Course:EOSC 475/ResearchProject/OMZ'sNitrogenCycle

Rozita Sabet Rasekh's wiki project:

The large concerns and insecurities about the Earth’s future climate have significantly increased the number of research conducted on the global biogeochemical cycles on land and in the oceans. The ocean ecosystem plays an important role in the carbon balance between the atmosphere and the deep ocean, but also is central in chemical and nutrient fluctuations that preserve the productivity of the ocean and greenhouse gas that fluxes to the atmosphere. One of the key regions in the oceans that helps to understand the oceans’ contribution to atmosphere greenhouse fluxes is the Oxygen Minimum Zone (OMZ). OMZs are also main areas to study the present unbalanced nitrogen cycle. Our knowledge about Nitrogen cycle has been changed since 1995 when Mulder and Van de Graaf simultaneously but in separate studies found out about anammox, a new biological process, by which ammonium and nitrite react to produce nitrogen gas. This finding affected the general understanding of nitrogen sink from fixed nitrogen that was thought to only involve Conventional denitrification. Now it has been estimated that anammox contributes up to 50% of nitrogen loss from OMZ in open oceans (Lam et al.). This will also change and affect what has been known about the marine nitrogen budget and whether it is currently balanced. Based on some marine studies, the rate of denitrification and anammox surpass that of the nitrogen fixation in ocean (Brandes et al.). If this assumption is true, then the biologically available nitrogen would be diminished from the ocean. However, the geochemical evidence does not support this (Ulloa et al.). In my paper I will discuss three major OMZs (off Namibia, Peru and in the Arabian Sea) and their microbial diversities. These regions have diverse microbial populations; however, two distinct anammox bacterial phylotypes are seemed to be dominant: Candidatus Scalindua clades 1 and 2 (Lam et al.). I will also discuss whether the denitification processes are overestimated in oceanic studies or the nitrogen fixation may have been underestimated.

1. Major OMZ regions

2. OMZ contribution to nitrogen sink

3. Microbial communities involved in OMZs

4. Is nitrogen budget fixed?

5. Nitrogen source and sink including nitrogen fixation and denitrification

6. Rates involved in nitrogen fixation and denitrification

OMZs and their major effects on the ocean ecosystem

OMZs are zones in ocean at depth of 200 to 1000 metres where the oxygen concentration is very low. The concentration of oxygen at the surface of seawater is very close to the atmosphere of the Earth. Aerobic bacteria few metres below the surface of water are able to feed from dissolved organic matters (DOMs). As these DMOs are consumed, the oxygen levels decrease in the water because of being used as an electron acceptor in the aerobic respiration process. On the other hand, deep down in the ocean the oxygen penetrates because of the thermohaline circulation and small levels of respiration due to low concentrations of dissolved organic matters. Therefore, in the intermediate level the oxygen concentration is lower than in the surface above and below (Wyrtki, 1962) which creates a large layer of oxygen depleted zone which has specific characteristics. The organisms capable or obligated to live in these environments have acquired special adaptations to limited oxygen levels. These areas do not support biodiversity of macrofauna nor the commercially valuable resources. However, large and diverse benthic communities such as sulphur-oxidizing bacteria, nitrate-respiring bacteria, protests ,and metazoans are well adapted to specifically low oxygen levels of these OMZs (Risgaard-Petersenetal.,2006). Although these OMZs with less than 20 µM of dissolved oxygen occupy only ~ 1% of the volume of the world oceans, they have large impact on global biogeochemical cycles, mainly on nitrogen cycle (Ulloa et al.).

Are OMZs rapidly expanding?

There are four major naturally occurring OMZs distributed globally. Permanent OMZs occupy ~8% of the total oceanic surface and include subsurfaces, focused in the tropical oceans: North and South Eastern Pacific (ETP); Arabian Sea (AS) and Bay of Bengale (BB) in the Northern Indian Ocean; less intense in the eastern Atlantic (SWACM, C, GG); another major OMZs are deeper in the subtropical eastern Pacific (ESTNP). In addition, two seasonal OMZs at high latitudes have been recognized in the West Bering Sea (WBS) and in the Gulf of Alaska (GA) (Paulmier and Ruiz-Pino, 2008). One of the major concerns with these regions is their rapid expansion in response to global warming which may decrease ventilation due to higher stratification and low oxygen solubility. In addition, human activities such as higher use of fertilizers have increased eutrophication and oxygen consumption which subsequently have increased these oxygen-poor waters (Paulmier and Ruiz-Pino, 2008). A large number of studies have been focused on measuring these OMZs.

Helly and his colleague are one the first people who provided a global quantification of naturally hypoxic continental margin floor (Helly et al.) They used hydrographic data to determine upper and lower OMZ depth boundaries and computed the area between these boundaries utilizing seafloor topography (Helly et al.). In their experiment, they measured the permanently hypoxic shelf and bathyal sea floor to be over one million km2 with dissolved oxygen < 0.5 ml L-1; They estimated that over half (59%) of these OMZs occurs in the northern Indian Ocean. It also seems there is strong inconsistency in the intensity, vertical position and thickness of the OMZ as a function of latitude in the eastern Pacific Ocean and as a function of longitude in the northern Indian Ocean (Helly et al.). In another experiment Stramma and his team constructed 50-year time series of dissolved-oxygen concentration for specific tropical oceanic regions by augmenting a historical database with recent measurements (Stramma et al.). Their results showed a vertical expansion of OMZs in the eastern tropical Atlantic and the equatorial Pacific during the past 50 years. Based on their measurements, in the 300- to 700-m layer of these regions the reduction of oxygen is about 0.09 to 0.34 micromoles per kilogram per year (Stramma et al.). The rapid reduction in oxygen levels may have dramatic effects on oceanic and coastal ecosystems. These observations are in agreement with previous climate model predictions of decrease in dissolved oxygen in the ocean (Matear et al. 2003 and Bopp et al. 2002).

Microbial diversity in OMZs

In recent years, there has been a large research on the microbial communities of the water columns associated with different OMZ regions. These researches are mostly focused on understanding the major players in the OMZ biogeochemical cycles and the metabolic pathways they are using. Another important aspect of these studies is to determine whether different OMZs support similar or distinct microbial communities in the world. For that, combination of different techniques and approaches such as flow cytometry, molecular techniques and biogeochemical approaches has been used.

Bacterioplanktons: In general, bacterioplanktons are abundant in surface water where their primary production takes place. However in OMZs, there is a secondary maximum of both phytoplankton bacteria and archaea at intermediate depths associated with the suboxic waters (Ulloa et al.) One commonly method utilized to determine the OMZ microbial diversity was to analyze the phylogenic relationship of sequences extracted from two clone libraries from OMZs and two from surface and deeper oxic waters based on their 16S rRNA gene fragments (Ulloa et al.).A distinct OMZ community was present in OMZs with high diversity compared to other regions (Castro-gonzález et al.). Cyanobacteria of genus Prochlorococcus and synechococcus have been found in the suboxic waters of upper boundary of the OMZ off northen Chile, where the light still penetrate for photosynthesis. To identify and classify these bacteria a combination of flow cytometry, high performance liquid chromatography (HPLC), dot blot hybridization and cloning and sequencing of the 16S rRNA genes has been performed. These picocyanobacteria seem to belong to new clades and to fix inorganic carbon well in OMZ waters at very low light intensities (Ulloa et al.).

Denitrifiers: Since denitrification can be done by diverse groups of bacteria that may be distantly related, using 16S rRNA approach does not help in looking at their diversities. Molecular surveys reveal that many distinct phylotypes are included in natural denitrifying assemblages (Braker et al., Jayakumar et al., and Scala et al.) Denitrification is the process of removal of fixed nitrogen through conventional denitrification or anammox. Major OMzs contain only small portion of total oceanic volume, but account for approximately 30% of the oceanic fixed nitrogen loss (Codispot et al.). A very common approach that often used in assessing the structure of the denitrifying bacteria is the combination of polymerase chain reaction, sequence and fragment analysis of clone libraries of nirS and nirK genes that encode for nitrate reductase , an enzyme responsible for important denitrification transformation steps (Jayakumar et al.) The results show that there is a shift in community structure along biogeochemical gradients such as dissolved oxygen, nitrate and nitrite (Ulloa et al.). The phylogenic analysis also reveals that the nirS genes are from presumably novel and yet uncultivated denitrifiers, which appear to be distantly related to those of the OMZ in the Arabian Sea (Ulloa et al. and Castro-González et al.).

Nitrifiers: The abundance of ammonia-oxidizing bacteria from the b-proteobacteria subclass (bAOB) has been studied within the OMZ (Ulloa et al.). The general approach is based on cloning and sequencing the genes encoding for the 16S ribosomal RNA and the ammonia monooxygenase enzyme active subunit (amoA). Sequences associated to Nitrosospira-like Cluster 1 dominated the 16S rRNA gene clone libraries constructed suboxic waters. Cluster 1 consists exclusively of yet uncultivated bAOB from marine environments (Ulloa et al.). All amoA sequences originated from the OMZ were either closely related to cultured Nitrosospira spp. from Cluster 0 or to other yet-uncultured bAOB from soil and aerated–anoxic Orbal process plant, and therefore different from those found in oxic waters. Thus the OMZ seems to have a distinct group of bAOB at the functional level (Molina et al.)

Anammox bacteria: Recently the presence of anammox (anaerobic ammonium oxidation) process in the suboxic waters off northern Chile has been confirmed (Thamdrup et al. 2006). In one study, Ulloa and his colleagues constructed 16S rRNA clone libraries from the same OMZ waters off northern Chile and Peru and found the presence of Planctomycetes closely associated with those found in the Black Sea and Benguela upwelling ecosystem (Kuypers et al. 2005). They have also used fluorescence in-situ hybridization (FISH) to quantify the abundance of anammox bacteria and determine their distribution in the water column and found that they are consistent with the anammox rate measurements being measured in the region and elsewhere (Ulloa et al.)

Of all the biogeochemical cycles, nitrogen is the one most closely associated with microbes. The flux of nitrogen between the oceans and the atmosphere is very critical for the continuity of life on Earth because it is an important factor in biological processes and is a fundamental element of biomass. Major processes in nitrogen cycle include nitrogen fixation, mineralization, nitrification, and denitrification. Nitrogen fixation is primarily done by bacteria and includes the conversion of nitrogen gas to ammonia (N2 + 6 H+ + 6 e− → 2 NH3). Mineralization includes the conversion of the organic nitrogen into ammonium which is also done by bacteria or some fungi. Nitrification usually involves two steps. In the primary step, ammonium is oxidized to nitrite via bacteria such as the Nitrosomonas species. Other bacteria such as the Nitrobacter, further oxidize the nitrite to into nitrate. Denitrification, on the other hand is the reduction of nitrates back into nitrogen gas, completing the nitrogen cycle. Bacteria species such Pseudomonas and Clostridium use nitrate as an electron acceptor during anaerobic conditions and reduce it back to nitrogen gas (this process is performed in some steps). Another biological process that directly converts nitrite and ammonium into nitrogen gas is anammox (Van de Graaf et al. 1996).

Is nitrogen budget fixed?

Nitrogen availability is an important factor that limits primary production in many parts of the ocean. It creates a link between carbon and nitrogen fluxes in marine life. Although many studies have been conducted to understand nitrogen cycle, still there are many areas of the cycle that remain unclear. For example it is not very clear how the nitrogen fixation relates to other processes involved in the cycle. There are some studies that suggest nitrogen fixation and denitrification may act together to control the concentration of nitrate in the ocean (Gruber 2004). Any global-scale decoupling of these two processes can change the primary production in the ocean (Zehr 2008); some recent studies suggest that nitrogen budget is not balanced at all (Codispoti 2007). Nitrogen fixation and assimilation into a biologically active pool in to the upper parts of the ocean is a way to quantify the new nitrogen added to the system (Montoya 2010). A very powerful method has been introduced for determining the fate of the new nitrogen added to the biological pool. This is done by measuring the nitrogen isotopic composition of the shells of planktonic foraminifera (Ren et al.). Nitrogen isotope studies are good approaches that help to clarify some of these areas of confusions. In this approach the natural abundance (δ15N) of the stable isotope 15N which varies as a result of biologically mediated isotopic discrimination is used to quantify the sources of nitrogen that are involved in contemporary pelagic ecosystems (montoya 2008). The isotopic δ15N in a low concentration is incorporated as a new nitrogen to the ocean via nitrogen fixation, whereas denitrification removes combined nitrogen, so the δ15N concentration increases as a result of old nitrogen removal (Galbraith et al. and McIlvin et al.).

Although denitrification is an essential part of the nitrogen cycle, it reduces the amount of fixed nitrogen that is also essential in controlling the primary production and respiration in global oceanic system. We do not really know that whether or not the nitrogen budget is balanced or should it be in balance, but a long –term imbalance can cause a global change. Therefore, it is important to be able to measure the rate at which the fixed nitrogen is lost. Until 1995, only conventional denitrification was known to be involved in biological loss of fixed nitrogen, which mostly takes place in the OMZs and sediments in the ocean. By discovery of anammox process in 1995 all the rate estimations of fixed nitrogen loss were understood to be underestimated (Mulder et al. and Van de Graaf et al. 1995). Anammox bacteria seem to be a phylogenetically narrow group and primarily autotrophs (Jetten et al.). Since the studies on anammox rates shows equal or even higher rates compared to conventional denitrification rates, all the previous estimations of nitrogen budget are subject to change (Ward et al.).

In one study Thamdrup and Dalsgaard by using the isotope-pairing method were able to detect anammox. This process enables the oceanographers to distinguish between anammox and denitrification and estimate their rates separately (Thamdrup and Dalsgaard, 2002). Incubation with 15N-labled nitrate or ammonium confirmed that during anammox process, nitrogen gas is produced through one to one pairing of nitrogen nitrate and ammonium. This observation separates annamox from denitrification process (Thamdrup and Dalsgaard 2006). These results revealed that in spite of the fact that denitrification in the sediments accounts for the large sinks for fixed nitrogen in the ocean, anammox also could equally be important in this regard (Bender et al., Christensen and Thamdrup and Dalsgaard 2006).

In their study Lam and his team also measured that in the Eastern Tropical South Pacific OMZ, high percentage of nitrogen gas obtained from anammox is from nitrate reduction and less from aerobic ammonia oxidation (lam et al.). They used stable-isotope pairing method which was consistent with the analysis from functional gene expression. They also argued that because the OMZ s are enlarging, it is important to understand the more accurate nitrogen-loss pathways to be able to understand how nitrogen cycle changes in the future because of the climate change (Lam et al.). Kuypers and his colleagues also found that in the OMZ waters of Benguela upwelling system, the anaerobic oxidation of ammonium by nitrate yielding nitrogen gas is the main process of nitrogen loss (Kuypers). In another study, Dalsgaard and his team were also interested in determining whether the the anammox reaction was responsible for significant amount of nitrogen gas production in the anoxic waters of Golfo Dulce. Their results showed this reaction is important in this area (Dalsgaard et al.). All these results and also other similar findings might imply that this reaction is globally important sink for oceanic nitrogen.

Rates involved in nitrogen fixation and denitrification

Because terrestrial nitrous oxide distribution, biological productivity, the ocean‘s capability to fix carbon dioxide and nitrogen fixation are very closely linked, it is important to precisely estimate oceanic fixed-N budget (Codispoti et al. 2006, and Falkowski). There is also a concern about the state of the oceanic fixed-N budget recently (Gruber and Sarmiento, 1997; Codispoti et al.; Gruber, 2004). Measurements of the nitrogen gas produced through conventional denitrification, being aware of anammox reaction and better understanding of OMZs and their distribution and contribution to the nitrogen cycle imply that the oceanic denitrification rate is higher than 400 TgNa−1 (Brandes et al.). However, the nitrogen fixation estimate is about 160 TgNa−1 (Codispoti et al., 2005; Deutsch et al., 2005 and 2007; Gruber, 2004). This rate might have been underestimated since it is only based on the observations and models constructed on the photic zone and coastal sediments nitrogen fixation, whereas nitrogen fixation also occurs in sub-euphotic waters and sediments by heterotrophs and lithotrophs (Capone, 2001; Zehr et al., 1998 and 2006). Subtracting the rate of nitrogen fixation from the dinitrification rates gives a value of about 200 TgNa−1 (Codispoti et al. 2006). It estimation suggests that there is a large deficit in the oceanic fixed nitrogen (Codispoti et al. 2006). The amount of the deficit seems to contradict with noticeable limits of carbon dioxide in the atmosphere and sedimentary δ15N records that suggest homeostasis during the Holocene (Codispoti et al. 2006). In addition, the ratio of nitrogen to phosphate in the ocean seems to be close to the canonical Redfield biological uptake ratio of 16 (by N and P atoms) which can be understood to show the presence of a powerful feed-back system that forces the system towards a balance (Codispoti et al. 2006).

The bottom line is that it is very difficult to look at literatures on nitrogen cycle and not to find the phrases like ‘the future studies may resolve all these uncertainties‘. This is not very surprising because the truth is there is a lot to learn. For example, even though the OMZs are known to be major global nitrogen sinks, the relative contribution of denitrifiers versus anammox bacteria is still not determined. Moreover, the ecology and diversity of microorganisms in OMZs is unknown. The future seems to be full of surprises since there are a large area of research that can be done on this fascinating ecosystem.

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Deutsch C, Sarmiento JL, Sigman DM, Gruber N, Dunne JP(2007) Spatial coupling of nitrogen inputs and losses in the ocean. Nature 445:163–167. In this study, Deutsch at al. they measured the rates of nitrogen fixation in the world’s ocean based on their effect on N and P concentrations in surface waters using an ocean circulation model.

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Kuypers, M. M. M., G. Lavik, D. Woebken, M. Schmid, B. M. Fuchs, R. Amann, B. B. Jørgensen & M. S. M. Jetten. 2005. Massive nitrogen loss from the Benguela upwelling system through anaerobic ammonium oxidation. Proceedings of the National Academy of Sciences USA, 102, 6478-6483. This group argued that in the OMZ waters of Benguela upwelling system, the anaerobic oxidation of ammonium by nitrate yielding nitrogen gas is the main process of nitrogen loss.

Lam, P., G. Lavik, M. M. Jensen, J. van de Vossenberg, M. Schmid, D. Woebken, D. Gutiérrez, R. Amann, M. S. M. Jetten, and M. M. M. Kuypers. 2009. Revising the nitrogen cycle in the Peruvian oxygen minimum zone. Proc. Natl. Acad. Sci. USA 106:4752-4757. In their article they discuss that in the Eastern Tropical South Pacific OMZ, high percentage of NO2− obtained from anammox is from nitrate reduction and less from aerobic ammonia oxidation. They used stable-isotope pairing method which was consistent with the analysis from functional gene expression. They also argue that because the OMZ s are enlarging, it is important to understand the more accurate nitrogen-loss pathways to be able to understand how nitrogen cycle changes in the future because of the climate change.

McIlvin MR, Altabet MA (2005) Chemical conversion of nitrate and nitrite to nitrous oxide for nitrogen and oxygen isotopic analysis in freshwater and seawater. Anal Chem 77:5589–5595. In this study McIlvin et al. investigated the effect of chloride concentration on the performance of the chemical reduction method for measurement of the nitrogen isotopic ratio (δ15N) in NO3 in natural waters.

Thamdrup B, Dalsgaard T. Production of N2 through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments. Appl Environ Microbiol. 2002;68:1312–1318. This study is one of the early studies that found an anaerobic oxidation of ammonium that involves in nitrate reduction, which result in nitrogen gas production in marine sediments. In this process nitrogen gas is produced from the reaction between nitrogen from nitrate and ammonium, which is a distinct process from denitrification. The process is similar to anammox that occur in wastewater bioractors.

Ulloa, O., L. Belmar, L. Farias, M. Castro-Gonzalez, A. Galan, P. Lavin, V. Molina, S. Ramirez, F. Santibanez, and H. Stevens (2006) Microbial communities and their biogeochemical role in the water column of the oxygen minimum zone in the eastern South Pacific. Gayana (Zool.) 70(1), 83–86. This group has argued that the OMZ has distinct microbial communities that are involved nitrogen cycle. Examples include denitrifiers,nitrifiers and anammox bacteris.

Van Mooy, B. A. S., Keil, R. G. & Devol, A. H. (2002) Impact of suboxia on sinking particulate organic carbon: Enhanced carbon flux and preferential degradation of amino acids via denitrification. Geochim. Cosmochim. Acta. 66, 457-465. Their studies suggests that microbes degrading organic carbon under OMZ via denitrification preferentially use amino acids that have higher ratio of N.

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