Applications and impacts of stable isotope probing for analysis of microbial interactions

Abstract

CitationApplications and impacts of stable isotope probing for analysis of microbial interactions. 2014, 98 (11) AbstractProbing the interactions between microbes and their environment with stable isotopes became a powerful technique over the last years. While quadruple mass spectrometry or isotope ratio mass spectrometry (IRMS) require at least 300.000 bacterial cells, analysis at the single-cell level is possible with secondary ion mass spectrometry or Raman microspectrometry. The latter two techniques however need enrichments of more than 10 atom-% while IRMS can deal with 0.0001 atom-%. To find out who eats what one has to discern between the different species in a community. Several methods have been introduced to discern between the different taxa in microbial communities, e.g. by using fatty acids as biomarkers, density centrifugation of DNA/RNA or fluorescent in situ hybridization (FISH) with phylogenetic probes. While the biomarker approach can be coupled with the high sensitivity of the IRMS, the DNA approach gives in general a better phylogenetic resolution of the metabolic active microbes. A combination of both is the separation via coupling of FISH-probes to magnetic beads or fluorescent assisted cell sorting (FACS) of stained cells leading to fractions which can be analyzed by IRMS. Applying these techniques over a time course can reveal the metabolic kinetics and food webs. In this review the different methods are presented with examples and their advantages and disadvantages are discussed. An outlook on the combination of the various techniques and their applications in microbial ecology is given.

The application of Raman spectroscopy for the detection and identification of microorganisms

“…If there is no biomarker (like carotenoids or cytochromes) at hand to follow metabolic activities in bacteria or the interaction between microbes and the environment, then one elegant approach is the incorporation of isotopically labeled substrates into the bacteria (named stable isotope labeling, SIP) followed by the detection or characterization of actively incorporating microbes. Raman microscopy can easily be used to detect isotopic ratios even on a single‐cell level, although a rather high enrichment is needed and the precision is low compared with other modalities like nano‐secondary ion mass spectrometry or isotope ratio mass spectrometry . The Raman spectral biomarkers can be chosen as needed and are not confined to the frequently used red shifts of the phenylalanine band at 1001 cm −1 or the C–H peaks between 2800 and 3100 cm −1 .…”

“…Raman microscopy can easily be used to detect isotopic ratios even on a single-cell level, although a rather high enrichment is needed and the precision is low compared with other modalities like nano-secondary ion mass spectrometry or isotope ratio mass spectrometry. [48] The Raman spectral biomarkers can be chosen as needed and are not confined to the frequently used red shifts of the phenylalanine band at 1001 cm À1 or the C-H peaks between 2800 and 3100 cm À1 .…”

Stable Isotope Probing Approaches to Study Anaerobic Hydrocarbon Degradation and Degraders

“…The technical features and possible applications of the different SIP techniques have been extensively reviewed [Abraham, 2014;Evershed et al, 2006;Friedrich, 2006;Lueders, 2015;Musat et al, 2012;Neufeld et al, 2007a, b;Radajewski et al, 2003;Seifert et al, 2012;Wagner, 2009;Whiteley et al, 2006] and are also summarized in a book [Murrell and Whiteley, 2011]. The different SIP technologies can be differentiated by their performance and inherent technological features.…”

“…A number of SIP methods have been described which considerably differ in terms of sensitivity, precision and requirements [Abraham, 2014;Murrell and Whiteley, 2011] ( table 1 ). As assimilated stable isotopes are incorporated into the whole biomass, specific biomolecules ('biomarkers') are used for detecting and/or quantifying the flux of the label into biomass fractions, such as amino acids (AA) [Richnow et al, 2000], phospholipid-derived fatty acids (PLFA) [Annweiler et al, 2000;Boschker et al, 1998], DNA (desoxyribonucleic acid) [Radajewski et al, 2000], RNA (ribonucleic acid) [Manefield et al, 2002] or proteins [Jehmlich et al, 2008].…”

“…Pure cultures, specific consortia, undefined enrichment cultures, environmental samples, in situ NanoSIMS 1 0.1 atom% [Abraham, 2014] Intermediate (target molecule: 16S rRNA) the density of DNA/RNA fragments posed by stable isotope labeling. Thus, DNA/RNA-SIP provides by far the best phylogenic resolution, where labeling can be screened for thousands of taxa per gradient, especially when nextgeneration sequencing of ribosomal genes or metagenomics is used [Aoyagi et al, 2015;Chen and Murrell, 2010].…”