The Effects of Inorganic Carbon and Irradiance on the Photosynthetic Performance and Growth of the Macroalga Sargassum horneri

Abstract

Sargassum horneri is a highly productive macroalgal species capable of assimilating dissolved inorganic carbon (DIC) and converting CO₂ into carbohydrates, making it a promising solution for carbon capture and biomass enhancement. Owing to its wide distribution and natural abundance, the utilization of S. horneri may help mitigate rising oceanic CO₂ concentrations. This study evaluated the combined effects of inorganic carbon availability (2000, 4000, and 8000 μM NaHCO₃) and irradiance (100, 150, and 200 µmol photons m⁻² s⁻¹) on photosynthetic performance and growth across short-term (24 h) and long-term (4-week) experimental trials. Carbon enrichment and light intensity interacted to significantly influence growth and carbon assimilation. The highest growth rate (35.83 ± 3.95%) was observed under 8000 μM DIC (0.75 g L⁻¹) at 200 µmol photons m⁻² s⁻¹, corresponding to an optimal mean growth condition of 19 ± 0.04% (p < 0.05). These findings demonstrate that elevated inorganic carbon enhances photosynthetic efficiency by supplying sufficient substrate for carbon fixation, thereby supporting the feasibility of Sargassum horneri as a viable species for CO₂ absorption and carbon capture applications.

1. Introduction

Rising levels of anthropogenic carbon emissions have significantly altered global biogeochemical processes, with the world’s oceans absorbing a substantial proportion of excess atmospheric CO₂ [1]. This influx of dissolved carbon directly influences carbonate system dynamics, water chemistry, and the ecological interactions of marine organisms, including Sargassum horneri, a pelagic brown macroalga of ecological and functional importance [2]. S. horneri plays a vital role in coastal ecosystems by contributing to primary productivity, enhancing water quality, and influencing parameters such as alkalinity, dissolved oxygen, and temperature regulation [3].

Native to the northwestern Pacific, S. horneri displays a broad biogeographical range and frequently forms large drifting mats, commonly referred to as “golden tides.” These floating assemblages serve as nursery grounds and feeding habitats for diverse coastal species, while also impacting community structures and ecosystem function [4,5,6]. Numerous studies highlight S. horneri as a highly functional macroalga rich in sulfated polysaccharides with notable anti-inflammatory, antioxidant, antitumor, and antiallergenic properties, demonstrating proven benefits in aquaculture species such as Litopenaeus vannamei through enhanced growth and immunity [7]. Brown algae, including S. horneri, have also been identified as effective bioindicators of heavy metal pollution [1], and are widely recognized for their applications in feed, fertilizers, biofuel production, and commercially valuable bioactive compounds such as fucoidan, fucoxanthin, and alginates [8,9,10]. These traits indicate S. horneri to be a promising macroalgal species for sustainable cultivation and utilization in environmental mitigation and biotechnological development [1,8].

Sargassum horneri exhibits high productivity and rapid growth, supported by buoyant gas-filled vesicles that maintain thallus flotation and promote extensive dispersal. Individuals can reach up to 5 m in length within a year [5]. Growth and productivity are influenced by environmental factors such as light intensity and quality, temperature, and nutrient availability. Photosynthesis drives carbon assimilation and biomass accumulation, converting light energy into chemical energy stored as glucose derived from inorganic carbon uptake [11,12]. Brown algae possess characteristic pigment suites that include chlorophyll a, chlorophyll c, fucoxanthin, β-carotene, and xanthophylls which exhibit broad absorption spectra and enhance light harvesting efficiency [13,14].

Photosynthetic capacity in macroalgae is governed by the Photosynthetic Electron Transport Chain (PETC), with performance typically quantified through the electron transport rate (ETR). Electron flow through Photosystems II and I generate the proton gradient necessary for ATP synthesis, while NADPH production supports carbon fixation via the Calvin–Benson cycle [15,16]. In dark-adapted states, CO₂ assimilation is mediated within the chloroplast stroma by the enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), which catalyzes carbon fixation through the C₃ metabolic pathway [17]. Marine macroalgae, including S. horneri, have evolved Carbon-Concentrating Mechanisms (CCMs) that enhance intracellular CO₂ availability. These mechanisms include enzymatic conversion of bicarbonate (HCO₃⁻) to CO₂ and carbonic anhydrase activity that elevates CO₂ concentration near the Rubisco active site [15]. Some studies further report the involvement of C₄-associated enzymes—such as pyruvate or orthophosphate dikinase (PPDK), which may contribute to photoprotection and mitigate photoinhibition under high-light stress [18,19,20,21]. Together, these adaptations regulate photosynthetic efficiency and support the ecological success of S. horneri, while also presenting opportunities for industrial-scale biomass production.

To better understand the interactive effects of light intensity and inorganic carbon availability on S. horneri, the present study conducted both a short-term (24 h; Trial 1) and long-term (4 weeks; Trial 2) experiment. The objectives were as follows:

• To evaluate the influence of inorganic carbon on Photosystem II function and photosynthetic efficiency;
• To quantify growth performance and biomass accumulation in response to varying light and carbon conditions;
• To assess how DIC and total alkalinity influence CO₂ assimilation in Sargassum horneri.

2. Materials and Methods

2.1. Experimental Design

The study consisted of two sequential experimental phases: a short-term trial (24 h) to assess initial physiological responses and methodological feasibility, followed by a long-term trial conducted over four weeks. A 3 × 3 × 3 factorial design was implemented using three inorganic carbon concentrations (2000, 4000, and 8000 µmol/kg NaHCO₃) and three irradiance levels (100, 150, and 200 µmol photons m⁻² s⁻¹), a total of 9 treatments replicated three times (n = 27). This design allowed assessment of the interactive effects of dissolved inorganic carbon (DIC) availability and light intensity on photosynthetic performance, carbon assimilation, and growth. Data collection encompassed chlorophyll fluorescence measurements and analyses of DIC, pH, and total alkalinity (TA).

2.2. Experimental Sampling and Procedure

Naturally occurring Sargassum horneri thalli were collected from the northern coast of Taiwan in April 2024. Samples were transported to the National Taiwan Ocean University Aquatic Center Laboratory, cleaned of epiphytes and debris, and acclimated for seven days in a 1 m³ fiberglass tank maintained at 20 °C under continuous aeration and ambient light. Experimental treatments consisted of 30.0 ± 0.5 g fresh weight portions placed individually into nine 20 L culture vessels maintained at 20 °C with continuous aeration and artificial LED illumination. PES nutrient medium was added at a ratio of 30 mL per 1000 mL seawater. Irradiance was quantified using a UPRtek MK350S handheld spectrometer, and target light levels (100, 150, 200 μmol photons m⁻² s⁻¹) were applied with a ±10% allowable variance. Inorganic carbon treatments were prepared by mixing Na₂CO₃ and NaHCO₃ in a 1:2 mass ratio, at 0, 0.35, and 0.75 g L⁻¹, corresponding to nominal DIC levels of 2000, 4000, and 8000 µmol/kg, respectively. Each week, temperature, pH, salinity, and dissolved oxygen were measured using a WQC-30 portable water quality meter (Hach, Ireland). At each sampling point (24 h for the short-term trial and weekly for the long-term trial), 250 mL water samples were collected, stored at –4 °C, and used for DIC and TA analyses. Following each weekly sampling, culture media were replaced with fresh natural seawater supplemented with PES medium, following the procedures described in [20,22].

2.3. Chlorophyll Fluorescence

The photosynthetic efficiency was evaluated using a portable Diving-PAM pulse-amplitude-modulated fluorometer (Heinz Walz GmbH, Effeltrich, Germany, 2019) with WinControl 3.30 software. Samples were dark-adapted for 20 min before measurements.

Equation (1). Maximum Quantum Yield of PSII

$${\displaystyle \frac{{\mathit{F}}_{\mathit{v}}}{{\mathit{F}}_{\mathit{m}}}}={\displaystyle \frac{{\mathit{F}}_{\mathit{m}}-{\mathit{F}}_{\mathit{o}}}{{\mathit{F}}_{\mathit{m}}}}$$

(1)

where F₀ is the minimal fluorescence yield, Fₘ is the maximal fluorescence under a saturating pulse, and Fᵥ (variable fluorescence) represents the reduction state of PSII reaction centers. A saturating pulse of 809–814 µmol photons m⁻² s⁻¹ for 1 s was applied to obtain maximum fluorescence values following [23].

Equation (2). Relative Electron Transport Rate

$$\mathbf{r}\mathbf{E}\mathbf{T}\mathbf{R}=\mathit{P}\mathit{A}\mathit{R}\times \mathit{Y}\left(\mathit{I}\mathit{I}\right)\times 0.84\times 0.5$$

(2)

where PAR is photosynthetically active radiation, Y(II) is the effective quantum yield of PSII, 0.84 represents the assumed light absorptance, and 0.5 denotes equal energy distribution between PSI and PSII.

Light-response (rapid light) curves were generated to determine rETR, ETR_max, and related photosynthetic parameters following methods described in [21,24].

2.4. Inorganic Carbon Analysis

DIC and TA were quantified at the conclusion of each experimental trial, as described in [25,26,27,28]. Water samples were collected in 250 mL polyethylene bottles, preserved with 200 µL HgCl₂, and stored at 4 °C until analysis.

Equation (3). Dissolved Inorganic Carbon

$$\mathbf{D}\mathbf{I}\mathbf{C}=\left[{\mathbf{C}\mathbf{O}}_2\left(\mathit{a}\mathit{q}\right)\right]+\left[{\mathbf{H}}_2\mathbf{C}{\mathbf{O}}_3\right]+\left[\mathbf{H}\mathbf{C}{\mathbf{O}}_3^{-}\right]+\left[\mathbf{C}{{\mathbf{O}}_3^2}^{-}\right]$$

(3)

For total organic carbon (TOC) analysis, 150 mL amber glass bottles were filled with culture water and acidified with three drops of H₂SO₄ to enhance conductivity. TA was determined via titration following standard carbonate chemistry protocols [24,29], enabling conversion among bicarbonate, carbonate, and hydroxide species. All analyses were conducted at a controlled temperature of 20 °C and pH 8.

Algal biomass from each treatment (n = 27) was dried to a constant weight at 60 °C and homogenized for elemental analysis using a CHN analyzer (Shimadzu Corp., Kyoto, Japan) to determine tissue carbon content pursual to [30,31].

Equation (4). Carbon Concentration

$$\mathbf{C}={\displaystyle \frac{\mathit{g}}{\mathit{L}}}\left(\mathbf{g}{\mathbf{L}}^{-1}\right)$$

(4)

2.5. Biomass and Growth Determination

Growth was assessed by measuring initial (W₁) and final (W₂) wet weights of algal samples following [31,32].

Equation (5). Relative Growth Rate (RGR)

$$\mathbf{R}\mathbf{G}\mathbf{R}={\displaystyle \frac{\mathbf{ln}\left({\mathit{W}}_2\right)-\mathbf{ln}\left({\mathit{W}}_1\right)}{\mathsf{\Delta }\mathit{t}}}\left(\mathbf{d}\mathbf{a}{\mathbf{y}}^{-1}\right)$$

(5)

Equation (6). Growth Percentage (%)

$$\left(\mathbf{\%}\right)\mathbf{G}\mathbf{r}\mathbf{o}\mathbf{w}\mathbf{t}\mathbf{h}=\left({\displaystyle \frac{{\mathit{W}}_2-{\mathit{W}}_1}{{\mathit{W}}_2}}\right)\times 100\mathbf{\%}$$

(6)

Growth rates were derived for each replicate under all light and carbon treatments and averaged for each treatment level.

2.6. Statistical Analysis

Statistical analyses were conducted using SPSS v26.0 (IBM SPSS Statistics, Chicago, IL, USA). A two-way ANOVA was performed to assess the main and interactive effects of irradiance and inorganic carbon concentration on measured parameters. Tukey’s post hoc test was used to identify significant pairwise differences at p ≤ 0.05. Normality and homogeneity of variance were evaluated using Shapiro–Wilk and Levene’s tests, respectively. All data are reported as mean ± standard deviation (SD), and statistically significant differences are denoted by lowercase superscript letters.

3. Results and Discussion

Given an increased interest in marine vegetation acting as carbon sinks for anthropogenic CO₂, this experiment found Sargassum horneri to be highly useful for the uptake of dissolved inorganic carbon for utilization and conversion into functional organic compounds for enhanced biomass and growth [33,34].

3.1. The Photosynthetic Performance of S. horneri

The interactions of light intensity and inorganic carbon influenced growth with an optimal yield condition (

Table 1. A one-way analysis of variance, ANOVA, of the factors influencing growth over a long-term period (T2) with the interactions of light (irradiance) and inorganic carbon.

Treatments	Total Organic Carbon	pH	O2 (mg L−1)	Salinity	Weight (g)	ETRm (μmol photons m−2 s−1)
L1_C1	0.618 ± 0.004 c	9.12 ± 0.14 b	7.17 ± 3.12 a	3.10 ± 0.17 ab	4.63 ± 0.35 abc	19.5 ± 0.46 b
L1_C2	0.374 ± 0.006 b	9.18 ± 0.03 b	7.13 ± 1.33 a	3.03 ± 0.15 ab	4.46 ± 0.06 ab	12.9 ± 1.69 b
L1_C3	0.371 ± 0.002 ab	8.95 ± 0.05 a	8.07 ± 0.86 a	3.30 ± 0.17 abc	4.49 ± 0.06 ab	19.0 ± 5.21 a
L2_C1	0.370 ± 0.005 ab	9.14 ± 0.12 b	7.20 ± 0.26 a	3.00 ± 0.00 a	4.88 ± 0.47 abc	17.5 ± 2.21 b
L2_C2	0.368 ± 0.002 ab	9.21 ± 0.04 b	7.83 ± 0.21 a	3.07 ± 0.12 ab	4.60 ± 0.25 bc	15.7 ± 0.43 b
L2_C3	0.401 ± 0.006 b	8.89 ± 0.09 a	8.87 ± 1.16 a	3.27 ± 0.15 abc	5.14 ± 0.09 c	20.1 ± 1.92 a
L3_C1	0.378 ± 0.030 ab	9.19 ± 0.02 b	6.93 ± 0.51 a	3.13 ± 0.15 ab	4.71 ± 0.49 abc	18.3 ± 1.67 b
L3_C2	0.394 ± 0.003 b	9.20 ± 0.08 b	7.77 ± 0.67 a	3.47 ± 0.06 c	4.24 ± 0.37 c	14.2 ± 5.98 b
L3_C3	0.343 ± 0.065 a	8.93 ± 0.06 a	9.43 ± 4.30 a	3.17 ± 0.08 ab	5.16 ± 0.05 c	18.6 ± 4.44 a

Notes: L1, L2, L3: Light intensity levels (100, 150, and 200 μmol photons m⁻² s⁻¹). C1, C2, C3: Inorganic carbon concentrations (2000, 4000, and 8000 μmol/kg) ± mean ± standard deviation, SD (variation among replicates). Superscript letters (a, b, c) indicate significant difference.

), as shown by L2_C3 or (150 μmol photons m⁻² s⁻¹) and high carbon (8000 μmol/kg); a similar trend with dissolved oxygen O₂ levels (Figure 2b) ranging from approximately 6.9–9.4 mgL⁻¹ depicting a higher photosynthetic O₂ evolution was also shown under both higher light intensity and moderate carbon concentration, indicating a greater efficiency of Carbon Capture Utilization and Storage (CCSU) subject to varying environmental conditions leading to adaptation strategies [15,35]. Although salinity levels remained consistent among all treatments, photoinhibition was shown at the highest light level of 200 μmol photons m⁻² s⁻¹; likewise, the treatment conditions with higher amounts of inorganic carbon indicate that S. horneri benefits from carbon enrichment coupled with moderate irradiance; comparably, floating algae require a greater exposure to high levels of light and temperature with lower levels of dissolved CO₂, which may be associated with the activation of the C4 pathway [36,37].

Significant variability was observed among treatments in total organic carbon (TOC), pH, dissolved oxygen (O₂), salinity, biomass (Wt.), and maximum electron transport rate (ETRmax). TOC values ranged from 0.34 ± 0.07 to 0.62 ± 0.004, with the highest concentration recorded under treatment L1_C1, which differed significantly from the mid- and high-light carbon treatments (p < 0.05). pH remained generally elevated across all treatments (8.89–9.21), while O₂ levels increased under higher carbon and irradiance conditions, reaching up to 9.43 ± 4.30 mgL⁻¹ in L3_C3, indicating enhanced photosynthetic O₂ evolution [37]. Biomass accumulation showed treatment-specific differences, with the greatest final wet weight observed in L2_C3 (5.14 ± 0.086 g), which was significantly higher (p < 0.05) than several low-light treatments. Maximum electron transport rates (ETRmax) ranged from (12.95 ± 1.69 to 20.08 ± 1.92), with peak values occurring under L2_C3, demonstrating that moderate irradiance paired with high levels of inorganic carbon promoted the highest photosynthetic performance (Figure 1). Generally, this trial indicates that intermediate light levels (L2) combined with elevated inorganic carbon levels (C3) produced the most favorable physiological and photosynthetic response in Sargassum horneri.

Growth in terms of biomass accumulation was statistically significant with the highest value determined at (L2_C3) or 150 m⁻² s⁻¹ 8000 μmol/kg; comparably, the lowest was 5.14 ± 0.09 (L3_C2) or 200 m⁻² s⁻¹ 4000 μmol/kg: 4.24 ± 0.37, indicating an ideal growth condition of moderate light couple with higher carbon input. The rETR increased asymptotically with rising PAR in all treatments, reflecting enhanced photochemical activity as photon availability increased. Algae grown under high-irradiance conditions (200 µmol photons m⁻² s⁻¹) consistently demonstrated the highest rETR values across the PAR range, indicating greater photosynthetic capacity and improved utilization of light energy.

Carbon enrichment further modulated photochemical efficiency, with 4000 and 8000 µmol/kg treatments supporting higher rETR at elevated PAR compared to 2000 µmol/kg. These trends reflect strong light-dependent enhancement of photosynthetic electron transport, with inorganic carbon playing a secondary but stimulatory role under high photon flux densities [37,38].

On completion of the short-term trial (24 h), the average carbon ratio of dried Sargassum horneri tissues remained relatively uniform across treatments (approximately 0.65–0.70; Figure 2a), with no statistically significant differences among the various light or inorganic carbon levels [39,40]. In contrast, dissolved oxygen (DO) levels exhibited greater variability among treatments (Figure 2b), ranging from approximately (6 to 10 mgL⁻¹). The highest mean DO concentration (9.43 ± 4.30 mgL⁻¹) occurred under 150 µmol photons m⁻² s⁻¹ and 8000 μmol/kg DIC, whereas the lowest value (6.93 ± 0.51 mgL⁻¹) was observed under 200 µmol photons m⁻² s⁻¹ with 8000 μmol/kg DIC. The broad error range associated with the highest DO values suggests substantial metabolic fluctuation, potentially attributable to transient changes in photosynthetic activity. Conversely, the reduced DO production at the highest irradiance level may reflect photoinhibition or light-induced stress, which can limit oxygen evolution despite ample inorganic carbon availability [39,41].

Growth rate (Figure 3a) increased noticeably under higher irradiance and elevated inorganic carbon concentrations. The combination of 200 µmol photons m⁻² s⁻¹ and 8000 μmol/kg DIC (L3_C3) yielded the highest growth rate (~30%) (

Table 2. Relative growth rate (RGR), percentage growth, and organic carbon ratio across light × carbon treatments in Sargassum horneri over a long-term period (T2) with interactions of light (irradiance) and inorganic carbon.

Treatment	RGR (day−1)	Growth (%)	Organic Carbon Ratio (Algal Wt.)
L1_C1	0.004	22.30	0.62
L1_C2	0.004	22.26	0.37
L1_C3	0.005	22.80	0.37
L2_C1	0.009	25.47	0.37
L2_C2	0.006	23.59	0.37
L2_C3	0.006	23.83	0.40
L3_C1	0.009	25.47	0.38
L3_C2	0.012	27.78	0.39
L3_C3	0.009	25.83	0.34

Notes: L1, L2, L3: Light intensity levels (100, 150, and 200 μmol photons m⁻² s⁻¹). C1, C2, C3: Inorganic carbon concentrations (2000, 4000, and 8000 μM).

), representing a statistically significant enhancement relative to other treatments. This pattern indicates that increased carbon availability, when paired with high light, facilitates greater rates of carbon fixation and assimilation, thereby promoting more rapid biomass production [37].

Figure 3b further supports this trend, as higher irradiance and inorganic carbon levels resulted in increased organic carbon accumulation within algal tissues. This enhancement reflects elevated photosynthetic carbon assimilation and storage under carbon-enriched, high-light conditions. Conversely, lower organic carbon ratios under weak irradiance indicate reduced photosynthetic activity and slower buildup of organic reserves [42]. Together, these results demonstrate that stronger irradiance combined with high inorganic carbon availability maximizes both biomass accumulation and organic carbon storage, reinforcing the species’ capacity for effective carbon assimilation under enriched conditions.

Sargassum horneri demonstrated substantial photo physiological plasticity across the tested irradiance and inorganic carbon regimes. The stability of maximum quantum yield (Fv/Fm; Figure 4a and Figure 5a) across treatments indicates that photosystem II (PSII) remained functionally robust, with no evidence of photoinhibition or stress-related decline. Enhanced ETRmax values under moderate to high irradiance (150–200 µmol photons m⁻² s⁻¹) combined with elevated inorganic carbon availability (4000–8000 µmol kg⁻¹) reflect greater electron transport capacity and improved photochemical energy conversion efficiency. This irradiance–carbon combination likely represents an optimal physiological range supporting high photosynthetic performance and biomass accumulation in S. horneri [21].

ETRmax values (Figure 4b) increased from approximately 10 to 25 µmol photons m⁻² s⁻¹, with the highest rates consistently observed under moderate–high light and mid-to-high carbon availability. In contrast, the lowest ETRmax values (Figure 5b), denoted by the superscript (‘c’), occurred under low-light conditions (100 µmol photons m⁻² s⁻¹) despite moderate carbon supply (4000 µmol kg⁻¹), indicating photon limitation. Collectively, these patterns confirm that ETRmax is a reliable indicator of the photosynthetic activity of PSII and that S. horneri relies exclusively on irradiance, with carbon enrichment providing secondary enhancement under sufficient light [43]. This outcome suggests that Sargassum horneri exhibits enhanced electron transport activity and therefore higher photosynthetic efficiency when both irradiance and inorganic carbon are available at moderate to high levels [37,44]. However, beyond this range, neither extremely high carbon concentrations nor further increases in light intensity produced additional gains in ETRmax, indicating the presence of a physiological saturation threshold in the photosynthetic apparatus of S. horneri [45]. Comparable trends have been reported in other macroalgae; for example, dissolved CO₂ enrichment significantly enhanced pigment content and biomass accumulation in Colaconema formosanum when supplemented with 1 gL⁻¹ inorganic carbon [20,22]. Such responses highlight the importance of environmental conditions in shaping algal photo-acclimation strategies [17,35].

Floating macroalgae, like S. horneri, often experience elevated light and temperature alongside relatively low dissolved CO₂, conditions that may induce alternative carbon assimilation pathways such as enhanced C₄ like metabolism to maintain efficient uptake and fixation [19,45]. Light is a dominant driver of biomass production in S.horneri with increases in RGR from the lowest to the highes light levels L1–L3 (Table 2) irrespective of carbon levels. Although some studies have argued that atmospheric CO₂ influx is largely independent of photosynthetic carbon fixation, other analyses suggest that increases in external CO₂ can stimulate carboxylation in C₃ species until a saturation point is reached, beyond which photosynthetic efficiency declines due to biochemical limitations; indicating an altered carbon storage rather than rapid growth, by a higher carbon ratio at L1_C1 (Table 2) [28,29,46,47]. Further investigation into S. horneri’s photosynthetic capacities including responses to variable light spectra, fluctuating irradiance, and differing carbonate chemistry would provide valuable insights into its acclimation mechanisms [36,37]. Such research would not only advance understanding of its physiological ecology but also inform assessments of its stress tolerance, cultural suitability, and broader adaptive potential within dynamic marine environments [27,48].

3.2. The Effects of Inorganic Carbon on S. horneri Culture Medium

Variations in irradiance (100, 150, and 200 µmol photons m⁻² s⁻¹) and dissolved inorganic carbon (DIC; 2000, 4000, and 8000 µM) produced measurable differences in total alkalinity (TA), pH, dissolved oxygen (O₂), and salinity in the culture medium (Table 1). Notable fluctuations in pH and TA (Figure 6a,b) suggest dynamic carbonate system responses that may have influenced DIC availability, particularly through enhanced utilization of bicarbonate (HCO₃⁻) during dark-adapted and light-driven CO₂ fixation (Figure 3a) [45]. These changes corresponded with increases in biomass, indicating a positive relationship between inorganic carbon enrichment and S. horneri growth.

DIC enrichment at 8000 µmol/kg produced the most consistent treatment effects across all irradiance levels, demonstrating a strong light × carbon interaction during the T1 trial. This trend is reflected in (Figure 6a,b), which show a clear relationship between inorganic carbon concentration, irradiance, pH, and seawater carbonate chemistry [49,50]. DIC increased predictably with carbon additions, with the lowest concentrations observed at 2000 µmol/kg (‘c’), intermediate levels at 4000 µmol/kg (‘b’), and highest at 8000 µmol/kg (‘a’), a pattern that remained consistent across all light treatments. Despite these differences, pH values remained relatively stable (8.89–9.21), indicating only minor fluctuations in response to carbon enrichment.

The stability of pH suggests strong natural buffering capacity of seawater and active CO₂ uptake by S. horneri during photosynthesis [25,26,27,49]. As inorganic carbon availability increased, pH remained near neutral-alkaline (~8.0–9.0), consistent with efficient removal of CO₂ and HCO₃⁻ from the medium during carbon fixation and associated oxygen evolution [30,51]. TA values further demonstrated that irradiance exerted only minimal influence on carbonate speciation, with bicarbonate (HCO₃⁻) remaining the dominant form of inorganic carbon available to S. horneri [28]. A similar pattern was observed in the long-term experiment (T2; Figure 7), where DIC increased with carbon additions and pH again remained stable between 8.5 and 9.0. At the highest irradiance level (200 µmol photons m⁻² s⁻¹), elevated carbon availability promoted both greater photosynthetic uptake and consistent chemical conditions, suggesting balanced carbonate dynamics. These results indicate that during photosynthesis, S. horneri actively consumes HCO₃⁻, thereby reducing its proportion in the medium and shifting the carbonate system toward increased CO₃²⁻ formation [31,52]. Light intensity amplified this effect slightly, reflecting enhanced photosynthetic carbon assimilation under stronger irradiance [44,53]. Overall, the combined patterns of DIC, TA, and pH demonstrate that Sargassum horneri relies heavily on bicarbonate as a carbon substrate, efficiently regulates carbonate equilibrium during growth, and plays a functional role in seawater carbonate cycling and alkalinity maintenance—a key mechanisms supporting its potential in marine carbon capture and biogeochemical regulation [31,54].

Across all nine treatment combinations (Figure 8), bicarbonate (HCO₃⁻) constituted the dominant fraction of the dissolved inorganic carbon (DIC) pool, representing approximately 65–70% of total inorganic carbon. Carbonate ions (CO₃²⁻) accounted for 25–30%, while dissolved CO₂ contributed less than 10%. These proportions remained relatively stable across varying irradiance and inorganic carbon concentrations, reflecting the intrinsic carbonate equilibrium of seawater in which bicarbonate is the predominant carbon species at pH values near 8 [28]. This distribution aligns with typical marine conditions, where most of the carbon exists as HCO₃⁻ rather than free CO₂ due to seawater buffering and equilibrium constraints and partitioning among CO₂, HCO₃⁻, and CO₃²⁻ [25,26,27].

Previous research has shown that bicarbonate acquisition varies considerably among macroalgal species and is often linked to physiological adaptation [38]. For example, in Colaconema formosanum, shifts in salinity and inorganic carbon enrichment significantly affected pigment synthesis and growth [20,22]. In the present study, total alkalinity (TA), stable pH values, and controlled temperature collectively determined the availability of DIC in the culture medium, which in turn influenced both carbon uptake and photosynthetic performance [32,55]. These outcomes suggest that, within the tested range, the relative proportions of carbon species are governed primarily by carbonate chemistry rather than irradiance levels. Although irradiance did not markedly alter the chemical speciation of DIC, it remains a key driver of macroalgal physiology [37,38,44]. Light availability directly influences growth, reproductive capacity, and metabolic performance in Sargassum horneri, consistent with findings reported for the related brown alga Hizikia fusiformis, which exhibits enhanced growth under optimal photorespiratory and photo-inhibitory conditions [33,34,48,56]. Similar studies on C. formosanum demonstrated that supplementation with inorganic carbon growth increased by approximately 18%, driven by enhanced pigment production and carbon assimilation [20,22,57].

Under controlled laboratory conditions these findings determined that S. horneri growth and photosynthetic efficiency are strongly influenced by the absolute availability of inorganic carbon but only minimally affected by irradiance-induced shifts in carbonate speciation [2,28,58]. Instead, irradiance primarily regulates metabolic activity—such as rETR, ETRmax, and biomass accumulation [37], while the chemical balance among carbon species remains governed by seawater carbonate equilibrium [25,28].

Finally, field-based validation remains necessary to confirm whether these laboratory-derived physiological responses apply to natural S. horneri populations or large-scale cultivation settings, where hydrodynamics, turbulence, diurnal cycles, and nutrient availability may further modify carbonate chemistry and carbon-use strategies [49,59].

4. Conclusions

Our experimental study revealed that Sargassum horneri is an efficient and resilient macroalgal species capable of sustaining photosynthetic efficiency, regulating carbonate use, and generating biomass across a range of light and carbon conditions with a maximum growth rate of 35–36% with the inclusion of 0.75 g/L of dissolved inorganic carbon. Light intensity paired with elevated inorganic carbon maximized growth and electron transport efficiency, while moderate conditions maintained a higher performance without signs of stress at the highest RGR of 0.012 day^–1 alongside a 27.8 % increase in growth at optimum conditions. These traits of strong carbon assimilation, stable photo physiology, oxygen production, and the ability to alter the surrounding carbonate chemistry support the ecological role of S. horneri as both a fast-growing primary producer and a potential contributor to coastal carbon sequestration and bioremediation in carbon-enriched, high-light environments.