Quantitative information on the ecophysiology of individual microorganisms is generally limited because it is difficult to assign specific metabolic activities to identified single cells. Here, we develop and apply a method, Halogen In Situ HybridizationSecondary Ion Mass Spectroscopy (HISH-SIMS), and show that it allows simultaneous phylogenetic identification and quantitation of metabolic activities of single microbial cells in the environment. Using HISH-SIMS, individual cells of the anaerobic, phototropic bacteria Chromatium okenii, Lamprocystis purpurea, and Chlorobium clathratiforme inhabiting the oligotrophic, meromictic Lake Cadagno were analyzed with respect to H 13 CO3 ؊ and 15 NH4 ؉ assimilation. Metabolic rates were found to vary greatly between individual cells of the same species, showing that microbial populations in the environment are heterogeneous, being comprised of physiologically distinct individuals. Furthermore, C. okenii, the least abundant species representing Ϸ0.3% of the total cell number, contributed more than 40% of the total uptake of ammonium and 70% of the total uptake of carbon in the system, thereby emphasizing that numerically inconspicuous microbes can play a significant role in the nitrogen and carbon cycles in the environment. By introducing this quantification method for the ecophysiological roles of individual cells, our study opens a variety of possibilities of research in environmental microbiology, especially by increasing the ability to examine the ecophysiological roles of individual cells, including those of less abundant and less active microbes, and by the capacity to track not only nitrogen and carbon but also phosphorus, sulfur, and other biological element flows within microbial communities.anaerobic phototrophs ͉ nanoSIMS

Oceanic subtropical gyres are considered biological deserts because of the extremely low availability of nutrients and thus minimum productivities. The major source of nutrient nitrogen in these ecosystems is N 2 -fixation. The South Pacific Gyre (SPG) is the largest ocean gyre in the world, but measurements of N 2 -fixation therein, or identification of microorganisms involved, are scarce. In the 2006/2007 austral summer, we investigated nitrogen and carbon assimilation at 11 stations throughout the SPG. In the ultra-oligotrophic waters of the SPG, the chlorophyll maxima reached as deep as 200 m. Surface primary production seemed limited by nitrogen, as dissolved inorganic carbon uptake was stimulated upon additions of 15 N-labeled ammonium and leucine in our incubation experiments. N 2 -fixation was detectable throughout the upper 200 m at most stations, with rates ranging from 0.001 to 0.19 nM N h À1 . N 2 -fixation in the SPG may account for the production of 8-20% of global oceanic new nitrogen. Interestingly, comparable 15 N 2 -fixation rates were measured under light and dark conditions. Meanwhile, phylogenetic analyses for the functional gene biomarker nifH and its transcripts could not detect any common photoautotrophic diazotrophs, such as, Trichodesmium, but a prevalence of c-proteobacteria and the unicellular photoheterotrophic Group A cyanobacteria. The dominance of these likely heterotrophic diazotrophs was further verified by quantitative PCR. Hence, our combined results show that the ultra-oligotrophic SPG harbors a hitherto unknown heterotrophic diazotrophic community, clearly distinct from other oceanic gyres previously visited.

The nitrogen cycling of Lake Cadagno was investigated by using a combination of biogeochemical and molecular ecological techniques. In the upper oxic freshwater zone inorganic nitrogen concentrations were low (up to approximately 3.4 microM nitrate at the base of the oxic zone), while in the lower anoxic zone there were high concentrations of ammonium (up to 40 microM). Between these zones, a narrow zone was characterized by no measurable inorganic nitrogen, but high microbial biomass (up to 4 x 10(7) cells ml(-1)). Incubation experiments with (15)N-nitrite revealed nitrogen loss occurring in the chemocline through denitrification (approximately 3 nM N h(-1)). At the same depth, incubations experiments with (15)N(2)- and (13)C(DIC)-labelled bicarbonate, indicated substantial N(2) fixation (31.7-42.1 pM h(-1)) and inorganic carbon assimilation (40-85 nM h(-1)). Catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and sequencing of 16S rRNA genes showed that the microbial community at the chemocline was dominated by the phototrophic green sulfur bacterium Chlorobium clathratiforme. Phylogenetic analyses of the nifH genes expressed as mRNA revealed a high diversity of N(2) fixers, with the highest expression levels right at the chemocline. The majority of N(2) fixers were related to Chlorobium tepidum/C. phaeobacteroides. By using Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy (HISH-SIMS), we could for the first time directly link Chlorobium to N(2) fixation in the environment. Moreover, our results show that N(2) fixation could partly compensate for the N loss and that both processes occur at the same locale at the same time as suggested for the ancient Ocean.

Summary The globally abundant, uncultured unicellular cyanobacterium UCYN‐A was recently discovered living in association with a eukaryotic cell closely related to a prymnesiophyte. Here, we established a double CAtalysed Reporter Deposition‐Fluorescence In Situ Hybridization (CARD‐FISH) approach to identify both partners and provided quantitative information on their distribution and abundance across distinct water masses along a transect in the North Atlantic Ocean. The N2 fixation activity coincided with the detection of UCYN‐A cells and was only observed in oligotrophic (< 0.067 N boldO bold3 −  μM and < 0.04 P boldO bold4 3 −  μM) and warm (> 18°C) surface waters. Parallel 16S ribosomal RNA gene analyses among unicellular diazotrophs indicated that only UCYN‐A cells were present. UCYN‐A cells were associated with an algal partner or non‐associated using the double CARD‐FISH approach. We demonstrated that UCYN‐A cells living in association with Haptophyta were the dominant form (87.0 ± 6.1%), whereas non‐associated UCYN‐A cells represented only a minor fraction (5.2 ± 3.9%). Interestingly, UCYN‐A cells were also detected living in association with unknown single‐celled eukaryotes in small amounts (7.8 ± 5.2%), presumably Alveolata. The proposed ecological niche of UCYN‐A as an oligotrophic, mesophilic and obligate symbiotic nitrogen‐fixing microorganism is evident for the North Atlantic Ocean.