Dataset: cyanobacteria N2 fixation
Deployment: lab_Hutchins_07-12_diazotrophs

The seven isolates were grown across a pCO2 range in triplicate steady-state semi-continuous cultures at 28 degrees C on a 12 h dark:12 h light cycle using cool white fluorescent bulbs at 120 µmol photons m-2 s-1, in 0.2 µm-filtered, microwave-sterilized artificial seawater enriched with 20 µM phosphate and Aquil trace metals and vitamins, but with no fixed nitrogen (Hutchins et al. 2007, Fu et al. 2008). Cultures were diluted every 2-3 days based on their growth rates, and were sampled when acclimation to experimental conditions was verified by statistically invariant growth rates over 10-15 generations. For Trichodesmium KO4-20, H9-4, and 2174 and Crocosphaera WH0401, WH0003 and WH8501 experimental CO2 concentrations were 100ppm, 190ppm, 280ppm, 750ppm, 1500ppm, and 2000ppm. The CO2 response curve for Trichodesmium GBR was obtained from data from a previous experiment (Hutchins et al. 2007) at concentrations of 150ppm, 370ppm, 750ppm, 1250ppm and 1500 ppm. Gentle bubbling with certified commercial air/CO2 mixtures (Praxair) was used to obtain these seawater concentrations, which were verified by dissolved inorganic carbon (DIC) and pH measurements (Hutchins et al. 2007, Fu et al. 2008). Triplicate preserved 25 mL DIC samples (200 µL 5% HgCl2) were stored in borosilicate flasks at 4 degrees C for <7 days until analysis. Total DIC was measured coulomb-metrically (model CM 140, UIC, Joliet, IL, USA) and pH was monitored daily using a microprocessor pH meter (NBS system) with three point buffer calibrations. Certified pCO2 values were verified using measured DIC and pH values with CO2 SYS software (http://cdiac.ornl.gov/ftp/co2sys/). Since measured values never deviated >1-2% from commercial certified values, pCO2 is reported as the latter value.

Rates of N2 fixation were measured at the same time of day for each culture (during the dark period for Crocosphaera and light period for Trichodesmium) with the acetylene reduction method using a 3:1 ratio to convert ethylene production to N2 fixation, and were normalized to culture chlorophyll a levels. Microscope counts were used to calculate cell-specific exponential growth rates (µ)( N_T=N₀e^uT, where N is the initial cell density, N_T is the cell density one day later, and T is one day) (Hutchins et al. 2007, Fu et al. 2008).

CO2 response curves for N2 fixation rates in each of the triplicate cultures for each isolate in each pCO2 treatment were fitted to Michaelis-Menten (1913) rectangular hyperbolic saturation equation curves (N2 fixation rate = Vmax * pCO2 / (K1/2 + pCO2)) using SigmaPlot software (SPSS), including determination of kinetic constants and curve correlation coefficients. Means and standard deviations of K1/2 (ppmv CO2) and Vmax (µmol N mg Chl a-1 h-1) values from the triplicate response curves are reported; significance of differences in K1/2 and Vmax values between isolates were tested using one-way ANOVA, followed by Fisher's Least Significant Difference to compare the mean of one group with the mean of another with SPSS statistics software (Hutchins et al. 2007, Fu et al. 2008).  Multi-variate Principle Coordinate Analysis (PCoA) and Hierarchical Clustering were used to analyze the variance between all treatments and replicates in order to group them using their K1/2 and Vmax values as metrics of their responses to the CO2 treatments (Ramette 2007).

References
Fu, F.-X., Mulholland, M.R., Garcia, N., Beck, A., Bernhardt, P.W., Warner, M.E., Sañudo-Wilhelmy, S.A. and Hutchins, D.A.  2008. Interactions between changing pCO2, N2 fixation, and Fe limitation in the marine unicellular cyanobacterium Crocosphaera. Limnology and Oceanography 53 (6): 2472- 2484. DOI: 10.4319/lo.2008.53.6.2472

Hynes, A.M., Webb, E.A., Doney, S.C., and Waterbury, J.B. 2012. Comparison of cultured Trichodesmium (Cyanophyceae) with species characterized from the field. Journal of Phycology 48: 196-210. DOI: 10.1111/j.1529-8817.2011.01096.x

Hutchins, D. A., Fu, F.-X., Zhang, Y., Warner, M. E., Feng, Y. et al.  2007. CO2 control of Trichodesmium N2 fixation, photosynthesis, growth rates, and elemental ratios:  Implications for past, present, and future ocean biogeochemistry. Limnology and Oceanography 52: 1293-1304. DOI: 10.4319/lo.2007.52.4.1293

Michaelis, L., and Menten, M. M. 1913. Die Kinetik der Invertinwirkung. Biochem. Z. 49: 333–369.

Ramette, Alban. 2007. Multivariate analyses in microbial ecology. FEMS Microbiol. Ecol. 62: 142–160. DOI: 10.1111/j.1574-6941.2007.00375.x

Webb, E. A., Ehrenreich, I. M., Brown, S. L., Valois, F. W., and Waterbury, J. B. 2009. Phenotypic and genotypic characterization of multiple strains of the diazotrophic cyanobacterium, Crocosphaera watsonii, isolated from the open ocean. Environmental Microbiology 11:338-348. DOI: 10.1111/j.1462-2920.2008.01771.x