Cultivation of Nannochloropsis sp. using narrow beam angle light emitting diode in an internally illuminated photobioreactor
© The Author(s) 2016
Received: 11 January 2016
Accepted: 29 June 2016
Published: 7 July 2016
This paper reports on the growth condition of Nannochloropsis sp. in an annular column-type photobioreactor (PBR) using light-emitting diode as an internal illumination.
The microalgae growth in the 20-L batch culture mode under mixed blue (450 nm) and red (660 nm) light-emitting diode (LED) in various conditions such as photoperiod and light intensity (controlled by supplied current) was monitored. Compact-type 5-W LED module with narrow beam angle (radiation pattern) was installed in the PBR so as to obtain higher intensity and deeper penetration to the culture.
Based on the PBR dimension with optical path length 120 mm, the minimum light intensity required at the PBR tank inner surface at initial stage of cultivation was approximately 350–370 mol m−2s−1, while mean light intensity derived was 140–160 mol m−2s−1. Photoperiod ratio of light:dark at 18:6 h provided better results compared to 12:12 in terms of final cell density achieved. Efficiency of light utilization was calculated to be 9.0 × 109 cell/mol photon (0.49 g/mol photon), while biomass volumetric productivity was 0.04 g L−1day−1.
Microalgae have an outstanding performance compared to the terrestrial plants based on its high multiplication rate and high lipid content (Rawat et al. 2013). One of its potential is to be used as biomass source for biodiesel production (Chisti 2007). Although successfully developed, open pond cultivation method which generates low density culture is faced with a tough situation to meet the low price requirement of biodiesel. In recent years, the trend of mass cultivating microalgae has shifted from open pond system to closed photobioreactor (PBR) system. PBR technology has attracted the attention of industrial and scientific communities observed by the increasing numbers of scientific research papers published over the past 30 years (Olivieri et al. 2014). Among the reasons for this shift are to reduce contamination problem and achieve higher cell density. Nevertheless, issues such as temperature rising, inconsistent solar rays, weather fluctuation, photoinhibition due to excessive light intensities, night time biomass losses are among problems that remain to be improved in the outdoor PBR.
In regards to light parameters, indoor PBR using artificial lights may offer some solutions to overcome the problems in the outdoor PBR which depends to sunlight. With rapid development of highly energy efficient LEDs which are increasingly cheaper and consume less energy than ordinary lamps, the potential of using artificial lights in PBR and its economic feasibility need to be reviewed (Fu et al. 2012). Consequently, the cost and energy consumption are factors that need to be seriously considered (Blanken et al. 2013). It seems that the high cost of PBR with artificial lights may not be an appropriate solution at this moment, but researchers are continuing to study and improve it as there are potentials to develop it as a standalone production system or as inoculation system for various applications.
Nannochloropsis sp. is among the marine microalgae that has been widely studied in aquaculture sector (Alsull et al. 2012; Bong et al. 2013), and currently being actively researched for biodiesel production worldwide (Quinn et al. 2012; Pegallapati and Nirmalakhandan 2013). The main pigment for photosynthesis in Nannochloropsis sp. is chlorophyll a, while chlorophyll b and c are completely lacking. In relation to this, light quality plays an important role in photoautotrophic cultivation of microalgae. Photosynthetic pigments such as chlorophylls are highly react to a particular light spectrum bandwidth; such as blue at 450–475 nm and red at 630–675 nm (Richmond 2004). Previous studies on a lab scale have identified the potential of blue and red LED as artificial light source for microalgae growth (Teo et al. 2014a, b). Similar findings revealed that optimum growth of C. vulgaris (Atta et al. 2013) and Nannochloropsis sp. (Ra et al. 2016) were obtained when cultivated in blue LED light, while other suggested the usage of red LED light as the optimum wavelength for Chlorella sp. growth (Yan et al. 2016). In addition, yellow light with supplemental of minimal blue light was found to be effective to maximize Chlamydomonas reinhardtii productivity by lowering the biomass specific light absorption rate (de Mooij et al. 2016). Variations of lighting strategies, PBR volume and configurations made it difficult to determine the basic method of choosing the best lighting parameters, although it is clear that monochromatic light especially blue and red provide better performance than wide spectrum light in general energy consumption.
Another critical parameter is light intensity (quantity), whereby it varies with its position and time in the cultivation process of microalgae (Huang et al. 2012) due to the nature of microalgae as an efficient light receiver. In lab scale experiment with small volume of culture and short light path, incident light intensity is commonly used as the light quantity parameter to determine the optimum condition (Wahidin et al. 2013; Teo et al. 2014b). However, in up-scale or large volume cultivation, mean light intensity is a better indication of light condition; light intensity at the illumination surface, IS and opposite surface, BS (end of light path) need to be considered (Ogbonna and Tanaka 2000). Light distribution inside the PBR is very important to ensure light energy to biomass conversion is at optimum condition.
The common approach in utilizing sunlight receiving for an outdoor PBR is to design it with high surface/volume ratio (SVR) (Posten 2009). Tubular and flat panels PBR have good properties in terms of high SVR for utilizing solar rays (Grima et al. 1996; Zijffers et al. 2010; Silva Benavides et al. 2013), while bubble column is not widely preferred. However, when considering artificial light as the light source for PBR, huge amount of energy would be needed to achieve intensity and uniform distribution of light similar to the sunlight. We are suggesting that this approach need to be reviewed for indoor application. As an example, LED is usually mounted in a closely packed array form to illuminate the flat panel PBR surface (Lee and Palsson 1994). In this case, the intensity of standard LED chip with wide angle (110°–120°) may not be sufficient to penetrate culture depth of more than 20 mm, so a PBR with short optical path length would be preferable. Yet, it would be difficult to illuminate the whole panel surface as PBR volume increase (up-scale project). So, an alternative method which is suitable for up-scale PBR is necessary, while at the same time there must be consideration for practical light quantity in high volume cultivation.
In general, bubble column has the advantage for up-scale project as it can easily accommodate large volume, efficient contact between the gas and liquid phases, relatively low cost for setup and operating, well accepted for other industrial usage and minimal maintenance is required. Although it is not popular as an outdoor PBR, there is potential to develop it as an indoor PBR with artificial light. In regards to microalgae cultivation in bubble column as indoor type, internal illumination method was found to be better in efficiency compared to external illumination (Pegallapati et al. 2012). This approach would also facilitate the daily operation in monitoring the microalgae growth since the lightings are placed internally. Few types of lightings have been proposed before such as using fluorescent light, optical fibre and others (Ogbonna et al. 1996; Csogor et al. 2001; Suh and Lee 2001; Xue et al. 2013). Light conversion efficiency is the main concern for PBR with artificial light, however due to varieties in each artificial light optical properties, it is quite difficult to have an equal comparison. The balance of microalgae growth and lightings energy, lightings layout, microalgae cell density are among parameters that need to be considered when using the artificial lights in PBR.
Therefore the aim of this study is to evaluate the performance of Nannochloropsis sp. cultivation in an internally illuminated PBR with LED. It is a primary scale-up (20 L) from previous lab study (0.5 L) which used red and blue LED to cultivate microalgae. The use of narrow beam angle LED was proposed in order to penetrate the deep culture (120 mm) in the bubble column, as normal wide angle LED has limitation in this aspect. The experimental data obtained can be utilized for further scale up (100 L) of an internally illuminated PBR using LED in future work.
The LED luminaire consisted of four LED bars mounted together to top and bottom plate. The LED bar was installed with three units of 5 W LED module, each package contained a total of 16 LED chips with combination of blue 450 nm and red 660 nm in quantity ratio 8:8 and intensity ratio approximately 60:40 %. The overall optical beam angle was designed to be at 55° (narrow angle) to achieve high light intensity through the built-in lens design. The LED luminaire was powered by using either adjustable current supply (200–340 mA) or a constant current driver (350 mA) throughout the experiments. The heat from LED was dissipated through the bar itself which is fabricated from aluminium material.
Culture and medium
Nannochloropsis sp. strain was originally obtained from the culture collection of Borneo Marine Research Institute (BMRI), Universiti Malaysia Sabah, Malaysia. The Nannochloropsis sp. cells were cultured in sterilized seawater (autoclaved at 1–2Bar, 121 °C) and enriched with Walne’s medium which contains: 100 g NaNO3, 1.3 g FeCl3·6H2O; 0.36 g MnCl2·4H2O; 33.6 g H3BO3; 45 g Na2·EDTA; 20 g NaH2PO4·2H2O; 2.1 g ZnCl2; 2 g CoCl2·6H2O; 0.9 g (NH4)6Mo7O24·4H2O; 2 g CuSO4·5H2O; 0.001 g Vitamin B12; 0.001 g Vitamin B1 and 0.2 μg Biotin per liter.
Each experiment was conducted in a 20 L working volume using batch culturing method. Experiment 1 was conducted at regulated current between 200 and 340 mA (an increment of 20 mA in every 2 days) with maximum intensity of 200–220 μmol m−2s−1 at BS, photo period of 12:12 h (light:dark) and initial cell density of 2.7 × 106 cell/mL (3.59 g/L). Experiments 2 and 3 were conducted by providing each LED package with a constant current of 350 mA with maximum intensity of 350–370 μmol m−2s−1 at BS, while photo period was set at 12:12 and 18:6 h respectively. Initial cell density for experiment 2 and 3, was reduced to within 1.9–2.0 × 106 cell/mL (2.52–2.66 g/L), after we realized that margin of light intensity was not sufficient to be increased more due to LED current at 500 mA causing heat issue for this prototype.
Measurement of photosynthetic photon flux density (PPFD)
Optimum light intensity is critical for the growth of microalgae, but the attenuation of light is very much influenced by the culture depth. Light intensity or PPFD was measured using a quantum sensor connected to Light Scout Dual Solar quantum light meter, in unit of μmol m−2s−1. PPFD evolution of the light intensity was confirmed by taking measurements at the illumination surface (outer surface of internal acrylic column), IS until opposite surface (inner surface of PBR tank), BS at incremental distance of 15 mm and fix height (highest light intensity point). Due to quantum sensor probe size/height (from base to sensor surface) itself is 15 mm, the PPFD measurement at inner surface BS was represented by the distance at 105 mm, instead of 120 mm. Throughout the experiments, PPFD at BS was monitored every 2 days at eight equally spaced radial locations. Each four points were representing the intensity of light region (LED beam covered region) and dark region (adjacent region where LED beam was limited) respectively. Since the LED module radiation pattern was narrow in angle (focus effect), the boundary of light and dark region can be clearly identified visually. Each LED module covered approximately an area of 130 × 130 mm (0.0169 m2) square boundaries at BS, resulting total coverage of light region from four LED bar (12 modules) equal to about 40 % (0.2028 m2), while dark region about 60 % (0.3222 m2) from total PBR inner surface area which is 0.525 m2. The dark region is defined as the area which received less than 70 % of peak intensity at BS. The effect of different ratio of light/dark region was not investigated in this study based on two reasons; (1) Increasing the light/dark region may not feasible due to space constraint, LED heat issue and cost factor, (2) Decreasing the light/dark region may effect to the microalgae growth due to lack of light by reduction of SVR. Figure 1 shows the schematic view of the PBR.
Measurement of microalgae growth and lipid content
The amount of lipid in Nannochloropsis sp. was determined according to the method described previously by Teo et al. (2014a) using improved Nile red staining method (Chen et al. 2011) and Perkin Elmer LS-55 fluorescence spectrophotometer. For the Nile red staining, 50 μL of Nile red (9-diethyl-amino-5H-benzo[a]phenoxazine-5-one; Sigma, USA) in acetone representing a concentration of 0.1 mg mL−1 was added to the 1 mL of sample. The mixture was pretreated using a microwave oven for 1 min. The excitation and emission wavelengths for the fluorescence were 490 and 585 nm respectively.
Measurement of light utilization efficiency
Results and discussions
Light intensity (PPFD) inside the PBR
Relationship of light intensity and photoperiod on Nannochloropsis sp. growth and lipid production
In these experiments, it was observed that the cell density graph intersected with PPFD graph at around 140–160 μmol m−2s−1 (refer to Fig. 4a–c), which is proposed to be the mean light intensity, I mean (μmol m−2s−1) of this PBR. Alternatively by using Eq. (6), where I 0 is determined as 2861 μmol m−2s−1 (actual measurement value exceeded the quantum meter range), K is assumed as 0.15 m2 g−1, C is biomass concentration at the intersection graph (2.7 × 106 cell/mL or 148 g/m3), and L at 0.12 m; light intensity, I can be calculated as 199 μmol m−2s−1. However this calculation is merely based on assumption of K value which may vary over the course of batch and depends on the spectrum of light being used. In conclusion, the mean light intensity from the graphs intersection point such as in Fig. 4c was preferably chosen for experiment 3, which is 140 μmol m−2s−1 as the mean light intensity, and the efficiency of light utilization, Y x,E of this PBR was calculated as 9.0 × 109 cell/mol photon (0.49 g/mol photon).
Summary of the current work and previous (lab scale) results
PPFD at BS (μmol m−2s−1)
Photoperiod light:dark (hours)
Spec. growth rate, μ (day−1)
Div. rate, k (day−1)
Initial cell density (×106 cell/mL)
Max. cell density (×106 cell/mL)
Total volume (L)
Teo et al. (2014b)
Comparison of the current work and other researcher results
Micro- algae speciesb
Biomass Productivity (g L−1day−1)
PPFD (μmol m−2s−1)
This study (IIC)
Pegallapati and Nirmalakhandan (2013)
Chiu et al. (2009)
Zittelli et al. (2003)
Zittelli et al. (2000)
The cultivation of Nannochloropsis sp. in internally illuminated PBR using combination of blue and red LED, with narrow beam angle was successfully performed. It was found that higher growth rate achieved at higher light intensity 350–370 μmol m−2s−1 and longer photoperiod 18:6. Usage of high intensity LED with narrow beam angle possibly helped in improving microalgae cultivation in deep culture and high volume such as bubble column PBR, provided that optimum initial cell density, lighting parameter and cultivation method to be reviewed.
MT, CLT, AI, AMY carried out the overall experiments, computational experiments and drafted the manuscript. All authors read and approved the final manuscript.
Financial support from Universiti Teknologi Malaysia (Flagship Grant/QJ13000.2409.02G33) and Q.J130000.21A2.02E32 for this research is gratefully acknowledged.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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