Dataset: Nitrogen and carbon fixation rates and ratios

The Trichodesmium erythraeum GBR strain used in this project was a tropical strain collected and isolated from the Great Barrier Reef (Fu and Bell 2003). In this study, the cultures were maintained with autoclaved artificial seawater by adding phosphate (10 μM), vitamins and trace metals as suggested by Aquil recipe (Garcia et al. 2011). Cool white fluorescent bulbs were used to provide a 12h dark: 12h light cycle at 150 μmol photons m-2s-1. Cultures were grown in acid-washed 120-ml plastic jars fitting into the thermal block that provides an even temperature gradient.

According to Fu et al. (2014), the temperature limit of this strain is 18-32°C, while the optimal growth range is 24-28°C. Within the optimal range, the growth of Trichodesmium erythraeum was at plateau stage, therefore, thermal variations that fall wholly within this range was expected to have negligible effects. 22°C and 30°C represents the “cold” and “warm” phases of the variation cycle. For each constant temperature, one or two variable treatments were used simultaneously, each with an average temperature equal to the corresponding constant temperature. There was an “intense” 18-26°C variable treatment and a “mild” 20-24°C variable treatment for 22°C. For 30°C, only one variable treatment “28-32°C” was used, because an “intense” one periodically exceeding the strain’s upper temperature limit was likely to kill the cultures.

For all the treatments, semi-continuous incubation methods were applied and dilution was conducted every four days. In each 4-day cycle, the first 48 hours of variable treatments were at a lower temperature (respectively 18, 20 and 28°C) and the second 48 hours were at a higher temperature (respectively 26, 24 and 32°C). In order to investigate the interactions between phosphate availability and thermal variation, there were triplicate bottles for phosphorus-replete (10 μmol/L) and phosphorus-limiting (0.2 μmol/L) conditions under the 5 constant and variable temperature treatments above.

Semi-continuous incubation was maintained until steady state was reached. Data on specific growth rates, nitrogen and carbon fixation rates were collected and analyzed. There were three sampling points in each cycle: the initial point (0 hour after the dilution and transfer to LT phase), the middle point (48 hours after the dilution, end of LT phase) and the final point (96 hours after the dilution, end of HT phase). For variable temperature treatments, nitrogen and carbon fixation data at the middle and final points and the average values of these two phases were compared to the corresponding data from the constant treatment.

Nitrogen fixation measurements. N2-fixation rate was determined using the Acetylene Reduction method (Capone 1993) by gas chromatography with a Shimadzu gas chromatograph GC-8a (Shimadzu Scientific Instruments). 10 ml of culture was added to a 27 ml serum vial. The vial was then air-tighten and 2 ml air was extracted. Then 2 ml of acetylene (C2H2) was added to the headspace of each vial. There is a theoretical 3:1 ratio (mol C2H2 to mol N2 reduced) to calculate the N2 fixation based on the rates of ethylene production (Montoya et al. 1996). Ethylene production was measured by injecting 200 μl of headspace to the GC device at 4-5 h intervals over the entire 12 h light period (Tuit et al. 2004). For each treatment, 3-6 replicates were incubated under the same light and temperature conditions. After total N2-fixation rates are measured, the cell count, PON and POC of each sample were measured and used to normalize N2-fixation rates.

C fixation rates. To measure uptake rates of carbon and iron, 0.2 μCi 14C-NaH14CO3 was added to 30 ml subsamples from each replicate (specific activity for final solutions was roughly 0.25 kBq/ml; PerkinElmer). The background dissolved inorganic carbon in the medium was determined by DIC measurements. Samples were then incubated for 24 h or select time under their respective experimental conditions, and filtered onto GF/F filters. To correct for filter adsorption, 30 ml of cultures from each treatment (10ml from each replicate bottle) was filtered immediately after adding equal amounts of NaH14CO3. All filters were rinsed with artificial seawater. Filters were then placed in 7 ml scintillation vials in the dark overnight after adding 4 ml scintillation fluid. To determine the total radioactivity (TA), 1 μCi 14C-NaH14CO3 together with 100 μl Phenylalanine was placed in identical scintillation vials with the addition of 4 ml scintillation solution. 14C radioactivity was measured using liquid scintillation counting (Perkin Elmer) for TA, blanks and samples (Xu et al. 2014).

Elemental stoichiometry. Elemental ratios were obtained by measuring particulate organic carbon and nitrogen (POC and PON).  For particulate organic carbon and nitrogen (POC and PON), a large volume (determined by the biomass) was filtered onto a pre-combusted (500 °C, 2-3 h) GF/F filter, which was then wrapped in aluminum foil and dried at 55 °C. POC and PON were analyzed on a Costech Elemental Analyzer using methionine and atropine as references to calibrate the system at the beginning and between measurements of every twelve samples (Fu et al. 2007).

Methodology References:

Fu, F., & Bell, P. (2003). Factors affecting N2 fixation by the cyanobacterium trichodesmium sp. GBRTRLI101. FEMS Microbiology Ecology, 45(2), 203-209.

Fu, F., Warner, M. E., Zhang, Y., Feng, Y., & Hutchins, D. A. (2007). Effects of increased temperature and CO2 on photosynthesis, growth, and elemental ratios in marine Synechococcus and Prochlorococcus (cyanobacteria) 1. Journal of Phycology, 43(3), 485-496.

Fu, F., Yu, E., Garcia, N. S., Gale, J., Luo, Y., Webb, E. A., & Hutchins, D. A. (2014). Differing responses of marine N2 fixers to warming and consequences for future diazotroph community structure. Aquatic Microbial Ecology, 72(1), 33-46.

Garcia, N. S., Fu, F., Breene, C. L., Bernhardt, P. W., Mulholland, M. R., Sohm, J. A., & Hutchins, D. A. (2011). Interactive effects of irradiance and CO2 on CO2 fixation and N2 fixation in the diazotroph Trichodesmium erythraeum (cyanobacteria) 1. Journal of Phycology, 47(6), 1292-1303.

Ihnken, S., Roberts, S., & Beardall, J. (2011). Differential responses of growth and photosynthesis in the marine diatom Chaetoceros muelleri to CO2 and light availability. Phycologia, 50(2), 182-193.

Tuit, C., Waterbury, J., & Ravizza, G. (2004). Diel variation of molybdenum and iron in marine diazotrophic cyanobacteria. Limnology and Oceanography, 49(4), 978-990.

Xu, K., Fu, F., & Hutchins, D. A. (2014). Comparative responses of two dominant Antarctic phytoplankton taxa to interactions between ocean acidification, warming, irradiance, and iron availability. Limnology and Oceanography, 59(6), 1919-1931.