Dataset: The Long Term Phytoplankton Evolution Experiment: Culture Analysis

Detailed methods can be found in the manuscript "Marine phytoplankton and heterotrophic bacteria rapidly adapt to future pCO2 conditions in experimental co-cultures". A summary of major methods are provided here.

The phytoplankton used in this study as well as the media in which they were grown are Prochlorococcus MIT9312 (PEv medium), Synechococcus CC9311 (SEv medium), Synechocystis PCC6803 (SEv medium), Thalassiosira oceanica CCMP1005 (FEv medium), and Emiliania huxleyi CCMP371 (FEv medium). All media types were derived from media commonly used to cultivate each organism detailed in "Algal Culturing Techniques" edited by Andersen. Prior to use in experiments, phytoplankton cultures were rendered clonal and axenic, then mixed with pure cultures of the heterotrophic bacterium Alteromonas EZ55, obtained by streaking for isolation on YTSS agar.

Cultures were experimentally evolved for approximately 500 generations at 22 degrees Celisius (°C) under approximately 75 micromoles photons per square meter per second (µmol photons m-2 s-1) in acid-washed conical-bottom glass tubes with airtight caps where the carbonate system was manipulated by the addition of acid or base to achieve present-day (400 parts per million (ppm)) or year 2100 (800 ppm) pCO2 conditions. Phytoplankton growth was measured every 48 hours using a Guava HT1 flow cytometer equipped with a 488 nanometer (nm) laser. When phytoplankton cell densities crossed a cutoff value, cultures were diluted 26-fold into fresh media, representing log₂26 or 4.7 generations per transfer. Samples from each lineage were cryopreserved every 25 generations and again at the end of the experiment.

At the end of the evolution period, we subcultured clonal evolved Alteromonas strains by spread-plating evolved cultures on YTSS agar and selecting single, isolated colonies for growth in YTSS broth. Prochlorococcus was rendered axenic by the addition of 100 micrograms per milliliter (µg mL-1) streptomycin. All growth experiments were initiated by mixing axenic Prochlorococcus with a specific Alteromonas clone (or else remaining axenic) and acclimating the co-culture for 3 transfer cycles (approximately 14 generations) at the target pCO2 concentration. Growth was then monitored by flow cytometry as described above for at least 3 subsequent transfers under constant conditions.