Microorganisms are responsible for driving the biogeochemical cycling of elements on Earth. Despite their importance and vast diversity, the taxonomic identity of the microorganisms involved in any specific process has usually been confined to that small fraction of the microbiota that has been isolated and cultivated. The recent coupling of molecular biological methods with stable-isotope abundance in biomarkers has provided a cultivation-independent means of linking the identity of bacteria with their function in the environment. Here we show that 13C-DNA, produced during the growth of metabolically distinct microbial groups on a 13C-enriched carbon source, can be resolved from 12C-DNA by density-gradient centrifugation. DNA isolated from the target group of microorganisms can be characterized taxonomically and functionally by gene probing and sequence analysis. Application of this technique to investigate methanol-utilizing microorganisms in soil demonstrated the involvement of members of two phylogenetically distinct groups of eubacteria; the alpha-proteobacterial and Acidobacterium lineages. Stable-isotope probing thus offers a powerful new technique for identifying microorganisms that are actively involved in specific metabolic processes under conditions which approach those occurring in situ.

The results of published and unpublished experiments investigating the impacts of elevated [CO2] on the chemistry of leaf litter and decomposition of plant tissues are summarized. The data do not support the hypothesis that changes in leaf litter chemistry often associated with growing plants under elevated [CO2] have an impact on decomposition processes. A meta-analysis of data from naturally senesced leaves in field experiments showed that the nitrogen (N) concentration in leaf litter was 7.1% lower in elevated [CO2] compared to that in ambient [CO2]. This statistically significant difference was: (1) usually not significant in individual experiments, (2) much less than that often observed in green leaves, and (3) less in leaves with an N concentration indicative of complete N resorption. Under ideal conditions, the efficiency with which N is resorbed during leaf senescence was found not to be altered by CO2 enrichment, but other environmental influences on resorption inevitably increase the variability in litter N concentration. Nevertheless, the small but consistent decline in leaf litter N concentration in many experiments, coupled with a 6.5% increase in lignin concentration, would be predicted to result in a slower decomposition rate in CO2-enriched litter. However, across the assembled data base, neither mass loss nor respiration rates from litter produced in elevated [CO2] showed any consistent pattern or differences from litter grown in ambient [CO2]. The effects of [CO2] on litter chemistry or decomposition were usually smallest under experimental conditions similar to natural field conditions, including open-field exposure, plants free-rooted in the ground, and complete senescence. It is concluded that any changes in decomposition rates resulting from exposure of plants to elevated [CO2] are small when compared to other potential impacts of elevated [CO2] on carbon and N cycling. Reasons for experimental differences are considered, and recommendations for the design and execution of decomposition experiments using materials from CO2-enrichment experiments are outlined.

Increased atmospheric nitrogen (N) deposition is known to reduce plant diversity in natural and semi-natural ecosystems, yet our understanding of these impacts comes almost entirely from studies in northern Europe and North America. Currently, we lack an understanding of the threat of N deposition to biodiversity at the global scale. In particular, rates of N deposition within the newly defined 34 world biodiversity hotspots, to which 50% of the world's floristic diversity is restricted, has not been quantified previously. Using output from global chemistry transport models, here we provide the first estimates of recent (mid-1990s) and future (2050) rates and distributions of N deposition within biodiversity hotspots. Our analysis shows that the average deposition rate across these areas was 50% greater than the global terrestrial average in the mid1990s and could more than double by 2050, with 33 of 34 hotspots receiving greater N deposition in 2050 compared with 1990. By this time, 17 hotspots could have between 10% and 100% of their area receiving greater than 15 kg N ha À1 yr À1 , a rate exceeding critical loads set for many sensitive European ecosystems. Average deposition in four hotspots is predicted to be greater than 20 kg N ha À1 yr À1 . This elevated N deposition within areas of high plant diversity and endemism may exacerbate significantly the global threat of N deposition to world floristic diversity. Overall, we highlight the need for a greater global approach to assessing the impacts of N deposition.

Processes in the soil remain among the least well-characterized components of the carbon cycle. Arbuscular mycorrhizal (AM) fungi are ubiquitous root symbionts in many terrestrial ecosystems and account for a large fraction of photosynthate in a wide range of ecosystems; they therefore play a key role in the terrestrial carbon cycle. A large part of the fungal mycelium is outside the root (the extraradical mycelium, ERM) and, because of the dispersed growth pattern and the small diameter of the hyphae (<5 micrometers), exceptionally difficult to study quantitatively. Critically, the longevity of these fine hyphae has never been measured, although it is assumed to be short. To quantify carbon turnover in these hyphae, we exposed mycorrhizal plants to fossil ("carbon-14-dead") carbon dioxide and collected samples of ERM hyphae (up to 116 micrograms) over the following 29 days. Analyses of their carbon-14 content by accelerator mass spectrometry (AMS) showed that most ERM hyphae of AM fungi live, on average, 5 to 6 days. This high turnover rate reveals a large and rapid mycorrhizal pathway of carbon in the soil carbon cycle.

Summary• The flux of pulse-derived 13 C from upland pasture plants to the external mycelium of their arbuscular mycorrhizal (AM) symbionts was traced and quantified over a 7-d post-labelling period.• Mesh cores, which allowed in-growth of native AM mycelium but were impenetrable to roots, were inserted into unlimed and limed plots and the surrounding vegetation was exposed to 13 CO 2 at ambient CO 2 concentrations.• Release of 13 CO 2 from cores colonized by AM mycelium peaked 9 -14 h after labelling and declined within 24 h after severance of mycelial connections to roots. Between 5 and 8% of carbon lost by plants was respired by AM mycelium over the first 21 h after labelling. Liming increased the amount of carbon fixed by plants and subsequently allocated to fine roots and AM mycelium.• The results demonstrate for the first time under field conditions that AM mycelia provide a rapid and important pathway of carbon flux from plants to the soil and atmosphere.

Well-drained non-agricultural soils mediate the oxidation of methane directly from the atmosphere, contributing 5 to 10% towards the global methane sink. Studies of methane oxidation kinetics in soil infer the activity of two methanotrophic populations: one that is only active at high methane concentrations (low affinity) and another that tolerates atmospheric levels of methane (high affinity). The activity of the latter has not been demonstrated by cultured laboratory strains of methanotrophs, leaving the microbiology of methane oxidation at atmospheric concentrations unclear. Here we describe a new pulse-chase experiment using long-term enrichment with 12CH4 followed by short-term exposure to 13CH4 to isotopically label methanotrophs in a soil from a temperate forest. Analysis of labelled phospholipid fatty acids (PLFAs) provided unambiguous evidence of methane assimilation at true atmospheric concentrations (1.8-3.6 p.p.m.v.). High proportions of 13C-labelled C18 fatty acids and the co-occurrence of a labelled, branched C17 fatty acid indicated that a new methanotroph, similar at the PLFA level to known type II methanotrophs, was the predominant soil micro-organism responsible for atmospheric methane oxidation.

Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 35) microcosms that were exposed to 13 CH 3 OH or 13 CH 4 . Distinct 13 C-labelled DNA ( 13 C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the 13 C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the 13 C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH 3 OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the β-subclass of the Proteobacteria. Analysis of AOB-selective 16S rDNA amplification products identifiedNitrosomonas and Nitrosospira sequences in the 13 C-DNA fractions, suggesting certain AOB assimilated a significant proportion of 13 CO 2 , possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the 13 C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the β-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the byproducts of methylotrophic carbon metabolism. Results from the 13 CH 3 OH and 13 CH 4 SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ.

Plant roots harbor a large diversity of microorganisms that have an essential role in ecosystem functioning. To better understand the level of intimacy of root-inhabiting microbes such as arbuscular mycorrhizal fungi and bacteria, we provided 13 CO2 to plants at atmospheric concentration during a 5-h pulse. We expected microbes dependent on a carbon flux from their host plant to become rapidly labeled. We showed that a wide variety of microbes occurred in roots, mostly previously unknown. Strikingly, the greatest part of this unsuspected diversity corresponded to active primary consumers. We found 17 bacterial phylotypes co-occurring within roots of a single plant, including five potentially new phylotypes. Fourteen phylotypes were heavily labeled with the 13 C. Eight were phylogenetically close to Burkholderiales, which encompass known symbionts; the others were potentially new bacterial root symbionts. By analyzing unlabeled and 13 C-enriched RNAs, we demonstrated differential activity in C consumption among these root-inhabiting microbes. Arbuscular mycorrhizal fungal RNAs were heavily labeled, confirming the high carbon flux from the plant to the fungal compartment, but some of the fungi present appeared to be much more active than others. The results presented here reveal the possibility of uncharacterized root symbioses.ribosomal RNA ͉ stable isotope probing ͉ symbiosis ͉ arbuscular mycorrhiza ͉ endophytes P lants are the dominant primary producers in most terrestrial ecosystems. In the soil, they are escorted by a myriad of microorganisms living freely or in intimate interaction with their roots (1, 2). These microorganisms can be pathogenic, parasitic, saprotrophic, or mutualistic. Among the root symbionts, arbuscular mycorrhizal (AM) fungi are well known and have been observed colonizing the roots of most plant species in many ecosystems (3). Recent studies show that high diversity is the norm even where plant diversity is low (4-6). These AM fungi are biotrophs, unable to grow in the absence of a living plant, and often display a broad host range although there is growing evidence for differences in host preference (5-8). They have been demonstrated to improve plant mineral nutrition (3) and stress resistance (3). AM fungi are important for the global carbon cycle because up to 20% of photoassimilates can be translocated to them (9). We also know that the diversity of AM fungi can determine plant community structure and ecosystem productivity (10). The plant-bacteria symbioses are variably documented. The best studied symbiosis is the rhizobiumlegume interaction, but a single plant root can harbor a large variety of fungi (1) and bacteria (2), as well as several different archaea (2). So far we have no information about the functions of most of these root-living microbes. The strategy chosen herein, stable isotope probing (SIP)-RNA analysis, enabled us to highlight an unsuspected diversity of microbes living in roots. We identified microbes that are active and direct utilizers of photosynthetic carbon ...