Abstract
Traditional approaches for the assessment of physiological responses of microbes in the environment rely on bulk filtration techniques that obscure differences among populations as well as among individual cells. Here, were report on the development on a novel micro-scale sampling device, referred to as the “Single-probe,” which allows direct extraction of metabolites from living, individual phytoplankton cells for mass spectrometry (MS) analysis. The Single-probe is composed of dual-bore quartz tubing which is pulled using a laser pipette puller and fused to a silica capillary and a nano-ESI. For this study, we applied Single-probe MS technology to the marine dinoflagellate Scrippsiella trochoidea, assaying cells grown under different illumination levels and under nitrogen (N) limiting conditions as a proof of concept for the technology. In both experiments, significant differences in the cellular metabolome of individual cells could readily be identified, though the vast majority of detected metabolites could not be assigned to KEGG pathways. Using the same approach, significant changes in cellular lipid complements were observed, with individual lipids being both up- and down-regulated under light vs. dark conditions. Conversely, lipid content increased across the board under N limitation, consistent with an adjustment of Redfield stoichiometry to reflect higher C:N and C:P ratios. Overall, these data suggest that the Single-probe MS technique has the potential to allow for near in situ metabolomic analysis of individual phytoplankton cells, opening the door to targeted analyses that minimize cell manipulation and sampling artifacts, while preserving metabolic variability at the cellular level.
Highlights
Marine phytoplankton contribute ca. 45 petagrams carbon per annum to net primary production (NPP) (Falkowski et al, 1998)
Whether phytoplankton are nutrient limited in the environment has long attracted the attention of oceanographers who are trying to understand the controls on NPP, given the large impact that limitation may have on the structure of marine ecosystems
The main aims of this study were to develop a single-cell-based metabolomic methodology that could be applied to individual algal cells and to demonstrate that this technology can detect physiological responses to environmental stimuli
Summary
Marine phytoplankton contribute ca. 45 petagrams carbon per annum to net primary production (NPP) (Falkowski et al, 1998). Phytoplankton are important drivers of several global biogeochemical cycles, notably of elements that are components of cellular biomass, including carbon (C), nitrogen (N), phosphorus (P), silicate (Si), and others. Phytoplankton biomass can be broadly approximated from environmental variables, such as nutrient concentrations, sea surface temperature, and solar irradiance (Antoine et al, 1996; Falkowski et al, 1998). At a more granular level, such as the cellular response of individual phytoplankton to dynamic oceanographic conditions, requisite adaptations are often not well-understood. Whether phytoplankton are nutrient limited in the environment has long attracted the attention of oceanographers who are trying to understand the controls on NPP, given the large impact that limitation may have on the structure of marine ecosystems
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