N transformation rates in the ODZ
Anammox occurs in both sediments and the water column, and its contribution to total fixed N loss varies widely. On cruises to the Eastern Tropical South Pacific (ETSP) in 2005 and to the Arabian Sea in 2007, we measured denitrification and anammox using two different methods to investigate the rates and relative importance of the two processes. One method is the conventional exetainer method (Dalsgaard et al 2005), in which small volume incubations are first flushed with helium to allow a greater sensitivity for the determination of enriched N2 after an incubation with 15N tracers. The second uses large volume (~8.5 L) gas tight trace metal clean bags, with minimal perturbation to the gases. In both methods, we found that denitrification was the overwhelmingly dominant N loss process in the Arabian Sea (Ward et al. 2009; Bulow et al. 2010), but that anammox was more important in the ETSP (Ward et al 2009) (Figure 1). We suspect that the Arabian Sea and the ETSP may not be fundamentally different, but rather that the apparent differences reflect the overall control of both processes through the episodic supply of organic matter from productive surface waters. Using extensive datasets of 15N tracer incubation experiments, we plan to investigate the coupling of all the major N transformation processes in the OMZ with data assimilation and modeling. Future cruises to two of the major OMZs, the Eastern Tropical North and South Pacific, are scheduled for 2012 and 2013.
Figure 1. Left panel: Denitrification and anammox rates at the four stations in Figure 1. Amx-ex = anammox rates measured in exetainers; Denit-ex = denitrification rate measured in exetainers; Amx-bb = anammox rates measured in 8-L bags; Denit-bb = denitrification rates measured in 8-L bags. ☻ = no rate detected; Ο = not measured. Error bars = standard error of the slope of 29N2 or 29N2 plus 30N2 accumulation in exetainer incubations.
Right panel: Depth distribution of total denitrifier nirS genes (Tot nirS), a dominant nirS clade (Dom nirS) and anammox 16S rRNA genes (Amx) at the four stations in Figure 1. Error bars indicate standard deviations of triplicate Q-PCR assays. (From Ward et al. 2009)
Molecular ecology of N cycle microorganisms in the ODZ
The distribution and diversity of the microbes that catalyze not only N loss, but N fixation and nitrification, should provide additional constraints on the distribution of N transformation processes within and proximate to the ODZs. Organisms responsible for N removal include the heterotrophic canonical denitrifiers and the autotrophic anammox bacteria. Denitrifying bacteria belong to two groups based on which nitrite reductase enzyme (a key step in the denitrification pathway) they utilize, encoded by the genes nirS and nirK. Our results show that the microbes catalyzing denitrification are diverse in the ODZs (Jayakumar et al. 2009a). With increasing denitrification, the abundance of both nirS and nirK genes increases and the diversity decreases. Based on nitrite reductase gene abundance, within the core of the ODZs, the abundance of denitrifiers is comparable to the total cell abundance in surface waters. Also, based on gene abundance and distribution, nirS is more abundant than nirK in ODZs. Both genes show dynamic changes that suggest denitrifiers undergo blooms within the OMZ (Jayakumar et al. 2009b), which is consistent with the variation in denitrifrication rates that we ascribe to episodic carbon fluxes. (Figure 2) The nirS denitrifiers are often undetectable outside the ODZs, while nirK is widely distributed even outside the ODZs. Anammox bacteria are also often undetectable outside the ODZs, and their abundance is an order of magnitude lower than that of denitrifiers within the ODZs. Based on our studies and several other studies, the anammox bacteria in the ODZs appear to be virtually monophyletic and are closely related to the Scalindua sp.
A. Cartoon representing relative concentrations of nitrate, nitrite and molecular nitrogen as a function of stage of denitrification.
B. Variation in diversity (H’) with stage of denitrification. Data include nirK ( r ) and nirS ( ¯ ), coastal (Jayakumar et al. 2004 ) and open ocean (Arabian Sea 2005), and samples from both bag incubations and in situ conditions.
Nitrogen fixation associated with the ODZ
Recently it has been hypothesized that the major source of fixed N to the ocean (N-fixation) is closely coupled to N removal in the ODZs (Deutsch et al. 2007) and that current N-fixation rates are underestimates. This led us to investigate novel N-fixing capability in the vicinity of and within the ODZ, zones where N loss occurs. Our result show the presence of the nifH gene, the diagnostic gene in the N-fixing pathway, at all depth intervals investigated, right from surface waters to the core of the ODZ. nifH genes amplified from the Arabian Sea include those affiliated with the Trichodesmium clade, a filamentous diazotroph that is considered the major N fixer worldwide. No sequences closely related to those of the single cell cyanobacterial N fixers were detected. We also detected nifH genes belonging to diverse clades of proteo bacteria. nifH gene fragments amplified from within the ODZs belonged mostly to alpha and gamma Proteobacterial clades and a few from beta and heterotrophic bacterial clades typical of anaerobic environments. nifH genes were not only detected, but some of these proteobacterial nifH genes are also actively being expressed (nifH mRNA was detected) in the ODZ where active N removal also occurs.
Nitrification in ODZ regions
Ammonia oxidation to nitrite is the first step of nitrification, followed by nitrite oxidation to nitrate, performed by the mutually exclusive groups of ammonia-oxidizing organisms (AOO, including ammonia-oxidizing bacteria (AOB) and Archaea (AOA)), and nitrite-oxidizing bacteria (NOB). As currently understood, AOB and AOA are autotrophs that make a living by oxidizing ammonia, and recent findings agree that AOA are generally more abundant than AOB in seawater, and that they are responsible for most of the ammonia oxidation in the marine environment. Thus it is surprising that a strong correlation between amoA gene copy number and nitrification rate is not always observed, suggesting that further research into the regulation of nitrification and the metabolism of AOO is warranted.
amoA gene abundance suggests high abundances of AOA below the euphotic zone between 400–1000 m but it is unclear whether these archaea are actively oxidizing ammonia (Bouskill et al 2011). Preliminary data suggest that a significant fraction of crenarchaeota within this depth range is autotrophic, but there is some debate as to what proportion oxidize ammonia.
Nitrification rates, as well as the relationships between rates and nitrifier abundance (both archaeal and bacterial) were investigated on a cruise in the Arabian Sea in 2007. Ammonia-oxidation rates were measured directly using 15N-NH4+ stable isotope additions in gas-impermeable trace metal clean trilaminate bags at in situ temperature. Ammonia oxidation rates were similar to previous open-ocean measurements, ranging from undetectable to 21.6±0.1 nmol l-1 d-1. The highest rates at each station occurred at the primary nitrite maximum (above the OMZ), and rates were very low at depths greater than 900 m. (Figure 3) The depth distribution of nitrification parallels that of the flux distribution modeled on the Martin curve, and indicates that nitrification is closely linked to organic matter remineralization. Both AOA and AOB amoA were detected above, within, and below the OMZ using quantitative PCR, and the AOA were 35-216 times more abundant. Nitrification rates were not directly correlated to AOA or AOB amoA abundance . Although very low ammonia oxidation rates were observed at depth, high abundances of AOA were present, suggesting that either they grow very very slowly, or they may subsist with some alternative metabolism (Newell et al., 2011).
Figure 3. Analytical curve fit to the measured ammonia oxidation rates. A power function (Martin et al. 1987) was fit to ammonia oxidation rates from every sample depth (three stations in the OMZ) combined into one profile and used to calculate the integrated nitrification rate (Newell et al., 2011).