*2.4. Measurement of NO3-N Uptake Rate*

Following the culture experiment, 0.2 g (FW) of *G. lemaneiformis* and/or *S. horneri* were randomly selected from each tank for NO3-N uptake measurements. They were transferred into conical flasks containing 200 mL of sterilized seawater enriched with 25% PESI medium and gently shaken for 2 h using a horizontal oscillator. The light and temperature conditions were the same as those described for the culture experiment. For each treatment, the media before and after the culture experiment were separately collected and the NO3-N concentrations were determined by the cadmium column reduction method and molybdenum blue method with ultraviolet absorption spectrophotometer, respectively [46,47]. The NO3-N uptake rate was calculated by Equation (2):

$$\mathbf{U}\_{\rm N} = (\mathbf{C}\_0 - \mathbf{C}\_t) \times \mathbf{V} \;/\ \; \text{(T} \times \text{W)}\tag{2}$$

where UN is the NO3-N uptake rate, C0 is the initial concentration of NO3-N (mg L−1), Ct is the final concentration of NO3-N (mg L<sup>−</sup>1) following the experiment, V is the medium volume (mL), T is the experiment period, and W is the FW of samples (g).

#### *2.5. Measurement of Chlorophyll a (Chl a)*

0.1 g (FW) of *G. lemaneiformis* was used to extract the Chl *a* in each replicate of all the experimental treatments. These samples were ground using liquid nitrogen, then 3 mL of phosphate buffer solution (0.1 M, pH = 6.8) was added. After transferring the samples into tubes, they were centrifuged for 30 min at 4000 rpm at 4 ◦C. Subsequently, 8 mL of dimethylformamide (DMF) was added to the precipitates obtained following centrifugation and maintained at 4 ◦C for 1 d. Extracts were then centrifuged for 10 min at 6000 rpm at 4 ◦C. The supernatant was collected and the absorption was determined at 750, 664, 647, and 625 nm. The Chl *a* content (mg g<sup>−</sup>1) was calculated by Equation (3):

$$\text{Chl } a = \left[ 12.65 \times (\text{A}\_{664} - \text{A}\_{750}) - 2.99 \times (\text{A}\_{647} - \text{A}\_{750}) - 0.04 \times (\text{A}\_{625} - \text{A}\_{750}) \right] \times \text{V\_e } / \left( \text{I} \times \text{W} \times 1000 \right) \tag{3}$$

where A750–625 is the absorption of extracts at 750, 664, 647, and 625 nm, Ve is the DMF volume (mL), I is the optical path in the cuvette (cm), and W is the FW of the samples (g).

0.25 g (FW) of *S. horneri* was used to extract the Chl *a* in each replicate of all the experimental treatments. These samples were placed into 2 mL of dimethyl sulfoxide for 5 min, and the supernatant absorption was determined at 665, 631, 582, and 480 nm. Subsequently, the same samples were placed into 3 mL of acetone for 2 h. The supernatant was moved to a 10 mL tube, then 1 mL of methanol and 1 mL of distilled water were added. The supernatant absorption was determined at 664, 631, 581, and 470 nm. The Chl *a* content (mg g<sup>−</sup>1) was calculated by Equation (4):

$$\text{Chl } a = \left[ \left( \text{A}\_{66\%} \ne 72.8 \right) \times \text{V}\_1 + \left( \text{A}\_{664} \ne 73.6 \right) \times \text{V}\_2 \right] / \left( \text{I} \times \text{W} \right) \tag{4}$$

where A665 and A664 are the absorption rates of the extracts at 665 and 664 nm, respectively, V1 is the volume of the extract at the first extraction process (mL), V2 is the volume of extract at the second extraction process (mL), I is the optical path in the cuvette (cm), and W is the FW of the samples (g).

#### *2.6. Measurements of Soluble Protein and Carbohydrate*

For all the experimental treatments, 0.1 g (FW) of samples from each replicate were homogenized with a pestle and mortar and 0.9 mL of phosphate buffer solution (0.1 M, pH = 7.4). The extract was centrifuged for 30 min at 12,000 rpm at 4 ◦C. The supernatant absorption at 595 nm was recorded using an ultraviolet spectrophotometer. The soluble proteins of samples were evaluated using Coomassie Brilliant Blue G-250 dye (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and bovine albumin [48].

An amount (0.1 g FW) of the samples from each replicate were ground in 2 mL of distilled water and diluted to 10 mL after adding 2 mL of MgCO3 suspension liquid. The mixture was centrifuged for 5 min at 4000 rpm at 4 ◦C. Then, 1 mL of supernatant was moved to a tube and diluted to 2 mL. After that, 8 mL of anthrone reagent was added. The mixture was bathed in boiled water for 10 min and cooled to room temperature. The absorption at 620 nm was recorded and the mixture was standardized. The soluble carbohydrate content was measured according to [49].

#### *2.7. Statistical Analysis*

For both species, one-way ANOVA was applied to analyze the RGR, net photosynthetic rate, NO3-N uptake rate, and the contents of biochemical compositions among different culture models (mono- or co-cultures with three BDRs). Prior to these analyses, the variances of the data were subjected to homogeneity tests. Duncan's multiple range test was used if significant differences were detected (*p* < 0.05). Statistical analyses were carried out using SPSS 26.0 software.

## **3. Results**
