Using stable isotopes as an indicator of activity is one of the more popular and robust ways to bridge the gap between microorganisms and their function in ecological processes. In environmental microbiology, stable isotope probing (SIP) is applied by incubating a sample with a highly-labelled substrate (including C, N, H or O (but not P)) that can be incorporated into the biomass of metabolically active cells \cite{Angel_2019,Dumont_2005}. The identity/community profile of the labelled organisms may then be determined using sequencing which allows to draw causal ecological interpretations of the microorganisms active in assimilatory processes of interest. Organisms labelled through SIP may further be detected and identified on a single-cell level using Raman microspectroscopy or NanoSIMS with FISH \cite{Musat_2016,Wang_2016}.
Other recent advances in linking microorganisms to functions include so-called 'next-generation physiology' approaches \cite{Hatzenpichler_2020}. Similar to SIP, these methods require the introduction of a labelled or noncanonical molecule into the sample for the detection of metabolically active organisms. The use of 'heavy water' labeling has become a recent popular approach for universal targeting of all active organisms in the environment using 18O-H2O \cite{Aanderud_2011,Schwartz_2007,Schwartz_2016} or deuterium oxide (D2O)\cite{Li_2019,Eichorst_2015}, either alone or in combination with other isotopes. The assimilation of 18O-H2O into DNA can be used to deduce microbial growth rates \cite{Hungate2015,Schwartz_2016}, whereas heavy water (D2O) can be detected in the newly synthesized lipids of active cells \cite{Li_2019}. Combined with identification of taxa of interest through amplicon sequencing represent powerful tools to take the next step in soil ecological research.
Amplicon sequencing may also be combined with with BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) to target only the fraction of cells within a soil sample that is translationally active in situ \cite{Couradeau_2019,Reichart_2020}. In BONCAT or Substrate Analog Probing (SAP), a modified version of an amino acid or substrate is incubated with the sample, which is followed by a click-chemistry reaction to add a fluorescent probe \cite{Hatzenpichler_2015a}. The use of modified indicator molecules opens new avenues for detecting metabolically active cells in the context of environmental samples, however the application to soil remains limited to a few studies \cite{Couradeau_2019,Reichart_2020}. Coupling these labeling approaches to cell sorting via fluorescence activated cell sorting (FACS) \cite{Couradeau_2019} or Raman-activated cell sorting (RACS) \cite{Lee_2019}, provides a non-destructive alternative to NanoSIMS for identifying the metabolically active organisms. Thus, allowing the labeled fraction of cells to be targeted for downstream sequencing. Additionally, combining these labeling approaches with cell sorting and sequencing may circumvent challenges associated with exogenous DNA.
In addition to labeling experiments, metagenomic and metatranscriptomic sequencing are increasingly being used to describe the functional gene diversity and expression in various environmental samples \citep{leaves}. These approaches remain promising for improving the link between organisms and their ecological roles, although both sequencing and bioinformatic costs for gaining functionally relevant insights into ecosystem processes by these approaches are typically magnitudes higher than those needed for analyzing amplicon sequencing data. Amplicon sequencing can certainly also be a valuable tool for planning of more targeted metagenomic or metatranscriptomic studies to investigate taxonomic composition, functional potential and/or gene expression in the community context.