*2.1. Microalgae Culture Condition*

The stock of the microalgae species *I. zhanjiangensis* was obtained from the Microalgae Laboratory of the College of Oceanography at Hainan University (Haikou, China). For the enrichment of the culture media, the nutrient medium Ningbo 3# was dissolved in filtered and sterilized seawater (29 PSU) with the composition per 1 L of: 100 mg NaNO3, 10 mg NaH2PO4, 2.5 mg FeSO4, 10 mg EDTA-2Na, 0.25 mg MnSO4, 0.5 × <sup>10</sup>−<sup>3</sup> <sup>μ</sup>g vitamin B12, and 6 μg vitamin B1.

The microalgae were cultured in 5 L flat-bottom glass flasks at 25 ± 1 ◦C, with the illumination provided by light-emitting diodes (LED, 191.8 μmoles/m2/s) with a 14:10 h light/dark photoperiod. To enhance growth and prevent the algae from settling, continuous aeration was applied using air stones at 20 L/min. The initial culture media inoculation of the microalgae was 100,000 cells/mL, and the illumination was immediately provided using green (495–530 nm), blue (450–480 nm), red (615–650 nm), white (450–465 nm), and yellow (580–595 nm) light-emitting diodes. Each illumination treatment was set separately to avoid any light interference from the neighboring treatments and set in triplicate.

#### *2.2. Measurement of I. Zhanjiangensis Growth*

The growth of *I. zhanjiangensis* cultured in the experiment was measured using cell density and biomass (dry weight) to precisely determine the growth pattern of the microalgae. Microalgae cell density was determined daily by counting the cell number using a hemacytometer under a white-light field microscope and was presented as the number of cells/mL. Cell dry weight was estimated by filtering 20 mL of cultured microalgae using a pre-weighed Whatman GF/C filter (47 mm ∅), washed three times with filtered seawater, and dried to constant weight. Samples were always collected at the same time of the day.

Productivity (*Px*) and specific growth rate (*μ*) of *I. zhangjiangensis* were calculated according to the following Formulae [44]:

$$P\_X = \left(X\_m - X\_i\right) \left(T\_c\right)^{-1} \tag{1}$$

$$
\mu = (\ln X\_m - \ln X\_i) \,\mathrm{(T\_c)}^{-1} \tag{2}
$$

where, *Xi* = initial biomass concentration (g/L), *Xm* = maximum biomass concentration (g/L), and *Tc* = cultivation time related to the maximum biomass concentration (days).

#### *2.3. Measurement of Photosynthetic Pigments, Protein, and Carbohydrate Content*

Chlorophyll concentration (Chl) content was determined using a modified method from Jeffrey and Humphrey [45]. Briefly, 20 mL samples were withdrawn from the culture flasks and transferred to opaque plastic bottles, kept away from light, and warmed to room temperature, then filtered using Whatman GF/C filters (25 mm ∅). After filtration, the filters containing the microalgae biomass were folded and stored separately at −2 ◦C. Chl extraction was carried out by grinding the filters in 90% acetone (2–4 mL) in a glass homogenizer on an ice bath under low-light conditions for up to 1 min. After grinding, the Chl extracts were transferred to a graduated and stoppered centrifuge tube and rinsed with 10 mL of acetone 90% (10 mL + dead volume of filter). The extract was then centrifuged for 10 min at 500 g. After completion of centrifugation, the absorbance of the supernatant (OD) was measured at 750, 664, 647, and 630 nm against a 90% acetone blank. The concentrations of Chl-*a*, Chl-*b*, and the total Chl were calculated according to the following Equations [45,46]:

$$C\_{\rm chla} = (11.85 \ E\_{664} - 1.54 \ E\_{647} - 0.08 \ E\_{630}) \times 10/\text{V} \tag{3}$$

$$C\_{\rm chlb} = (21.03 \ E\_{647} - 5.43 \ E\_{664} - 2.66 \ E\_{630}) \times 10/\text{V} \tag{4}$$

$$\mathbb{C}\_{tch1} = (20.21 \, E\_{647} + 8.02 \, E\_{664}) \times 10/\text{V} \tag{5}$$

where, *Cchla*, *Cchlb*, and *Ctch1* are the concentrations of Chl-*a*, Chl-*b*, and the total Chl (mg/L), and V is the filtered volume (mL).

The protein content of each sample was determined by the Lowry method [47]. Carbohydrates were measured by the phenol sulfuric acid method [48].

#### *2.4. Statistical Analysis*

Significant differences (*p* < 0.05) among variables were first identified using the *t*-test. Before analysis, data were tested for normality using Kolmogorov–Smirnov's test and for homogeneity of variance using Cochran's C test. All statistical analyses were performed using DPS14.5 software (Hangzhou Rui Feng Information Technology Co., Ltd., Hangzhou, China).

#### **3. Results**
