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Fed-batch strategy for enhancing cell growth and C-phycocyanin production of Arthrospira (Spirulina) platensis under phototrophic cultivation
Accepted Manuscript Fed-batch strategy for enhancing cell growth and C-phycocyanin production of Arthrospira (Spirulina) platensis under phototrophic cultivation Youping Xie, Yiwen Jin, Xianhai Zeng, Jianfeng Chen, Yinghua Lu, Keju Jing PII: DOI: Reference:
S0960-8524(14)01819-7 http://dx.doi.org/10.1016/j.biortech.2014.12.073 BITE 14395
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
23 October 2014 20 December 2014 22 December 2014
Please cite this article as: Xie, Y., Jin, Y., Zeng, X., Chen, J., Lu, Y., Jing, K., Fed-batch strategy for enhancing cell growth and C-phycocyanin production of Arthrospira (Spirulina) platensis under phototrophic cultivation, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.12.073
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Fed-batch strategy for enhancing cell growth and C-phycocyanin production of Arthrospira (Spirulina) platensis under phototrophic cultivation
Youping Xie 1,2, Yiwen Jin 2, Xianhai Zeng3,4, Jianfeng Chen1, Yinghua Lu 2,4, Keju Jing 2,4,*
1
College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108,
China 2
Department of Chemical and Biochemical Engineering, College of Chemistry and
Chemical Engineering, Xiamen University, Xiamen 361005, China 3 4
College of Energy, Xiamen University, Xiamen 361005, China Key Laboratory for Chemical Biology of Fujian Province, Xiamen University,
Xiamen 361005, China
* Corresponding author: Keju Jing Tel: +86-592-2186038 Fax: +86-592-2186038 Email: [email protected]
Abstract The C-phycocyanin generated in blue-green algae Arthrospira platensis is gaining commercial interest due to its nutrition and healthcare value. In this study, the light intensity and initial biomass concentration were manipulated to improve cell growth and C-phycocyanin production of Arthrospira platensis in batch cultivation. The results show that low light intensity and high initial biomass concentration led to increased C-phycocyanin accumulation. The best C-phycocyanin productivity occurred when light intensity and initial biomass concentration were 300 µmol/m2/s and 0.24 g/L, respectively. The fed-batch cultivation proved to be an effective strategy to further enhance C-phycocyanin production of Arthrospira platensis. The results indicate that C-phycocyanin accumulation not only requires nitrogen-sufficient condition, but also needs other nutrients. The highest C-phycocyanin content (16.1%), production (1034 mg/L) and productivity (94.8 mg/L/d) were obtained when using fed-batch strategy with 5 mM medium feeding.
Keywords: Arthrospira platensis; C-phycocyanin (C-PC); initial biomass concentration; light intensity; fed-batch
1. Introduction C-phycocyanin (C-PC), a phycobiliprotein is a water-soluble and colored protein component of the photosynthetic light-harvesting antenna complexes present in cyanobacteria, red algae, and cryptomonads (Eriksen, 2008; Kuddus et al., 2013). Due to its antioxidant, anti-inflammatory, hepatoprotective and radical-scavenging properties, C-PC is widely used in food, cosmetics and pharmaceuticals (Belay, 2002; Bermejo et al., 2008; Romay et al., 2003). Also C-PC is well suited as a fluorescent reagent for diagnostic applications because it is nontoxic and has a high molar extinction coefficient and fluorescence quantum yield (Kuddus et al., 2013; Sekar & Chandramohan, 2008). Arthrospira, a type of cyanobacteria, has a high growth rate with as high as 25% (w/w) C-PC content in the biomass, and thus is considered as a promising and commercial C-PC source (Ciferri, 1983). The worldwide production of Arthrospira has grown significantly in recent years, with greater than 3000 tons dry biomass that are produced worldwide annually and used for animal feed additives and health food products (Kuddus et al., 2013). In general, the C-PC productivity combines the dual effects of C-PC content and biomass productivity is the most important indicator to evaluate the efficiency of C-PC production in the commercial process. In order to improve C-PC productivity, several attempts were made in the literature by varying the growth metabolism of Arthrospira, such as phototrophic (Chen et al., 2013; Chojnacka & Noworyta, 2004; Zeng et al., 2012), mixotrophic (Chen & Zhang, 1997; Chojnacka & Noworyta, 2004) and heterotrophic cultivation (Chojnacka & Noworyta, 2004; Muhling et al., 2005). However, when compared to mixotrophic and heterotrophic cultivation, phototrophic cultivation without organic substrates and contamination risks, along with the added benefit of mitigating CO2 emissions, seems to be preferable in terms of commercial feasibility for C-PC production. Therefore, commercial C-PC production is currently produced using phototrophic cultures of Arthrospira in open ponds and raceways (Eriksen, 2008; Kuddus et al., 2013). Moreover, improved productivities of phototrophic Arthrospira cultures have
been obtained in closed photobioreactors (Carlozzi, 2003; Chen et al., 2013; Zitelli et al., 1996). The volumetric biomass productivity obtained from closed photobioreactors is five to 20 times above what is obtained in open raceways (Eriksen, 2008), which may also favour closed photobioreactors for commercial systems. However, the cost of C-PC production in closed photobioreactors still needs to be reduced significantly if it is to be economically feasible on an industrial scale. Recently, some literature have pointed out the photobioreactor type and culture conditions (e.g., initial pH, seawater media, CO2 supply, nitrogen concentration and light intensity) could significantly affect C-PC accumulation and the biomass accumulation of Arthrospira (Chen et al., 2013; Leema et al., 2010; Zeng et al., 2012). In additions, culture strategies (e.g., fed-batch, semi-batch and continuous cultivation) have already been applied to improve pigments, lipid and carbohydrate production of microalgae (Ho et al., 2013b; Hsieh & Wu, 2009; Xie et al., 2013). However, no efforts have yet been made to evaluate fed-batch strategy for enhancing the C-PC production of Arthrospira platensis under phototrophic conditions. Therefore, this study was firstly aimed to optimize the light intensity and initial biomass concentration for the C-PC production of Arthrospira platensis. Moreover, the feasibility of fed-batch strategies to improve the production of C-PC was also assessed.
2. Material and methods 2.1. Microalgal strain and its preculture conditions The microalgal strain Arthrospira platensis WH879 used in this study was obtained from the Institute of Hydrobiology (IHB) in Wuhan, China. The medium used for the preculture of the strain was Zarrouk medium (Duerr et al., 1997; Zarrouk, 1966) consisting of (per liter): 16.8 g NaHCO3, 0.5 g K2HPO4, 2.5 g NaNO3, 1 g K2SO4, 1 g NaCl, 0.2 g MgSO4.2H2O, 0.04 g CaCl4.2H2O, 0.01 g FeSO4.7H2O, 0.08 g EDTA and 1 ml of trace metal solution. The trace metal solution consisted of (per liter): 2.86 g H3BO3, 1.81 g MnCl4.4H2O, 0.222 g ZnSO4.4H2O, 0.0177 g Na2MoO4,
0.079 g CuSO4.5H2O. The microalga was regularly grown at 28°C±0.5°C for 8-12 days with a continuous supply of 2.5% CO2 at an aeration rate of 0.2 vvm. The microalgal culture was illuminated 24 hours a day with a light intensity of approximately 60 µmol/m2/s which 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 cultivate the Arthrospira platensis was a 1-liter glass vessel (15.5 cm in length and 9.5 cm in 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 microalga was pre-cultured and inoculated into the PBR with an inoculums size of 0.08-0.24 g/L. The medium used for strain culture in the PBR was Zarrouk medium. The PBR were operated at 28°C, pH 9.0, and an agitation rate of 400 rpm under a light intensity of approximately 75-450 µmol/m2/s. Serving as the sole carbon source, 2.5% CO2 with 0.2 vvm was fed into the microalgal culture continuously during cultivation. Liquid samples were collected from the culture broth at set time intervals to determine the cell concentration, pH, C-PC content and residual nitrate concentration.
2.3. Operation of fed-batch strategy The fed-batch cultivation was started on a batch culture with 0.24 g/L inoculums size and 300 µmol/m2/s light intensity. 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 5.0 mM and 10 mM. The timing for starting new pulse-feeding was when the nitrate concentration in the culture broth was nearly depleted. Liquid samples were collected from the culture broth at set time intervals to determine the cell concentration, pH, C-PC content and residual nitrate concentration.
2.4. Determination of cell growth and CO 2 fixation rate Microalgae biomass concentration was determined by measuring the optical density of the sample at a wavelength of 680 nm (denoted as OD680) using a UV/Vis spectrophotometer (model U-2001, Hitachi, Tokyo, Japan) after proper dilution with deionized water. The OD680 values were converted to biomass concentration via appropriate calibration between OD680 and dry cell weight:
(
Biomass (g/L) = 0.584 ⋅ OD680 − 2.367 R2 = 0.99
)
The specific growth rate of microalgal culture was obtained by following calculation:
µ (d -1 ) =
d ln X dt
max
(i.e. maximum solpe from lnX versus t plot)
where X and t indicate the biomass concentration (g/L) and cultivation time (day), respectively. 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 DCW/L) within a cultivation time of ∆ t (d).
2.5. Determination of nitrate concentration The nitrate concentration in the medium was determined by a colorimetric method described in Ho et al. (2013b). The calibration between the absorbance and nitrogen concentration was established using sodium nitrate as the standard.
2.6. Determination of C-phycocyanin content A fixed amount (0.06 g) of the lyophilized biomass was mixed with 10 ml of 0.15 M phosphate buffer (pH = 7.0) and maintained at 4°C for 36 hours. The cell
debris was then removed by centrifugation at 8,000 rpm, and the supernatant (blue color) was collected. The absorbance of crude extract was measured with UV/Vis spectrophotometer at the wavelengths of 620 nm and 652 nm for calculating the concentration of C-PC according to the following equation (Bennett & Bogorad, 1973): C-PC (g/L) =
OD 620 − 0.474 ⋅ OD 652 5.34
3. Results and discussion 3.1. Time-course performance on cell growth and C-PC production The cellular composition of microalgae usually varies with the cell growth phase (Ho et al., 2012; Mandal & Mallick, 2009; Xie et al., 2013), and thus it is necessary to harvest the microalgal cells at the optimal timing to ensure maximal C-PC productivity for commercialization. In this work, Arthrospira platensis was cultivated in a batch culture for around 16 days to investigate variation in C-PC production. As shown in Fig. 1, the C-PC content and productivity increased simultaneously along with cell growth and nitrogen consumption, and the maximal values (10.3±0.3 mg/g and 38.0±1.0 mg/L/d) were obtained at the beginning of nitrogen depletion. While under nitrogen depletion conditions, both of them decreased significantly with the prolonged cultivation time. This trend is well in agreement with the report of Chen et al. (2013). It has been suggested that C-PC belongs to a family of phycobiliproteins which have obtained a secondary role as intracellular nitrogen storage compounds that are mobilized for other purposes in times of nitrogen shortage (Eriksen, 2008). This is also supported by several studies, which have demonstrated that nitrogen depletion causes a reduction in protein content, along with an enhancement of energy-rich compounds, such as lipids and carbohydrates (Ho et al., 2012; Mandal & Mallick, 2009). The possible reason could be that under nitrogen depletion the intracellular proteins was degraded as a source of nitrogen to maintain the metabolic functions of microalgae, and utilized for synthesis essential cell structures to against environment stress. Therefore, in order to attain the maximal C-PC content and productivity, the
beginning of nitrogen depletion should be the best time for harvesting microalgal biomass during the batch cultivation.
3.2. Effect of light intensity on cell growth and C-PC production For phototrophic cultivation, the light intensity not only strongly influences the cell growth of microalgae, but also changes the levels of light-related molecules in photosynthetic system (Markou et al., 2012; Xie et al., 2013). To investigate the effect of light intensity on cell growth and C-PC production of Arthrospira platensis, different light intensities (75-450 µmol/m2/s) were examined in this study. As shown in Fig. 2a, both the biomass productivity and specific growth rate of Arthrospira platensis increased significantly when the light intensity was increased from 75 to 300 µmol/m2/s. However, a sharp decrease was observed when light intensity was further increased up to 450 µmol/m2/s, indicating that this light intensity falls within the light inhibition region (Xue et al., 2011). This could be directly supported by the observation of changing cells color from green to white during cultivation in the first 3 days (data not shown). Thus, 300 µmol/m2/s seems to be the optimal light intensity for the growth of Arthrospira platensis, with the highest biomass productivity of 436.3±6.2 mg/L/d and specific growth rate of 1.02±0.04 d -1. To obtain high biomass productivity, the light intensity should be increased properly but a low intensity of light was more favorable for the C-PC accumulation. As shown in Fig. 2b, the maximum C-PC content of Arthrospira platensis decreased significantly from 18.4±0.4 to 2.2±0.2% as the light intensity was increased from 75 to 450 µmol/m2/s. It is known that C-PC is also an antenna pigment used by mainly cyanobacteria and eukaryotic algae to increase efficiency of photosynthesis by collecting light energy at wavelength where chlorophylls poorly absorb, and transfering the energy in high efficiency to chlorophyll a in the thylakoid membranes (Eriksen, 2008; Kuddus et al., 2013; Sun et al., 2006). The decrease in C-PC content at higher light intensity would be because the necessary amount of light energy is completely absorbed by a growing number of cells, thus the cells require a lower
content of cellular C-PC. Similarly, Markou et al. (2012) reported that higher light intensity results to lower chlorophyll and protein content of Arthrospira platensis. Chen et al. (2013) also reported an increase of up to 27% in C-PC content, when the light intensity of the culture decreased from 1300 to 100 µmol/m2/s. As for C-PC productivity, Fig. 2b shows that there was a slight increase as the light intensity increased until photo-inhibition occurred. This trend is well in agreement with the report of Chen et al. (2013). The highest C-PC productivity of 40.0±0.6 mg/L/d was obtained when using a light intensity of 300 µmol/m2/s. The optimization of light intensity is achieved due to combine the dual effects on C-PC content and biomass productivity. However, the optimal light intensity obtained from this study is inconsistent with the findings of Chen et al. (2013), which reported the maximal C-PC productivity of Arthrospira platensis was obtained at 700 µmol/m2/s. Zeng et al. (2012) also got an excellent yield of C-PC using 200 µmol/m2/s but did not evaluate different light intensities. The reasons for this discrepant result might be due to differences in bioreactor configurations and culture strategy. However, in any case, it should be noted that the observation in regard to the photo-bleaching of the algae might suggest an analytical method to set the optimal light intensity.
3.3. Effect of initial biomass concentration on cell growth and C-PC production Initial biomass concentration (IBC) is an important factor that affects the viability and productivity of microalgae in batch cultivation. Thus, the effect of IBC on cell growth and C-PC production of Arthrospira platensis was investigated, and the results are shown in Fig. 3. The maximum specific growth rate of Arthrospira platensis decreased significantly with an increase in the IBC. Conversely, the biomass productivity increased with increasing IBC from 0.08 to 0.16 g/L, while further increases in IBC to 0.24 g/L did not lead to better biomass productivity (Fig. 3a). These results are similar to that observed by Hu et al. (1997), who reported that an exponential decrease in specific growth rate and a positive skewed pattern in biomass
productivity were associated with an increase in cell density of Monodus subterraneus and Arthrospira platensis. Lu et al. (2012) also reported that the maximum growth rate of Chlorella sorokiniana in a lower inoculum culture was 1.38- to 2.42-fold increase when compared to the highest inoculum size culture. Increasing IBC in a certain range led to a shorten in the growth cycle of microalgae by avoiding the lag phase, resulting in the higher biomass productivity (Ho et al., 2013a; Ma et al., 2013), although the values of final cell density were almost the same under different IBC (data not shown). However, when the IBC is higher than a certain threshold level, irradiance levels lower than the light saturation level of the photosynthetic organism may induce light limitation, resulting in a reduction in biomass productivity (Hu et al., 1997; Lu et al., 2012). As shown in Fig. 3b, a considerable increase in C-PC content from a basal level of 9.2±0.3% to 11.6±0.4% was observed when IBC was increased up to 0.24 g/L, indicating that a higher IBC is more preferred for C-PC accumulation of Arthrospira platensis. Considering the fact that cell density could alter the average irradiance by cell absorption and self-shading, so when using high IBC, the average irradiance (the amount of light/unit of cell) was lower than that using low IBC. As a result, the size of light-harvesting antenna complex should be increased to absorb more light energy, resulting in higher C-PC content. Moreover, Fig. 3b also shows that the C-PC productivity of Arthrospira platensis increased nearly 70% when the IBC was raised from 0.08 to 0.24 g/L. The highest C-PC productivity of Arthrospira platensis (66.8± 1.5 mg/L/d) was obtained with an IBC of 0.24 g/L. This result can be mainly contributed to greatly improve in C-PC content and shorten the culture time to reach the maximum C-PC content when properly increasing IBC. The decrease in biomass productivity, however, was lower than the increase in C-PC content, resulting in a net increase of the C-PC productivity. However, as taking account of process economics, increasing IBC also requires a proportional increase in capital and operating resources (Zemke et al., 2013). Therefore, it is need a trade-off between seed cost and production efficiency to achieve low-cost and large-scale microalgae cultivation for C-PC production with Arthrospira platensis.
3.4. Improvement of C-PC production of Arthrospira platensis by using fed-batch operation As indicated in Fig.1, the maximal C-PC content and productivity were obtained at the beginning of nitrogen depletion. It has been suggested that C-PC accumulation is highly dependent on the level of residual nitrogen concentration (Chen et al., 2013). Moreover, although increasing average irradiance (the amount of light/unit of cell) enhanced the cell growth rate, it reduced C-PC content. Thus, it is necessary to develop an effective strategy that could enhance biomass production and simultaneously achieve a relatively high C-PC content. Fed-batch operation was often viewed as an effective strategy to control the nutrient concentration in the culture broth and enhance biomass production via prolonging cell growth phase (Hsieh & Wu, 2009; Sánchez-Luna et al., 2004; Xie et al., 2013). Accordingly, trying to produce a high amount of biomass with high C-PC content, the fed-batch operation was performed. The start-up of the fed-batch process was the same as that used for the batch operation with 0.24 g/L IBC and 300 µmol/m2/s light intensity. When the initial nitrate was nearly depleted, the 100-fold concentrated nitrate solution was then pulse-fed into the culture to reach a nitrate concentration of 5.0 mM and 10 mM, and the results are shown in Fig. 4 and Table 1. The biomass production and productivity of Arthrospira platensis were quite similar in both cases. The C-PC content as well as C-PC productivity in 5 mM nitrate feeding was nearly 15% higher than that of 10 mM nitrate feeding. When using 10 mM nitrate feeding, C-PC content was just the same as that obtained with batch cultivation. Chen et al. (2013) reported that biomass productivity and C-PC productivity decreased when initial nitrogen concentration was higher than 45 mM. Meanwhile, C-PC content was observed at a constant range when increasing initial nitrogen concentration from 30 to 90 mM under batch cultivation. These seem to indicate that controlling the nitrogen concentration at a lower level in the fed-batch cultivation was more favorable for C-PC accumulation. Similarly, Xie et al. (2013) also reported that a higher lutein content was obtained in a fed-batch culture fed with relatively lower nitrogen concentration. The reason for this might be because
when controlling the nitrogen concentration at a low but non-limiting level, nitrogen should be used first and foremost to synthesis light-related molecules and light-harvesting proteins in photosynthetic system. Moreover, in order to evaluate whether controlling other components in the medium have a positive or negative influence on C-PC production, the fed-batch operation with 5 mM medium feeding was also investigated, and the results are shown in Fig. 5 and Table 1. After initial nitrate concentration was depleted, both nitrate feeding and medium feeding led to a continuous increase in biomass concentration, while the biomass concentration obtained from medium feeding was slightly higher than that of nitrate feeding. Moreover, the biomass production in fed-batch cultivation was nearly 30-40% higher than that of batch cultivation (Table 1). Similarly, Hsieh & Wu (2009) reported that urea feeding could effectively enhance the biomass production of Chlorella sp.. Xie et al. (2014) also reported that fed-batch with medium feeding could achieve a relatively higher biomass concentration of Desmodesmus sp. F51 when compared to the fed-batch with nitrate feeding. In addition, Fig.4a and Fig. 5 show that the C-PC content of Arthrospira platensis increased with the prolonged time of fed-batch operation, and the highest values obtained were 13.4±0.5 and 16.1±0.2%, for nitrate feeding (5 mM) and medium feeding (5 mM), occurring at the cultivation times of 9.8 and 10.9 d, respectively. After that, the C-PC content, as well as nitrate consumption rate, showed a downward trend in both case. This could be attributed to the lower cell viability with the prolonged cultivation time. When using 5 mM medium feeding, the maximum C-PC content was around 20% higher than that of 5 mM nitrate feeding, indicating that C-PC accumulation not only requires nitrogen-sufficient condition, but also needs other nutrients. C-PC belongs to a family of phycobiliproteins which are some of the most abundant proteins in many cyanobacteria and algae (Eriksen, 2008). Several studies have demonstrated that nutrients depletion, such as S, P and N, would cause a decrease in protein content of microalgae (Ho et al., 2012; Markou et al., 2012; Procházková et al., 2013). Thus, when using 5 mM nitrating feeding, some nutrients in culture broth would decrease to deprivation condition with the prolonged
cultivation time, resulting in a relatively lower C-PC content than that obtained from using medium feeding. Moreover, when compared with the batch cultivation, the C-PC content obtained from 5 mM medium feeding enhanced around 40%, which was within the upper range of C-PC content reported in previous literature (e.g., 7.0-18.5%) (Table 2). As a result, the increased biomass with high C-PC content led to the ultimate enhancement of C-PC production in fed-batch cultivation with 5 mM medium feeding. The highest C-PC production obtained from medium feeding reached to 1034.8±7.5 mg/L, which was nearly 98% higher than that obtained from the conventional batch process (Table 1). Furthermore, the highest C-PC productivity in 5 mM medium feeding was 94.8±1.4 mg/L/d, which was not only around 42% higher than that in batch cultivation, but also higher than the results in most of the related studies (ranging from 14 to 125 mg/L/d) (Table 2). Therefore, fed-batch with medium feeding is indeed a very effective way for enhancing C-PC production of Arthrospira platensis.
4. Conclusions The effects of light intensity and initial biomass concentration on cell growth and C-PC production of Arthrospira platensis were investigated under the batch phototrophic cultivation. The average irradiance affects C-PC content and cell growth rate in an opposite way. Controlling a lower nitrogen level in fed-batch cultivation was more favorable for C-PC accumulation. The fed-batch cultivation with 5 mM medium feeding was proved to be an effective method to further enhance the C-PC production, giving the highest C-PC content, titer, and productivity of 16.1%, 1034 mg/L, and 94.8 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 the National High Technology Research and Development Program 863, China (No. 2014AA021701).
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20. Mandal, S., Mallick, N., 2009. Microalga Scenedesmus obliquus as a potential source for biodiesel production. Appl. Microbiol. Biotechnol. 84(2), 281-291. 21. Markou, G., Chatzipavlidis, I., Georgakakis, D., 2012. Effects of phosphorus concentration and light intensity on the biomass composition of Arthrospira (Spirulina) platensis. World J. Microbiol. Biotechnol. 28(8), 2661-2670. 22. Muhling, M., Belay, A., Whitton, B.A., 2005. Screening Arthrospira (Spirulina) strains for heterotrophy. J. Appl. Phycol. 17, 129-135. 23. Procházková, G., Brányiková, I., Zachleder, V., Brányik, T., 2013. Effect of nutrient supply status on biomass composition of eukaryotic green microalgae. J. Appl. Phycol. 26, 1359-1377. 24. Romay, C., Gonzalez, R., Ledon, N., Remirez, D., Rimbau, V., 2003. C-phycocyanin: a biliprotein with antioxidant, anti-inflammatory and neuroprotective effects. Curr. Protein Pept. Sc. 4, 207-216. 25. Sánchez-Luna, L.D., Converti, A., Tonini, G.C., Sato, S., de Carvalho, J.C.M., 2004. Continuous and pulse feedings of urea as a nitrogen source in fed-batch cultivation of Spirulina platensis. Aquacult. Eng. 31(3-4), 237-245. 26. Sekar, S., Chandramohan, M., 2008. Phycobiliproteins as a commodity: trends in applied research, patents and commercialization. J. Appl. Phycol. 20, 113-136. 27. Sun, L., Wang, S., Qiao, Z., 2006. Chemical stabilization of the phycocyanin from cyanobacterium Spirulina platensis. J. Biotechnol. 121(4), 563-569. 28. Xie, Y.P., Ho, S.H., Chen, C.Y., Chen, C.N.N., Liu, C.C., Ng, I.S., Jing, K.J., Yang, S.C., Chen, C.H., Chang, J.S., Lu, Y.H., 2014. Simultaneous enhancement of CO2 fixation and lutein production with thermo-tolerant Desmodesmus sp. F51 using a repeated fed-batch cultivation strategy. Biochem. Eng. J. 86, 33-40. 29. Xie, Y., Ho, S.H., Chen, C.N., Chen, C.Y., Ng, I.S., Jing, K.J., Chang, J.S., Lu, Y., 2013. Phototrophic cultivation of a thermo-tolerant Desmodesmus sp. for lutein production: Effects of nitrate concentration, light intensity and fed-batch operation. Bioresource. Technol. 144, 435-444. 30. Xue, S., Su, Z., Cong, W., 2011. Growth of Spirulina platensis enhanced under intermittent illumination. J. Biotechnol. 151(3), 271-277.
31. Zarrouk, C., 1966. Contribution a l'etude d'une cyanobacterie: influence de divers facteurs physiques et chimiques sur la croissance et la photosynthese de Spirulina maxima (Setchell et Gardner) Geitler, University of Paris, Paris. 32. Zemke, P.E., Sommerfeld, M.R., Hu, Q., 2013. Assessment of key biological and engineering design parameters for production of Chlorella zofingiensis (Chlorophyceae) in outdoor photobioreactors. Appl. Microbiol. Biotechnol. 97(12), 5645-5655. 33. Zeng, X., Danquah, M.K., Zhang, S., Zhang, X., Wu, M., Chen, X.D., Ng, I.S., Jing, K., Lu, Y., 2012. Autotrophic cultivation of Spirulina platensis for CO2 fixation and phycocyanin production. Chem. Eng. J. 183, 192-197. 34. Zitelli, G.C., Tomasello, V., Pinzani, E., Tredici, M.R., 1996. Outdoor culture of Arthrospira platensis during autumn and winter in temperate climate. J. Appl. Phycol. 8, 293-301.
Table 1 Effect of different fed-batch strategies on cell growth, CP-C content and C-PC production of Arthrospira platensis (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; temperature, 28°C; light intensity, 300 µmol/m2/s; nitrate concentration, 30 mM).
Nitrate/Medium
Biomass
Biomass
C-PC
C-PC
C-PC
feeding
production
productivity
content
productivity
production
concentration
(g/L)
(mg/L/d)
(%)
(mg/L/d)
(mg/L)
0
4.82±0.05
576.3±5.2
11.6±0.4
66.8±1.5
523.0±3.2
5 mM (Nitrate)
6.18±0.04
592.9±3.5
13.4±0.5
79.6±0.6
782.5±5.3
10 mM (Nitrate)
6.20±0.06
594.2±7.2
11.7±0.3
69.1±1.2
714.0±8.2
5 mM (Medium)
6.78±0.07
588.2±6.8
16.1±0.2
94.8±1.4
1034.8±7.5
The values are calculated at the time of maximal C-PC productivity. Data shown are the averages of two runs ± SD (standard deviation).
Table 2 Comparison of the performance of C-PC content, production and productivity of Arthrospira platensis with that obtained in the related reports under phototrophic cultivation.
C-PC
C-PC
C-PC
Content
production
productivity
(%)
(mg/L)
(mg/L/d)
Operation Microalgae strains
Reference
strategies Arthrospira platensis
Batch
7.0
210
64
Zitelli et al., 1996
Arthrospira platensis
Batch
7.0
350
92
Carlozzi, 2003
Arthrospira platensis
Batch
4.8
350
14
Leema et al., 2010
Arthrospira platensis
Batch
16.8
1220
67.8
Zeng et al., 2012
Arthrospira platensis
Batch
12-12.6
400-700
110-125
Chen et al., 2013
Arthrospira platensis
Fed-batch
16.1±0.2
1034.8±7.5
94.8±1.4
This study
Figure Legends Figure 1 Time-course profiles of biomass concentration, nitrate concentration, C-PC content and C-PC productivity during the batch growth of Arthrospira platensis (operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; light intensity, 150 µmol/m2/s; temperature, 28°C; nitrate concentration, 30 mM; inoculums size, 0.08 g/L). Figure 2 The performance of cell growth and C-PC production of Arthrospira platensis under different light intensities: (a) maximum specific growth rate and biomass productivity, (b) C-PC content and C-PC productivity. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; temperature, 28°C; nitrate concentration, 30 mM; inoculums size, 0.08 g/L). Data are the averages of two runs and the error bars are standard deviations. Figure 3 The performance of cell growth and C-PC production of Arthrospira platensis under different initial biomass concentrations: (a) biomass production and biomass productivity, (b) C-PC content and C-PC productivity. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; temperature, 28°C; nitrate concentration, 30 mM; light intensity, 300 µmol/m2/s). Data are the averages of two runs and the error bars are standard deviations. Figure 4 Time-course profiles of biomass concentration, nitrate concentration, C-PC content and C-PC productivity of Arthrospira platensis with (a) 5 mM nitrate feeding and (b) 10 mM nitrate feeding (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; temperature, 28°C; light intensity, 300 µmol/m2/s). Figure 5 Time-course profiles of biomass concentration, nitrate concentration, C-PC content and C-PC productivity of Arthrospira platensis with 5 mM medium feeding (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; temperature, 28°C; light intensity, 300 µmol/m2/s).
Nitrate concentration (g/L) 3.0 6
2.5 5
2.0
1.5
1.0
Biomass concentration (g/L)
15
4
3
0.5 1
0.0 0 10
0 4 8
Time (d) 12 16 0
C-PC content (%)
20
40
30
20
2 5
10
0
C-PC productivity (mg/L/d)
Figure 1
50
Maximum C-PC content (%)
Biomass productivity (mg/L/d)
15
10
5
0 75 150 300 450
Light intensity (mol/m /s)
2 -1
(a)
400 1.2
300 0.9
200 0.6
100 0.3
0 0.0
(b) 40
20
30
20
10
0
Maximum specific growth rate (d )
500
Maximum C-PC productivity (mg/L/d)
Figure 2 1.5
Maximum C-PC content (%) 0 0.08
600 1.2
400 0.9
0.6
200 0.3
0 0.0
(b) 80
10 60
40
5 20
0.12 0.16 0.20
Initial biomass concentration (g/L) 0.24 0
Maximum Specific growth rate (d )
-1
(a)
Maximum C-PC productivity (mg/L/d)
Biomass productivity (mg/L/d)
Figure 3 1.5
Nitrate concentration (g/L) 2.0
0.0
1.5
1.0
0.5
0.0
10
4
2
0
0
0
0
3
3
6 9
6
9
Time (d) 12
8
12
0
2.5
2.0
6 10
4
2
0
60
40
5
60
40
5 20
0
C-PC productivity (mg/L/d)
2.5
C-PC productivity (mg/L/d)
0.5 6
C-PC content (%)
8
C-PC content (%)
1.0
Biomass concentration (g/L)
1.5
Biomass concentration (g/L)
Nitrate concentration (g/L)
Figure 4
100
(a) 15 80
20
0
Time (d) 100
(b) 15 80
Nitrate concentration (g/L) 2.0
1.5
1.0
0.5
0.0
Biomass concentration (g/L)
2.5
6 10
4
2
0 0 3 6 9
Time (d) 12 0
60
40
5 20
0
C-PC productivity (mg/L/d)
8
C-PC content (%)
Figure 5
100
15 80
Highlights
Arthrospira platensis WH879 shows high potential as an C-phycocyanin producer
The average irradiance affects C-PC content and cell growth rate in an opposite way
Controlling a lower nitrogen level was more favorable for C-PC accumulation Fed-batch cultivation with medium feeding effectively enhanced the C-PC production
S0960-8524(14)01819-7 http://dx.doi.org/10.1016/j.biortech.2014.12.073 BITE 14395
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
23 October 2014 20 December 2014 22 December 2014
Please cite this article as: Xie, Y., Jin, Y., Zeng, X., Chen, J., Lu, Y., Jing, K., Fed-batch strategy for enhancing cell growth and C-phycocyanin production of Arthrospira (Spirulina) platensis under phototrophic cultivation, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.12.073
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A revised manuscript (BITE-D-14-05994) submitted to Bioresource Technology (All the changes are marked with yellow highlight)
Fed-batch strategy for enhancing cell growth and C-phycocyanin production of Arthrospira (Spirulina) platensis under phototrophic cultivation
Youping Xie 1,2, Yiwen Jin 2, Xianhai Zeng3,4, Jianfeng Chen1, Yinghua Lu 2,4, Keju Jing 2,4,*
1
College of Biological Science and Engineering, Fuzhou University, Fuzhou 350108,
China 2
Department of Chemical and Biochemical Engineering, College of Chemistry and
Chemical Engineering, Xiamen University, Xiamen 361005, China 3 4
College of Energy, Xiamen University, Xiamen 361005, China Key Laboratory for Chemical Biology of Fujian Province, Xiamen University,
Xiamen 361005, China
* Corresponding author: Keju Jing Tel: +86-592-2186038 Fax: +86-592-2186038 Email: [email protected]
Abstract The C-phycocyanin generated in blue-green algae Arthrospira platensis is gaining commercial interest due to its nutrition and healthcare value. In this study, the light intensity and initial biomass concentration were manipulated to improve cell growth and C-phycocyanin production of Arthrospira platensis in batch cultivation. The results show that low light intensity and high initial biomass concentration led to increased C-phycocyanin accumulation. The best C-phycocyanin productivity occurred when light intensity and initial biomass concentration were 300 µmol/m2/s and 0.24 g/L, respectively. The fed-batch cultivation proved to be an effective strategy to further enhance C-phycocyanin production of Arthrospira platensis. The results indicate that C-phycocyanin accumulation not only requires nitrogen-sufficient condition, but also needs other nutrients. The highest C-phycocyanin content (16.1%), production (1034 mg/L) and productivity (94.8 mg/L/d) were obtained when using fed-batch strategy with 5 mM medium feeding.
Keywords: Arthrospira platensis; C-phycocyanin (C-PC); initial biomass concentration; light intensity; fed-batch
1. Introduction C-phycocyanin (C-PC), a phycobiliprotein is a water-soluble and colored protein component of the photosynthetic light-harvesting antenna complexes present in cyanobacteria, red algae, and cryptomonads (Eriksen, 2008; Kuddus et al., 2013). Due to its antioxidant, anti-inflammatory, hepatoprotective and radical-scavenging properties, C-PC is widely used in food, cosmetics and pharmaceuticals (Belay, 2002; Bermejo et al., 2008; Romay et al., 2003). Also C-PC is well suited as a fluorescent reagent for diagnostic applications because it is nontoxic and has a high molar extinction coefficient and fluorescence quantum yield (Kuddus et al., 2013; Sekar & Chandramohan, 2008). Arthrospira, a type of cyanobacteria, has a high growth rate with as high as 25% (w/w) C-PC content in the biomass, and thus is considered as a promising and commercial C-PC source (Ciferri, 1983). The worldwide production of Arthrospira has grown significantly in recent years, with greater than 3000 tons dry biomass that are produced worldwide annually and used for animal feed additives and health food products (Kuddus et al., 2013). In general, the C-PC productivity combines the dual effects of C-PC content and biomass productivity is the most important indicator to evaluate the efficiency of C-PC production in the commercial process. In order to improve C-PC productivity, several attempts were made in the literature by varying the growth metabolism of Arthrospira, such as phototrophic (Chen et al., 2013; Chojnacka & Noworyta, 2004; Zeng et al., 2012), mixotrophic (Chen & Zhang, 1997; Chojnacka & Noworyta, 2004) and heterotrophic cultivation (Chojnacka & Noworyta, 2004; Muhling et al., 2005). However, when compared to mixotrophic and heterotrophic cultivation, phototrophic cultivation without organic substrates and contamination risks, along with the added benefit of mitigating CO2 emissions, seems to be preferable in terms of commercial feasibility for C-PC production. Therefore, commercial C-PC production is currently produced using phototrophic cultures of Arthrospira in open ponds and raceways (Eriksen, 2008; Kuddus et al., 2013). Moreover, improved productivities of phototrophic Arthrospira cultures have
been obtained in closed photobioreactors (Carlozzi, 2003; Chen et al., 2013; Zitelli et al., 1996). The volumetric biomass productivity obtained from closed photobioreactors is five to 20 times above what is obtained in open raceways (Eriksen, 2008), which may also favour closed photobioreactors for commercial systems. However, the cost of C-PC production in closed photobioreactors still needs to be reduced significantly if it is to be economically feasible on an industrial scale. Recently, some literature have pointed out the photobioreactor type and culture conditions (e.g., initial pH, seawater media, CO2 supply, nitrogen concentration and light intensity) could significantly affect C-PC accumulation and the biomass accumulation of Arthrospira (Chen et al., 2013; Leema et al., 2010; Zeng et al., 2012). In additions, culture strategies (e.g., fed-batch, semi-batch and continuous cultivation) have already been applied to improve pigments, lipid and carbohydrate production of microalgae (Ho et al., 2013b; Hsieh & Wu, 2009; Xie et al., 2013). However, no efforts have yet been made to evaluate fed-batch strategy for enhancing the C-PC production of Arthrospira platensis under phototrophic conditions. Therefore, this study was firstly aimed to optimize the light intensity and initial biomass concentration for the C-PC production of Arthrospira platensis. Moreover, the feasibility of fed-batch strategies to improve the production of C-PC was also assessed.
2. Material and methods 2.1. Microalgal strain and its preculture conditions The microalgal strain Arthrospira platensis WH879 used in this study was obtained from the Institute of Hydrobiology (IHB) in Wuhan, China. The medium used for the preculture of the strain was Zarrouk medium (Duerr et al., 1997; Zarrouk, 1966) consisting of (per liter): 16.8 g NaHCO3, 0.5 g K2HPO4, 2.5 g NaNO3, 1 g K2SO4, 1 g NaCl, 0.2 g MgSO4.2H2O, 0.04 g CaCl4.2H2O, 0.01 g FeSO4.7H2O, 0.08 g EDTA and 1 ml of trace metal solution. The trace metal solution consisted of (per liter): 2.86 g H3BO3, 1.81 g MnCl4.4H2O, 0.222 g ZnSO4.4H2O, 0.0177 g Na2MoO4,
0.079 g CuSO4.5H2O. The microalga was regularly grown at 28°C±0.5°C for 8-12 days with a continuous supply of 2.5% CO2 at an aeration rate of 0.2 vvm. The microalgal culture was illuminated 24 hours a day with a light intensity of approximately 60 µmol/m2/s which 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 cultivate the Arthrospira platensis was a 1-liter glass vessel (15.5 cm in length and 9.5 cm in 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 microalga was pre-cultured and inoculated into the PBR with an inoculums size of 0.08-0.24 g/L. The medium used for strain culture in the PBR was Zarrouk medium. The PBR were operated at 28°C, pH 9.0, and an agitation rate of 400 rpm under a light intensity of approximately 75-450 µmol/m2/s. Serving as the sole carbon source, 2.5% CO2 with 0.2 vvm was fed into the microalgal culture continuously during cultivation. Liquid samples were collected from the culture broth at set time intervals to determine the cell concentration, pH, C-PC content and residual nitrate concentration.
2.3. Operation of fed-batch strategy The fed-batch cultivation was started on a batch culture with 0.24 g/L inoculums size and 300 µmol/m2/s light intensity. 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 5.0 mM and 10 mM. The timing for starting new pulse-feeding was when the nitrate concentration in the culture broth was nearly depleted. Liquid samples were collected from the culture broth at set time intervals to determine the cell concentration, pH, C-PC content and residual nitrate concentration.
2.4. Determination of cell growth and CO 2 fixation rate Microalgae biomass concentration was determined by measuring the optical density of the sample at a wavelength of 680 nm (denoted as OD680) using a UV/Vis spectrophotometer (model U-2001, Hitachi, Tokyo, Japan) after proper dilution with deionized water. The OD680 values were converted to biomass concentration via appropriate calibration between OD680 and dry cell weight:
(
Biomass (g/L) = 0.584 ⋅ OD680 − 2.367 R2 = 0.99
)
The specific growth rate of microalgal culture was obtained by following calculation:
µ (d -1 ) =
d ln X dt
max
(i.e. maximum solpe from lnX versus t plot)
where X and t indicate the biomass concentration (g/L) and cultivation time (day), respectively. 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 DCW/L) within a cultivation time of ∆ t (d).
2.5. Determination of nitrate concentration The nitrate concentration in the medium was determined by a colorimetric method described in Ho et al. (2013b). The calibration between the absorbance and nitrogen concentration was established using sodium nitrate as the standard.
2.6. Determination of C-phycocyanin content A fixed amount (0.06 g) of the lyophilized biomass was mixed with 10 ml of 0.15 M phosphate buffer (pH = 7.0) and maintained at 4°C for 36 hours. The cell
debris was then removed by centrifugation at 8,000 rpm, and the supernatant (blue color) was collected. The absorbance of crude extract was measured with UV/Vis spectrophotometer at the wavelengths of 620 nm and 652 nm for calculating the concentration of C-PC according to the following equation (Bennett & Bogorad, 1973): C-PC (g/L) =
OD 620 − 0.474 ⋅ OD 652 5.34
3. Results and discussion 3.1. Time-course performance on cell growth and C-PC production The cellular composition of microalgae usually varies with the cell growth phase (Ho et al., 2012; Mandal & Mallick, 2009; Xie et al., 2013), and thus it is necessary to harvest the microalgal cells at the optimal timing to ensure maximal C-PC productivity for commercialization. In this work, Arthrospira platensis was cultivated in a batch culture for around 16 days to investigate variation in C-PC production. As shown in Fig. 1, the C-PC content and productivity increased simultaneously along with cell growth and nitrogen consumption, and the maximal values (10.3±0.3 mg/g and 38.0±1.0 mg/L/d) were obtained at the beginning of nitrogen depletion. While under nitrogen depletion conditions, both of them decreased significantly with the prolonged cultivation time. This trend is well in agreement with the report of Chen et al. (2013). It has been suggested that C-PC belongs to a family of phycobiliproteins which have obtained a secondary role as intracellular nitrogen storage compounds that are mobilized for other purposes in times of nitrogen shortage (Eriksen, 2008). This is also supported by several studies, which have demonstrated that nitrogen depletion causes a reduction in protein content, along with an enhancement of energy-rich compounds, such as lipids and carbohydrates (Ho et al., 2012; Mandal & Mallick, 2009). The possible reason could be that under nitrogen depletion the intracellular proteins was degraded as a source of nitrogen to maintain the metabolic functions of microalgae, and utilized for synthesis essential cell structures to against environment stress. Therefore, in order to attain the maximal C-PC content and productivity, the
beginning of nitrogen depletion should be the best time for harvesting microalgal biomass during the batch cultivation.
3.2. Effect of light intensity on cell growth and C-PC production For phototrophic cultivation, the light intensity not only strongly influences the cell growth of microalgae, but also changes the levels of light-related molecules in photosynthetic system (Markou et al., 2012; Xie et al., 2013). To investigate the effect of light intensity on cell growth and C-PC production of Arthrospira platensis, different light intensities (75-450 µmol/m2/s) were examined in this study. As shown in Fig. 2a, both the biomass productivity and specific growth rate of Arthrospira platensis increased significantly when the light intensity was increased from 75 to 300 µmol/m2/s. However, a sharp decrease was observed when light intensity was further increased up to 450 µmol/m2/s, indicating that this light intensity falls within the light inhibition region (Xue et al., 2011). This could be directly supported by the observation of changing cells color from green to white during cultivation in the first 3 days (data not shown). Thus, 300 µmol/m2/s seems to be the optimal light intensity for the growth of Arthrospira platensis, with the highest biomass productivity of 436.3±6.2 mg/L/d and specific growth rate of 1.02±0.04 d -1. To obtain high biomass productivity, the light intensity should be increased properly but a low intensity of light was more favorable for the C-PC accumulation. As shown in Fig. 2b, the maximum C-PC content of Arthrospira platensis decreased significantly from 18.4±0.4 to 2.2±0.2% as the light intensity was increased from 75 to 450 µmol/m2/s. It is known that C-PC is also an antenna pigment used by mainly cyanobacteria and eukaryotic algae to increase efficiency of photosynthesis by collecting light energy at wavelength where chlorophylls poorly absorb, and transfering the energy in high efficiency to chlorophyll a in the thylakoid membranes (Eriksen, 2008; Kuddus et al., 2013; Sun et al., 2006). The decrease in C-PC content at higher light intensity would be because the necessary amount of light energy is completely absorbed by a growing number of cells, thus the cells require a lower
content of cellular C-PC. Similarly, Markou et al. (2012) reported that higher light intensity results to lower chlorophyll and protein content of Arthrospira platensis. Chen et al. (2013) also reported an increase of up to 27% in C-PC content, when the light intensity of the culture decreased from 1300 to 100 µmol/m2/s. As for C-PC productivity, Fig. 2b shows that there was a slight increase as the light intensity increased until photo-inhibition occurred. This trend is well in agreement with the report of Chen et al. (2013). The highest C-PC productivity of 40.0±0.6 mg/L/d was obtained when using a light intensity of 300 µmol/m2/s. The optimization of light intensity is achieved due to combine the dual effects on C-PC content and biomass productivity. However, the optimal light intensity obtained from this study is inconsistent with the findings of Chen et al. (2013), which reported the maximal C-PC productivity of Arthrospira platensis was obtained at 700 µmol/m2/s. Zeng et al. (2012) also got an excellent yield of C-PC using 200 µmol/m2/s but did not evaluate different light intensities. The reasons for this discrepant result might be due to differences in bioreactor configurations and culture strategy. However, in any case, it should be noted that the observation in regard to the photo-bleaching of the algae might suggest an analytical method to set the optimal light intensity.
3.3. Effect of initial biomass concentration on cell growth and C-PC production Initial biomass concentration (IBC) is an important factor that affects the viability and productivity of microalgae in batch cultivation. Thus, the effect of IBC on cell growth and C-PC production of Arthrospira platensis was investigated, and the results are shown in Fig. 3. The maximum specific growth rate of Arthrospira platensis decreased significantly with an increase in the IBC. Conversely, the biomass productivity increased with increasing IBC from 0.08 to 0.16 g/L, while further increases in IBC to 0.24 g/L did not lead to better biomass productivity (Fig. 3a). These results are similar to that observed by Hu et al. (1997), who reported that an exponential decrease in specific growth rate and a positive skewed pattern in biomass
productivity were associated with an increase in cell density of Monodus subterraneus and Arthrospira platensis. Lu et al. (2012) also reported that the maximum growth rate of Chlorella sorokiniana in a lower inoculum culture was 1.38- to 2.42-fold increase when compared to the highest inoculum size culture. Increasing IBC in a certain range led to a shorten in the growth cycle of microalgae by avoiding the lag phase, resulting in the higher biomass productivity (Ho et al., 2013a; Ma et al., 2013), although the values of final cell density were almost the same under different IBC (data not shown). However, when the IBC is higher than a certain threshold level, irradiance levels lower than the light saturation level of the photosynthetic organism may induce light limitation, resulting in a reduction in biomass productivity (Hu et al., 1997; Lu et al., 2012). As shown in Fig. 3b, a considerable increase in C-PC content from a basal level of 9.2±0.3% to 11.6±0.4% was observed when IBC was increased up to 0.24 g/L, indicating that a higher IBC is more preferred for C-PC accumulation of Arthrospira platensis. Considering the fact that cell density could alter the average irradiance by cell absorption and self-shading, so when using high IBC, the average irradiance (the amount of light/unit of cell) was lower than that using low IBC. As a result, the size of light-harvesting antenna complex should be increased to absorb more light energy, resulting in higher C-PC content. Moreover, Fig. 3b also shows that the C-PC productivity of Arthrospira platensis increased nearly 70% when the IBC was raised from 0.08 to 0.24 g/L. The highest C-PC productivity of Arthrospira platensis (66.8± 1.5 mg/L/d) was obtained with an IBC of 0.24 g/L. This result can be mainly contributed to greatly improve in C-PC content and shorten the culture time to reach the maximum C-PC content when properly increasing IBC. The decrease in biomass productivity, however, was lower than the increase in C-PC content, resulting in a net increase of the C-PC productivity. However, as taking account of process economics, increasing IBC also requires a proportional increase in capital and operating resources (Zemke et al., 2013). Therefore, it is need a trade-off between seed cost and production efficiency to achieve low-cost and large-scale microalgae cultivation for C-PC production with Arthrospira platensis.
3.4. Improvement of C-PC production of Arthrospira platensis by using fed-batch operation As indicated in Fig.1, the maximal C-PC content and productivity were obtained at the beginning of nitrogen depletion. It has been suggested that C-PC accumulation is highly dependent on the level of residual nitrogen concentration (Chen et al., 2013). Moreover, although increasing average irradiance (the amount of light/unit of cell) enhanced the cell growth rate, it reduced C-PC content. Thus, it is necessary to develop an effective strategy that could enhance biomass production and simultaneously achieve a relatively high C-PC content. Fed-batch operation was often viewed as an effective strategy to control the nutrient concentration in the culture broth and enhance biomass production via prolonging cell growth phase (Hsieh & Wu, 2009; Sánchez-Luna et al., 2004; Xie et al., 2013). Accordingly, trying to produce a high amount of biomass with high C-PC content, the fed-batch operation was performed. The start-up of the fed-batch process was the same as that used for the batch operation with 0.24 g/L IBC and 300 µmol/m2/s light intensity. When the initial nitrate was nearly depleted, the 100-fold concentrated nitrate solution was then pulse-fed into the culture to reach a nitrate concentration of 5.0 mM and 10 mM, and the results are shown in Fig. 4 and Table 1. The biomass production and productivity of Arthrospira platensis were quite similar in both cases. The C-PC content as well as C-PC productivity in 5 mM nitrate feeding was nearly 15% higher than that of 10 mM nitrate feeding. When using 10 mM nitrate feeding, C-PC content was just the same as that obtained with batch cultivation. Chen et al. (2013) reported that biomass productivity and C-PC productivity decreased when initial nitrogen concentration was higher than 45 mM. Meanwhile, C-PC content was observed at a constant range when increasing initial nitrogen concentration from 30 to 90 mM under batch cultivation. These seem to indicate that controlling the nitrogen concentration at a lower level in the fed-batch cultivation was more favorable for C-PC accumulation. Similarly, Xie et al. (2013) also reported that a higher lutein content was obtained in a fed-batch culture fed with relatively lower nitrogen concentration. The reason for this might be because
when controlling the nitrogen concentration at a low but non-limiting level, nitrogen should be used first and foremost to synthesis light-related molecules and light-harvesting proteins in photosynthetic system. Moreover, in order to evaluate whether controlling other components in the medium have a positive or negative influence on C-PC production, the fed-batch operation with 5 mM medium feeding was also investigated, and the results are shown in Fig. 5 and Table 1. After initial nitrate concentration was depleted, both nitrate feeding and medium feeding led to a continuous increase in biomass concentration, while the biomass concentration obtained from medium feeding was slightly higher than that of nitrate feeding. Moreover, the biomass production in fed-batch cultivation was nearly 30-40% higher than that of batch cultivation (Table 1). Similarly, Hsieh & Wu (2009) reported that urea feeding could effectively enhance the biomass production of Chlorella sp.. Xie et al. (2014) also reported that fed-batch with medium feeding could achieve a relatively higher biomass concentration of Desmodesmus sp. F51 when compared to the fed-batch with nitrate feeding. In addition, Fig.4a and Fig. 5 show that the C-PC content of Arthrospira platensis increased with the prolonged time of fed-batch operation, and the highest values obtained were 13.4±0.5 and 16.1±0.2%, for nitrate feeding (5 mM) and medium feeding (5 mM), occurring at the cultivation times of 9.8 and 10.9 d, respectively. After that, the C-PC content, as well as nitrate consumption rate, showed a downward trend in both case. This could be attributed to the lower cell viability with the prolonged cultivation time. When using 5 mM medium feeding, the maximum C-PC content was around 20% higher than that of 5 mM nitrate feeding, indicating that C-PC accumulation not only requires nitrogen-sufficient condition, but also needs other nutrients. C-PC belongs to a family of phycobiliproteins which are some of the most abundant proteins in many cyanobacteria and algae (Eriksen, 2008). Several studies have demonstrated that nutrients depletion, such as S, P and N, would cause a decrease in protein content of microalgae (Ho et al., 2012; Markou et al., 2012; Procházková et al., 2013). Thus, when using 5 mM nitrating feeding, some nutrients in culture broth would decrease to deprivation condition with the prolonged
cultivation time, resulting in a relatively lower C-PC content than that obtained from using medium feeding. Moreover, when compared with the batch cultivation, the C-PC content obtained from 5 mM medium feeding enhanced around 40%, which was within the upper range of C-PC content reported in previous literature (e.g., 7.0-18.5%) (Table 2). As a result, the increased biomass with high C-PC content led to the ultimate enhancement of C-PC production in fed-batch cultivation with 5 mM medium feeding. The highest C-PC production obtained from medium feeding reached to 1034.8±7.5 mg/L, which was nearly 98% higher than that obtained from the conventional batch process (Table 1). Furthermore, the highest C-PC productivity in 5 mM medium feeding was 94.8±1.4 mg/L/d, which was not only around 42% higher than that in batch cultivation, but also higher than the results in most of the related studies (ranging from 14 to 125 mg/L/d) (Table 2). Therefore, fed-batch with medium feeding is indeed a very effective way for enhancing C-PC production of Arthrospira platensis.
4. Conclusions The effects of light intensity and initial biomass concentration on cell growth and C-PC production of Arthrospira platensis were investigated under the batch phototrophic cultivation. The average irradiance affects C-PC content and cell growth rate in an opposite way. Controlling a lower nitrogen level in fed-batch cultivation was more favorable for C-PC accumulation. The fed-batch cultivation with 5 mM medium feeding was proved to be an effective method to further enhance the C-PC production, giving the highest C-PC content, titer, and productivity of 16.1%, 1034 mg/L, and 94.8 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 the National High Technology Research and Development Program 863, China (No. 2014AA021701).
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Table 1 Effect of different fed-batch strategies on cell growth, CP-C content and C-PC production of Arthrospira platensis (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; temperature, 28°C; light intensity, 300 µmol/m2/s; nitrate concentration, 30 mM).
Nitrate/Medium
Biomass
Biomass
C-PC
C-PC
C-PC
feeding
production
productivity
content
productivity
production
concentration
(g/L)
(mg/L/d)
(%)
(mg/L/d)
(mg/L)
0
4.82±0.05
576.3±5.2
11.6±0.4
66.8±1.5
523.0±3.2
5 mM (Nitrate)
6.18±0.04
592.9±3.5
13.4±0.5
79.6±0.6
782.5±5.3
10 mM (Nitrate)
6.20±0.06
594.2±7.2
11.7±0.3
69.1±1.2
714.0±8.2
5 mM (Medium)
6.78±0.07
588.2±6.8
16.1±0.2
94.8±1.4
1034.8±7.5
The values are calculated at the time of maximal C-PC productivity. Data shown are the averages of two runs ± SD (standard deviation).
Table 2 Comparison of the performance of C-PC content, production and productivity of Arthrospira platensis with that obtained in the related reports under phototrophic cultivation.
C-PC
C-PC
C-PC
Content
production
productivity
(%)
(mg/L)
(mg/L/d)
Operation Microalgae strains
Reference
strategies Arthrospira platensis
Batch
7.0
210
64
Zitelli et al., 1996
Arthrospira platensis
Batch
7.0
350
92
Carlozzi, 2003
Arthrospira platensis
Batch
4.8
350
14
Leema et al., 2010
Arthrospira platensis
Batch
16.8
1220
67.8
Zeng et al., 2012
Arthrospira platensis
Batch
12-12.6
400-700
110-125
Chen et al., 2013
Arthrospira platensis
Fed-batch
16.1±0.2
1034.8±7.5
94.8±1.4
This study
Figure Legends Figure 1 Time-course profiles of biomass concentration, nitrate concentration, C-PC content and C-PC productivity during the batch growth of Arthrospira platensis (operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; light intensity, 150 µmol/m2/s; temperature, 28°C; nitrate concentration, 30 mM; inoculums size, 0.08 g/L). Figure 2 The performance of cell growth and C-PC production of Arthrospira platensis under different light intensities: (a) maximum specific growth rate and biomass productivity, (b) C-PC content and C-PC productivity. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; temperature, 28°C; nitrate concentration, 30 mM; inoculums size, 0.08 g/L). Data are the averages of two runs and the error bars are standard deviations. Figure 3 The performance of cell growth and C-PC production of Arthrospira platensis under different initial biomass concentrations: (a) biomass production and biomass productivity, (b) C-PC content and C-PC productivity. (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; temperature, 28°C; nitrate concentration, 30 mM; light intensity, 300 µmol/m2/s). Data are the averages of two runs and the error bars are standard deviations. Figure 4 Time-course profiles of biomass concentration, nitrate concentration, C-PC content and C-PC productivity of Arthrospira platensis with (a) 5 mM nitrate feeding and (b) 10 mM nitrate feeding (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; temperature, 28°C; light intensity, 300 µmol/m2/s). Figure 5 Time-course profiles of biomass concentration, nitrate concentration, C-PC content and C-PC productivity of Arthrospira platensis with 5 mM medium feeding (Operating conditions: CO2 concentration, 2.5%; CO2 flow rate, 0.2 vvm; temperature, 28°C; light intensity, 300 µmol/m2/s).
Nitrate concentration (g/L) 3.0 6
2.5 5
2.0
1.5
1.0
Biomass concentration (g/L)
15
4
3
0.5 1
0.0 0 10
0 4 8
Time (d) 12 16 0
C-PC content (%)
20
40
30
20
2 5
10
0
C-PC productivity (mg/L/d)
Figure 1
50
Maximum C-PC content (%)
Biomass productivity (mg/L/d)
15
10
5
0 75 150 300 450
Light intensity (mol/m /s)
2 -1
(a)
400 1.2
300 0.9
200 0.6
100 0.3
0 0.0
(b) 40
20
30
20
10
0
Maximum specific growth rate (d )
500
Maximum C-PC productivity (mg/L/d)
Figure 2 1.5
Maximum C-PC content (%) 0 0.08
600 1.2
400 0.9
0.6
200 0.3
0 0.0
(b) 80
10 60
40
5 20
0.12 0.16 0.20
Initial biomass concentration (g/L) 0.24 0
Maximum Specific growth rate (d )
-1
(a)
Maximum C-PC productivity (mg/L/d)
Biomass productivity (mg/L/d)
Figure 3 1.5
Nitrate concentration (g/L) 2.0
0.0
1.5
1.0
0.5
0.0
10
4
2
0
0
0
0
3
3
6 9
6
9
Time (d) 12
8
12
0
2.5
2.0
6 10
4
2
0
60
40
5
60
40
5 20
0
C-PC productivity (mg/L/d)
2.5
C-PC productivity (mg/L/d)
0.5 6
C-PC content (%)
8
C-PC content (%)
1.0
Biomass concentration (g/L)
1.5
Biomass concentration (g/L)
Nitrate concentration (g/L)
Figure 4
100
(a) 15 80
20
0
Time (d) 100
(b) 15 80
Nitrate concentration (g/L) 2.0
1.5
1.0
0.5
0.0
Biomass concentration (g/L)
2.5
6 10
4
2
0 0 3 6 9
Time (d) 12 0
60
40
5 20
0
C-PC productivity (mg/L/d)
8
C-PC content (%)
Figure 5
100
15 80
Highlights
Arthrospira platensis WH879 shows high potential as an C-phycocyanin producer
The average irradiance affects C-PC content and cell growth rate in an opposite way
Controlling a lower nitrogen level was more favorable for C-PC accumulation Fed-batch cultivation with medium feeding effectively enhanced the C-PC production