Significance of N 2 fixation in dissolved fractions of organic nitrogen

Introduction Conclusions References Tables Figures


Introduction
Over the last three decades, the global budget of oceanic fixed nitrogen (NO − 3 , NO − 2 , NH + 4 , particulate organic nitrogen (PON), and dissolved organic nitrogen (DON)) has been studied extensively to investigate the primary production in the ocean (e.g., Wada et al., 1975;Codispoti and Christensen, 1985;Gruber and Sarmiento, 1997;Brandes and Devol, 2002).The total fixed nitrogen is predominantly controlled by the total influx of fixed nitrogen through N 2 fixation and by the total outflux of fixed nitrogen through denitrification (Codispoti et al., 2001;Brandes and Devol, 2002;Deutsch et al., 2004).
The two most commonly used incubation methods to estimate the N 2 fixation rate are the 15 N 2 tracer method and the C 2 H 2 reduction method.Because the former method is used to measure the 15 N uptake rate from 15 N 2 to the PON fraction (Montoya et al., 1996), the estimated N 2 fixation rate may be underestimated if a considerable amount of N is released into the DON fraction during cultivation (Bronk and Glibert, 1991;Glibert and Bronk, 1994).Glibert and Bronk (1994) performed culture experiments using Trichodesmium spp.and found that the rates of release of fixed N release into the DON fractions accounted for 50% of the total N 2 fixation rates on an average.Therefore, in addition to PON, one should determine the initial and final values of the concentration and nitrogen isotopic composition (δ 15 N=( 15 N/ 14 N) sample /( 15 N/ 14 N) AirN 2 −1) of DON in each incubation bottle in order to estimate the total N 2 fixation rates using the 15 N 2 tracer method.However, it is difficult to determine the δ 15 N value of DON in natural samples using the conventional elemental analyzer isotope ratio mass spectrometry (EA-IRMS) techniques (Mulholland et al., 2004;Meador et al., 2007).
On the other hand, the C 2 H 2 reduction method is used to measure the C 2 H 4 production rate through the reduction of C 2 H 2 by nitrogenase (Capone, 1993).However, this method requires the use of a conversion factor to convert the observed C 2 H 2 reduction rates to N 2 fixation rates.While the theoretical reduction ratio of C 2 H 2 :N 2 is 3:1 (mol:mol) (Montoya et al., 1996;Postgate, 1998), little evidence has been found to support the accuracy of this ratio under natural conditions Figures

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Printer-friendly Version Interactive Discussion (Mulholland et al., 2006;Tsunogai et al., 2008).Nitrogenase-dependent H 2 evolution, which is inhibited by C 2 H 2 , results in deviations from this theoretical stoichiometry (Robson and Postgate, 1980;Mulholland et al., 2006Mulholland et al., , 2007)).Therefore, the conversion factor is generally determined using the same field measurements by comparing the N 2 fixation rate calculated using both the C 2 H 2 reduction method and 15 N 2 -labeled PON method.Although Capone and Montoya (2001) recommended a conversion factor of 4, Mulholland et al. (2006) showed that the factor was variable with values ranging from 3.7 to 15.7 even in experiments conducted on a daily basis.Furthermore, it is impossible to estimate total N 2 fixation rates using the conversion factors estimated by the 15 N-labeled PON method.Therefore, it is impossible to estimate the quantitative values of the total N 2 fixation rates by the C 2 H 2 reduction method.
The only way to solve the above mentioned problem is to determine the δ 15 N values of DON and PON during 15 N 2 tracer incubation.Recent developments in highsensitivity δ 15 N analysis of organic nitrogen have now enabled us to determine the δ 15 N values of DON (Tsunogai et al., 2008).This is the first report on the estimation of the total N 2 fixation rates in the ocean, including the DON fractions.

Sampling and methods
Both the collection and incubation of water samples were performed onboard the R/V Co. Ltd., Tokyo, Japan) was injected into each bottle using a gas-tight syringe.The bottles were gently shaken and then incubated in thermostatic baths on a deck covered with screens to simulate the in situ temperature and light intensity for periods ranging from 12 to 72 h.Although the duration of incubation was variable, the incubation was mainly performed during diurnal periods (24, 48, or 72 h) to avoid the bias caused by the day-night cycle on the N 2 fixation rate.However, two samples were incubated for three different periods of 12, 36, and 60 h during the KH06-2 expedition and may have been biased by the day-night cycle.Therefore, we corrected the bias by adopting the relative variations between 36 and 12 h (corresponding to 24 h incubation), or those between 60 and 12 h (corresponding to 48 h incubation).
Immediately after incubation, the suspended particles (PON fraction) in each incubated water sample were collected on a pre-combusted ( 450• C for 4 h) Whatman GF/F filter (pore size=0.7 µm) by gentle vacuum filtration.The pressure difference was strictly controlled to be <100 mm Hg to avoid the leakage of small particles from the filters.The <0.7-µm filtrate (herein considered to be the DON fraction) was collected in a light-resistant polyethylene bottle (100 mL) and frozen until analysis.The suspended particles collected on the filter were further washed with filtered clean seawater, placed in a plastic case, frozen instantaneously, and stored in a deep freezer (−80  et al. (2008).This method involves the oxidation/reduction methods such as the oxidation of PON or DON to nitrate using persulfate (Knapp et al., 2005;Tsunogai et al., 2008), reduction of nitrate to nitrite using spongy cadmium, and further reduction of nitrite to nitrous oxide using sodium azide.The total recovery rate of N was around 90% for the samples.The blank level was <10 nmol N for PON (corresponding to Figures

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Printer-friendly Version Interactive Discussion 0.02 µmol N L −1 when the filtrate volume was 500 mL) and <1.0 µmol N L −1 for DON.
All the data presented herein had already been corrected for the blank contributions.
The standard deviation of the sample measurements was less than 0.3‰ for samples containing more than 50 nmol N, and less than 0.5‰ for those containing more than 20 nmol N.For the DON samples, we also needed to correct by subtracting the contributions of nitrate, nitrite, and ammonium.However, we neglected these contributions because their concentrations, which were quantified by using an AutoAnalyzer (AACS II; Bran+Luebbe), were low (mostly below detection levels) at the studied sites (Table 1).The concentrations of PON and DON ranged from 0.11 to 0.60 µmol N L −1 and from 4.0 to 7.0 µmol N L −1 , respectively (Table 2).Details will be discussed elsewhere.
The total N 2 fixation rate was calculated for each incubation bottle using the results for both the concentration and δ and agitated three times at 4800 rpm for 50 s.The samples were incubated at 70 • C for 60 min.After incubation, the XS buffer was transferred to a 1.5-mL microtube, vortexed for 10 s, and placed on ice for 30 min.Cell debris was removed by centrifugation at 15 000 g at 5 • C for 15 min; the supernatants were then decanted into another 1.5-mL microtube with the same amount of isopropanol.The samples were incubated at room temperature for 10 min, and the precipitated DNA was pelleted by centrifugation at 15 000 g for 15 min at 5 • C. Isopropanol was decanted, and the DNA pellets were washed with 70% ethanol, vacuum dried, and resuspended in 100 µL of 10 mM Tris-HCl (pH 8.5).The obtained samples were stored at −20 • C until further analysis.
Quantitative PCR (qPCR) assays targeting partial nifH fragments were carried out with a Thermal Cycler Dice Real Time System (TP800; TaKaRa) using primers and TaqMan probes designed by Church et al. (2005); they determined five nifH phylotypes including the cyanobacteria Crocosphaera spp.(termed Group B), an uncultivated phylotype termed Group A that was presumed to be a unicellular cyanobacterium, Trichodesmium spp., heterocystous cyanobacteria, and g-proteobacteria in the North Pacific Ocean.For each set of primers and probes set, standard curves were derived using duplicate or triplicate serial dilutions of linearized pUC18 plasmids (TaKaRa) containing the positive control insert.The number of molecules of a plasmid was estimated from the amount of DNA according to the equation derived by Short and Zehr (2005).
The PCR amplification mixture solution (25 µL) contained 12.5 µL of Premix Ex Taq those stations during the late summer expeditions (Table 1).However, the column-integrated quantities of PO 3− 4 were also small at Stns. 5, 6, and 7 (KH06-2) during the early summer expedition, where active spp.during early summer.Therefore, explanations other than the limited availability of nutrients may be required to explain active N 2 fixation during the early summer.Owing to the ability to store P, Trichodesmium spp. is active for a few months after PO 3− 4 deficiency (Thompson et al., 1994;Moutin et al., 2005).Therefore, during early summer, the N 2 fixers were using the stored P supplied during winter or spring, while they may exhaust the stored P during late summer when we found the direct links between P-availability and the N 2 fixation rate.In conclusion, the estimated values of N 2 fixation rates for the PON fractions were representative of those observed in the western North Pacific area during each season.The δ 15 N values of initial PON also supported our conclusions (Table 2).The lower δ 15 N values of PON (+0‰ and +0.9‰; Stns. 1 and 5 during the KH06-2 expedition, +0.8‰ and +1.5‰; Stns.16 and 22 during KH08-2 expedition) were found at the station where the higher N 2 fixation rate in the PON fractions was observed.Because the PON derived from N 2 fixation had nearly 0‰ of δ 15 N derived from atmospheric N 2 (Minagawa and Wada, 1986;Carpenter et al., 1997;Montoya et al., 2002), the geographical variations in δ 15 N of PON also support the significant N 2 fixation in the PON fractions.

N 2 fixation rates in the DON fractions
The areal N 2 fixation rates estimated in this study for the DON fractions (ranging from <0.5 to 54 µmol N m −2 d −1 ) accounted for 50% (ranging from <10 to 84%) of the total N 2 fixation rates on average (Table 2).Glibert and Bronk (1994) also estimated that the rates of N release into the DON fractions could account for 50% on average of the total N 2 fixation rates in cultured Trichodesmium spp.Furthermore, Mulholland et al. (2006) estimated the rates of release of fixed nitrogen into DON fractions in the ocean on the basis of the discrepancies in the N 2 fixation rates estimated by the 15 N 2 tracer method and the C 2 H 2 reduction method for the same samples, and they found the DON fraction to comprise 52% (ranging from 9.1% to 81%) of the total N 2 fixation Introduction

Conclusions References
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Full Screen / Esc Printer-friendly Version Interactive Discussion by using the theoretical conversion factor of 3 for the C 2 H 2 reduction method.Both the average N 2 fixation rate for the DON fractions within the total N 2 fixation rate and the range of the variation estimated in this study corresponded well with the estimated rate and variation in past studies.The estimated N 2 fixation rates in the DON fractions estimated in this study may be highly reliable for estimating those in the ocean.
The discrepancies in the estimates for the N 2 fixation rates between the 15 N 2 tracer method and C 2 H 2 reduction method have been noted in the western North Pacific region as well.Using the C 2 H 2 reduction method, Kitajima et al. (2009) found high N 2 fixation rates of 0.5-12 nmol N L −1 d −1 in the western North Pacific region; this range is more than twice as high as that estimated for the PON fractions in this study (0.4-4.7 nmol N L −1 d −1 during the KH06-2 expedition) based on the 15 N 2 tracer method.
It is difficult to attribute the differences in the estimations to the seasonal variations since both experiments were performed during the same early summer season (May to June).Because the C 2 H 2 reduction method resulted in higher N 2 fixation rates in comparison with the 15 N 2 tracer method for the PON fractions when the N 2 fixation in the DON fractions was significant, the systemic difference between the estimates obtained by the two different methods for the same region implies that the N 2 fixation rates for the DON fractions were almost as significant as those for the PON fractions.The δ 15 N values of initial DON also support our conclusions.Within the whole δ 15 N values of initial DON in surface water (ranging from +5.5 to +8.8‰, Table 2), which agree with those reported in a previous study in the Central Pacific region (Meador et al., 2007)

Mechanisms of N 2 fixation in DON fractions
The significant N 2 fixation observed in the DON fractions can be explained by the following two mechanisms: (1) active N 2 fixation in the DON fraction by small plankton such as bacterioplankton and/or picoplankton and (2) active secondary release of N into the DON fractions from the PON fractions, which includes recently fixed nitrogen such as viral cell lysis (Hewson et al., 2004), grazing (O'Neil et al., 1996), cell death (Berman-Frank et al., 2004), or direct release of N-compounds (Glibert and Bronk, 1994).
In particular, we focused on the mechanisms of the large N 2 fixation rate in the DON fractions at high latitudes (Fig. 3).In several previous studies, it has been observed that γ-proteobacteria in waters are characterized by being both cooler in temperature and richer in nutrients than waters where the usual cyanobacterial N 2 fixers are dominant (Bird et al., 2005;Langlois et al., 2005;Church et al., 2008).In particular, Church et al. (2008) found that the nifH gene was actively expressed in γ-proteobacterial phylotypes at stations far north up to 44 • N in the north Eastern Pacific region.Because bacterioplankton and/or picoplankton having sizes ranging from ∼0.2 to ∼2 µm could pass through the GF/F filter (pore size: 0.7 µm) and mix with the DON fractions, they could cause active N 2 fixation in the DON fractions.The abundance of nifH gene copies determined by the quantified PCR method, however, indicated that the dominant N 2 fixer at stations at high latitudes and showing active N 2 fixation in DON fractions was Trichodesmium spp.(Fig. 4).Although we could not directly compare the number of nifH gene copies with the N 2 fixation rates (Zehr et al., 2007), the large N 2 fixation rates observed in the DON fractions at high latitudes could be attributed to active secondary release of N into the DON fractions from recently fixed nitrogen by Trichodesmium spp.Glibert and Bronk (1994)  fractions corresponded well with our results for the natural samples.However, further studies are essential to confirm the precise mechanisms of active N 2 fixation in DON fractions.

Implication for total N 2 fixation flux in ocean
In previous studies, the oceanic N 2 fixation rates were estimated to be in the range from 80-140 Tg N yr −1 on the basis of the data estimated by the 15 N 2 tracer method (Brandes et al., 2007); however, these estimates only accounted for the N 2 fixation rates in the PON fractions.Using the present data, we could estimate the total N 2 fixation rates more accurately by correcting the past estimates.Using a roughly average N 2 fixation of 50% in the DON fractions that were underestimated in previous studies over oceans worldwide, the revised N 2 fixation inputs should be increased to 160-280 Tg N yr −1 .Codispoti et al. (2001) estimated the total influx and outflux of fixed nitrogen to be 287 and 482 Tg N yr −1 , respectively, for the current global fixed nitrogen budget in oceans; in this budget, the outflux exceeds the influx by ∼200 Tg N yr −1 .The revised influx reduces the imbalance in the global fixed nitrogen budget.However, as observed during this study, the N 2 fixation rates in the DON fractions could be highly variable on different temporal and spatial scales.Further studies should be conducted to estimate the N 2 fixation rate in the DON fractions more accurately.

Conclusions
We found significant N 2 fixation signal in the < 0.7 µm fraction, typically characterized as dissolved organic nitrogen (DON) in the western North Pacific region; N 2 fixation in these fractions had been ignored in previous studies.As a result, N 2 fixation in the DON fractions accounted for 50% (ranging from < 10% to 84%) of the total N 2 fixation rates on an average.The abundance of nifH gene copies determined by quantified PCR method indicated that the large N 2 fixation rates observed in the DON fractions at high Full indicate that the values almost balance with the total fixed nitrogen budget.However, subsequent • C) until analysis.For quantitative polymerase chain reaction (qPCR) assays targeting partial nifH fragments, seawater (1 L) was filtered onto 25-mm Supor filters (pore size: 0.2 µm, Pall Corporation) under gentle vacuum (<100 mm Hg).The obtained filters were frozen in a deep freezer (−80• C) until analysis.The concentrations and δ 15 N values of both PON and DON, including those incubated under 15 N 2 addition, were analyzed using the method developed by Tsunogai 15 N values of PON/DON.15N enrichment was clearly observed over time in most of the samples of PON and DON incubated under 15 N 2 addition (Fig.1); this result indicates that part of the recently fixed-nitrogen was transferred into DON pools during the incubation experiment.The vertical distributions of the N 2 fixation rates estimated from PON fractions during the KH06-2 expedition are shown in Fig.2.The profiles indicate that the N 2 fixation rates at the water surface were high and that these rates linearly decreased to nearly zero at depths of ca. 100 m.Therefore, we calculated the areal N 2 fixation rates by integrating the N 2 fixation rates on a volume from surface to 100 m depths, assuming linear attenuation toward zero with depths up to 100 m including the stations where the estimation was limited to the surface.If the increased δ 15 N values estimated from the incubation experiments were less than 2‰, we classified the N 2 fixation rates as less than the detection limit and presented the maximum value.DNA extraction was performed according to the method ofShort and Zehr (2005) with slight modifications.In brief, 600 µL of XS buffer (1% potassium ethyl xanthogenate; 100 mM Tris-HCl, pH 7.4; 1 mM EDTA, pH 8.0; 1% sodium dodecylsulfate; 800 mM ammonium acetate) and ca.0.2 g of 0.1-mm glass beads were added to the vials containing the filters.The vials were placed in a bead beater (BioSpec Products)

(
Perfect Real Time, TaKaRa), 0.05 µL of each primer (final conc.: 0.2 µM), 0.1 µL of the probe (final conc.: 0.4 µM), 11.3 µL of sterile Milli-Q water, and 1 µL of DNA template.In each qPCR run, environmental DNA and no template controls (i.e.sterile Milli-Q water) were also prepared in duplicate or triplicate.The thermal cycling reactions were carried out as follows: 95• C for 10 s, and 50 cycles of 95• C for 5 s followed by 60 rates in the PON fractionsThe areal N 2 fixation rates estimated from the PON fractions varied from <1-160 µmol N m −2 d −1 during the three expeditions undertaken in the western North Pacific (Table2).Using the traditional 15 N 2 tracer method for the PON fractions, Shiozaki et al. (2009) estimated the areal N 2 fixation rates in the western North Pacific region to be 29-152 µmol N m −2 d −1 in early spring.The areal N 2 fixation rates estimated from the PON fractions in the present study during the early summer expedition (KH06-2 cruise; 22-160 µmol N m −2 d −1 ) agreed well with those reported by Shiozaki et al. (2009).On the other hand, the areal N 2 fixation rates estimated during the late summer expeditions (KH07-2 and KH08-2 cruises; <20 µmol N m −2 d −1 ) were lower than those estimated during the early summer expeditions.The concentration of the sea surface chlorophyll-a was lower on the late summer expeditions (0.01 to 0.05 µg L −1 ) than during the early summer expeditions (0.07 to 0.21 µg L −1 ), indicating that the late summer expeditions coincided with the post-blooming season, when nutrients are limited.Thus, the observed difference in the areal N 2 fixation rates could be attributed to the seasonal variations in the N 2 fixation rates (Sa ñudo-Wilhelmy et al., 2001;Moutin et al., 2005).The lowest observed N 2 fixation rates at the two stations during the late summer expeditions (Stns.19 and 20, KH08-2) (Table2) could also be attributed to the lack of nutrients in post-blooming season, because the observed column-integrated quantities (from the surface to the depth of 100 m) of PO 3Stn.20) were the smallest for N 2 fixation (>22 µmol N m −2 d −1 ) was observed in the PON fractions.Moutin et al. (2005) also found few direct links between the P availability and accumulation of TrichodesmiumFigures , the lowest δ 15 N value of DON (+5.5‰;Stn.16 during the KH08-2 expedition) was found at the station where the highest N 2 fixation rate in the DON fractions was observed.Because the fixation of atmospheric N 2 (δ 15 N=0‰) produces organic nitrogen with δ 15 N values similar to atmospheric N 2 (Bourbonnais et al., 2009), the geographical variations in δ 15 N of DON also support the significant N 2 fixation in the DON fractions.In conclusion, the estimated significant N 2 fixation rates in the DON fractions represented those in the western North Pacific region.
also found that the rates of release of N from cultured Trichodesmium spp.into the DON fractions could account for 50% of the total N 2 fixation rates on an average.The release rate of N into the DON

Fig. 4 .
Fig. 4. Abundance of nifH gene copies (bar charts: left axis) and the N 2 fixation rates (line charts: right axis) during the KH08-2 expedition.The dark gray, white, light gray, and black bars denote the results for Trichodesmium spp., heterocystous diazotroghs, nanoplanktonic diazotrophs, and picoplanktonic diazotrophs, respectively.The circles and squares on the line charts represent the total N 2 fixation rates (µmol N m −2 d −1 ), and the rates in the DON fractions, respectively.

Table 1 .
attributed to active secondary N release processes to DON fractions from recently fixed-nitrogen by Trichodesmium.The new total N 2 fixation flux including N 2 fixation in DON fractions has possibility to double the original estimates; therefore, the revised influx may reduce the imbalance in the global oceanic fixed nitrogen budget.Introduction Locations of sampling stations as well temperature (SST), salinity, and concentrations of chlorophyl-a and nutrients at surface (5 m depth).Introduction