However, even using automated platforms, Raman-activated cell sorting currently can only achieve sorting rates of up to 500 cells/h.
Laser tweezers can then be used to separate functionally active cells from the microbial community. An alternative to SIP targeted at a specific assimilatory pathway is to label all anabolically active cells in a sample with deuterated water ( 2H 2O) and visualize heavy isotope uptake into individual cells using Raman microspectroscopy. SIP experiments have been limited by their need for isotope-labeled molecules that can be prohibitively expensive or unavailable because of compositional complexity. Traditionally, metagenomic predictions about the ecophysiology of microbes have often been tested using stable isotope probing (SIP), which can determine cellular activity through incorporation of growth substrates. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria.
Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions.
In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. Metagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems.
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