Summary: A stable isotope probing (SIP) approach was used to study aerobic methane‐oxidizing bacteria (methanotrophs) in lake sediment. Oligotrophic Lake Stechlin was chosen because it has a permanently oxic sediment surface. 16S rRNA and the pmoA gene, which encodes a subunit of the methane monooxygenase enzyme, were analysed following the incubation of sediment with ¹³ CH ₄ and the separation of ¹³ C‐labelled DNA and RNA from unlabelled nucleic acids. The incubation with ¹³ CH ₄ was performed over a 4‐day time‐course and the pmoA genes and transcripts became progressively labelled such that approximately 70% of the pmoA genes and 80% of the transcripts were labelled at 96 h. The labelling of pmoA mRNA was quicker than pmoA genes, demonstrating that mRNA‐SIP is more sensitive than DNA‐SIP; however, the general rate of pmoA transcript labelling was comparable to that of the pmoA genes, indicating that the incorporation of ¹³ C into ribonucleic acids of methanotrophs was a gradual process. Labelling of Betaproteobacteria was clearly seen in analyses of 16S rRNA by DNA‐SIP and not by RNA‐SIP, suggesting that cross‐feeding of the ¹³ C was primarily detected by DNA‐SIP. In general, we show that the combination of SIP approaches provided valuable information about the activity and growth of the methanotrophic populations and the cross‐feeding of methanotroph metabolites by other microorganisms.

The isotope enrichment factors (ε) in Methanosaeta concilii and in a lake sediment, where acetate was consumed only by Methanosaeta spp., were clearly less negative than the ε usually observed for Methanosarcina spp. The fraction of methane produced from acetate in the sediment, as determined by using stable isotope signatures, was 10 to 15% lower when the appropriate ε of Methanosaeta spp. was used.