Simultaneous enhancement of CO2 fixation and lutein production with thermo-tolerant Desmodesmus sp. F51 using a repeated fed-batch cultivation strategy

Biochemical Engineering Journal 86 (2014) 33–40

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Regular article

Simultaneous enhancement of CO2 fixation and lutein production with thermo-tolerant Desmodesmus sp. F51 using a repeated fed-batch cultivation strategy You-Ping Xie a , Shih-Hsin Ho b , Chun-Yen Chen c , Ching-Nen Nathan Chen d,e , Chen-Chun Liu f , I.-Son Ng a , Ke-Ju Jing a , Sheng-Chung Yang g , Chi-Hui Chen g , Jo-Shu Chang c,f,h,∗ , Ying-Hua Lu a,i,∗∗ a

Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, PR China Organization of Advanced Science and Technology, Kobe University, Kobe, Japan c University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan d Institute of Marine Biology, National Sun Yat-sen University, Kaohsiung 804, Taiwan e Asia-Pacific Ocean Study Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan f Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan g Department of Energy and Agile System, Metal Industries Research and Development Centre, Kaohsiung 811, Taiwan h Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan 701, Taiwan i Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, PR China b

a r t i c l e

i n f o

Article history: Received 20 November 2013 Received in revised form 4 February 2014 Accepted 23 February 2014 Available online 2 March 2014 Keywords: Microalgae, Lutein Fed-batch culture Desmodesmus sp. Bioreactors Bioprocess design

a b s t r a c t This study aimed to improve lutein production using a thermo-tolerant lutein-rich microalga Desmodesmus sp. F51. To achieve this goal, four fed-batch cultivation strategies were investigated for CO2 fixation and lutein production of Desmodesmus sp. F51. Among them, Fed-batch IV showed the best performance, giving the highest CO2 fixation rate and lutein productivity of 1582.4 mg/L/d and 3.91 mg/L/d, respectively. Both increasing the light intensity and limiting the nutrients led to a lower carotenoids content in the microalga, with a higher proportion of lutein and lower proportion of ␤-carotene being obtained in the carotenoids. The carotenoid present in the biomass was mainly lutein, accounting for 50–66% of total carotenoids. Repeated operations of the Fed-batch IV strategy could effectively improve CO2 fixation and lutein production of Desmodesmus sp. F51, giving the best results of 1826.0 mg/L/d, and 4.61 mg/L/d, respectively. This performance is better than most of the previously reported values. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Lutein is one of the most widely used carotenoids, as a food supplement or pharmaceutical treatment, due to its effectiveness with regard to protecting the eyes and potential ability to prevent or ameliorate the effects of various degenerative human diseases [1]. A daily lutein intake of 1 mg/kg body weight was suggested by the European Food Safely Authority (EFSA) [2], while the lutein

∗ Corresponding author at: Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan. Tel.: +886 6 2757575x62651; fax: +886 6 235714. ∗∗ Corresponding author at: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, PR China. Tel.: +86 592 2183751; fax: +86 592 2186400. E-mail addresses: [email protected] (J.-S. Chang), [email protected] (Y.-H. Lu). http://dx.doi.org/10.1016/j.bej.2014.02.015 1369-703X/© 2014 Elsevier B.V. All rights reserved.

market was estimated at nearly $233 million in 2010, and expected to reach $309 million in 2018 [3]. Marigolds are currently the primary commercial lutein production source. However, there is an increasing interest in using microalgae as an alternative source of lutein, due to the higher lutein content and productivity compared to marigolds [1]. Using phototrophic microalga strains to produce lutein is preferable due to the high lutein content [4,5], with the additional benefit of CO2 fixation [6]. The literature shows that the cell growth rate, lutein content and lutein productivity of some microalgae are mainly influenced by the culture conditions, such as light intensity [7] and nitrogen concentration [4], as well as the operation modes, such as batch [4], fed-batch [8], semi-batch [9], and continuous cultivation [10]. Under phototrophic conditions, light energy is used to drive photosynthesis reactions, thereby playing an important role in the cell growth of microalgae. However, the culture conditions usually affect the lutein content and biomass

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productivity of microalgae in an opposite way [1]. For example, high irradiance usually enhances microalgae cell growth rate, but could simultaneously cause a decrease in the lutein content [7,8]. A nitrogen-sufficient condition is required for cellular accumulation of lutein due to the requirement of continued synthesis of light-harvesting proteins under optimal growth conditions [11]. Moreover, our previous study showed that fed-batch cultivation with pulse-feeding of 2.2 mM nitrate was effective in enhancing the lutein production of Desmodesmus sp. F51 [8]. However, retardation of cell growth, due to light limitation and nutrient deficiency with prolonged cultivation time, led to reductions in the nitrate consumption rate, cell growth rate and lutein accumulation rate. It has been suggested that either optimizing the feeding rate of the concentrated medium [12], or making stepwise increases in light intensity [13], could be used to enhance biomass productivity in fed-batch cultivation. On the other hand, several studies demonstrate that semi-batch operation is a relatively straightforward way to maintain the culture at the exponential phase, resulting in higher biomass productivity and CO2 fixation ability [6,14]. Moreover, compared with other cultivation modes, semi-batch cultivation has greater potential to achieve low-cost and large-scale microalgae cultivation [14]. However, no efforts have yet been made to evaluate combined strategies (e.g., semi-batch operation combined with fed-batch cultivation, or repeated fed-batch cultivation) for simultaneously increasing CO2 fixation and lutein production of microalgae. In this study, Desmodesmus sp. F51, a thermo-tolerant luteinrich microalga [8], was selected as the target strain to explore engineering strategies to improve lutein production efficiency. The influence of several cultivation strategies on the biomass production and lutein accumulation was investigated, with the aim of optimizing the fed-batch process. An innovative strategy combining semi-batch operation and fed-batch cultivation was further assessed for its effectiveness in improving both CO2 fixation and lutein production simultaneously.

2. Material and methods 2.1. Microalgal strain and its preculture conditions The thermo-tolerant Desmodesmus sp. F51, isolated from southern Taiwan, was obtained from Prof. Ching-Nen Nathan Chen’s Laboratory at National Sun Yat-sen University, Taiwan. The medium used for the preculture of the strain was modified Bold 3 N medium [8]. The microalga was regularly grown at a temperature of 26–28 ◦ C for 4–5 days with a continuous supply of 2.5% CO2 at an aeration rate of 0.2 vvm. The microalgal culture was illuminated 24 h a day with a light intensity of approximately 60 ␮mol/m2 /s. The light intensity was measured by a Li-250 Light Meter with a Li-190SA pyranometer sensor (Li-COR Inc., Lincoln, Nebraska, USA).

2.2. Operation of photobioreactor The photobioreactor (PBR) used to grow the target microalgal strain was a 1-L glass tubular vessel (15.5 cm in length and 9.5 cm in inner diameter) equipped with an external light source (14 W TL5 tungsten filament lamps, Philips Co., Taipei, Taiwan) mounted on both sides of the PBR. The Desmodesmus sp. F51 strain was precultured and then inoculated into the photobioreactor with an inoculum size of 70 mg/L. The microalga was grown in the PBR with modified Bristol’s medium according to the procedures described in Xie et al. [8].

2.3. Operation of fed-batch cultivation The fed-batch process was started up on batch mode with an initial nitrate concentration of 8.8 mM and a light intensity of approximately 600 ␮mol/m2 /s. When the initial nitrate was depleted, the 100-fold concentrated nitrate solution or concentrated fresh medium was then pulse-fed into the culture to reach a nitrate concentration of 2.2 mM. The timing for starting new pulsefeeding was when the nitrate concentration in the medium was nearly depleted. The fed-batch process was also operated with a stepwise increase of light intensity. In this process, the initial light intensity was set at 300 ␮mol/m2 /s, and was then increased at a rate of 150 ␮mol/m2 /s per day until reaching the maximal intensity of 1050 ␮mol/m2 /s. 2.4. Operation of repeated fed-batch cultivation The process was started with the fed-batch cultivation with medium feeding and stepwise increases in light intensity, as described above. When the cell concentration reached about 6.0 g/L, 35–40% volume of the culture broth was replaced by sterile deionized water, and then the concentrated fresh medium was added into the culture to reach a nitrate concentration of 2.2 mM. After culture broth replacement, the cultivation was operated in the same manner as the fed-batch process with medium feeding. The timing for starting a new cycle was when the microalgal biomass concentration recovered to approximately 6.0 g/L. The liquid sample was collected from the culture broth before and after medium replacement to determine microalgae cell concentration, pH, lutein content, and nitrate concentration. 2.5. Determination of cell growth and CO2 fixation rate Microalgae biomass concentration was determined by measuring the optical density of the sample at a wavelength of 685 nm (denoted as OD685 ) using a UV/Vis spectrophotometer (model U2001, Hitachi, Tokyo, Japan) after proper dilution with deionized water. The OD685 values were converted to biomass concentration via appropriate calibration between OD685 and dry cell weight, and the conversion factor was determined as 1.0 OD685 = 0.30 ± 0.02 g/L. The biomass productivity (Pbiomass ) during the culture period was calculated from the equation: Pbiomass (g DCW/L/d) =

X t

where X is the variation of biomass concentration (g/L) within a cultivation time of t (d).   The CO2 fixation rate PCO2 was calculated as follows:

 PCO2 (mg/L/d) = Pbiomass Ccarbon

MCO2



MC

where Pbiomass is the biomass productivity (mg/L/d), Ccarbon is the carbon content of the biomass (g/g) determined with an elemental analyzer (Elementar Vario EL III), MCO2 is the molar mass of CO2 , and MC is the molar mass of carbon. 2.6. Determination of nitrate concentration The nitrate concentration in the medium was determined by a colorimetric method described in Ho et al. [6]. The calibration between the absorbance and nitrogen concentration was established using sodium nitrate as the standard.

Y.-P. Xie et al. / Biochemical Engineering Journal 86 (2014) 33–40

3. Results and discussion

8

6

Biomass (g/L)

(a)

Fed-batch I Fed-batch II Fed-batch III Fed-batch IV

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3.1. Effect of fed-batch cultivation strategies on cell growth and CO2 fixation

5 4 3 2 1 0

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0.85 0.80 0.75 0.70 0.65 0.60 0.55 0

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Time (d) Fig. 1. Time-course profiles of (a) biomass concentration, (b) overall biomass productivity of Desmodesmus sp. F51 with different fed-batch cultivation strategies (operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; temperature, 35 ◦ C). Arrows indicate the time of increasing light intensity (increase in an interval of 150 ␮mol/m2 /s).

2.7. Determination of total carotenoids content and carotenoids composition Total carotenoids content was determined by using the procedures described by Chan et al. [15]. The optical density of the resulting solution was measured at a wavelength of 444 nm in a spectrophotometer. The amount of total carotenoids was calculated as follows: Ct = 4.32OD444 − 0.0439 After extraction of total carotenoids, the composition in the extracts was analyzed using high performance liquid chromatography (Finnigan Surveyor, Thermo Scientific, Germany), following the same conditions and procedures proposed by Taylor et al. [16]. The carotenoids were detected by measuring absorbance at 450 nm. Lutein and ␤-carotene were identified using authentic standards (Sigma). The lutein productivity (Plutein ) was determined based on the calculation indicated below:

Plutein (mg/L/d) =

Our recent study showed that fed-batch cultivation with pulsefeeding of 2.2 mM nitrate appeared to effectively enhance biomass production, while simultaneously achieving relatively high lutein content [8]. However, light limitation and nutrient deficiency with prolonged cultivation time occurred during the operation, resulting in lower cell growth, thus leading to a lower biomass productivity and lutein production rate. To identify a more suitable fed-batch strategy for lutein production, four fed-batch cultivation strategies were investigated, as follows: (1) fed-batch cultivation with pulsefeeding of concentrated nitrate solution (denoted as Fed-batch I), (2) fed-batch cultivation with pulse-feeding of concentrated fresh medium (denoted as Fed-batch II), (3) fed-batch cultivation with pulse-feeding of concentrated nitrate solution and stepwise increases in light intensity (denoted as Fed-batch III), and (4) fedbatch cultivation with pulse-feeding of concentrated fresh medium and stepwise increases in light intensity (denoted as Fed-batch IV). Fig. 1 depicts the time–course profiles of cell growth and overall biomass productivity of strain F51 under different fed-batch cultivation strategies. It is clearly observed that before initiating the operation of the fed-batch culture (the initial 2.5 days of cultivation was carried out on batch mode), stepwise increases in light intensity from 300 ␮mol/m2 /s to 600 ␮mol/m2 /s (Fed-batches III and IV) gave a comparable cell growth and biomass productivity to those seen with a fixed light intensity of 600 ␮mol/m2 /s (Fed-batches I and II). This result indicates that an initial light intensity of 300 ␮mol/m2 /s is sufficient for cell growth. However, as the concentrations of both microalgal cells and products increase, light shielding effects may occur. Theoretically, the enhancement of light intensity from 300 to 600 ␮mol/m2 /s could thus maintain the high growth rate of the cells [17]. Indeed, our recent work indicated that at the beginning of the cultivation, the specific growth rate resulting from 300 and 450 ␮mol/m2 /s illumination was very similar to that from 600 ␮mol/m2 /s, whereas using light irradiation of 300 and 450 ␮mol/m2 /s led to a lower cell growth rate after 1.5 days cultivation, due primarily to the self-shading effect arising from the increase in cell density [8]. In this study, it was also observed that after 3 days cultivation, the biomass concentration obtained from using stepwise increases of light intensity from 300 to 1050 ␮mol/m2 /s (i.e., Fed-batch III (nitrate feeding) and IV (medium feeding)) was higher than that obtained from using a fixed light intensity of 600 ␮mol/m2 /s (i.e., Fed-batch I (nitrate feeding) and II (medium feeding), respectively) (Fig. 1a). Similar behavior was also reported by Cheirsilp and Torpee [13], showing that fed-batch cultivation with increasing light intensity is effective in avoiding the self-shading effects, thereby enhancing the cell growth of marine Chlorella sp. and Nannochloropsis sp. Moreover, Fig. 1b shows that the biomass productivity began to drop after cultivation for 2.5, 5.3, 2.5 and 7.2 days in Fed-batch I, II, III and IV operations, respectively. When compared with nitrate feeding (Fed-batch I and III), fed-batch with medium feeding (Fedbatch II and IV) achieved a relatively higher biomass concentration, which is consistent with the findings of Lababpour et al. [12], who reported that fed-batch cultivating with concentrated medium addition resulted in an increase in cell concentration of Haematococcus pluvialis. Bezerra et al. [14] also found that the depletion of some limiting nutrients likely decreased the cell productivity of

cumulative microalgae biomass production (g) × lutein content (mg/g) working volume (L) × cultivation time (d)

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Table 1 Performance of biomass production, CO2 fixation, and lutein production of Desmodesmus sp. F51 with different fed-batch cultivation strategies. Operation strategies

Biomass production (g/L)

Fed-batch I Fed-batch II Fed-batch III Fed-batch IV

3.26 4.23 3.69 6.28

± ± ± ±

0.05 0.05 0.04 0.06

Biomass productivity (g/L/d) 706.2 778.8 718.8 879.1

± ± ± ±

8.6 8.7 6.3 9.5

CO2 fixation rate (mg/L/d) 1214.7 1382.5 1265.1 1582.4

± ± ± ±

14.8 15.5 11.1 17.1

Lutein content (mg/g) 5.05 4.72 5.02 4.44

± ± ± ±

0.20 0.05 0.10 0.09

Lutein production (mg/L) 16.5 19.6 18.5 27.8

± ± ± ±

0.5 0.4 0.4 0.6

Maximal lutein productivity (mg/L/d) 3.56 3.67 3.61 3.91

± ± ± ±

0.10 0.08 0.07 0.12

The values are calculated at the time when the maximal lutein productivity was reached. Data are the averages of two runs ± SD (standard deviation).

Arthrospira platensis. The above findings indicate that nutrients, as well as light intensity, are the key factors affecting cell growth as the cultivation time is prolonged. As shown in Table 1, the highest biomass productivity of strain F51 was obtained with Fed-batch IV (879.1 mg/L/d), followed by with Fed-batch II (778.8 mg/L/d), Fed-batch III (718.8 mg/L/d), and Fed-batch I (706.2 mg/L/d). The reason why Fed-batch I resulted in lower biomass productivity was probably due to the light limitation and nutrient deficiency. The fed-batch cultivation associated with medium feeding (Fedbatch II) and stepwise increase in light intensity (Fed-batch III), individually, led to a slightly higher biomass productivity than that of Fed-batch I. However, a significant increase in biomass productivity was observed when using Fed-batch IV. This indicates that the combined effects of medium feeding and stepwise increases in light intensity on the growth of the microalgal cells are much more significant than the individual effects of the two factors (e.g., Fedbatches II and III). As a result, the highest biomass concentration of strain F51 obtained in Fed-batch IV (6.28 g/L) was nearly 30–90% higher than that obtained from other fed-batch strategies examined in this work (ranging from 3.26 g/L to 4.23 g/L) (Table 1). Since microalgal-CO2 fixation involves photoautotrophic growth of cells, the CO2 fixation capability of microalgae would positively correlate with their cell growth rate [6]. Therefore, the highest CO2 fixation rate was obtained from using Fed-batch IV (1582.4 mg/L/d). This CO2 fixation performance is also superior to that obtained from using other cultivation strategies (Table 2). Therefore, strain F51 with Fed-batch IV cultivation seems to be promising in terms of both biomass production and the potential for CO2 reduction. 3.2. Effects of fed-batch cultivation strategies on nitrate consumption and lutein production Our recent work indicated that strain F51 exhibited great potential for lutein production, in which the nitrogen availability as well as irradiance supply were closely related to the accumulation of lutein [8]. Therefore, the effects of the various fed-batch cultivation strategies on nitrate consumption and lutein production were investigated in the current work. As shown in Fig. 2, the nitrate consumption rate was almost the same for all groups during cultivation in the first 3 days, while it began to decrease after cultivation for 3.5, 4.5, and 4 days, respectively, for Fed-batch I, II, and III. However,

for Fed-batch IV the nitrate consumption remained stable as the cultivation time was prolonged. These findings suggest that both light limitation and nutrient deficiency could lead to a decrease in nitrate consumption rate, resulting in the lower cell growth rate as shown in Fig. 1. These findings are also quite similar to those observed in related studies. For instance, Chen et al. [21] reported that in the case of the growth of Spirulina platensis, using appropriately high light intensities usually led to higher cell growth rates, which also increased the nitrogen consumption rate. In addition, several studies have demonstrated that relatively high nutrient concentrations, such as those of iron, phosphorus and zinc, are required to support the nitrogen consumption of marine phytoplankton [22,23]. On the other hand, Fig. 2 shows that the lutein content increased during the early stage of fed-batch cultivation, but it started to decline after cultivation for 4.6, 5.3, and 5.1 days, respectively, for Fed-batches I–III. This phenomenon could be attributed to poor cell growth due to light limitation and nutrient deficiency. It has been suggested that primary xanthophylls, such as lutein and ␤-carotene, are structural and functional components of the photosynthetic apparatus of the cell, and therefore are associated with cell growth [11]. As shown in Fig. 2, the best timing for harvesting microalgal cells to achieve the maximal lutein content and productivity was different for the four Fed-batch strategies. For example, the best timing to harvest the cells for Fed-batches I and IV was the cultivation time of 4.6 and 7.0 days, respectively. The best harvesting timing for cell harvesting with high lutein productivity seems to greatly depend on the combined effects of how fast the cells grow, how fast the cells accumulate lutein, and how fast the nitrate content in the medium is depleted. Consequently, Fig. 2 clearly demonstrates that using Fed-batch IV (medium feeding combined with stepwise increases in light intensity) as the operation mode could not only enhance cell growth of strain F51, but also lead to a slight increase in lutein content as the cultivation time was prolonged. As shown in Table 1, the maximum lutein contents of strain F51 are quite similar when using nitrate feeding strategy (Fed-batch I and III), and around 6–13% higher than the lutein content obtained from using medium feeding (Fed-batch II and IV). Moreover, Figs. 2a and c also show that the maximum lutein content was obtained when the nitrate consumption rate started to decrease. These results suggest that

Table 2 Comparison of biomass productivity and CO2 fixation rate of Desmodesmus sp. F51 with those obtained from other microalgae species reported in the literature under phototrophic cultivation. Microalgae strains

Operation strategies

Biomass productivity (mg/L/d)

CO2 fixation rate (mg/L/d)

References

C. Vulgaris LEB-104 C. vulgaris P12 T. subcordiform Spirulina platensis S. obliquus CNW-N S. obliquus CNW-N S. obliquus CNW-N Desmodesmus sp. F51 Desmodesmus sp. F51

Batch Two-stage Batch Batch Batch Semi-batch Continuous Fed-batch IV Repeated Fed-batch IV

129.3 485.4 825.1 995 554.5 883.8 876.5 879.1 ± 9.5 1043.4 ± 60.6

251.6 912.5 1323.6 1650 959.2 1546.7 1532.5 1582.4 ± 17.1 1826.0 ± 106.1

[18] [19] [20] [21] [6] [6] [6] This study This study

Y.-P. Xie et al. / Biochemical Engineering Journal 86 (2014) 33–40

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Fig. 2. Time-course profiles of nitrate consumption, lutein content and lutein productivity of Desmodesmus sp. F51 with different fed-batch cultivation strategies (operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; temperature, 35 ◦ C).

Different microalgae strains usually have different carotenoids composition [25]. For photoautotrophic microalgae, the irradiance supply and nutrient availability can strongly affect the cell growth, as well as the accumulation of carotenoids [7]. It is thus interesting to know how the carotenoids composition of Desmodesmus sp. F51 varies under the four different fed-batch operation strategies. HPLC analysis revealed the presence of neoxanthin, violaxanthin, lutein, ␣-carotene and ␤-carotene in Desmodesmus sp. F51 (data not shown), which were identified by comparing their retention times and visible spectra with data from the literature [16].

Others β -carotene

Lutein Carotenoids content

100 10 80 8 60 6 40

4

20

2

0 Fe

a d-b

tch

I Fe

a d-b

tch

II Fe

a d-b

tch

Carotenoids content (mg/g)

3.3. Effects of fed-batch cultivation strategies on carotenoids composition

A similar carotenoids composition was observed in Scenedesmus obliquus CNW-N [15]. Specific carotenoids (lutein and ␤-carotene) were further quantified with the authentic standards involved in the processes of excess solar energy dissipation [24]. Fig. 3 illustrates the profiles of carotenoids content and composition of Desmodesmus sp. F51 under the four fed-batch cultivation strategies. The carotenoids content slightly decreased when using nitrate feeding (Fed-batches I and III), while a 9.0–13.0% enhancement in lutein proportion and a 1.3–1.6% decrease in ␤-carotene proportion was seen when compared with the results obtained from using medium feeding (Fed-batch II and IV). Similarly, fed-batch cultivation combined with stepwise increases in light intensity

Carotenoids profile (%, out of total carotenoids)

some nutrients at relatively low concentrations could promote the accumulation of lutein content. Similarly, a recent report also showed that an ideal copper concentration of 0.2 mM could enable maximum intracellular lutein accumulation while sustaining cell growth of Coccomyxa onubensis [24]. However, in this study, it is difficult to evaluate which component(s) in the medium positively influenced the lutein accumulation of strain F51. Although the lutein content obtained from Fed-batch IV was not the highest among the four fed-batch operation strategies examined in this work, the lutein production (27.8 mg/L) obtained from Fedbatch IV was nearly 40–70% higher than that obtained from the other three fed-batch strategies. This result can be mainly contributed to the highest biomass production with Fed-batch IV (Table 1). In addition, the highest lutein productivity of strain F51 was also obtained with Fed-batch IV (3.91 mg/L/d), followed by Fedbatch II (3.67 mg/L/d), Fed-batch III (3.61 mg/L/d) and Fed-batch I (3.56 mg/L/d). Therefore, Fed-batch IV appears to be the best strategy for lutein production of strain F51.

0

I II Fe

a d-b

t ch

IV

Fig. 3. Effect of different fed-batch cultivation strategies on carotenoids content and carotenoids composition of Desmodesmus sp. F51.

Y.-P. Xie et al. / Biochemical Engineering Journal 86 (2014) 33–40

3.4. Repeated fed-batch cultivation As mentioned above, although fed-batch cultivation (in particular, Fed-batch IV) was shown to be effective with regard to optimizing the biomass production, CO2 fixation rate and lutein production of Desmodesmus sp. F51, the good performance cannot be maintained when the cultivation time is prolonged, due to the negative effects of light limitation and nutrient deficiency. A strategy for maintaining satisfactory lutein production during longterm cultivation of microalgae (e.g., Desmodesmus sp. F51) is thus needed [24]. As shown in Figs. 1 and 2d, after the light intensity reached 1050 ␮mol/m2 /s (or the biomass concentration reached approximately 4.0 g/L), the biomass and lutein productivity significantly increased. The maximal biomass and lutein productivities occurred at the biomass concentration of around 6.0 g/L, while the performance slightly decreased as the cultivation time was further prolonged. In order to prolong the lutein production phase, as well as to achieve sustainable lutein production, a repeated operation version of Fed-batch IV strategy was carried out. In this new operation mode, the microalgae culture was first grown using Fedbatch IV strategy. To maintain the high productivity, the operation mode was then changed to repeated operation mode by replacing 35–40% volume of culture broth with a solution containing sterile deionized water and concentrated fresh medium to adjust the biomass concentration in the resulting culture to 4.0 g/L (Fig. 4). After replacement, the cultivation was operated in the same manner as the Fed-batch IV operation. The timing for starting a new repeated cycle was when the microalgal biomass concentration

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Lutein productivity (mg/L/d)

Biomass (g/L)

(Fed-batches III and IV) also led to a lower total carotenoids content but a higher proportion of lutein and a lower proportion of ␤-carotene. Therefore, among the four fed-batch cultivation strategies tested, the lowest carotenoids content of 7.74 mg/g was obtained using Fed-batch III (fed-batch cultivation with nitrate feeding and stepwise increases in light intensity), along with the highest proportion of lutein (66.0%) and the lowest proportion of ␤-carotene (9.3%) (Fig. 3). Nutrients limitation may enhance the synthesis of secondary carotenoids (astaxanthin) [7], whereas most of carotenoids in strain F51 are primary carotenoids, which should be associated with cell growth [11], and their accumulation would be enhanced under the nutrient sufficient conditions. Moreover, the reduction in carotenoids content when using the strategy of stepwise increases in light intensity could be due to the decrease in the size of the light-harvesting antenna complex under high light intensity [26]. It has been suggested that only lutein binding to the L1 site is common to all light-harvesting antenna proteins. This plays an important role in the quenching of triplet chlorophyll states under excess light, while the occupancy of the other xanthophyll binding sites differs in each of the other light-harvesting antenna proteins [27,28]. In chloroplasts, one of the major pathways for the formation of reactive oxygen species (ROS) is energy transfer from triplet chlorophyll to molecular oxygen, leading to the formation of singlet oxygen [29], and the deactivation of singlet oxygen is provided by ␤-carotene in the PSII reaction center to avoid photooxidative damage to the photosynthetic apparatus [30]. Therefore, it is reasonable that proportions of lutein and ␤-carotene in the total carotenoids show opposite trends. However, regardless of the cultivation strategies used, the compositions of the carotenoids produced by Desmodesmus sp. F51 are in general quite similar. Lutein is the most abundant carotenoid, accounting for 50–66% of the total carotenoids. This lutein content is also higher than that reported for most other microalgae, including Dunaliella salina (15%) [31], Chlamydomonas acidophila (34.7%) [32], Chlorella zofingiensis (40–52%) [7], and Muriellopsis sp. (50%) [9]. These results further demonstrate the potential of using Desmodesmus sp. F51 as a lutein producer in practical applications.

Lutein content (mg/g)

38

1

1

0

0 0

2

4

6

8

10

Time (d) Fig. 4. Time–course profiles of (a) biomass concentration and nitrate consumption, (b) lutein content and lutein productivity of Desmodesmus sp. F51 by using the repeated Fed-batch IV strategy.

recovered to approximately 6.0 g/L (Fig. 4). The theory behind this operation strategy is as follows: Figs. 1 and 2d show that after the light intensity reached 1050 ␮mol/m2 /s (or the biomass concentration reached approximately 4.0 g/L), the biomass and lutein productivity significantly increased. The maximal biomass and lutein productivities were achieved when the biomass concentration was around 6.0 g/L, but the performance slightly decreased as the cultivation time was further extended. Therefore, in order to maintain the high productivity, when the cell concentration reached 6.0 g/L, the operation mode was then changed to repeated fed-batch operation by replacing 35–40% of the culture broth with the cell-free medium to adjust the biomass concentration in the resulting culture back to 4.0 g/L. Fig. 4a shows that the cell growth rate and nitrate consumption rate remained nearly constant during the five cycles employed. The lutein content and lutein productivity also did not vary significantly in each cycle (Fig. 4b). These results indicate that the repeated Fed-batch IV strategy is an effective approach for practical lutein production with microalgae. Moreover, as shown in Table 2, the biomass productivity (1043.4 mg/L/d) and CO2 fixation rate (1826.0 mg/L/d) of strain F51 obtained from the repeated fed-batch strategy are not only higher than those of Fed-batch IV, but also higher than the results obtained from most related studies. These excellent results mean that the strain F51 is a good potential candidate for practical applications of CO2 fixation. It was also found that the lutein content obtained in the repeated fed-batch strategy is almost the same as that of Fed-batch IV, but the lutein productivity of the repeated fed-batch operation is nearly 10% higher than that of Fed-batch IV, due to the enhancement of biomass productivity (Table 3).

Y.-P. Xie et al. / Biochemical Engineering Journal 86 (2014) 33–40

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Table 3 Comparison of the performance of lutein content and productivity of Desmodesmus sp. F51 with that obtained from other microalgae species reported in the literature under phototrophic cultivation. Microalgae strains

Operation strategies

Cultivation time (days)

Lutein content (mg/g)

Lutein productivity (mg/L/d)

References

Muriellopsis sp. Muriellopsis sp. Chlorella zofingiensis Muriellopsis sp. Scenedesmus almeriensis Scenedesmus almeriensis Scenedesmus sp. Chlorella sorokiniana (wild type) Chlorella sorokiniana (Mr-16) mutant) Desmodesmus sp. F51 Desmodesmus sp. F51

Batch Continuous Batch Semi-batch Continuous Continuous Batch Batch Batch Fed-batch IV Repeated Fed-batch IV

4–7 4 (Daylight period) 5–8 17 5 5 11 10 10 7.2 10

5.5 4.3 3.4 4–6 5.5 5.3 2.0–2.5 3.0 5.0 4.44 ± 0.09 4.42 ± 0.06

0.8–1.4 7.2 3.00 1 4.77 3.80 0.44 2.50 4.20 3.91 ± 0.12 4.61 ± 0.26

[25] [33] [7] [9] [10] [34] [5] [4] [4] This study This study

Although the lutein content obtained in strain F51 is within the range of values reported in the literature, the average lutein productivity of strain F51 (4.61 mg/L/d) obtained from using the repeated fed-batch strategy is among the best performances (Table 3). Therefore, this strategy is indeed a very effective way of enhancing both the CO2 fixation rate and lutein productivity of Desmodesmus sp. F51. Moreover, the repeated fed-batch strategy may also be utilized with other products, such as C-phycocyanin [21] and other proteins [35], which also require nitrogen-sufficient or nutrientsufficient conditions to accumulate. Nevertheless, the proposed strategy should still be examined in large-scale outdoor cultivation to further evaluate its feasibility with regard to commercial applications. In summary, lutein content and productivity of microalgae depend not only on microalgae species [25], but also on the operation mode of the cultivation system. Our results show that fed-batch cultivation can effectively enhance the lutein content, but the performance cannot be stably maintained for a long-term cultivation [8]. In contrast, semi-batch cultivation is a relatively straightforward way to maintain exponential growth phase of the culture to achieve higher biomass productivity but cannot easily improve the lutein content. Thus, a repeated fed-batch operation was proposed to combine the advantages of the two operation modes to further enhance the lutein productivity with relatively stable operation. Using continuous cultivation of microalgae can also enhance the biomass productivity [10], which is beneficial for lutein production. However, continuous microalgae culture still has several difficulties in its large scale control. Since microalgae grow very slowly and are very sensitive to the environmental factors, using continuous culture would markedly increase the risk of contamination and is also more difficult to control at a steady-state operation [14]. Therefore, due to the advantages of easier operation, better stability, and higher efficiency, the proposed repeated fed-batch operation seems to have greater potential over the continuous culture to achieve low-cost and large-scale microalgae cultivation for lutein production with the Desmodesmus sp. F51 strain. 4. Conclusions This work demonstrated that the CO2 fixation and lutein production with Desmodesmus sp. F51 can be significantly improved by using fed-batch strategies. Cultivation under increasing light intensity and/or nutrients limitation resulted in a lower carotenoids content, but a higher proportion of lutein, which is the primary carotenoid of strain F51. The repeated Fed-batch IV operation effectively enhanced both CO2 fixation and lutein production of Desmodesmus sp. F51 during long-term cultivation. The highest CO2 fixation rate and lutein productivity obtained in this work were

1826.0 mg/L/d and 4.61 mg/L/d, respectively, which are higher than most of the previously reported values.

Acknowledgments The authors gratefully acknowledge the financial support this project has received from Taiwan’s National Science Council (under grant number NSC 101-2221-E-006-094-MY3, 102-3113-P-006016 and 101-3113-P-110-003). This research also received funding from the Headquarters of University Advancement at the National Cheng Kung University, which is sponsored by the Ministry of Education of the People’s Republic of China. This work was also supported by the National Natural Science Foundation of China (No. 31071488), the National Marine Commonwealth Research Program, China (no. 201205020-2), and the Fundamental Research Funds for the Central Universities, China (No. 201212G004).

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