Sediment microbial assemblage structure is modified by marine polychaete gut passage.

Invertebrate activities in sediments, predominantly the redistribution of particles and porewater, are well-known to regulate the structure of associated microbial assemblages; however, relatively little attention has been given to the effects of sediment ingestion, gut passage and excretion by deposit-feeding invertebrates. Here, we use high-throughput sequencing and quantitative PCR to examine how passage through the gut of the marine polychaete Hediste diversicolor affects the structure of bacterial and archaeal assemblages and the abundance of nitrogen cycling taxa. We show that the digestive tract of H. diversicolor contains unique transitory microbial assemblages that, during gut passage, become more like the surrounding sediment assemblages. Enrichment of similar microbial taxa in both the hindgut and the burrow wall suggest that these transitory gut assemblages may influence the composition of the local sediment community. The hindgut of H. diversicolor also forms a reservoir for unique ammonia-oxidising archaeal taxa. Furthermore, distinct microbial assemblages on external polychaete surfaces suggest that deposit-feeding invertebrates act as vectors that transport microbes between sediment patches. Collectively, these findings suggest that the passage of sediment and associated microbial assemblages through the gut of deposit feeding invertebrates is likely to play a significant role in regulating sediment microbial assemblages and biogeochemical functioning.

Polychaete mucopolysaccharide alters sediment microbial diversity and stimulates ammonia-oxidising functional groups.

Sediment nitrogen cycling is a network of microbially mediated biogeochemical processes that are vital in regulating ecosystem functioning. Mucopolysaccharides (mucus) are produced by many invertebrates and have the potential to be an important source of organic carbon and nitrogen to sediment microorganisms. At present, we have limited understanding of how mucopolysaccharide moderates total sediment microbial communities and specific microbial functional groups that drive nitrogen cycling processes. To start addressing this knowledge gap, sediment slurries were incubated with and without Hediste diversicolor mucus. Changes in dissolved inorganic nitrogen (ammonia, nitrite and nitrate) concentrations and bacterial and archaeal community diversity were assessed. Our results showed that mucopolysaccharide addition supported a more abundant and distinct microbial community. Moreover, mucus stimulated the growth of bacterial and archaeal ammonia oxidisers, with a concomitant increase in nitrite and nitrate. Hediste diversicolor mucopolysaccharide appears to enhance sediment nitrification rates by stimulating and fuelling nitrifying microbial groups. We propose that invertebrate mucopolysaccharide secretion should be considered as a distinct functional trait when assessing invertebrate contributions to sediment ecosystem function. By including this additional trait, we can improve our mechanistic understanding of invertebrate-microbe interactions in nitrogen transformation processes and provide opportunity to generate more accurate models of global nitrogen cycling.

Prokaryotic niche partitioning between suspended and sinking marine particles.

Suspended particles are major organic carbon substrates for heterotrophic microorganisms in the mesopelagic ocean (100-1000 m). Nonetheless, communities associated with these particles have been overlooked compared with sinking particles, the latter generally considered as main carbon transporters to the deep ocean. This study is the first to differentiate prokaryotic communities associated with suspended and sinking particles, collected with a marine snow catcher at four environmentally distinct stations in the Scotia Sea. Amplicon sequencing of 16S rRNA gene revealed distinct prokaryotic communities associated with the two particle-types in the mixed-layer (0-100 m) and upper-mesopelagic zone (mean dissimilarity 42.5% ± 15.2%). Although common remineralising taxa were present within both particle-types, gammaproteobacterial Pseudomonadales and Vibrionales, and alphaproteobacterial Rhodobacterales were found enriched in sinking particles up to 32-fold, while Flavobacteriales (Bacteroidetes) favoured suspended particles. We propose that this niche-partitioning may be driven by organic matter properties found within both particle-types: K-strategists, specialised in the degradation of complex organic compounds, thrived on semi-labile suspended particles, while generalists r-strategists were adapted to the transient labile organic contents of sinking particles. Differences between the two particle-associated communities were more pronounced in the mesopelagic than in the surface ocean, likely resulting from exchanges between particle-pools enabled by the stronger turbulence.

Methane stimulates massive nitrogen loss from freshwater reservoirs in India.

The fate of the enormous amount of reactive nitrogen released to the environment by human activities in India is unknown. Here we show occurrence of seasonal stratification and generally low concentrations of dissolved inorganic combined nitrogen, and high molecular nitrogen (N2) to argon ratio, thus suggesting seasonal loss to N2 in anoxic hypolimnia of several dam-reservoirs. However, 15N-experiments yielded low rates of denitrification, anaerobic ammonium oxidation and dissimilatory nitrate reduction to ammonium-except in the presence of methane (CH4) that caused ~12-fold increase in denitrification. While nitrite-dependent anaerobic methanotrophs belonging to the NC10 phylum were present, previously considered aerobic methanotrophs were far more abundant (up to 13.9%) in anoxic hypolimnion. Methane accumulation in anoxic freshwater systems seems to facilitate rapid loss of reactive nitrogen, with generally low production of nitrous oxide (N2O), through widespread coupling between methanotrophy and denitrification, potentially mitigating eutrophication and emissions of CH4 and N2O to the atmosphere.

Adaptability as the key to success for the ubiquitous marine nitrite oxidizer Nitrococcus.

Nitrite-oxidizing bacteria (NOB) have conventionally been regarded as a highly specialized functional group responsible for the production of nitrate in the environment. However, recent culture-based studies suggest that they have the capacity to lead alternative lifestyles, but direct environmental evidence for the contribution of marine nitrite oxidizers to other processes has been lacking to date. We report on the alternative biogeochemical functions, worldwide distribution, and sometimes high abundance of the marine NOB Nitrococcus. These largely overlooked bacteria are capable of not only oxidizing nitrite but also reducing nitrate and producing nitrous oxide, an ozone-depleting agent and greenhouse gas. Furthermore, Nitrococcus can aerobically oxidize sulfide, thereby also engaging in the sulfur cycle. In the currently fast-changing global oceans, these findings highlight the potential functional switches these ubiquitous bacteria can perform in various biogeochemical cycles, each with distinct or even contrasting consequences.

Methane oxidation coupled to oxygenic photosynthesis in anoxic waters.

Freshwater lakes represent large methane sources that, in contrast to the Ocean, significantly contribute to non-anthropogenic methane emissions to the atmosphere. Particularly mixed lakes are major methane emitters, while permanently and seasonally stratified lakes with anoxic bottom waters are often characterized by strongly reduced methane emissions. The causes for this reduced methane flux from anoxic lake waters are not fully understood. Here we identified the microorganisms and processes responsible for the near complete consumption of methane in the anoxic waters of a permanently stratified lake, Lago di Cadagno. Interestingly, known anaerobic methanotrophs could not be detected in these waters. Instead, we found abundant gamma-proteobacterial aerobic methane-oxidizing bacteria active in the anoxic waters. In vitro incubations revealed that, among all the tested potential electron acceptors, only the addition of oxygen enhanced the rates of methane oxidation. An equally pronounced stimulation was also observed when the anoxic water samples were incubated in the light. Our combined results from molecular, biogeochemical and single-cell analyses indicate that methane removal at the anoxic chemocline of Lago di Cadagno is due to true aerobic oxidation of methane fuelled by in situ oxygen production by photosynthetic algae. A similar mechanism could be active in seasonally stratified lakes and marine basins such as the Black Sea, where light penetrates to the anoxic chemocline. Given the widespread occurrence of seasonally stratified anoxic lakes, aerobic methane oxidation coupled to oxygenic photosynthesis might have an important but so far neglected role in methane emissions from lakes.

Comparison of colonoscopic performance between medical and nurse endoscopists: a non-inferiority randomised controlled study in Asia.

OBJECTIVE: To test the hypothesis that trained nurse endoscopists are not inferior to medical endoscopists in finding adenomas during colonoscopy. DESIGN: This is a prospective, randomised, single-blind, non-inferiority study comparing nurses with medical endoscopists in performing screening colonoscopy. The nurse endoscopists had been trained according to the British Joint Advisory Group on GI Endoscopy curriculum and had completed at least 140 colonoscopic procedures prior to the study. The primary endpoint was the adenoma detection rate. Secondary endpoints included the caecal intubation rate, intubation time, complication rate, patient pain and satisfaction scores. RESULTS: We enrolled and analysed a total of 731 patients over a 15-month period. At least one adenoma was found in 159 (43.8%) of 363 patients by nurse endoscopists and 120 (32.7%) of 367 patients by medical endoscopists and a proportion difference of +11.1% compared with the medical endoscopists (95% CI 4.1% to 18.1%). The withdrawal time was, however, significantly longer among nurses (998 vs 575 s, p<0.001). After adjusting for differences in a regression analysis, colonoscopy by nurses was associated with a lower adenoma detection rate (OR 0.475: 95% CI 0.311 to 0.725). Nurse endoscopists had a lower caecal intubation rate (97.3% vs 100%), received better pain and satisfaction scores and had a high rate of patient acceptance. CONCLUSIONS: In this pragmatic trial, nurses can perform screening colonoscopy but require a longer procedural time to achieve a comparable adenoma detection rate as medical endoscopists. TRIAL REGISTRATION NUMBER: NCT01923155.

Responses of the coastal bacterial community to viral infection of the algae Phaeocystis globosa.

The release of organic material upon algal cell lyses has a key role in structuring bacterial communities and affects the cycling of biolimiting elements in the marine environment. Here we show that already before cell lysis the leakage or excretion of organic matter by infected yet intact algal cells shaped North Sea bacterial community composition and enhanced bacterial substrate assimilation. Infected algal cultures of Phaeocystis globosa grown in coastal North Sea water contained gamma- and alphaproteobacterial phylotypes that were distinct from those in the non-infected control cultures 5 h after infection. The gammaproteobacterial population at this time mainly consisted of Alteromonas sp. cells that were attached to the infected but still intact host cells. Nano-scale secondary-ion mass spectrometry (nanoSIMS) showed ∼20% transfer of organic matter derived from the infected (13)C- and (15)N-labelled P. globosa cells to Alteromonas sp. cells. Subsequent, viral lysis of P. globosa resulted in the formation of aggregates that were densely colonised by bacteria. Aggregate dissolution was observed after 2 days, which we attribute to bacteriophage-induced lysis of the attached bacteria. Isotope mass spectrometry analysis showed that 40% of the particulate (13)C-organic carbon from the infected P. globosa culture was remineralized to dissolved inorganic carbon after 7 days. These findings reveal a novel role of viruses in the leakage or excretion of algal biomass upon infection, which provides an additional ecological niche for specific bacterial populations and potentially redirects carbon availability.

High rates of denitrification and nitrous oxide emission in arid biological soil crusts from the Sultanate of Oman.

Using a combination of process rate determination, microsensor profiling and molecular techniques, we demonstrated that denitrification, and not anaerobic ammonium oxidation (anammox), is the major nitrogen loss process in biological soil crusts from Oman. Potential denitrification rates were 584±101 and 58±20 µmol N m(-2) h(-1) for cyanobacterial and lichen crust, respectively. Complete denitrification to N2 was further confirmed by an (15)NO3(-) tracer experiment with intact crust pieces that proceeded at rates of 103±19 and 27±8 µmol N m(-2) h(-1) for cyanobacterial and lichen crust, respectively. Strikingly, N2O gas was emitted at very high potential rates of 387±143 and 31±6 µmol N m(-2) h(-1) from the cyanobacterial and lichen crust, respectively, with N2O accounting for 53-66% of the total emission of nitrogenous gases. Microsensor measurements revealed that N2O was produced in the anoxic layer and thus apparently originated from incomplete denitrification. Using quantitative PCR, denitrification genes were detected in both the crusts and were expressed either in comparable (nirS) or slightly higher (narG) numbers in the cyanobacterial crusts. Although 99% of the nirS sequences in the cyanobacterial crust were affiliated to an uncultured denitrifying bacterium, 94% of these sequences were most closely affiliated to Paracoccus denitrificans in the lichen crust. Sequences of nosZ gene formed a distinct cluster that did not branch with known denitrifying bacteria. Our results demonstrate that nitrogen loss via denitrification is a dominant process in crusts from Oman, which leads to N2O gas emission and potentially reduces desert soil fertility.

The metagenome of the marine anammox bacterium 'Candidatus Scalindua profunda' illustrates the versatility of this globally important nitrogen cycle bacterium.

Anaerobic ammonium-oxidizing (anammox) bacteria are responsible for a significant portion of the loss of fixed nitrogen from the oceans, making them important players in the global nitrogen cycle. To date, marine anammox bacteria found in marine water columns and sediments worldwide belong almost exclusively to the 'Candidatus Scalindua' species, but the molecular basis of their metabolism and competitive fitness is presently unknown. We applied community sequencing of a marine anammox enrichment culture dominated by 'Candidatus Scalindua profunda' to construct a genome assembly, which was subsequently used to analyse the most abundant gene transcripts and proteins. In the S. profunda assembly, 4756 genes were annotated, and only about half of them showed the highest identity to the only other anammox bacterium of which a metagenome assembly had been constructed so far, the freshwater 'Candidatus Kuenenia stuttgartiensis'. In total, 2016 genes of S. profunda could not be matched to the K. stuttgartiensis metagenome assembly at all, and a similar number of genes in K.stuttgartiensis could not be found in S. profunda. Most of these genes did not have a known function but 98 expressed genes could be attributed to oligopeptide transport, amino acid metabolism, use of organic acids and electron transport. On the basis of the S. profunda metagenome, and environmental metagenome data, we observed pronounced differences in the gene organization and expression of important anammox enzymes, such as hydrazine synthase (HzsAB), nitrite reductase (NirS) and inorganic nitrogen transport proteins. Adaptations of Scalindua to the substrate limitation of the ocean may include highly expressed ammonium, nitrite and oligopeptide transport systems and pathways for the transport, oxidation, and assimilation of small organic compounds that may allow a more versatile lifestyle contributing to the competitive fitness of Scalindua in the marine realm.

Benthic nitrogen loss in the arabian sea off pakistan.

A pronounced deficit of nitrogen (N) in the oxygen minimum zone (OMZ) of the Arabian Sea suggests the occurrence of heavy N-loss that is commonly attributed to pelagic processes. However, the OMZ water is in direct contact with sediments on three sides of the basin. Contribution from benthic N-loss to the total N-loss in the Arabian Sea remains largely unassessed. In October 2007, we sampled the water column and surface sediments along a transect cross-cutting the Arabian Sea OMZ at the Pakistan continental margin, covering a range of station depths from 360 to 1430 m. Benthic denitrification and anammox rates were determined by using (15)N-stable isotope pairing experiments. Intact core incubations showed declining rates of total benthic N-loss with water depth from 0.55 to 0.18 mmol N m(-2) day(-1). While denitrification rates measured in slurry incubations decreased from 2.73 to 1.46 mmol N m(-2) day(-1) with water depth, anammox rates increased from 0.21 to 0.89 mmol N m(-2) day(-1). Hence, the contribution from anammox to total benthic N-loss increased from 7% at 360 m to 40% at 1430 m. This trend is further supported by the quantification of cd(1)-containing nitrite reductase (nirS), the biomarker functional gene encoding for cytochrome cd(1)-Nir of microorganisms involved in both N-loss processes. Anammox-like nirS genes within the sediments increased in proportion to total nirS gene copies with water depth. Moreover, phylogenetic analyses of NirS revealed different communities of both denitrifying and anammox bacteria between shallow and deep stations. Together, rate measurement and nirS analyses showed that anammox, determined for the first time in the Arabian Sea sediments, is an important benthic N-loss process at the continental margin off Pakistan, especially in the sediments at deeper water depths. Extrapolation from the measured benthic N-loss to all shelf sediments within the basin suggests that benthic N-loss may be responsible for about half of the overall N-loss in the Arabian Sea.

Nitrite oxidation in the Namibian oxygen minimum zone.

Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in (15)N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (≤372 nM NO(2)(-) d(-1)) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ~9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO(3)(-) was re-oxidized back to NO(3)(-) via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways.

Heterotrophic organisms dominate nitrogen fixation in the South Pacific Gyre.

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(-1). 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 γ-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.

A randomized comparison of ultrathin and standard colonoscope in cecal intubation rate and patient tolerance.

BACKGROUND: Complete colonoscopy examination cannot be performed in as many as 10% of cases. The new 9.2-mm ultrathin colonoscope (UTC) with an extra bending section may improve procedure tolerance and allow improvement in colonoscopy completion rate compared with a 12.9-mm standard colonoscope (SC). OBJECTIVE: To compare the performance of the 9.2-mm UTC with that of the 12.9-mm SC. DESIGN: Prospective, randomized, controlled trial. SETTING: Academic endoscopic unit. PATIENTS: Subjects 18 years and older undergoing their first colonoscopy. INTERVENTION: Subjects were randomized to either the UTC or SC group. MAIN OUTCOME MEASUREMENTS: First and rescue successful cecal intubation rates, subject satisfaction scores, and sedation requirements were compared. RESULTS: A total of 1121 patients (56% women, mean age 53.6 years) were randomized to the UTC group (n = 551) or the SC group (n = 570). There was no statistically significant difference in the first successful cecal intubation rate between the UTC and SC groups (98.9% vs 97.4%, P = .057). The mean (standard deviation) dose of midazolam and pethidine used was significantly lower in the UTC group (2.65 [0.65] mg vs 2.82 [0.85] mg, P < .001 and 27.6 [7.4] mg vs 29.7 [9.6] mg, P < .001, respectively). The mean (standard deviation) patient satisfaction score was similar between groups (6.99 [2.89] vs 7.04 [3.06], P = .762). Of the 21 patients (1.9%) with an incomplete initial colonoscopy (6 in the UTC group and 15 in the SC group), all 6 in the UTC group had their procedure completed with an SC. Eleven of 15 patients in the SC group had their procedures completed with a UTC in the same session. LIMITATIONS: Low failure rate may mask any difference between the 2 colonoscopes as a rescue instrument. CONCLUSIONS: The 9.2-mm UTC has performance characteristics similar to those of an SC in Chinese subjects undergoing their first colonoscopy performed by experienced and trainee endoscopists. ( CLINICAL TRIAL REGISTRATION NUMBER: NCT01142167.).

Intensive nitrogen loss over the Omani Shelf due to anammox coupled with dissimilatory nitrite reduction to ammonium.

A combination of stable isotopes ((15)N) and molecular ecological approaches was used to investigate the vertical distribution and mechanisms of biological N(2) production along a transect from the Omani coast to the central-northeastern (NE) Arabian Sea. The Arabian Sea harbors the thickest oxygen minimum zone (OMZ) in the world's oceans, and is considered to be a major site of oceanic nitrogen (N) loss. Short (<48 h) anoxic incubations with (15)N-labeled substrates and functional gene expression analyses showed that the anammox process was highly active, whereas denitrification was hardly detectable in the OMZ over the Omani shelf at least at the time of our sampling. Anammox was coupled with dissimilatory nitrite reduction to ammonium (DNRA), resulting in the production of double-(15)N-labeled N(2) from (15)NO(2)(-), a signal often taken as the lone evidence for denitrification in the past. Although the central-NE Arabian Sea has conventionally been regarded as the primary N-loss region, low potential N-loss rates at sporadic depths were detected at best. N-loss activities in this region likely experience high spatiotemporal variabilities as linked to the availability of organic matter. Our finding of greater N-loss associated with the more productive Omani upwelling region is consistent with results from other major OMZs. The close reliance of anammox on DNRA also highlights the need to take into account the effects of coupling N-transformations on oceanic N-loss and subsequent N-balance estimates.

Microbial nitrogen cycling processes in oxygen minimum zones.

Oxygen minimum zones (OMZs) harbor unique microbial communities that rely on alternative electron acceptors for respiration. Conditions therein enable an almost complete nitrogen (N) cycle and substantial N-loss. N-loss in OMZs is attributable to anammox and heterotrophic denitrification, whereas nitrate reduction to nitrite along with dissimilatory nitrate reduction to ammonium are major remineralization pathways. Despite virtually anoxic conditions, nitrification also occurs in OMZs, converting remineralized ammonium to N-oxides. The concurrence of all these processes provides a direct channel from organic N to the ultimate N-loss, whereas most individual processes are likely controlled by organic matter. Many microorganisms inhabiting the OMZs are capable of multiple functions in the N- and other elemental cycles. Their versatile metabolic potentials versus actual activities present a challenge to ecophysiological and biogeochemical measurements. These challenges need to be tackled before we can realistically predict how N-cycling in OMZs, and thus oceanic N-balance, will respond to future global perturbations.

Oxygen sensitivity of anammox and coupled N-cycle processes in oxygen minimum zones.

Nutrient measurements indicate that 30-50% of the total nitrogen (N) loss in the ocean occurs in oxygen minimum zones (OMZs). This pelagic N-removal takes place within only ~0.1% of the ocean volume, hence moderate variations in the extent of OMZs due to global warming may have a large impact on the global N-cycle. We examined the effect of oxygen (O(2)) on anammox, NH(3) oxidation and NO(3)(-) reduction in (15)N-labeling experiments with varying O(2) concentrations (0-25 µmol L(-1)) in the Namibian and Peruvian OMZs. Our results show that O(2) is a major controlling factor for anammox activity in OMZ waters. Based on our O(2) assays we estimate the upper limit for anammox to be ~20 µmol L(-1). In contrast, NH(3) oxidation to NO(2)(-) and NO(3)(-) reduction to NO(2)(-) as the main NH(4)(+) and NO(2)(-) sources for anammox were only moderately affected by changing O(2) concentrations. Intriguingly, aerobic NH(3) oxidation was active at non-detectable concentrations of O(2), while anaerobic NO(3)(-) reduction was fully active up to at least 25 µmol L(-1) O(2). Hence, aerobic and anaerobic N-cycle pathways in OMZs can co-occur over a larger range of O(2) concentrations than previously assumed. The zone where N-loss can occur is primarily controlled by the O(2)-sensitivity of anammox itself, and not by any effects of O(2) on the tightly coupled pathways of aerobic NH(3) oxidation and NO(3)(-) reduction. With anammox bacteria in the marine environment being active at O(2) levels ~20 times higher than those known to inhibit their cultured counterparts, the oceanic volume potentially acting as a N-sink increases tenfold. The predicted expansion of OMZs may enlarge this volume even further. Our study provides the first robust estimates of O(2) sensitivities for processes directly and indirectly connected with N-loss. These are essential to assess the effects of ocean de-oxygenation on oceanic N-cycling.

GeneFISH--an in situ technique for linking gene presence and cell identity in environmental microorganisms.

Our knowledge concerning the metabolic potentials of as yet to be cultured microorganisms has increased tremendously with the advance of sequencing technologies and the consequent discoveries of novel genes. On the other hand, it is often difficult to reliably assign a particular gene to a phylogenetic clade, because these sequences are usually found on genomic fragments that carry no direct marker of cell identity, such as rRNA genes. Therefore, the aim of the present study was to develop geneFISH - a protocol for linking gene presence with cell identity in environmental samples, the signals of which can be visualized at a single cell level. This protocol combines rRNA-targeted catalysed reporter deposition - fluorescence in situ hybridization and in situ gene detection. To test the protocol, it was applied to seawater samples from the Benguela upwelling system. For gene detection, a polynucleotide probe mix was used, which was designed based on crenarchaeotal amoA clone libraries prepared from each seawater sample. Each probe in the mix was selected to bind to targets with up to 5% mismatches. To determine the hybridization parameters, the T(m) of probes, targets and hybrids was estimated based on theoretical calculations and in vitro measurements. It was shown that at least 30%, but potentially the majority of the Crenarchaeota present in these samples harboured the amoA gene and were therefore likely to be catalysing the oxidation of ammonia.

Co-occurrence of denitrification and nitrogen fixation in a meromictic lake, Lake Cadagno (Switzerland).

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.

Revising the nitrogen cycle in the Peruvian oxygen minimum zone.

The oxygen minimum zone (OMZ) of the Eastern Tropical South Pacific (ETSP) is 1 of the 3 major regions in the world where oceanic nitrogen is lost in the pelagic realm. The recent identification of anammox, instead of denitrification, as the likely prevalent pathway for nitrogen loss in this OMZ raises strong questions about our understanding of nitrogen cycling and organic matter remineralization in these waters. Without detectable denitrification, it is unclear how NH(4)(+) is remineralized from organic matter and sustains anammox or how secondary NO(2)(-) maxima arise within the OMZ. Here we show that in the ETSP-OMZ, anammox obtains 67% or more of NO(2)(-) from nitrate reduction, and 33% or less from aerobic ammonia oxidation, based on stable-isotope pairing experiments corroborated by functional gene expression analyses. Dissimilatory nitrate reduction to ammonium was detected in an open-ocean setting. It occurred throughout the OMZ and could satisfy a substantial part of the NH(4)(+) requirement for anammox. The remaining NH(4)(+) came from remineralization via nitrate reduction and probably from microaerobic respiration. Altogether, deep-sea NO(3)(-) accounted for only approximately 50% of the nitrogen loss in the ETSP, rather than 100% as commonly assumed. Because oceanic OMZs seem to be expanding because of global climate change, it is increasingly imperative to incorporate the correct nitrogen-loss pathways in global biogeochemical models to predict more accurately how the nitrogen cycle in our future ocean may respond.