Effect of Red and Blue Leds on the Production of Phycocyanin By

Journal of Science and Technology in Lighting Vol.41, 2017

J-STAGE Advanced published date: December 4, 2017, doi: 10.2150/jstl.IEIJ160000597

Paper Effect of Red and Blue LEDs on the Production of Phycocyanin by Spirulina Platensis Based on Photosynthetically Active Radiation Feng TIAN*, **, David BUSO*, Tongming WANG***, Manuel LOPES*, Urbain NIANGORAN*, **** and Georges ZISSIS*,†

* LAPLACE, UPS, Université de Toulouse, 118 route de Narbonne, 31062 Toulouse Cedex 9, France ** State Key Laboratory of Automotive Safety and Energy, Tsinghua University, Beijing 100084, China *** Laboratoire des Interactions Plantes-Microorganismes, (INRA, UMR441-CNRS, UMR2594), Castanet-Tolosan F-31326, France **** Laboratoire Image Instrumentation et Spectroscopie, Institut National Polytechnique Félix Houphouet Boigny de Yamous- soukro, Yamoussoukro, Côte d’Ivoire

Received December 1, 2016, Accepted October 31, 2017

ABSTRACT Phycocyanin (PC) is a kind of valuable pigment extracted from spirulina platensis (S. platensis). Light environment is one of the most important factors on the production of PC. Using light-emitting diode (LED) light sources, the S. platensis was cultured with ﬁve different ratios of red and blue photons. Pho- tosynthetically Active Radiation (PAR) was precisely controlled by the function relationship between PAR, junction temperature and forward current. The comparative analysis shows that blue light is conducive to improve the mass fraction of PC, but the total production per incubator is lower than the one obtained under red light.

KEYWORDS: spirulina platensis, phycocyanin, light emitting diode, PAR

1. Introduction It has been clarified that blue and red light are the PC is a photosynthetic pigment, which falls into C most important radiation for plant photosynthesis5, 6), and R types. The former was found in cyanobacteria, but this mechanism may not apply for microalgae. PC and the latter was found in red algae and cryptophytes. has much higher content than other pigments and it is PC is an accessory pigment of chlorophyll, which ap- indispensable for S. platensis photosynthesis. Neverthe- pears blue, absorbs orange and red light, and transmits less, functions of mixed spectra on the production of PC light energy during the process of photosynthesis. Over are seldom reported. Until now, there are some reports the past decades, PC has been used as natural edible demonstrating the effect of single colored light on bio- pigment, cosmetics, medicine and fluorescent reagent1). mass and pigment production of S. platensis on the pho- Until now, its new functions such as antitumor, anti- toautotrophic cultivation with artificial LEDs including oxidant, anti-allergy and improving the organism im- red, yellow, green or blue colors7–9). munity are being gradually explored2, 3). For example, Wang et al.7) showed that Red LED pro- LED is a kind of solid state light source. As the latest moted the highest specific growth rate while blue LED lighting source, it has been quickly developed in green- showed the least efficiency in the conversion of photon houses due to the advantages of high light efficiency, to biomass. Chainapong et al.10) focused on different long lifetime, narrow spectrum radiation, cool light, concentrations of photosynthetic pigments produced by robustness and so on. S. platensis in photoautrophic and mixotrophic condi- Instead of using photon energy, PAR between tions with different light quality generated by filtered 400–700 nm is often used to characterize photosynthesis sun light. In their study they used white light as well efficiency driven by photons. PAR includes photosyn- as colored light. They showed that white light exhibit thetic photon flux (PPF) and yield photon flux (YPF)4). the maximum biomass concentration in mixotrophic YPF is a more accurate measure of a horticulture light cultures while the content of pigments was reduced. ability to drive photosynthesis of plants. As there is no Kim et al.9) showed that red and green light produce relative quantum efficiency curve for spirulina platensis, significantly higher growth rate while green and blue PPF was preferred to measure the light intensity in this produce the higher photosynthetic pigment content. study. As biomass production and content of pigments

† Corresponding Author: Georges Zissis [email protected]

148 The Illuminating Engineering Institute of Japan Journal of Science and Technology in Lighting Vol.41, 2017

doi: 10.2150/jstl.IEIJ160000597

Figure 1 Experimental setup for incubators with five different proportions of red and blue photons (Incubator size: 30(L)∗15(W)∗20(D) cm). like PC are strongly correlated and dependent on A kind of aquarium made with transparent glass was the culture environment as well as light condition, in used as incubator. Six liters of Zarrouk medium were this paper, we used mixed colors at different ratios to filled for the cultivation. As shown in Figure 1, two LED investigate the conjugated effect on biomass and PC plates were fixed beside the incubator with a distance of production. Moreover, to approach operational condi- 1 cm to the walls of incubator. Twenty-four LEDs were tion of intensive culture in bioreactor, 9L incubators adopted for each incubator, and evenly distributed on were used instead of small volume incubators (typically both sides. The PAR of each incubator was 74.42µmol/ 500mL flask). (m2·s) tested by a spectroradiometer (specbos 1201) in We originally used mixed spectra with five differ- an integrating sphere (diameter: 25 cm). Five different ent proportions of blue and red photons to cultivate S. proportions of red and blue photons are set to B : R=4 : 0 platensis in order to explore the effects of blue and red (only blue Figure 1.a), B : R=3 : 1 (three quarter of blue spectrum on the production of PC. Twenty-four high PAR and one quarter of red PAR, Figure 1.b), B : R=2 : 2 power LEDs were adopted for each incubator. Three- (equal blue and red PAR, Figure 1.b), B : R=1 : 3 (three dimensional relationships between the PAR, forward quarter of red PAR and one quarter of blue PAR, Fig- current (If) and junction temperature (Tj) were obtained ure 1.b) and B : R=0 : 4 (only red Figure 1.c), respectively. to get accurate PAR with the help of spectroradiometer A total of five incubators were used with the preceding (specbos 1201), an integrating sphere and temperature settings. controlled chamber and module. The experiment was repeated three times. The S. platensis produced by the first experiment was con- 2. Materials and methods sidered as the first generation, including five different 2.1 Culture conditions kinds corresponding five incubators. For the second ex- The microorganism used was Spirulina platensis periment, we inoculated from the first generation, and UTEX LB 2340 from Natron Lake, which was grown the third experiment was inoculated from the second photoautotrophically in Zarrouk medium11). The strain generation. The other conditions remained the same. culture was grown in 80 L of glass container at 32.5±1°C with continuous illumination provided by white tubular 2.2 Accurate PAR control method fluorescent lamps (Osram T5 HE ES 13W/840 G5 Lu- According to the characteristics of LED, the forward milux), and agitated by a circulating pump. For inocula- current (If) and junction temperature (Tj) are two key tion, we took certain amount of the strain culture, and parameters to get accurate PAR. Normally, Tj and for- filtered it by 30 µm strainer, then diluted the S. platensis ward voltage (Vf) of LED have a linear relationship. We with Zarrouk to an Optical Density at 600 nm (OD600) used 10 mA pulse current, produced by a sourcemeter of 0.180. (keithley 2602), to measure Vf at different temperatures The experiment was performed in greenhouse with in a temperature controlled chamber. Then the relation- an air conditioner. The temperature of culture medium ship between Tj and Vf was extracted. for S. platensis kept the same as strain culture at Then, LED PAR was measured in function of the 32.5±1°C. Wave maker pumps were used to agitate the junction temperature at different current levels. In culture solution. The flow velocity was 5000L/h. The order to accurately describe PAR for each incuba- pH value increased from the beginning of 8.5 to the end tor, single LED was measured by a spectroradiom- of less than 11 during the culture process. eter (specbos 1201) in an integrating sphere (diameter: Blue and red LEDs were selected with the same size 25 cm) with a small temperature controlled module (3.45∗3.45∗2.00 (H) mm). The peak wavelengths were assembled in the center. The parameters of LEDs mea- 458 nm and 625 nm, and viewing angle 135° and 130° at sured by integrating sphere were more accurate and 50% current value, respectively. Both of the LEDs had believable than a quantum sensor in the experimental a rated current of 350 mA and a maximum current of conditions. The purpose is to quantify the PAR and 1000 mA. provide a reference, which can improve the repeatabil-

149 The Illuminating Engineering Institute of Japan Journal of Science and Technology in Lighting Vol.41, 2017

doi: 10.2150/jstl.IEIJ160000597

-1 ity of the experiment. The PAR of each LED was 74.42 content of allophycocyanin (in mg/mL ); X3 is the con- 2 -1 µmol/(m ·s). With the same quantity and distribution of tent of phycoerythrobilin (in mg/mL ); X4 is the mass LEDs, each incubator can get the same PAR. The only fraction of PC (g/100 g); A is the absorbance of corre- diﬀerence presented in the experiment was the color of sponding wavelengths (620 nm, 652 nm and 562 nm); V is LEDs. The other conditions were maintained the same the constant volume of test samples (mL); m is the dry between diﬀerent experiments. Measurements were weight (g) of test samples. performed each 5 min in order to keep Tj constant. On the basis of the relationship between Tj and Vf, the true 3. Results and discussion test Tj was available in the integrating sphere. Thus, 3.1 Growth curve of spirulina the three-dimensional relationships between the PAR, After 5 days of continuous illumination we got the

Tj and If were obtained as Figure 2. growth curves of spirulina presented in Figure 3. The Through the surface fitting tool of Matlab, the fitting specific growth rate under red LEDs was much faster functions for blue and red LEDs are as Eq. (1) and (2) than blue. When the culture was illuminated only with (with 95% confidence bounds, R-square≧0.9997). So the red light (B : R=0 : 4), final OD600=1.324 was 7.4 times accurate PAR can be easily determined by setting If higher than the initial OD600 of 0.180. This implied that and Tj. red light was considered as the most efficient emission required for algal photosynthesis, which resulted in the PAR =+1.141 0.0071⋅⋅TI + 192.7 blue j f highest biomass accumulation. The result was parallel 2 7) 8) −0.4178 ⋅⋅−TIjf 54.82 ⋅ I f (1) to the observations of Wang et al. and Chen et al. . For an equivalent ratio of red and blue PAR (B : R=2 : 2), final PARred =−2.585 + 0.0173 ⋅+TIj 180.7 ⋅f OD600 was 1.031. For ratios of three quarter of blue −0.7728 ⋅⋅−TI 33.8 ⋅ I2 (2) jf f PAR and one quarter of red PAR (B : R=3 : 1) and ratios of three quarter of red PAR and one quarter of blue 2.3 Phycocyanin content detection PAR (B : R=1 : 3), almost the same growth rate was ob- Certain amount of dry spirulina sample was dissolved served. Under blue light (B : R=4 : 0) a minimum biomass by phosphate buffer solution (pH=7), shattered by the is obtained, and OD600 was only twice the original. The ultrasonic wave, and placed in the refrigerator (-20°C) results indicate that the combinations of red and blue for 12 h to precipitate out PC, then centrifuged 15 min LEDs had an intermediate effect for photosynthesis. Blue at 3000 rpm. The supernatant was used to measure the absorbance at 620 nm, 652 nm and 562 nm by spectro- photometry12). The functions for the mass fraction of phycocyanin (MFPC) are as follows:

XA1 =0.187·620 − 0.089· A652 (3)

XA2 =0.196·652 − 0.041· A620 (4)

X3 =0.104· A562 −− 0.251· XX1 0.088· 2 (5)

X4=( XXXV 123 ++)· ·100/( m ·1000) (6)

-1 Where: X1 is the content of PC in mg/mL ; X2 is the Figure 3 Growth curves of S. platensis for phycocyanin.

Figure 2 Three-dimensional relationship between photosynthetically active radiation (PAR) (µmol/s·sqm) versus junction tempera-

ture (Tj) (°C) and forward current If (A) of the blue (a) and red (b) LEDs.

150 The Illuminating Engineering Institute of Japan Journal of Science and Technology in Lighting Vol.41, 2017

doi: 10.2150/jstl.IEIJ160000597

Table 1 Test data for mass fraction of phycocyanin in the experiment.

A562 A620 A652 X1 X2 X3 X4 (%) B : R =4 : 0 0.952 1.839 0.789 0.274 0.079 0.023 31.355 B : R =3 : 1 0.657 1.292 0.581 0.190 0.061 0.015 22.175 B : R =2 : 2 0.412 0.797 0.378 0.115 0.041 0.010 13.921 B : R =1 : 3 0.393 0.761 0.371 0.109 0.042 0.010 13.383 B : R =0 : 4 0.488 0.954 0.449 0.138 0.049 0.012 16.586 TFL 0.622 1.213 0.518 0.181 0.052 0.015 20.608

Figure 5 Total average production of PC per incubator in three generations.

not been measured in this study, but it can reasonably assume that in the case of red light, the lower MFPC may be explained by a nitrogen deficiency. This could be explained by the priority of biosynthesis. Biomass accumulation has a higher priority than the production of PC. Besides, PC also serves as an alternative nitrogen Figure 4 Mass fraction of phycocyanin under different propor- storage, so the MFPC may rapidly decrease when nitro- 13) tions of red and blue photons in three generations. gen deficiency occurs in Zarrouk medium . However, under blue light, the growth rate of S. platensis is much light does not perform well for biomass accumulation. slower than that under red light, which is favorable to produce high MFPC. 3.2 Mass fraction of phycocyanin (MFPC) Compared with the second and third generations, The test data and results for MFPC are shown in MFPC of the first generation was the lowest under Table 1 and Figure 4. MFPC under TFL (Tubular blue and combinations of blue and red light. MFPC of Fluorescent Lamp) illumination was used as a reference the second and third generations had the same trend to

(MFPCTFL=20.608%). It shows that the maximum aver- increase with one exception that the third generation age MFPC under blue light is about 2.3 times of that of red light had a lower MFPC. It can be inferred that under red light. The average MFPC decreased from blue light promote the MFPC in different generations. 30.382% under blue light conditions to 12.996% under But MFPC does not show this trend under the red light. red light conditions. But the average MFPC under The total average production of PC (TPPC) is shown B : R =2 : 2, 1 : 3 and 0 : 4 did not show significant difference in Figure 5. Although blue light is conducive to produce (respectively 13.983%, 13.427% and 12.996%). high MFPC, TPPC is only 66.4% of the TPPC obtained These results are in line with previous work. Chen et under red light illumination. B : R=3 : 1 and red light al.8) who found that a higher biomass results in a lower (B : R=0 : 4) could be a better choice to produce more PC specific PC production rate. It is also confirmed by Kim under the light conditions. et al.9) who found that the growth rate for the group with red LED treatment was significantly higher than 4. Concluding remark that with blue LED treatment, and the photosynthetic Based on the accurate PAR control, red and blue pho- pigment content was higher in the blue LED group tons have different effects on the production of PC in S. than in the red LED group, especially for phycobilipro- platensis. Blue light is conducive to the synthesis of PC, tein. Our result showed a similar trend: the faster the and can be used to get high mass fraction of PC. Red biomass increased, the smaller the MFPC was. light promotes more photosynthesis, so the total pro- Nitrogen is essential element for S. platensis. Indeed duction of PC per incubator is higher than that under considerable nitrogen is consumed for the develop- blue light. In order to get the maximum PC, B : R=3 : 1 ment of chlorophyll a and photosynthesis, so the rapid and red light can be better choices in the test condi- increase of biomass caused depletion of the nitrogen tions. Proper adjustment of the ratio between red and under red light8, 13). On the other side, PC development blue lights can achieve the desired production of PC. requires also nitrogen. Nitrogen consumption rate has

151 The Illuminating Engineering Institute of Japan Journal of Science and Technology in Lighting Vol.41, 2017

doi: 10.2150/jstl.IEIJ160000597

Acknowledgments (2007). (8) Chen, H. B., Wu, J. Y., Wang, C. F., Fu, C.-C., Shieh, We appreciate the help and support from Biosentec C.-J., Chen, C.-I., Wang, C.-Y. and Liu, Y.-C.: Model- company for this study. It is also supported by China ing on chlorophyll a and phycocyanin production Scholarship Council (CSC). by Spirulina platensis under various light-emitting diodes, Biochem. Eng. J., 53-1, pp. 52–56 (2010). References (9) Kim, N. N., Shin, H. S., Park, H. G., Lee, J., Kil, G.-S. and Choi, C. Y.: Profiles of photosynthetic pigment (1) Eriksen, N. T.: Production of phycocyanin—A pig- accumulation and expression of photosynthesis- ment with applications in biology, biotechnology, related genes in the marine cyanobacteria Syn- foods and medicine, Appl. Microbiol. Biotechnol., echococcus sp.: Effects of LED wavelengths, Bio- 80-1, pp. 1–14 (2008). technol. Bioproc. Eng., 19-2, pp. 250–256 (2014). (2) Estrada, J. P., Bescos, P. B. and Del Fresno, A. V.: (10) Chainapong, T., Traichaiyaporn, S. and Deming, R. Antioxidant activity of different fractions of Spi- L.: Effect of light quality on biomass and pigment rulina platensis protean extract, Il farmaco, 56-5-7, production in photoautotrophic and mixotrophic pp. 497–500 (2001). cultures of Spirulina platensis, J. Agr. Technol., 8-5, (3) Sarada, D. V. L., Kumar, C. S. and Rengasamy, R.: pp. 1593–1604 (2012). Purified C-phycocyanin from Spirulina platensis (11) Zarrouk, C.: Contribution a L’etude D’une Ciano- (Nordstedt) Geitler: A novel and potent agent phycee: Influence de Divers Facteurs Physiques against drug resistant bacteria, World J. Microbiol. Et Chimiques Sur la Croissance Et la Photosyn- Biotechnol., 27-4, pp. 779–783 (2011). these de Spirulina Maxima (Setch. Et Garndner) (4) da Costa, G. J. and Cuello, J. L.: The phytometric Geitler, Faculte des Sciences, Universite de Paris system: A new concept of light measurement for (1966). plants, J. Illum. Eng. Soc., 33-1, pp. 34–42 (2004). (12) SN/T 1113–2002: Method for determination of (5) Hogewoning, S. W., Trouwborst, G., Maljaars, H., phycocyanin and chlorophyiis in spirulina powder Poorter, H., van Ieperen, W. and Harbinson, J.: for import and export [S], Standards Press of Chi- Blue light dose–responses of leaf photosynthesis, na (2002). morphology, and chemical composition of Cucumis (13) Lee, S.-H., Lee, J. E., Kim, Y. and Lee, S.-Y.: The sativus grown under different combinations of red production of high purity phycocyanin by Spiruli- and blue light., J. Exp. Bot., 61-11, pp. 3107–3117 na platensis using light-emitting diodes based two- (2010). stage cultivation, Appl. Biochem. Biotechnol., 178-2, (6) Chen, C.-C., Huang, M.-Y., Lin, K.-H., Wong, S.-L., pp. 382–395 (2016). Huang, W.-D. and Yang, C.-M.: Effects of light quality on the growth, development and metabolism of Part of this work was supported by European Union rice seedlings (Oryza sativa L.), Res. J. Biotechnol., and partially funded by the European Regional Devel- 9, pp. 15–24 (2014). opment Fund (ERDF). (7) Wang, C. Y., Fu, C. C. and Liu, Y. C.: Effects of us- All or part of this work was presented at 15th Inter- ing light-emitting diodes on the cultivation of Spi- national Symposium on the Science and Technology of rulina platensis, Biochem. Eng. J., 37-1, pp. 21–25 Lighting (LS15), May 2016, Kyoto, Japan.

152 The Illuminating Engineering Institute of Japan