Dataset: Synechococcus elongatus CCMP1629 in nitrate-limited and nutrient-replete cultures

The culture was grown in either a nitrate-limited or nutrient-replete continuous culture system on a 14:10 light:dark cycle of illumination at temperatures of 10, 15, 20, 25, and 30°C. The irradiance during the photoperiod was either 50 or 300 umol photons m–2 s–1. Photosynthetically active radiation (400–700 nm) was measured with a Biospherical Instruments model QSL 2100 quantum sensor. Temperature was controlled to within 0.1°C by circulating water from a Haake model DC10 temperature-controlled water bath through the outer jacket of the polycarbonate reaction chamber. The dilution rate of the growth chamber was controlled with a peristaltic pump (Masterflex Model 77200-60) to within ± 0.002 per day. The CO2 concentration in the laboratory was monitored with an infrared absorption-based CO2 meter (CO2METER.com, model AZ-004) calibrated at 0 and 400 ppm CO2 with a standard gas mixture.

Experiments were carried out at 400 uatm pCO2 from January 2016 until November 2017. Experiments at 1000 uatm pCO2 were carried out from September 2017 until November 2018. In each case, the first set of experiments was carried out under nutrient-replete conditions to determine what the nutrient-saturated growth rates were at the given irradiance, temperature, and pCO2. The nitrate-limited experiments at the same irradiance, temperature, and pCO2 were then initiated as soon as the nutrient-replete growth rate had been determined. The growth chambers were autoclaved glass reaction flasks. The nitrate-limited experiments were run in a growth chamber with a working volume of 2143 ±5 mL. The nutrient-replete experiments were run in an identical growth chamber but with a working volume of 1306 ±4 mL that was reduced by lowering the level of the overflow tube (see Figure S1). At a fixed medium pumping rate, the growth rates in the nutrient-replete system were therefore 2143/1306 = 1.64 times the growth rates in the nitrate-limited system. High growth rates could therefore be maintained in the nutrient-replete system without sterile filtering new medium reservoirs at frequent intervals.

When samples were not being collected from the growth chamber, air containing either 400 or 1000 ppm CO2 was bubbled into the growth chamber through a sterile air filter and a three-way valve, as shown in Figure 1.

When the chemostat was not being sampled, the three-way valve was turned so that the sterile air bubbled into the bottom of the growth chamber. Bubbling with sterile air created a pressure in the headspace that was sufficient to force medium through the overflow line. Sterile medium from the nutrient reservoir was continuously dripped into the growth chamber at a controlled rate by the peristaltic pump (Figure 1). When it was time to sample the chemostat, the overflow line was clamped, and pressure rapidly built up in the headspace. Within about 5 seconds, the three-way valve was then rotated 90° so that culture could flow up the aeration tube to the three-way valve and then out the collection tube. In this way samples could be collected from the growth chamber within a matter of seconds and without introducing non-sterile air into the headspace. After collection of a sample, the three-way valve was rotated back to its normal position and the clamp on the overflow line was removed.

Fig. 1, see Supplemental Files.

The system was judged to be in steady state when cell counts, measured with a Beckman Coulter model Z1 particle counter, had been reproducible to within ± 2% for at least 4 doubling times. Apart from measurements of cell counts, all sampling was done after the steady state condition had been reached. Chlorophyll a concentrations were determined from 50-mL samples collected on glass fiber (GF/F) filters and extracted in methanol. The absorbances were measured at 664 and 750 nm with a Cary Model 50 spectrophotometer. Samples were collected for particulate carbon (PC) and particulate nitrogen (PN) by filtering replicate 50-mL samples from the growth chamber onto GF/F glass fiber filters, which were then stored frozen. The filters were dried at 60°C for 24 hours prior to analysis with an Exeter Analytical model CE-440 elemental analyzer. The pH was measured with a Thermo Spectronic Helios spectrophotometer, as described in SOP 6b by Dickson, et al 2007 with minor modifications, and with a Hach SensION model PH31 pH meter calibrated with standards on the total pH scale, prepared as per Millero, et al. (Marine Chemistry 44[2]: 143–152 [1993]), with minor modifications.

The growth medium consisted of artificial seawater with a total alkalinity of 2365 µeq L–1. For the nitrate-limited chemostats, nutrient concentrations corresponded to f/2 medium, with the exception of nitrate, which was added at a concentration of 20 micromolar, and trace metals, which were added at the concentrations specified by Sunda and Hardison (Limnology & Oceanography 52[6]: 2496–2506 [2007]). For the nutrient-replete chemostats, the medium was identical, except that nitrate concentrations were 883 micromolar, the concentration specified for f/2 medium. The medium was sterile filtered (0.2 micron) into a 40-liter glass carboy that had been previously autoclaved. Cell concentrations in the nitrate-limited and nutrient-replete growth chambers were similar at the same irradiance, temperature, and pCO2. Therefore, nitrate concentrations in the growth chamber under nutrient-replete conditions were therefore in excess of 800 micromolar.

When operation of the chemostat had consumed most of the contents of one 40-liter carboy, another carboy was autoclaved and 40 liters of fresh medium were sterile filtered into it. The old carboy was then replaced with the new carboy using sterile techniques to make the necessary connection to the growth chamber. To ensure that there was no perturbation to the steady state in the growth chamber, carboys were never changed during sampling. Carboys were always changed between steady states. Sampling at a given steady state was never initiated unless there was sufficient medium in the carboy to complete all sampling at that steady state.

Table 1, see Supplemental Files.

Samples for analysis of particulate carbon, particulate nitrogen (i.e., CHN), and chlorophyll a were collected in triplicate every two hours from the start to the end of the 14-h photoperiod (a total of 7 time points). Because this sampling would have exceeded the dilution rate of the chemostat in all cases, sampling had to take place over a period as long as 12 days. The protocol required that the volume of culture withdrawn for a sample be replaced by fresh medium before another sample could be taken. At low growth rates, this criterion required that 50-mL samples be collected at 4-hour intervals. The sampling was therefore staggered on alternate days. For example, on day one, single samples were collected at times 0, 4, 8, and 12 hours, and on day two samples were collected at times 2, 6, 10, and 14 hours. Collecting triplicate samples for both CHN and chlorophyll a analysis in such cases required that sampling extend over a period of 12 days.

After the sampling for CHN and chlorophyll a had been completed, short-term (5-minute) photosynthesis versus irradiance curves were measured at the start, middle, and end of the photoperiod. For these experiments, triplicate 5-mL aliquots from the growth chamber were added to liquid scintillation vials pre-inoculated with 0.5 microcuries of C-14 bicarbonate. The vials were incubated at irradiances of 55, 80, 120, 150, 200, 250, 300, and 350 micro-mol photons m–2 s–1 for 5 minutes. Fixation was stopped by adding 0.5 mL of 1 N HCl to the vials, and samples were allowed to vent for 48 hours. The activity of C-14 in the samples was then determined by counting on a Packard Tri-Carb model 3100 TR liquid scintillation counter. Total alkalinity was determined using the open-cell titration method described in SOP 3b by Dickson et al. (2007), using a pH electrode and meter (Hach SensION model PH31). Briefly, 100 mL of culture was brought to 25°C and initially titrated to a pH of 3.50 with standardized HCl (Fisher) diluted to 0.10 N in 0.6 M NaCl. The solution was then stirred vigorously for 5–10 minutes. Afterwards, the solution was further titrated stepwise with 50-µL additions of the HCl solution to ultimately achieve a pH of 3.00. The total alkalinity was then calculated based on the relative potential of the electrode after each acid addition and on the cumulative volume of acid added at each step. DIC concentrations were then calculated from temperature, salinity, total alkalinity, and pH using the equations in Zeebe and Wolf-Gladrow, CO2 in Seawater: Equilibrium, Kinetics, Isotopes.

Three functions, a simple hyperbola, a negative exponential, and a hyperbolic tangent were assessed to determine which one best described the relationship between irradiance and photosynthetic rate. Based on a least squares criterion, the hyperbolic tangent (tanh) consistently gave the best description of the data.

P = Pmax*tanh(Ea/ Pmax)                                                                  (1)

Here, E is the irradiance with units of umol photons m–2 s–1, Pmax is the light-saturated photosynthetic rate with units of grams carbon per gram chlorophyll a per hour, and a is the initial slope of the photosynthesis-irradiance curve (i.e., the slope in the limit as E ® 0). Values of Pmax and a were determined by least squares (Figure S2).

Fig. 2, see Supplemental Files.

The calculated values of a were converted to units of m2 (carbons/photon) g−1 chl a

by multiplying by 106 to convert mmoles to moles, dividing by 3600 to convert hours to seconds, and dividing by 12 to convert grams of carbon to moles of carbon.

The dark-adapted Fv/Fm ratios were measured in triplicate for each continuous culture in steady state at the start, middle, and end of the photoperiod. Fv/Fm measurements were made with a PSI AquaPen C100 with the manufacturer’s supplied plastic cuvettes containing 4 mL of culture each. Dark-adaptation of the culture samples was achieved by wrapping each of three cuvettes in aluminum foil and incubating them for 30–40 minutes in the same thermoregulated water bath used to control the temperature of the growth chamber. Afterwards, Fv/Fm was measured in a darkened room.