Apportioning bacterial carbon source utilization in soil using ¹⁴ C isotope analysis of FISH-targeted bacterial...

An unresolved need in microbial ecology is methodology to enable quantitative analysis of in situ microbial substrate carbon use at the population level. Here, we evaluated if a novel combination of radiocarbon-labelled substrate tracing, fluorescence in situ hybridisation (FISH) and fluorescence-activated cell sorting (FACS) to sort the FISH-targeted population for quantification of incorporated radioactivity (¹⁴ C-FISH-FACS) can address this need. Our test scenario used FISH probe PSE1284 targeting Pseudomonas spp. (and some Burkholderia spp.) and salicylic acid added to rhizosphere soil. We examined salicylic acid-¹⁴ C fate (mineralized, cell-incorporated, extractable and non-extractable) and mass balance (0-24 h) and show that the PSE1284 population captured ∼ 50% of the Nycodenz extracted biomass ¹⁴ C. Analysis of the taxonomic distribution of the salicylic acid biodegradation trait suggested that PSE1284 population success was not due to conservation of this trait but due to competitiveness for the added carbon. Adding 50KBq of ¹⁴ C sample^-1 enabled detection of ¹⁴ C in the sorted population at ∼ 60-600 times background; a sensitivity which demonstrates potential extension to analysis of rarer/less active populations. Given its sensitivity and compatibility with obtaining a C mass balance, ¹⁴ C-FISH-FACS allows quantitative dissection of C flow within the microbial biomass that has hitherto not been achieved.