GLOBAL BIOGEOCHEMICAL CYCLES, VOL. 26, GB3022, doi:10.1029/2011GB004096, 2012
Nitrogen fixation and identification of potential diazotrophs in the Canadian Arctic Marjolaine Blais,1,2 Jean-Éric Tremblay,1 Anne D. Jungblut,3,4 Jonathan Gagnon,1 Johannie Martin,1 Mary Thaler,5 and Connie Lovejoy5 Received 20 April 2011; revised 27 July 2012; accepted 29 July 2012; published 7 September 2012.
[1] Global gaseous nitrogen (N2) fixation rates may be underestimated and data is lacking from many regions without conspicuous diazotrophic cyanobacteria, such as cold oceans. We estimated N2 fixation rates at diverse sites in the Canadian Arctic, including the mouth of the Mackenzie River, the offshore Beaufort Sea, Lancaster Sound, Baffin Bay and a river influenced fjord. We also identified potential diazotrophic communities using a targeted survey of the nifH gene. Nitrogen fixation rates ranged from 0.02 nmol N L1 d1 in Baffin Bay to 4.45 nmol N L1 d1 in the Mackenzie River plume. Sequences recovered from the nifH gene survey belonged mainly to Cluster III, a group of nifH sequences associated with diverse microorganisms, with some a- and g-proteobacteria nifH genes at most sites. Cyanobacteria nifH genes with best matches to Nostocales, which are common in Arctic freshwaters, were recovered from the marine Beaufort Sea. The geographic pattern of N2 fixation rates and nifH gene identities suggest that the Mackenzie River is the source of a diazotrophic community that contributes new nitrogen to the nitrogen-depleted surface waters of the Beaufort Sea. This first record of N2 fixation at high latitudes refines our understanding of the global nitrogen budget. Citation: Blais, M., J.-É. Tremblay, A. D. Jungblut, J. Gagnon, J. Martin, M. Thaler, and C. Lovejoy (2012), Nitrogen fixation and identification of potential diazotrophs in the Canadian Arctic, Global Biogeochem. Cycles, 26, GB3022, doi:10.1029/2011GB004096.
1. Introduction [2] Global estimates of nitrogen fluxes indicate that the oceanic nitrogen cycle is not in balance [Codispoti et al., 2001]. Over long-time scales, the main source of biologically available nitrogen to the ocean is gaseous nitrogen (N2) fixation by diazotrophic (N2-fixing) cyanobacteria. Conversely, the major loss of this fixed nitrogen from the ocean to the atmosphere is mediated by other bacteria and occurs via denitrification, which ultimately releases N2 after the dissimilatory reduction of nitrate (NO 3 ), or via the anaerobic oxidation of ammonium (NH+4 ) [Ward et al., 2007]. Estimates of denitrification, in both the water column and in seafloor sediments, are globally greater than N2 fixation rates 1 Département de Biologie, Quebec-Océan, and Takuvik, Université Laval, Quebec, Quebec, Canada. 2 Now at Institut des Sciences de la Mer de Rimouski, Université du Quebec à Rimouski, Rimouski, Quebec, Canada. 3 Centre d’Études Nordiques and Département de Biologie, Université Laval, Quebec, Quebec, Canada. 4 Now at Department of Botany, Natural History Museum, London, UK. 5 Département de Biologie, Quebec-Océan, Takuvik, and Institut de Biologie Intégrative et des Systèmes, Université Laval, Quebec, Quebec, Canada.
Corresponding author: J.-É. Tremblay, Département de Biologie, Québec-Océan, and Takuvik, Université Laval, 1045 Ave. de la Médecine, Quebec, QC G1V 0A6, Canada. (
[email protected]) ©2012. American Geophysical Union. All Rights Reserved. 0886-6236/12/2011GB004096
implying that the ocean is losing nitrogen to the atmosphere [Brandes and Devol, 2002; Mahaffey et al., 2005; Codispoti, 2007; Gruber and Galloway, 2008]. [3] A substantial portion of global denitrification occurs in the sediments of the wide shallow shelves that dominate the western Arctic seascape [Devol et al., 1997; Chang and Devol, 2009]. The ensuing loss of nitrogen to the atmosphere is thought to lower the N:P ratio of Pacific origin waters that transit through the Beaufort Sea and eventually enter the North Atlantic Ocean [Yamamoto-Kawai et al., 2006]. The low molar N:P ratio, which is below nine at the onset of the growing season in the southeast Beaufort Sea [Tremblay et al., 2008], could favor diazotrophy in the western Arctic Ocean, in the same manner as further south, in the Atlantic Ocean [Karl et al., 2002; Voss et al., 2004; Yamamoto-Kawai et al., 2006]. Nitrogen input from N2 fixation could also explain the continued drawdown of dissolved inorganic carbon and soluble reactive phosphorus following the depletion of NO 3 in the surface mixed layer of the Beaufort Sea [Tremblay et al., 2008]. [4] The most conspicuous diazotrophs in the ocean are cyanobacteria and include Trichodesmium spp. and several uncultivated groups, which mostly occur in tropical and subtropical oceans [Goebel et al., 2010; Moisander et al., 2010] and at low temperate latitudes [LaRoche and Breitbarth, 2005; Needoba et al., 2007; Langlois et al., 2008]. Diazotrophic cyanobacteria are rare in polar marine waters [Vincent, 2000; Tremblay et al., 2009; Lovejoy et al., 2011] and N2 fixation seems unlikely within the Arctic Ocean.
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However, bacteria other than cyanobacteria found in coastal and estuarine environments [Moisander et al., 2008; Foster et al., 2009; Rees et al., 2009] and in some parts of the open ocean [Falcón et al., 2004; Church et al., 2008; Langlois et al., 2008] have the capacity to fix N2. [5] To our knowledge, N2 fixation has never been reported from the Arctic Ocean and only once north of 30 N in the coastal temperate Atlantic Ocean [Mulholland et al., 2012]. Any N2 fixation in the Arctic Ocean would alter the current paradigm of the nitrogen balance between Pacific and Atlantic oceans and explain some of the discrepancy between input and removal estimates of fixed nitrogen. Nitrogen fixation could also potentially influence regional new production, which is the portion of primary production supported by allochthonous nitrogen and balanced by vertical carbon export under steady state conditions [Eppley and Peterson, 1979]. We addressed this data gap by combining rate measurements and nifH gene surveys to assess the potential for N2 fixation in the Arctic Ocean.
2. Materials and Methods 2.1. Sampling [6] This study was carried out aboard the Canadian icebreaker CCGS Amundsen in the coastal (Mackenzie Shelf ) and offshore Beaufort Sea (19 July to 3 August 2008 and 31 July to 13 August 2009) and in Lancaster Sound and Baffin Bay (7–25 September 2008) (Figure 1). Stations that were too shallow for the ship in the Mackenzie River plume and Kugmallit Bay were accessed by helicopter or barge and the surface samples were collected directly into a clean carboy. At all other stations, water was collected with a rosette sampler equipped with twenty-four 12-L Niskin type bottles (OceanTest Equipment Inc.), a conductivity-temperaturedepth (CTD) profiler (SBE-911, Sea-Bird Inc.), a fluorometer (Seapoint Sensors Inc.) and a sensor measuring photosynthetically available radiation (PAR; QCP2300, Biospherical Instruments Inc.). [7] Samples for N2 fixation assays and molecular analyses in 2008 were collected from the surface or ≈5 m when collected with the rosette sampler. In 2009, N2 fixation assays were conducted on samples collected from ≈5 m and the subsurface chlorophyll maximum (SCM, between 30 and 57 m) to investigate diazotrophic potential deeper in the water column. The seawater was passed through a 200 mm Nitex mesh to eliminate large particles and zooplankton and then collected into 20 L acid-washed carboys that had been rinsed 3 times with sample water prior to filling. 2.2. Nitrogen Fixation Assays [8] The 15N2 tracer method was selected since it is more sensitive than the acetylene (C2H2) reduction method and is preferred for oligotrophic systems with low diazotrophic activity [Montoya et al., 1996]. In 2008, water for the N2 fixation assays was immediately dispensed into 250 mL clear glass bottles, which were filled to overflowing to avoid air bubbles. A gas-tight syringe (SGE Analytical Science) was used to inject 1 mL of 15N2 (≥98 atom% 15N; Cambridge Isotope Laboratories) through twin-valve bottle caps, leading to an enrichment of ca. 16–20 atom%. Immediately after 15N2 addition, T0 bottles were filtered under gentle vacuum (0.7 mm size fraction generally accounted for >90% of the total N2 fixation at both depths sampled for estuarine and marine stations of the Beaufort Sea (Table 4). 3.3. Temperature Effect [21] In 2009, N2 fixation rates in the estuarine samples were higher at 7 C compared to 2 C. The slope of the linear regression of N2 fixation differed significantly from unity (t = 4.679, df = 11, p = 0.001, conformity T-test, Systat 11.0) (Figure 4). The corresponding Q10, which is the rate of change of a biological reaction following a 10 C increase in temperature, was estimated to be 1.44 using: Q10 ¼ 1:2ð10=T2T 1Þ
b
Averages and standard deviations (parentheses) were calculated using all stations. Bottom depth is indicated. c Data not analyzed due to high turbidity of samples. d Cyano = Cyanobacteria. e Only one station sampled. The value in parentheses is the standard error of replicates.
-c 0.21 0.65 (0.11) 0.50 0.29 (0.18) 0.42 -c 3.76 0.85 (0.44) 0.14 0.29 (0.16) 0.09 1b 4b 46 (5) 15 21 (5) 44.9 4.40 10.30 28.63 (1.31) 31.17 30.55 (1.00) 30.13 19.4 15.0 7.0 (1.2) 0.2 2.1 (1.8) 1.5 Mackenzie River Kugmallit Bay Beaufort Seaa Lancaster Sound Baffin Baya Gibbs Fjord
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toward Baffin Bay; Gibbs Fjord values were relatively higher than those of offshore Baffin Bay (Table 1). Within the Beaufort Sea region, cyanobacteria concentrations were extremely low with values of ca. 400 cells ml1 in the Mackenzie River plume and less than 100 cell ml1 elsewhere (Table 1). concentrations [18] Surface temperatures and NO 3 concentrations decreased while surface salinity and PO3 4 increased from the Mackenzie River plume to the offshore Beaufort Sea in 2008 (Table 1). Moreover, CTD data from 2009 revealed that vertical PAR attenuation was much greater on the shallow shelf (stations with bottom depth 98% similar to Nostoc commune Vaucher (UTEX 584) [Wright et al., 2001], a terrestrial (freshwater) species. N2 fixation has been recorded in Arctic Nostocales dominated microbial mats [Vincent, 2000] and Waleron et al. [2007] also recovered sequences belonging to Oscillatoriales with closest affinities to freshwater species in the marine Beaufort Sea, but not to those found in the Baltic Sea or saline lakes. Alternatively, windblown cyanobacteria could be transported offshore [Harding et al., 2011]. The lack of cyanobacteria sequences from the river plume at the time of sampling could also be an artifact due to poor coverage in the libraries [Zehr and Capone, 1996].
Figure 5. Relative abundance of each bacterial nifH gene cluster retrieved for the Mackenzie River plume, Kugmallit Bay, marine Beaufort Sea, Baffin Bay and Gibbs Fjord. Sample size (n) corresponds to the number of correct nif H sequences retrieved in each clone library. 8 of 13
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Figure 6. Maximum likelihood phylogenetic trees of nifH-derived nucleotide sequences for a-proteobacteria. Clones recovered in this study are in bold type and number in brackets indicates the total number of sequences per OTU retrieved when more than one sequence was recovered. Relationships were bootstrapped 1000 times and values >50% are shown. The origin of environmental sequences is indicated in the name of the sequence. [31] Irrespective of the observation of nifH cyanobacteria sequences offshore, we did not recover cyanobacteria sequences from the estuarine Beaufort Sea and in Gibbs Fjord, where highest N2 fixation rates were recorded. Cluster III nifH genes are not common in brackish or marine surface water [Church et al., 2005; Langlois et al., 2005; Moisander et al., 2007]. However, Langlois et al. [2008] noted that Cluster III tended to be more abundant at the more northerly stations in the North Atlantic Ocean. Cluster III sequences retrieved here from the Arctic were not highly
similar to sequences recovered elsewhere from either oceanic or terrestrial biomes. Within Cluster III, most sequences retrieved in the estuarine Beaufort Sea clustered more closely with terrestrial-derived sequences while sequences from Baffin Bay clustered with brackish and marinederived sequences. Although less abundant, the a- and g-proteobacteria that commonly dominate heterotrophic diazotrophic assemblages in oceans [Falcón et al., 2004; Langlois et al., 2008] were also present at most stations. Interestingly, a- and g-proteobacteria phylogenies revealed
Figure 7. Maximum likelihood phylogenetic trees of nifH-derived nucleotide sequences for g-proteobacteria. Clones recovered in this study are in bold type and number in brackets indicates the total number of sequences per OTU retrieved when more than one sequence was recovered. Relationships were bootstrapped 1000 times and values >50% are shown. The origin of environmental sequences is indicated in the name of the sequence. 9 of 13
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Figure 8. Maximum likelihood phylogenetic trees of nifH-derived nucleotide sequences for Cluster III. Clones recovered in this study are in bold type and number in brackets indicates the total number of sequences per OTU retrieved when more than one sequence was recovered. Relationships were bootstrapped 1000 times and values >50% are shown. The origin of environmental sequences is indicated in the name of the sequence. that the estuarine Beaufort Sea sequences were closest to freshwater environmental sequences while the only marine g-proteobacteria sequence was recovered in Gibbs Fjord, which is less influenced by freshwater runoff compared to the Beaufort Sea region. 4.3. Mackenzie River Influence on N2 Fixation Rates [32] Overall, the majority of N2 fixation activity was detected in a region highly influenced by freshwater runoff, and was close to detection limits in most of the predominantly marine Baffin Bay. In the Beaufort region, the rapid decrease in diazotrophic activity away from sites influenced by the Mackenzie River, suggests that the river could be the source of active diazotrophs to the western coastal Canadian Arctic Ocean. The gene survey was consistent with this view but must be interpreted carefully. However, the exponential decrease of diazotrophic activity with increasing salinity away from the river cannot be explained strictly by the dilution of the river assemblage since such pattern should be linear. In the estuarine Beaufort Sea, where highest N2 fixation rates were recorded, all OTUs except one were
associated with non-cyanobacterial diazotrophs. The Mackenzie River is the largest single source of land-derived organic matter to the Arctic Ocean [O’Brien et al., 2006] and suspended organic material fuels high marine bacterial productivity in the coastal Beaufort Sea [Garneau et al., 2006]. Such a large input of organic matter could facilitate heterotrophic diazotrophy. Organic matter could also promote diazotrophy in the estuarine Beaufort Sea by its positive effect on the uptake of molybdenum [Howarth and Cole, 1985; Stal et al., 1999] and decreasing organic matter may contribute to the exponential decrease of N2 fixation rates away from the Mackenzie River plume. 4.4. Contribution of Arctic N2 Fixation to Global Nitrogen and Carbon Cycles [33] A mean annual denitrification rate of 1 mmol N m2 1 d has been estimated for the western Arctic shelf sediment of the Bering, Chukchi and Beaufort seas [Devol et al., 1997]. We estimate that the upper limit of N2 fixation rates integrated down to the SCM in the marine Beaufort Sea, assuming uniform rates from the surface to the SCM, could
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Figure 9. Maximum likelihood phylogenetic trees of nifH-derived nucleotide sequences for cyanobacteria. Clones recovered in this study are in bold type and number in brackets indicates the total number of sequences per OTU retrieved when more than one sequence was recovered. Relationships were bootstrapped 1000 times and values >50% are shown. The origin of environmental sequences is indicated in the name of the sequence. reach 0.0065 mmol N m2 d1, which is over 2 orders of magnitude less than denitrification rates. Clearly the Arctic Ocean plays a significant role in global denitrification [Devol et al., 1997; Chang and Devol, 2009] and its contribution to the reverse flux appears rather small. However, our results indicate that N2 fixation in the Arctic Ocean and surrounding seas should not be dismissed out of hand, especially considering the lack of measurements adjacent to the other major rivers that enter the Arctic Ocean. [34] The ecological importance of heterotrophic N2 fixation is well recognized in seagrass beds, where the release of newly fixed nitrogen by heterotrophic bacteria supports plant growth [Welsh, 2000]. A similar process could contribute to new phytoplankton production on Arctic shelves via direct release by diazotrophs or via the recycling of fixed nitrogen by bacteria and grazers. In the Beaufort Sea during early autumn 2002 and 2003, estimates of new primary production based on phytoplankton cell size ranged from 14 to 25 mg C m2 d1 [Brugel et al., 2009]. Applying a mean molar C:N ratio of 7.3 for this region [Tremblay et al., 2008], N2 fixation-based new primary production would be equivalent to 0.6 mg C m2 d1 in the surface layer of the marine Beaufort Sea. It would thus represent up to 4.3% of new primary production in the Beaufort Sea. The contribution of N2 fixation to new primary production within the western Canadian Arctic Ocean, and consequently to the export of carbon toward the deep sea, is presently small but not negligible.
5. Conclusions [35] This study demonstrates that substantial N2 fixation occurs on a coastal shelf of the High Arctic, an area thought to be unsuitable for diazotrophic organisms until now, and shows the potential for N2 fixation in the Arctic Ocean and adjacent seas. This input of fixed N may compensate partly
for sediment denitrification on shelves, thereby reducing the excess of phosphorus in Pacific-derived waters entering the Atlantic Ocean [Yamamoto-Kawai et al., 2006]. Even a small contribution of the Arctic Ocean to global N2 fixation could affect the oceanic N balance and the carbon cycle [Falkowski, 1997]. [36] The potential for N2 fixation in the Arctic Ocean has yet to be completely explored and its contribution to the nitrogen cycle requires further attention. The Russian Arctic seas are influenced by several major rivers and potentially contribute to N2 fixation in the eastern Arctic. Ongoing climate change causing increases in temperature [Intergovernmental Panel on Climate Change, 2007] and river discharge, notably of the Mackenzie River [YamamotoKawai et al., 2009], may favor N2 fixation in the Arctic Ocean and could eventually lead to a revised global N budget. This study also provided baseline information by which to assess future alteration of the nitrogen cycle in the rapidly changing Arctic Ocean. [37] Acknowledgments. This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the ArcticNet Network Center of Excellence (NCE) and is a contribution to the scientific program of Québec-Océan and three international polar year (IPY) research programs: Circumpolar Flaw Lead System Study (CFL), Canada’s Three Oceans (C3O) and Malina. Marjolaine Blais received a graduate scholarship from NSERC and financial support from the Northern Scientific Training Program. We thank the captains and crews of the CCGS Amundsen for their invaluable support in the field. We are indebted to Véronique Lago and Dominique Boisvert for the collection and analysis of physical data, to Mariane Berrouard for field logistics, to Jessie Motard-Côté and Tommy Harding for help during laboratory analysis, and to Michel Gosselin for access to pico- and nano-cyanobacteria data. We thank Jon Zehr and Michel Gosselin for insightful discussions and comments on an earlier draft of the manuscript.
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