Growth, pigment content, antioxidant activity, and phytoene desaturase gene expression in Caulerpa lentillifera grown under different combinations of blue and red light-emitting diodes

Light-emitting diodes (LEDs) have been developed as light sources for indoor algae cultivation. In this study, we determined the effects of blue and red light ratios on weight gain, pigment content, antioxidant activity, and phytoene desaturase (PDS) gene expression in Caulerpa lentillifera. Caulerpa lentillifera was grown under six LED light treatments: white (W), red (R, 660 nm, 100%), blue (B, 447 nm, 100%), 1B1R (50% blue + 50% red), 1B2R (33.3% blue + 66.7% red), and 1B5R (16.7% blue + 83.3% red). Our results showed that red light improved the weight gain percentage of C. lentillifera, whereas red light with a low proportion of blue light (1B5R, 16.7%; blue + 83.3% red) led to a significantly higher weight gain percentage and photosynthetic pigment (Chl a + Chl b) content than the other LED light treatments. Increasing the proportion of blue light negatively affected weight gain and the photosynthetic pigment contents. However, blue LED light induced significantly higher transcription of the PDS gene, which coincided with increased β-carotene accumulation and antioxidant activity in C. lentillifera. These results indicated that the ratio of blue to red light is important for the indoor cultivation of C. lentillifera, and 100 μmol photons m⁻² s⁻¹ of 1B5R LED light with a 12/12 h light-dark photoperiod resulted in good growth and development.

Keywords: Caulerpa lentillifera; Chlorophyta; Light-emitting diodes; Red and blue light ratio; Phytoene desaturase

Edible green algae from the genus Caulerpa (also known as "sea grapes" because they form tiny spherical beads that are closely packed together on the stem) have gained recognition in the international seafood industry because of their high nutritional value (Zubia et al. [46]). Caulerpa has relatively high levels of polysaccharides, which have been identified as having medicinal value because of their antiviral (Ghosh et al. [15]), antitumoral (Ji et al. [20]), immunostimulant (Maeda et al. [29]), antinociceptive and anti-inflammatory properties (Ribeiro et al. [34]). They also contain high levels of zinc, iron, cobalt and selenium, which are required nutrients (Gaillande et al. [13]). To date, commercial aquaculture production has primarily involved Caulerpa lentillifera which is imported from the Philippines and Vietnam to Japan and Taiwan (Gaillande et al. [13]). To achieve a better control of the quality and quantity of C. lentillifera, some optimal cultivation conditions have been investigated. For example, C. lentillifera can tolerate wide fluctuations in salinity, but the optimal salinity was suggested at be 35 psu (Deraxbudsarakom et al. [9]; Wang [42]; Guo et al. [16]). Caulerpa lentillifera was reported be distributed in the tropical and subtropical seas, and lower temperatures (< 18 °C) led to softening and decay and reduced productivity. Optimal temperatures ranging from 20 to 28 °C were suggested for the growth of C. lentillifera (Friedlander et al. [12]; Guo et al. [16]). In addition, C. lentillifera is a green seaweed adapted to a low-light environment and higher irradiance has negative effects on its growth rate and photosynthetic pigment contents (Guo et al. [16]). Therefore, indoor cultivation involving controlled salinity, temperature, lighting, and nutrients could be a cost-effective and energy-efficient option to meet the requirements of C. lentillifera for growth.

Light is an essential factor in the growth and photosynthesis of algae in indoor cultivation systems. Light-emitting diodes (LEDs) are an advanced and economical technology compared with traditional fluorescent lamps. Each LED device has a narrow emission spectrum (Virgili et al. [41]), thus allowing for the precise control of light requirements for photomorphogenesis studies and light-based plant responses. In various spectra, red light (maximum absorption at 642–663 nm) generally induces plant growth, and blue light (maximum absorption at 430–453 nm) affects photosynthesis (Okamoto et al. [30]). A synergetic effect was observed when mixtures of blue and red LEDs were used to irradiate land plants (Yorio et al. [44]; Heo et al. [17]). However, the use of LED light sources in macroalgae research is uncommon. Studies on macroalgae growth under different light sources have reported that red light can increase the ratio of fucoxanthin to chlorophyll a (Chl a) and chlorophyll c in Laminaria hyperborea (Dring [10]), whereas red light can be utilized as a reproductive inductor to produce tetraspores in Gracilaria birdiae (Barufi et al. [4]). Blue light can increase thallus growth in Pyropia haitanensis (Wu [43]) but suppresses the growth of Gracilaria tikvahiae (Kim et al. [22]). Le et al. ([25]) reported that Ulva pertusa shows high levels of antioxidant activity under blue light. Kim et al. ([22]) demonstrated that dichromatic and trichromatic LED arrangements can yield more biomass than monochromatic LEDs. According to the above studies, the arrangement of LED light sources can regulate algae growth and photosynthetic pigment synthesis, and the effects appear to vary by species. The manner in which macroalgae respond to changes in the ratio of blue to red light is unclear because most LED-related studies have simply compared macroalgal growth under specific ratios of blue and red LEDs, thus resulting in inconsistent results. To evaluate the effects of light on the specific physiological responses of algae, more detailed information is needed on the regulation of the related biosynthesis pathways. For example, carotenoids are powerful antioxidants that are induced by high-light stress to protect cells against photooxidative processes. Phytoene desaturase (PDS), which converts the first two desaturation steps of phytoene, has been considered a rate-limiting enzyme in the carotenoid biosynthetic pathway (Chamovitz et al. [7]; Steinbrenner and Linden [38]; Liu et al. [26]). Previous studies have proposed the use of blue light to upregulate the biosynthesis pathway gene of PDS in the microalgae Haematococcus pluvialis (Ma et al. [28]) and Phaeodactylum tricornutum (Srinivasan et al. [37]). Monitoring the expression of the PDS gene would assist in identifying how cells regulate carotenoid biosynthesis under various light conditions.

This study aimed to determine the effects of different ratios of blue and red LEDs on the growth characteristics, morphological changes, photosynthetic pigment contents, and antioxidant activities of C. lentillifera. In particular we examined whether the PDS gene plays a protective role or causes damage under different LED light sources. The results of this study are expected to provide baseline information for the design of artificial lighting sources in closed macroalgae production systems.

Healthy samples of C. lentillifera were provided by Chensheng Aquatic Products Technology Co., Ltd. (Taitung, Taiwan). The culture medium was composed of sterilized seawater (salinity 30 psu). Water motion was provided by aeration, and the culture seawater was refreshed every 4 days. Caulerpa lentillifera was incubated in a 40 cm × 50 cm × 50 cm fiber-reinforced plastic incubator at 25 ± 1 °C under a photosynthetic photon flux density (PPFD) of 60 μmol photons m−2 s−1 provided by cool-white fluorescent lamps operating in a 12:12 h light/dark (L/D) cycle. PPFD was measured using a LI-COR spherical quantum sensor (LI-250A Light Meter, LI-COR Inc., USA).

Three grams (approximately 4 cm) of fresh weight fronds of C. lentillifera was transferred to a glass beaker containing 1 L of filtered seawater (five replicates per light treatment) for a starting biomass density of 3 g FW L−1 at 25 ± 1 °C. Caulerpa lentillifera was grown in general culture conditions for 1 day prior to the experiments. Intact and healthy fronds were selected for the subsequent experimental treatments. Different light sources were independently established and positioned vertically. Six LED light sources were utilized: white (W), red (R, 660 nm, 100%), blue (B, 447 nm, 100%), 1B1R (50% blue + 50% red), 1B2R (33.3% blue + 66.7% red), and 1B5R (16.7% blue + 83.3% red). The optimal light source was determined by the weight gain percentage, and the experimental procedure investigated three photointensities, namely, 50, 100, and 150 μmol photons m−2 s−1, and three photoperiods, namely, 8 L/16D, 12 L/12D, and 16 L/8D. The experiments were performed for 12 days and measured every 4 days.

The LED lamps were purchased from NuPolar-Lights Optoelectronics Co. Ltd. (New Taipei City, Taiwan), and the spectral distribution was initially measured at 25 cm from the LED lighting sources to the top of the pots and at five points (center and four edges of each tray holding the pots) by using a spectroradiometer (LI-1800; LI-COR, USA) which showed the relative spectral distribution (Fig. 1).

Graph: Fig. 1 Relative spectral distribution of white, red, blue, and the three ratios of red to blue LED light used in this study. a white (W), b red (R, 660 nm, 100%), c blue (B, 447 nm, 100%), d 1B1R (50% blue + 50% red), e 1B2R (33.3% blue + 66.7% red), and f 1B5R (16.7% blue + 83.3% red). The spectral distribution was initially measured at 25 cm from the LED lighting sources to top of the pots and at five points

The weight gain percentage was calculated as [(Wt–W0)/W0] × 100%, where W0 is the initial fresh weight, and Wt is the fresh weight after t days.

A simple and rapid extraction procedure was applied to minimize the exposure of the samples to light, heat, and air. Fresh samples were ground using a mortar and pestle under liquid nitrogen. A 0.1 g sample of fine powder was weighed and extracted using 20 mL of acetone at 4 °C in darkness for 24 h. The extracted solution was filtered through 0.45-μm syringe filters before being injected into a high-performance liquid chromatograph (Waters Alliance e2695 HPLC, USA). All experiments were performed in dim light. The Chl a, chlorophyll b (Chl b) and β-carotene contents were analyzed as described in Zvezdanović et al. ([47]), with modifications. The separations were performed on a Hypersil MOS-2 C8 column (100 mm × 4.6 mm, 3 μm) at 25 °C. The mobile phase consisted of methanol and acetonitrile. A linear gradient program was used with a flow rate of 0.4 mL min−1 consisting of 0–12 min from 10 to 50% acetonitrile, followed by 12–15 min at 50% acetonitrile, 15–20 min from 50 to 10% acetonitrile, and 4 min at 10% acetonitrile. An injection volume of 5 μL was used. The ultraviolet-visible spectra were simultaneously recorded on a DAD detector (Waters Photodiode Array Detector, PDA 2998, USA) at three detection wavelengths (λdet): 430 nm for Chl a, 453 nm for Chl b, and 478 nm for β-carotene. The photosynthetic pigment content was the sum of Chl a and Chl b.

Fresh samples were ground using a mortar and pestle under liquid nitrogen. A 0.1 g sample of fine powder was weighed and extracted using 20 mL of acetone at 4 °C in darkness for 24 h. The extract was centrifuged at 5000×g for 10 min and was then used to perform the 2,2′-azino-bis3-ethylbenzothiazoline-6-sulfonic (ABTS) radical cation decolorization assay and determine the reducing power. The ABTS radical anion scavenging assay was performed using the method of Re et al. ([33]), and the reducing power was determined by using the method of Oyaizu ([31]).

For RNA extraction fresh samples were harvested, frozen under liquid nitrogen, and ground using a mortar and pestle. A 0.1 g sample of fine powder was resuspended in RLT buffer (Qiagen, USA) with 1% v/v β-mercaptoethanol (Sigma-Aldrich, USA), followed by supersonic disruption (VCX600, Sonics & Materials, USA). The total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) according to the instruction manual. The residual genomic DNA in the RNA extract was removed by column digestion with the RNase-Free DNase Set (Qiagen) during RNA purification. The concentration of the total RNA was determined with a spectrophotometer (ND-1000, NanoDrop, USA) at 260 nm.

The total RNA (0.2 μg) was reverse-transcribed to cDNA by using a Superscript III kit (Invitrogen, USA). For the cloning of the PDS gene, degenerate primers (Table 1) designed from conserved regions in cyanobacteria, algae and plants were used. Polymerase chain reaction (PCR) amplification was performed using Taq DNA Polymerase Mater Mix RED (Ampliqon, Denmark). PCR products were purified from an agarose gel and then cloned into pGEM-T vectors (Promega). The sequencing of the cloned fragments was performed using an ABI Prism 3730 DNA sequencer by Genomics (Taipei, Taiwan). A sequence of the PDS gene was obtained and deposited in GenBank under accession number MN395292. This sequence was translated to a deduced amino acid sequence by using the Bioedit Sequence Alignment Editor (version 7.2.5; Department of Microbiology, North Carolina State University, Raleigh, NC, USA). The amino acid sequence was aligned with PDS sequences from other organisms by using ClustalW (Thompson et al. [40]), and the resulting alignment was used to construct a maximum likelihood phylogenetic tree with 100 bootstrap replicates in the MEGA 7 program (Kumar et al. [24]).

Primers used in this study

a nucleotide single-letter code; A, adenosine; C, cytosine; G, guanine; T, thymine; R, A/G; Y, C/T; D, G/A/T; N, A/T/C/G

The real-time (RT) PCR analysis was performed according to Kang et al. ([21]). The reaction mix contained 5 μL of tenfold diluted cDNA, 300 nM forward and reverse primers (Table 1), and 2 × SYBR Green PCR Master Mix (Applied Biosystems) which are mixed in a final volume of 20 μL. All reactions were performed in duplicate. The gene-specific primers for the 18S rRNA and PDS genes were designed using Primer Express 3 (Applied Biosystems). RT-PCR was performed on an Applied Biosystems 7500 Real-Time System (Applied Biosystems). No template controls were included in each RT-PCR run for quality control. Amplicons were also confirmed by melting temperature analysis from 60 to 95 °C. The relative levels of the amplified mRNA were calculated according to the 2–ΔΔCT method (Livak and Schmittgen [27]) by using 18S rRNA for normalization.

All data are expressed as the means ± standard deviation (n = 5). The results obtained from the one-way analysis of variance and the Tukey tests were used to analyze the differences among treatments in SPSS. The significance level was set at P < 0.01.

We first investigated the effects of different ratios of red to blue light on the growth of C. lentillifera for 12 days under 100 μmol photons m−2 s−1 and a 12 L/12D photoperiod (Fig. 2a). The W, R, 1B1R, 1B2R and 1B5R treatments were more effective for weight gain percentage than the B light treatment at days 4, 8 and 12. The morphological observations showed that the R, 1B1R, 1B2R, and 1B5R treatments mainly stimulated the growth of stolons and rhizoids and had little effect on branches. The R, 1B2R and 1B5R treatments showed no significant difference in weight gain percentage. Among them, 1B5R most obviously promoted stolon growth, and the tissues were also relatively strong. In contrast, the branches started to shrink after 8 days under B light and then turned white and became soft (Fig. 2b). Hortensteiner ([18]) reported a loss of green color and chlorophyll in plants during senescence; thus, when more than half of the Caulerpa fragment became discolored, the fragment was considered dead (Gao et al. [14]). When the proportion of blue light increased, C. lentillifera showed a lower weight gain percentage; this trend is similar to that reported by Son and Oh ([36]), who observed that increasing blue light levels negatively affected lettuce growth. Folta and Childers ([11]) similarly demonstrated that strawberry plant growth was higher when illuminated with a combination of blue and red lights than when illuminated with red or blue light alone. Choi et al. ([8]) also reported that a combination of blue and red lights yielded the highest production of fruit compared with respective treatments with a red or blue light alone. Kinoshita et al. ([23]) found that the responses of higher plants to blue light allow the plants to capture light energy efficiently. Our results suggested that red light served as a major light source that improved the weight gain percentage and that an additional low proportion of blue light (1B5R, 16.7%; blue + 83.3% red) led to a higher weight gain percentage than other light source combinations.

Graph: Fig. 2 Weight gain percentage (a) and morphology (b) of C. lentillifera cultivated under white and different red/blue ratio LED lights at 100 μmol photons m−2 s−1 and 12 L/12D photoperiod for 12 days. The data are presented as the mean ± SD (n = 5). Letters indicate significant differences within each experiment at P < 0.01

Chlorophylls and carotenoids are the predominant plastid pigments in higher plants. Adams et al. ([1]) indicated that chlorophylls play a central and indispensable role in photosynthetic light harvesting and energy transduction, and light stress is known to decrease chlorophyll levels. The sum of Chl a and Chl b content indicates photosynthetic activity (Bollivar [6]), and β-carotene content was represented as photostimulus (Zhang et al. [45]). We analyzed the Chl a + Chl b (photosynthetic pigment) and β-carotene contents of C. lentillifera under white and different red/blue LED ratios.

Figure 3a shows that photosynthetic pigment contents varied with different blue/red LED ratios with 100 μmol photons m−2 s−1 under a photoperiod of 12 L/12D. The photosynthetic pigment contents all decreased under light treatments compared with the contents on day 0. This may be due to the damage caused by cutting C. lentillifera into cultivatable sizes. The light conditions directly affected C. lentillifera at days 8 and 12, and the photosynthetic pigment content gradually increased as the ratio of blue light decreased, with the values under the B and 1B1R treatments decreasing significantly compared with those of other treatments (Fig. 3a). This suggests that excessive blue light causes photosynthetic pigment damage. However, the growth rate of the macroalga Pyropia haitanensis was reportedly higher when grown under blue rather than red LED light (Wu [43]). This indicates that different macroalga species respond differently to light quality possibly because both blue and red wavelengths are required for promotion of photosynthesis, but the role of each wavelength differs somewhat (Banaś et al. [3]).

Graph: Fig. 3 The contents of photosynthetic pigments (Chl a + Chl b) (a) and β-carotene (b) in C. lentillifera cultivated under white and different red/blue ratio LED lights at 100 μmol photons m−2 s−1 and a 12 L/12D photoperiod for 12 days. The data are presented as the mean ± SD (n = 5). Letters indicate significant differences within each experiment at P < 0.01

β-carotene is the major carotenoid in algae. It plays a central role in photosystem II, blue light harvesting, the transfer of energy to photosystem reaction centers, and protection of the photosynthetic apparatus against photooxidative damage by reducing the formation of reactive oxygen species (ROS) under light stimulation (Telfer [39]). Thus, we analyzed the β-carotene content of C. lentillifera to understand the effects of the different light sources. We found that the β-carotene content decreased under the LED treatments compared with that on day 0. This suggests that the decrease was also caused by cutting damage. At days 4 and 8 the β-carotene contents under all light treatments were not significantly different. However, the β-carotene content was significantly higher under blue light treatment at day 12 (Fig. 3b). Blue light is an effective activator of carotenoid accumulation in plants and β-carotene can protect the photosynthetic apparatus against photooxidative damage. Zhang et al. ([45]) used blue light to stimulate improved β-carotene accumulation, but it can also be used to evaluate the effects of light sources (Telfer [39]). Figures 2a and b and 3a and b show that blue LED light increased β-carotene accumulation but decreased growth and Chl a and Chl b pigment contents after 8 days. Therefore, long-term cultivation with a blue light may be ineffective for C. lentillifera cultivation.

The antioxidant-promoting activities of LED lighting in closed plant production systems have received increasing research attention because antioxidants remove harmful ROS that cause aging and chronic diseases in humans. Our study involved the effects of different ratios of red to blue light on ABTS antioxidant activity and the reducing power of C. lentillifera. Caulerpa lentillifera did not exhibit high ABTS antioxidant activity or reducing power under the combined blue and red light treatments, and no significant differences were observed between these parameters under W and R light treatments. Only blue light significantly increased the ABTS antioxidant activity and reducing power at days 4, 8, and 12 (Fig. 4). Although B light enhanced the antioxidant activities of C. lentillifera, this effect may be the product of a defense mechanism against light damage (Telfer [39]). In the results for C. lentillifera growth (Fig. 2) and photosynthetic pigment content (Fig. 3), B light proved unsuitable for the long-term irradiation of C. lentillifera. Thus, we suggest that the increase in antioxidant activity may be due to an increase in light stress, which was detrimental to algal growth. Le et al. ([25]) demonstrated that the macroalgae U. pertusa showed high levels of antioxidant activities under blue light. However, little is known about the potential influence of light on carotenoid synthesis and antioxidant activities in macroalgae. Srinivasan et al. ([37]) and Ma et al. ([28]) reported that blue light can upregulate the PDS gene, which encodes an important enzyme in the carotenoid biosynthesis pathway (Chamovitz et al. [7]; Steinbrenner and Linden [38]; Qin et al. [32]). Thus, we analyzed the effects of different LED light sources on PDS gene expression.

Graph: Fig. 4 The ABTS antioxidant activity (a) and reducing power (b) measured in C. lentillifera cultivated under white and different red/blue ratio LED lights at 100 μmol photons m−2 s−1 and a 12 L/12D photoperiod for 12 days. The data are presented as the mean ± SD (n = 5). Letters indicate significant differences within each experiment at P < 0.01

With degenerate primers for the PDS gene, a cDNA fragment of 440 base pairs was amplified from C. lentillifera. After losing its primer sequences, this gene fragment contained an open reading frame of 139 amino acids. The conserved domains of PDS (PLN02612, TIGR02731) were detected by the BLAST server of the National Center for Biotechnology Information. Phylogenetic analysis indicated that this sequence was grouped with PDS in green algae (Fig. 5). To evaluate the influence of light on the expression of the PDS gene, the transcript levels were measured by quantitative RT-PCR normalized against 18S rRNA. The diel variations in PDS expression were examined under a light-dark cycle. The transcript levels of PDS increased with time and reached higher levels during the light period but decreased during the dark period (Fig. 6a). This result was consistent with reports of higher transcript levels of this gene under light conditions in green algae (Steinbrenner and Linden [38]; Im et al. [19]). To compare the effects of light sources on the regulation of PDS gene expression with incubation time, the transcript level of the PDS gene was measured during the light period (6 h after the lights came on). To evaluate the effects of different light sources on PDS expression, C. lentillifera was grown under different light treatments for 12 days. The upregulation of PDS mRNA was detected under red and blue light sources at day 4, and significantly higher PDS gene transcript levels were detected under blue light than under other light sources at days 8 and 12 (Fig. 6b). The enhanced expression of the PDS gene was also detected under blue light in microalgae (Bohne and Linden [5]; Im et al. [19]; Ma et al. [28]). Our results indicate that blue light illumination upregulated the transcription of the PDS gene, which in turn triggered an increase in the accumulation of β-carotenoids in C. lentillifera (Fig. 3b).

Graph: Fig. 5 Phylogenetic tree of phytoene desaturase amino acid sequences in cyanobacteria, algae and higher plants. Species names and GenBank accession numbers are given. The cyanobacteria branch is used as the outgroup. The numbers at the nodes are bootstrap values based on 100 resamplings, and clades receiving over 60% bootstrap support are shown. The scale bar represents an estimated number of amino acid substitutions per site

Graph: Fig. 6 The effect of light on the transcript levels of PDS in C. lentillifera under a light-dark cycle. The filled horizontal bars indicate the dark period (a). Time course of transcript levels under different light sources (b). Error bars represent one standard deviation of the tube replicates

The optimization of light quality is important in indoor cultivation techniques for C. lentillifera. We gathered the statistical results for the weight gain percentage, photosynthetic pigment content, β-carotene content, ABTS antioxidant activity and reducing power of C. lentillifera cultivated under different LED lights for 12 days. The data show that the 1B5R LED light treatment was a better condition, followed by the white, red, 1B1R/1B2R, and blue LED treatments (Table 2). Thus, we used 1B5R to determine the changes in C. lentillifera parameters under various irradiance (50, 100, and 150 μmol photons m_SP_−2_sp_ s_SP_−1_sp_) and photoperiods (8 L/16D, 12 L/12D, and 16 L/8D).

The statistical results for weight gain percentage, photosynthetic pigment content, β-carotene content, ABTS antioxidant activity, and reducing power of C. lentillifera cultivated under different LED lights at 12 days

The same uppercase letters indicate no significant difference between treatments (ANOVA followed by Tukey's test, P < 0.01)

In the irradiance test, C. lentillifera was cultivated under 1B5R LED light at 50, 100, and 150 μmol photons m−2 s−1 and a photoperiod of 12 L/12D for 12 days. The weight gain percentage was higher at 100 μmol photons m−2 s−1 than that at 50 and 150 μmol photons m−2 s−1 (Fig. 7a). Furthermore, the photosynthetic pigment content of the 100 μmol photons m−2 s−1 group was higher than that of the 150 μmol photons m−2 s−1 group, and no significant difference from the 50 μmol photons m−2 s−1 group was evident (Fig. 7b). Only the 150 μmol photons m−2 s−1 group caused the photostimulus to increase the reducing power at day 12 (Fig. 7c and d). After 12 days of culture, no morphological differences were observed between the 50 and 100 μmol photons m−2 s−1 groups, and the branches in the 100 μmol photons m−2 s−1 group were harder. In contrast, C. lentillifera in the 150 μmol photons m−2 s−1 group turned white, and parts of the fragments fell off and became soft. This may have been caused by a photooxidation reaction inside the cell due to excess light that could not be absorbed by the photosynthetic apparatus (Richmond [35]).

Graph: Fig. 7 The weight gain percentage, pigment content and antioxidant activity of C. lentillifera cultivated under 1B5R LED light for 12 days with different irradiance and photoperiods. The weight gain percentage (a), pigment content (b), ABTS antioxidant activity (c), and reducing power (d) of C. lentillifera under 50, 100, and 150 μmol photons m−2 s−1. The weight gain percentage (e), photosynthetic pigment content (f), ABTS antioxidant activity (g), and reducing power (h) of C. lentillifera under 8 L/16D, 12 L/12D, and 16 L/8D. The data are presented as the mean ± SD (n = 5). Letters indicate significant differences within each experiment at P < 0.01

In the photoperiod tests, C. lentillifera was cultivated under 1B5R LED light at 100 μmol photons m−2 s−1 with 3 photoperiod cycles of 8 L/16D, 12 L/12D, and 16 L/8D for 12 days. Larger fronds with higher water content were observed at a photoperiod of 12 L/12D, and significantly higher weight gain percentages were also obtained in the 12 L/12D group than in the other groups at days 8 and 12 (Fig. 7e). In contrast, the algal tissues turned white and became soft under the photoperiod of 16 L/8D, and the photosynthetic pigment contents were also significantly lower than those in the other 2 photoperiods (Fig. 7f). However, the different photoperiods were not associated with significant differences in photostimulus (ABTS antioxidant activity and reducing power variety) at days 8 and 12 (Fig. 7g and h). The difference in reducing power at day 4 was presumed to be caused by adaptation to the environment (Fig. 7h). Furthermore, the photosynthetic pigment contents were higher under low-light irradiance (50 μmol photons m−2 s−1) and a short photoperiod (8 L/16D). These results suggested that low-light induced the synthesis of larger photosynthetic units, presumably to aid light harvesting; at high-light levels, the algae synthesized smaller photosynthetic units presumably to prevent photo damage (Ak et al. [2]). Our results indicated that 100 μmol photons m−2 s−1 of 1B5R light with a photoperiod of 12 L/12D resulted in good growth and development in C. lentillifera.

Red and blue LEDs had positive effects on the growth and antioxidant activity of C. lentillifera, respectively. Red light was effective in stimulating the growth of C. lentillifera. In contrast, blue light increased β-carotene accumulation and antioxidant activity in C. lentillifera. However, increasing the blue light ratio negatively affect weight gain and photosynthetic pigments. It appears that a mixture of red light with low proportion of blue light enhances both growth and yield in closed macroalga production systems.

This work was supported by TIARF106A1-001 from the Taiwan International Algae Research Fund.

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