The sensitivity of marine N2 fixation to dissolved inorganic nitrogen

The dominant process adding nitrogen (N) to the ocean, di-nitrogen (N2) fixation, is mediated by prokaryotes (diazotrophs) sensitive to a variety of environmental factors. In particular, it is often assumed that consequential rates of marine N2 fixation do not occur where concentrations of nitrate (NO−3) and/or ammonium (NH+4) exceed 1μM because of the additional energetic cost associated with assimilating N2 gas relative to NO−3 or NH+4. However, an examination of culturing studies and in situ N2 fixation rate measurements from marine euphotic, mesopelagic, and benthic environments indicates that while elevated concentrations of NO−3 and/or NH+4 can depress N2 fixation rates, the process can continue at substantial rates in the presence of as much as 30μM NO−3 and/or 200μM NH+4. These findings challenge expectations of the degree to which inorganic N inhibits this process. The high rates of N2 fixation measured in some benthic environments suggest that certain benthic diazotrophs may be less sensitive to prolonged exposure to NO−3 and/or NH+4 than cyanobacterial diazotrophs. Additionally, recent work indicates that cyanobacterial diazotrophs may have mechanisms for mitigating NO−3 inhibition of N2 fixation. In particular, it has been recently shown that increasing phosphorus (P) availability increases diazotroph abundance, thus compensating for lower per-cell rates of N2 fixation that result from NO−3 inhibition. Consequently, low ambient surface ocean N:P ratios such as those generated by the increasing rates of N loss thought to occur during the last glacial to interglacial transition may create conditions favorable for N2 fixation and thus help to stabilize the marine N inventory on relevant time scales. These findings suggest that restricting measurements of marine N2 fixation to oligotrophic surface waters may underestimate global rates of this process and contribute to uncertainties in the marine N budget.


INTRODUCTION
Phytoplankton growing in the sunlit surface ocean (euphotic zone) produce organic matter via photosynthesis at a rate of ∼50 Pg C year −1 (Westberry et al., 2008), and thus play an important role in the global carbon (C) cycle. However, phytoplankton growth in the euphotic zone is commonly limited by the availability of nutrients such as nitrogen (N); consequently, the processes that add and remove N to and from the ocean, respectively, influence the C cycle and thus climate. Unlike the physical processes that supply other biologically necessary elements like phosphorus (P) and iron (Fe) to the ocean (such as atmospheric deposition and fluvial inputs), the dominant process adding N to the ocean, di-nitrogen (N 2 ) fixation, is unique in that it is biologically mediated. While N 2 gas is unavailable to most organisms, certain groups of prokaryotes known as diazotrophs have the enzymatic ability to reduce dissolved N 2 to ammonium (NH + 4 ) and assimilate it into their biomass. The ultimate fate of N in diazotrophic biomass is to be cycled into more bio-available forms, including nitrate (NO − 3 ), that serve as the primary source of assimilative N for non-diazotrophic phytoplankton and bacteria in the ocean. In spite of the fundamental importance of N 2 fixation in the global C cycle and in supporting the base of the food web, the locations and rates of N 2 fixation in the ocean are poorly known.
Uncertainty in the rates of marine N 2 fixation contributes to ambiguity as to whether the modern marine N budget is balanced. Some estimates suggest that rates of N fluxes to the ocean only compensate for one third to one half of the fluxes of N out of the ocean (Codispoti et al., 2001;Codispoti, 2007), while constraints from paleoceanographic and modeling studies indicate that the marine N budget has been balanced to within ∼10% over at least the Holocene (Brandes and Devol, 2002;Deutsch et al., 2004). Assuming that the marine N budget is essentially balanced, the discrepancy in N flux estimates requires that rates of marine N 2 fixation are underestimated and/or that rates of N loss are overestimated. The constraint of an approximately balanced marine N budget also implies that there are feedback mechanisms allowing N 2 fixation and denitrification, the dominant pathway by which N is lost from the ocean, to respond to each other on relatively short (i.e., ≤1000 years) timescales. Currently, both the size of the fluxes of N to and from the ocean, as well as the nature of potential feedback mechanisms that maintain a balanced marine N budget, remain ill-defined.
While improved knowledge of the marine N cycle requires a multifaceted approach, characterizing the physical and chemical sensitivities of marine diazotrophs to various environmental conditions provides constraints on regions of the ocean that may support diazotrophy. A better understanding of the sensitivities of marine diazotrophs may also reveal mechanisms by which marine N 2 fixation can respond to changes in rates of marine denitrification. However, our ability to describe the sensitivities of N 2 fixation depends on the degree to which we understand and have characterized the diversity of marine diazotrophs, an understanding presently limited by the small number of marine diazotrophs isolated for manipulative culture-based experiments.
The majority of marine N 2 fixation has historically been attributed to the filamentous, non-heterocystous cyanobacteria Trichodesmium spp. resident in the warm, stratified, and nutrient-depleted regions of the surface ocean (Carpenter, 1983;Capone et al., 1997Capone et al., , 2005. However, the past decade has seen a number of challenges to the paradigm that N 2 fixation by Trichodesmium spp., especially in the tropical North Atlantic, is the primary source of N to the global ocean. For example, molecular tools have identified novel diazotrophs present in environments with physical and/or chemical characteristics different from their more well-studied counterparts in tropical and subtropical seas (Zehr et al., 2001(Zehr et al., , 2008Montoya et al., 2004;Langlois et al., 2008;Moisander et al., 2010;Fernandez et al., 2011). Additionally, indirect evidence such as remote sensing (Westberry et al., 2005;Westberry and Siegel, 2006) and geochemical modeling (Deutsch et al., 2007) describes geographic distributions of N 2 fixers, including Trichodesmium spp., that differs from our expectation of oligotrophic dominance. Finally, a number of both in situ and culture-based studies challenge some long-held notions of diazotrophic sensitivities to nutrients, including the degree to which inorganic N inhibits N 2 fixation. All of these findings raise the possibility that the geographic distribution and sensitivities of marine diazotrophs may be different than previously thought. As recognition of both the breadth of oceanic conditions supportive of diazotrophy and the diversity of marine diazotrophs increases, so too does the possibility that considerable rates of N 2 fixation occur in environments beyond the surface waters of the oligotrophic gyres. If so, global marine N 2 fixation rates may be greater than previously estimated.
In spite of an incomplete knowledge of marine diazotroph diversity, environmental and culture-based observations can establish criteria consistent with diazotrophic success. Environmental factors that are known to regulate marine diazotrophy include light (Carpenter et al., 1993;Milligan et al., 2007;Breitbarth et al., 2008), temperature (Chen et al., 1998;Breitbarth et al., 2007;Stal, 2009), oxygen (Robson and Postgate, 1980;Capone and Budin, 1982;Stal and Heyer, 1987), and metal availability (Rueter et al., 1990;Berman-Frank et al., 2001;Kustka et al., 2003;Chappell and Webb, 2010;Saito et al., 2011). Here, the sensitivity of marine diazotrophs to dissolved inorganic N (DIN), in particular NO − 3 and NH + 4 , is evaluated, and evidence for the inhibition of N 2 fixation by DIN in (1) the euphotic zone, (2) the sub-euphotic zone, and (3) benthic marine environments, is reviewed. In particular, the question of whether significant rates of N 2 fixation can occur when ambient DIN concentrations are significant, i.e., ≥1 µM, is examined. The findings of this review are that: (1) reports of substantial rates of N 2 fixation in euphotic and benthic environments with ≥1 µM DIN indicate that elevated DIN does not necessarily preclude large N 2 fixation fluxes; (2) certain benthic marine diazotrophs may be less sensitive to chronic exposure to elevated concentrations of DIN than diazotrophs in the euphotic zone; (3) while benthic N 2 fixation is widespread and can occur at significant rates, global estimates are poorly known, likely contributing significant uncertainty to global estimates of marine N 2 fixation fluxes, and, (4) euphotic zone diazotrophs may respond to changes in ambient N:P ratios, providing a potential mechanism for diazotrophs to respond to changes in denitrification rates and thus to stabilize the marine N inventory. These findings are investigated below.

NUTRIENT INHIBITION OF EUPHOTIC ZONE N 2 FIXATION
There are three primary lines of evidence for the inhibition of marine N 2 fixation by inorganic N. The first results from circumstances associated with the origins of marine diazotrophic research. Before molecular tools became widely available, our understanding of marine diazotrophs was largely limited to the study of macroscopic cyanobacteria that could be readily identified and manipulated in field and culture-based studies. The most conspicuous and well-studied marine diazotroph, Trichodesmium spp., has predominantly been observed in warm, nutrient depleted regions of the surface ocean (Carpenter, 1983;Capone et al., 1997Capone et al., , 2005. The association of Trichodesmium spp. with these environmental characteristics, and the strong bias of studies of marine diazotrophs towards Trichodesmium spp., has perhaps unintentionally lead to the expectation that other marine diazotrophs will share the same environmental preferences. The second line of evidence for DIN inhibition of N 2 fixation comes from calculations showing that it requires ∼25% more energy to reduce N 2 (87 kcal) than NO − 3 (69 kcal) to NH + 4 (Falkowski, 1983). Together with the majority of field observations of diazotrophs from nutrient-depleted tropical surface waters, this additional energetic cost has lead to the assumption that significant rates of N 2 fixation do not occur in marine environments with ≥1 µM DIN.
The third line of evidence for the inhibition of N 2 fixation by DIN comes from culture studies of marine diazotrophs that test the effects of short-term and/or chronic exposure to NO − 3 and NH + 4 (e.g., Ohki et al., 1991;Mulholland et al., 2001;Fu and Bell, 2003;Holl and Montoya, 2005) (Table 1) (the numerous studies of DIN inhibition of fresh water diazotrophs are not reviewed here). These studies have demonstrated that NH + 4 is more effective at inhibiting N 2 fixation than NO − 3 (Ohki and Fujita, 1982;Ohki et al., 1991;Mulholland et al., 2001), presumably because of the larger energetic cost associated with assimilating N 2 vs. NH + 4 than with assimilating N 2 vs. NO − 3 . Additionally, these studies have shown that chronic exposure to both NO − 3 and NH + 4 more strongly inhibits N 2 fixation than does short-term (i.e., less than 24 h) exposure (Ohki et al., 1991;Mulholland et al., 2001;Fu and Bell,  2003; Milligan et al., 2007;Dekaezemacker and Bonnet, 2011;Sandh et al., 2011;Knapp et al., 2012) (Table 1). Supporting these observations of depressed N 2 fixation rates, physiological changes in Trichodesmium have also been documented when cultures are grown with NO − 3 as a source of assimilative N instead of dissolved N 2 gas. After chronic exposure of Trichodesmium cultures to 100 µM NO − 3 (Milligan et al., 2007) demonstrated a downregulation of Mehler activity relative to cultures grown on N 2 gas, while (Sandh et al., 2011) found an inhibition of nitrogenase expression and diazocyte development. These effects of NO − 3 on diazotroph physiology suggest that chronic exposure to DIN has a greater impact on N 2 fixation rates than does short-term exposure.
The relatively small impact on N 2 fixation rates by short-term exposure to NO − 3 ( Table 1) has implications for proposed mechanisms for diazotrophs to acquire limiting nutrients such as P. For example, short-term exposure to DIN could take place during the vertical migration of Trichodesmium spp. (Capone et al., 1990) showed that nitrogenase in Trichodesmium spp. is synthesized each morning prior to the initiation of nitrogenase activity. Consequently, the downward migration of Trichodesmium spp. at night (Villareal and Carpenter, 1990) to acquire P (Villareal and Carpenter, 2003) at the top of the nutricline (where NO − 3 is also present) might not strongly depress peak daytime N 2 fixation rates in Trichodesmium spp. if exposure to NO − 3 is brief and occurs at night before new nitrogenase is synthesized. While studies of the effects of DIN inhibition on marine diazotrophs have largely been restricted to Trichodesmium spp., recent culturing work suggests that Crocosphaera has similar sensitivities to short-term vs. chronic NO − 3 exposure (Dekaezemacker and Bonnet, 2011;Knapp et al., 2012). Given the similarity in response of Trichodesmium and Crocosphaera spp. and the limited genetic divergence in nitrogenase amino acid sequences in marine diazotrophic cyanobacteria (Zehr, 2011), the smaller effect of short-term vs. long-term DIN exposure on N 2 fixation rates may be common among other diazotrophic cyanobacteria as well.
Culturing studies clearly show that DIN can inhibit N 2 fixation; however most inhibition studies have been performed with concentrations of N and/or P in the culture media that exceed those typically found in the euphotic zone ( Table 1). This discrepancy between nutrient concentrations in the environment and in cultures leaves open the possibility that culturing studies overestimate the degree to which DIN inhibits N 2 fixation in the environment. Recent culturing work using concentrations of NO − 3 and PO 3− 4 typically found in the euphotic zone show that chronic exposure of Trichodesmium and Crocosphaera to 5 to 16 µM NO − 3 depresses N 2 fixation rates relative to cultures grown with no NO − 3 , but that N 2 fixation did not stop even in cultures amended with as much as 16 µM NO − 3 (Knapp et al., 2012). Moreover, the same work showed that higher concentrations of PO 3− 4 can offset NO − 3 inhibition of per-cell N 2 fixation rates by increasing diazotroph abundance. Consequently, the volume-integrated rate of N 2 fixation in treatments grown with 5.0 µM NO − 3 and 1.0 µM PO 3− 4 was comparable to the volumeintegrated rate of N 2 fixation in treatments not amended with NO − 3 and grown with 0.5 µM PO 3− 4 (Knapp et al., 2012). The finding of increased diazotroph abundance as a function of increasing P availability is consistent with the well-recognized role that P availability plays in regulating the biomass of microbes [e.g., (Elser et al., 2007;Loladze and Elser, 2011;Scott et al., 2012)]. Investigations into variability in phytoplankton biomass N:P ratios indicate that P is preferentially used to create new biomass (e.g., in DNA) whereas N is required both for the production of new biomass as well as for the production of proteins, especially associated with resource acquisition (Klausmeier et al., 2004;Loladze and Elser, 2011). Consequently, the results of (Knapp et al., 2012) documenting a two-to three-fold greater abundance of both the diazotrophs Crocosphaera watsonii and Trichodesmium erythraeum in batch cultures grown with 1.0 vs. 0.5 µM PO 3− 4 are perhaps unsurprising. What is surprising is that the increase in diazotrophic biomass was sufficient to offset the lower per-trichome rates of N 2 fixation resulting from inhibition by 5.0 µM NO − 3 . This work shows that NO − 3 present at typical www.frontiersin.org October 2012 | Volume 3 | Article 374 | 5 surface ocean concentrations does not necessarily preclude N 2 fixation fluxes comparable to those observed in NO − 3 -depleted environments, and suggests that field and numerical modeling investigations of marine N 2 fixation that exclude surface ocean environments with ≥1 µM NO − 3 may overlook potentially significant regions of N 2 fixation.
Additionally, the work of (Knapp et al., 2012) identifies a potential mechanism for euphotic zone diazotrophs to respond to changes in surface ocean concentrations of NO − 3 and PO 3− 4 . Specifically, while it has been assumed that low ambient N:P ratios (a condition created by denitrification occurring below the euphotic zone) would stimulate higher rates of N 2 fixation (Haug et al., 1998;Deutsch et al., 2004), no mechanism has been proposed for how a diazotroph would sense and respond favorably to lower N:P ratios. The results of Knapp et al. (2012) describe how the separate physiological effects of relatively high concentrations of P (i.e., increased diazotroph abundance) and relatively low concentrations of N (i.e., lessened NO − 3 inhibition of N 2 fixation) together can create conditions that can support significant N 2 fixation fluxes. While relatively low N and high P concentrations have distinct effects on diazotrophs, combining these effects results in a perceived advantage for diazotrophs growing in environments with low ambient N:P ratios and may provide a feedback mechanism for diazotrophs to respond to increases in denitrification and thus help stabilize the marine N inventory. This finding also has implications for diazotroph biogeography, and suggests that significant abundances and/or N 2 fixation fluxes may not be restricted to oligotrophic surface waters such as the North Atlantic, but may occur in more nutrient-replete regions of the surface ocean such as the surface waters overlying ODZs where rates of N loss are high.
Indeed, these culture-based results are consistent with recent field observations by (Fernandez et al., 2011;Sohm et al., 2011) who document N 2 fixation rates of 0.1-7.5 nmol N L −1 d −1 in surface ocean waters with 5-20 µM NO − 3 ( Table 2), although molecular analyses indicate that this fixation was carried out by diazotrophs other than Trichodesmium or Crocospahera spp. These rates of N 2 fixation in NO − 3 -replete coastal waters are comparable to the range in N 2 fixation rates measured at Station ALOHA in the North Pacific gyre of 0.5-11 nmol N L −1 d −1 (Church et al., 2009) and where surface NO − 3 concentrations are consistently ≤100 nM (Fujieki et al., 2011). Similarly, (Halm et al., 2012) found higher euphotic zone rates of N 2 fixation in regions of the South Pacific gyre with higher concentrations of NO − 3 (as well as PO 3− 4 ) compared to more oligotrophic regions of the gyre, i.e., average N 2 fixation rates of 1.5 ± 0.3 nmol N L −1 d −1 vs. 0.4 ± 0.3 nmol N L −1 d −1 , respectively. These N 2 fixation rate measurements are supported by numerous other field observations documenting significant abundances of and/or N 2 fixation by diazotrophs including Trichodesmium spp. in other near-shore locations (Lenes et al., 2001;White et al., 2007;Rodier and Le Borgne, 2008;Grosse et al., 2010;Rodier and Le Borgne, 2010;Bombar et al., 2011) (Table 2).
These reports of substantial rates of N 2 fixation in NO − 3 -bearing surface waters, especially in upwelling and coastal regions, underscore the potential bias of prior field campaigns documenting N 2 fixation predominantly in the nutrient-depleted oligotrophic gyres, and suggest that N 2 fixation may have a broader geographic distribution in marine euphotic waters that episodically and/or chronically have significant DIN concentrations. Indeed, the strains of Trichodesmium erythraeum commonly used in culture studies, i.e., NIBB1067 and IMS101, were collected from the coastal waters of Japan and North Carolina, respectively (Ohki and Fujita, 1982;Prufert-Bebout et al., 1993), where surface water DIN concentrations are at least episodically elevated. That Trichodesmium spp. are frequently found in coastal waters that can have relatively high DIN concentrations is relevant considering that recent remote sensing (Westberry and Siegel, 2006) and geochemical modeling (Deutsch et al., 2007) studies have predicted high abundances of diazotrophs and/or rates of N 2 fixation in regions of the surface ocean with NO − 3 concentrations consistently ≥5 µM (Garcia et al., 2010). The results reviewed here suggest that NO − 3 is not as inhibitive of N 2 fixation by euphotic-zone diazotrophs as previously thought, especially if P and the necessary trace metals are abundant, and have implications for field studies documenting marine N 2 fixation fluxes as well as for the parameterization of N 2 fixation in models.

NUTRIENT INHIBITION OF MESOPELAGIC N 2 FIXATION
While there are only a handful of reports of N 2 fixation occurring in the mesopelagic (i.e., sub-euphotic) water column, advances in molecular techniques capable of identifying diazotrophs and the improved sensitivity of mass spectrometers for detecting the incorporation of labeled 15 N 2 into suspended particulate organic N (PN susp ) have improved our ability to evaluate N 2 fixation in this environment. It is expected that N 2 fixation in this portion of the water column would be carried out by diazotrophs that have substantially different physiologies than those living in the euphotic zone: mesopelagic diazotrophs require a different energy source than their photosynthetic counterparts, they need to tolerate lower temperatures, and due to the higher concentrations of NO − 3 below the base of the euphotic zone, they would also presumably be less inhibited by NO − 3 . Perhaps unsurprisingly then, diazotrophs collected from mesoand bathypelagic waters contain nifH sequences distinct from euphotic zone diazotrophs. In samples collected from the deep North Pacific (Mehta et al., 2003(Mehta et al., , 2005 identified a number of nifH sequences associated with methanogens and anaerobic sulfate reducers from hydrothermal vent systems, and was able to document growth and N 2 fixation in a culture of thermophilic archeal methanogens (Mehta and Baross, 2006). (Hewson et al., 2007) identified nifH genes in samples collected throughout the water column of the Sargasso Sea and detected nifH in mesoand abyssopelagic samples more consistently than in euphotic zone samples, suggesting the potential for diazotrophy below the euphotic zone. However, (Hewson et al., 2007) recovered nifH sequences of the cyanobacterial diazotrophs Trichodesmium thiebautii and Crocosphaera watsonii at 250 and 1000 m, respectively, demonstrating that the nifH associated with diazotrophs active in other environments persists upon transport to the deep ocean in a reasonably robust form, as has been recently reported for RuBisCO (Orellana and Hansell, 2012). However, (Hewson Table 2 (Jayakumar et al., 2012) found both nifH DNA and cDNA sequences associated with strictly anaerobic proteobacteria in samples collected from the oxygen minimum zone of the Arabian Sea, also indicating potential activity of diazotrophs in sub-euphotic zone waters.
In addition to the molecular studies described above, two recent reports document relatively low rates of N 2 fixation in mesopelagic samples collected from coastal environments. In the NO − 3 -rich coastal waters of the Eastern Tropical South Pacific (ETSP), (Fernandez et al., 2011) measured N 2 fixation both in the euphotic zone and in mesopelagic waters, including in the core of the local oxygen deficient zone (ODZ) where they reported rates of 1.3 nmol N L −1 d −1 ( Table 2). While (Fernandez et al., 2011) recovered numerous nifH sequences, they amplified no cyanobacterial phylotypes in surface or subsurface waters; instead most of the nifH sequences aligned with Cluster I, and to a lesser extent, Cluster III nifH genes, including representatives of anaerobic sulfate reducers. In mesopelagic samples collected in the California Borderland Basins (i.e., San Pedro and Santa Monica Basins) (Hamersley et al., 2011) report similar N 2 fixation rates of 0.07 µmol N m −3 d −1 ( Table 2). The most common nifH phylotype recovered by (Hamersley et al., 2011) was from the UCYN-A group found both in surface and mesopelagic samples. Additionally, (Hamersley et al., 2011) recovered heterotrophic nifH sequences in mesopelagic samples from Cluster I as well as a number of Cluster III sequences that correspond to strict anaerobes, including alpha-and gamma-proteobacteria, as well as sulfate reducing bacteria (SRB). While both (Fernandez et al., 2011;Hamersley et al., 2011) suggest that diazotrophy in these mesopelagic environments may be associated with oxygen deficiency, the similarity of some mesopelagic nifH sequences to those of diazotrophs found both in surface waters and in benthic environments (see below) raises the possibility that some of the diazotrophs recovered in these near-shore mesopelagic samples are introduced via sinking particles (from the euphotic zone) or via nepheloid layer from sediments to the water column further offshore. Given that a number of the phylotypes collected by (Hamersley et al., 2011) are similar to sequences from microbial mats and/or to cultivated strains of strict anaerobes, a condition not met in the water column of the San Pedro Basin where ambient oxygen concentrations are ∼11 µM, it raises the possibility that sedimentary microbes are resuspended and then detected in mesopelagic waters.
The determination of N 2 fixation rates in mesopelagic waters presents unique analytical challenges as it depends on the incorporation of 15 N 2 by living, active diazotrophs into particulate organic matter that can then be analyzed by combustion on an isotope ratio mass spectrometer (Montoya et al., 1996). Even with increasingly sensitive instrumentation, the concentration of PN susp in mesopelagic waters is extremely low. Thus, even with "large volume," i.e., 4 L, incubations and given a typical detection limit of ∼1.4 µmol N for GC-MS systems commonly used to analyze these samples (e.g., http://stableisotopefacility.ucdavis. edu/), a PN susp concentration of ∼0.35 µM is required to generate a signal above typical analytical detection limits. Since most open-ocean PN susp concentrations are only this high within the euphotic zone, and then decrease sharply in the mesopelagic (i.e., PN susp concentrations at 300 m at BATS and HOT are 0.05 µM) (Michaels and Knap, 1996;Fujieki et al., 2011), even larger volume incubations and/or more sensitive analytical approaches are required to reliably to detect N 2 fixation rates in these waters. While PN susp concentrations in mesopelagic waters of near-shore environments are higher than those in the oligotrophic ocean, e.g., (Hamersley et al., 2011) report PN susp of 0.23 and 0.25 µM for their samples collected at 500 and 850 m, respectively, ensuring that mesopelagic samples have sufficient PN susp to generate a signal above detection limits remains a significant challenge for documenting mesopelagic N 2 fixation rates. Moreover, it is not clear that improving incubation techniques to increase 15 N 2 gas solubility (Mohr et al., 2010) will improve the ability to measure mesopelagic N 2 fixation rates, as this modification does not increase the initial quantity of PN susp in a mesopelagic sample. Given the very low PN susp concentration in mesopelagic waters, great care must be taken to quantify blanks for these incubations and to demonstrate that N 2 fixation rates generated by these methods contain a sufficient quantity of N to exceed analytical detection limits. Consequently, it may be warranted to view the water-column integrated mesopelagic N 2 fixation rates of 55 µmol N m −2 d −1 in the California Borderland Basins (Hamersley et al., 2011) and 5.4 ± 2.4 µmol N m −2 d −1 in the ETSP (Fernandez et al., 2011), and their potential to help resolve global marine N budget imbalances, as provisional estimates until supporting measurements confirm the activity of N 2 fixation in mesopelagic environments. If these early reports of relatively low N 2 fixation rates in sub-euphotic zone waters ( Table 2) are broadly characteristic of mesopelagic environments, they may be the consequence of NO − 3 inhibition. A better understanding of the capacity of mesopelagic environments to support diazotrophy will benefit from methodological and analytical improvements of in situ N 2 fixation rate measurements, as well as successful culturing of microbes recovered from these environments.

NUTRIENT INHIBITION OF BENTHIC MARINE N 2 FIXATION
From intertidal cyanobacterial mats to dark muds, and from low to high latitudes, numerous reports from diverse marine ecosystems demonstrate that benthic diazotrophy is widespread (Capone, 1983 and references therein). N 2 fixation in marine sediments has received renewed attention based on evidence that the net flux of N 2 gas in certain coastal sediments may have changed from efflux, via denitrification, to influx, via N 2 fixation, potentially forced by climate change (Fulweiler et al., 2007). Due to the high concentrations of NO − 3 and/or NH + 4 that can accumulate as a result of organic matter degradation, N 2 fixation in benthic environments presents perhaps the greatest challenge to the expectation for DIN to inhibit diazotrophy. Table 2 includes the small subset of all studies documenting benthic marine N 2 fixation that reported both N 2 fixation rates as well as concentrations of ambient NO − 3 and/or NH + 4 that exceeded 1 µM. While the culture-based studies described above indicate that NH + 4 significantly depresses N 2 fixation rates in Trichodesmium and Crocosphaera spp.,
Given that the highest rates of N loss in the ocean occur in marine sediments (Brandes and Devol, 2002), it is perplexing that both N 2 fixation and denitrification have frequently been observed in the same sediments (Haines et al., 1981;Slater and Capone, 1984;Joye and Paerl, 1993;Currin et al., 1996;An and Joye, 2001;Gardner et al., 2006;Lee and Joye, 2006;Fulweiler et al., 2007;McCarthy et al., 2008;Bertics et al., 2012;Rao and Charette, 2012). Indeed, Azospirillum, a bacteria associated with seagrasses (Patriquin, 1978) is thought to carry out both denitrification and N 2 fixation (Bothe et al., 1981). These observations raise the question: if N 2 fixation is an energetically costly process whose role is to provide a source of assimilatory N to the ecosystem, and if diazotrophs are inhibited by DIN, why does N 2 fixation happen at significant rates in benthic environments rich in DIN and that also support denitrification?
Benthic diazotrophy has been investigated with a variety of biological and geochemical tools that together indicate that benthic N 2 fixation is carried out by a diverse suite of microbes at environmentally significant rates ( Table 2). Many benthic N 2 fixation rates have been measured using acetylene reduction, and concerns have been raised regarding its use in these environments because of the capacity for acetylene to inhibit other microbial processes including denitrification, methanogenesis, methane oxidation, sulfate reduction, nitrification, and even N 2 fixation [ (Capone, 1983) and references therein]. In spite of these and other more general concerns regarding the limitation of methods to measure absolute rates of benthic microbial processes, including the high degree of spatial heterogeneity due to microsites and steep geochemical gradients on millimeter spatial scales, benthic diazotrophy has been validated using 15 N 2 assimilation (Patriquin and Knowles, 1972;Burris, 1976;Carpenter et al., 1978;Capone and Budin, 1982;Dekas et al., 2009) and net N 2 gas flux measurements made using membrane inlet mass spectrometry (MIMS) (An and Joye, 2001;Gardner et al., 2006;Fulweiler et al., 2007;McCarthy et al., 2008;Rao and Charette, 2012). Based on visual identification of diazotrophs and differences in N 2 fixation rates between light and dark incubations, cyanobacteria are thought to contribute to N 2 fixation fluxes in intertidal microbial mat consortia (Joye and Paerl, 1993;Currin et al., 1996;Lee and Joye, 2006). Additionally, a number of benthic studies have used molybdate amendment experiments to inhibit sulfate reduction and have simultaneously inhibited N 2 fixation in the same sediments; such experiments have been used to attribute N 2 fixation in certain benthic marine environments to SRB (Gandy and Yoch, 1988;Welsh et al., 1996;Nielsen et al., 2001;Burns et al., 2002;Steppe and Paerl, 2002;Bertics et al., 2010). Molecular tools have also verified the presence of nif genes, and thus the metabolic potential for N 2 fixation, in various benthic marine microbes including in SRB (Burns et al., 2002;Paerl, 2002, 2005;Dekas et al., 2009;Bertics et al., 2010Bertics et al., , 2012, anaerobic methane-oxidizing archaea (Dekas et al., 2009), and benthic cyanobacteria (Steppe and Paerl, 2005;Bertics et al., 2010).
Previous studies provide some insight into the role of DIN in regulating N 2 fixation and denitrification in some benthic environments. Specifically, Paerl, 1993, 1994) established seasonality in patterns of N 2 fixation and denitrification in Tomales Bay, CA sediments that are consistent with studies documenting DIN inhibition of N 2 fixation. Paerl, 1993, 1994) observed that when ambient benthic DIN concentrations were relatively low, N 2 fixation rates were high and denitrification rates were low, but when runoff or other sources introduced NO − 3 to sediments, denitrification rates increased and N 2 fixation rates decreased. These observations from Tomales Bay indicate both that denitrification is NO − 3 limited and that N 2 fixation is inhibited by NO − 3 . The sensitivity of benthic N 2 fixation and denitrification rates to changes in ambient DIN concentration in Tomales Bay has been replicated in manipulated core studies and observed in other benthic N cycling studies. For example, in the estuarine sands of Waquoit Bay, MA (Rao and Charette, 2012) documented net N 2 fixation, and suggested that denitrification occurring elsewhere in the estuary removes DIN, permitting N 2 fixation to proceed downstream. Similarly, in a study of N 2 fixation rates associated with seagrass roots in a French estuary (Welsh et al., 1996) observed peak N 2 fixation rates when ambient NH + 4 concentrations reached their annual minima of 190 µM, relative to the peak concentration of 290 µM.
Many of these studies also document complex interactions between oxygen, DIN, and/or organic carbon, and their relationship with N 2 fixation and/or denitrification rates in benthic environments. For example, (Fulweiler et al., 2007) attributed a change from net denitrification to net N 2 fixation in Narragansett Bay, RI sediments to a decrease in the organic matter flux to the sediments due to diminished winter-spring blooms in the Bay. (Fulweiler et al., 2007) tested this hypothesis, observing a change from net N 2 fixation to net denitrification after adding organic matter to incubated sediment cores that had previously shown net N 2 fixation. In the past, benthic remineralization of winter-spring bloom material in Narragansett Bay provided a source of DIN to the sediment and overlying water column, which is nutrient-poor in summers; presumably the reduction in the magnitude of the organic matter flux to Narragansett Bay sediments corresponds to a reduced DIN flux to the sediments, and is proposed by Fulweiler et al. (2007) to be the cause of the switch to net N 2 fixation from net denitrification.
Observations of decreased rates of benthic N 2 fixation when ambient DIN concentrations increase, either because of runoff or remineralization, are generally consistent with the observations described above that show that DIN inhibits, but does not stop, pelagic diazotrophy. However, the observations of decreased benthic N 2 fixation rates when DIN concentrations increase are not consistent with other observations of high rates of benthic N 2

Frontiers in Microbiology | Aquatic Microbiology
October 2012 | Volume 3 | Article 374 | 10 fixation in dark, NH + 4 -rich environments [e.g., (Haines et al., 1981;Capone, 1982;Welsh et al., 1996;Bertics et al., 2010)]. Some previous studies of benthic N 2 fixation have suggested that oxygen and organic carbon availability also play a role in mitigating DIN inhibition (Yoch and Whiting, 1986;McGlathery et al., 1998). Another explanation for why N 2 fixation may occur at considerable rates in DIN-rich benthic environments invokes a role for N 2 fixation that is entirely different from providing a source of assimilatory N to the ecosystem. Specifically, there is evidence that in the presence of high concentrations of NH + 4 benthic N 2 fixation can serve as a sink for excess electrons to help bacteria achieve redox balance, especially in the absence of a viable Calvin-Benson-Bassham pathway (Joshi and Tabita, 1996;Tichi and Tabita, 2000). Ultimately, sensitivity studies of benthic diazotrophs to these parameters are limited by the lack of isolated diazotrophs for manipulative culture studies.
A better understanding of the sensitivities of the diverse suite of benthic diazotrophs to oxygen, organic carbon and DIN is critical for refining models of benthic N cycling, and in particular determining whether marine sediments are a net source or sink of fixed N to the marine environment. While marine sediments are normally considered a net sink for fixed N (Seitzinger, 1988), a variety of reports show that some benthic environments can be a net source of bioavailable N at least on seasonal timescales, if not annually, as well (Currin et al., 1996;Lee and Joye, 2006;Fulweiler et al., 2007;McCarthy et al., 2008). Moreover, if environmental conditions change to favor diazotrophy (e.g., Fulweiler et al., 2007), it is plausible that even if marine sediments do not overwhelmingly become a source of fixed N, they might at least not be as large of a sink as previously thought. Benthic N 2 fixation deserves more attention as it is a poorly constrained term in the global marine N budget; the process is not always included in marine N budget estimates, although (Capone, 1983) estimated it may contribute 15 Tg N year −1 , which would increase some estimates of N fluxes to the marine environment by 10-15% (Brandes and Devol, 2002).

CONCLUSIONS
Rates of the dominant fluxes of N to and from the ocean are highly uncertain, leaving open the question of whether the modern marine N budget is balanced. Some estimates suggest that rates of N fluxes to the ocean only compensate for one-third to one-half of the fluxes of N out of the ocean (Codispoti et al., 2001;Codispoti, 2007), while paleoceanographic and modeling studies require a balanced N budget, implying that either rates of N 2 fixation are underestimated, and/or that rates of N loss are overestimated (Brandes and Devol, 2002;Deutsch et al., 2004). One potential liability in previous estimates of N fluxes to the ocean is the assumption that the highest rates of marine N 2 fixation occur in the warm, nutrient-depleted regions of the surface ocean. However, culture and field evidence reviewed here indicates that low concentrations of NO − 3 and/or NH + 4 (≤1 µM) are not a strict requirement for high rates of marine N 2 fixation, and that numerical models using this as a criteria for significant diazotroph abundance and/or N 2 fixation fluxes may not accurately represent diazotroph sensitivities to DIN. Generally, the best-studied cyanobacterial diazotrophs show little inhibition by short-term exposure to inorganic N. Instead, depressed rates of N 2 fixation occur after long-term exposure of diazotrophs to elevated concentrations of DIN, although long-term exposure does not necessarily stop N 2 fixation. Recent field and culturing work has shown that NO − 3 concentrations commonly found in marine surface waters, i.e., up to 20 µM, do not preclude rates of N 2 fixation comparable to those measured in the NO − 3depleted surface waters of the North Pacific gyre (Fernandez et al., 2011;Sohm et al., 2011). Moreover, field and culture evidence suggests that well-studied cyanobacterial diazotrophs such as Trichodesmium spp. are more tolerant of NO − 3 than previously assumed, especially when P is relatively abundant. Together with molecular evidence documenting novel diazotrophs in cooler euphotic zone waters [e.g., (Needoba et al., 2007;Moisander et al., 2010)], these observations imply that surface waters other than those in the warm, nutrient-poor oligotrophic gyres may support substantial rates of N 2 fixation, and that overlooking these potential diazotrophic contributions may compound uncertainties in the marine N budget, as well as modeled estimates of global marine diazotroph distributions and rates of N 2 fixation.
While the nascent case for significant N 2 fixation fluxes by mesopelagic diazotrophs is ambiguous, it is clear that N 2 fixation occurs in diverse benthic environments at significant rates in the presence of DIN concentrations in excess of 100 µM. Benthic N 2 fixation is peculiar in that it presents the strongest challenge to DIN inhibition of N 2 fixation, and because it often occurs in environments that also support high rates of N loss via denitrification and/or anammox. While traditionally it has been thought that benthic environments represent a net loss of bioavailable N from the marine ecosystem, previous work has shown that the net flux of N 2 gas to or from the sediments varies seasonally, and may be sensitive to environmental perturbations that may accelerate due to anthropogenic activities. These observations underscore the importance of developing and testing models of what controls benthic N 2 fixation (and denitrification) to generate more robust estimates of benthic N fluxes.
Our current understanding of the sensitivity of even the most well studied marine diazotrophs is incomplete, and we have considerably more to learn about diazotrophs that have only recently been identified using molecular tools. These are critical uncertainties to resolve if we are to understand how the marine N inventory can remain balanced on 100-1000 year time scales. Better constraints of diazotroph sensitivities will help us understand N cycle changes in the past, and to predict future changes as atmospheric carbon dioxide concentrations and temperatures increase and potentially stimulate N 2 fixation by Trichodesmium (Breitbarth et al., 2007;Hutchins et al., 2007;Levitan et al., 2007;Ramos et al., 2007;Levitan et al., 2010), if not other diazotrophs as well.