In this study, two strains of P. haitanensis, W28-42 (a narrow strain) and WO15-4 (a wide strain), were obtained from the Laboratory of Germplasm Improvements and Applications of Pyropia in Fujian Province. The sporophytic filaments (the conchocelis stage) were cultured at 29°C under a light intensity of 10–20 µmol m⁻² s⁻¹ with a photoperiod of 8 h light/16 h dark until maturity. The mature conchocelis were then cultured at 21°C with aeration to promote the release of conchospores capable of developing into gametophytic blades (the thallus stage). Healthy, intact thalli with a length of 5 ± 1 cm were randomly selected, with three thalli placed into each 1 L sterilized glass flask for cultivation.

The thalli were cultured at 21 ± 1°C in nutrient-enriched natural seawater prepared according to Provasoli’s seawater medium (Starr and Zeikus, 1993). The media were aerated during cultivation, and the photoperiod was 12 h light/12 h dark. In our preliminary experiments, P. haitanensis thalli were cultured at six gradient light intensity conditions (5, 50, 200, 500, 700 and 1000 μmol m^–2 s^–1). However, thalli failed to survive under light intensities of 700 and 1000 μmol m^–2 s^–1. Therefore, only four light intensity gradients (5, 50, 200, and 500 μmol m^–2 s^–1) were selected for the formal experiments. Each light treatment contained three biological replicates. Samples were harvested after seven days of cultivation.

After seven days of cultivation, 200 mL of the medium was collected from each glass flask and filtered using pre-combusted Whatman GF/F filters (diameter: 25 mm, pore size: 0.7 µm) under a low vacuum. The filter membranes were dried before the analysis of particulate organic carbon (POC) and stored at –20°C to prevent microbial decomposition of the POC. The filtrate (50 mL) was collected into pre-treated brown glass bottles and immediately stored at –20°C for the determination of dissolved organic carbon (DOC). All glassware was combusted in a muffle furnace at 450°C for four hours, soaked in 0.1 mol/L HCl solution for over 24 h, and finally rinsed with ultrapure water.

On the final day of cultivation, the thalli were harvested from the culture bottles, and surface water was removed with absorbent gauze. The fresh weight of the thalli was measured using an analytical balance. The thalli were then dried in an oven at 60°C to a constant weight, recorded as the dry weight. The dried algae were stored at –20°C for the determination of elemental contents in the tissue.

The length (L), width (W), fresh weight (FW), and dry weight (DW) of the thalli were measured before and after seven days of cultivation. The formulas for the specific growth rate of fresh weight (FW-SGR), the daily growth rate of fresh weight (FW-DGR), and the length (L-DGR) and width (W-DGR) were as follows:

where the lowercase numbers 0 and 7 indicate that the parameters were measured on days 0 and 7, respectively.

The concentration of DOC was determined with a Shimadzu TOC-VCPH analyzer (TOC5000A; Shimadzu, Kyoto, Japan) through high-temperature catalytic oxidation at 720°C. The POC samples were dried at a constant temperature of 60°C before analysis and were measured using a Costech ECS CHNSO elemental analyzer. The net release rates (mg·g^–1 d^–1 DW) of OC (as DOC and POC) by P. haitanensis were calculated as

where δOC represents the increase in organic carbon concentration (mg/L) over seven days of culture, and V and DW are the volume of seawater (L) and the initial dry weight of the algal tissue (g), respectively.

The algal samples were dried at a constant temperature of 60°C and then ground into a powdered form. The C and N contents of the algal tissue were obtained by combustion and were measured using a Costech ECS CHNSO elemental analyzer (Costech, USA). The tissue P content was measured by acid-persulfate digestion and subsequent soluble reactive phosphate analysis using an Automatic Discrete Analyzer (CleverChem380, DeChem-Tech, Germany). The obtained C, N, and P contents were converted into molar concentrations, and the molar ratios of C:N, C:P, and N:P for different treatment groups were calculated.

All values represent the mean of three replicates for each treatment. Error bars in the figures depict standard deviations. The differences between treatment groups were analyzed using one-way analysis of variance (ANOVA). All statistical analyses were carried out using SPSS 24.0 (International Business Machines Corporation, USA).

Light intensity had a significant effect on the growth rates of strains W28–42 and WO15-4 (P < 0.05, Figures 1 , 2 ). All four treatments displayed initial increases in growth rate followed by decreases with increasing light intensity, reaching a maximum at 200 μmol m^–2 s^–1. The specific growth rates of fresh weight (FW-SGR) of W28–42 were always higher than those of WO15-4, but the difference was only significant at a light intensity of 200 μmol m^–2 s^–1 (P < 0.05, Figure 2A ). Interestingly, the daily changes in fresh weight (FW-DGR) showed a different trend compared with FW-SGR; the FW-DGR of W28–42 was always lower than that of WO15-4, except for the lowest light treatment (P < 0.01, Figure 2B ). Similarly, the daily changes in length (L-DGR) and width (W-DGR) showed an opposite trend. The L-DGR of W28–42 was significantly higher than that of WO15-4 (P < 0.001, Figure 2C ) under exposure to light intensities of 50–500 μmol m^–2 s^–1, but the W-DGR of W28–42 was significantly lower than that of WO15-4 (P < 0.001, Figure 2D ).

The carbon accumulation rates of algal tissue and the release rates of DOC and POC of the two strains under four light intensities were measured to investigate the distribution of photosynthetically fixed carbon by the metabolic activity of P. haitanensis. The tissue C accumulation rates significantly increased with light intensity (P < 0.05, Figure 3A ), and no significant differences were found between the two strains concerning the rates of tissue C accumulation and DOC release (P > 0.05, Figures 3A, B ). The DOC release rates of the two strains showed different responses to light intensity, with the rate of strain W28–42 increasing with light intensity, while strain WO15–4 was not affected by the light intensity ( Figure 3B ). The POC release rates of both strains increased with light intensity (P < 0.05, Figure 3C ). In addition, except for the light intensity of 5 μmol m^–2 s^–1, the POC release rates of strain W28–42 were much higher than those of strain WO15-4 (P < 0.001, Figure 3C ).

The production rates of the three types of organic C were positively correlated with the specific growth rates of the algae ( Figure 4 ). In particular, the coefficient of determination (R²) between tissue C accumulation and specific growth rate ranged from 0.7169 to 0.8364, with the high R² values indicating a relatively close relationship between these two parameters ( Figure 4A ). Meanwhile, R² values between DOC release and specific growth rate were relatively lower, ranging only from 0.2824 to 0.3665 ( Figure 4B ). R² values between the POC release rate and specific growth rate exceeded 0.6 ( Figure 4C ), indicating a significant positive correlation between these two parameters.

To further explore the effects of light intensity on the nutrient elemental composition in the tissue of P. haitanensis, we measured the C, N, and P contents in the algal tissue. The results showed that significant differences in the contents of the three nutrient elements under different light intensities. Specifically, the tissue C content of both strains significantly increased with the enhancement of light intensity (P < 0.05). The C content of the W28–42 strain increased by 29.3–34.5%, while the C content of the WO15–4 strain increased by 31.2–34.6% ( Figure 5A ). However, the tissue N and P contents were either negatively (P < 0.05) affected or unaffected (P > 0.05) by increasing light intensity ( Figures 5B, C ). There were significant differences between the two strains in the contents of C, N, and P at the two lower light intensities, with no differences under the two higher light intensities ( Figures 5A–C ).

We calculated the ecological stoichiometric ratios based on the elemental contents. The results showed significant differences in the stoichiometric ratios of P. haitanensis under different light intensities. Specifically, the C:N ratios were significantly higher under high-light conditions than under low-light conditions (P < 0.05, Figure 5D ). In addition, the tissue C:P ratio ( Figure 5E ) and tissue N:P ratio ( Figure 5F ) of both strains showed a trend of initially increasing and then decreasing with the increase in light intensity (P < 0.05). At the lowest light intensity (5 μmol m^–2 s^–1), the differences between the two strains in the C:N, C:P, and N:P ratios were not significant. Under 50 μmol m^–2 s^–1, only the C:N ratio showed a significant difference between the two strains (P < 0.01, Figure 5D ). Under the two higher light intensities (200 and 500 μmol m^–2 s^–1), the C:N ratio of strain W28–42 tended to be higher than that of strain WO15-4, while the C:P and N:P ratios showed opposite trends between the strains ( Figures 5D–F ).

At the lowest light intensity of 5 μmol m^–2 s^–1, the C:P and N:P ratios of both strains of P. haitanensis were much lower than the Redfield ratio (106:16:1), and growth under the other light intensities tended to be higher than the Redfield ratio ( Figures 5E, F ).

The C:N ratio of algal tissue increased linearly with increasing N content (R² = 0.8610, Figure 6A ), but not with increasing P content (R² = 0.1859, Figure 6B ). The C:P ratio showed a relatively weak negative correlation with N content (R² = 0.3459) ( Figure 6C ) and a relatively stronger correlation with P content (R² = 0.5005) ( Figure 6D ), indicating the complex regulatory role of light intensity on the elemental stoichiometric ratios of P. haitanensis and its ecological significance.

The results of the correlation analysis indicated that the overall trends for the two strains were similar ( Figures 7A, B ). There were significant positive correlations between light intensity and the four measures of growth rate, tissue C content, C:N ratio, and tissue C accumulation rate. However, light intensity was negatively correlated with the N and P contents of the algal tissue. The production of DOC, POC, and tissue C was positively correlated with the four measures of growth rate and tissue C content. There were significant differences between the two strains regarding the correlations among tissue P content. For strain WO15-4, tissue P content was negatively correlated with the four measures of growth rate and tissue C content ( Figure 7B ); however, strain W28–42 showed weak positive correlations ( Figure 7A ). The tissue P content of WO15–4 exhibited negative correlations with the C:N, C:P, and N:P ratios as well as the production of DOC, POC, and tissue C, but strain W28–42 displayed opposite or weak correlations. These results reveal a strain-specific difference in phosphorus metabolism under different light intensities.

It is generally believed that light, as a key environmental factor, plays a crucial role in regulating the photosynthetic physiology and growth rate of seaweed (Lichtenthaler and Wellburn, 1983). The impact of light on the growth rate of marine algae has been extensively studied in various species. Despite the large differences in research subjects and study periods, the overall trends and mechanisms have been fairly consistent. In this study, there were significant correlations between the growth rate of P. haitanensis and light intensity. As light intensity increased, the growth rate of P. haitanensis showed an initially increased and then decreased trend ( Figure 1 ), a pattern that aligns with findings from studies on other seaweed species, such as Sargassum fusiforme (Endo et al., 2023). This phenomenon indicates that light intensity is a key environmental factor affecting seaweed growth. Specifically, under a light intensity of 200 μmol m^–2 s^–1, the fresh weight growth rate of P. haitanensis significantly exceeded the values observed under the lowest and highest light intensities, regardless of strain ( Figures 1 , 2 ). These results suggested that there exists an optimal light intensity range for P. haitanensis.

Particularly, under the light intensity of 200 μmol m^–2 s^–1, although the relative growth rate (specific growth rate) of W28–42 was higher than that of WO15-4, the absolute growth rate (daily growth rate) showed the opposite trend. This could be attributed to the difference in morphology between the two strains; the initial fresh weight of the narrow strain W28–42 was much lower than that of the wide strain WO15-4. Interestingly, the daily growth rates in length and width showed strain-specific responses to changing light intensity. Under exposure to light intensities of 50–500 μmol m^–2 s^–1, the growth in length of W28–42 was greater than that of WO15-4, while the opposite trend was observed for width ( Figure 2 ). Such a phenomenon indicates an allometric relationship and has been intensively studied (Brown et al., 2004; West et al., 2001).

Macroalgae release a substantial amount of DOC into the environment during growth, meaning that they have significant carbon sequestration potential (Ortega et al., 2019; Raven, 2018). Meanwhile, the DOC released by autotrophs is an important allochthonous carbon source for heterotrophic bacteria, enhancing the detrital food chain within ecosystems (Ho et al., 2024). In this way, the extracellular release of DOC supports the functional health of coastal ecosystems by delivering photosynthetically fixed carbon to higher trophic levels through the microbial loop, thereby maintaining ecosystem function (Hall et al., 2024). Numerous studies have demonstrated a significant influence of environmental factors on the release of DOC from seaweeds (Paine et al., 2021); this poses challenges for assessing their potential for carbon sequestration. Among the relevant environmental factors, light can be considered the most important for seaweeds since it influences many important metabolic pathways and regulates growth, development, and photosynthetic processes. Given that almost all of the organic carbon released from seaweeds originates from photosynthetically fixed carbon, the release of DOC by seaweeds is considered highly regulated by light intensity (Haas et al., 2010).

Recent studies have reported that the DOC release by seaweed Sargassum thunbergii and marine macrophyte communities is highly light-dependent, in another words, rely on photosynthetic activity (Jiménez-Ramos et al., 2023; Zhao et al., 2023). However, in this study, we observed that the DOC release rate of P. haitanensis strain W28–42 was significantly greater under higher light intensities than under low light conditions, but light intensity did not have a significant effect on the DOC release of strain WO15-4 ( Figure 3 ). Moreover, a recent study found that different life stages (thallus vs conchocelis) of P. haitanensis showed varied responses in DOC release to changing light conditions (Xu et al., 2022). In addition, considering the complex interactions between environmental factors, the present and previous studies highlight the challenges in quantifying the effects of environmental factors on DOC release and the coastal carbon cycle mediated by diverse seaweed species and strains.

In the present study, we attempted to explain the mechanisms resulting in the strain-specific DOC release rates of P. haitanensis in response to changes in light intensity. First, differences in photosynthetic physiological characteristics among strains could be the primary factor contributing to the different DOC release rates under varying light intensity. This is because the carbon in DOC originates from the carbon fixed through photosynthesis. According to the “overflow hypothesis”, DOC is released from algae when the photosynthetic rate exceeds the rate necessary for growth (Paine et al., 2021). However, this hypothesis applies only to strain W28-42, as a linear relationship between growth rate and DOC release was observed in this strain ( Figure 4B ). Different strains of seaweed may have different photosynthetic efficiencies, pigment contents, and antioxidant systems, all of which influence the metabolism and reallocation of organic carbon within and outside the tissue. Second, significant differences in P metabolism between the two strains ( Figure 7 ) may also contribute to the variation in DOC release. Nutrient supply is a key factor in the release of DOC from seaweed (Paine et al., 2021), and this could influence the effect of light intensity (Mueller et al., 2016). In addition, a recent study found variation in the C, N, and P composition of seaweed tissue and the released organic matter, indicating that elemental metabolism may be directly related to the release of organic matter from seaweed (Chen et al., 2020).

The coupling between the biogeochemical cycling of elements and the service functions of ecosystems is one of the core issues in ecological research (Schröter et al., 2005). In 1934, Redfield discovered that the ratio of carbon (C), nitrogen (N), and phosphorus (P) in planktonic organisms in the ocean was roughly 106:16:1, closely mirroring the C:N:P ratio found in seawater (Redfield, 1934). This finding suggested that these three elements played a pivotal role in the biogeochemical cycles within oceanic ecosystems. By altering external environmental conditions, it is possible to influence the absorption rates of C, N, and P by marine organisms, thereby affecting the stoichiometric ratios of chemical elements. Both a recent study (Xu et al., 2022) and the present study found that an increase in light intensity significantly enhanced the C:N and C:P ratios of P. haitanensis thalli. This could be attributed to that the increase in light intensity enhancing the tissue C content but tending to decrease the tissue N and P contents ( Figure 5 ). Changing the light intensity also altered the C:N ratio of the microalga Coccomyxa subellipsoidea by influencing the cell growth rate, nitrogen consumption, and carbon allocation (Liu and Wei, 2023). Similarly, the C:N and C:P ratios of phytoplankton communities increased with light intensity (Dickman et al., 2006). These results suggest that there are unequal effects of light intensity on the metabolism of C versus other elements of P. haitanensis and other algae. Further studies are needed to explore the underlying mechanisms.

In addition, significant differences were observed in the C:N, N:P and C:P ratios between the two strains of P. haitanensis ( Figure 5 ). These strain-specific responses of elemental ratios to changing light intensity may be useful for the selection of new varieties. The altered elemental ratio can subsequently affects algal growth and carbon sequestration capacity, as well as play a pivotal role in ecosystem biogeochemical cycling (Hessen et al., 2013).