Dynamic secondary ion mass spectrometry imaging of microbial populations utilizing C-labelled substrates in pure culture and in soil

We demonstrate that dynamic secondary ion mass spectrometry (SIMS)-based ion microscopy can provide a means of measuring (13)C assimilation into individual bacterial cells grown on (13)C-labelled organic compounds in the laboratory and in field soil. We grew pure cultures of Pseudomonas putida NCIB 9816-4 in minimal media with known mixtures of (12)C- and (13)C-glucose and analysed individual cells via SIMS imaging. Individual cells yielded signals of masses 12, 13, 24, 25, 26 and 27 as negative secondary ions indicating the presence of (12)C(-), (13)C(-), (24)((12)C(2))(-), (25)((12)C(13)C)(-), (26)((12)C(14)N)(-) and (27)((13)C(14)N)(-) ions respectively. We verified that ratios of signals taken from the same cells only changed minimally during a approximately 4.5 min period of primary O(2)(+) beam sputtering by the dynamic SIMS instrument in microscope detection mode. There was a clear relationship between mass 27 and mass 26 signals in Pseudomonas putida cells grown in media containing varying proportions of (12)C- to (13)C-glucose: a standard curve was generated to predict (13)C-enrichment in unknown samples. We then used two strains of Pseudomonas putida able to grow on either all or only a part of a mixture of (13)C-labelled and unlabelled carbon sources to verify that differential (13)C signals measured by SIMS were due to (13)C assimilation into cell biomass. Finally, we made three key observations after applying SIMS ion microscopy to soil samples from a field experiment receiving (12)C- or (13)C-phenol: (i) cells enriched in (13)C were heterogeneously distributed among soil populations; (ii) (13)C-labelled cells were detected in soil that was dosed a single time with (13)C-phenol; and (iii) in soil that received 12 doses of (13)C-phenol, 27% of the cells in the total community were more than 90% (13)C-labelled.