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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.54 No.3 pp.89-93
DOI : https://doi.org/10.14397/jals.2020.54.3.89

Semicylindrical Column Photobioreactor for Cultivation of Spirulina platensis

Chang-Su Lee1, Do-Wook Woo1,2, Gyeong-In Lee1,2, Jong-Hee Kwon1,2,*
1Division of Applied Life Sciences (BK21 plus), Gyeongsang National University, Jinju 52828, Republic of Korea
2Department of Food Science & Technology and Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea
*Corresponding author: Jong-Hee Kwon Tel: +82-55-772-1901 Fax: +82-55-772-1909 E-mail: jhkwon@gnu.ac.kr
April 17, 2020 ; May 25, 2020 ; June 22, 2020

Abstract


Microalgae and cyanobacteria are photosynthetic microorganism used as human nutrients. Light is the most important factor affecting the biomass accumulation and production of bioactive compound by photosynthetic microorganisms. However, excessive light can suppress their growth by damaging the photosynthetic machinery such as Photosystem II (PS2). Therefore, an LED-based illumination system that is used for cultivation of photosynthetic cells needs a device to appropriately regulate the light level, but the addition of an electronic dimmer increases the initial costs. Spirulina is one of the promising and representative photosynthetic microorganisms. In the present study, we describe the use of a new semicylindrical column photobioreactor for cultivation of Spirulina. The amount of incident light reaching the photobioreactor was easily adjusted by changing angle of the LED in relation to the flat surface of the bioreactor. Arrangement of the LED bar at a 45° angle led to the highest growth and chlorophyll content, and led to no retardation of cell growth during the initial growth phase.



초록


    Introduction

    There is increasing interest in the photosynthetic cyanobacterium Spirulina (currently classified as Arthrospira platensis) because these cells can produce bioactive compounds that are economically valuable and have various therapeutic properties. In particular, these cells consist of 60−70% protein, and have abundant minerals, fatty acids (e.g., γ-linolenic acid), polysaccharides, pigments (chlorophyll, carotenoids, phycobilins), and nearly all essential vitamins (Ali & Saleh, 2012;Belay et al., 1993;Estrada et al., 2001;Holman & Malau‐Aduli, 2013). Most commercial production of Spirulina is currently performed in open ponds, which are inexpensive, easy to operate, and rely upon sunlight as a free source of energy (Borowitzka, 1999). However, open ponds do not allow high biomass productivity, because of the limited control over environmental parameters, such as light, temperature, and contaminants, leading to low photosynthetic efficiency (Brennan & Owende, 2010). Growing photosynthetic microorganisms in a photobioreactor (PBR) allows greater control of environmental parameters (Moheimani et al., 2011;Pulz, 2001;Singh & Sharma, 2012).

    Light is the major source of energy for the growth of photosynthetic cells. However, too much light can induce oxidation stress, resulting in growth inhibition and even irreversible inactivation of the photosynthetic machinery (Aro et al., 1993;Schulze et al., 2017). Meanwhile, photosynthetic organisms also have mechanisms that protect them from photodamage (Fujita et al., 1994;Joshua & Mullineaux, 2004). Light emitting diodes (LEDs) are commonly used in current custom PBR systems. When LEDs are used as an artificial light source, a dimmer device is needed to prevent excess irradiation during the initial cultivation stage. However, the use of an electronic dimmer could increase the initial costs for commercial microalgal cultivation. Thus, researchers have developed PBRs with different geometries and sizes for the more effective utilization of light (Hoekema et al., 2006;Kumar et al., 2011;Pulz & Scheibenbogen, 1998).

    In the present study, a new vertical column photobioreactor with semi-cylinder shape was developed and introduced for the cultivation of photosynthetic microorganism Spirulina. The productivity in photobioreactor is directly coupled with its ability to harvest light and to distribute it into the culture volume. Due to the geometry of semi-cylinder, our new-type column photobioreactor provide flat surface areas which could have better light penetration than round surface. Besides, without any additional dimming device, amount of incident light could be adjusted by changing angle of LEDs bar which is set toward flat surface of our reactor. This new light attenuation system was tested for the reliable biomass and pigment production from Spirulina. Here, we showed the new type semicylindrical column photobioreactor is very practical for accumulation of biomass and chlorophyll from Spirulina.

    Materials and Methods

    1. The setup of semi-cylinder photobioreactor and light intensity control

    A new columnar PBR was designed to improve light utilization during the photosynthetic cultivation of Spirulina (Fig. 1). This column PBR has a semicylindrical geometry (height: 60 cm, radius: 10 cm, working volume: 5 L). The light supply consisted of a single LED bar with a rotator that was aimed toward the flat vertical panel of the PBR at a distance of 12 cm. The total amount of incident light reaching the surface of the PBR was easily controlled by changing the angle of the LED bar. The photosynthetic photon flux density (PPFD) was measured using a Li-Cor LI-189 2 π PAR quantum sensor. The PBR was efficiently mixed by aeration, using a constant flow of 50 mL min-1 of ambient air that passed through PTFE filter (0.2 μm pore size) that are embedded in the middle-bottom of the reactor. Cell growth was monitored by measuring optical density and dry cell weight. The initial pH was 8.0 and the temperature was maintained at 25 °C.

    2. Cultivation of Spirulina platensis

    Arthrospira platensis KCTC AG40101 cells (hereafter A. platensis or Spirulina) was cultivated in the semicylindrical column PBR using standard SOT medium (16.8 g L-1 NaHCO3, 0.5 g L-1 K2HPO4, 2.5 g L-1 NaNO3, 1.0 g L-1 K2SO4, 1.0 g L-1 NaCl, 0.2 g L-1 MgSO4・7H2O, 0.04 g L-1 CaCl2・2H2O, 0.01 g L-1 FeSO4・7H2O, 0.08 g L-1 EDTA, and 1 mL of micronutrients). The micronutrient solution consisted of 2.86 g L-1 H3BO3, 1.81 g L-1 MnCl2・4H2O, 0.222 g L-1 ZnSO4・4H2O, 0.0177 g L-1 Na2MoO4, and 0.079 g L-1 CuSO4・5H2O. Pre-cultures were maintained in transparent 50 mL culture flasks with a photosynthetic photon flux density (PPFD) of 50 μmol photons m-2 s-1 PAR. Prior to inoculation, the prepared medium was sterile-filtered, and the reactor was chemically sterilized by incubation with 0.03% sodium hypochlorite for 1 h. After washing the reactor with sterile water 2 times, the sterile-filtered medium was added, and the pre-culture was then inoculated. During cultivation, the angle of the LED bar was set at 15°, 45°, 60°, or 90° to adjust the PPFD (Fig. 1).

    3. Dry cell weight and Optical cell density

    Culture samples were passed through filter paper and then dried at 80 ºC for 24 h to determine dry biomass. The difference between the final weight and initial weight of the filter paper was considered as total biomass. During cultivation, the optical density of each sample was measured by UV/Vis spectrophotometer (JASCO-V-730) at 750 nm and 680 nm.

    4. Measurement of chlorophyll content

    Cells (1 mL samples) were centrifuged at 10,000 rpm for 15 min and collected. Then 1 mL of 90% methanol was added, and the cells were the homogenized and stored at 4°C for 1 h. Samples were then centrifuged again at 10,000 rpm for 1 min. A UV/Vis spectrophotometer (JASCO-V-730) was used to measure absorbance at 665 nm and 650 nm, and the chlorophyll content (mg L-1) was calculated as 16.5 × A665nm + 8.5 × A650nm.

    Results and Discussion

    1. Comparing light efficiency according to surface type of photobioreactor

    Flat-bed PBRs usually have greater light efficiency than fully cylindrical column PBRs because flat panels have large illuminated surfaces and a more uniform distribution of light throughout the culture. However, the comparison of light efficiency among different reactor types but with the same geometric and cultivation conditions has not yet been performed.

    Our new semicylindrical column PBR has a flat vertical panel that lies opposite to a round vertical panel, and we compared illumination at flat vertical panel with at round vertical panel in the new PBR using the same mixing conditions. We applied a PPFD of 50 μmol m-2 s-1 PAR to each surface in the new PBR during the cultivation of A. platensis. After 22 days, the biomass concentration and chlorophyll concentration were about 1.28-times greater at case of illumination at flat panel in semicylindrical PBR (Fig. 2). Although the total area of round panel receiving light from the LED panel was larger, photosynthetic productivity was greater when using the flat vertical panel in semicylindrical PBR (Fig. 2). Considering that the hydrodynamic conditions and PPFD were the same, light reflection at the round surface of the semicylindrical PBR may have reduced photosynthetic productivity.

    2. A simple control of light intensity by changing the angle of LED bar toward flat-panel

    In PBR systems, light is sometimes regarded as the “fourth phase”, in addition to the liquid phase (fluid medium), solid phase (the cell), and the gas phase (Posten, 2009). Depending on the light supply, different regions within a PBR can be considered as productive light zones (sufficient light for photosynthesis) and unproductive dark zones (insufficient light for photosynthesis). On the other hand, too much light can lead to photoinhibition and inactivation of the photosynthetic machinery (Aro et al., 1993;Grima et al., 1996;Sandnes et al., 2005). It is therefore important to optimize the light conditions when culturing photosynthetic microorganisms.

    An electronic dimmer is commonly used to adjust the level of light from LEDs, but this entails a high initial investment. In the present study, we used a single adjustable LED bar to alter the PPFD with a semicylindrical column PBR for irradiation during Spirulina cultivation. We adjusted the PPFD by simply changing the angle of the LED bar relative to the flat vertical panel of the PBR (Fig. 1). We measured PPFD at each angle of the LED bar (from 0° to 90°, at 15° increments), from left edge to right edge at a distance of 2.5 cm (Fig. 3). An angle of 90° and 75° provided symmetrical PPFD, but the PPFD was asymmetrical for angles of 60° and below (Fig. 3). At 60° and less, the highest PPFD was at 12.5 cm from left edge of the bioreactor. The highest PPFD measured (179 μmol m-2 s-1 PAR) was at 10 cm from the left edge when the LED bar was at 90° (Fig. 3a). We also calculated the integrated PPFD as the average of the 7 given points (Fig. 1, Fig. 3b). The highest integrated PPFD occurred when the LED bar had an angle of 90°. Interestingly, an angle of 0° led to a PPFD of 77.7 ± 53.03 μmol m-2 s-1 PAR, almost half of the highest integrated PPFD. As expected, the integrated PPFD decreased as angle of the LED bar decreased. However, the deviation of PPFD among the given 7 points increased as angle of LED bar decreased (Fig. 3b).

    These results indicated that changing the angle of the LED bar was a simple method for adjusting the PPFD that does not require an electronic dimmer. It is thus possible to precisely control the PPFD by the simple addition of a movable part. Our semicylindrical column PBR has a single movable part (Fig. 1).

    3. Validation of photosynthetic growth and chlorophyll formation in hybrid photobioreactor with new light control

    The PPFD affects the growth of photosynthetic microorganisms. To validate the cost-efficient cultivation of a photosynthetic microorganism in our semicylindrical PBR using LEDs without an electronic dimming system, we measured Spirulina growth and chlorophyll synthesis when the LED bar was at different angles (Fig. 4). If the PPFD is too weak, photosynthesis and growth will decline; if the PPFD is too strong, growth will decline and irreversible inactivation of the photosynthetic apparatus will occur.

    The results indicated that Spirulina biomass was greatest for angles of 60° and 45°, and lower for an angle of 15° (because of insufficient light) and an angle of 90° (because of excessive light and photoinhibition) (Fig. 4a). The same pattern appeared when measuring chlorophyll content (Fig. 4b). The lowest chlorophyll level for angle of 15° may be due a low growth. Under excessive light intensity, chlorophyll can become damaged or disintegrated, resulting in overall lower chlorophyll contents. These results are in agreement with the well-known correlation of chlorophyll content with light condition.

    LEDs are very useful semiconductor light sources. The effective application of LEDs for cultivation of photosynthetic microorganisms requires a cost-efficient device to control the PPFD (Jo and Tayade, 2014). Although changing the distance between a PBR and the light resource is often used to adjust PPFD without an electronic dimming system, this often requires a large space and cannot provide dynamic light conditions. In our system, we easily adjusted the PPFD during the cultivation of Spirulina by changing the angle of an LED bar, a system that has minor space requirements. In addition, adding more rotating parts to the LED bar could provide a more precise control of PPFD.

    In conclusion, we introduced a semicylindrical column PBR in which the PPFD can be easily altered by changing the ori- entation of an LED bar. In particular, simple changes in the angle of the LED bar relative to the flat vertical surface of this PBR effectively altered the PPFD, making an electronic dimmer unnecessary. Our comparison of illumination at flat vertical panel with at round vertical panel in the new PBR with same geometric conditions indicated the light reflection is one of important factor impacting on accumulation of biomass and chlorophyll contents. Growth and chlorophyll content were optimal when the LED bar was at an angle of 45° relative to the flat vertical surface of the semicylindrical PBR.

    Acknowledgments

    This research was supported by the Basic Science Research Program, through the National Research Foundation of Korea (NRF), and was funded by the Ministry of Science, ICT & Future Planning (2015R1C1A1A01054303).

    Figures

    JALS-54-3-89_F1.gif

    Geometry of the 5-L semicylindrical column PBR. The rotation point of the LED bar is 5 cm from the flat vertical panel. Gray triangles indicate possible areas of irradiation when the LED bar is at different angles.

    JALS-54-3-89_F2.gif

    (a) Biomass and (b) chlorophyll accumulation of A. platensis cells grown under the same PPFD (50 μmol m-2 s-1 PAR) at flat vertical panel and at round panel in the new semicylindrical column PBR. Here and below, values are the averages of three independent measurements with standard errors.

    JALS-54-3-89_F3.gif

    PPFD at individual locations (a) and integrated PPFD (b) in the semicylindrical column PBR with the LED bar at different angles. Bars (mean ± SEM) with different letters are significantly different (p < 0.05). Data were analyzed using one-way ANOVA followed by Duncan’s test.

    JALS-54-3-89_F4.gif

    (a) Growth and (b) chlorophyll accumulation of A. platensis cells grown in the semicylindrical column PBR with the LED bar at different angles. Bars (mean ± SEM) with different letters are significantly different (p < 0.05). Data were analyzed using one-way ANOVA followed by Duncan’s test.

    Tables

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