Table of Contents

• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Cyanobacteria N2 fixation responses to pCO2 using four Trichodesmium isolates representing three species and three Crocosphaera watsonii strains, isolated from locations across the North and South Pacific and Atlantic Oceans (Webb et al. 2009, Hynes et al. 2012) and spanning the taxonomic diversity within each genus.

Detailed methods and results will be available in the following publication (see Figures 1 and 2):
Hutchins, D.A., Fu, F.-X., Webb, E.A. and Tagliabue, A.  (In press). Taxon-specific response of marine nitrogen fixers to elevated carbon dioxide concentrations. Nature Geoscience.

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

BCO-DMO re-arranged data formatted as separate tables into one dataset. Parameter names were changed to conform with BCO-DMO conventions.

From NSF award abstract:

The importance of marine N2 fixation to present ocean productivity and global nutrient and carbon biogeochemistry is now universally recognized. Marine N2 fixation rates and oceanic N inventories are also thought to have varied over geological time due to climate variability and change. However, almost nothing is known about the responses of dominant N2 fixers in the ocean such as Trichodesmium and unicellular N2 fixing cyanobacteria to past, present and future global atmospheric CO2 regimes. Our preliminary data demonstrate that N2 and CO2 fixation rates, growth rates, and elemental ratios of Atlantic and Pacific Trichodesmium isolates are controlled by the ambient CO2 concentration at which they are grown. At projected year 2100 pCO2 (750 ppm), N2 fixation rates of both strains increased 35-100%, with simultaneous increases in C fixation rates and cellular N:P and C:P ratios. Surprisingly, these increases in N2 and C fixation due to elevated CO2 were of similar relative magnitude regardless of the growth temperature or P availability. Thus, the influence of CO2 appears to be independent of other common growth-limiting factors. Equally important, Trichodesmium growth and N2 fixation were completely halted at low pCO2 levels (150 ppm), suggesting that diazotrophy by this genus may have been marginal at best at last glacial maximum pCO2 levels of ~190 ppm. Genetic evidence indicates that Trichodesmium diazotrophy is subject to CO2 control because this cyanobacterium lacks high-affinity dissolved inorganic carbon transport capabilities. These findings may force a re-evaluation of the hypothesized role of past marine N2 fixation in glacial/interglacial climate changes, as well as consideration of the potential for increased ocean diazotrophy and altered nutrient and carbon cycling in the future high-CO2 ocean.

We propose an interdisciplinary project to examine the relationship between ocean N2 fixing cyanobacteria and changing pCO2. A combined field and laboratory approach will incorporate in situ measurements with experimental manipulations using natural and cultured populations of Trichodesmium and unicellular N2 fixers over range of pCO2 spanning glacial era to future concentrations (150-1500 ppm). We will also examine how effects of pCO2 on N2 and C fixation and elemental stoichiometry are moderated by the availability of other potentially growth-limiting variables such as Fe, P, temperature, and light. We plan to obtain a detailed picture of the full range of responses of important oceanic diazotrophs to changing pCO2, including growth rates, N2 and CO2 fixation, cellular elemental ratios, fixed N release, photosynthetic physiology, and expression of key genes involved in carbon and nitrogen acquisition at both the transcript and protein level.

This research has the potential to evolutionize our understanding of controls on N2 fixation in the ocean. Many of our current ideas about the interactions between oceanic N2 fixation, atmospheric CO2, nutrient biogeochemistry, ocean productivity, and global climate change may need revision to take into account previously unrecognized feedback mechanisms between atmospheric composition and diazotrophs. Our findings could thus have major implications for human society, and its increasing dependence on ocean resources in an uncertain future. This project will take the first vital steps towards understanding how a biogeochemically-critical process, the fixation of N2 in the ocean, may respond to our rapidly changing world during the century to come.