The field water collected from a sampling station (SNUTY) located in Buksin Bay off Tongyeong was incubated under hypoxic environment of 1.5 mg O₂ L⁻¹ (Eom et al. 2025). Using two consecutive single-cell isolations, four clonal cultures were established from the species that were dominant under hypoxic conditions: two dinoflagellate species Prorocentrum triestinum PTTY2405 and Amphidinium carterae ACTY2405, and two diatom species Cylindrotheca closterium CyClTY2405 and Chaetoceros curvisetus ChCuTY2405 (Table 1). All isolated cultures were incubated at 22°C with 30 μmol photons m⁻² s⁻¹ using cool white fluorescent light under a 14 h : 10 h light : dark cycle. The cells of the dinoflagellate species P. triestinum PTTY2405 and A. carterae ACTY2405 were maintained in an f/2 medium without silicate, whereas the diatom species Cy. closterium CyClTY2405 and Ch. curvisetus ChCuTY2405 were cultured in an f/2 medium with silicate (Guillard and Ryther 1962).

Experiment 1 measured the growth rates of two dinoflagellate species P. triestinum PTTY2405 and A. carterae ACTY2405, and two diatom species Cy. closterium CyClTY2405 and Ch. curvisetus ChCuTY2405 under five target DO concentrations (0.5, 1.0, 1.5, 4.0, and >7.0 mg O₂ L⁻¹), respectively (Fig. 1A). The experiment was conducted at 22°C under an irradiance of 30 μmol photons m⁻² s⁻¹ with a 14 h : 10 h light : dark (LD) cycle. This irradiance was selected to represent the light condition under which the four experimental species were isolated as dominant species during hypoxic incubation (Eom et al. 2025), as well as the low-light environment reported in hypoxic coastal bottom waters (Ok et al. 2023). Moreover, this photoperiod approximates the natural summer LD cycle in Tongyeong, the sampling site where hypoxia frequently occurs during summer (National Institute of Fisheries Science 2015–2025, Sunrise-Sunset 2026).

The cultures assigned to >7.0 mg O₂ L⁻¹ were placed on the shelves outside an oxygen-controlled glovebox in a temperature-controlled chamber (Fig. 1A). In addition, three control bottles containing filtered seawater—using the same bottle type, volume, temperature, and salinity—were established to determine whether the water under ambient oxygen conditions successfully maintained DO concentrations above 7.0 mg O₂ L⁻¹. In contrast, the cultures targeted for lower DO concentrations were incubated inside the oxygen-controlled glovebox (Fig. 1A). The low DO concentrations were established using the method described in Eom et al. (2025). A pre-mixed gas cylinder containing 400 parts per million of carbon dioxide (CO₂) balanced with N₂ was connected to the glovebox equipped with a Coy Oxygen Controller (Coy Laboratory Products, Grass Lake, MI, USA), which regulated gas release to maintain the target DO and stable pH throughout the experiment (Eom et al. 2025). In addition, a magnetic stir bar was placed in each bottle to promote homogeneous mixing, thereby reducing diffusion boundary layers around cells (Gao 2021). Hence, stirring minimized the localized changes in oxygen concentration caused by photosynthesis and respiration during the LD cycle, respectively, and ensured a uniform distribution of physical and chemical parameters within the culture. After thorough mixing, the DO concentration and water temperature were periodically monitored and recorded using a calibrated OBOD sensor (Multilab IDS ProOBOD; YSI Inc., Yellow Springs, OH, USA) connected to a Multilab 4010-3W (YSI Inc.). By monitoring DO concentration, the oxygen concentration in the glovebox was adjusted accordingly to maintain the target DO concentration.

Before the main incubation, cultures were pre-incubated for acclimation to the target DO concentrations. Each culture was first maintained for 5 d at the next higher DO concentration (i.e., 4.0 mg O₂ L⁻¹ for the 1.5 mg O₂ L⁻¹ treatment) and then for 2 d at the target DO concentration before starting the 5-d experiment (Supplementary Fig. S1A & B). The cultures during the pre-incubation period were maintained in triplicate using wide-mouth 1,000-mL polycarbonate (PC) bottles, and the one with the highest cell density on day 7 of pre-incubation was selected as the stock culture for the main incubation. All PC bottles used during acclimation and the 5-d incubation period of the growth rate experiment were left opened to allow gas exchange between the culture and the ambient atmosphere or controlled atmosphere within the glovebox. The main incubation was performed in triplicate using wide-mouth 500-mL PC bottles with a working volume of 300 mL to provide shallow water depth and wide openings to facilitate atmosphere mixing to maintain the target DO. Moreover, the initial carbon biomass was targeted at ≤250 ng C mL⁻¹, corresponding to 837–1,256 cells mL⁻¹ for P. triestinum PTTY2405, 1,155–1,569 cells mL⁻¹ for A. carterae ACTY2405, 936–2,788 cells mL⁻¹ for Cy. closterium CyClTY2405, and 1,055–1,758 cells mL⁻¹ for Ch. curvisetus ChCuTY2405 across all DO treatments (Supplementary Table S1). For target DO concentrations of 0.5, 1.0, 1.5, and 4.0 mg O₂ L⁻¹, DO in all four experimental species was continuously maintained within the ranges of 0.44–0.54, 0.97–1.04, 1.48–1.54, and 3.99–4.08 mg O₂ L⁻¹, respectively. For DO concentration of >7.0 mg O₂ L⁻¹, it ranged from 7.02 to 11.03 mg O₂ L⁻¹ (Supplementary Fig. S2A–D).

The stock cultures and media were acclimated to the target DO to minimize DO fluctuations at the start of the experiment. Subsequently, equal volumes of the stock cultures were inoculated into the prepared media. During the experiment, 5-mL aliquots were collected daily from each experimental bottle. Samples were then fixed with Lugol’s solution (final concentration: 5%). The cells were examined in triplicate 1-mL Sedgewick-Rafter counting chambers, and all or ≥200 cells of each target species were enumerated.

Experiment 2 assessed the NPR and DRR of the same four phytoplankton species under the same five target DO concentrations (Fig. 1B). The acclimation process followed the same pre-incubation protocol as Experiment 1, totaling 7 d (Supplementary Fig. S1A). To set up Experiment 2, the same stock cultures from Experiment 1 were used. For the main incubation, separate 12-h treatments were conducted at 22°C: one setup under continuous light at 30 μmol photons m⁻² s⁻¹ for NPR assessment, and another setup in continuous dark at 0 μmol photons m⁻² s⁻¹ for DRR (Supplementary Fig. S1C).

Experiment 2 targeted the initial carbon biomass of ≤500 ng C mL⁻¹ (depending on the density of the pre-incubated stock culture), at which the target DO concentration could be maintained without excessive bubbling, thereby minimizing bubbling-induced stress (Supplementary Table S1). For each species at each DO concentration, triplicate biological oxygen demand (BOD) bottles containing cells were established to measure NPR and DRR, respectively, alongside triplicate cell-free filtrate control bottles for each condition. In total, 12 BOD bottles were prepared for each species at each DO concentration (Fig. 1B). All BOD bottles used during the 12-h incubation period of the NPR and DRR experiments were tightly closed with borosilicate glass robotic stoppers and securely covered with polypropylene snap caps. The experiment setup assigned to >7.0 mg O₂ L⁻¹ were assembled outside the oxygen-controlled glovebox in a temperature-controlled chamber, whereas the lower DO experiments were assembled inside the oxygen-controlled glovebox to maintain the target DO concentration. The DO concentration and water temperature were monitored in both experimental and control BOD bottles at hours 0 and 12 under both light and dark conditions (30 and 0 μmol photons m⁻² s⁻¹, respectively) using the same OBOD sensor from the pre-incubation, connected to a Multilab 4010-3W (YSI Inc.). To minimize the effects of background DO fluctuations, the DO concentration change in the control BOD bottles from hour 0 to 12 was subtracted from that in the experimental BOD bottles. In this study, the hourly rates of net photosynthesis and dark respiration were assumed to be linear over the 12-h incubation period. After measuring the DO, 5-mL aliquots were collected at both the initial and final incubation hours, fixed with Lugol’s solution, and cells were enumerated as described in Experiment 1.

To measure the swimming speeds of the two dinoflagellate species, P. triestinum PTTY2405 and A. carterae ACTY2405, under DO concentrations of 0.5, 1.5, and >7.0 mg O₂ L⁻¹, the acclimation process was conducted according to the pre-incubation protocols used in Experiments 1 and 2. After acclimation, cultures were carefully transferred to 50-mL culture flasks without headspace and sealed with Parafilm to limit gas exchange. Each flask containing a given species at a target DO concentration was then placed under a dissecting microscope equipped with a video analyzing system (SRD-1673DN; Samsung, Seoul, Korea) and a CCD camera (Sony 3CCD ExwareHAD; Sony, Tokyo, Japan). The video camera was focused on a single circular field of view within the culture flask. After the culture flasks were placed on the microscope stage and allowed to stand for 30 min to minimize water movement, swimming cells of P. triestinum PTTY2405 and A. carterae ACTY2405 were recorded at magnifications of 20× and 25×, respectively. To determine the average and maximum swimming speeds, 40 cells of each species at each target DO concentration were tracked after the first 15 min of recording. Single-frame playback at 1/8× speed for P. triestinum and 1/2× speed for A. carterae was used to trace individual cell trajectories with a rolling digital tape measure. After the linear displacement of each cell was measured, swimming speed was calculated using elapsed time corrected for playback speed and microscope magnification.

The specific growth rate (μ, d⁻¹) for each species was calculated as:

, where C₀ is the cell density on initial day (cells mL⁻¹); C_t is the cell density on final day (cells mL⁻¹); and t is the incubation time (d).

The NPR (pmol O₂ cell⁻¹ h⁻¹) or DRR (pmol O₂ cell⁻¹ h⁻¹) was calculated as:

, where DO_{cell 12h or 0h} is the DO concentration of experimental bottles (with cells) at hour 12 or 0 (mg O₂ L⁻¹); DO_{fil 12h or 0h} is the DO concentration of control bottles (without cells, filtrate only) at hour 12 or 0 (mg O₂ L⁻¹); CD_{avg 0,12h} is the average cell density of experimental bottles (with cells) between hour 0 and 12 (cells mL⁻¹); 10⁶/32 is the conversion factor to express the rate as pmol O₂ cell⁻¹ h⁻¹ when DO is in mg O₂ L⁻¹ and cell density is in cells mL⁻¹.

To determine the differences in growth rate, NPR, and DRR across five different DO concentrations, the assumptions of normality and homogeneity of variances were examined using the Shapiro-Wilk and Levene’s median tests, respectively. If the data met normality and homoscedasticity, a one-way analysis of variance (ANOVA) followed by a post-hoc Tukey’s honestly significant difference test was performed (Tukey 1949). If the data were not normally distributed, a non-parametric Kruskal-Wallis test was performed with a post-hoc Mann-Whitney U test with Bonferroni correction (Mann and Whitney 1947, Kruskal and Wallis 1952). All analyses were conducted using SPSS ver. 29.0 (IBM-SPSS Inc., Armonk, NY, USA). Moreover, linear regressions were conducted to examine the relationships between growth rate, NPR, and DRR and DO concentration, as well as the relationships among the rates. All the regression analyses were performed using Origin version 2024b (OriginLab Corporation, Northampton, MA, USA).

The measured DO concentrations in P. triestinum PTTY2405 bottles at each target DO concentration (0.5, 1.0, 1.5, and 4.0 mg O₂ L⁻¹) ranged from 0.44–0.53, 0.98–1.04, 1.50–1.54, and 4.00–4.05 mg O₂ L⁻¹, respectively, indicating that the target DO concentrations were well maintained (Supplementary Fig. S2A). Furthermore, the measured DO concentration at the target DO concentration of >7.0 mg O₂ L⁻¹ ranged from 7.02–7.95, as the bottles were allowed to mix with ambient air, for which the measured DO concentration was 7.60–7.92 mg O₂ L⁻¹ in the control bottles containing filtered seawater in the same setting (Supplementary Fig. S2A). During the 5-d incubation period, the average cell density at 0.5 mg O₂ L⁻¹ decreased, while that at 1.0 to >7.0 mg O₂ L⁻¹ continuously increased with increasing DO concentration (Supplementary Fig. S2E). The growth rate of P. triestinum PTTY2405 at 0.5 mg O₂ L⁻¹ was −0.34 d⁻¹, but it continuously increased to 0.65 d⁻¹ at >7.0 mg O₂ L⁻¹ (Table 2, Fig. 2A). The growth rates were significantly affected by the DO concentrations (Fig. 2A, Supplementary Table S2).

The measured DO in A. carterae ACTY2405 bottles at 0.5 to 4.0 mg O₂ L⁻¹ were well maintained, ranging from 0.50–0.53, 1.00–1.04, 1.48–1.52, and 3.99–4.02 mg O₂ L⁻¹, respectively (Supplementary Fig. S2B). Furthermore, the measured DO at >7.0 mg O₂ L⁻¹ ranged from 7.06–8.91 mg O₂ L⁻¹ due to mixing with ambient air, for which the measured DO concentration was 7.60–7.92 mg O₂ L⁻¹ in the control bottles (Supplementary Fig. S2B). During the 5-d incubation period, the average cell density continuously increased with increasing DO concentrations (Supplementary Fig. S2F). The growth rate of A. carterae ACTY2405 at 0.5 mg O₂ L⁻¹ was 0.44 d⁻¹, and it continuously increased to 0.65 d⁻¹ at >7.0 mg O₂ L⁻¹ (Table 2, Fig. 2B). The growth rates were significantly affected by the DO concentrations, but pairwise comparisons showed no significant differences among individual DO concentrations (Fig. 2B, Supplementary Table S2).

The measured DO concentrations in Cy. closterium CyClTY2405 bottles at 0.5 to 4.0 mg O₂ L⁻¹ were well maintained ranging from 0.48–0.54, 0.98–1.04, 1.49–1.53, and 4.00–4.08 mg O₂ L⁻¹, respectively (Supplementary Fig. S2C). Furthermore, the measured DO concentration at >7.0 mg O₂ L⁻¹ ranged from 7.06–8.90 mg O₂ L⁻¹ due to mixing with ambient air, for which the measured DO concentration was 7.41–7.96 mg O₂ L⁻¹ in the control bottles (Supplementary Fig. S2C). During the 5-d incubation period, the average cell density continuously increased with increasing DO concentrations up to 1.5 mg O₂ L⁻¹, after which it remained relatively constant (Supplementary Fig. S2G). The growth rate of Cy. closterium CyClTY2405 was 0.88 d⁻¹ at 0.5 mg O₂ L⁻¹ and increased with DO concentration, reaching 1.44 d⁻¹ at 1.5 mg O₂ L⁻¹ (Table 2, Fig. 2C). The growth rates were significantly affected by the DO concentrations (Fig. 2C, Supplementary Table S2).

The measured DO concentrations in Ch. curvisetus ChCuTY2405 at 0.5 to 4.0 mg O₂ L⁻¹ were well maintained, ranging from 0.49–0.53, 0.97–1.03, 1.48–1.52, and 3.99–4.03 mg O₂ L⁻¹, respectively (Supplementary Fig. S2D). Furthermore, the measured DO concentration at >7.0 mg O₂ L⁻¹ ranged from 7.05–11.03 mg O₂ L⁻¹ due to mixing with ambient air, for which the measured DO concentration was 7.41–7.96 mg O₂ L⁻¹ in the control bottles (Supplementary Fig. S2D). During the 5-d incubation period, the average cell density continuously increased with increasing DO concentrations up to 1.5 mg O₂ L⁻¹, beyond which it slightly declined (Supplementary Fig. S2H). The growth rate of Ch. curvisetus ChCuTY2405 was 0.45 d⁻¹ at 0.5 mg O₂ L⁻¹, continuously increased with DO concentration to 0.78 d⁻¹ at 1.5 mg O₂ L⁻¹, and slightly decreased to 0.66 d⁻¹ at >7.0 mg O₂ L⁻¹, respectively (Table 2, Fig. 2D). The growth rates were significantly affected by the DO concentrations (Fig. 2D, Supplementary Table S2).

During the 0.5-d incubation period, the average DO concentration in P. triestinum PTTY2405 BOD bottles under light incubation (BOD_L) decreased at the target DO concentration of 0.5 mg O₂ L⁻¹, whereas it increased at 1.0 to >7.0 mg O₂ L⁻¹ after correction with the control (filtrate) bottles (Supplementary Fig. S2I). The average cell density increased by 5–50% during the incubation period at 1.0 to >7.0 mg O₂ L⁻¹ but decreased by 37% at 0.5 mg O₂ L⁻¹ (Supplementary Fig. S2M). The NPR of P. triestinum PTTY2405 was negative at 0.5 mg O₂ L⁻¹, increased continuously with increasing DO concentrations to reach a maximum of 0.60 pmol O₂ cell⁻¹ h⁻¹ at 1.5 mg O₂ L⁻¹, and slightly decreased thereafter (Table 3, Fig. 3A). The NPRs were significantly affected by the DO concentrations (Fig. 3A, Supplementary Table S2).

During the 0.5-d incubation period, the average DO concentration in P. triestinum PTTY2405 BOD bottles under dark incubation (BOD_D) decreased at all target DO concentrations from 0.5 to >7.0 mg O₂ L⁻¹ after control correction (Supplementary Fig. S2Q). The average cell density at all target DO concentrations decreased up to 43% during the incubation period (Supplementary Fig. S2U). The DRR of P. triestinum PTTY2405 was close to zero at 0.5 mg O₂ L⁻¹ and continuously increased with increasing DO concentrations, reaching a maximum of 0.27 pmol O₂ cell⁻¹ h⁻¹ at >7.0 mg O₂ L⁻¹ (Table 3, Fig. 4A). The DRRs were significantly affected by the DO concentrations, but pairwise comparisons showed no significant differences among individual DO concentrations (Fig. 4A, Supplementary Table S2).

For A. carterae ACTY2405, the average DO concentration in BOD_L increased at all target DO concentrations (Supplementary Fig. S2J), while the average cell density increased by 10–44% during the incubation period (Supplementary Fig. S2N). The NPR of A. carterae ACTY2405 increased continuously with increasing DO concentrations to reach a maximum of 0.74 pmol O₂ cell⁻¹ h⁻¹ at 1.5 mg O₂ L⁻¹ before decreasing (Table 3, Fig. 3B). The NPRs were significantly affected by the DO concentrations (Fig. 3B, Supplementary Table S2).

For A. carterae ACTY2405, the average DO concentration in BOD_D decreased at all target DO concentrations (Supplementary Fig. S2R). The average cell density increased up to 13% during incubation period at 1.0 to >7.0 mg O₂ L⁻¹ but decreased by 3% at 0.5 mg O₂ L⁻¹ (Supplementary Fig. S2V). The DRR of A. carterae ACTY2405 continuously increased with increasing DO concentrations, reaching a maximum of 0.19 pmol O₂ cell⁻¹ h⁻¹ at >7.0 mg O₂ L⁻¹ (Table 3, Fig. 4B). The DRRs were significantly affected by the DO concentrations (Fig. 4B, Supplementary Table S2).

For Cy. closterium CyClTY2405, the average DO concentration in BOD_L increased at all target DO concentrations (Supplementary Fig. S2K), and the average cell density increased 17–55% (Supplementary Fig. S2O). The NPR of Cy. closterium CyClTY2405 increased continuously with increasing DO concentrations to reach a maximum of 0.75 pmol O₂ cell⁻¹ h⁻¹ at 1.5 mg O₂ L⁻¹, and decreased thereafter (Table 3, Fig. 3C). The NPRs were significantly affected by the DO concentrations (Fig. 3C, Supplementary Table S2).

For Cy. closterium CyClTY2405, the average DO concentration in BOD_D decreased at all target DO concentrations (Supplementary Fig. S2S), and the average cell density decreased by 1–9% (Supplementary Fig. S2W). The DRR of Cy. closterium CyClTY2405 continuously increased with increasing DO concentrations, reaching a maximum of 0.19 pmol O₂ cell⁻¹ h⁻¹ at >7.0 mg O₂ L⁻¹ (Table 3, Fig. 4C). The DRRs were significantly affected by the DO concentrations (Fig. 4C, Supplementary Table S2).

For Ch. curvisetus ChCuTY2405, the average DO concentration in BOD_L increased at all target DO concentrations (Supplementary Fig. S2L), and the average cell density increased by 6–40% (Supplementary Fig. S2P). The NPR of Ch. curvisetus ChCuTY2405 increased continuously with increasing DO concentrations to reach a maximum of 0.66 pmol O₂ cell⁻¹ h⁻¹ at 1.5 mg O₂ L⁻¹, and decreased thereafter (Table 3, Fig. 3D). The NPRs were significantly affected by the DO concentrations (Fig. 3D, Supplementary Table S2).

For Ch. curvisetus ChCuTY2405, the average DO concentration in BOD_D decreased at all target DO concentrations (Supplementary Fig. S2T), and the average cell density decreased up to 25% (Supplementary Fig. S2X). The DRR of Ch. curvisetus ChCuTY2405 continuously increased with increasing DO concentrations, reaching a maximum of 0.19 pmol O₂ cell⁻¹ h⁻¹ at >7.0 mg O₂ L⁻¹ (Table 3, Fig. 4D). The DRRs were significantly affected by the DO concentrations (Fig. 4D, Supplementary Table S2).

The average (±standard error [SE], n = 40) and maximum swimming speeds of P. triestinum PTTY2405 at 0.5 mg O₂ L⁻¹ were 250.8 (±9.8) and 369 μm s⁻¹, respectively (Fig. 5A). Under 1.5 and >7.0 mg O₂ L⁻¹, the average and maximum swimming speeds were 256.9 (±9.0) and 406 μm s⁻¹, and 297.3 (±11.3) and 532 μm s⁻¹, respectively. The swimming speeds of P. triestinum PTTY2405 were significantly affected by the DO concentrations (Kruskal-Wallis, H₂ = 9.58, p < 0.01).

The average (±SE, n = 40) and maximum swimming speeds of A. carterae ACTY2405 at 0.5 mg O₂ L⁻¹ were 79.6 (±3.5) and 114 μm s⁻¹, respectively (Fig. 5B). Under 1.5 and >7.0 mg O₂ L⁻¹, the average and maximum swimming speeds were 77.1 (±2.5) and 125 μm s⁻¹, and 79.8 (±3.5) and 137 μm s⁻¹, respectively. The swimming speeds of A. carterae ACTY2405 were not significantly affected by the DO concentrations (Kruskal-Wallis, H₂ = 0.27, p = 0.87).

The results of this study indicated that phototrophic dinoflagellate species required higher oxygen concentrations to reach their maximum growth rates compared to diatom species. Phototrophic dinoflagellate species showed their maximum growth rates under normoxic conditions, which is consistent with the results reported for Alexandrium fraterculus AFYS1309 and Scrippsiella lachrymosa SLBS1703 (Table 4, Fig. 6A). However, data for both strains of A. carterae (ACTY2405 and CCMP1314) from this study and Bausch et al. (2019) showed that growth rates did not differ significantly among individual DO concentrations. Amphidinium carterae is a benthic species (Rodrigues and Patil 2022), that can be frequently exposed to hypoxic bottom waters. In fact, A. carterae was the dominant dinoflagellate species observed in field waters from Tongyeong after 15 d of hypoxic incubation (unpublished data). Furthermore, Amphidinium sp. HYA002, an endosymbiont of a marine flatworm where the environment inside the host is low-oxic, exhibited similar growth rates under hypoxia and normoxia (Kitaya et al. 2008). Thus, although most phototrophic dinoflagellate species grow better under normoxia, those known to inhabit low-oxygen environments may not show significant differences in growth rates across DO concentrations.

Unlike phototrophic dinoflagellate species, diatom species in this study reached their maximum growth rates at 1.5 mg O₂ L⁻¹. Consistent with these results, Thalassiosira weissflogii CCMP1336 showed an increased growth rate under hypoxia (1.8 mg O₂ L⁻¹) compared to normoxia (8.2 mg O₂ L⁻¹) (Table 4, Fig. 6A) (Sun et al. 2022). In contrast, Phaeodactylum tricornutum CCAP 1055, Skeletonema costatum, and Thalassiosira pseudonana CCMP1335 exhibited decreased growth rates under more severe hypoxia (0.7, 0.5, and 1.3 mg O₂ L⁻¹, respectively) compared to normoxia (7.4, 7.0, and 8.0 mg O₂ L⁻¹) (Table 4, Fig. 6A) (Wu et al. 2012, Zhao et al. 2022, Chen et al. 2025). These differences in growth rates may result from the varied target DO concentrations used to define hypoxia across studies. Indeed, the present study demonstrated that growth rates can either decrease (at 0.5 mg O₂ L⁻¹) or increase (at 1.5 mg O₂ L⁻¹) relative to normoxia, depending on the specific hypoxic DO concentration. Thus, the low resolution of DO concentrations in previous studies may account for the contrasting growth responses observed under hypoxia, highlighting the need for further studies conducted across a finer gradient of DO concentrations.

In the present and previous studies, changes in growth rates under hypoxia were determined (Fig. 7). Under hypoxic conditions, Sk. costatum showed a linear increase in growth rate from 0.5 to 2.0 mg O₂ L⁻¹, although only two measurements were available (Wu et al. 2012). Among the phytoplankton species, P. triestinum PTTY2405 exhibited the fastest increase in growth rate over the 0.5 to 2.0 mg O₂ L⁻¹ range. Cells of P. triestinum PTTY2405 had the highest active swimming speeds among the experimental species, even under hypoxia. Furthermore, when comparing the ratio of NPR to DRR between 0.5 and 1.5 mg O₂ L⁻¹, P. triestinum PTTY2405 showed the lowest ratio at 0.5 and 1.0 mg O₂ L⁻¹, and the second lowest ratio at 1.5 mg O₂ L⁻¹. This likely results in a higher respiration rate relative to the photosynthesis rate, suggesting greater sensitivity in the growth of P. triestinum PTTY2405 to oxygen decline under hypoxia.

When excluding Sk. costatum due to differences in experimental conditions between studies, A. carterae ACTY2405 exhibited the slowest increase in growth rate under hypoxia, indicating the lowest sensitivity of growth rates to hypoxia among the four species examined in this study (Fig. 7). A. carterae is a benthic species, and the culture has been established under hypoxic conditions. Furthermore, when comparing the ratio of NPR to DRR from 0.5 to 1.5 mg O₂ L⁻¹, A. carterae ACTY2405 showed the second-highest ratio at 1.5 mg O₂ L⁻¹, and the highest ratio at 1.0 mg O₂ L⁻¹. This may suggest that A. carterae ACTY2405 maintains a relatively high NPR compared with its respiration rate even at low DO concentration, contributing to its low sensitivity of growth rate to hypoxia.

In this study, to minimize oxygen shock, a stepwise 7-d pre-incubation protocol was used; cultures of all experimental species were maintained at the next higher DO concentration for 5 d, followed by a 2-d acclimation at the target DO. During this 2-d pre-incubation period, all four species underwent approximately one or fewer cell divisions at each target DO. Because severe hypoxic conditions can increase mortality in phytoplankton species, this brief 2-d pre-incubation was specifically designed to prevent excessive cell death and maintain sufficient cell densities required to initiate the main experiments. Furthermore, to minimize the influence of transient stress responses on our results, growth rates were calculated over the subsequent 5-d experimental incubation period. In addition, cell densities exhibited consistent increases or decreases throughout this 5-d incubation period (Supplementary Fig. S2), suggesting that the observed growth responses reflected the target DO concentrations. Nevertheless, the potential effects of the 2-d pre-incubation period should be considered when interpreting the findings of the present study.

To minimize bubbling-induced stress during the growth experiments, DO concentrations were maintained by using low initial cell densities, gentle stirring with a magnetic stirrer, and a shallow water depth. For the >7.0 mg O₂ L⁻¹ experiment, all bottles were incubated under ambient air conditions. Thus, DO concentrations may not have been maintained at comparable levels when cellular oxygen production from photosynthesis exceeded air exchange driven by stirring. When oxygen exceeds saturation, growth inhibition may occur in phytoplankton species (Gao et al. 2022). Therefore, this potential growth inhibition should be considered as one of the factors contributing to the reduced growth rate at >7.0 compared to 1.5 mg O₂ L⁻¹.

Phytoplankton growth and photosynthetic responses to low oxygen vary significantly depending on irradiance and species. Under low light, low oxygen may either stimulate growth through increased CO₂-concentrating mechanism (CCM) and enhanced light energy utilization, or suppress growth through reduced carbon fixation depending on species (Chen et al. 2021, 2024, Sun et al. 2022). Under high light, low oxygen may suppress growth by impairing Photosystem II (Pruder and Bolton 1980). These contrasting responses highlight species-specific interactions between oxygen availability and irradiance. In addition, temperature interacts with irradiance to influence phytoplankton growth responses (Edwards et al. 2016). Therefore, irradiance and temperature should be considered when comparing results among studies.

By combining data from the present and previous studies, this study identified the maximum NPR across different DO concentrations, whereas prior studies only compared two or three DO concentrations (Table 4, Fig. 6B). In the present study, all four experimental species showed the highest NPR at 1.5 mg O₂ L⁻¹. Hence, the results imply that photosynthetic activity was not a limiting factor for growth at ≤1.5 mg O₂ L⁻¹ for all experimental species.

Theoretically, reduced oxygen in water increases the likelihood of CO₂ binding to the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), thereby enhancing photosynthetic activity in phytoplankton (Pruder and Bolton 1980, Raven and Larkum 2007, Gao and Campbell 2014). Consistent with the findings in this study, culture of Th. weissflogii CCMP1336 and natural diatom-dominated phytoplankton assemblages showed higher NPR and net primary productivity at 1.8 mg O₂ L⁻¹ than under normoxia (Sun et al. 2022). However, A. carterae CCMP1314 and Th. pseudonana CCMP1335 showed decreased NPR under low-oxic or hypoxic conditions compared to normoxic conditions (Table 4, Fig. 6B) (Bausch et al. 2019, Chen et al. 2025). The varying NPR responses to DO concentrations may result from the low resolution of previous studies, which accounted for only one DO concentration under hypoxia. Thus, future studies exploring other phototrophic dinoflagellate and diatom species at higher DO resolution are recommended. Furthermore, there was a positive correlation between NPR and growth rate in both phototrophic dinoflagellate and diatom species (Fig. 8A & B). Photosynthesis is the basis of phytoplankton survival (Peng et al. 2022), and the inability of P. triestinum PTTY2405 to maintain photosynthetic activity under 0.5 mg O₂ L⁻¹ may have led to its negative growth rate. Despite the theoretical advantage of reduced oxygen for RuBisCO activity, severe hypoxia may impair photosynthetic activity by suppressing mitochondrial respiration, which requires oxygen as the final electron acceptor (Banti et al. 2013). This suppression would reduce ATP availability for CCMs and photochemical processes (Burlacot and Peltier 2023, Intarasit and Inwongwan 2026). As extremely hypoxic waters have been shown to reduce photosynthetic yield (F_v/F_m) (Stauffer et al. 2013), future studies should include F_v/F_m measurements in addition to NPR to better evaluate the relationship between oxygen concentration and photosynthetic activity.

Combining the results from the present study and previous studies, all diatom and phototrophic dinoflagellate species showed the highest DRR at >7.0 mg O₂ L⁻¹ (Table 4, Fig. 6C). Moreover, there was a positive correlation between DRR and growth rate in phototrophic dinoflagellate species across different DO concentrations (Fig. 8C). The pelagic dinoflagellate P. triestinum PTTY2405 showed the highest DRR among the four experimental species at >7.0 mg O₂ L⁻¹, suggesting a high oxygen demand for respiration. Notably, P. triestinum PTTY2405 exhibited approximately three- to four-fold higher maximum and average swimming speeds than the benthic dinoflagellate A. carterae ACTY2405 across DO concentrations from >7.0 to 0.5 mg O₂ L⁻¹ (Fig. 5). This high oxygen demand from fast motility can result in a high energy demand to maintain cell viability. Indeed, dinoflagellates exhibit high cellular respiration rates, which may in part reflect the energetic costs of motility (Langdon 1993, Pitcher and Probyn 2016). While the cost of motility in dinoflagellates is generally thought to constitute only a small fraction (≤ 1% or less) of total metabolic expenditure—with swimming speeds mostly at 10 body lengths s⁻¹ or less—it may impose a substantially higher metabolic cost (ca. 1–10%) in species with high swimming speeds, such as P. triestinum, which can reach approximately 40 body lengths s⁻¹ (Crawford 1992). Similarly, Al. fraterculus AFYS1309 and Sc. lachrymosa SLBS1703, which also exhibited negative growth rates at 0.5 mg O₂ L⁻¹, have relatively high maximum swimming speeds of 680 and 786 μm s⁻¹, respectively (Eom et al. 2024). In addition to swimming, flagella uncovered by the cell wall may increase metabolic costs due to osmoregulation, while a large amount of genetic material may further increase costs associated with cellular turnover (Rizzo 2003, López-Sandoval et al. 2014 and references therein). Collectively, these inherently high basal metabolic costs in dinoflagellates may contribute to their high respiration rates; however, direct measurements at the species level remain limited, and future studies are needed to specify the relative contributions of these factors to metabolic expenditure under different oxygen concentrations.

On the other hand, there was no correlation between DRR and growth rate in diatom species across different DO concentrations (Fig. 8D). Cy. closterium is a benthic species exhibiting very slow gliding speeds of 0.7 μm s⁻¹ (Apoya–Horton et al. 2006), while members of the genus Chaetoceros are reported to be non-motile (Thomas et al. 2013). Thus, diatom species may have lower respiratory demands associated with metabolic activities such as swimming compared to phototrophic dinoflagellate species. Consequently, diatom species may achieve their maximum growth rates at 1.5 mg O₂ L⁻¹ where photosynthesis is sufficient.

Hypoxia affects the structure and function of protist communities, including phytoplankton (Rocke et al. 2013, Santoferrara et al. 2022, Ok et al. 2023, Eom et al. 2025). Hence, understanding the effects of oxygen on phytoplankton physiological rates is critical for investigating changes in the structure and function of coastal ecosystems under various DO concentrations. The present study provides novel insights into phytoplankton responses of growth, net photosynthesis, and dark respiration under varying DO concentrations by identifying, for the first time, the specific DO concentrations at which maximum rates are reached. In addition, this study also reports, for the first time, the swimming speeds of phototrophic dinoflagellate species across different DO concentrations. Except for P. triestinum PTTY2405 at 0.5 mg O₂ L⁻¹, all experimental species showed tolerance to varying degrees of hypoxic conditions, as supported by their positive growth rates. Changes in photosynthetic and respiration rates in these four phytoplankton species can support survival across different DO concentrations, each with its own metabolic trade-offs. Such responses are similar to the physiological plasticity observed in response to other environmental stressors such as temperature and salinity (Anneville et al. 2018, Orizar and Lewandowska 2025). To further elucidate the mechanisms underlying these responses, comparative experiments between strains exposed to recurring hypoxic conditions and those without such exposure are recommended to determine whether these changes in physiological rates are driven by adaptation or plasticity. Therefore, future studies should include multiple DO concentrations to achieve higher resolution in assessing the responses of physiological rates across DO concentrations.