Amal Jayakumar

Denitrification as the dominant nitrogen loss process in the Arabian Sea

Primary production in over half of the world’s oceans is limited by fixed nitrogen availability. The main loss term from the fixed nitrogen inventory is the production of dinitrogen gas (N2) by heterotrophic denitrification or the more recently discovered autotrophic process, anaerobic ammonia oxidation (anammox). Oceanic oxygen minimum zones (OMZ) are responsible for about 35% of oceanic N2 production and up to half of that occurs in the Arabian Sea. Although denitrification was long thought to be the only loss term, it has recently been argued that anammox alone is responsible for fixed nitrogen loss in the OMZs. Here we measure denitrification and anammox rates and quantify the abundance of denitrifying and anammox bacteria in the OMZ regions of the Eastern Tropical South Pacific and the Arabian Sea. We find that denitrification rather than anammox dominates the N2 loss term in the Arabian Sea, the largest and most intense OMZ in the world ocean. In seven of eight experiments in the Arabian Sea denitrification is responsible for 87–99% of the total N2 production. The dominance of denitrification is reproducible using two independent isotope incubation methods. In contrast, anammox is dominant in the Eastern Tropical South Pacific OMZ, as detected using one of the isotope incubation methods, as previously reported. The abundance of denitrifying bacteria always exceeded that of anammox bacteria by up to 7- and 19-fold in the Eastern Tropical South Pacific and Arabian Sea, respectively. Geographic and temporal variability in carbon supply may be responsible for the different contributions of denitrification and anammox in these two OMZs. The large contribution of denitrification to N2 loss in the Arabian Sea indicates the global significance of denitrification to the oceanic nitrogen budget.

Environmental factors determining ammonia‐oxidizing organism distribution and diversity in marine environments

Ammonia-oxidizing bacteria (AOB) and archaea (AOA) play a vital role in bridging the input of fixed nitrogen, through N-fixation and remineralization, to its loss by denitrification and anammox. Yet the major environmental factors determining AOB and AOA population dynamics are little understood, despite both groups having a wide environmental distribution. This study examined the relative abundance of both groups of ammonia-oxidizing organisms (AOO) and the diversity of AOA across large-scale gradients in temperature, salinity and substrate concentration and dissolved oxygen. The relative abundance of AOB and AOA varied across environments, with AOB dominating in the freshwater region of the Chesapeake Bay and AOA more abundant in the water column of the coastal and open ocean. The highest abundance of the AOA amoA gene was recorded in the oxygen minimum zones (OMZs) of the Eastern Tropical South Pacific (ETSP) and the Arabian Sea (AS). The ratio of AOA : AOB varied from 0.7 in the Chesapeake Bay to 1600 in the Sargasso Sea. Relative abundance of both groups strongly correlated with ammonium concentrations. AOA diversity, as determined by phylogenetic analysis of clone library sequences and archetype analysis from a functional gene DNA microarray, detected broad phylogenetic differences across the study sites. However, phylogenetic diversity within physicochemically congruent stations was more similar than would be expected by chance. This suggests that the prevailing geochemistry, rather than localized dispersal, is the major driving factor determining OTU distribution.

Rapid nitrous oxide cycling in the suboxic ocean

More N2O is no laughing matter Because N2O is a potent greenhouse gas, tracking its sources and sinks—including those from natural processes—is imperative. Babbin et al. developed an isotopic tracer method to measure biological N2O reduction rates directly in the Eastern Tropical North Pacific Ocean. Incomplete denitrification results in the rapid cycling and net accumulation of N2O. As oxygen minimum zones expand in the global ocean, more N2O may enter the atmosphere than previously expected. Science, this issue p. 1127 Nitrous oxide undergoes rapid biological cycling in the ocean’s oxygen minimum zones. Nitrous oxide (N2O) is a powerful greenhouse gas and a major cause of stratospheric ozone depletion, yet its sources and sinks remain poorly quantified in the oceans. We used isotope tracers to directly measure N2O reduction rates in the eastern tropical North Pacific. Because of incomplete denitrification, N2O cycling rates are an order of magnitude higher than predicted by current models in suboxic regions, and the spatial distribution suggests strong dependence on both organic carbon and dissolved oxygen concentrations. Furthermore, N2O turnover is 20 times higher than the net atmospheric efflux. The rapid rate of this cycling coupled to an expected expansion of suboxic ocean waters implies future increases in N2O emissions.

Diversity, distribution, and expression of diazotroph nifH genes in oxygen-deficient waters of the Arabian Sea

The Arabian Sea oxygen minimum zone (OMZ), the largest suboxic region in the worlds oceans, is responsible for up to half of the global mesopelagic fixed nitrogen (N) loss from the ocean via denitrification and anammox. Dinitrogen (N(2)) fixation is usually attributed to cyanobacteria in the surface ocean. Model prediction and physiological inhibition of N(2) fixation by oxygen, however, suggest that N(2) fixation should be enhanced near the oxygen-deficient zone (ODZ) of the Arabian Sea. N(2) fixation and cyanobacterial nifH genes (the gene encoding dinitrogenase reductase) have been reported in surface waters overlying the Arabian Sea ODZ. Here, water samples from depths above and within the Arabian Sea ODZ were examined to explore the distribution, diversity, and expression of nifH genes. In surface waters, nifH DNA and cDNA sequences related to Trichodesmium, a diazotroph known to occur and fix N(2) in the Arabian Sea, were detected. Proteobacterial nifH phylotypes (DNA but not cDNA) were also detected in surface waters. Proteobacterial nifH DNA and cDNA sequences, as well as nifH DNA and cDNA sequences related to strictly anaerobic N -fixers, were obtained from oxygen-deficient depths. This first report of nifH gene expression in subsurface low-oxygen waters suggests that there is potential for active N(2) fixation by several phylogenetically and potentially metabolically diverse microorganisms in pelagic OMZs.

Ammonia and nitrite oxidation in the Eastern Tropical North Pacific

Nitrification plays a key role in the marine nitrogen (N) cycle, including in oceanic oxygen minimum zones (OMZs), which are hot spots for denitrification and anaerobic ammonia oxidation (anammox). Recent evidence suggests that nitrification links the source (remineralized organic matter) and sink (denitrification and anammox) of fixed N directly in the steep oxycline in the OMZs. We performed shipboard incubations with 15N tracers to characterize the depth distribution of nitrification in the Eastern Tropical North Pacific (ETNP). Additional experiments were conducted to investigate photoinhibition. Allylthiourea (ATU) was used to distinguish the contribution of archaeal and bacterial ammonia oxidation. The abundance of archaeal and β-proteobacterial ammonia monooxygenase gene subunit A (amoA) was determined by quantitative polymerase chain reaction. The rates of ammonia and nitrite oxidation showed distinct subsurface maxima, with the latter slightly deeper than the former. The ammonia oxidation maximum coincided with the primary nitrite concentration maximum, archaeal amoA gene maximum, and the subsurface nitrous oxide maximum. Negligible rates of ammonia oxidation were found at anoxic depths, where high rates of nitrite oxidation were measured. Archaeal amoA gene abundance was generally 1 to 2 orders of magnitude higher than bacterial amoA gene abundance, and inhibition of ammonia-oxidizing bacteria with 10 μM ATU did not affect ammonia oxidation rates, indicating the dominance of archaea in ammonia oxidation. These results depict highly dynamic activities of ammonia and nitrite oxidation in the oxycline of the ETNP OMZ.

Nitrous oxide production by nitrification and denitrification in the Eastern Tropical South Pacific oxygen minimum zone

The Eastern Tropical South Pacific oxygen minimum zone (ETSP-OMZ) is a site of intense nitrous oxide (N2O) flux to the atmosphere. This flux results from production of N2O by nitrification and denitrification, but the contribution of the two processes is unknown. The rates of these pathways and their distributions were measured directly using 15N tracers. The highest N2O production rates occurred at the depth of peak N2O concentrations at the oxic-anoxic interface above the oxygen deficient zone (ODZ) because slightly oxygenated waters allowed (1) N2O production from both nitrification and denitrification and (2) higher nitrous oxide production yields from nitrification. Within the ODZ proper (i.e., anoxia), the only source of N2O was denitrification (i.e., nitrite and nitrate reduction), the rates of which were reflected in the abundance of nirS genes (encoding nitrite reductase). Overall, denitrification was the dominant pathway contributing the N2O production in the ETSP-OMZ.

Community composition of ammonia-oxidizing archaea from surface and anoxic depths of oceanic oxygen minimum zones.

Ammonia-oxidizing archaea (AOA) have been reported at high abundance in much of the global ocean, even in environments, such as pelagic oxygen minimum zones (OMZs), where conditions seem unlikely to support aerobic ammonium oxidation. Due to the lack of information on any potential alternative metabolism of AOA, the AOA community composition might be expected to differ between oxic and anoxic environments. This hypothesis was tested by evaluating AOA community composition using a functional gene microarray that targets the ammonia monooxygenase gene subunit A (amoA). The relationship between environmental parameters and the biogeography of the Arabian Sea and the Eastern Tropical South Pacific (ETSP) AOA assemblages was investigated using principal component analysis (PCA) and redundancy analysis (RDA). In both the Arabian Sea and the ETSP, AOA communities within the core of the OMZ were not significantly different from those inhabiting the oxygenated surface waters above the OMZ. The AOA communities in the Arabian Sea were significantly different from those in the ETSP. In both oceans, the abundance of archaeal amoA gene in the core of the OMZ was higher than that in the surface waters. Our results indicate that AOA communities are distinguished by their geographic origin. RDA suggested that temperature (higher in the Arabian Sea than in the ETSP) was the main factor that correlated with the differences between the AOA communities. Physicochemical properties that characterized the different environments of the OMZ and surface waters played a less important role, than did geography, in shaping the AOA community composition.

Revisiting nitrification in the Eastern Tropical South Pacific: A focus on controls

Nitrification, the oxidation of ammonium ( NH4+) to nitrite ( NO2−) and to nitrate ( NO3−), is a component of the nitrogen (N) cycle internal to the fixed N pool. In oxygen minimum zones (OMZs), which are hotspots for oceanic fixed N loss, nitrification plays a key role because it directly supplies substrates for denitrification and anaerobic ammonia oxidation (anammox), and may compete for substrates with these same processes. However, the control of oxygen and substrate concentrations on nitrification are not well understood. We performed onboard incubations with 15N-labeled substrates to measure rates of NH4+ and NO2− oxidation in the eastern tropical South Pacific (ETSP). The spatial and depth distributions of NH4+ and NO2− oxidation rates were primarily controlled by NH4+ and NO2− availability, oxygen concentration, and light. In the euphotic zone, nitrification was partially photoinhibited. In the anoxic layer, NH4+ oxidation was negligible or below detection, but high rates of NO2− oxidation were observed. NH4+ oxidation displayed extremely high affinity for both NH4+ and oxygen. The positive linear correlations between NH4+ oxidation rates and in situ NH4+ concentrations and ammonia monooxygenase subunit A (amoA) gene abundances in the upper oxycline indicate that the natural assemblage of ammonia oxidizers responds to in situ NH4+ concentrations or supply by adjusting their population size, which determines the NH4+ oxidation potential. The depth distribution of archaeal and bacterial amoA gene abundances and N2O concentration, along with independently reported simultaneous direct N2O production rate measurements, suggests that AOA were predominantly responsible for NH4+ oxidation, which was a major source of N2O production at oxygen concentrations > 5 µM.

Distribution and Relative Quantification of Key Genes Involved in Fixed Nitrogen Loss From the Arabian Sea Oxygen Minimum Zone

The Arabian Sea (AS) oxygen minimum zone (OMZ) is one of the largest pelagic low-oxygen environments in the open ocean. It is responsible for the removal of up to 60 Tg of nitrogen annually, roughly an eighth of the global fixed nitrogen sink. Although denitrification has long been believed to be the major process responsible for fixed nitrogen loss from the oceans, recent studies show that anaerobic ammonium oxidation (anammox) is potentially a more important process involved. We have investigated the phylogeny of both anammox and denitrifying microbes in the AS, and here we report on their diversity in terms of their characteristic genes. Denitrifiers were targeted using nirS and nirK genes and anammox bacteria with the 16S rRNA gene. The nirK gene was amplified from all the samples, but nirS gene could only be detected when nitrite was present. The distribution of phylotypes was related to the concentration of nitrite and the apparent stage of denitrification. Most nirK or nirS genes from the AS had low identities with other published sequences. The closest identities were to sequences from other water column denitrifying environments rather than sedimentary or terrestrial environments. Phylogenetic analysis of the nirS and nirK genes revealed overall lower diversity than the very high diversities reported from estuarine and sedimentary environments. 16S rRNA partial gene sequences revealed very limited diversity among anammox sequences. All the anammox sequences were >98% identical to each other and were similar to sequences from other marine and water column environments, which are distantly related to Scalindua sp. Quantification using quantitative polymerase chain reaction assays showed that both nirS genes and anammox 16S rRNA genes were more abundant at depths with higher nitrite concentrations. The presence and abundance of genes indicative of both processes suggest that both canonical denitrification and anammox likely occur in the OMZ of the AS. However, the abundance of the nirS gene, one of the genes responsible for canonical denitrification, was an order of magnitude higher than the abundance of the anammox 16S gene, indicating that denitrifiers are numerically dominant in this environment. The greater diversity of nirS and nirK genes relative to anammox genes both among and within stations also suggests that the denitrifier assemblages are more dynamic in response to environmental conditions.

Biological nitrogen fixation in the oxygen-minimum region of the eastern tropical North Pacific ocean

Biological nitrogen fixation (BNF) was investigated above and within the oxygen-depleted waters of the oxygen-minimum zone of the Eastern Tropical North Pacific Ocean. BNF rates were estimated using an isotope tracer method that overcame the uncertainty of the conventional bubble method by directly measuring the tracer enrichment during the incubations. Highest rates of BNF (~4 nM day−1) occurred in coastal surface waters and lowest detectable rates (~0.2 nM day−1) were found in the anoxic region of offshore stations. BNF was not detectable in most samples from oxygen-depleted waters. The composition of the N2-fixing assemblage was investigated by sequencing of nifH genes. The diazotrophic assemblage in surface waters contained mainly Proteobacterial sequences (Cluster I nifH), while both Proteobacterial sequences and sequences with high identities to those of anaerobic microbes characterized as Clusters III and IV type nifH sequences were found in the anoxic waters. Our results indicate modest input of N through BNF in oxygen-depleted zones mainly due to the activity of proteobacterial diazotrophs.