Fig. 2.

C-D signature region of Raman spectra is conserved in diverse microorganisms. (A) Representative Raman spectra of microorganisms cultivated in media amended with various percentages of heavy water. Characteristic C-D and C-H regions are shaded in gray. N. moscoviensis and N. gargensis did not grow with 100% D₂O. Both methanogens were only grown at 30% D₂O and not tested at other levels. (B) NanoSIMS quantification of cellular D incorporation (at%) of different strains when grown in 30% D₂O (and E. coli with 0% D₂O). Each point is a measurement of a single cell, and box plots indicate quartiles. Note that only a few cells of N. moscoviensis became active after incubation (low-active/nonactive cells are shown in gray), a feature we have also observed by other techniques in medium without heavy water. M. smithii cultures produced extracellular substances enriched in D that were excluded from the analysis. Autotrophic organisms (M. smithii, N. gargensis, and N. moscoviensis) were significantly more enriched in D than heterotrophic organisms (E. coli, B. subtilis, and B. thuringiensis) when cultivated in the presence of the same D₂O concentration (30% D₂O; ANOVA, P < 0.001).