Optimization of a high‐throughput phenotyping method for chain‐forming phytoplankton species

Abstract

Modern equipment facilitates phenotyping of hundreds of strains of unicellular organisms by culturing and monitoring growth in microplates. However, in the field of phytoplankton ecology, automated monitoring of growth is not often done and this method has not been tested for many species. To meet the demand for a high‐throughput technique for monitoring growth of chain‐forming phytoplankton species, we have assessed and optimized a method commonly used for other microorganisms. Skeletonema marinoi is a pelagic chain‐forming diatom, and we have acquired growth patterns in four different treatments (i.e., low and high light, low and high nutrient concentrations) when cultured in multi‐well plates. Due to the unexpected heterogeneity in growth rates and maximum cell densities observed between wells (spatial) and runs (temporal), a set of models was fitted to the obtained phenotypic data to correct for these biases. Models were tested for robustness on two replicate multi‐strain experiments including 23 different strains. Using the model accounting for temporal and spatial bias, we could reliably determine changes in growth rate caused by nutrient treatments as well as differences in cell density as a response to nutrient availability and light treatment. This method can facilitate high‐throughput phenotyping of hundreds of strains, which is often a bottleneck in characterizing the ecology and capacity for adaptation of chain‐forming phytoplankton.

Friends With Benefits: Exploring the Phycosphere of the Marine Diatom Skeletonema marinoi

“…Cultures were centrifuged (1500 × g , 5 min), washed once in fresh EGM, and dissolved in 20 mL EGM. Cell density of washed pre-cultures was measured (Gross et al, 2018) and then used to prepare experimental cultures with a starting concentration of 25 000 cells mL –1 . Bacterial cultures (5 mL) were grown in Difco marine broth (BD Biosciences, United States) overnight with shaking a day before experiment.…”

Marine microalgae for outdoor biomass production—A laboratory study simulating seasonal light and temperature for the west coast of Sweden

“…In the secondary screening, triplicate cultures of 20 ml were inoculated from the working cultures at a cell density of 0.5-1 relative fluorescence units (RFU) (~5-10 × 10 4 cells ml −1 ) and incubated in the same conditions as during reactivation, for 6-8 days. Algal growth was monitored by changes in chlorophyll fluorescence expressed in RFU using a Varioscan™ Flash Multimode Reader (Thermo Scientific) in multi-well microplates, as described by Gross et al (2018). The fluorescence wavelength settings were 425 nm for excitation and 680 nm for emission detection, and the samples were dark adapted for 10 min at room temperature prior to the measurement.…”

“…where RFUt2 and RFUt1 are the relative chlorophyll fluorescence at two consecutive measurements between days 2 and 6 of the exponential growth. The conversion from RFU to cell number was done according to Gross et al (2018). Strains that displayed at least 21 RFU (~2 × 10 6 cells ml −1 ) were selected for the third screening.…”

High single‐cell diversity in carbon and nitrogen assimilations by a chain‐forming diatom across a century

“…Growth curves were established by daily sampling of 700 μl for measurements of relative fluorescence unit (RFU) on multi-well plates using a Varioscan™ Flash Multimode Reader (ThermoScientific) with fluorescence settings at 425 nm excitation and 680 nm emission detection wavelengths. These measurements provide relative estimates of in vivo chlorophyll a concentrations (method further described in Gross et al, 2018). Cell abundance and size were analysed by microscopy (Olenina et al, 2006).…”