Artificial light source selection in seaweed production: growth of seaweed and biosynthesis of photosynthetic pigments and soluble protein

Seaweed species and cultivation

The juveniles of two seaweed species, Sargassum fusiforme, and Ulva pertusa, were collected from Dongtou Island, Wenzhou City, Zhejiang Province, China (27°41′42″ N, 1211°1′06″E). For acclimation, all the collected seaweeds were maintained in glass jars containing filtered, sterile natural seawater (33‰), and the temperature of seawater was controlled at 16 °C by a chiller (LS16-600, JLLN, Shenzhen, China). Illumination was provided by fluorescent lamps (120 µmol photons m⁻²s⁻¹, 12 h of light followed by 12 h of darkness). After 3 days of acclimation, healthy individual seaweeds were selected and used in the following experiments.

Light sources

Four LEDs emitting white, red, green, and blue light were used as artificial light sources for seaweed cultivation, providing 120 µmol photons m⁻²s⁻¹ on the surface of seaweed thalli. The LED lighting system was assembled in PVC tubes (height: 25 cm, diameter: 20 cm), with flexible LED light belts (length: 3 m) affixed to the inner walls of each PVC tubes (Fig. 1). All the LED lighting diodes (Opple Co. Ltd., Shanghai, China) were driven by a 220V power supply. The light was supplied for 12 h every day from 6:00 a.m to 6:00 p.m. Light spectra were measured with an optical spectrum analyzer (CMS-2S, Inventfine Co. Ltd., Hangzhou, China). Light intensity was measured using an MQ-200 Quantum Separate Sensor (Apogee Instruments, USA). Residual energy ratio (Rer) underwater was measured following Chiang’s method (Table S2) (Chiang, Chen & Chen, 2011). Among the four LED lights, the red LED light has the lowest Rer value, followed by the white, green, and blue light, respectively (Table S2).

Emission spectral distribution of light sources

The peak wavelengths of the red, green, and blue LEDs were 632 nm, 517 nm, and 462 nm, respectively, and all the peaks are narrow (80–100 nm) (Fig. 2). White LED had a continuous spectrum with two peaks in the blue and green light region.

Light incubation experiments

The cultivation lasted for 18 days. Five juvenile individual seaweeds (approx. 5 g fresh weight) were placed into one cylindrical plastic bottle (1000 ml, diameter: 100 mm). Three replicate bottles were placed inside the PVC tubes for light treatments (Fig. 1) (Kim et al., 2015). In total, 15 juveniles of U. pertus and 12 juveniles of S. fusiforme were set in a PVC tube. The LED lights were controlled independently. During the experimental period, both S. fusiforme and U. pertusa were cultivated in filtered and sterile natural seawater. The nutrients, phosphate (PO₄³⁻) and nitrate (NO₃⁻) were renewed every second day. The PO₄³⁻ and NO₃⁻ concentrations of seawater were 15 µmol L⁻¹ and 150 µmol L⁻¹, respectively. The seawater was sufficiently aerated by an air pump. The temperature in the aquarium was kept at 16 °C.

Growth

Specific growth rate (SGR) was calculated following the method used in our previous study (Xiao et al., 2015): (1)${\displaystyle SGR=ln\left(\frac{W_t}{W_0}\right)\times t^{-1}\times 100}$ where W ₀ is the initial algal biomass, and W_t is the algal biomass after t days of cultivation. Fresh weights (FW) of S. fusiforme and U. pertusa were measured every second day.

Photosynthetic pigment and soluble protein

The photosynthetic pigment Chlorophyll a and carotenoid content were measured. Chlorophyll a was extracted using acetone (90%) neutralized with sodium carbonate, as described in Jeffrey & Humphrey (1975). Carotenoid concentrations were detected following Seely, Duncan, and Vidaver (Seely, Duncan & Vidaver, 1972). Chlorophyll a and carotenoid were measured every second day by spectrophotometer (Inesa 722S, Shanghai, China). The soluble protein concentrations were also determined spectrophotometrically at 595 nm by Coomassie brilliant blue method every second day (Bradford, 1976).

Data analysis

Differences between light treatments were tested for each species separately using one-way ANOVA with a significant level of p < 0.05. Tukey’s test was used for the post hoc test. Data normality was checked by the shapiro-wilk test and variances homogeneity were checked by Levene’s test. Differences between data that did not pass the normality test were analyzed by Kruskal–Wallis H test (non-parametric). Data with differences that did not pass Levene’s test were analyzed by Welch’s ANOVA, where the Games-Howell test was used for post hoc test. Statistical tests were performed with SPSS (version 19.0).

Specific growth rate

For both seaweed species (Ulva pertusa and Sargassum fusiforme), white-LED light stimulated seaweed growth and the light colors of LEDs differed in their influences on seaweed growth. The white light stimulated the growth of U. pertusa (average SGR = 5.16 ±  0.88% d⁻¹) significantly, compared to the green light (4.07 ± 0.64% d⁻¹) (Fig. 3, p < 0.05). As for S. fusiforme, SGR decreased following the sequence of white LED light (3.21 ± 1.10% d⁻¹) >green and blue LED light (2.44 ± 1.13 and 2.35 ± 0.66% d⁻¹, p > 0.05) >red LED light (1.34 ± 0.39% d⁻¹, p < 0.05).

Photosynthetic pigments and soluble protein

The LED lights induced changes in pigments and soluble protein synthesis in the two seaweed species. For instance, the Chl a concentration of U. pertusa treated with red LED light (1.21 ± 0.15 mg g⁻¹) was higher than those treated with white (0.92 ± 0.19 mg g⁻¹, adjusted p = 0.004, p = 0.001), green (1.00 ± 0.20 mg g⁻¹, adjusted p = 0.040, p = 0.007) and blue LED lights (1.06 ± 0.13 mg g⁻¹, adjusted p = 0.491, p = 0.082) (Fig. 4). For S. fusiforme, Chl a concentration of the seaweed were the same in all the light treatments. Carotenoid concentrations shared similar levels in different LED groups for both U. pertusa and S. fusiforme (p > 0.05). The concentrations of soluble protein showed no significant difference among four LED groups in U. pertusa and S. fusiforme (Fig. 5).

Light driven shifts in seaweed growth

For both seaweed species U. pertusa (green algae) and S. fusiforme (brown algae), the experimental seedlings achieved the highest growth rate under white LED lighting, which is consistent with previous studies (Tovar et al., 2000; Kim et al., 2015). This may be partially explained by the broad light wavelengths of white light (from 430 nm to 630 nm) (Fig. 2). White LED light, with the ability to provide spectrum comparable of sunlight (Glemser et al., 2016), is capable of supporting C and N metabolism in seaweeds (Tsekos et al., 2002; Korbee, Figueroa & Aguilera, 2005). White LED light with a broad continuous emission spectrum, is also providing a higher luminous efficiency compared to a fluorescent white-light source (Pimputkar et al., 2009). S. fusiforme cultivated under red LED lights had a significantly lower growth rate compared to those treated with white LED lights. S. fusiforme contains fucoxanthin, one of the brown algae carotenoids that plays an important role in photosynthesis (Terasaki et al., 2005; Xiao et al., 2012). The main absorption peaks of fucoxanthin are in the blue region (about 455 nm) (Wang et al., 2005). Thus, red light is not likely to be utilized at high efficiency by S. fusiforme. Another species of brown algae Sargassum horneri also showed slower growth than the individuals cultivated in white or blue LED lights (Miki et al., 2017). Interestingly, red LED light has been widely applied in the cultivation of microalgae and terrestrial plants (Goins et al., 1997; Poudel, Kataoka & Mochioka, 2008) (see also Table S1). However, a negative influence of red LED light on S. fusiforme growth was found in our experiment. This hinted again, the importance to further investigate more seaweed species since the influence of light quality appeared to be highly species-dependent.

Through the 18 days of cultivation, U. pertusa seemed to maintain a relatively fast and stable growth under all the LED lights with constant growth rates, while S. fusiforme held a higher growth rate in the last days than the earlier days (Fig. 3). Ulva species (i.e., U. lactuca and U. prolifera) grow faster than many other macroalgal species (Pedersen, Borum & Fotel, 2010; Tang et al., 2021). Also, the simple morphology of U. pertusa leads to an easier adaptation to the environment (including changes in the light condition). On the contrary, S. fusiforme may take a longer time for adaptation when cultured in changed light conditions (Fig. 3). Another brown alga Sargassum horneri, which was morphologically similar to S. fusiforme, showed a similar growth pattern when cultured in LED lights (Miki et al., 2017). The thalli from S. horneri were found to absorb red light in low efficiency, we suspect that the utilization rate of red light may also be lower for S. fusiforme thalli, leading to a lower growth rate under red LED lights (Matsui, Ohgai & Murase, 1994; Miki et al., 2017).

Light-driven shifts in seaweed biosynthesis

The accumulation of photosynthetic pigments and soluble protein in U. pertusa and S. fusiforme were also influenced by light sources. Chl a concentration was significantly higher in U. pertusa under red LED light as compared to white and green LED lights. Similar to our findings, several other green algae Ulva prolifera and Ulva lactuca held higher Chl a content in red LED cultivation compared to white and blue LED lights (Takada et al., 2011; Gong, Liu & Zou, 2020). However, U. pertusa was found to synthesize less Chl a and form smaller chloroplast under red light, as compared to blue and white lights (Muthuvelan, Noro & Nakamura, 2002; Le et al., 2018). The higher Chl a concentration per fresh weight of U. pertusa may be derived from the restrained biomass accumulation under red light.

As for carotenoid and soluble protein content, no significant difference was found among the four light treatments for both S. fusiforme and U. pertusa in our study. Previously, the red light was found to promote carotenoid synthesis in a green algae Dunaliella salina to reduce reactive oxygen species formation and increase anti-oxidant level (Xu & Harvey, 2019a; Xu & Harvey, 2019b).

Seaweed cultivation in fields using supplementary LED lights

Seaweeds play an important role in food and feed supply (Makkar et al., 2016). LED lights could stimulate the growth and increase the yield of specific seaweeds as compared to traditional fluorescent light (Kim, Choi & Lee, 2019; Gong, Liu & Zou, 2020). The growth and biochemical composition of seaweed were affected by the light quality, indicating the potential for using artificial light to increase the yield and proportion of high-value biomolecules in seaweed aquaculture (Table S1). There are plenty of commercially available LED devices, and underwater LED lighting has been developed for many years, which makes LED cultivation systems easy to be established for both land and offshore seaweed cultivation (Hardy et al., 2008; Shen et al., 2013). Our indoor experiment showed that white LEDs were favored in the cultivation of U. pertusa and S. fusiforme because white LED promoted seaweed growth while protein production was maintained at a constant level. White LED has also a relatively good ability to penetrate underwater (Table S2). Nevertheless, the turbidity of water is still an important factor to be considered in fields. For seaweed cultivation on lands, water renewal or flow water are usually applied, so it is relatively easy to keep the water clean. But for seaweed growing in a natural water body, such as for seaweed-based ecosystem restoration, turbidity and other environmental factors need to be considered in the future study.