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.

Summary 1. Groundwater ecosystems offer vast and complex habitats for diverse microbial communities. Here we review the current status of groundwater microbial biodiversity research with a focus on Bacteria and Archaea and on the prospects of modern techniques for enhancing our understanding of microbial biodiversity patterns and their relation to environmental conditions. 2. The enormous volume of the saturated terrestrial underground forms the largest habitat for microorganisms on earth. Up to 40% of prokaryotic biomass on earth is hidden within this terrestrial subsurface. Besides representing a globally important pool of carbon and nutrients in organisms, these communities harbour a degree of microbial diversity only marginally explored to date. 3. Although first observations of groundwater microbiota date back to Antonie van Leeuwenhoek in 1677, the systematic investigation of groundwater microbial biodiversity has gained momentum only within the last few decades. These investigations were initiated by an increasing awareness of the importance of aquifer microbiota for ecosystem services and functioning, including the provision of drinking water and the degradation of contaminants. 4. The development of sampling techniques suitable for microbiological investigations as well as the application of both cultivation‐based and molecular methods has yielded substantial insights into microbial communities in contaminated aquifers, whereas knowledge of microbial biodiversity in pristine habitats is still poor at present. 5. Several novel phylogenetic lineages have been described from groundwater habitats, but to date no clearly ‘endemic’ subsurface microbial phyla have been identified. The future will show if the rather low diversity generally found in pristine oligotrophic aquifers is a fact or just a result of low abundances and insufficient resolution of today’s methods. Refined approaches complemented by statistically rigorous applications of biodiversity estimates are urgently needed. 6. Factors identified to control microbial diversity in aquifers include spatial heterogeneity, temporal variability and disturbances such as pollution with chemical anthropogenic contaminants. Although first insights into the importance of individual biogeochemical processes may be obtained from surveys of microbial diversity within functional groups, direct links to groundwater ecosystem functioning have rarely been established so far.

Stable-isotope probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-isotope-labeled carbon (13C) or nitrogen (15N) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids (DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4-5 days, with much of the time being committed to the ultracentrifugation step.

The population dynamics of Archaea after flooding of an Italian rice field soil were studied over 17 days. Anoxically incubated rice field soil slurries exhibited a typical sequence of reduction processes characterized by reduction of nitrate, Fe 3؉ , and sulfate prior to the initiation of methane production. Archaeal population dynamics were followed using a dual approach involving molecular sequence retrieval and fingerprinting of small-subunit (SSU) rRNA genes. We retrieved archaeal sequences from four clone libraries ( Only the relative abundance of Methanosarcinaceae (182 bp) increased, roughly doubling from 15 to 29% of total archaeal gene frequency within the first 11 days, which was positively correlated to the dynamics of acetate and formate concentrations. Our results indicate that a functionally dynamic ecosystem, a rice field soil after flooding, was linked to a relatively stable archaeal community structure.