Table of Contents

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

Trichodesmium erythraeum growth rates per day for all replicates in each treatment calculated from cell counts. Trichodesmium erythraeum was grown in biological triplicate under two CO2 regimes (380 and 750 uatm pCO2) and four different nutrient regimes (replete, phosphorus-limited,iron-limited, iron/phosphorus colimited). 

These data were utilized in the following publications:

Walworth, N. G., Fu, F. X., Webb, E. A., Saito, M. A., Moran, D., Mcllvin, M. R. & Hutchins, D. A. (2016a). Mechanisms of increased Trichodesmium fitness under iron and phosphorus co-limitation in the present and future ocean. Nature Communications, 7, 12081. doi: 10.1038/ncomms12081

Walworth, N. G., Lee, M. D., Suffridge, C., Qu, P., Fu, F., Saito, M. A., ... & Hutchins, D. A. (2018). Functional genomics and phylogenetic evidence suggest genus-wide cobalamin production by the globally distributed marine nitrogen fixer Trichodesmium. Frontiers in Microbiology, 9, 189. doi: 10.3389/fmicb.2018.00189

Genetic sequences from the Trichodesmium erythraeum used in this experiment can be accessed at The National Center for Biotechnology Information (NCBI) under the BioProject PRJNA312342.

Walworth, N. G., Lee, M. D., Fu, F. X., Hutchins, D. A., ... & Webb, E. A.(2016b). Molecular and physiological evidence of genetic assimilation to high CO2 in the marine nitrogen fixer Trichodesmium. Proceedings of the National Academy of Sciences, 113(47), E7367-E7374. doi: 10.1073/pnas.1605202113

Related Dataset: Trichodesmium proteomes and Hi CO2 adaptation

The following are excerpts from the Nature Communication paper Walworth et al. 2016a. Please refer to this reference for more details of the methodology used to generate these data.

"We grew the nitrogen-fixing cyanobacterium Trichodesmium for 1 year under Fe/P co-limitation following 7 years of both low and high CO2 selection. "

"Growth rates were calculated according to microscopic cell counts for reported values, or using in vivo chlorophyll fluorescence measurements with a Turner 10 AU fluorometer for semi-daily dilution calculations in real time during the experiments. Comparisons between cell counts and in vivo-based growth rates revealed no significant differences between the two methods of assessing biomass changes." 

A picogram carbon/µm filament was used for all replicates in each treatment (cell size proxy).

Description from NSF award abstract:
This study will employ a novel combination of experimental evolution techniques and state-of-the-art molecular methods to yield unique insights into adaptive changes in the keystone marine cyanobacteria Trichodesmium and Crocosphaera in response to selection by high CO2. Several studies have suggested that N2-fixation rates of the biogeochemically-critical cyanobacteria Trichodesmium and Crocosphaera may increase dramatically in the future high CO2 ocean, but these have all used the same limited set of cultured isolates and considered cells only briefly acclimated to elevated CO2. The investigator's new results, however, demonstrate that a broad diversity of high- and low-CO2 adapted ecotypes exists within each diazotroph genus. Furthermore, in a preliminary four year experimental evolution study with Trichodesmium, the PIs observed large adaptive responses following 500-700 generations of selection by high CO2- but in a completely unexpected way. All of the six replicate high CO2-adapted cell lines exhibited strong constitutive up-regulation of N2 fixation rates. These very elevated N2 fixation rates continued, even though the cultures have were switched back to low-CO2 conditions for many months. Expression of the nif operon and N assimilatory genes was also up-regulated in these cell lines, as is expression of many intergenic regions of the genome.

The investigators hypothesize that constitutive up-regulation of cellular N2 fixation systems may be a common adaptive response of both Trichodesmium and Crocosphaera under extended selection by elevated CO2. This project will test this hypothesis in a four-year experimental evolution study to determine the adaptive responses of both high- and low-CO2 specialized ecotypes of these two diazotrophs to increased CO2.

The investigators will grow representative high- and low-CO2 adapted ecotypes from each genus in well-replicated cell lines at 380 ppm and 750 ppm CO2 for up to 1000 generations. Periodically, they will perform "switch" experiments to measure N2 and CO2 fixation rates and growth rates of high CO2-selected cell lines grown briefly (one week) at low CO2, and vice versa. These switch experiments will allow screening for cell lines which exhibit adaptive changes in phenotypically-expressed rate parameters, such as those observed in the preliminary Trichodesmium study. Evolutionary mechanisms in the CO2-selected cell lines will be examined by comparison of changes in their genomes, transcriptomes, and proteomes over time relative to reference genomes, using frozen samples archived monthly during the preceding selection period. Examination of these molecular and biochemical changes will be coordinated with an in-depth array of physiological and biogeochemical analyses. This combined approach will allow an evaluation of potential adaptive mechanisms in diazotrophic cyanobacteria ranging from indel, duplication, single nucleotide polymorphism, and transposition mutations to altered putative non-coding RNA expression, protein expression, and post-translational protein modifications, and then allow the investigators to link these mechanisms directly with their potential impacts on ecosystem-level biogeochemical processes like N2 and CO2 fixation. Finally, the research team will determine how long term selection by high CO2 affects the iron and phosphorus requirements of Trichodesmium and Crocosphaera, since constitutive up-regulation of N2 fixation would also have major implications for limitation of diazotrophs by these two critical nutrients in the future high CO2 ocean.