**4. Discussion**

#### *4.1. Specific Growth Rate and Biomass Productivity of I. zhangjiangensis*

The present study examined the effect of different colored LED illumination on the productivity, Chl, protein, and carbohydrate content in the indoor culture of microalgae *I. zhangjiangensis*. The highest specific growth rate was observed under the red light, 1.5 fold higher when compared to the white light. Whereas the highest biomass observed under the white light was 2.0-fold higher than that under the red light (Table 1). This observation seems contradictory; however, it is possible that the size of the cells in the microalgae cultured under white light was larger than under the other light colors. Unfortunately, cell size data were not collected at any stage during the experimental period to fully support this idea. Nevertheless, it has been reported that light quality regulates the cell size of the microalgae [49]. For instance, the cell sizes of the green microalgae *C. reinhardtii* grown under blue light were 1.3 and 1.6 times larger than under white and red light sources, respectively, due to a delay in cellular division processes [50]. Additionally, the light intensity has resulted in cell enlargement by a factor of 2.5 in these species, compared to 1.9 in *D. quadricauda*, and only 1.3 in *P. kessleri*. The smaller increase in cell size in *P. kessleri* was compensated for by a 13.6-fold daily increase in cell number under optimal conditions, as compared with a 9.7-fold increase in *C. reinhardtii* and *D. quadricauda* [35]. Another study using different strains of *Chlorella* sp. revealed that the light spectrum had a significant influence on microalgae cell size. The largest and smallest cells were observed under blue and red lights, respectively [51]. Such an increase in cell size is a specific response of organisms that divide by multiple fission and thus can respond to better growth conditions beyond a simple increase in the cell number. The larger cell size is a mechanism that supports better growth in the next cell cycle, possibly leading to better productivity [35]. Nevertheless, it should be emphasized that although the value of the specific growth rate is important, the most relevant parameter related to potential largescale microalgae production systems is biomass productivity, which was highest when *I. zhangjiangensis* was cultivated under white light. It has been proven that multi-chromatic white light, whether provided by a fluorescent lamp or by LED, is more advantageous than monochromatic light for promoting the growth of *Isochrysis* sp. [42,52,53], which can also be supported by our results. A similar observation has already been reported in the productivity of *Nannochloropsis* sp. cultured under different colored lights, where pink and white lights exhibited higher biomass productivity [54]. These results are probably related to the differences in energy provided by light and captured by the photosynthetic apparatus of the photosynthetic microorganisms [55]. Particularly, between 380 and 750 nm, the energy content is sufficient to produce chemical changes in the absorbing molecules, as happens throughout the photosynthetic pathways prevailing in the microalgae [56].

#### *4.2. The Effect of Different LED Colors on the Pigment Content of I. zhangjiangensis*

Microalgae, similar to plants, capture light energy (light-harvesting antennas) and produce electrons in the reaction center of the photosystems. For efficient photosynthesis, preserving an excitation balance between the two photosystems (PSI and PSII) is of prime importance. To serve this purpose, microalgae possess specific light-harvesting antennas to expand the available light wavelength. Certain groups of algae contain accessory pigments that help in efficiently harvesting light for photosynthesis [54]. Green algae, in particular, possess a chlorophyll–protein complex which is comprised of Chl-*a* and *b* and carotenoids for carrying out the photosynthesis [57]. Chl molecules absorb light energy and transfer this energy to the photochemical reaction centers presented in algae, cyanobacteria, and higher plants by PSI and PSII, where charge separation occurs. Upon illumination, two electrons are extracted from water, mostly (O2 is evolved), and transferred through a chain of electron carriers to produce one molecule of NADPH2 (nicotinamide adenine dinucleotide hydrogen). Simultaneously, protons are transported from an external space (stroma) into the intra-thylakoid space (lumen), forming a pH gradient. According to Mitchel's chemiosmotic hypothesis, the gradient drives ATP synthesis, which is catalyzed by the protein complex called ATPase or ATP synthase—a reaction called photophosphorylation [58]. In the present investigation, for *I. zhangjiangensis*, the highest Chl content was attained under green light, while blue light resulted in the lowest Chl content, indicating that different light qualities can evoke different levels of photosynthetic pigments. Green light has promoted the production of Chl in *Chlorella vulgaris* [46], and also allowed improvement of *S. platensis* growth [10,59]. A different result has been reported for *I. galbana*. The highest contents of Chl-*a*, Chl-*c*, and Car were obtained under white light, while blue light resulted in the lowest pigment contents [53]. The same report described the highest light absorption in cells cultivated under blue light, but the photochemical reaction was lowered. Cells cultivated under blue and red lights were, respectively, restricted by downregulated photosynthetic efficiency and sufficient light absorption. Meanwhile, green light showed an increase in photosynthetic efficiency, associated with a light absorption close to that for cells exposed to white light, suggesting that green light promotes the photosynthesis of *I. galbana* by balancing the light absorption and utilization [53]. Light with a shorter wavelength, for example, blue light, has a higher probability to cause growth photo-inhibition by striking the light-harvesting complex of cells at its peak electrical energy due to its high energy [60].

Studies have shown that infrared light can cause cell damage [61], while in some multicellular algae, blue light can significantly increase the content of algal photosynthetic pigments, increasing photosynthetic efficiency and ultimately, the growth rate [62,63]. The content of photosynthetic pigments in *I. zhangjiangensis* is the highest under green light, which is conducive to the accumulation of its biomass, while the content of photosynthetic pigments is the lowest under blue light. A previous study reported a contrasting result: low-intensity blue light reduced the pigment content of *Chaetoceros gracilis* but increased it in *I. galbana* [64]. The effect of light qualities on the high-value pigments has also been reported in five microalgae strains from three distinct lineages [33]. In the Rhodophyte *Rhodella* sp., the Chl-*a* levels obtained under red and white LEDs were higher than those reached under green and blue illumination for medium and high intensity. Similarly, the diatom *Stauroneis* sp. also returned a higher Chl-*a* content under medium white light intensities. Contrariwise, in the chlorophyte *K. aperta* and *B. submarina*, the Chl content was significantly higher under blue and green lights at high and medium intensities, returning two-fold higher Chl-*a* compared to red and white LEDs. The cultivation of *Phaeothamnion* sp. under high-intensity blue LEDs also induced a significant increase in Chl-*a*. In general, responses of each strain to different colored LEDs were generally speciesspecific. These results indicate that the growth performance of different microalgae under different light qualities is not consistent, which may be due to the different compositions of the pigment system of different microalgae, resulting in different requirements for light quality in photosynthesis. It is also known that the content of the photosynthetic pigments increases as light intensity decreases [65]. In the case of *P. kessleri*, the concentrations

of Chl-*a* and *b* and carotenoids decreased with increasing light intensity. Cultures of *C. reinhardtii* and *D. quadricauda* maintained similar levels of photosynthetic pigments at low light intensities, but their concentration increased with the time of cultivation at the highest light intensity [35]. This increase in the photosynthetic pigments is attributed to the need of the microorganisms to improve their photosynthetic efficiency and capture as much energy as possible from light [28]. Considering this, a conclusion can be drawn that when Chl content is high under a specific light color, it does not necessarily imply that this illumination provides adequate amounts of energy for biomass synthesis, and this may be the case of *I. zhangjiangensis* cultured under green light. In addition, light-harvesting ability and energy-transfer processes also differed between green algae species. This has been demonstrated by observing the delayed fluorescence spectra in *C. reinhardtii* and *C. variabilis* cells grown under different light qualities. Both types of green algae primarily modified the associations between light-harvesting chlorophyll protein complexes (LHCs) and photosystems (PSII and PSI) [57].
