The biomass stoichiometry of microbial communities are important indicators of ecosystem processes. For approximately 30 years the technique of energy dispersive spectroscopy (EDS) using an electron microscope has been employed, by a scant number of laboratories, to examine elemental ratios in biological systems. However, the limitations of the technique, primarily the difficulty of reliable data acquisition and data processing, have limited its widespread application in the field. Yet, the power of the technique within the fields of microbiology and microbial ecology is undeniable – single-cell measurements of carbon, nitrogen, and phosphorus (among other elements) within unfixed and untreated cells. We evaluated the benefits of single-cell stoichiometry over typical bulk measures of microbial C:N:P ratios. Employing EDS analysis, on a JOEL SEM calibrated using Acetyl-CoA and adenosine diphosphate as C:N:P standards, combined with ‘bulk’ analyses we have examined cell populations in batch culture throughout a canonical batch growth experiment. In addition, we demonstrate the stoichiometric flexibilities of known bacterial strains grown on defined media with a range of C:P provided. Investigations into the interrelatedness of phylogeny and stoichiometric flexibility were also performed on known bacterial isolates within and outside of the Sphingomonadaceae to determine the cellular C:N:P response to changing environmental C:N:P.
Results/Conclusions
Calibration factors for carbon, nitrogen, and phosphorus were generated using acetyl-CoA and validated with adenosine diphosphate. C:N:P ratios for bacteria, taken during exponential growth, were subsequently obtained by EDS and matched those obtained by “bulk” analyses – thereby validating the technique. We detected both intra- and inter-cellular variability which is lost with bulk analyses. Visual detection of (predicted) polyphosphate bodies is a clear example of intracellular C:N:P variability. Inter-cell variability was demonstrated when cellular C:N:P was measured throughout growth in batch culture. Variability was highest during lag- and stationary-phase, while exponential-phase cultures were stoichiometrically-restricted. In addition, all strains demonstrated a change in cellular C:P that corresponded to media C:P. However, not all strains responded with the same flexibility – and this plasticity, or lack thereof, was not linked to phylogeny. We will continue to pursue this line of questioning to determine, using a larger strain library, if stoichiometry and/or stoichiometric plasticity have a phylogenetic signal. Finally, pursuing EDS analysis on mock and real communities with simultaneous phylogenetic labeling will permit determination of in situ C:N:P ratios of specific phylogenetic groups; which is currently impossible using bulk techniques.