Table 1.

Selected techniques of microbial ecology that are suitable for characterization of MET

Technique	Principle & marker	Information	Selected recommended references and examples
16S rRNA gene sequencing	The DNA of the microbial sample is extracted, and the 16S rRNA gene is partially amplified and sequenced. Depending on the applied approach this can cover a suitable small region, for example, in the range of 100‐200 bp dependent on choice of primers, (Liu et al., 2007; Soergel et al., 2012; Klindworth et al., 2013).	The obtained results can be interpreted in a phylogeny dependent as well as independent way. For the first, the obtained sequences are compared to databases (e.g., using the Ribosomal Database Project (Cole et al., 2014)). The sequences are assigned on different phylogenetic levels going down to genera, dependent on the respective sequences, their length and the available information in the databases. For phylogeny independent analysis, individual sequences are grouped into OTUs (e.g., 97% sequence identity although this is not always representative of a single species). The diversity of the microbial community sample is then characterized based on the number of different phylogenetic groups or OTUs (richness) and their relative abundances (evenness). The technique is suitable to get a general impression of the phylogenetic composition of a microbial community and allows monitoring reactor microbiomes over time and in response to changes in process parameters.	Gilbert et al., (2012); Ishii et al., (2014); Yarza et al., (2014)
16S rRNA gene fingerprinting (e.g. T‐RFLP)	The DNA of the microbial sample is extracted, and the 16S rRNA gene is partially amplified. Depending on the applied approach this can cover nearly the complete gene (Schütte et al., 2008; Lefebvre et al., 2010). Depending on the chosen technique, the fingerprint consists of fragments which are representative of sequence characteristics, for example, position of cutting sites for restriction endonucleases.	The obtained results are interpreted in a phylogeny independent way. The fragments of the fingerprint represent a certain group of organisms that have similar sequence characteristics, for example, the same position of a cutting site for a restriction endonuclease. Each fragment (e.g., peak in a T‐RFLP profile) represents one OTU. The diversity of the microbial community sample is then characterized based on the number of different OTUs (richness) and their relative abundances (evenness). The technique is suitable to get a general impression of the diversity of a microbial community and allows monitoring reactor microbiomes over time and in response to changes in process parameters although phylogenetic information is not provided.	Marzorati et al., (2008); Schütte et al., (2008); Koch et al., (2014b)
Cytometric fingerprinting	Cytometric fingerprinting is a single cell‐based approach that utilizes optical characteristics (cell size, DNA content after staining) of individual microbial cells to characterize a microbial community sample.	The optical characteristics are independent of the phylogenetic background of the cells. Complex microbial communities are characterized in a simple and rapid way. The changes in the cytometric fingerprint are, similar to 16S rRNA fingerprinting, representative of changes in the community composition and allow monitoring reactor microbiomes over time and in response to changes in process parameters.	Koch et al., (2013); Günther et al., (2016)
Metagenomics, Metatranscriptomics, Metaproteomics	The entire DNA, RNA or expressed protein content of a microbial community is analysed.	The results reflect the genes and their expression products that reveal the presence of certain metabolic capacities. Including also abundance information, potential metabolic pathways in the microbial community can be identified and allocated to individual species.	Ishii et al., (2013); Wöhlbrand et al., (2013); Vanwonterghem et al., (2016)
Fluorescence in situ hybridization (FISH)	FISH is a single cell‐based approach that utilizes phylogenetic information in form of a target specific, fluorescently labelled probe that hybridizes to the DNA or RNA within the cells. Therefore, a priori knowledge about the potential relevant microorganisms in a microbial community (e.g., 16S rRNA gene sequencing) is recommended.	The technique allows detection and enumeration of bacteria based on a specific phylogenetic marker and can reveal the spatial organization of the cells, for example, cell density and different layers within a biofilm.	Amann and Fuchs (2008); Mielczarek et al., (2013); Shrestha et al., (2013)
NanoSIP/nanoSIMS	The assimilation of substrates marked with stable isotopes (e.g., 13C, 15N, 34S or 2H) in microbial biomass is visualized on single cell level in combination with a phylogenetic marker. A priori knowledge about the potential relevant microorganisms and their potentially utilized substrates is recommended.	The results reflect metabolic activity in combination with phylogenetic as well as spatial information on single cell level.	McGlynn et al., (2015); Musat et al., (2016)
Electrochemical microcosm	Small scale BES can be set up for characterizing specific functions of electroactive biofilms.	Under defined conditions, the microbial activity can be investigated including utilization of specific substrates as well as detailed mechanisms of the microorganism–electrode interaction.	Pous et al., (2014)