Hydrostatic pressure impedes the degradation of sinking copepod carcasses and fecal pellets.

Fast-sinking zooplankton carcasses and fecal pellets appear to contribute significantly to the vertical transport of particulate organic carbon (POC), partly because of low temperature that decreases microbial degradation during the descent into the deep ocean. Increasing hydrostatic pressure could further reduce the degradation efficiency of sinking POC, but this effect remains unexplored. Here, the degradation of carcasses and fecal pellets of the abundant marine copepod Calanus finmarchicus was experimentally studied as a function of pressure (0.1-100 MPa). Samples were either exposed to elevated pressure in short 1-day incubations or a gradual pressure increase, simulating continuous particle sinking during a 20-day incubation. Both experiments revealed gradual inhibition of microbial respiration in the pressure range of 20-100 MPa, corresponding to 2-10-km depth. This suggests that hydrostatic pressure impedes carbon mineralization of fast-sinking carcasses and fecal pellets and enhances the deep-sea deposition rate of zooplankton-derived organic material.

Respiration by "marine snow" at high hydrostatic pressure: Insights from continuous oxygen measurements in a rotating pressure tank.

It is generally anticipated that particulate organic carbon (POC) for most part is degraded by attached microorganisms during the descent of "marine snow" aggregates toward the deep sea. There is, however, increasing evidence that fresh aggregates can reach great depth and sustain relatively high biological activity in the deep sea. Using a novel high-pressure setup, we tested the hypothesis that increasing levels of hydrostatic pressure inhibit POC degradation in aggregates rapidly sinking to the ocean interior. Respiration activity, a proxy for POC degradation, was measured directly and continuously at up to 100 MPa (corresponding to 10 km water depth) in a rotating pressure tank that keeps the aggregates in a sinking mode. Model diatom-bacteria aggregates, cultures of the aggregate-forming diatom Skeletonema marinoi, and seawater microbial communities devoid of diatoms showed incomplete and complete inhibition of respiration activity when exposed to pressure levels of 10-50 and 60-100 MPa, respectively. This implies reduced POC degradation and hence enhanced POC export to hadal trenches through fast-sinking, pressure-exposed aggregates. Notably, continuous respiration measurements at ≥50 MPa revealed curved instead of linear oxygen time series whenever S. marinoi was present, which was not captured by discrete respiration measurements. These curvatures correspond to alternating phases of high and low respiration activity likely connected to pressure effects on unidentified metabolic processes in S. marinoi.

Freshwater copepod carcasses as pelagic microsites of dissimilatory nitrate reduction to ammonium.

A considerable fraction of freshwater zooplankton was recently found to consist of dead specimens that sink to the lake bottom. Such carcasses host intense microbial activities that may promote oxygen depletion at the microscale. Therefore, we tested the hypothesis that sinking zooplankton carcasses are microsites of anaerobic nitrogen cycling that contribute to pelagic fixed-nitrogen loss even in the presence of ambient oxygen. Incubation experiments were performed with the ubiquitous copepods Eudiaptomus sp. and Megacyclops gigas at different ambient oxygen levels that sinking carcasses encounter during their descent in stratified lakes. 15N-stable-isotope incubations revealed intense carcass-associated anaerobic nitrogen cycling only at low ambient oxygen levels (<25% air saturation). Dissimilatory nitrate reduction to ammonium (DNRA) dominated over denitrification and thus the potential for fixed-nitrogen loss was low. Consistent with this partitioning of anaerobic nitrogen cycling, the relative abundance of the carcass-associated marker gene for DNRA (nrfA) was ∼20-400 times higher than that for denitrification (nirS). Additionally, the relative nrfA and nirS abundances were ∼90-180 times higher on copepod carcasses than in lake water. This functional distinctiveness of carcass-associated bacterial communities was further substantiated by 16S rDNA-based fingerprinting. We conclude that the unique bacterial communities and microenvironments provided by zooplankton carcasses influence pelagic nitrogen cycling in lakes, but mainly at seasonally low ambient O2 levels in the bottom water.

Intracellular nitrate in sediments of an oxygen-deficient marine basin is linked to pelagic diatoms.

Intracellular nitrate is an important electron acceptor in oxygen-deficient aquatic environments, either for the nitrate-storing microbes themselves, or for ambient microbial communities through nitrate leakage. This study links the spatial distribution of intracellular nitrate with the abundance and identity of nitrate-storing microbes in sediments of the Bornholm Basin, an environmental showcase for severe hypoxia. Intracellular nitrate (up to 270 nmol cm-3 sediment) was detected at all 18 stations along a 35-km transect through the basin and typically extended as deep as 1.6 cm into the sediment. Intracellular nitrate contents were particularly high at stations where chlorophyll contents suggested high settling rates of pelagic primary production. The depth distribution of intracellular nitrate matched that of the diatom-specific photopigment fucoxanthin in the upper 1.6 cm and calculations support that diatoms are the major nitrate-storing microbes in these sediments. In contrast, other known nitrate-storing microbes, such as sulfide-oxidizing bacteria and foraminifers, played only a minor role, if any. Strikingly, 18S rRNA gene sequencing revealed that the majority of the diatoms in the sediment were pelagic species. We conclude that intracellular nitrate stored by pelagic diatoms is transported to the seafloor by settling phytoplankton blooms, implying a so far overlooked 'biological nitrate pump'.

Effect of settled diatom-aggregates on benthic nitrogen cycling.

The marine sediment hosts a mosaic of microhabitats. Recently it has been demonstrated that the settlement of phycodetrital aggregates can induce local changes in the benthic O2 distribution due to confined enrichment of organic material and alteration of the diffusional transport. Here, we show how this microscale O2 shift substantially affects benthic nitrogen cycling. In sediment incubations, the settlement of diatom-aggregates markedly enhanced benthic O2 and NO3- consumption and stimulated NO2- and NH4+ production. Oxygen microprofiles revealed the rapid development of anoxic niches within and underneath the aggregates. During 120 h following the settling of the aggregates, denitrification of NO3- from the overlying water increased from 13.5 µmol m-2 h-1 to 24.3 µmol m-2 h-1, as quantified by 15N enrichment experiment. Simultaneously, N2 production from coupled nitrification-denitrification decreased from 33.4 µmol m-2 h-1 to 25.9 µmol m-2 h-1, probably due to temporary inhibition of the benthic nitrifying community. The two effects were of similar magnitude and left the total N2 production almost unaltered. At the aggregate surface, nitrification was, conversely, very efficient in oxidizing NH4+ liberated by mineralization of the aggregates. The produced NO3- was preferentially released into the overlying water and only a minor fraction contributed to denitrification activity. Overall, our data indicate that the abrupt change in O2 microdistribution caused by aggregates stimulates denitrification of NO3- from the overlying water, and loosens the coupling between benthic nitrification and denitrification both in time and space. The study contributes to expanding the conceptual and quantitative understanding of how nitrogen cycling is regulated in dynamic benthic environments.

Intracellular Nitrate of Marine Diatoms as a Driver of Anaerobic Nitrogen Cycling in Sinking Aggregates.

Diatom-bacteria aggregates are key for the vertical transport of organic carbon in the ocean. Sinking aggregates also represent pelagic microniches with intensified microbial activity, oxygen depletion in the center, and anaerobic nitrogen cycling. Since some of the aggregate-forming diatom species store nitrate intracellularly, we explored the fate of intracellular nitrate and its availability for microbial metabolism within anoxic diatom-bacteria aggregates. The ubiquitous nitrate-storing diatom Skeletonema marinoi was studied as both axenic cultures and laboratory-produced diatom-bacteria aggregates. Stable 15N isotope incubations under dark and anoxic conditions revealed that axenic S. marinoi is able to reduce intracellular nitrate to ammonium that is immediately excreted by the cells. When exposed to a light:dark cycle and oxic conditions, S. marinoi stored nitrate intracellularly in concentrations >60 mmol L-1 both as free-living cells and associated to aggregates. Intracellular nitrate concentrations exceeded extracellular concentrations by three orders of magnitude. Intracellular nitrate was used up within 2-3 days after shifting diatom-bacteria aggregates to dark and anoxic conditions. Thirty-one percent of the diatom-derived nitrate was converted to nitrogen gas, indicating that a substantial fraction of the intracellular nitrate pool of S. marinoi becomes available to the aggregate-associated bacterial community. Only 5% of the intracellular nitrate was reduced to ammonium, while 59% was recovered as nitrite. Hence, aggregate-associated diatoms accumulate nitrate from the surrounding water and sustain complex nitrogen transformations, including loss of fixed nitrogen, in anoxic, pelagic microniches. Additionally, it may be expected that intracellular nitrate not converted before the aggregates have settled onto the seafloor could fuel benthic nitrogen transformations.

Direct Nitrous Oxide Emission from the Aquacultured Pacific White Shrimp (Litopenaeus vannamei).

UNLABELLED: The Pacific white shrimp (Litopenaeus vannamei) is widely used in aquaculture, where it is reared at high stocking densities, temperatures, and nutrient concentrations. Here we report that adult L. vannamei shrimp emit the greenhouse gas nitrous oxide (N2O) at an average rate of 4.3 nmol N2O/individual × h, which is 1 to 2 orders of magnitude higher than previously measured N2O emission rates for free-living aquatic invertebrates. Dissection, incubation, and inhibitor experiments with specimens from a shrimp farm in Germany indicated that N2O is mainly produced in the animal's gut by microbial denitrification. Microsensor measurements demonstrated that the gut interior is anoxic and nearly neutral and thus is favorable for denitrification by ingested bacteria. Dinitrogen (N2) and N2O accounted for 64% and 36%, respectively, of the nitrogen gas flux from the gut, suggesting that the gut passage is too fast for complete denitrification to be fully established. Indeed, shifting the rearing water bacterial community, a diet component of shrimp, from oxic to anoxic conditions induced N2O accumulation that outlasted the gut passage time. Shrimp-associated N2O production was estimated to account for 6.5% of total N2O production in the shrimp farm studied here and to contribute to the very high N2O supersaturation measured in the rearing tanks (2,099%). Microbial N2O production directly associated with aquacultured animals should be implemented into life cycle assessments of seafood production. IMPORTANCE: The most widely used shrimp species in global aquaculture, Litopenaeus vannamei, is shown to emit the potent greenhouse gas nitrous oxide (N2O) at a particularly high rate. Detailed experiments reveal that N2O is produced in the oxygen-depleted gut of the animal by bacteria that are part of the shrimp diet. Upon ingestion, these bacteria experience a shift from oxic to anoxic conditions and therefore switch their metabolism to the anaerobic denitrification process, which produces N2O as an intermediate and dinitrogen (N2) gas as an end product. The N2O/N2 production ratio is unusually high in the shrimp gut, because denitrification cannot be fully established during the short gut passage time of food-associated bacteria. Nitrous oxide emission directly mediated by L. vannamei contributes significantly to the overall N2O emission from aquaculture facilities.

Anaerobic Nitrogen Turnover by Sinking Diatom Aggregates at Varying Ambient Oxygen Levels.

In the world's oceans, even relatively low oxygen levels inhibit anaerobic nitrogen cycling by free-living microbes. Sinking organic aggregates, however, might provide oxygen-depleted microbial hotspots in otherwise oxygenated surface waters. Here, we show that sinking diatom aggregates can host anaerobic nitrogen cycling at ambient oxygen levels well above the hypoxic threshold. Aggregates were produced from the ubiquitous diatom Skeletonema marinoi and the natural microbial community of seawater. Microsensor profiling through the center of sinking aggregates revealed internal anoxia at ambient 40% air saturation (∼100 µmol O2 L(-1)) and below. Accordingly, anaerobic nitrate turnover inside the aggregates was evident within this range of ambient oxygen levels. In incubations with (15)N-labeled nitrate, individual Skeletonema aggregates produced NO2 (-) (up to 10.7 nmol N h(-1) per aggregate), N2 (up to 7.1 nmol N h(-1)), NH4 (+) (up to 2.0 nmol N h(-1)), and N2O (up to 0.2 nmol N h(-1)). Intriguingly, nitrate stored inside the diatom cells served as an additional, internal nitrate source for dinitrogen production, which may partially uncouple anaerobic nitrate turnover by diatom aggregates from direct ambient nitrate supply. Sinking diatom aggregates can contribute directly to fixed-nitrogen loss in low-oxygen environments in the ocean and vastly expand the ocean volume in which anaerobic nitrogen turnover is possible, despite relatively high ambient oxygen levels. Depending on the extent of intracellular nitrate consumption during the sinking process, diatom aggregates may also be involved in the long-distance export of nitrate to the deep ocean.

Nitrate Storage and Dissimilatory Nitrate Reduction by Eukaryotic Microbes.

The microbial nitrogen cycle is one of the most complex and environmentally important element cycles on Earth and has long been thought to be mediated exclusively by prokaryotic microbes. Rather recently, it was discovered that certain eukaryotic microbes are able to store nitrate intracellularly and use it for dissimilatory nitrate reduction in the absence of oxygen. The paradigm shift that this entailed is ecologically significant because the eukaryotes in question comprise global players like diatoms, foraminifers, and fungi. This review article provides an unprecedented overview of nitrate storage and dissimilatory nitrate reduction by diverse marine eukaryotes placed into an eco-physiological context. The advantage of intracellular nitrate storage for anaerobic energy conservation in oxygen-depleted habitats is explained and the life style enabled by this metabolic trait is described. A first compilation of intracellular nitrate inventories in various marine sediments is presented, indicating that intracellular nitrate pools vastly exceed porewater nitrate pools. The relative contribution by foraminifers to total sedimentary denitrification is estimated for different marine settings, suggesting that eukaryotes may rival prokaryotes in terms of dissimilatory nitrate reduction. Finally, this review article sketches some evolutionary perspectives of eukaryotic nitrate metabolism and identifies open questions that need to be addressed in future investigations.

Anaerobic methane oxidation coupled to denitrification is the dominant methane sink in a deep lake.

Anaerobic methane oxidation coupled to denitrification, also known as "nitrate/nitrite-dependent anaerobic methane oxidation" (n-damo), was discovered in 2006. Since then, only a few studies have identified this process and the associated microorganisms in natural environments. In aquatic sediments, the close proximity of oxygen- and nitrate-consumption zones can mask n-damo as aerobic methane oxidation. We therefore investigated the vertical distribution and the abundance of denitrifying methanotrophs related to Candidatus Methylomirabilis oxyfera with cultivation-independent molecular techniques in the sediments of Lake Constance. Additionally, the vertical distribution of methane oxidation and nitrate consumption zones was inferred from high-resolution microsensor profiles in undisturbed sediment cores. M. oxyfera-like bacteria were virtually absent at shallow-water sites (littoral sediment) and were very abundant at deep-water sites (profundal sediment). In profundal sediment, the vertical distribution of M. oxyfera-like bacteria showed a distinct peak in anoxic layers that coincided with the zone of methane oxidation and nitrate consumption, a strong indication for n-damo carried out by M. oxyfera-like bacteria. Both potential n-damo rates calculated from cell densities (660-4,890 µmol CH4⋅m(-2)⋅d(-1)) and actual rates calculated from microsensor profiles (31-437 µmol CH4⋅m(-2)⋅d(-1)) were sufficiently high to prevent methane release from profundal sediment solely by this process. Additionally, when nitrate was added to sediment cores exposed to anoxic conditions, the n-damo zone reestablished well below the sediment surface, completely preventing methane release from the sediment. We conclude that the previously overlooked n-damo process can be the major methane sink in stable freshwater environments if nitrate is available in anoxic zones.

Effect of high electron donor supply on dissimilatory nitrate reduction pathways in a bioreactor for nitrate removal.

The possible shift of a bioreactor for NO3(-) removal from predominantly denitrification (DEN) to dissimilatory nitrate reduction to ammonium (DNRA) by elevated electron donor supply was investigated. By increasing the C/NO3(-) ratio in one of two initially identical reactors, the production of high sulfide concentrations was induced. The response of the dissimilatory NO3(-) reduction processes to the increased availability of organic carbon and sulfide was monitored in a batch incubation system. The expected shift from a DEN- towards a DNRA-dominated bioreactor was not observed, also not under conditions where DNRA would be thermodynamically favorable. Remarkably, the microbial community exposed to a high C/NO3(-) ratio and sulfide concentration did not use the most energy-gaining process.

High spatial resolution of distribution and interconnections between Fe- and N-redox processes in profundal lake sediments.

The Fe and N biogeochemical cycles play key roles in freshwater environments. We aimed to determine the spatial positioning and interconnections of the N and Fe cycles in profundal lake sediments. The gradients of O2, NO3(-), NH4(+), pH, Eh, Fe(II) and Fe(III) were determined and the distribution of microorganisms was assessed by most probable numbers and quantitative polymerase chain reaction. The redox zones could be divided into an oxic zone (0-8 mm), where microaerophiles (Gallionellaceae) were most abundant at a depth of 7 mm. This was followed by a denitrification zone (6-12 mm), where NO3(-)-dependent Fe(II) oxidizers and organoheterotrophic denitrifiers both reduce nitrate. Lastly, an iron redox transition zone was identified at 12.5-22.5 mm. Fe(III) was most abundant above this zone while Fe(II) was most abundant beneath. The high abundance of poorly crystalline iron suggested iron cycling. The Fe and N cycles are biologically connected through nitrate-reducing Fe(II) oxidizers and chemically by NOx(-) species formed during denitrification, which can chemically oxidize Fe(II). This study combines high resolution chemical, molecular and microbiological data to pinpoint sedimentary redox zones in which Fe is cycled between Fe(II) and Fe(III) and where Fe and N-redox processes interact.

Microscopic oxygen imaging based on fluorescein bleaching efficiency measurements.

Photobleaching of the fluorophore fluorescein in an aqueous solution is dependent on the oxygen concentration. Therefore, the time-dependent bleaching behavior can be used to measure of dissolved oxygen concentrations. The method can be combined with epi-fluorescence microscopy. The molecular states of the fluorophore can be expressed by a three-state energy model. This leads to a set of differential equations which describe the photobleaching behavior of fluorescein. The numerical solution of these equations shows that in a conventional wide-field fluorescence microscope, the fluorescence of fluorescein will fade out faster at low than at high oxygen concentration. Further simulation showed that a simple ratio function of different time-points during a fluorescence decay recorded during photobleaching could be used to describe oxygen concentrations in an aqueous solution. By careful choice of dye concentration and excitation light intensity the sensitivity in the oxygen concentration range of interest can be optimized. In the simulations, the estimation of oxygen concentration by the ratio function was very little affected by the pH value in the range of pH 6.5-8.5. Filming the fluorescence decay by a charge-coupled-device (ccd) camera mounted on a fluorescence microscope allowed a pixelwise estimation of the ratio function in a microscopic image. Use of a microsensor and oxygen-consuming bacteria in a sample chamber enabled the calibration of the system for quantification of absolute oxygen concentrations. The method was demonstrated on nitrifying biofilms growing on snail and mussel shells, showing clear effects of metabolic activity on oxygen concentrations.

Dissimilatory nitrate reduction by Aspergillus terreus isolated from the seasonal oxygen minimum zone in the Arabian Sea.

BACKGROUND: A wealth of microbial eukaryotes is adapted to life in oxygen-deficient marine environments. Evidence is accumulating that some of these eukaryotes survive anoxia by employing dissimilatory nitrate reduction, a strategy that otherwise is widespread in prokaryotes. Here, we report on the anaerobic nitrate metabolism of the fungus Aspergillus terreus (isolate An-4) that was obtained from sediment in the seasonal oxygen minimum zone in the Arabian Sea, a globally important site of oceanic nitrogen loss and nitrous oxide emission. RESULTS: Axenic incubations of An-4 in the presence and absence of oxygen and nitrate revealed that this fungal isolate is capable of dissimilatory nitrate reduction to ammonium under anoxic conditions. A ¹5N-labeling experiment proved that An-4 produced and excreted ammonium through nitrate reduction at a rate of up to 175 nmol ¹5NH4⁺ g⁻¹ protein h⁻¹. The products of dissimilatory nitrate reduction were ammonium (83%), nitrous oxide (15.5%), and nitrite (1.5%), while dinitrogen production was not observed. The process led to substantial cellular ATP production and biomass growth and also occurred when ammonium was added to suppress nitrate assimilation, stressing the dissimilatory nature of nitrate reduction. Interestingly, An-4 used intracellular nitrate stores (up to 6-8 µmol NO3⁻ g⁻¹ protein) for dissimilatory nitrate reduction. CONCLUSIONS: Our findings expand the short list of microbial eukaryotes that store nitrate intracellularly and carry out dissimilatory nitrate reduction when oxygen is absent. In the currently spreading oxygen-deficient zones in the ocean, an as yet unexplored diversity of fungi may recycle nitrate to ammonium and nitrite, the substrates of the major nitrogen loss process anaerobic ammonium oxidation, and the potent greenhouse gas nitrous oxide.

Chironomus plumosus larvae increase fluxes of denitrification products and diversity of nitrate-reducing bacteria in freshwater sediment.

Benthic invertebrates affect microbial processes and communities in freshwater sediment by enhancing sediment-water solute fluxes and by grazing on bacteria. Using microcosms, the effects of larvae of the widespread midge Chironomus plumosus on the efflux of denitrification products (N2O and N2+N2O) and the diversity and abundance of nitrate- and nitrous-oxide-reducing bacteria were investigated. Additionally, the diversity of actively nitrate- and nitrous-oxide-reducing bacteria was analyzed in the larval gut. The presence of larvae increased the total effluxes of N2O and N2+N2O up to 8.6- and 4.2-fold, respectively, which was mostly due to stimulation of sedimentary denitrification; incomplete denitrification in the guts accounted for up to 20% of the N2O efflux. Phylotype richness of the nitrate reductase gene narG was significantly higher in sediment with than without larvae. In the gut, 47 narG phylotypes were found expressed, which may contribute to higher phylotype richness in colonized sediment. In contrast, phylotype richness of the nitrous oxide reductase gene nosZ was unaffected by the presence of larvae and very few nosZ phylotypes were expressed in the gut. Gene abundance of neither narG, nor nosZ was different in sediments with and without larvae. Hence, C. plumosus increases activity and diversity, but not overall abundance of nitrate-reducing bacteria, probably by providing additional ecological niches in its burrow and gut.

Response of the ubiquitous pelagic diatom Thalassiosira weissflogii to darkness and anoxia.

Thalassiosira weissflogii, an abundant, nitrate-storing, bloom-forming diatom in the world's oceans, can use its intracellular nitrate pool for dissimilatory nitrate reduction to ammonium (DNRA) after sudden shifts to darkness and anoxia, most likely as a survival mechanism. T. weissflogii cells that stored 4 mM (15)N-nitrate consumed 1.15 (±0.25) fmol NO3 (-) cell(-1) h(-1) and simultaneously produced 1.57 (±0.21) fmol (15)NH4 (+) cell(-1) h(-1) during the first 2 hours of dark/anoxic conditions. Ammonium produced from intracellular nitrate was excreted by the cells, indicating a dissimilatory rather than assimilatory pathway. Nitrite and the greenhouse gas nitrous oxide were produced at rates 2-3 orders of magnitude lower than the ammonium production rate. While DNRA activity was restricted to the first few hours of darkness and anoxia, the subsequent degradation of photopigments took weeks to months, supporting the earlier finding that diatoms resume photosynthesis even after extended exposure to darkness and anoxia. Considering the high global abundance of T. weissflogii, its production of ammonium and nitrous oxide might be of ecological importance for oceanic oxygen minimum zones and the atmosphere, respectively.

Role of diatoms in the spatial-temporal distribution of intracellular nitrate in intertidal sediment.

Intracellular nitrate storage allows microorganisms to survive fluctuating nutrient availability and anoxic conditions in aquatic ecosystems. Here we show that diatoms, ubiquitous and highly abundant microalgae, represent major cellular reservoirs of nitrate in an intertidal flat of the German Wadden Sea and are potentially involved in anaerobic nitrate respiration. Intracellular nitrate (ICNO3) was present year-round in the sediment and was spatially and temporally correlated with fucoxanthin, the marker photopigment of diatoms. Pyrosequencing of SSU rRNA genes of all domains of life confirmed that ICNO3 storage was most likely due to diatoms rather than other known nitrate-storing microorganisms (i.e., large sulfur bacteria and the eukaryotic foraminifers and gromiids). Sedimentary ICNO3 concentrations reached up to 22.3 µmol dm(-3) at the sediment surface and decreased with sediment depth to negligible concentrations below 5 cm. Similarly, the ICNO3/fucoxanthin ratio and porewater nitrate (PWNO3) concentrations decreased with sediment depth, suggesting that ICNO3 of diatoms is in equilibrium with PWNO3, but is enriched relative to PWNO3 by 2-3 orders of magnitude. Cell-volume-specific ICNO3 concentrations in a diatom mat covering the sediment surface during spring were estimated at 9.3-46.7 mmol L(-1). Retrieval of 18S rRNA gene sequences related to known nitrate-storing and nitrate-ammonifying diatom species suggested that diatoms in dark and anoxic sediment layers might be involved in anaerobic nitrate respiration. Due to the widespread dominance of diatoms in microphytobenthos, the total nitrate pool in coastal marine sediments may generally be at least two times larger than derived from porewater measurements and partially be recycled to ammonium.

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.

Shell biofilm-associated nitrous oxide production in marine molluscs: processes, precursors and relative importance.

Emission of the greenhouse gas nitrous oxide (N2 O) from freshwater and terrestrial invertebrates has exclusively been ascribed to N2 O production by ingested denitrifying bacteria in the anoxic gut of the animals. Our study of marine molluscs now shows that also microbial biofilms on shell surfaces are important sites of N2 O production. The shell biofilms of Mytilus edulis, Littorina littorea and Hinia reticulata contributed 18-94% to the total animal-associated N2 O emission. Nitrification and denitrification were equally important sources of N2 O in shell biofilms as revealed by (15) N-stable isotope experiments with dissected shells. Microsensor measurements confirmed that both nitrification and denitrification can occur in shell biofilms due to a heterogeneous oxygen distribution. Accordingly, ammonium, nitrite and nitrate were important drivers of N2 O production in the shell biofilm of the three mollusc species. Ammonium excretion by the animals was found to be sufficient to sustain N2 O production in the shell biofilm. Apparently, the animals provide a nutrient-enriched microenvironment that stimulates growth and N2 O production of the shell biofilm. This animal-induced stimulation was demonstrated in a long-term microcosm experiment with the snail H. reticulata, where shell biofilms exhibited the highest N2 O emission rates when the animal was still living inside the shell.