in

# Enhancement of diatom growth and phytoplankton productivity with reduced O2 availability is moderated by rising CO2

### Field studies

Photosynthetic carbon fixation was investigated at eight different stations in the Pearl River estuary of the South China Sea (Fig. 1a and Supplementary Table 1), where the phytoplankton assemblages were dominated by diatoms45 during the time of our investigation (June 2015). Samples were collected from 10 to 20 m depths and transferred immediately into 50 mL quartz tubes and sealed to prevent gas exchange. The samples were inoculated with 100 μL of 5 μCi (0.185 MBq) NaH14CO3 solution for 2.15 h. All the incubations were carried out under incident solar radiation, attenuated with neutral density filters to simulate light intensities at the sampling depths, and the temperature was controlled with flow-through surface seawater.

After incubation, the cells were filtered onto glass-fiber filters (25 mm, Whatman GF/F, USA) and stored at −20 ° C until measurement, during which the filters were exposed to HCl fumes overnight and dried (20 °C, 6 h) to remove unincorporated NaH14CO3 as CO2. The incorporated radioactivity was measured by liquid scintillation counting (LS 6500, Beckman Coulter, USA), and photosynthetic carbon fixation rates were estimated as previously reported46. Since the measurements were carried out under varying and low light levels similar to in situ levels at depths of 10 and 20 m, we normalized the photosynthetic rates to light intensity (μmol C (μg Chl a)−1 h−1 (μmol photons m−2 s−1)−1) to obtain the light use efficiency of photosynthesis (PLUE). This was done to allow for a meaningful comparison among different stations according to the linear relationship of photosynthetic carbon fixation under low solar irradiance levels46, which lies within the range of sunlight levels used in the present fieldwork (<100 μmol photons m−2 s−1).

Field DO, chlorophyll a (Chl a) concentration and nutrients were measured as described previously5,47. Briefly, field DO was manually measured on board using the Winkler titration method48. The Chl a content was measured with a Turner Designs Model 10 Fluorometer. The nitrogen (NOX, NO3 + NO2), NH4+, and SiO32− concentrations were measured with a nutrient-autoanalyzer (Quickchem 8500, Lachat Instruments, USA) following the description of Kirkwood et al.49. This equipment has detection limits of 0.014 and 0.075 μM for NOX and SiO32−, respectively.

Dissolved inorganic carbon (DIC) concentrations at investigated stations were estimated based on measured salinity and the relationship between salinity and DIC concentrations in the published literature50 in the same area of the Pearl River estuary during the same season. CO2 concentration and pHT were calculated using CO2SYS software51, using the equilibrium constants K1 and K2 for carbonic acid dissociation52.

### Mesocosm studies

Surface seawater (0–1 m) with natural plankton assemblages was sampled from a harbor near the Dongshan Swire Marine Station of Xiamen University (23.65o N, 117.49o E) with an acid-cleaned plastic bucket, filtered (180 μm) to remove large grazers, and transported to the station within 1 h. The incubation system used 30-liter cylindrical polymethyl methacrylate tanks (n = 3), which allowed 91% PAR transmission and were water-jacketed for temperature control with a re-circulating cooler (running water). We set two O2 and two CO2 levels with three pO2:pCO2 combinations: (1) ambient O2 (AO, ~213 μM) & ambient CO2 (AC, ~13 μM), AOAC; (2) low O2 (LO, ~57 μM) & ambient CO2, LOAC; (3) low O2 & high CO2 (HC, ~27 μM), LOHC. The presented O2 and CO2 concentrations are average values across the entire experiment. N2, CO2, and air were mixed proportionally to create different and stable pO2:pCO2 combinations in the gas stream. The incubation tanks were continuously aerated (0.5 L min−1) under incident solar radiation. The O2 concentration was measured (20:00) with a precise single-channel fiber optic oxygen sensor (Microx 4, PreSence, Germany) every day. CO2 concentrations of seawater were calculated from daily measured pHNBS (20:00) and TA measured every other day using CO2SYS software. The pH was determined according to Dickson (2010)53 with a high-quality pH meter (Orion StarA211, Thermo, USA) which was calibrated with standard National Bureau of Standards (NBS) buffer solutions (Hanna). The pHNBS values were converted to pHTotal (pHT) using the CO2SYS software as described above.

For nutrient measurements, water samples were stored in 80-mL polycarbonate bottles, instantly frozen, and stored at −20 °C until analysis. Samples for silicate determination were fixed with 1‰ chloroform and preserved at 4 °C. Nutrients were measured with an AA3 Auto-Analyzer (Bran-Luebbe, GmbH, Germany) with detection limits of 0.08, 0.08, and 0.16 μM for NOX, PO43−, and SiO32−, respectively.

Samples for analysis of Chl a and other pigments were filtered onto glass-fiber filters (25 mm, Whatman GF/F, USA) which were immediately preserved in liquid nitrogen until analysis. Measurement was conducted with a high-performance liquid chromatography system (UltiMate 3000, ThermoFisher Scientific, USA) after filters were submerged in N, N-dimethylformamide and then mixed 1:1 (V:V) with 1-M ammonium acetate54. Chlorophyll a and other pigments were identified by their retention times and quantified using peak areas and standard curves. Quantification was performed with standards purchased from DHI Water & Environment, Hørsholm, Denmark. Chemotaxonomic analysis was carried out using CHEMTAX software55,56.

To measure gross and net primary productivity, respectively, seawater samples were inoculated with 200 μL of 10 μCi (0.37 MBq) NaH14CO3 solution (ICN Radiochemicals, USA) for 2 h (gross) and with 100 μL of 5 μCi (0.185 MBq) NaH14CO3 solution for 24 h (net). All the incubations were carried out under incident solar radiation in a flow-through water bath to obtain a uniform temperature. Photosynthetic carbon fixation rates in the mesocosm experiment were estimated as described above.

Photosynthetic fluorescence parameters were measured with a fluorescence induction and relaxation system (In-Situ FIRe, Satlantic, NS Canada). NPQ was estimated by the equation of Genty et al.57:

$${{{{{rm{NPQ}}}}}}=({{{{{{rm{F}}}}}}}_{{{{{{rm{md}}}}}}}{-{{{{{rm{F}}}}}}}_{{{{{{rm{m}}}}}}}hbox{‘}){/{{{{{rm{F}}}}}}}_{{{{{{rm{m}}}}}}}hbox{‘} ,$$

(1)

where Fmd is the maximal fluorescence measured before sunrise and Fm’ is the effective yield at 11:00 a.m. under incident sunlight.

### Diatom culture studies

The diatom Thalassiosira weissflogii (CCMP 1336) was incubated in artificial seawater prepared according to the Aquil* medium recipe58, and was cultured semi-continuously in polycarbonate bottles. Cultures were incubated at 20 °C in a plant growth chamber (HZ100LG, Ruihua, Wuhan, China) and illuminated with cool white fluorescent light at 200 μmol photons m−2 s−1 (measured by a US-SQS/WB spherical micro quantum sensor; Walz, Germany) with a 12:12 h light:dark cycle. The maximum cell concentration was maintained below 5000 cells mL-1 by diluting the cultures every 24 h with newly prepared medium, equilibrated with the target O2 and CO2 levels, in order to maintain a stable range of dissolved O2 (DO) and carbonate chemistry in the culture without aeration (Supplementary Fig. 3). To avoid the cells settling, the bottles were shaken gently every 2 h during the daytime (0800–2000).

The diatom cells were acclimated to four treatments with two levels of CO2 (ambient and high CO2) and two levels of O2 (ambient and low O2), respectively. In order to create the ambient O2 & ambient CO2 seawater (AOAC, ~254 μM O2, ~15 μM CO2) or ambient O2 & high CO2 seawater (AOHC, ~256 μM O2, ~33 μM CO2), we aerated the medium with ambient air or CO2-enriched air using a CO2 enricher (CE-100, Ruihua, Wuhan, China). In order to maintain low O2 conditions and to sustain constant carbonate chemistry, pure nitrogen was introduced into the headspace of bottles containing seawater with different CO2 concentrations, so that the O2 in the water was displaced, and reduced O2 & ambient CO2 (LOAC, ~58 μM O2, ~14 μM CO2) or reduced O2 & high CO2 (LOHC, ~56 μM O2, ~36 μM CO2) conditions were achieved (Supplementary Fig. 3e, f and Supplementary Table 6).

The dissolved O2 and pH of seawater were measured before and after diluting the culture medium (Supplementary Fig. 3 and Supplementary Table 6). The dissolved O2 was measured with a Clark-type oxygen electrode (Hansatech, UK). Parameters of the seawater carbonate system (Supplemental Table 6) were calculated from pH and TA with CO2SYS software, and the pHNBS values were converted to pHTotal (pHT) using the CO2SYS software as described above. Photosynthesis vs CO2 curves (n = 3) and other parameters (n = 3) were obtained from two separate experiments under the same experimental conditions after the cells had acclimated for at least nine generations (see Supplemental Fig. 3 for detail).

Cell concentrations were measured with a Counter Particle Count and Size Analyzer (Z2, Beckman Coulter, USA) before and after the dilutions every 24 h. The cells had acclimated for at least nine generations before the growth rate was measured. The specific growth rate (μ, d−1) was calculated as

$${{{{{rm{mu }}}}}}=({{{{{{rm{lnN}}}}}}}_{1}-{{{{{{rm{lnN}}}}}}}_{0})/({t}_{1}{-t}_{0}),$$

(2)

where N1 and N0 represent cell concentrations at t1 (before the dilution) and t0 (initial or just after the dilution), respectively.

A Clark-type oxygen electrode was used to measure mitochondrial respiration (after acclimation for ~13 generations) under the conditions of pH, O2 levels, and temperature used for growth, and the oxygen consumption rates were monitored in the dark (~10 min). About 6–8 × 105 cells were harvested by gentle vacuum filtration (<0.01 MPa) onto polycarbonate membrane filters (1.2 μm, Millipore, Germany). These cells were then re-suspended in seawater (2 mL) buffered with 20 mM Tris (without introducing additional DIC into media, pHT = 8.00 for AC of 14 μM and pHT = 7.70 for HC of 34 μM) to maintain stable pH in the media. Tris-buffered seawaters were flushed with pure nitrogen and ambient air to achieve the culture O2 levels.

During the measurements of photosynthetic O2 evolution and photorespiration, 5–6 × 105 cells were harvested after acclimation for ~18 generations and re-suspended as above. Photosynthetic O2 evolution was tested under growth O2 levels (~255 μM for AO and ~57 μM for LO), and photorespiration (Supplementary Fig. 5) was estimated as the difference in photosynthetic O2 evolution of the cells under reduced (~25 μM) and culture (~255 μM for AO and ~57 μM for LO) O2 conditions, an approach which has been used widely26,59. However, this method might have overestimated the absolute value of photorespiration to some extent because of the ignored mitochondrial respiration rates at different O2 levels. Therefore, we re-estimated the photorespiration (Fig. 6c) using the differences of dark-respiration rates between the samples measured under ~25 μM O2 and growth O2 conditions (~255 μM O2 for AO and ~57 μM O2 for LO), assuming that the mitochondrial respiration rates for the cells grown under the treatments were the same under light and darkness. To obtain the reduced or ambient levels of O2, pure nitrogen gas or ambient air were bubbled into Tris-buffered seawater (20 mM, pHT = 8.00 for AC of about 14 μM and pHT = 7.70 for HC of about 34 μM). Light intensity and temperature were the same as in the growth experiment.

Inhibition of photosynthetic O2 evolution by acetazolamide (AZ)60, an inhibitor of periplasmic carbonic anhydrase (eCA), was determined with a Clark-type oxygen electrode under culture conditions. We added the AZ dissolved in 0.05 mM NaOH at a final concentration of 100 μM; an equal amount of 0.05 mM NaOH was added as a control treatment. The cells used for this test had been acclimated to the growth O2 and CO2 levels for about ten generations, and ~5 × 105 cells were harvested and re-suspended in 2 mL seawater buffered with 20 mM Tris to maintain the CO2 partial pressures as mentioned above. O2 levels were achieved and controlled as above.

The photosynthesis vs CO2 curves was determined with a Clark-type oxygen electrode under standard conditions commonly used for CCM studies19. Approximately 4–10 × 105 cells were harvested as above after acclimation for approximately nine generations and were re-suspended in DIC-free seawater (2 mL) medium buffered with 20 mM Tris (pHT = 8.00). The concentrations of DIC in the seawater were then adjusted by adding sodium bicarbonate solution, and the final DIC concentration reached to 8 mM. DIC (μM) values were converted to CO2 (μM) with CO2SYS software. All the cells from different treatments were measured under the same standard conditions (pHT = 8.00, light intensity = 400 μmol photons m−2 s−1, O2 was in the range of 50–200 μM, and the temperature was controlled at 20 ± 0.1 °C). CO2 acquisition efficiency was calculated as

$${{{{{rm{CO}}}}}}_{2};{{{{{rm{acquisition}}}}}};{{{{{rm{efficiency}}}}}}={V}_{{{{{{rm{max}}}}}}}/{K}_{0.5}({{{{{{rm{CO}}}}}}}_{2}),$$

(3)

where Vmax and K0.5 were calculated by fitting the photosynthetic O2 evolution rates at various CO2 concentrations with the Michaelis–Menten formula.

Measurements of chlorophyll fluorescence parameters were carried out with a pulse amplitude modulated (PAM) fluorometer (XE-PAM, Walz, Effelrich, Germany) after the cells had acclimated for ~12 generations. Effective photosystem II (PSII) quantum yield of photosystem (Yield) was measured with an actinic light level of 226 μmol photons m−2 s−1 (similar to that of the culture level). Nonphotochemical quenching (NPQ) was also measured at this actinic light intensity.

Approximately 5–8 × 105 cells were harvested (~18 generations) for measuring elemental composition. Particulate organic carbon (POC) and particulate organic nitrogen (PON) were determined by filtering cells on the pre-combusted (450 °C for 6 h) GF/F filters (25 mm, Whatman), storing at −80 °C before measuring. Filters were treated with HCl fumes to remove inorganic carbon and dried before analysis on a CHNS elemental analysizer (vario EL cube, Elementar, Germany). Biogenic silica (BSi) was determined by the spectrophotometric method61, and the cells were harvested onto Polycarbonate filters (1.2 μm, Millipore, Germany). Production of POC, PON, and BSi was calculated by multiplying the cellular content by specific growth rate.

### Statistics and reproducibility

The data are expressed in raw form, or presented as means ± standard deviation (SD) with n = 3 (triplicate cultures or mesocosms). We used one-way ANOVA to assess significant differences among the treatments. Prior to analyses, data were checked for homoscedasticity. If required, data were Ln transformed, and then LSD test was used for post hoc investigation. If the data, even after transformation, did not meet the assumption for equal variance, Games–Howell tests were chosen for post hoc investigation. Linear fitting analysis was conducted with Pearson correlation analysis (two-tailed). Partial Correlation Analysis was employed to explore the net correlation between DO and photosynthetic light use efficiency in the Pearl River estuary investigation. Parameters including pHT, cultured temperature, DIN, SiO32−, DIC, and CO2 were under control. A 95% confidence level was used in all analyses.

### Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Source: Ecology - nature.com