Journal of Methods Microbiological Journal of Microbiological Methods 31 (1998) 99–111 Direct fingerprinting of metabolically active bacteria in environmental samples by substrate specific radiolabelling and lipid analysis * Peter Roslev , Niels Iversen, Kaj Henriksen Environmental Engineering Laboratory, Aalborg University, Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark Accepted 1 September 1997 Abstract Substrate specific radio assays were used for enumeration and fingerprinting of microorganisms in environmental samples. 14 Direct fingerprinting was based on incorporation of C-labelled substrates into microbial lipids. A radioactive fingerprint 14 was obtained by subsequent radio analysis of whole sample phospholipid ester-linked fatty acids ( C-PLFA fingerprint). 14 14 This approach provided a C-PLFA fingerprint of the organism actively metabolizing the added C-labelled substrate. Labelled and unlabelled PLFAs were analysed as methyl ester derivatives by gas–liquid chromatography with flame 14 14 ionization detection. The presence of C-PLFAs were determined by collection of CO produced after combustion of the 2 fatty acids. Additional analysis of the microbial community was carried out by analysis of the radioactivity assimilated into 14 14 poly-b-hydroxyalkanoates relative to that assimilated into total phospholipids ( C-PHA / C-PL ratio). The number of 14 14 organisms involved in the degradation of a C-labelled substrate was estimated using a C-most-probable-number 14 14 technique. These different C-based methods were evaluated by studying [ C]methane oxidation in agricultural soil, and 14 [ C]phenanthrene degradation in activated sludge and marine sediment. The radio assays resulted in distinct fingerprints of the bacterial populations capable of degrading the different radiolabelled substrates. Manipulation of the incubation conditions (e.g., oxygen status) resulted in significant changes in population specific metabolic activity and labelling pattern. 14 Phenotypically related microorganisms appeared to dominate [ C]phenanthrene degradation in activated sludge and marine 14 sediment under oxic conditions. Anaerobic [ C]phenanthrene degraders in activated sludge produced a very different 14 C-PLFA fingerprint. In methane enriched agricultural soil, aerobic methane oxidation was dominated by organisms most 14 similar to the Type I methanotrophic bacteria. Several of the findings obtained by the C-PLFA analysis could not have been established on the basis of conventional PLFAs analysis alone. The results suggest that variations of this simple 14 C-fingerprinting method may be applicable to studies of substrate metabolism in mixed microbial communities. Direct fingerprinting based on substrate specific radiolabelling may also aid in phenotypic characterization of heterotrophic microorganisms without the need for enrichment or cultivation.  1998 Elsevier Science B.V. 14 14 Keywords: Radioactive Fingerprinting; Phospholipid ester-linked fatty acids (PLFA); C-MPN; C-PLFA; Phenotypic Characterization; Active Bacteria; Methane; Phenanthrene * Corresponding author: Tel.: 145 96 358505; fax: 145 98 142555; e-mail: i5pr@civil.auc.dk 0167-7012 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII S0167-7012(97)00094-8