Tim Kalvelage

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 (O2) on anammox, NH3 oxidation and NO3 − reduction in 15N-labeling experiments with varying O2 concentrations (0–25 µmol L−1) in the Namibian and Peruvian OMZs. Our results show that O2 is a major controlling factor for anammox activity in OMZ waters. Based on our O2 assays we estimate the upper limit for anammox to be ∼20 µmol L−1. In contrast, NH3 oxidation to NO2 − and NO3 − reduction to NO2 − as the main NH4 + and NO2 − sources for anammox were only moderately affected by changing O2 concentrations. Intriguingly, aerobic NH3 oxidation was active at non-detectable concentrations of O2, while anaerobic NO3 − reduction was fully active up to at least 25 µmol L−1 O2. Hence, aerobic and anaerobic N-cycle pathways in OMZs can co-occur over a larger range of O2 concentrations than previously assumed. The zone where N-loss can occur is primarily controlled by the O2-sensitivity of anammox itself, and not by any effects of O2 on the tightly coupled pathways of aerobic NH3 oxidation and NO3 − reduction. With anammox bacteria in the marine environment being active at O2 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 O2 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.

Giant hydrogen sulfide plume in the oxygen minimum zone off Peru supports chemolithoautotrophy

In Eastern Boundary Upwelling Systems nutrient-rich waters are transported to the ocean surface, fuelling high photoautotrophic primary production. Subsequent heterotrophic decomposition of the produced biomass increases the oxygen-depletion at intermediate water depths, which can result in the formation of oxygen minimum zones (OMZ). OMZs can sporadically accumulate hydrogen sulfide (H2S), which is toxic to most multicellular organisms and has been implicated in massive fish kills. During a cruise to the OMZ off Peru in January 2009 we found a sulfidic plume in continental shelf waters, covering an area >5500 km2, which contained ∼2.2×104 tons of H2S. This was the first time that H2S was measured in the Peruvian OMZ and with ∼440 km3 the largest plume ever reported for oceanic waters. We assessed the phylogenetic and functional diversity of the inhabiting microbial community by high-throughput sequencing of DNA and RNA, while its metabolic activity was determined with rate measurements of carbon fixation and nitrogen transformation processes. The waters were dominated by several distinct γ-, δ- and ε-proteobacterial taxa associated with either sulfur oxidation or sulfate reduction. Our results suggest that these chemolithoautotrophic bacteria utilized several oxidants (oxygen, nitrate, nitrite, nitric oxide and nitrous oxide) to detoxify the sulfidic waters well below the oxic surface. The chemolithoautotrophic activity at our sampling site led to high rates of dark carbon fixation. Assuming that these chemolithoautotrophic rates were maintained throughout the sulfidic waters, they could be representing as much as ∼30% of the photoautotrophic carbon fixation. Postulated changes such as eutrophication and global warming, which lead to an expansion and intensification of OMZs, might also increase the frequency of sulfidic waters. We suggest that the chemolithoautotrophically fixed carbon may be involved in a negative feedback loop that could fuel further sulfate reduction and potentially stabilize the sulfidic OMZ waters.

Carbon, nitrogen and O(2) fluxes associated with the cyanobacterium Nodularia spumigena in the Baltic Sea.

Photosynthesis, respiration, N2 fixation and ammonium release were studied directly in Nodularia spumigena during a bloom in the Baltic Sea using a combination of microsensors, stable isotope tracer experiments combined with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorometry. Cell-specific net C- and N2-fixation rates by N. spumigena were 81.6±6.7 and 11.4±0.9 fmol N per cell per h, respectively. During light, the net C:N fixation ratio was 8.0±0.8. During darkness, carbon fixation was not detectable, but N2 fixation was 5.4±0.4 fmol N per cell per h. Net photosynthesis varied between 0.34 and 250 nmol O2 h−1 in colonies with diameters ranging between 0.13 and 5.0 mm, and it reached the theoretical upper limit set by diffusion of dissolved inorganic carbon to colonies (>1 mm). Dark respiration of the same colonies varied between 0.038 and 87 nmol O2 h−1, and it reached the limit set by O2 diffusion from the surrounding water to colonies (>1 mm). N2 fixation associated with N. spumigena colonies (>1 mm) comprised on average 18% of the total N2 fixation in the bulk water. Net NH4+ release in colonies equaled 8–33% of the estimated gross N2 fixation during photosynthesis. NH4+ concentrations within light-exposed colonies, modeled from measured net NH4+ release rates, were 60-fold higher than that of the bulk. Hence, N. spumigena colonies comprise highly productive microenvironments and an attractive NH4+ microenvironment to be utilized by other (micro)organisms in the Baltic Sea where dissolved inorganic nitrogen is limiting growth.

Nitrogen isotope effects induced by anammox bacteria.

Significance Nitrogen is essential for all organisms and limits primary production in the ocean. It is mainly lost from low-oxygen environments by the activity of microorganisms that convert fixed nitrogen to N2 gas. The isotopic composition of nitrogen species can be used to assess nitrogen sinks in the environment, but its use in biogeochemical studies can be fully exploited only if the isotope discrimination that is associated with the respective nitrogen-converting pathways is known. This study reveals the wide range of nitrogen isotope effects of anaerobic oxidation of ammonium (anammox), a major player in marine fixed nitrogen loss, reconciling experimental data with nitrogen isotope signatures observed in the ocean. Nitrogen (N) isotope ratios (15N/14N) provide integrative constraints on the N inventory of the modern ocean. Anaerobic ammonium oxidation (anammox), which converts ammonium and nitrite to dinitrogen gas (N2) and nitrate, is an important fixed N sink in marine ecosystems. We studied the so far unknown N isotope effects of anammox in batch culture experiments. Anammox preferentially removes 14N from the ammonium pool with an isotope effect of +23.5‰ to +29.1‰, depending on factors controlling reversibility. The N isotope effects during the conversion of nitrite to N2 and nitrate are (i) inverse kinetic N isotope fractionation associated with the oxidation of nitrite to nitrate (−31.1 ± 3.9‰), (ii) normal kinetic N isotope fractionation during the reduction of nitrite to N2 (+16.0 ± 4.5‰), and (iii) an equilibrium N isotope effect between nitrate and nitrite (−60.5 ± 1.0‰), induced when anammox is exposed to environmental stress, leading to the superposition of N isotope exchange effects upon kinetic N isotope fractionation. Our findings indicate that anammox may be responsible for the unresolved large N isotope offsets between nitrate and nitrite in oceanic oxygen minimum zones. Irrespective of the extent of N isotope exchange between nitrate and nitrite, N removed from the combined nitrite and nitrate (NOx) pool is depleted in 15N relative to NOx. This net N isotope effect by anammox is superimposed on the N isotope fractionation by the co-occurring reduction of nitrate to nitrite in suboxic waters, possibly enhancing the overall N isotope effect for N loss from oxygen minimum zones.

Aerobic Microbial Respiration In Oceanic Oxygen Minimum Zones

Oxygen minimum zones are major sites of fixed nitrogen loss in the ocean. Recent studies have highlighted the importance of anaerobic ammonium oxidation, anammox, in pelagic nitrogen removal. Sources of ammonium for the anammox reaction, however, remain controversial, as heterotrophic denitrification and alternative anaerobic pathways of organic matter remineralization cannot account for the ammonium requirements of reported anammox rates. Here, we explore the significance of microaerobic respiration as a source of ammonium during organic matter degradation in the oxygen-deficient waters off Namibia and Peru. Experiments with additions of double-labelled oxygen revealed high aerobic activity in the upper OMZs, likely controlled by surface organic matter export. Consistently observed oxygen consumption in samples retrieved throughout the lower OMZs hints at efficient exploitation of vertically and laterally advected, oxygenated waters in this zone by aerobic microorganisms. In accordance, metagenomic and metatranscriptomic analyses identified genes encoding for aerobic terminal oxidases and demonstrated their expression by diverse microbial communities, even in virtually anoxic waters. Our results suggest that microaerobic respiration is a major mode of organic matter remineralization and source of ammonium (~45-100%) in the upper oxygen minimum zones, and reconcile hitherto observed mismatches between ammonium producing and consuming processes therein.

Aquatic Respiration Rate Measurements at Low Oxygen Concentrations

Despite its huge ecological importance, microbial oxygen respiration in pelagic waters is little studied, primarily due to methodological difficulties. Respiration measurements are challenging because of the required high resolution of oxygen concentration measurements. Recent improvements in oxygen sensing techniques bear great potential to overcome these limitations. Here we compare 3 different methods to measure oxygen consumption rates at low oxygen concentrations, utilizing amperometric Clark type sensors (STOX), optical sensors (optodes), and mass spectrometry in combination with 18-18O2 labeling. Oxygen concentrations and consumption rates agreed well between the different methods when applied in the same experimental setting. Oxygen consumption rates between 30 and 400 nmol L−1 h−1 were measured with high precision and relative standard errors of less than 3%. Rate detection limits in the range of 1 nmol L−1 h−1 were suitable for rate determinations in open ocean water and were lowest at the lowest applied O2 concentration.

Oxygen minimum zone cryptic sulfur cycling sustained by offshore transport of key sulfur oxidizing bacteria

Members of the gammaproteobacterial clade SUP05 couple water column sulfide oxidation to nitrate reduction in sulfidic oxygen minimum zones (OMZs). Their abundance in offshore OMZ waters devoid of detectable sulfide has led to the suggestion that local sulfate reduction fuels SUP05-mediated sulfide oxidation in a so-called “cryptic sulfur cycle”. We examined the distribution and metabolic capacity of SUP05 in Peru Upwelling waters, using a combination of oceanographic, molecular, biogeochemical and single-cell techniques. A single SUP05 species, UThioglobus perditus, was found to be abundant and active in both sulfidic shelf and sulfide-free offshore OMZ waters. Our combined data indicated that mesoscale eddy-driven transport led to the dispersal of UT. perditus and elemental sulfur from the sulfidic shelf waters into the offshore OMZ region. This offshore transport of shelf waters provides an alternative explanation for the abundance and activity of sulfide-oxidizing denitrifying bacteria in sulfide-poor offshore OMZ waters.The presence and activity of sulfide-oxidizing denitrifying bacteria in sulfide-poor offshore oxygen minimum zone waters remains unclear. Here, the authors combine oceanography, molecular, biogeochemical and single-cell techniques to examine their distribution, metabolic capacity, and origins.