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Next-generation physiology approaches to study microbiome function at single cell level


The function of cells in their native habitat often cannot be reliably predicted from genomic data or from physiology studies of isolates. Traditional experimental approaches to study the function of taxonomically and metabolically diverse microbiomes are limited by their destructive nature, low spatial resolution or low throughput. Recently developed technologies can offer new insights into cellular function in natural and human-made systems and how microorganisms interact with and shape the environments that they inhabit. In this Review, we provide an overview of these next-generation physiology approaches and discuss how the non-destructive analysis of cellular phenotypes, in combination with the separation of the target cells for downstream analyses, provide powerful new, complementary ways to study microbiome function. We anticipate that the widespread application of next-generation physiology approaches will transform the field of microbial ecology and dramatically improve our understanding of how microorganisms function in their native environment.

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Fig. 1: Examples of traditional approaches to study microbial physiology.
Fig. 2: Next-generation physiology workflow to study microorganisms.
Fig. 3: Reporters and their associated Raman spectral fingerprints in microbial next-generation physiology.


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The authors are grateful to A. Kohtz for generating the Raman data depicted in Fig. 3. They thank R. Gerlach and H. Smith (Montana State University), J. Hemp (University of Utah), members of the Hatzenpichler laboratory – A. Kohtz, M. Lynes and N. Reichart – and the three reviewers for critical comments that improved the manuscript. Next-generation physiology research in the Hatzenpichler laboratory is supported through grants by the Gordon and Betty Moore Foundation (GBMF5999) and the National Science Foundation (MCB award 1817428 and RII Track-2 FEC award 1736255), as well as an Early Career Fellowship by the National Aeronautics and Space Administration to R.H. (80NSSC19K0449). Montana State University’s Confocal Raman microscope was acquired with support by the National Science Foundation (DBI-1726561) and the M.J. Murdock Charitable Trust (SR-2017331).

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R.H. designed the concept for this Review. All authors wrote the manuscript.

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Correspondence to Roland Hatzenpichler.

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Nature Reviews Microbiology thanks Wei Huang, Aaron Wright and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Synonymous with the microbial community; all of the microscopic organisms, including archaea, bacteria, unicellular eukaryotes and their viruses, within a sample.


An observable characteristic of an organism that is manifested on a molecular, cellular or population level. A phenotype of a cell varies over time and with changing physicochemical conditions.


Molecules or chemical motifs that can be specifically traced within the cell; ideally, the reporter group is entirely absent from the target cell under natural conditions.


The sets of genes or the entire genome of an organism.


The functioning of a cell in its native habitat under a given set of conditions, including interactions with other cells and the abiotic environment.


The random shotgun sequencing of DNA from a sample containing more than one genotype.

Single cell genomics

An individual cell is separated from a microbiome and its genome is amplified and sequenced.


(MAR). A method that detects uptake of radioactively labelled substrates into cells through formation of silver grains after exposure to a photographic emulsion. MAR is limited in its widespread application because of its dependency on isotopes with a suitable half-life, its low throughput and its destructive nature.

Nano-scale secondary ion mass spectroscopy

(nanoSIMS). A technique that expels secondary ions from a sample surface through a focused ion beam in high vacuum, extracts them by an electric field and analyses them by time-of-flight mass spectrometry. nanoSIMS provides unrivalled sensitivity and spatial resolution but has very low throughput and destroys the sample.

Quantitative stable isotope probing

(qSIP). A technique that separates isotopically heavy biomolecules (for example, 13C-containing DNA) from unlabelled molecules by buoyant density centrifugation. By collecting multiple density fractions and determining their taxonomic and genetic make-up, taxon-specific isotope enrichments can be calculated.

Next-generation physiology

Any approach enabling study into the physiology of an individual cell in a microbiome in a non-destructive way, thus enabling physical separation of this cell based on its phenotype for further downstream applications.

Click chemistry

A summary term for a range of reactions with a high thermodynamic driving force and extremely high yields and reaction efficiencies. The term is often used synonymously for azide–alkyne cycloaddition reactions, which are the most commonly used type of click chemistry reactions in biology.

Raman-activated cell sorting

(RACS). A set of techniques that combines Raman spectral acquisition with single cell separation.

Fluorescence in situ hybridization

(FISH). A technique that uses single-stranded DNA probes and fluorescence microscopy to visualize cells based on their taxonomic identity (ribosomal RNA FISH) or gene expression (mRNA FISH).

Intact cells

Cells that have not been exposed to a chemical fixative (such as formaldehyde or ethanol) that might interfere with downstream analyses (such as cultivation or DNA sequencing).

Metabolically active

A cell carrying out specific metabolic function (such as redox activity or activity of a specific enzyme); this term is agnostic about whether this activity leads to the build-up of new biomass (that is, anabolic activity).

Anabolically active

Performing de novo synthesis of specific macromolecules (for example, DNA, RNA, proteins and lipids).

Silent region

The area in the Raman spectrum of a cell that is free of background interference from cellular vibrations (~1,800–2,700 cm–1).


A molecule carrying a functional group that is amenable to azide–alkyne click chemistry.

Bioorthogonal reaction

A reaction that does not interfere with biological processes; it can be used to label a cell or molecule with a reporter.

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Hatzenpichler, R., Krukenberg, V., Spietz, R.L. et al. Next-generation physiology approaches to study microbiome function at single cell level. Nat Rev Microbiol 18, 241–256 (2020).

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