Stable Isotope Probing
SIP is a relatively new approach to assessing microbial populations actively involved in metabolizing a compound of interest. The technique pioneered by J.C. Murrells’ group is based on feeding natural populations (microcosms) a heavy (13C) isotope of a C1 compound of interest (methane, methanol, etc.), followed by separation of a labeled (heavy) fraction of DNA by isopycnic centrifugation. The heavy DNA fraction is enriched in sequences representing the organisms that have consumed the labeled compound and turned it into cell constituents, including DNA. The nature of these organisms is revealed via PCR-amplification of genes of interest, using universal or group-specific phylogenetic PCR primers, or using primers targeting functional genes. The SIP technique has been so far employed to target populations active in utilization of methane, methanol, methylamine, formaldehyde, formate and chloromethane. It is understood that SIP experiments should be conducted in conditions as closely resembling the conditions in situ as possible, to obtain a true picture of the activities and processes native to a given environment.
We traced methylotrophic members of the sediment microbial community by addition of [13C]-labelled substrates (methane, methanol, methylamine, formaldehyde, formate) in microcosms, followed by the analysis of heavy 13C-DNA. As reflected by the analysis of 16S rRNA directly isolated from the Lake Washington sediment, a high diversity of microorganisms are active in situ.