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Stable isotope probing of RNA has enthused researchers right from its first introduction in 2002. The concept of a labelling-based detection of process-targeted microbes independent of cellular replication or growth has allowed for a much more direct handle on functionally relevant microbiota than by labelling of other biomarkers. This has led to a widespread application of the technology, and breakthroughs in our understanding of carbon flow in natural microbiomes, autotrophic and heterotrophic physiologies, microbial food webs, host–microbe interactions and environmental biotechnology. Recent studies detecting labelled mRNA demonstrate that RNA-SIP is not limited to the analysis of rRNA, but is currently developing towards an approach for accessing targeted transcriptomes. In combination with next-generation sequencing and other methodological advances, RNA-SIP will continue to deliver invaluable insights into the functioning of microbial communities. [ABSTRACT FROM AUTHOR]

A novel approach was developed to follow the successive utilization of organic carbon under anoxic conditions by microcalorimetry, chemical analyses of fermentation products and stable-isotope probing ( SIP). The fermentation of 13 C-labeled glucose was monitored over 4 weeks by microcalorimetry in a stimulation experiment with tidal-flat sediments. Based on characteristic heat production phases, time points were selected for quantifying fermentation products and identifying substrate-assimilating bacteria by the isolation of intact ribosomes prior to rRNA- SIP. The preisolation of ribosomes resulted in rRNA with an excellent quality. Glucose was completely consumed within 2 days and was mainly fermented to acetate. Ethanol, formate, and hydrogen were detected intermittently. The amount of propionate that was built within the first 3 days stayed constant. Ribosome-based SIP of fully labeled and unlabeled rRNA was used for fingerprinting the glucose-degrading species and the inactive background community. The most abundant actively degrading bacterium was related to Psychromonas macrocephali (similarity 99%) as identified by DGGE and sequencing. The disappearance of Desulfovibrio-related bands in labeled rRNA after 3 days indicated that this group was active during the first degradation phase only. In summary, ribosome-based SIP in combination with microcalorimetry allows dissecting distinct phases in substrate turnover in a very sensitive manner. [ABSTRACT FROM AUTHOR]

A toluene-degrading microbial consortium was enriched directly in a BTEX-contaminated aquifer under sulfate-reducing conditions using in situ microcosms consisting of toluene-loaded activated carbon pellets. Degradation of toluene and concomitant sulfide production by the consortium was subsequently demonstrated in laboratory microcosms. The consortium was physiologically and phylogenetically characterized by isotope tracer experiments using nonlabeled toluene, [13C]-α-toluene or [13C7]-toluene as growth substrates. Cells incubated with [13C]-α-toluene or [13C7]-toluene incorporated 8–15 at.%13C and 51–57 at.%13C into total lipid fatty acids, respectively, indicating a lower specific incorporation of 13C from [13C7]-toluene. In order to identify the toluene-assimilating bacteria, the incorporation of carbon from both [13C]-α-toluene and [13C7]-toluene into rRNA was analyzed by stable isotope probing. Time and buoyant density-resolved 16S rRNA gene-based terminal restriction fragment length polymorphism profiles, combined with cloning and sequencing, revealed that an uncultured bacterium (99% sequence similarity) related to the genus Desulfocapsa was the main toluene-degrading organism in the consortium. The ratio of the respective terminal restriction fragments changed over time, indicating trophic interactions within this consortium. [ABSTRACT FROM AUTHOR]

Here, we present a detailed functional and phylogenetic characterization of an iron-reducing enrichment culture maintained in our lab with benzene as sole carbon and energy source. We used DNA-stable isotope probing to identify microbes within the enrichment most active in the assimilation of 13C-label. When 12C6- and 13C6-benzene were added as comparative substrates, marked differences in the quantitative buoyant density distribution became apparent especially for uncultured microbes within the Gram-positive Peptococcaceae, closely related to environmental clones retrieved from contaminated aquifers world wide and only distantly related to cultured representatives of the genus Thermincola. Prominent among the other constituents of the enrichment were uncultured Deltaproteobacteria, as well as members of the Actinobacteria. Although their presence within the enrichment seems to be stable they did not assimilate 13C-label as significantly as the Clostridia within the time course of our experiment. We hypothesize that benzene degradation in our enrichment involves an unusual syntrophy, where members of the Clostridia primarily oxidize benzene. Electrons from the contaminant are both directly transferred to ferric iron by the primary oxidizers, but also partially shared with the Desulfobulbaceae as syntrophic partners. Alternatively, electrons may also be quantitatively transferred to the partners, which then reduce the ferric iron. Thus our results provide evidence for the importance of a novel clade of Gram-positive iron-reducers in anaerobic benzene degradation, and a role of syntrophic interactions in this process. These findings shed a totally new light on the factors controlling benzene degradation in anaerobic contaminated environments.The ISME Journal (2007) 1, 643–653; doi:10.1038/ismej.2007.73; published online 13 September 2007 [ABSTRACT FROM AUTHOR]

Methane is formed on rice roots mainly by CO2 reduction. The present study aimed to identify the active methanogenic populations responsible for this process. Soil-free rice roots were incubated anaerobically under an atmosphere of H2/13CO2 or N2/13CO2 with phosphate or carbonate (marble) as buffer medium. Nucleic acids were extracted and fractionated by caesium trifluoroacetate equilibrium density gradient centrifugation after 16-day incubation. Community analyses were performed for gradient fractions using terminal restriction fragment polymorphism analysis (T-RFLP) and sequencing of the 16S rRNA genes. In addition, rRNA was extracted and analysed at different time points to trace the community change during the 16-day incubation. TheMethanosarcinaceaeand the yet-uncultured archaeal lineage Rice Cluster-I (RC-I) were predominant in the root incubations when carbonate buffer and N2 headspace were used. The analysis of[13C]DNA showed that the relative 16S rRNA gene abundance of RC-I increased whereas that of theMethanosarcinaceaedecreased with increasing DNA buoyant density, indicating that members of RC-I were more active than theMethanosarcinaceae. However, an unexpected finding was that RC-I was suppressed in the presence of high H2 concentrations (80%, v/v), which during the early incubation period caused a lower CH4 production compared with that with N2 in the headspace. Eventually, however, CH4 production increased, probably because of the activity ofMethanosarcinaceae,which became prevalent. Phosphate buffer appeared to inhibit the activity of theMethanosarcinaceae, resulting in lower CH4 production as compared with carbonate buffer. Under these conditions,Methanobacteriaceaewere the prevalent methanogens. Our study suggests that the active methanogenic populations on rice roots change in correspondence to the presence of H2 (80%, v/v) and the type of buffer used in the system. [ABSTRACT FROM AUTHOR]

Stable isotope probing (SIP) is a novel technique to characterize structure and in situ function of active microbial populations, which is based on the incorporation of 13C-labelled substrates into nucleic acids. Here, we have traced methylotrophic members of a rice field soil microbial community, which became active upon continuous addition of 13C-methanol (<22 mM) as studied in microcosms. By combining rRNA- and DNA-based SIP, as well as domain-specific real-time PCR detection of templates in fractions of centrifugation gradients, we were able to detect 13C-labelled bacterial rRNA after 6 days of incubation. Fingerprinting and comparative sequence analysis of ‘heavy’ bacterial rRNA showed that mostly members of the Methylobacteriaceae and a novel clade within the Methylophilaceae formed part of the indigenous methylotrophic community. Over time, however, the Methylophilaceae were enriched. Unexpectedly, nucleic acids of eukaryotic origin were detected, mostly in intermediately 13C-labelled gradient fractions. These eukaryotes were identified as fungi mostly related to Fusarium and Aspergillus spp., and also Cercozoa, known as predatory soil flagellates. The detection of fungi and protozoa in 13C-enriched nucleic acid fractions suggests a possible involvement in either direct assimilation of label by the fungi, or a food web, i.e. that primary 13C-methanol consuming methylotrophs were decomposed by fungi and grazed by protozoa. [ABSTRACT FROM AUTHOR]

Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of ‘light’ and ‘heavy’ nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems. [ABSTRACT FROM AUTHOR]

Microbial ecologists have long sought to associate the transformation of compounds in the environment with the microbial clades responsible. The development of stable isotope probing (SIP) has made this possible in many ecological and biotechnological contexts. RNA-based SIP technologies represent a significant leap forward for culture-independent ‘functional phylogeny’ analyses, where specific consumption of a given compound carrying a 13C signature can be associated with the small subunit ribosomal RNA molecules of the microbes that consume it. Recent advances have led to the unequivocal identification of microorganisms responsible for contaminant degradation in engineered systems, and to applications enhancing our understanding of carbon flow in terrestrial ecosystems. [Copyright &y& Elsevier]

Raman microspectroscopy is a prime tool to characterize the molecular and isotopic composition of microbial cells. However, low sensitivity and long acquisition times limit a broad applicability of the method in environmental analysis. In this study, we explore the potential, the applicability, and the limitations of stable isotope Raman microspectroscopy (SIRM), resonance SIRM, and SIRM in combination with surface-enhanced Raman scattering (SERS) for the characterization of single bacterial cells. The latter two techniques have the potential to significantly increase sensitivity and decrease measurement times in SIRM, but to date, there are no (SERS-SIRM) or only a limited number (resonance SIRM) of studies in environmental microbiology. The analyzed microorganisms were grown with substrates fully labeled with the stable isotopes 13C or 2H and compounds with natural abundance of atomic isotopes (12C 98.89% or 1H 99.9844%, designated as 12C or 1H, respectively). Raman bands of bacterial cell compounds in stable isotope-labeled microorganisms exhibited a characteristic red-shift in the spectra. In particular, the sharp phenylalanine band was found to be an applicable marker band for SIRM analysis of the Deltaproteobacterium strain N47 growing anaerobically on 13C-naphthalene. The study of G. metallireducens grown with 13C- and 2H-acetate showed that the information on the chromophore cytochrome c obtained by resonance SIRM at 532 nm excitation wavelength can be successfully complemented by whole-organism fingerprints of bacteria cells achieved by regular SIRM after photobleaching. Furthermore, we present here for the first time the reproducible SERS analysis of microbial cells labeled with stable isotopes. Escherichia coli strain DSM 1116 cultivated with 12C- or 13C-glucose was used as a model organism. Silver nanoparticles synthesized in situ were applied as SERS media. We observed a reproducible red-shift of an adenine-related marker band from 733 to 720 cm-1 in SERS spectra for 13C-labeled cells. Additionally, Raman measurements of 12C/13C-glucose and -phenylalanine mixtures were performed to elucidate the feasibility of SIRM for nondestructive quantitative and spatially resolved analysis. The performed analysis of isotopically labeled microbial cells with SERS-SIRM and resonance SIRM paves the way toward novel approaches to apply Raman microspectroscopy in environmental process studies. [ABSTRACT FROM AUTHOR]