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

The marine cyanobacterium Synechococcus elongatus clone CCMP1629 was grown in continuous culture systems to study the interactive effects of temperature, irradiance, nutrient limitation, and the partial pressure of CO₂ (pCO₂) on its growth and physiological characteristics. The culture was grown in either a nitrate-limited or nutrient-replete continuous culture system on a 14:10 light:dark cycle of illumination with light provided by a bank of fluorescent lamps. The irradiance on the growth chamber during the photoperiod was either 300 or 50 micro mol quanta per square meter per second. 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 thermoregulated water bath through the outer jacket of the polycarbonate reaction/growth chamber. Experiments were carried out in 5°C increments from 20°C to 45°C. The dilution rate of the growth chamber was controlled with a peristaltic pump (Masterflex Model 77200-60) to within ± 0.002 per day. The partial pressure of CO₂ was regulated by bubbling air with the desired partial pressure of CO₂ (either 400 or 1000 ppm) into the growth chamber. The pCO₂ in the growth medium was calculated from measurements of pH and total alkalinity. 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.

Initial experiments were carried out beginning January 2016 through December 2017 under nutrient-replete conditions to determine what the nutrient-saturated growth rates were at the given irradiance, temperature, and pCO₂. Once that growth rate had been determined, the nitrate-limited experiments were initiated with the same factors. 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 1, Supplemental Files section). 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. 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.

Cell counts were measured every day at the same time during the photoperiod with a Beckman Coulter model Z1 particle counter. The system was judged to be in steady state when cell counts 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 measured every 2 hours during the photoperiod. Triplicate 50-mL samples from the growth chamber were filtered onto glass fiber (GF/F) filters and extracted in methanol. The absorbances were measured at 664 and 750 nm with a Cary Model 50 spectrophotometer. Triplicate samples were likewise collected at 2-hour intervals during the photoperiod for particulate carbon (PC) and particulate nitrogen (PN) by filtering 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 sampling for chlorophyll and CHN samples extended over as many as 12 days because we required that the amount of sample withdrawn from the growth chamber never exceed the amount of fresh medium that would be added to the growth chamber during the subsequent two-hour interval. In some cases, this required taking one 50-mL sample every 4 hours, in which case collecting 3 chlorophyll samples and 3 CHN samples every two hours required sampling for a total of 12 days. On odd days one 50-mL sample was collected at times 0, 4, 8, and 12 hours after the start of the photoperiod and on even days at times 2, 6, 10, and 14 hours after the start of the photoperiod.

The growth medium consisted of artificial seawater with a total alkalinity of 2365microequivalents per liter (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 (2007). We found that the biomass produced with 20 micromolar nitrate in the nutrient reservoir was sufficiently low that the pCO₂ in the growth chamber did not deviate by more than ±10% from the targeted partial pressures. 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 in excess of 800 micromolar.

When operation of the chemostat had consumed most of the contents of one 40-liter reservoir, another carboy was autoclaved, and 40 liters of fresh medium were sterile filtered into it. The reservoir-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, carboys were only (and always) changed between steady states, e.g. sampling at a given steady state was never initiated unless there was sufficient medium in the carboy to complete all sampling for  that steady state. 

After the sampling for CHN and chlorophyll a had been completed at a given steady-state growth rate, additional samples were collected during the next three days so that short-term (5-minute) photosynthesis-versus-irradiance curves could be 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. A platform was set up in front of a bank of fluorescent lamps, and 20-mL glass vials were positioned on the platform at varying distances from the lamps so as to achieve irradiances of 55, 80, 120, 150, 200, 250, 300, and 350micromole photons per square meter per sec (µmol photons m−2 s−1), as measured with a quantum scalar light meter (Biospherical Instruments model QSL 2100). Three replicate vials were placed at each irradiance position. To account for background activity and any dark uptake, an additional three vials were wrapped in aluminum foil but otherwise treated in the same manner as the exposed vials. To each vial, a fixed volume (80–100 µL) of a 4.5-mL solution containing approximately 20 µCi of ¹⁴C (40–60 µCi/µmol NaH¹⁴CO₃; Perkin Elmer) at pH 12 was added. At the beginning of each assay, a 5.0-mL aliquot of culture was added to each vial in series, 5 seconds apart. The timing was controlled using a stopwatch, and each vial was incubated for a total of 5 minutes. At the conclusion of the 5-minute incubation, 0.5 mL of 1 N HCl was added to each vial to halt photosynthesis and convert any non-assimilated ¹⁴C to CO₂. The uncapped vials were then allowed to vent for a further 48 hours, after which 10 mL of scintillation cocktail (Ultima Gold XR, Perkin Elmer) was added. Additionally, to determine the total ¹⁴C activity that was added to each vial, the same volume of ¹⁴C solution was combined with 5.0 mL of filtered artificial seawater and 0.5 mL of 0.1 N NaOH, and the resulting solution was immediately capped. After addition of scintillation cocktail, the activity in each vial was measured for 5 minutes on a scintillation counter (Tri-Carb 3100TR, Perkin Elmer). Background activity was determined based on duplicate solutions containing 5 mL of filtered artificial seawater and 10 mL scintillation cocktail.

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. (1993), with minor modifications. 

[See the notepad doc for details and revisions]

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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.

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The cells were grown in either a nutrient-replete mode or nitrate-limited. For the nitrate-limited studies, the concentration of nitrate in the nutrient reservoir was 40 micro molar. The growth medium was artificial seawater enriched with f/2 nutrients with the exception of nitrate for the nitrate-limited studies. The medium was sterile filtered into a 40-liter nutrient reservoir and pumped into the growth chamber at a controlled rate with a peristaltic pump.

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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.

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.