Nitrogen fixation in temperate oceans is a potentially important, but poorly
understood process that may influence the marine nitrogen budget. This study
determined seasonal variations in nitrogen fixation and the diazotroph
community within the euphotic zone in the temperate coastal region of the
northwestern North Pacific. Nitrogen fixation as high as 13.6 nmol N L
The amount of bioavailable nitrogen introduced into the global ocean via
nitrogen fixation is considered to be roughly balanced at the large
spatiotemporal scale by nitrogen loss through denitrification, as indicated
by the sedimentary nitrogen isotope record during the Holocene epoch
(Brandes and Devol, 2002; Deutsch et al., 2004). However, rate measurement
data have revealed that denitrification far exceeds nitrogen fixation
(Codispoti, 2007). This discrepancy in the nitrogen balance has raised the
possibility that the current estimate of marine nitrogen fixation, which is
primarily based on data collected in tropical and subtropical oceans where
large cyanobacterial diazotrophs (e.g.,
The temperate coastal ocean is one of the regions where nitrogen fixation
rates have been understudied and potentially underestimated. Conventionally,
nitrogen fixation in temperate oceans has been assumed to be low because of
the relatively low temperatures (<
This study examined the seasonal variation in nitrogen fixation along two onshore–offshore transects in the interfrontal zone of the northwestern North Pacific. In this temperate region, physical, chemical, and biological properties vary widely between seasons (Shiozaki et al., 2014b) due to the confluence of three currents: the Kuroshio (warm current), the Tsugaru Warm Current, and Oyashio (cold current). Data on nitrogen fixation rates in the temperate Pacific are limited (Needoba et al., 2007), and to the best of our knowledge, the present study is the first to examine diazotrophy during all seasons in the temperate ocean. This study was conducted as part of a project to monitor the dynamics of the coastal ecosystem and the recovery thereof after the 2011 Tohoku-Oki Tsunami, which struck the region on 11 March 2011.
The experiments were conducted during six cruises in the temperate coastal
region of the western North Pacific. These cruises covered a full seasonal
cycle, including spring (KS-14-2_Mar, 14–19 March 2014),
early summer (KK-13-1_Jun, 24–29 June 2013), mid-summer
(KT-12-20_Aug, 7–12 August 2012), late summer
(KK-13-6_Sep, 14–21 September 2013), fall
(KT-12-27_Oct, 15–22 October 2012), and winter
(KT-13-2_Jan, 19–25 January 2013). Sampling stations were
located along the transect lines OT (39
Sampling locations in the northwestern North Pacific Ocean.
Temperature, salinity, and dissolved oxygen profiles of regions near the bottom floor were measured using a SBE 911-plus conductivity–temperature–pressure (CTD) system (Sea-bird Electronics, Bellevue, WA, USA). Water samples were collected in an acid-cleaned bucket and Niskin-X bottles. At offshore stations, samples for nutrient analysis were collected from 7–15 different depths in the upper 200 m, while at shallower (< 200 m) bay stations, samples were collected from 4–9 different depths in the entire water column, except at Station (Stn.) OT1 where only surface water samples were collected. Samples for DNA analysis and incubation experiments were collected from the surface at almost every station, and from depths corresponding to 10 and 1 % of the surface light intensities at Stns. OT4 and ON5. Light attenuation was determined using a submersible PAR sensor.
Samples for nutrient analysis were stored in 10 mL acrylic tubes and kept
frozen until onshore analyses. Nitrate, nitrite, ammonium, and phosphate
concentrations were determined using an AACSII auto-analyzer (Bran
Nitrogen fixation was determined by the
To examine the possibility of underestimation of nitrogen fixation as
determined by the bubble method (Mohr et al., 2010; Großkopf et al.,
2012), we compared the nitrogen fixation rates determined using the
To examine if sugar addition affected nitrogen fixation rates (Bonnet et
al., 2013; Rahav et al., 2013; Moisander et al., 2011), we determined
nitrogen fixation rates (the bubble method, see above) for surface seawater
samples (stations ON4 and OT6 during the KS-14-2_Mar cruise)
with and without addition of mannitol (final conc. 0.8
Pearson's correlation coefficient was used to examine the relationships
between nitrogen fixation activities and environmental variables including
temperature, nitrate, ammonium, phosphate, and the ratio of nitrate
Samples (0.38–1 L) for DNA analysis were filtered through 0.2
Summary of recovered
Numbers in parentheses indicate the number of sequences with > 97% similarity at the amino acid level to terrestrial diazotroph sequences.
The clone library analysis showed that UCYN-A,
Nitrogen fixation rates determined by the bubble and dissolution methods
were compared during the KK-13-6_Sep and
KS-14-2_Mar cruises (Fig. 2). Both methods failed to detect
nitrogen fixation in samples collected during the KS-14-2 cruise. During the
KK-13-6_Sep cruise, the nitrogen fixation rates determined by
the dissolution method were significantly higher (1.5–2.2 fold) than those
determined by the bubble method at Stns. OT6 and ON5 (
Nitrogen fixation rates estimated simultaneously by the
According to the temperature–salinity (TS) diagram proposed by Hanawa and Mitsudera (1987), both the offshore and bay waters collected during this investigation mostly belonged mostly to either the surface layer water system (SW) or the Tsugaru Warm Current water system (TW) (Fig. 3). Exceptions included the waters collected from the 1 % light depth (119 m) at Stn. ON5 during the KT-13-2_Jan cruise (classified as the Oyashio water system (OW)) and those collected at the surface of OT5 during the KS-14-2_Mar cruise (classified as the Coastal Oyashio water system (CO)). These water classifications based on the TS diagram were generally consistent with the geostrophic current field of the investigated region (Fig. S1). Based on these results, it was assumed that surface waters collected during the same cruise in a particular season generally belonged to the same water system that was prevalent in the investigated region at the time of our sampling.
Temperature–salinity diagram at each sampling point. The water classification was defined by Hanawa and Mitsudera (1986). SW, KW, TW, OW, and CO denote the surface layer water system, Kuroshio water system, Tsugaru Warm Current water system, Oyashio water system, and Coastal Oyashio water system, respectively.
Sea surface temperatures (SSTs) (range of 1.5–24.3
Average
During the four cruises conducted in early summer (KK-13-1_Jun), mid-summer (KT-12-20_Aug), late summer
(KK-13-6_Sep), and fall (KT-12-27_Oct),
nitrogen fixation was measurable in most of the samples collected from
surface waters: the nitrogen fixation rates varied in the range of
0.33–13.6 nmol N L
Nitrogen fixation rates were determined for samples collected from different
depths (0–119 m) at Stns. OT4 and ON5 (Fig. 5). Nitrogen fixation was
detected in surface and deeper layers during four cruises conducted in early
summer (KK-13-1_Jun), mid-summer (KT-12-20_Aug),
late summer (KK-13-6_Sep), and fall (KT-12-27_Oct) (Fig. 4). Nitrogen fixation rates tended to be higher at the surface
than in the deeper layers during mid-summer (KT-12-20_Aug) and
late summer (KK-13-6_Sep (at Stn. OT4)), whereas this
vertical trend was less evident during fall (KT-12-27_Oct)
and early summer (KK-13-1_Jun). At Stn. OT4, nitrogen
fixation was detected even in the layers below the nitracline
(KT-12-27_Oct, depth
Time-series variations in the vertical profiles of temperature
[
Nitrogen fixation rates tended to increase with temperature (
Pearson's correlation matrix of N
Relationship between nitrogen fixation [nmol N L
Nitrogen fixation rates were negatively correlated with nitrate and
phosphate concentrations (
PCR reagents have been suggested to be a potential source of
The
The recovered cyanobacterial sequences belonged to
The
Average abundances of
Time-series variations in the vertical profiles of
Nitrogen fixation rates were measurable mainly from early summer to fall
when nitrate was generally depleted in sample seawaters, although there were
some exceptions. Our estimates of the nitrogen fixation rates
(0.33–13.6 nmol N L
In our study, spatiotemporal variability in nitrogen fixation rates appeared to be partly related to the Tsugaru Warm Current path. This current, which flows from the north (after passage through the Tsugaru Strait) to the study region (Fig. S1), may carry active diazotrophs and therefore enhance nitrogen fixation in our study region. This is supported by the fact that nitrogen fixation rates during individual cruises tended to be higher at Stn. OT4 than at Stn. ON5. These stations were located up- and down-stream of the Tsugaru Warm Current, respectively. In addition, variations in nitrogen fixation rates among stations and seasons might also be related to the extent of vertical mixing in the Tsugaru Warm Current. It has been suggested that vertical mixing may introduce iron-rich subsurface water to the surface of the Tsugaru Strait (Saitoh et al., 2008). Such input of iron may enhance nitrogen fixation rates. Consistent with this notion, our results showed that the nitrogen fixation rate was relatively high at Stn. OT4, where the nitracline was relatively deep.
Blais et al. (2012) proposed that nitrogen fixation can occur even in nutrient-replete waters, if large amounts of iron and organic materials are available for consumption by bacterial diazotrophs. In the present study, this possibility was examined by conducting mannitol addition experiments using surface seawaters collected during spring. These waters, which belong to the Oyashio Current system (Nishioka et al., 2007, 2011; Shiozaki et al., 2014b), were considered to be rich in iron during spring, as indicated by a previous study (iron conc., 0.79–8.46 nM; Nishioka et al. 2007). Despite potentially high iron concentrations, our results showed that nitrogen fixation was undetectable even after the mannitol addition, suggesting that, contrary to the Blais et al. proposition, diazotrophs remained inactive under our experimental settings.
Our data showed that nitrogen fixation rates were below the detection limit
during winter, spring, and late summer (KK-13-6_Sep), when
nitrate concentrations were high. These results were consistent with the
results of previous studies in the Pacific Ocean, which indicated that
nitrogen fixation rates were low or undetectable in DIN-replete waters
(Shiozaki et al., 2010). In contrast, Mulholland et al. (2012) reported
that, in temperate regions of the Atlantic Ocean, nitrogen fixation rates
were high even in DIN-replete (> 1
The qPCR analysis demonstrated that the target groups were quantifiable even
at stations at which their sequences were not recovered by the clone library
analysis, suggesting that the number of clones was not sufficient to capture
the diazotroph community structure on each cruise. Despite this limitation,
the sequences more frequently recovered in the clone library generally
corresponded to the most abundant group revealed by the qPCR analysis. For
example, UCYN-A was frequently recovered in the library during the
KT-12-20_Aug, KK-13-1_Jun, and
KK-13-6_Sep cruises; for these samples, the qPCR results
showed that UCYN-A was the most abundant group among the four examined.
Similarly, qPCR data indicated that
UCYN-A was detected in all seasons except spring (KS-14-2_Mar),
suggesting that this group of diazotrophs could be important agents of
nitrogen fixation in this region. Especially from early to late summer, the
abundance of UCYN-A was generally higher than that of
We detected
UCYN-B was not detected except at one station. This result is consistent
with previous knowledge. UCYN-B becomes abundant with increasing
temperature, similar to
In nitrate-rich water during winter and spring, Cluster III diazotrophs were
detected at most of the stations. Furthermore, from early summer to fall,
Many
This study demonstrated that nitrogen fixation can and does proceed at high
rates, depending on the season, in the temperate coastal region of the
northwestern North Pacific, although we failed to detect nitrogen fixation
in DIN-replete cold waters.
T. Shiozaki, T. Nagata, and K. Furuya designed the experiment and T. Shiozaki collected the samples at sea. T. Shiozaki determined nitrogen fixation and nutrient concentrations and analyzed satellite data sets. T. Shiozaki and M. Ijichi conducted the genetic analyses. T. Shiozaki prepared the manuscript with contributions from all co-authors.
We acknowledge K. Kogure, K. Hamasaki, A. Tsuda, Y. Tada, R. Fujimura, R. Kaneko,
H. Takasu, T. Yokokawa, K. Seike, and T. Kitahashi for their
assistance in the sample collection and analysis. We thank the captains,
crewmembers, and participants on board the R/V