Deoxygenation enhances photosynthetic performance and increases N₂ fixation in the marine cyanobacterium Trichodesmium under elevated pCO₂

Abstract

Effects of changed levels of dissolved O₂ and CO₂ on marine primary producers are of general concern with respect to ecological effects of ongoing ocean deoxygenation and acidification as well as upwelled seawaters. We investigated the response of the diazotroph Trichodesmium erythraeum IMS 101 after it had acclimated to lowered pO₂ (~60 μM O₂) and/or elevated pCO₂ levels (HC, ~32 μM CO₂) for about 20 generations. Our results showed that reduced O₂ levels decreased dark respiration significantly, and increased the net photosynthetic rate by 66 and 89% under the ambient (AC, ~13 μM CO₂) and the HC, respectively. The reduced pO₂ enhanced the N₂ fixation rate by ~139% under AC and only by 44% under HC, respectively. The N₂ fixation quotient, the ratio of N₂ fixed per O₂ evolved, increased by 143% when pO₂ decreased by 75% under the elevated pCO₂. Meanwhile, particulate organic carbon and nitrogen quota increased simultaneously under reduced O₂ levels, regardless of the pCO₂ treatments. Nevertheless, changed levels of O₂ and CO₂ did not bring about significant changes in the specific growth rate of the diazotroph. Such inconsistency was attributed to the daytime positive and nighttime negative effects of both lowered pO₂ and elevated pCO₂ on the energy supply for growth. Our results suggest that Trichodesmium decrease its dark respiration by 5% and increase its N₂-fixation by 49% and N₂-fixation quotient by 30% under future ocean deoxygenation and acidification with 16% decline of pO₂ and 138% rise of pCO₂ by the end of this century.

1. Introduction

Biological N₂ fixation by marine diazotrophs is an important source of “new” usable nitrogen to phytoplankton in oligotrophic surface waters (Karl et al., 1997; Bergman et al., 2013). The N₂-fixing cyanobacteria, Trichodesmium spp., contribute about half of the current estimate of global annual marine N₂ fixation, playing a pivotal role in global biogeochemical N/C cycles (Mahaffey et al., 2005; Sohm et al., 2011). The nitrogenase enzyme encoded by the nifHDK genes is critically sensitive to and can be inactivated by oxygen (O₂) (Staal et al., 2007; Hutchins and Sañudo-Wilhelmy, 2022). Most cyanobacterial N₂-fixers can avoid O₂ damage via temporal (fixing N₂ during night) or spatial (using heterocyst) strategies. However, Trichodesmium spp. do not possess such contrivances to avoid photosynthetically evolved O₂, since all cells of the trichomes have PSII (Lin et al., 1998; Bergman et al., 2013). It is known that Trichodesmium cells down-regulate its photosynthesis during midday to protect nitrogenase using various physiological pathways (e.g., the Mehler reaction, respiration and flavoprotein-mediated O₂ uptake) to sequestrate O₂ under light (Berman-Frank et al., 2001; Gallon, 2001; Milligan et al., 2007), which is considered as a metabolic strategy for the O₂-sensitive diazotroph (Luo et al., 2022).

Dissolved O₂ (DO) levels in the oceans have been declining due to global warming, and are expected to further decline during the 21th century (Schmidtko et al., 2017; Breitburg et al., 2018). Rising global temperatures decrease the solubility of O₂, enhancing stratification (hindering ventilation to deeper layers) of the upper mixing layers and increasing biological respiration, thus ultimately reduce DO in surface and subsurface oceans (Carstensen et al., 2014). Consequently, the oxygen-minimum zones (OMZ) have expanded horizontally and vertically along with progressive ocean deoxygenation (Stramma et al., 2008; Schmidtko et al., 2017). On the other hand, internal O₂ concentrations of Trichodesmium colonies or blooms ranged from 0 to 500 μM during dark and/or high-light periods, which is supposed to affect the nitrogenase activity (Paerl and Bebout, 1992; Eichner et al., 2019). Light stimulation of nitrogenase activity is most obvious at low O₂ concentrations, while it becomes gradually less important with increased levels of O₂ in Trichodesmium IMS 101 (Staal et al., 2007). Therefore, it is likely that the marine diazotrophs including Trichodesmium would benefit from ocean deoxygenation.

On the other hand, ocean acidification (OA) induced by continuous dissolution of anthropogenically emitted CO₂ into seawater is known to affect various phytoplankton species and ecological processes (Gao et al., 2020), which show positive and/or negative responses to OA under different environmental conditions (Boyd et al., 2018). Temperature, light, nutrients and UV radiation are known to modulate the influences of elevated pCO₂, resulting in negative in oligotrophic waters but in neutral or positive effects in nutrients-replete waters (Gao et al., 2022). Most cyanobacteria and microalgae, including Trichodesmium, have evolved efficient CO₂-concentrating mechanisms (CCMs) that actively uptake CO₂ and bicarbonate to increase the level of intracellular dissolved inorganic carbon for efficient photosynthesis (Giordano et al., 2005). Therefore, the energy saved by the downregulation of CCMs under elevated pCO₂ is expected to be reallocated to other metabolic processes (Kranz et al., 2010). Differential responses have also been documented in Trichodesmium. OA has been shown to promote the N₂ fixation and growth of Trichodesmium (Hutchins et al., 2007; Levitan et al., 2007; Garcia et al., 2011; Hutchins et al., 2013). However, it was also shown to result in insignificant or even negative effects on Trichodesmium, and the negative effects were more obvious under iron limitation (Shi et al., 2012; Hong et al., 2017; Zhang et al., 2019). It was suggested that the energy saved from downregulation of CCMs under elevated pCO₂ is not enough for the energy demand of increased N₂-fixation rates in Trichodesmium under OA conditions (Luo et al., 2019). While these disputable findings need to be mechanistically reconciled, effects of multiple drivers on the marine diazotroph should also be examined (Wake, 2019). Recently, it was shown that reduced O₂ availability modulated the effects of elevated pCO₂ on a diatom (Sun et al., 2022). Combined effects of ocean deoxygenation and acidification on Trichodesmium has yet to be investigated.

Since levels of pH/pCO₂ and O₂ in the center of Trichodesmium colonies fluctuate during a diel cycle due to night respiration and daytime photosynthesis (Eichner et al., 2019), its physiological performance can be naturally influenced by the changed levels of both O₂ and CO₂. In parallel, progressive ocean deoxygenation and acidification are covarying factors that affect marine organisms including the diazotrophs (Staal et al., 2007; Paulmier et al., 2011; Sun et al., 2022). Considering that low levels of O₂ can theoretically enhance carboxylation via decreasing oxygenation processes catalyzed by Rubisco and stimulate the activity of nitrogenase, we hypothesize that combination of ocean deoxygenation and acidification can synergistically enhance photosynthetic performance and N₂ fixation of Trichodesmium. Hence, we simulated different microenvironments by growing Trichodesmium erythraeum (T. erythraeum) strain IMS 101 under four different pO₂/pCO₂ combinations (ambient CO₂ & ambient O₂; ambient CO₂ & low O₂; high CO₂ & ambient O₂; high CO₂ & low O₂) in order to investigate the responses of Trichodesmium. In this work, we found that reduced O₂ concentration increased both photosynthetic performance and N₂ fixation rate and thus raised the particulate organic carbon and nitrogen even under elevated pCO₂ projected for future ocean acidification by the end of this century.

2. Materials and methods

2.1. Culture conditions

Trichodesmium erythraeum (strain IMS 101), originally isolated from the North Atlantic Ocean (Prufert-Bebout et al., 1993), was grown in YBCII medium without combined N prepared with autoclaved sterilized artificial seawater (Chen et al., 1996). Experiments were conducted in polycarbonate bottles under 160 μmol photons m⁻² s⁻¹ of PAR (measured by a Solar light sensor, PMA2100, United States) with a day-night cycle of 12: 12 h at 27 ± 0.5°C in an incubator (HP300G–C, Ruihua, China).

Triplicate experiments were carried out in a four treatments matrix of two levels of CO₂ (ambient and high CO₂) and two levels of O₂ (ambient and low O₂), respectively. T. erythraeum cells were maintained in the exponential growth phase by semicontinuous culture (diluted every 48 h). The chlorophyll a (Chl a) concentration was maintained within a range of 0.003–0.015 μg mL⁻¹ (Supplementary Figure S1), so that dissolved O₂ and carbonate chemistry in the cultures were little altered. The carbonate chemistry parameters were relatively stable before and after the dilution, the pH variations were less than 0.08 units under either the HC (elevated CO₂, 32 μM) or AC (ambient CO₂, 12 μM) treatments, along with 12 to 19% decrease of pCO₂ before the dilutions, and dissolved O₂ concentrations varied less than 10 μM (245–255 μM) and 30 μM (60–90 μM) under AO (ambient O₂) and LO (low O₂) treatments, respectively (Table 1; Supplementary Figure S2). Prior to dilution, fresh media were prepared with the target O₂ and CO₂ levels: ambient CO₂ & ambient O₂ (ACAO), ambient CO₂ & low O₂ (ACLO,), high CO₂ & ambient O₂ (HCAO), high CO₂ & low O₂ (HCLO) (Table 1). A customized CO₂/O₂ controlling device (CE-100DY, Ruihua, China) was employed to achieve the above O₂ and CO₂ levels. The bottles were shaken gently every 3 h during the daytime to ensure that the cyanobacterial filaments were in a suspended state.

2.2. Carbonate chemistry and dissolved O₂ parameter in cultures

The dissolved O₂ and pH of seawater were measured before and after dilution every 2 days. The dissolved O₂ was measured with a Clark-type oxygen electrode (Hansatech, United Kingdom). The pH_NBS was measured using a pH meter, which was three-point calibrated with NBS buffer. Total alkalinity (TA) was measured using the Gran potentiometric titration method (Gao et al., 2018). Dissolved inorganic carbon (DIC) and other carbonate chemistry parameters (Table 1) were calculated from pH_NBS and TA by using the CO2SYS software (Lewis et al., 1998).

2.3. Chl a concentration and specific growth rate

Trichodesmium erythraeum cells were gently filtered onto 25 mm glass-fiber filters (GF/F, Whatman), which was then extracted in pure methanol at 4°C for 24 h for Chl a determination. The extracts were centrifuged at 6000 g for 10 min, and the absorbances of the supernatants at 665 nm and 750 nm were measured using a UV–VIS Spectrophotometer (DU800, Beckman, United States). The Chl a concentration was calculated according to the equations Chl a (μg mL⁻¹) =12.9447 × (A₆₆₅ − A₇₅₀) × V1/V2 (Ritchie, 2006), where V1 and V2 represent the 100% methanol volume (mL) and filtered cell volume (mL), respectively. The specific growth rates (μ, d⁻¹) were determined from linear regressions of the natural log of Chl a vs. time during the exponential growth (Yi et al., 2020).

2.4. Dark respiration and net photosynthetic oxygen evolution

Dark respiration and net photosynthetic oxygen evolution were measured in the middle of the light period within 3 h using a Clarke-type electrode (Hansatech, United Kingdom). Cells were harvested by filtering onto polycarbonate membrane filters (5 μm, Millipore, Germany) under gentle vacuum pressure (<0.01 MPa). These cells were then re-suspended in seawater buffered with 20 mM Tris–HCl with a final Chl a concentration of approximately 0.5 μg mL⁻¹. The pH levels of the Tris buffered-medium were pre-adjusted by adding hydrochloric acid or sodium hydroxide to the same levels of the cultures (pH 7.83 for HC and 8.13 for AC), and the O₂ levels were achieved by flushing the medium with pure N₂. The resuspended cells were injected into an oxygen electrode vessel with a magnetic stirrer held in a water-jacked chamber (temperature controlled at 27°C) under the same level of light intensity. The respiration rate was estimated in darkness by covering the reaction chamber with aluminum foil. Photosynthetic O₂ evolution was determined under growth O₂ levels.

2.5. Nitrogen fixation rate

N₂-fixation rates were determined at the midpoint of the photoperiod with the acetylene reduction assay, assuming a ratio of 4: 1 to convert ethylene production to N₂ fixation (Capone, 1993). Although one time point measurement has been able to identify changes of N₂-fixation rate under different environmental conditions (Zhang et al., 2019), it may overlook other timepoint values of the N₂-fixation during the whole light period. For determination of the N₂-fixation rates, 5 ml of sample was added to a 13 mL vial, which was then sealed. After 1 mL of air was extracted from the headspace, 1 ml of acetylene (C₂H₂) was added to each vial. All replicates were mixed well and incubated for 2 h under the same temperature and light in the incubator as the growth conditions. The ethylene production was measured using a gas chromatograph with a flame ionization detector (Clarus 580, PerkinElmer, United States).

2.6. Particulate organic carbon and nitrogen (PON)

Cell samples for particulate organic carbon (POC) and particulate organic nitrogen (PON) were collected onto pre-combusted (450°C for 6 h) GF/F filters (Whatman), and were stored at −80°C before measuring. Filters were exposed to HCl fumes to remove inorganic carbon and dried at 60°C before analysis with a CHNS elemental analyzer (vario EL cube, Elementar, Germany).

2.7. Statistical analysis

The data were presented as means of three replicates ± SD (n = 3, triplicate independent cultures). One-way ANOVA and Tukey test were performed to analyze the statistical differences among different treatments. Differences were termed significant when p < 0.05. Before performing parametric tests, data were tested for homogeneity of variance (Levene test) and normality (ShapiroWilk test).

3. Results

3.1. Specific growth rate

Neither elevated pCO₂ (p = 0.9881) nor lowered pO₂ (p = 0.6284) significantly affected the specific growth rate of T. erythraeum (Figure 1). The specific growth rates were 0.534 ± 0.012 and 0.531 ± 0.043 d⁻¹ under AC, and were 0.525 ± 0.013 and 0.528 ± 0.038 d⁻¹ under HC, at the levels of ambient and lowered O₂, respectively (Figure 1).

Figure 1

3.2. Photosynthetic performance

Dark respiration decreased with lowered levels of O₂, whereas net and gross photosynthetic rate increased (Figure 2). Reduced O₂ levels (from 250 to 60 μM) significantly increased net photosynthetic rate by 66% under AC (631 vs. 379 μmol O₂ mg chl a⁻¹ h⁻¹, p < 0.0001) and by 89% under HC conditions (659 vs. 348 μmol O₂ mg chl a⁻¹ h⁻¹, p < 0.0001), respectively (Figure 2A). Nevertheless, no significant differences were found in the net photosynthetic rate between the AC and HC treatments (p = 0.9432) (Figure 2A), though the mean net photosynthetic rate of the cells grown under HC was higher by about 4% than that of AC under LO. Decreased O₂ levels to 60 μM (by 75%) significantly inhibited dark respiration by 40% under the AC (136 vs. 226 μmol O₂ mg chl a⁻¹ h⁻¹, p = 0.0002) and 39% under the HC (172 vs. 283 μmol O₂ mg chl a⁻¹ h⁻¹, p < 0.0001), respectively (Figure 2B), reflecting that Trichodesmium would decrease its dark respiration by 9% and increase its net photosynthetic rate by 14% with 16% pO₂ decline by the end of this century (Table S1). Meanwhile, the HC-acclimated cells had higher (by 25% at AO and by 27% at LO) dark respiration rate compared to that of AC cells regardless of O₂ levels (p = 0.0031 for AO, p = 0.0278 for LO). The reduced O₂ levels significantly increased gross photosynthetic rate by 27% (767 vs. 605 μmol O₂ mg chl a⁻¹ h⁻¹, p < 0.0019) under AC and by 32% (831 vs. 631 μmol O₂ mg chl a⁻¹ h⁻¹, p = 0.0005) under HC, respectively (Figure 2C). Meanwhile, HC treatments increased gross photosynthetic rate by 4% (p = 0.4844) under AO and by 8% (p = 0.1081) under LO compared to the AC-grown cells, though the difference was insignificant.

Figure 2

3.3. Nitrogen fixation rate

When T. erythraeum cells were grown under different O₂ levels, their N₂ fixation rates showed a negative relationship with the O₂ concentrations (Figure 3A, r² = 0.88). The N₂ fixation rate increased linearly by 9% with each 10% decline in the dissolved O₂ concentration. After the Trichodesmium cells acclimated to different levels of O₂ and pCO₂, lowering DO to about 60 μM increased the Chl a-specific N₂ fixation rate by ~139% (20.0 vs. 8.4 μmol N₂ mg chl a⁻¹ h⁻¹, p < 0.0001) under AC but only by 44% (27.5 vs. 19.2 μmol N₂ mg chl a⁻¹ h⁻¹, p < 0.0001) under the HC conditions (Figure 3B), indicating obvious less enhancement by the reduced O₂ treatment under the elevated pCO₂. The Trichodesmium cells grown in the HC treatment had higher N₂ fixation rates than those grown in the AC cultures regardless of the O₂ levels (p < 0.0001 for AO, p = 0.0001 for LO), being increased by 129% under AO and by 38% under LO, respectively. The ratio of N₂ fixation to the gross photosynthetic rate, as a proxy of the N₂-fixation quotient (NFQ), ranged from 0.014 to 0.033 (mol N₂: mol O₂) (Figure 3C). The lowered pO₂ treatment increased the NFQ by 89% under AC (p = 0.0016) and by only 10% under HC (p = 0.5104), and the elevated pCO₂ and low pO₂ treatment increased it by 143% compared to ACAO (p < 0.0001). Based on the results in this work, we estimated that Trichodesmium would increase its N₂-fixation by 14% and N₂-fixation quotient by 19% under future ocean deoxygenation with 16% decline of pO₂ till 2,100 under the current pCO₂ level (Table S1).

Figure 3

3.4. Particulate C and N quotas

Decreased O₂ levels to about 60 μM slightly raised the cellular particulate organic carbon (POC) by 16% (p = 0.0583) and particulate organic nitrogen (PON) by 15% (p = 0.0190) in the T. erythraeum cells ISM 101 grown under the AC (Figures 4A,B). Contrastingly, under the HC conditions, the reduced O₂ concentration increased POC by 44% (p = 0.0002) and PON by 45% (p < 0.0001), respectively (Figures 4A,B). Nevertheless, the cellular C:N ratios were about 6.8, being neither affected under the changed levels of pCO₂ (p = 0.0796) nor under the reduced levels of pO₂ (p = 0.9859) (Figure 4C).

Figure 4

4. Discussion

In the present study, the T. erythraeum cells acclimated to lowered pO₂ and elevated pCO₂ levels showed higher N₂-fixation quotient (ratio of N₂ fixed to gross O₂ production), being increased by 143% (with pO₂ declined bys 75%) and by 12% (with pO₂ declined by 10%) compared to ambient levels of pO₂ and pCO₂ (Figure 5). This could be attributed to decreased dark respiration and increased photosynthetic performance as well as increased N₂ fixation under the reduced levels of pO₂ and to increased rates of dark respiration and N₂ fixation under the elevated pCO₂ (Figures 2, 3). Although both low pO₂ and high pCO₂ treatments did not significantly affect the growth rates of T. erythraeum, its cellular particulate organic carbon (POC) and nitrogen (PON) contents increased under the lowered O₂ and elevated pCO₂ levels. Our data provided the experimental evidence that reduced O₂ levels increased N₂ fixation and photosynthetic performance by stimulating the activities of nitrogenase and Rubisco (Figure 5), enhancing POC and PON production in anoxic and/or hypoxic environments, where lowered levels of O₂ and pH (high pCO₂) are covarying drivers.

Figure 5

4.1. Responses of N₂ fixation, photosynthesis, and growth to deoxygenation

Nitrogenase, the key enzyme that catalyzes the reduction of N₂, is instantaneously and irreversibly inactivated by O₂via oxidative damage (Staal et al., 2007). Meanwhile, significantly decreased photosynthetic O₂ evolution coincided with increased respiratory CO₂ release during midday, that favored the N₂ fixation (Kranz et al., 2009). In the present work, reduced levels of O₂ in milieu significantly increased N₂-fixation and photosynthesis (Figures 2, 3). Since lowered O₂ concentration increased the net photosynthetic O₂ evolution, the dissolved O₂ in the cultures increased before the continual dilutions, which, nevertheless, did not significantly alter the difference in O₂ treatments between lowered (LO) and ambient (AO) pO₂ (Table 1). Therefore, the O₂ levels (AO: 245–255 μM; LO: 60–90 μM) during the growth period could still reflect LO and AO conditions. In photosynthesis, electrons transport via the photosynthetic and respiratory transport chains must have promoted production of ATP and reductant for N₂ fixation and CO₂ assimilation in Trichodesmium (Suggett et al., 2010; Eichner et al., 2019). While no decline in net photosynthetic O₂ evolution was found with enhanced dark respiration in this study (Figure 2), higher respiration rates during the photoperiod would decrease the photosynthetic O₂ evolution in Trichodesmium (Berman-Frank et al., 2001), which must partially favored the N₂ fixation. In the present work, the rates of dark respiration accounted for 37% in AO-grown and for 18% in LO-grown cells of the gross photosynthesis (Figures 2B,C), indicating that the role to remove O₂ by respiration was down-regulated under LO. Since the O₂ consumptions such as dark respiration were reduced under LO, and the net photosynthesis significantly increased (Figure 2), Mehler reaction must have been active in maintaining low intracellular O₂ to sustain the activity of the nitrogenase and promote energetically expensive N₂ fixation. Additionally, it is most likely that the Trichodesmium cells grown under low O₂ could have greater abundances and/or upregulated activity of the nitrogenase (Zehr et al., 1993). Such physiological responses can be responsible for increased values of the N₂-fixation quotient under the low O₂ conditions (Figure 3C), indicating that the N₂-fixation efficiency increased per photosynthetically evolved O₂ or C fixed under low O₂ and high CO₂ conditions.

4.2. Combined effects of deoxygenation and acidification

Although the cells of Trichodesmium grown under HC increased their N₂ fixation under both ambient and lowered pO₂ (Figure 3), their specific growth rates were unaffected compared to the AC-grown cells (Figures 1, 2). This contradicts to some of the previously reported results (Table 2), which show either enhanced or inhibited growth rates of Trichodesmium grown under future ocean acidification conditions. The likely reason responsible for such discrepancies between these studies (Table 2) could be that different light sources (different emission spectra) regulate the diazotroph’s response to elevated pCO₂ even under equal levels of PAR (Yi and Gao, 2022), though such hypothetical explanation should be based on N₂-fixation action spectra, which has not been documented. In addition, limitation of phosphate and iron ions as well as exposure to solar UV radiation could also result in negative or insignificant effects of future ocean acidification on the N₂-fixation of Trichodesmium (Hong et al., 2017; Yi and Gao, 2022; Zhang et al., 2022). In the present study, when the Trichodesmium cells acclimated to the acidified HC treatment with replete phosphate and iron, its photosynthesis and growth did not show significant changes, though its N₂-fixation increased by 129% under ambient and by 38% under the reduced pO₂, respectively (Figure 3). In addition, the HC treatment increased its POC significantly under the LO (Figure 4), though its photosynthesis did not show significant change (Figure 2). Such inconsistency between the photosynthetic responses and the POC production could be attributed to altered photosynthetic quotients (ratios of evolved O₂ to CO₂ fixed). In the present work, gross photosynthetic rates changed less compared to the net photosynthetic rates, since dark respiration decreased when net photosynthesis increased under the different combinations of pO₂ and pCO₂. On the other hand, the documented photosynthetic quotients of Trichodesmium ranged 1.28–2.60 (Kranz et al., 2010; Boatman et al., 2019). Therefore, photosynthetic O₂ evolution rates can hardly be taken as a proxy of C fixation, which must be responsible for the observed discrepancy between the POC production and photosynthetic O₂ evolution.

Carbon and nitrogen fixation are both energy-intensive processes that compete directly for the products of photosynthesis (Berman-Frank et al., 2001; Hutchins et al., 2007). Previously, the enhancement of N₂ fixation as well as growth rate of Trichodesmium at elevated pCO₂ levels has been considered as an indirect effect resulting from alleviation of C-limitation of CO₂ fixation (Hutchins et al., 2007) or attributed to saved energy from down-regulation of CCMs (Table 2). However, Shi et al. (2012) and Hong et al. (2017) showed that the N₂ fixation rate of T. erythraeum significantly decreased under the elevated pCO₂ projected for future acidification, which was attributed to reduced efficiency of nitrogenase, although the expression of the nitrogenase was enhanced in terms of proteomic responses (Zhang et al., 2019; Wen et al., 2022). Our data did not show significant effects of the elevated pCO₂ on the growth rate and photosynthesis in T. erythraeum, but its N₂ fixation rate and POC/PON were enhanced (Figures 2–4). It is likely that the energy saved from downregulation of CMMs under HC was not enough to bring significant increase of net photosynthesis and growth rate (Luo et al., 2019). Considering that phytoplankton can benefit from the elevated pCO₂ under light but suffer from the acidic stress in darkness, resulting in daytime enhanced and nighttime suppressed growth rates (Qu et al., 2021), the net effects of the HC treatment on growth should be holistically considered for the diel cycle and availability of nutrients (Table 2). We suggest that extra energy required to cope with the acidic stress under HC could be provided via increased dark respiration (Figure 2), compensating for the insufficient energy supply from the downregulation of CCMs in Trichodesmium cells grown under HC. In the present work, lowered levels of O₂ enhanced photosynthesis and POC production of Trichodesmium (Figures 2, 4). This could be attributed to suppressed respiration (Figure 2), which enables the cells to save more POC (Figure 4A).

4.3. Discrepancy between the N₂ fixation and PON production

Since one time point measurement might have overlooked daily N₂ fixation, the rates of N₂ fixation in this work could hardly be approximated to that of PON production (Figures 3, 4). When the N₂ fixation rate was integrated for the daytime, it turned out that PON production rate (Supplementary Figure S3) was higher than the integrated N₂ fixation per day, which was about one third of the PON production rates. Similar discrepancy was also reported by Zhang et al. (2019), whose daily PON production rate was about 3 times the daily integrated N₂ fixation. In the present work, increased proportion (15%) of PON is much less than the enhancement (139%) of N₂ fixation under LO compared to AO under ambient CO₂ concentration (Figures 3, 4), such inconsistency could be contributed to the fact that PON production reflects how much new fixed N is ultimately incorporated into the cells which covers several generations, while N₂ fixation rate measured within 2 h during the midday could only reflect the periodical performance during light period. Future studies should examine the diurnal variations of the N₂ fixation to look into the diel or diurnal responses. Meanwhile, increased availability of CO₂ (Kranz et al., 2010; Hutchins et al., 2015) and elongated light period (Cai and Gao, 2015) could alter the maximal rate of N₂ fixation from the mid-light period, the low O₂ treatment might have led to different levels of N₂ fixation rates at different time points during the light period, which could also be responsible for the inconsistency. On the other hand, since the cells can release 10 to 50% of the fixed N as dissolved organic N (DON) and / or NH₄⁺ into seawater (Mulholland et al., 2004; Konno et al., 2010; Lu et al., 2018), the proportional increase of PON could be less. The exudation of “new-fixed” N may be further increased at elevated pCO₂ (Hutchins et al., 2007), which is mirrored in our study in that the elevated pCO₂ enhanced the N₂ fixation but not changed the cellular PON under the ambient O₂ level (Figures 3, 4).

4.4. Implications for future ocean deoxygenation and acidification

The global oxygen loss has been suggested to be about 2% of the total ocean inventory per decade since 1960 (Schmidtko et al., 2017) and the O₂ concentration of ocean has been predicted to decline to about 200 μmol L⁻¹ by the end of the century (−5 μmol kg⁻¹ per decade) (Breitburg et al., 2018). In addition, upwelling-induced hypoxia events have been shown in sunlit layers in time-series observations (Gireeshkumar et al., 2017), and typhon-driven mixing would churn deep seawater of low O₂ and high CO₂ to surface layer. Therefore, diazotrophs and other phytoplankton are inevitably exposed to extreme low O₂ and high CO₂ conditions. This study provided the experimental evidence that combination of lowered pO₂ and pH along with elevated pCO₂ enhanced the photosynthesis and N₂-fixation of Trichodesmium. Under the future scenarios of ocean deoxygenation and acidification, as estimated in the present work, Trichodesmium would decrease its dark respiration by 5% and increase its N₂-fixation by 49% and N₂-fixation quotient by 30% with 16% decline of pO₂ and 138% rise of pCO₂ by the end of this century (Supplementary Table S1; Figure 5). Such estimation might overestimate the changed percentage, since increased POC and PON only accounted for 10% and by 8%, respectively (Figure 4). As increased temperature would also enhance iron use efficiency (Jiang et al., 2018) and stimulate dark respiration and then favors N₂-fixation, decreased O₂ availability along with ocean warming can further enhance the activity of the nitrogenase and increased N₂-fixation of Trichodesmium, as long as ocean warming does not surpass the thermal tipping point for its growth.

Funding

This study was supported by National Natural Science Foundation of China (nos. 41721005, 41720104005, and 41890803).

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