4 and urea. The biomass production using nitrate as nitrogen source was around 2–3-fold higher than that obtained from using NH4 and urea, indicating that nitrate is the favorable nitrogen source for the growth of S. obliquus FSP-3. This result was consistent with results obtained from the cultivation of other microalgal species. For example, the growth of Neochloris oleoabundans was better when using nitrate as the nitrogen source when compared with using NH4 and urea (Li et al., 2008). However, the other microalgal species, Ellipsoidion
Nitrogen concentration (mM)
0 8
4 6 3 4
2
2
1
0
Lutein content (mg/g DCW)
2.6. Determination of the lutein content of microalgal biomass
sp., had a higher growth rate with ammonia (Xu et al., 2001). Hence, the suitable nitrogen source for the microalgal growth is largely species-dependent (Hsieh and Wu, 2009). Ammonium is usually favorable over nitrate as a nitrogen source by microalgae due to no redox reaction and less energy required for its assimilation (Ramanna et al., 2014). However, the lowest biomass production obtained from S. obliquus FSP-3 with NH4 may be attributed to a sharp decrease in pH from 7.0 to around 5.0 during the assimilation of ammonium ions (data not shown). Wu et al. (2013) also reported similar results, showing that ammonium consumption by Monoraphidium sp. SB2 resulted in a pH decrease from 8.0 to 4.4 during the cultivation. On the other hand, urea dissociates to form CO2 and ammonium in solution via the urea amino hydrolase pathway (urease enzyme) (Ramanna et al., 2014). The lower biomass production with urea-N might be due to a relatively low urease activity of this strain. Moreover, Fig. 1 shows that the lutein content increased gradually along with the consumption of NH4 and urea, while the maximum lutein content was obtained at the beginning of nitrate depletion, while the lutein content started to decrease under the
Biomass concentration (g/L)
of the resulting mixture was measured at a wavelength of 570 nm and the value of absorbance was converted to urea concentration via appropriate calibration.
0 0
1
2
3
Time (d)
4
5
Lutein content (Nitrate) Lutein content (Ammonia) Lutein content (Urea) Biomass and Nitrogen (Nitrate) Biomass and Nitrogen (Ammonia) Biomass and Nitrogen (Urea)
Fig. 1. Time-course profiles of biomass concentration, nitrogen source concentration, lutein content and lutein productivity during the growth of S. obliquus FSP-3 using different nitrogen sources. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s).
Table 1 Effect of different nitrogen sources on biomass production, biomass productivity, lutein content and lutein productivity of S. obliquus FSP-3. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s). Nitrogen source
Biomass production (g/L)a
Biomass productivity (mg/L/day)a
Lutein content (mg/g)a
Lutein productivity (mg/L/day)a
Ca(NO3)2 Urea (NH4)2SO4
1.84 ± 0.08 0.96 ± 0.05 0.60 ± 0.04
942.0 ± 14.1 477.8 ± 8.4 286.3 ± 6.1
4.61 ± 0.11 2.18 ± 0.13 1.58 ± 0.08
4.35 ± 0.09 1.04 ± 0.07 0.45 ± 0.03
Data shown in this table are average values of triplicates ± SD (standard deviation). a The values were calculated at the time when the maximal lutein productivity was reached.
Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062
S.-H. Ho et al. / Bioresource Technology xxx (2014) xxx–xxx
20
4
15
3 2
10
1
5
Biomass concentration (g/L)
0
0 25
16mM Nitrate
5
20
4
15
3 2
10
1
5
Biomass concentration (g/L)
0
0 25
24mM Nitrate
5
20
4
15
3 2
10
1
5
0
0 0
1
2
3
Time (d)
4
5
5 4 3 2 1
Lutein content & productivity (mg/g, mg/L/d)
25
8mM Nitrate
5
0 5 4 3 2 1
Lutein contnt & productivity (mg/g, mg/L/d)
Biomass concentration (g/L)
Our previous study showed that nitrogen availability is a vital factor affecting the lutein accumulation of microalgae during
0 5 4 3 2 1
Lutein content & productvity (mg/g, mg/L/d)
3.2. Effect of nitrate concentration on cell growth and lutein content of S. obliquus FSP-3
Nitrate concentration (mM)
cultivation (Ho et al., 2014a). However, the nitrogen concentration in media used for producing lutein from S. obliquus FSP-3 was not optimized. Thus, in this study, modified Detmer’s Medium containing 8, 16 and 24 mM calcium nitrate, respectively, were examined. The time-course profiles of cell growth and lutein production obtained with different nitrate concentrations are shown in Fig. 2. As indicated in Fig. 2 and Table 2, the biomass concentration of S. obliquus FSP-3 improved significantly when the nitrate concentration increased from 8 to 24 mM. However, the biomass productivity was slightly lower when using higher nitrate concentration. This could be attributed to more severe light shading effects arising from the higher cell density obtained from using higher nitrate concentration. Moreover, Fig. 2 shows that in all different nitrate concentrations groups, the maximum lutein content occurred at the beginning of nitrate depletion, and increased slightly from 4.61 to 4.95 mg/g as nitrate concentration increased from 8 to 24 mM (Table 2). Similar result was also reported in other studies with other microalgal strains, showing an improvement in lutein content when increasing nitrogen concentration (Cordero et al.,
Nitrate concentration (mM)
nitrate depletion condition. It was suggested that nitrogen-sufficient condition was required for lutein accumulation (Xie et al., 2013). As shown in Table 1, the lutein content of S. obliquus FSP3 obtained from using nitrate as the nitrogen source was nearly 1–2 folds higher than that obtained from using NH4 and urea. In contrast, Del Campo et al. (2000) reported that the nitrogen sources (NaNO3, NH4Cl and NH4NO3) did not influence the lutein level in Muriellopsis sp. This means that the correlation of nitrogen source and lutein content may also be species-specific. Further, the highest lutein productivity (4.35 mg/L/day) was also obtained when using nitrate as nitrogen source. Thus, it is clear that among the three nitrogen sources examined, nitrate is the most favorable nitrogen source for both cell growth and lutein accumulation of S. obliquus FSP-3 under the culture conditions.
Nitrate concentration (mM)
4
0
6 Lutein content Lutein productivity Biomass concentration Nitrate concentration
Fig. 2. Time-course profiles of biomass concentration, nitrate concentration, lutein content and lutein productivity during the growth of S. obliquus FSP-3 using different nitrate concentrations. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s).
Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062
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S.-H. Ho et al. / Bioresource Technology xxx (2014) xxx–xxx
Table 2 Effect of different nitrate concentrations on biomass production, biomass productivity, lutein content and lutein productivity of S. obliquus FSP-3. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s). Nitrate concentration (mM)
Culture time (day)a
Biomass production (g/L)a
Biomass productivity (mg/L/day)a
Lutein content (mg/g)a
Lutein productivity (mg/L/day)a
8 16 24
1.9 3.8 4.9
1.84 ± 0.08 3.38 ± 0.14 4.30 ± 0.21
942.0 ± 14.1 872.1 ± 10.9 859.1 ± 11.3
4.61 ± 0.11 4.87 ± 0.16 4.95 ± 0.22
4.35 ± 0.09 4.25 ± 0.12 4.25 ± 0.14
Data shown in this table are average values of triplicates ± SD (standard deviation). a The values were calculated at the time when the maximal lutein productivity was reached.
2011; Xie et al., 2013). As the biomass concentration increased with the cultivation time under high nitrate concentration, the light may hardly penetrated to cell broth and the specific light intensity (i.e., the amount of light intensity received per cell) decreased sharply (Cheirsilp and Torpee, 2012). Thus, the size of the light-harvesting antenna complex should be increased to absorb more light energy, resulting in higher lutein content (Solovchenko et al., 2008). As shown in Table 2, the highest lutein productivity of S. obliquus FSP-3 (4.35 mg/L/day) was obtained with 8 mM nitrate concentration, which is slightly higher than that obtained with the other nitrate concentrations. This result might be due to the highest biomass productivity with 8 mM nitrate concentration. In addition, when using a nitrate concentration of 8 mM, the culture time required to reach the highest lutein content was significantly shorter than that required for other nitrate concentrations examined. This would benefit the economic feasibility of lutein production. When the nitrate concentration was decreased to 4 mM or 2 mM, the overall biomass productivity slightly dropped due to the presence of a longer lag phase in the early stage of the cultivation (data not shown). Moreover, low nitrate concentration is also not conducive to the accumulation of lutein because of the excess light exposure. Therefore, based on the results shown above, 8 mM seems to be the best nitrate concentration for lutein production of S. obliquus FSP-3. 3.3. Improvement of the lutein content of S. obliquus FSP-3 by twostage process with light-intensity shift Many environmental stresses can lead to changes in the biochemical composition and cell growth of microalgae, such as nitrogen depletion (Ho et al., 2013a), light intensity (Xie et al., 2013), and high salinity (Ho et al., 2014b). Among them, light intensity seems to be the most direct factor influencing the production of photosynthesis-associated pigments, such as lutein, while the effects of light intensity on lutein production of microalgae, as reported in the literature, have been very diverse. Sanchez et al. (2008) reported that elevating the light intensity could increase
the lutein content of Scenedesmus almeriensis. In contrast, Solovchenko et al. (2008) found that the lutein content increases upon the decrease of light supply. This difference might be associated with the function of lutein, which could play a role in either light protection or light energy capture (Demmig-Adams and Adams, 1996). Our recent work also investigated the relationship between the lutein content and irradiance of the S. obliquus FSP3 strain, and observed a higher lutein content when using lower light intensity (Ho et al., 2014a), while on the other hand higher light intensity is favorable for cell growth. Accordingly, in this study, a two-stage process with a switch in light intensity was attempted to enhance the lutein productivity of S. obliquus FSP-3. In the first stage, microalgae were cultivated under a higher light intensity of 300 lmol/m2/s to facilitate cell growth. At difference nitrogen consumption stages (i.e., 70%, 80%, 90% and 95% of nitrogen source consumption), the microalgae culture was shifted to lower irradiance (75 lmol/m2/s) in the second stage to stimulate the lutein accumulation until nitrogen starvation occurred (i.e., 100% nitrogen source consumption). Single-stage cultivation at a fixed light intensity of 300 lmol/m2/s was performed as the control. As indicated in Table 3, switching the light intensity on microalgae culture to a lower level of 75 lmol/m2/s could indeed lead to an significant increase in lutein content. In particular, the light intensity shift at 70% nitrogen consumption resulted in a lutein content increase from 4.20 to 5.04 mg/g. The light intensity shift activated at 80%, 90%, and 95% nitrogen consumption also showed a slight increase in the lutein content when compared to the control group (Table 3). However, because of the lower biomass productivity in the second stage due to lower irradiance on the culture, the overall lutein productivity in this two-stage operation is still markedly lower when compared to the control, in which the microalga was grown in a single-stage process with a constant light intensity of 300 lmol/m2/s. In summary, using the two-stage operation strategy, the lutein productivity was not improved when compared to the single-stage operation, whereas the lutein content could be promoted to a higher level when applying the light intensity shift strategy. The
Table 3 Effect of different light-intensity (from 300 lmol/m2/s to 75 lmol/m2/s) and switching timing (at 70%, 80%, 90% and 95% of nitrogen consumption) on biomass productivity, lutein content and lutein productivity of S. obliquus FSP-3. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s; nitrogen source, calcium nitrate at 8.0 mM). Percentage of nitrogen consumptiona (%)
Lutein accumulation timec (hr)
Biomass productivity (mg/L/day)
Lutein content (mg/g)
70% 80% 90% 95% Controlb
44.0 ± 2.1 16.5 ± 1.3 10.5 ± 1.1 3.0 ± 0.5 –
572.4 ± 19.1 765.3 ± 28.8 836.8 ± 24.7 901.2 ± 29.6 942.0 ± 14.1
4.20 ± 0.09 4.25 ± 0.13 4.41 ± 0.06 4.57 ± 0.16
Before light intensity shift
After light intensity shift
5.04 ± 0.15 4.89 ± 0.11 4.75 ± 0.09 4.69 ± 0.12 4.61 ± 0.11
Lutein productivity (mg/L/day)
2.88 ± 0.07 3.74 ± 0.10 3.97 ± 0.07 4.23 ± 0.14 4.35 ± 0.09
a
The switch point of light-intensity was based on the percentage of nitrogen consumption (%). Single-stage operation at a fixed light intensity of 300 lmol/m2/s without light intensity shift. The time interval starting from the shift of light intensity from 300 to 75 lmol/m2/s till the time when nitrogen starvation was reached (i.e., the time when 100% nitrate consumption occurred). b
c
Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062
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Table 4 Performance of semi-batch culture with different broth replacement ratios in terms of biomass productivity, lutein content and lutein productivity of S. obliquus FSP-3. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s; nitrogen source, calcium nitrate at 8.0 mM). Replacement ratio (%)
Time interval (hr)
Biomass productivity (mg/L/day)
Lutein content (mg/g)
Lutein productivity (mg/L/day)
10% 30% 50% 70% Batch
4±0 13.3 ± 1.2 22.5 ± 0.8 31.9 ± 1.2 48(2 days)
1304.8 ± 33.7 1159.8 ± 102.5 1118.2 ± 37.7 1087.2 ± 30.6 942.0 ± 14.1
4.61 ± 0.16 4.53 ± 0.21 4.42 ± 0.14 4.54 ± 0.17 4.61 ± 0.11
6.01 ± 0.24 5.25 ± 0.44 4.95 ± 0.27 4.94 ± 0.27 4.35 ± 0.09
2.2 10% replacement
3
2.0
2 1.9
1 0 0.6
0.8 30% replacement
5
2.0
4
1.8
3
1.6
2
1.4
1
0.5
1.0
1.5
2.0
2.4
Lutein content (mg/g DCW)
0.4
0 3.0
2.5 50% replacement
2.2
5
2.0
4
1.8 3
1.6 1.4
2
1.2
Lutein content (mg/g DCW)
Biomass concnetration (g/L)
0.2
2.2
0.0
1
1.0 0.8
0 0
Biomass concentration (g/L)
Lutein conent (mg/g DCW)
4
0.0
Biomass concentration (g/L)
5
2.1
1
2
3
4
5 6
70% replacement 5
2.0
4 1.5
3 2
1.0
Lutein content (mg/g DCW)
Biomass concentration (g/L)
Data shown in this table are average values of five replicates ± SD (standard deviation).
1 0.5 0 0
2
4
6
Time(d) Fig. 3. Profiles of cell growth and lutein content of S. obliquus FSP-3 cultivated with semi-continuous operation with intermittent replacement of culture broth with various replacement ratios (10%, 30%, 50%, 70%). (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.4 vvm; light intensity, 300 lmol/m2/s; nitrogen source, calcium nitrate at 8.0 mM).
Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062
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S.-H. Ho et al. / Bioresource Technology xxx (2014) xxx–xxx Table 5 Comparison of the performance of lutein content and lutein productivity in this study and in other related studies. Microalga source
Operation mode
Chlorella protothecoides Chlorella protothecoides Chlorella zofingiensis Chlorella zofingiensis Chlorella fusca Chlorella proboscideum Muriella aurantiaca Murielladecolor Muriellopsis sp. Neospongiococcus gelatinosum Desmodesmus sp. F2 Muriellopsis sp. Scenedesmus almeriensis Coccomyxa acidophila Scenedesmus obliquus FSP-3 Scenedesmus obliquus FSP-3 Scenedesmus obliquus FSP-3
Batch Batch Batch Batch Batch Batch Batch Batch Batch Batch Pulse-feeding Continuous Continuous Batch Batch Two-stage Semi-batch
Cultivating conditions Light supply
Metabolic mode
Absence of light Absence of light Continuous light Continuous light Continuous light Continuous light Continuous light Continuous light Continuous light Continuous light Continuous light Sunlight period Sunlight period Continuous light Continuous light Continuous light Continuous light
Heterotrophic Heterotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Autotrophic Mixotrophic Autotrophic Autotrophic Autotrophic
increase in lutein content per unit cell may lead to a decrease in the cost for product separation and purification, especially when the process is scaled up. Therefore, the proposed two-stage process may still have the niche. To further enhance the lutein content and productivity of the two-stage process, the light intensity employed in each stage and the duration time of the second stage may need to be optimized to achieve the best performance. 3.4. Improvement of lutein productivity of S. obliquus FSP-3 by using semi-batch operation From the results shown above, using two-stage operation could improve the lutein content of the FSP-3 strain but with a side-effect of lower biomass productivity. Therefore, another engineering strategy must be developed to further enhance the lutein productivity. As depicted in Fig. 2, in batch culture, using calcium nitrate dehydrate at a concentration of 8 mM as the nitrogen source resulted in the highest lutein productivity of 4.35 mg/L/ day. Therefore, this nitrogen source condition was adapted for conducting semi-continuous operations, aiming to enhance the biomass productivity, while maintaining the lutein content at a relatively high level (Chiu et al., 2009; Ho et al., 2013b). Before the semi-continuous operation, S. obliquus FSP-3 was cultivated for two days until the biomass concentration reached 1.9–2.1 g/L, at which the microalgal strain had the maximal lutein content and maximal lutein productivity, which has the same trend as earlier results shown in Fig. 2. The operation mode was then changed to semi-continuous cultivation by intermittent replacement of culture broth with fresh medium at different replacement ratios. As shown in Table 4, the replacement ratios included 10% replacement (at intervals of 4 h), 30% replacement (at intervals of 12–15 h), 50% replacement (at intervals of 21–24 h) and 70% replacement (at intervals of 30–34 h),. The new cycle was started when the biomass concentration again reached 1.9–2.1 g/L again (Fig. 3). As shown in Fig. 3, the results of semi-continuous operation, such as biomass productivity and lutein content, were consistent during the five cycles with four replacement ratios, suggesting that the operation of semi-continuous is stable and feasible for industrial production of lutein from S. obliquus FSP-3. Table 4 summarizes the biomass and lutein production performance of semibatch operation of S. obliquus FSP-3 under the four medium replacement ratios. There is no significant difference in lutein content for the semi-continuous cultures at all the four replacement ratios used, as the values fall within the range of 4.40–4.60 mg/g.
Lutein content (mg/g)
Lutein productivity (mg/L/day)
References
1.9 4.6 2.8 3.5 4.2 3.4 2.6 0.5 5.5 4.4 5.1 4.3 5.5 3.5 4.6 4.7 4.6
3.6 12.0 1.3 1.2 0.8 1.2 3.1 1.3 4.3 6.0 3.6 7.2 4.9 0.9 4.4 4.2 6.0
Wei et al. (2008) Shi et al. (2000) Del Campo et al. (2000) Del Campo et al. (2004) Del Campo et al. (2000) Del Campo et al. (2000) Del Campo et al. (2000) Del Campo et al. (2000) Del Campo et al. (2000) Del Campo et al. (2000) Xie et al. (2013) Sanchez et al. (2008) This study This study This study
However, semi-continuous operation with 10% medium replacement could achieve the highest biomass productivity and lutein productivity of 1304.8 and 6.01 mg/L/day, respectively. As shown in Table 5, the biomass productivity and lutein productivity are much higher than those obtained from batch culture (942.0 and 4.35 mg/L/day, respectively) and are also higher than most of the reported values in related studies (Del Campo et al., 2000; Hodaifa et al., 2009; Mandal and Mallick, 2009; Sanchez et al., 2008; Wei et al., 2008). To the best of our knowledge, this is the first study reporting the use of semi-continuous cultivation to improve lutein production from microalgae. By using this strategy, the highest lutein productivity of 6.01 ± 0.24 mg/L/day was achieved, which is approximately 40% higher than that obtained with the batch system. This demonstrates the feasibility of using semi-continuous process with 10% medium replacement ratio for lutein production from the lutein-rich S. obliquus FSP-3 strain. 4. Conclusions Calcium nitrate at a concentration of 8 mM was the favorable nitrogen source condition for both cell growth and lutein production of S. obliquus FSP-3. The two-stage operation with light intensity shift elevated the lutein content to 5.04 mg/g, but obtained lower lutein productivity due to a marked decrease in biomass productivity. The semi-continuous operation with 10% medium replacement ratio effectively enhanced microalgal biomass (1304.8 mg/L/day) and lutein productivity (6.01 mg/L/day), which are higher than most reported values. This semi-continuous strategy should be further assessed in outdoor scale-up cultivation to evaluate its feasibility in commercial production of microalgal lutein. Acknowledgements The authors gratefully acknowledge the support from the Top University Project of NCKU, Energy Technology Program for Academia of Bureau of Energy of Ministry of Economic Affairs, and from Taiwan’s Ministry of Science and Technology (MOST) under grant numbers 103-3113-P-006-006, 102-3113-P-110-011 and 1012221-E-006-209-MY3. References Astorg, P., 1997. Food carotenoids and cancer prevention: an overview of current research. Trends Food Sci. Technol. 8 (12), 406–413.
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Please cite this article in press as: Ho, S.-H., et al. Effects of nitrogen source availability and bioreactor operating strategies on lutein production with Scenedesmus obliquus FSP-3. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.10.062