1. Introduction
Microalgae have recently attracted noticeable research interest as a potential renewable source for several value-added products synthesis, including carbohydrates, proteins, lipids, and pigments. Due to their great diversity, better photosynthetic efficiency than higher plants, and low nutrient requirements, microalgae hold great promise for the co-production of high-value compounds, potentially used in a variety of biotechnological and commercial applications (pharmaceuticals, nutraceuticals, cosmetics, biofuels, animal feed, aquaculture, etc.). Moreover, due to their incomparable photosynthetically induced carbon assimilation rates and their high intracellular accumulation rates of lipids, microalgae carry enormous potential to contribute to a clean-energy future. In particular, microalgal biodiesel, a third-generation renewable biofuel, has recently received commercial interest, since, without overlooking the management practices for its environmentally sustainable production [
1], it presents some distinct advantages, including renewability, non-toxicity, and environmental friendliness [
2,
3]. However, it has become evident that with the current state-of-the-art technologies, a microalgae-based biodiesel production process is only conditional sustainable, and its economic feasibility is ensured only upon the adoption of the biorefinery concept as an operational strategy [
4]. The biorefinery concept is tightly related to the multiple-commodity production approach and exploitation of all biomass components simultaneously for commercial applications and energy recovery. However, such a production scheme requires understanding the biochemistry of the carbon storage metabolism in microalgae cells. Only then, and through a systematic handling of the cultivation process operating profile, will the efficient steering of microalgae cultures toward the overproduction of the desired products and sufficient improvement of the process productivity and ultimate economic viability potential be enabled [
5].
Both microalgae culture growth and their photosynthetic metabolite production rates are affected by many environmental factors, such as nutrient type and concentration [
6,
7], CO
2 availability [
8,
9], temperature [
10,
11], and light (wavelength, intensity, and photoperiod) [
12,
13,
14]. Many studies identify that under intensively or moderately stressed conditions, such as nutrient limitation/deprivation, increased/decreased light intensity, or high salt concentrations, microalgae cells develop survival strategies and undergo morphological and biochemical changes [
15,
16].
Light quality, in terms of both wavelength and intensity, represents one of the most important parameters for phototrophic microalgae systems, as it is the driving force for the photosynthesis and the regulation of several cellular processes. Light emitting diodes (LEDs), capable of producing monochromatic light, can be successfully used for microalgae cultivation to efficiently control the productivity of desirable intracellular metabolites. Photosynthetically active radiation (PAR) is the wavelength range between 400 and 700 nm that sufficiently supports the energy requirements of the photosynthesis process [
17,
18]. Photosynthesis is initiated when light is captured by light-harvesting antenna complexes composed of different light-capturing pigments [
19]. Chlorophylls (a and b) are the primary pigments essential for oxygenic photosynthesis in green algae, delivering the energy from the light photons to the molecules in the form of reducing potential, whereas phycobilins and carotenoids are accessory pigments. Carotenoids, in particular, regulate the protection of the photosynthetic apparatus, while phycobilins contribute to light harvesting by increasing the absorption spectrum of microalgal cells in low light conditions [
17,
19]. These pigments exhibit a characteristic color, as they absorb light in specific wavelengths (
Table 1). The use of light wavelengths that correspond to the two major absorption bands of chlorophyll a and b, blue (430–475 nm) and red (630–680 nm) [
20,
21], has recently received widespread attention for potentially enhancing the photosynthesis process in microalgae cultures [
17,
18].
Indeed, many experimental results indicate that blue and red light not only promote the photosynthetic rate when compared to white, but also cause variations to the regulation of several cellular metabolites and morphogenesis in algae. However, different conclusions about the influence of light wavelength on microalgae culture performance have been reported. Teo et al. (2014) claimed that blue light enhanced the culture growth and oil productivity for both
Tetraselmis sp. and
Nannochloropsis sp. when compared to red, red-blue, and white light [
22]. Similar results have been obtained by Das et al. (2011), who concluded that the use of blue light favored the biomass productivity of both mixotrophic and autotrophic cultures of
Nannochloropsis sp. when compared to other types of light (i.e., red, green, and white) [
23]. On the other hand, the use of red light as a tool to enhance the photosynthetic process has been advocated in several studies. When
Chlorella vulgaris was cultivated under white, red, green, and blue light, it exhibited the maximum growth rate when exposed to red light [
24]. Shu et al. (2011) also reported that both monocultures of
Chlorella sp. and mixed cultures with
Saccharomyces cerevisiae attained greater specific growth rates under red LED light [
25]. Kim et al. (2014) noticed that exposure of
C. vulgaris cultures to blue light led to significantly increased cell size, whereas red light resulted in a maximum number of smaller cells [
14]. The same observations were recorded by Oldenhof et al. (2006), who pointed out that when
Chlamydomonas reinhardtii cultures were subjected to blue light, cells were continuously growing for a longer period and attained larger sizes than under red light [
26]. Similar results were obtained by Koc et al. (2013), who found that
Chlorella kessleri cells grown under blue LED light were larger than those grown under red or white fluorescent light, while red light resulted in higher biomass concentration than blue LED light, even though the average cell size was smaller [
27]. In a study by Rendón et al. (2013), who tried to elucidate the effect of CO
2 supply and illumination of
C. vulgaris cultures at different light wavelengths, the highest biomass production was found when algal cultures were supplied with 8.5% CO
2 and exposed to white light [
28]. Furthermore, the use of white light was found to increase the production rate of
Scenedesmus sp. when compared to blue, green, and red light. Nonetheless, in the same study, when red and blue LED light were combined in different intensity ratios, remarkably higher biomass production rates were measured than with a single wavelength of light (even white light), irrespective of the ratio used to combine the light wavelengths [
29]. Abiusi et al. (2014) presented a complete study over the effect of different light wavelengths on cell size and cell cycle, growth rate, productivity, photosynthetic efficiency, and biomass composition of
Tetraselmis suecica. Whereas red and white light were the most effective regarding biomass productivity and photosynthetic efficiency, cultivation under red light attained the highest protein content, while cultures grown under blue and green light exhibited the highest lipid and carbohydrate content, respectively. This study also confirmed the suggestion that red light on microalgae cultures could lead to smaller cells [
30]. Finally, Mutaf et al. (2019), who recently investigated the effect of different culture media and different light policies on the cultivation of
Stichococcus bacillaris, reported that even if blue and red light did not play a critical role on the culture growth kinetics, the use of these two lights had a significant impact on chlorophyll accumulation and lipid profile [
31].
From the numerous studies surveyed, it was unambiguously concluded that the effect of the light on microalgae cell growth and preferred expression of desirable metabolites is most likely species- and culture condition-dependent, and currently the gained knowledge is still insufficient as a general pattern cannot be described yet. This might be due to the fact that very few studies examine microalgae cultures at the scale of a photo-bioreactor under well monitored and controlled conditions. On the other hand, the majority focus only on total microalgae biomass production and specific intracellular metabolites concentration, especially lipids, neglecting the effect of different light wavelengths on the synthesis of other, equally important and commercially exploitable cell products (proteins, carbohydrates, and pigments). The knowledge of total bioproducts accumulation rates is essential for screening the light spectrum effect on the cultivation of a specific microalgae strain and extracting significant conclusions about the impact of light wavelength from the biorefinery perspective.
In our previous study, the effect of five process variables (i.e., illumination flux, aeration rate, CO
2 supply rate, nitrogen concentration, and salinity) on the performance of
Stichococcus sp. cultures was thoroughly investigated [
32]. Precisely, employing the Taguchi design experiment method, the biomass production rate and the accumulation capacity of the microalgae population in carbohydrates, proteins, and lipids were correlated with the above key process variables, and the best operating policy that selectively maximizes the component of interest was explored. With this experimental background over the strain
Stichococcus sp., the aim of the present work is to quantitatively examine the effect of the light spectrum on the growth and bioproducts concentration (lipids, proteins, and carbohydrates) of the studied marine microalgae strain. To our knowledge, the influence of light spectrum on the growth and complete biochemical composition on the cultivation of
Stichococcus sp. in the scale of a photobioreactor system is elucidated for the first time. Thus,
Stichococcus sp. cultures are exposed to different combinations of cool white, red, and blue LED light in a lab-scale photobioreactor (PBR), with simultaneous re-adjustment of the already explored optimal operational variables, to unravel the potential of employing the proposed cell factories in a biorefinery plant.
2. Materials and Methods
2.1. Microalgae Strain and Pre-Culture Conditions
The marine strain
Stichococcus sp. (identified with the 18S-rDNA gene sequence analysis) was isolated from the Crete coastal area in Southern Greece.
Stichococcus is a genus of green algae (Chlorophyta) characterized by a simple morphology, with cell size ranging from 2 to 6 μm, organized in filamentous or unicellular structures [
33], with some species (e.g.,
S. bacillaris) being distinguished for their resistance to temperature, salinity and pH variation, their lipid content, as well as their capacity to remove efficiently heavy metals from wastewater [
32,
34,
35,
36].
Both for the stock cultures and the preparation of pre-cultures inoculating the bioreactor, cells were cultivated in Erlenmeyer flasks of 500 mL capacity with a working volume of 300 mL and incubated for 15 days at 25 °C in a shaking incubator (GFL 3031), at a constant shaking rate of 80 rpm. Pre-cultures were regenerated every 15 days in new flasks by inoculating fresh medium with the 15-day-old pre-culture at a volume ratio of approximately 1/20 to let the new pre-culture start with initial OD600nm around 0.25. Thus, it was guaranteed that all the bioreactors were inoculated with pre-culture cells of roughly the same history. Modified Bold Basal medium with a 3-fold increase of its nitrogen content and vitamin addition (3NBBM+V) was used both for pre-cultures and stock cultures. Precisely, the medium contained nutrients (in g/L): NaNO3 0.75, KH2PO4 0.175, K2HPO4 0.075, MgSO4·7H2O 0.075, CaCl2·2H2O 0.025, NaCl 0.025; trace elements (in mg/L): FeSO4·7H2O 0.60, Na2EDTA 3.92, Na2MoO4·2H2O 0.03, CoCl·6H2O 0.02, MnCl2·4H2O 0.25, ZnCl2 0.03; and vitamins (in mg/L): Thiamine-B1 1.2, Cobalamin-B12 0.01. All nutrients were autoclaved at 121 °C for 20 min in separate solutions. The trace elements and vitamin solutions were filter-sterilized using filters (Whatman Polytetrafluoroethylene- PTFE- syringe filters, 0.2 μm). The medium initial pH value was equal to 6.5 and was regulated by the phosphate buffer, already contained in the culture medium (i.e., KH2PO4/K2HPO4). Atmospheric air was sparged into the liquid medium through a filter with 0.20 μm pore size (Whatman PTFE syringe filters, 0.2 μm) at a flow rate of 0.2 L/min to ensure sufficient aeration. For the illumination of cultures, fluorescence lamps with warm daylight 3000 K (6500 lm) were used. A photoperiod of 16 h lighting followed by 8 h darkness was applied.
2.2. PBR Experiments
The experiments of
Stichococcus sp. cultures implemented in the present study to explore the synergistic role of different light spectrums in regulating either the culture growth rate or cell composition were performed in a lab-scale bench-top photobioreactor (FerMac 320, Electrolab Biotech Limited, UK), with a total capacity of 3 L. The research outcome of a preliminary study [
32], published by the same research group, was employed to determine the appropriate PBR operating profile. More specifically, given that the culture aeration rate, CO
2 supply rate, and initial nitrogen concentration in the culture medium were distinguished as the most important operating factors and examined for their contribution to the total variance of biomass and products concentration, their optimal values found in that work were adopted in the present study. Thus, in all the PBR experiments, separate feeding streams of atmospheric air and CO
2 (purity ≥ 99.9%) at a constant flow rate of 0.1 L/min and 2.5 mL/min, respectively, were filtered (Whatman PTFE filters, 0.2 μm) and sparged into the photobioreactor containing 2 L of medium (including the inoculum). The decision of the PBR agitation rate and intensity of the incident light is discussed separately below since these operating values were dynamically adjusted with the progress of the culture growth. The composition of the medium used for all the experiments in the PBR was the same as that used for the pre-cultures.
For the pH control, the PBR was equipped with a pH measuring electrode (F-695-B225-DK, Broadley-James, Irvine, CA, USA) immersed in the culture; automated addition of buffer solutions of NaOH (1M) and HCl (1M) was implemented by peristaltic pumps holding the pH at the set point value of 6.7 with a dead-band of 0.1. The dissolved oxygen concentration (D.O.) in the culture medium was also measured with a suitable selective electrode (D140-B120-PT-D9, Broadley-James, Irvine, CA, USA) and expressed as a percentage of the respective saturation value of the medium prior to the PBR inoculation. A spherical sensor (US-SQS Spherical Micro Quantum Sensor, Heinz Walz GmbH, Effeltrich, Germany), submerged inside the culture at a fixed point, was used for light intensity measurement. Temperature was maintained at 25 ± 0.1 °C using a bioreactor cooling-heating system, i.e., chilled water circuit via a cooling coil and heating via heater mat. The photoperiod was set to 16:8 h light/darkness. The outflow gas stream from the top of the bioreactor was directed through a gas analyzer (Model 902P O2/CO2 Analyser, Quantek Instruments, Grafton, MA, USA) to evaluate the CO2 quantity consumed and O2 produced, as an indication of the photosynthetic efficiency of the culture. A sufficient pre-culture volume was used to inoculate the PBR, resulting in initial OD@600nm and DCW (Dry Cell Weight) values of 0.25 and 0.03 (g/L), respectively.
2.3. Analytical Measurements
Frequent sampling and off-line analysis was applied to monitor the overall progress of the Stichococcus sp. cultures. Precisely, samples of 50–35 mL of culture broth were collected at 24-h intervals to perform measurements of both culture growth rate and accumulation rate of the products of interest. Cell growth, measured as optical density (OD) at 600 nm in a UV-Vis spectrophotometer (Lamda 35, Perkin Elmer, Akron, OH, USA), was assessed twice a day (once immediately after the completion of the dark period and then 8 h after the beginning of the illumination period) to provide accurate growth monitoring. Determination of dry biomass concentration, measured as dry cell weight (DCW), was carried out by filtering 5 mL of culture through a pre-weighted glass microfiber filter (Whatman 934-AH, pore diameter 0.2 μm), then dried at 50 °C overnight, and finally weighted at a high precision micro-balance (XP 105, Mettler Toledo, Columbus, OH, USA). Microscopic observations of the culture were carried out daily in an optical microscope (DM 2000, Leika, Wetzlar, Germany).
For the determination of chlorophylls (a) and (b) concentration, 2 mL samples of the culture were centrifuged and the precipitate was washed with distilled water three times. In total, 2 mL of pure methanol (99.8%) was then added to the cell pellet and the mixture was vortexed and left in dark at room temperature for 20 min. The amount of pigments was calculated (in mg/L) via Equations (1) and (2), with prior measurement of the supernatant absorbance at 652 and 665 nm in a UV-Vis spectrophotometer (Lamda 35, Perkin Elmer, Akron, OH, USA) [
37,
38]:
Lipids contained in microalgae cells were extracted, and lipid content was determined gravimetrically according to the Folch extraction protocol [
39]. More specifically, a sufficient quantity of culture broth was centrifuged at 7000×
g, and cell pellets were frozen at −20 °C and then lyophilized. In total, 2 mg of the freeze-dried algal biomass were treated with 1.5 mL of chloroform-methanol (2:1 v/v) solvent mixture and underwent sonication within an ice-bath for 15 min, at 50% of the sonicator’s maximum amplitude (Vibra Cell VC-505, Sonics & Materials, Newtown, CT, USA). The solution was centrifuged and the solvent was collected. The procedure was repeated three times with the submerged biomass to ensure total lipids extraction. Then, in the liquid solvent phase, an aqueous solution of KCl 0.88% (w/v) of volume equal to 20% of the final solvent volume was added, resulting in the formation of two phases. The upper phase was gently removed and 0.3 mL Pure Solvents Upper Phase mixture [
39] was added to the lower phase. After complete separation of the two phases, the upper phase was removed, and this step was repeated to ensure satisfactory washing of the crude extract. A sufficient amount of methanol was then added to the lower phase up to a final total volume of 4.5 mL. The derived single-phase mixture containing the extracted lipids was collected and the lipids dried overnight at 45 °C and were weighed with a precision microbalance (XP 105, Mettler Toledo, Columbus, OH, USA).
Proteins were extracted from 2 mg freeze-dried biomass upon treatment with 9.6 mL of aqueous solution of NaOH (0.5 M) containing 5% methanol (in volume fraction) and 0.4 mL phosphate buffer (0.05 M) and sonication in an ice-bath. Τhe cell suspension was homogenized for 10 min at 50% of the sonicator’s maximum amplitude (Vibra Cell VC-505, Sonics & Materials, Newtown CT, USA) to ensure cell breakage and protein release following the applied protocol [
40]. After homogenization, 5 mL of the aqueous solution (NaOH 0.5 M, 5% v/v MeOH) was added and samples were heated at 100 °C for 30 min under continuous stirring. The protein content was measured with the aid of a Micro-BCA kit (Thermo Scientific, Waltham, MA, USA) at a microplate spectrophotometer (ELx808 Microplate Reader, BioTek, Winooski, VT, USA) [
41]. The calibration curve was obtained with Bovine Serum Albumin (BSA) solutions of known concentrations.
For the measurement of the carbohydrates content, 2 mg of lyophilized algal biomass was redispersed in 1 mL HCl solution (2.5 M) and subsequently incubated at 100 °C for 3 h under continuous stirring to achieve cell membrane breakage and reduction of polysaccharides, oligosaccharides, and disaccharides to monosaccharides. After neutralization (2.5 M NaOH) and centrifugation, the phenol-sulfuric method was used to quantify the neutral monomeric sugar content as glucose equivalent by treating the unknown samples with 1 mL phenol solution 1% (w/v) and 5 mL H
2SO
4 96% (w/w) and measuring the absorption at 483 nm (Lamda 35, Perkin Elmer, Akron, OH, USA) [
42]. Solutions of D-glucose of known concentrations were used as reference standards for calculating the glucose concentration-absorption calibration curve.
Finally, the nitrate concentration at the culture medium was determined by measuring the absorbance of the supernatant at 220 nm using a UV–vis spectrophotometer (Lamda 35, Perkin Elmer, Akron, OH, USA) [
43].
3. Illumination Profile and Agitation Rate
Microalgae cultivation systems are strongly dependent on light availability. Supposing optimal temperature, sufficient nutrients, and CO
2 supply, limitation to the photosynthesis rate and thus biomass productivity falls to light insufficiency [
19]. There is a strong need to evaluate the potential of maximizing light utilization by microalgae to enhance their performance. One serious concern on continuous stirred tank photobioreactor units used for photoautotrophic microalgae cultures arises from the realization of light exponential attenuation upon its radial penetration from the surficial culture layer to the internal ones. As photons impinge on the reactor surface, they are absorbed by cells, scattered, and reflected, resulting in a decrease in light intensity as the distance from the PBR surface increases. Constant PBR illumination may lead to a reduction of biomass productivity, as the outermost layer of cells in the bioreactor are exposed to excessive intensity, resulting in photo-inhibition, whereas inadequate light intensity in the innermost layer of the culture, because of cells’ self-shading, becomes growth-limiting. For given geometric characteristics of bioreactor vessels, Carvalho et al. (2011) emphasized the need for intensive stirring in PBRs to increase the efficiency of the light usage by microalgae cells and to attain a uniform exposure of the microalgae cell population to the incident light [
16].
In the present study, different light spectrum profiles were investigated to potentially induce controlled biomass compositional changes of the marine strain
Stichococcus sp. The lighting policies were implemented under predefined optimal culture conditions and heuristically adjusted agitation and incident light flux profiles. Α custom-made lighting box, equipped with 19 cool white daylight LED lamps (Osram SubstiTube advanced, 10 W/6500 K, 1100 lm) placed symmetrically at its internal sides, was employed for the photobioreactor illumination in all the experiments. Five stripes of blue and five stripes of red LED light with a total length and intensity of 10.5 m and 75.6 W for each color were also amended in this structure, as shown in
Figure 1a,b. The emission spectrum of the light sources used in the present study, recorded via a mini-spectrometer (RC-VIS: C9407, Hamamatsu, Japan), are depicted in
Figure 2a,b. The employed white LED lamps produce a light spectrum with a sharp peak at 450 nm (blue light) and a broader distribution at the rest of the visible spectrum, thus emitting a cool white light. On the other hand, red and blue LED strips emit exclusively at the red and blue region of the light spectrum, with a sharp peak at 630 nm and 460 nm, respectively.
An innovative approach was adopted to mitigate the constraint of light saturation. Precisely, a time-escalating profile of illumination (4400–20,900 lm) was applied in all experiments during different culture stages by manually turning on additional lamps symmetrically around the bioreactor, in combination with a gradual increase of agitation in a range of 100–320 rpm. More specifically, in all experiments the initial culture agitation rate was selected equal to 100 rpm. This was increased to 150 rpm on the third day of the cultivation period, while from the fourth to the twelfth day it was increased in a stepwise mode by 10 rpm every day and by 20 rpm from the thirteenth to the sixteenth day (
Figure 3).
Three independent batch experiments were conducted in a bench-top photobioreactor in two replicates (mean values are reported). In the WL experiment, white lighting was exclusively applied throughout the 17-day cultivation period. The culture started with only four lamps on (4400 lm), and successively two (2200 lm) or three additional LED lamps (3300 lm) were set on at predefined time instants. In the WRL experiment, white and red lighting was applied throughout the 17-day cultivation period. The time schedule for the white lighting was identical to the WL, however the culture started with the red-light band being reinforced by five red LED strips. Finally, in the WBL experiment, white and blue lighting was applied throughout the 17-day cultivation period; the time schedule employed for the white and blue illumination was the same as the one imposed in the WRL. In
Table 2, the initial and final lighting conditions in all three experiments are reported, while the respective dynamic profiles are shown jointly with the agitation profile in
Figure 3 and discussed in the following section. Thus, it was expected that sufficient exposure of the cells to light was ensured, and the inhibitory effect of the cells’ self-shading phenomenon was confined.
5. Conclusions
The microalgae photosynthetic cultivation process, combining cell proliferation and biomass production and intracellular accumulation of particular products, turned out to be seriously dependent on the quality of available light in the culture. Thus, the utilization of suitable wavelengths of visible light could prove to be an effective strategy to guide microalgae cultures, targeting the overexpression of the desired intracellular metabolites. However, the decision over the level of exposure of the culture to the employed light by cautious selection of the luminous flux and culture mixing time profiles, in combination with the CO2 feeding, can only be considered as an integral part of such a strategy.
Stichococcus sp. could be successfully used as a potential cell factory for an integrated biorefinery plant, considering its ability to create high-density cell populations and efficiently accumulate three prominent metabolites, carbohydrates, proteins, and lipids. Using different light wavelengths on the cultivation of Stichococcus sp. can contribute to the directed photosynthetic production of different useful products, in conformity with the operational framework desired in a biorefinery plant. White LED light has been proved to favor the Stichococcus sp. culture growth up to large dry cell mass values, while it has been immediately associated with the intensive accumulation of carbohydrates by the cells. On the other hand, enhancing the white light spectrum with blue LED light triggers the overproduction of lipids, favoring the assimilation of CO2-carbon preferentially in the metabolic pathway of lipid synthesis, while the white light enhancement in the red band is a promising strategy to intensify the accumulation of proteins when sufficient nitrogen supply is ensured. Finally, this work indicated that both red and blue light in combination with white can dramatically increase chlorophylls concentrations.
The knowledge generated in the present research is expected to support the efficient scaling-up of the microalgae cultivation process, which is of primary importance for the development and establishment of a potential microalgae-based technology.