BGBiogeosciencesBGBiogeosciences1726-4189Copernicus PublicationsGöttingen, Germany10.5194/bg-16-167-2019Warming effect on nitrogen fixation in Mediterranean macrophyte sedimentsNitrogen fixation in Mediterranean macrophytesGarcias-BonetNeusneus.garciasbonet@kaust.edu.sahttps://orcid.org/0000-0001-6469-7167Vaquer-SunyerRaquelhttps://orcid.org/0000-0003-4507-0531DuarteCarlos M.MarbàNúriahttps://orcid.org/0000-0002-8048-6789King Abdullah University of Science and Technology, Red Sea Research
Center, Thuwal 23955-6900, Saudi ArabiaDepartment of Global Change Research, Institut Mediterrani d'Estudis Avançats, IMEDEA
(CSIC–UIB), 07190 Esporles, Mallorca,
SpainNeus Garcias-Bonet (neus.garciasbonet@kaust.edu.sa)17January20191611671758August201815August201823November201817December2018This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/This article is available from https://bg.copernicus.org/articles/16/167/2019/bg-16-167-2019.htmlThe full text article is available as a PDF file from https://bg.copernicus.org/articles/16/167/2019/bg-16-167-2019.pdf
The Mediterranean Sea is warming faster than the global ocean, with important
consequences for organisms and biogeochemical cycles. Warming is a major
stressor for key marine benthic macrophytes. However, the effect of warming
on marine N2 fixation remains unknown, despite the fact that the high
productivity of macrophytes in oligotrophic waters is partially sustained by
the input of new nitrogen (N) into the system by N2 fixation. Here,
we assess the impact of warming on the N2 fixation rates of three key
marine macrophytes: Posidonia oceanica, Cymodocea nodosa,
and Caulerpa prolifera. We experimentally measured N2
fixation rates in vegetated and bare sediments at temperatures encompassing
current summer mean (25 and 27 ∘C), projected summer mean (29 and
31 ∘C), and projected summer maximum (33 ∘C) seawater
surface temperatures (SSTs) by the end of the century under a scenario of
moderate greenhouse gas emissions. We found that N2 fixation rates
in vegetated sediments were 2.8-fold higher than in bare sediments at current
summer mean SST, with no differences among macrophytes. Currently, the
contribution of N2 fixation to macrophyte productivity could
account for up to 7 %, 13.8 %, and 1.8 % of N requirements for
P. oceanica, C. nodosa, and C. prolifera,
respectively. We show the temperature dependence of sediment N2
fixation rates. However, the thermal response differed for vegetated
sediments, in which rates showed an optimum at 31 ∘C followed by a
sharp decrease at 33 ∘C, and bare sediments, in which rates increased
along the range of the experimental temperatures. The activation energy and
Q10 were lower in vegetated than bare sediments, indicating the lower
thermal sensitivity of vegetated sediments. The projected warming is expected
to increase the contribution of N2 fixation to Mediterranean
macrophyte productivity. Therefore, the thermal dependence of N2
fixation might have important consequences for primary production in coastal
ecosystems in the context of warming.
Introduction
Global mean surface temperatures increased 0.85 ∘C from 1880 to
2012 and are projected to increase between 1 and 3.5 ∘C by the end
of the 21st century relative to preindustrial times (IPCC, 2013).
Similarly, heat waves are more frequent since the second half of the 20th
century in Europe, Asia, and Australia (IPCC, 2013; Perkins et al., 2012).
Oceans store most of the accumulated heat in the biosphere, warming at an
average rate of 0.11 ∘C per decade at the surface (up to 75 m of depth)
since 1970 (IPCC, 2013), with longer and more frequent marine heat waves over
the last century (Oliver et al., 2018). Warming is larger in small and
enclosed basins such as the Mediterranean Sea (Vargas-Yáñez et al.,
2008), which is warming at 0.61 ∘C per decade (Belkin, 2009), with
rapid migration of marine isotherms (Burrows et al., 2011) and increased
thermal extremes (Diffenbaugh et al., 2007). Specifically, the maximum
surface seawater temperature (SSTmax) in the Balearic Islands, in
the western Mediterranean Sea, is projected to increase by 3.4±1.3∘C by 2100 under a scenario of moderate greenhouse gas (GHG)
emissions (A1B scenario from the IPCC Special Report on Emissions Scenarios,
equivalent to the RCP6.0 scenario of the IPCC Fifth Assessment Report)
(Jordà et al., 2012), with important consequences for marine organisms
and ecosystems.
Seagrass ecosystems provide important ecosystem services, such as the
increase in diversity, the reduction of wave action, the protection of
the coast, the increase in water clarity by trapping suspended particles, and
climate change mitigation by acting as carbon sinks (Costanza et al., 1997;
Duarte, 2017; Fourqurean et al., 2012). In the Mediterranean Sea, the most
relevant seagrass species are Posidonia oceanica, an endemic
long-living seagrass, and Cymodocea nodosa, commonly found in the
eastern Mediterranean Sea and on the northeastern Atlantic coast. Similarly,
benthic green macroalgae, such as the autochthonous Mediterranean
Caulerpa prolifera, form highly productive ecosystems contributing
to the atmospheric CO2 sequestration (Duarte, 2017). However, these
coastal vegetated ecosystems are threatened by climate change at the global scale
(Duarte et al., 2018) and at the Mediterranean Sea scale (Marbà et al.,
2015). In particular, warming increases the mortality rates of P. oceanica (Marba and Duarte, 2010), which is predicted to be functionally
extinct by 2049 to 2100 due to warming (Chefaoui et al., 2018; Jordà et
al., 2012). Mesocosm experiments showed that C. nodosa is more
resistant to warming than P. oceanica (Olsen et al., 2012),
concurrent with thermal niche models (Chefaoui et al., 2016,
2018); however, a loss of 46.5 % in C. nodosa extension is
predicted by 2100 under the worst-case warming scenario (Chefaoui et al.,
2018). Although C. prolifera thrives well in warm waters, its
photosynthesis is inhibited at temperatures above 30 ∘C (Lloret et
al., 2008; Vaquer-Sunyer and Duarte, 2013), compromising its survival at
temperatures above this threshold.
Warming also affects metabolic processes driving biogeochemical cycles in
coastal benthic ecosystems. Warming enhances sediment sulfate reduction rates
(Robador et al., 2016), leading to an increase in sulfide accumulation in
coastal bare sediments (Sanz-Lázaro et al., 2011) and seagrass-colonized
sediments (Koch et al., 2007). In the Mediterranean Sea, heat waves and
warming trigger sulfide intrusion in P. oceanica shoots (García
et al., 2013), which has toxic effects on plant meristems (Garcias-Bonet et
al., 2008) and increases shoot mortality (Calleja et al., 2007).
Sanz-Lázaro et al. (2011) found that warming enhances sediment oxygen
uptake and CO2 emissions in coastal sediments, boosted by the
addition of labile organic matter, in a mesocosm experiment. Similarly,
warming together with eutrophication have been identified as main drivers of
hypoxia in a Mediterranean macroalgae Caulerpa prolifera meadow
(Vaquer-Sunyer et al., 2012). However, studies on the effect of warming on
atmospheric nitrogen fixation in coastal marine ecosystems are lacking.
Nitrogen (N2) fixation plays a fundamental role in balancing
nutrient budgets at the basin scale in the Mediterranean Sea, with most of
this N2 fixation associated with P. oceanica seagrass
meadows (Béthoux and Copin-Montégut, 1986). Indeed, endophytic
nitrogen-fixing bacteria have been detected in roots of P. oceanica
(Garcias-Bonet et al., 2012, 2016) and N2
fixation has been reported in leaves and roots of P. oceanica
(Agawin et al., 2016; Lehnen et al., 2016) and in situ incubations (Agawin
et al., 2017). Therefore, the high productivity of P. oceanica in
the oligotrophic Mediterranean waters is partially supported by N2
fixation. However, the magnitude of N2 fixation rates in the
rhizosphere of P. oceanica is still unknown, as is N2
fixation associated with other key macrophytes commonly found in the
Mediterranean Sea, such as C. nodosa and C. prolifera. In
addition, whether warming will affect N2 fixation rates is still
unknown.
Here, we test the hypothesis that N2 fixation rates in coastal
ecosystems is temperature dependent and will increase with the forecasted
warming. We do so by experimentally assessing the response of N2
fixation rates to warming in coastal Mediterranean vegetated ecosystems. We
focus specifically on the key macrophyte species most commonly found in the
Mediterranean Sea: two seagrass species (P. oceanica, C. nodosa) and one green macroalgae species (C. prolifera). We
experimentally measured N2 fixation rates in vegetated and bare
sediments at temperatures encompassing the current summer mean SST range (25 and
27 ∘C), the projected summer mean SST range (29 and 31 ∘C), and
projected summer SSTmax (33 ∘C) by the end of the
century under a scenario of moderate GHG emissions to assess (i) differences
between vegetated and bare sediments, (ii) differences among macrophyte
species, and (iii) the thermal dependence of sediment N2 fixation
rates.
Materials and methodsStudy site
The study was conducted with benthic communities sampled in Pollença Bay
(Mallorca, Spain), a bay located in the western Mediterranean Sea
(39∘53.792′ N, 3∘5.523′ E). The study site was selected
based on the coexistence of the three most commonly found macrophyte species
in the region, including two seagrasses (Posidonia oceanica and
Cymodocea nodosa) and one green macroalgae (Caulerpa prolifera). The three macrophytes grow close to each other in monospecific
patches at 5 m of depth. Mean (±SE) shoot density estimates were 699±444 and 604±136 shoot m-2 for P. oceanica and
C. nodosa, respectively (Marbà and Vaquer-Sunyer, unpublished).
The study was conducted in mid-June 2017 when in situ daily mean (±SE)
SST was 26.4±0.08∘C.
We sampled sediment colonized by these three macrophytes and the adjacent
bare sediment using sediment cores (50 cm in length and 4.5 cm in diameter).
We collected 16 sediment cores for each type of sediment. The vegetated
sediment cores were collected from the center of the macrophyte patches
between shoots or blades, collecting belowground plant material but avoiding
the collection of aboveground biomass. The bare sediment cores were collected
about 5 m away from the edge of the vegetated patches. We collected the
sediment samples by pushing the cores down into the sediment with the help of
a rubber hammer and carefully extracting at least 15 cm of undisturbed top
sediment. The cores were transported immediately to the laboratory. We
measured seawater salinity using a calibrated conductivity meter (ProfiLine
Cond 3310; WTW®, USA) and summer SST was
monitored and recorded in situ every 2 h from 2012 to 2017 using a Hobo
logger (Onset Computer Corporation®, MA,
USA). Full summer SST records are available for 2012, 2013, 2106, and 2017.
No data are available for 2015, and only partial temporal coverage is
available for 2014 (Fig. S1 in the Supplement). The summer mean (±SE)
SST varied from 26.29±0.05∘C in 2013 to 27.03±0.04∘C in 2017, with an average summer mean SST of 26.54±0.17∘C from 2012 to 2017. Average summer minimum and maximum SST
were 22.92 and 29.08 ∘C, respectively. The highest maximum SST was
29.67 ∘C and was registered in August 2017 (Table S1 in the
Supplement).
Nitrogen fixation rates
We measured sediment N2 fixation rates in an acetylene reduction
assay (Capone and Taylor, 1980) in P. oceanica, C. nodosa,
and C. prolifera vegetated sediments and the adjacent bare sediment
at five incubation temperatures: 25, 27, 29, 31, and 33 ∘C. The 25
and 27 ∘C temperature treatments represent the current summer mean
SST, covering the in situ recorded average summer mean SST of 26.54±0.2∘C (25 % percentile = 25.81 ∘C and 75 %
percentile = 27.61 ∘C) from 2012 to 2017. The 29 and
31 ∘C temperature treatments represent the range of the projected
summer mean SST by the end of the century under a scenario of moderate GHG
emissions equivalent to RCP6.0 by applying the projected mean SST increase of
2.8±1.1∘C in the region (Jordà et al., 2012) over the
summer mean SST registered in 2017 (27.03 ∘C). The 33 ∘C
temperature treatment represents the projected summer SSTmax by
the end of the century under a scenario of moderate GHG emissions equivalent
to RCP6.0 by applying the projected SSTmax increase of 3.4±1.3∘C (Jordà et al., 2012) over the summer SSTmax
of 29.67 ∘C already recorded in 2017. The sediment incubations were
run in five water baths (i.e., one per temperature treatment) equipped with
thermometers and heaters located in a stable temperature room. The target
temperature for each water bath was maintained using an IKS-AQUASTAR system,
which controlled and recorded the temperature every 10 min. During the
incubations, the temperature oscillation around the target temperatures
ranged from 0.3 to 0.7 ∘C, and the temperature accuracy was ±0.05∘C on average (Table S2).
Once in the laboratory, the sediment from the cores was extruded carefully
using a plunger, and the first 10 cm of sediment below the surface was
collected; the rest of the sediment in the core was discarded. For each
replicate, 80 mL of sediment together with the belowground biomass present
was placed in a 500 mL glass bottle. No aboveground biomass was included in
the incubation glasses. Then, we added 200 mL of autoclaved seawater and the
bottles were closed with a lid fitted with a gas-tight valve. Finally, we
added 20 mL of acetylene-saturated seawater through the gas-tight valve of
each bottle in order to achieve a final acetylene concentration of 4 mM. The
acetylene-saturated seawater was prepared according to Wilson et al. (2012).
We ran the sediment incubations in triplicate for each type of sediment and
each temperature treatment under dark conditions. The incubations lasted 24 h,
starting after the addition of acetylene-saturated seawater. We sampled the
headspace five times: at the start of the experiment and at 12, 17, 20, and
24 h since the onset of the experiment. Specifically, we withdrew 3 mL of
air from the headspace with a gas-tight syringe. The headspace air sample was
immediately injected into a 3 mL vacuum vial for further analysis of
ethylene concentration on a gas chromatographer equipped with a flame
ionization detector (FID-GC; Agilent 5890) using a PoraPLOT U GC column
(25 m × 0.53 mm × 20 µm; Agilent Technologies,
USA). We built a calibration curve using three ethylene standards of known
concentration (1.02, 10.13, and 99.7 ppm) and helium as a balance gas,
supplied by Carburos Metálicos S.A. (Palma de Mallorca, Spain). We
estimated the concentration of dissolved ethylene from the ethylene
concentration in the equilibrated air as described previously (Wilson et al.,
2012) and applied the solubility coefficient of ethylene according to
Breitbarth et al. (2004) as a function of temperature and
salinity.
We ran negative controls consisting of sediment without the addition of
acetylene-saturated seawater in order to confirm that ethylene was not
naturally produced by our samples and autoclaved seawater used in the
preparation of the incubations with the addition of acetylene-saturated seawater
in order to confirm that ethylene was not produced in the seawater. No
ethylene was produced in any of the negative controls. The ethylene production
rates were converted into N2 fixation rates by applying the common
ratio of 3 mol of acetylene to 1 mol of N2 (Welsh, 2000).
At the end of the incubation, we dried the sediment samples at 60 ∘C
and recorded the dry weight for further calculations. Moreover, we calculated
the sediment organic matter (OM) content of each replicate sediment sample by
loss on ignition (Dean Jr., 1974). The sediment N2 fixation rates
were first calculated by sediment dry weight and then standardized to surface
area integrated over 10 cm of sediment depth by taking into account the
sediment bulk density.
Statistical analysis
Differences in sediment OM content and bulk density among P. oceanica, C. nodosa, and C. prolifera vegetated sediments
and bare sediment were tested with the nonparametric Kruskal–Wallis test.
Differences in sediment N2 fixation rates among the four types of
sediment (P. oceanica, C. nodosa, and C. prolifera
vegetated sediments and bare sediment) were tested by Friedman test matching
by temperature treatment. Then, we tested the effect of temperature (as a
categorical explanatory variable with three levels: current summer mean SST range
(25 and 27 ∘C), projected summer mean SST range (29 and
31 ∘C), and projected summer SSTmax (33 ∘C))
and type of sediment (as a categorical explanatory variable with two levels:
vegetated and bare sediments) on sediment N2 fixation rates (our
response variable) after a log transformation to meet normality requirements
by a full factorial two-way ANOVA. Finally, differences in sediment
N2 fixation rates between vegetated and bare sediments were tested
by a nonparametric Mann–Whitney U test at three different temperature
ranges: current summer mean SST range (25 and 27 ∘C), projected
summer mean SST range (29 and 31 ∘C), and projected summer
SSTmax (33 ∘C). Moreover, we tested the thermal
dependence of sediment N2 fixation rates in vegetated and bare
sediments by fitting the Arrhenius function to estimate the activation energy
(Ea), derived from the linear regression between the natural
logarithm of N2 fixation rates and the inverse of the temperature
multiplied by the Boltzmann constant (Dell et al., 2011), and Q10, the
relative rate of increase in N2 fixation expected for a
10 ∘C temperature increase (Raven and Geider, 1988). The Q10
was calculated using the following equation (Raven and Geider, 1988):
Q10=e10EaRT2,
where R is the gas constant (8.314472 mol-1 K-1), T is the
mean absolute temperature across the range over which Q10 was measured
(K), and Ea is the activation energy (J mol-1). The activation
energy and Q10 of N2 fixation in vegetated sediments were
calculated using the increasing rates measured at four temperature treatments
(25, 27, 29, and 31 ∘C), while the declining rates measured at
33 ∘C were not included. The full range of temperature treatments
was used for bare sediments since no decline was detected. All statistical
analyses were performed using JMP (SAS Institute Inc., USA) and PRISM
(GraphPad Software Inc., USA) statistical software.
Organic matter content and bulk density in sediments colonized by
different macrophyte species and bare sediment in Pollença Bay (Mallorca)
in June 2017. Mean values (±SEM), ranges (minimum–maximum
values), and the sample size (N) are shown.
Sediment OM content was significantly different in the sediments colonized by
different macrophyte species (χ3,562=50.33, p<0.0001). Posidonia oceanica sediments had the highest OM content
(13.34±0.56 %), whereas bare sediments had the lowest OM content
(0.44±0.50 %, Table 1). Sediment bulk density differed among
sediment types (χ3,562=46.02, p<0.0001; Table 1).
Average N2 fixation rates in bare sediments were 0.06±0.01 nmol N g DW-1 h-1
(range from 0.01 to 0.09), while
average N2 fixation rates in vegetated sediments were 3-fold
greater at 0.19±0.03 nmol
N g DW-1 h-1 (range from 0.05 to 0.9), pooling all temperature treatments together.
Within the vegetated sediments, the maximum mean N2 fixation rate
was detected in C. prolifera (0.22±0.05 nmol N g DW-1 h-1), whereas the minimum mean
N2 fixation rate was measured in C. nodosa (0.15±0.04 nmol N g DW-1 h-1). The mean N2 fixation rate in
P. oceanica was 0.21±0.06 nmol N g DW-1 h-1.
Nitrogen fixation rates differed among the four different sediment types
(i.e., bare, P. oceanica, C. nodosa, and C. prolifera sediments) (χ3,562=10.68, p=0.005) when expressed
by sediment dry weight. However, once the rates were converted into aerial
basis, these differences were no longer significant (χ3,562=6.12, p>0.05) due to high variability in sediment bulk
densities. Sediment N2 fixation rates were independent of OM
content (linear regression, dfN= 1, dfD= 58, Pearson's
r= 0.19, p>0.05).
Nitrogen fixation rates in aerial basis were significantly higher in
vegetated sediments compared to bare ones (U=154, p<0.002)
when pooling all temperature treatments together, with sediments colonized by
macrophytes supporting, on average, twice the nitrogen fixation rate as
bare sediments (mean ± SE =3.86±0.53 and 1.77±0.20 mg
N m-2 d-1, respectively), considering all temperature treatments.
Temperature and type of sediment (vegetated and bare sediments) had a
significant effect on N2 fixation rates (two-way ANOVA; sediment
type F1,59=10.40, p<0.01; temperature F2,59=4.89, p<0.05), with no significant
interaction between them. Specifically, at the current summer SST range
(25–27 ∘C), N2 fixation rates in vegetated sediments
(3.15±0.48 mg N m-2 d-1) were significantly higher (U=
13, p<0.01) than those in bare sediments (1.14±0.3 mg
N m-2 d-1) (Fig. 1a). Similarly, at the projected summer mean SST
range (29–31 ∘C), N2 fixation rates in vegetated
sediments (5.25±1.17 mg N m-2 d-1) were significantly
higher (U=23, p<0.05) than the rates measured in bare
sediments (2.18±0.2 mg N m-2 d-1) (Fig. 1b). However,
N2 fixation rates did not differ between vegetated and bare
sediments at projected summer SSTmax (33 ∘C), with
N2 fixation rates of 2.49±0.248 and 2.21±0.15 mg
N m-2 d-1, respectively (Fig. 1c).
Box plot of the N2 fixation rates (expressed by area) of
sediments colonized by different macrophytes (as well as grouping all
vegetated sediments together) and bare sediment measured at the current summer
SST range (25–27 ∘C, a), the projected summer mean SST range
by 2100 under RCP6.0 scenario (29–31 ∘C, b), and the projected
summer SSTmax by 2100 under RCP6.0 (33 ∘C, c).
Boxes extend from the 25th to 75th percentiles, whiskers are calculated using the
interquartile distance (IQR) according to the Tukey method, lines inside
boxes represent the median, “+” represents the mean, and dots represent
individual values greater than the 75th percentile plus 1.5 X IQR.
Statistically significant differences are indicated by asterisks;
* indicates p<0.05 and ** indicates p<0.01.
The sample size (N) is also indicated.
In vegetated sediments, N2 fixation rates increased linearly with
temperature up to 31 ∘C (N2 fixation (nmol N g
DW-1 h-1)=-0.63+ 0.03 × temperature, R2=0.11, p<0.05), with a marked
decrease from 0.32±0.09 nmol N g DW-1 h-1 at
31 ∘C to 0.11±0.01 nmol N g DW-1 h-1 at
33 ∘C (Fig. 2). Nitrogen fixation rates in bare sediments increased
linearly with temperature up to 33 ∘C (N2 fixation (nmol
N g DW-1 h-1)=-0.11+ 0.01 × temperature, R2=0.51, p<0.005, Fig. 2). The associated activation energies
were 0.91±0.39 and 1.25±0.39 eV for vegetated and bare
sediments, respectively (Fig. 3). The associated Q10 values were 3.84±2.22 and 6.41±2.97 for vegetated and bare sediments, respectively.
Relationship of experimental incubation temperature and mean
sediment N2 fixation rates (expressed by sediment dry weight) in
vegetated (black dots) and bare sediments (open dots). Black and open dots
indicate mean values, and errors bars indicate standard error of the mean.
Individual replicate measurements of N2 fixation rates for each
macrophyte species are also shown in colored-coded dots; green dots
represent measurements on P. oceanica sediments, blue dots represent
measurements on C. nodosa sediments, and pink dots represent
measurements on C. prolifera sediments.
Discussion
The overall average N2 fixation rate found in Mediterranean
vegetated sediments at current summer mean SST (3.15±0.48 mg
N m-2 d-1) is within the range of rates reported for
sediments colonized by temperate seagrass species (from 1.2 to 6.5 mg N m-2 d-1 in Zostera marina sediments in the North Sea
(McGlathery et al., 1998) and on the northwest Atlantic coast (Capone, 1982)
and from 0.1 to 7.3 mg N m-2 d-1 in Zostera noltii on
the northeast Atlantic coast; Welsh et al., 1996). However, N2
fixation rates are lower than the rates reported for tropical and subtropical
seagrass species (see references in Welsh, 2000). The overall N2
fixation rates in vegetated sediments are higher than in bare sediments,
consistent with the long-recognized role of marine plants in enhancing
N2 fixation rates (Capone, 1988). Specifically, the vegetated
sediments supported 3- to 4-fold higher N2 fixation rates than bare
sediments at the current summer mean SST range when expressed by area and by
sediment dry weight, respectively.
Arrhenius plot for N2 fixation rates in vegetated (black
dots) and bare sediments (grey dots), showing the linear regression between
ln N2 fixation rates and the inverse of the temperature multiplied
by the Boltzmann constant (1/kT) for
vegetated (black solid line) and bare (grey solid line) sediments.
The N2 fixation rates we measured in P. oceanica sediments
at current summer mean SST (2.86±1.26 mg N m-2 d-1) are
higher than the rates reported in summer at similar seawater temperature by
Agawin et al. (2017) using benthic bell-jar chambers containing P. oceanica shoots and the underlying sediment (ranging from 0.06 to 1.51 mg
N m-2 d-1). However, the different methodological approaches make
comparisons difficult: while sediment slurries might slightly overestimate
rates due to sediment structure disturbance and increases in organic matter
availability, incubation chambers might underestimate rates due to poor
diffusion of acetylene into the sediment (Welsh, 2000). Nevertheless, the
N2 fixation rates in bare sediments at current summer mean SST
(1.14±0.31 mg N m-2 d-1) are very similar to those
measured by benthic bell-jar in bare sediment adjacent to a P. oceanica meadow (from 0.01 to 1.99 mg N m-2 d-1) (Agawin et
al., 2017), suggesting that these differences in N2 fixation rates
in P. oceanica sediment might also be due to variability among
sites. The N2 fixation rates in C. nodosa and C. prolifera sediments reported here are the first reports, to the best of our
knowledge, for these two important Mediterranean macrophyte species. Indeed,
the analysis of sediment N stocks in a C. nodosa meadow in the
Mediterranean Sea suggested that N2 fixation might be contributing
to enhancing the N stocks compared to bare sediments (Pedersen et al., 1997).
The similar stable N isotope composition of C. nodosa tissues and
those of P. oceanica in the Mediterranean (Fourqurean et al., 2007)
also suggests that they use similar sources of nitrogen. The N2
fixation rates at current summer mean SST in C. prolifera sediments
found here (0.17±0.04 nmol N g DW-1 h-1) are similar to
the sediment N2 fixation rates associated with the invasive
C. taxifolia in Monaco (0.12±0.09 nmol
N g DW-1 h-1) but 20-fold lower than the N2 fixation
rates reported for C. taxifolia in France (3.96±1.99 nmol
N g DW-1 h-1) (Chisholm and Moulin, 2003).
Although the sediments colonized by these three macrophyte species hold
similar rates, the contribution of sediment N2 fixation to the
productivity of each plant is different. Taking into account the average net
production (2.63 and 1.47 g DW m-2 d-1 for P. oceanica
and C. nodosa, respectively (Duarte and Chiscano, 1999), and
5.16 g DW m-2 d-1 for C. prolifera, Marbà,
unpublished) and the tissue nitrogen content (from 1.55 % to 1.63 %
for P. oceanica, from 1.91 % to 2.28 % for C. nodosa (Duarte, 1990; Fourqurean et al., 2007), and from 3 % to 4.9 %
for C. prolifera; Morris et al., 2009), the mean measured sediment
N2 fixation rates detected at current summer mean SST (25 and
27 ∘C) could account for 6.7 % to 7 %, 11.5 % to
13.8 %, and 1.1 % to 1.8 % of the nitrogen requirements for
P. oceanica, C. nodosa, and C. prolifera,
respectively. The calculated contribution of N2 fixation to
seagrass growth requirements falls within the range of the N2
fixation contributions reported for temperate seagrasses, ranging from
5 % to 12 % for Z. marina and Z. noltii,
respectively (Welsh, 2000). The calculated contribution of N2
fixation to fulfilling the macrophyte growth requirements points to N2
fixation as partially supporting the high productivity of these primary
producers in Mediterranean oligotrophic waters.
We experimentally demonstrate that N2 fixation in coastal sediments
is thermal dependent, both in vegetated and bare sediments. Despite the fact that a formal
experimental demonstration was lacking, the N2 fixation thermal
dependence reported here is in agreement with the higher rates typically
measured in warm tropical and subtropical meadows compared to the rates
reported in temperate and cold seagrass systems (Herbert, 1999; McGlathery,
2008; Welsh, 2000). The thermal dependence, as reflected by the activation
energy and Q10, for N2 fixation rates was, however, higher in
bare sediments than in vegetated sediments, possibly due to different
bacterial communities. Westrich and Berner (1988) also found that sulfate
reduction exhibited a more pronounced thermal dependence in sediments
supporting lower rates. The activation energies for N2 fixation in
vegetated sediments (0.91±0.4 eV or 87.8±37.6 KJ mol-1)
and in bare sediments (1.25±0.4 eV or 120.6±37.6 KJ mol-1) are within the range of the activation energy reported
for sediment sulfate reduction (range from 36 to 132 KJ mol-1; Robador
et al., 2016; Westrich and Berner, 1988) and for sediment organic matter
degradation (range from 54 to 125 KJ mol-1; Middelburg et al., 1996).
The Q10 values associated with sediment N2 fixation (3.84±2.22 and 6.41±2.97 for vegetated and bare sediments, respectively) are
higher than those reported for sediment sulfate reduction (from 1.6 to 3.4; Robador et al., 2016),
but still similar to values associated with
organic matter degradation (from 2.2 to 6.3; Middelburg et al., 1996).
Moreover, the thermal response differed for vegetated sediments, in which
N2 fixation rates showed an optimum at 31 ∘C, followed by a
sharp decrease at 33 ∘C, and bare sediments, in which N2
fixation rates increased along the range of experimental temperatures tested
here. The thermal response of N2 fixation in vegetated sediments
found here is similar to the thermal response reported for N2
fixation in soil crusts (Zhou et al., 2016) and seagrass rhizosphere
(Garcias-Bonet et al., 2018), with an increase in rates up to 30 and
29 ∘C, respectively, and a marked decrease in rates at temperatures
above the optimum. The forecasted warming by the end of the century could
potentially increase N2 fixation rates by 36.7 % in vegetated
sediments and 46.8 % in bare sediments. However, the decrease in
N2 fixation rates in vegetated sediments at 33 ∘C would
imply a reduction of a third in the contribution of N2 fixation to
the macrophyte productivity during heat waves. The forecasted warming could
also affect other biogeochemical processes in coastal sediments, such as
sulfate reduction (Robador et al., 2016), anaerobic ammonium oxidation, and
denitrification (Garcias-Bonet et al., 2018; Nowicki, 1994), among others,
and therefore potential synergic or antagonistic effects may occur.
Responses of sediments colonized by different macrophyte species may also
differ due to differences in the lability of their OM and nutrient stocks
associated with differences in C : N : P ratios (Enríquez et al.,
1993; Lanari et al., 2018). Similarly, losses of seagrass coverage by
heat waves could lead to an increase in CO2 emissions by increased
remineralization of C stocks (Arias-Ortiz et al., 2018).
The thermal dependence of N2 fixation in vegetated sediments found
here might have important consequences for primary production in coastal
ecosystems in the context of warming. This may not be the case for P. oceanica, as this species is projected to be critically compromised to the
extent that functional extinction is possible, with projected Mediterranean
warming rates by 2050–2100 (Chefaoui et al., 2018; Jordà et al., 2012).
However, in order to draw general conclusions on the effect of warming on
N2 fixation in coastal ecosystems, the thermal dependence found
here needs be tested for a diversity of seagrass ecosystems. Similarly, our
results from experimental temperature treatments did not account for
potential acclimation and adaptation of microbial communities to warming,
which should also be tested. Moreover, N2 fixation is likely to be
subjected to other environmental controls that may change, either in an
additive, synergistic, or antagonistic manner, with warming, so predicting
N2 fixation rates in a future warmer coastal ocean remains
challenging.
Conclusions
Mediterranean macrophyte meadows are sites of intense N2 fixation
rates twice as high as those in adjacent bare sediments. As these rates
increase with warming, actual warming of the Mediterranean Sea is expected
to lead to enhanced sediment N2 fixation rates, with future
warming leading to further increases in N2 fixation rates up to
33 ∘C in bare sediments and 31 ∘C, followed by a decrease at
higher temperatures in vegetated sediments. However, more work covering a
larger area is needed to confirm a generalized warming effect on sediment
N2 fixation.
The dataset is provided in the attached Supplement
(Table S3).
The supplement related to this article is available online at: https://doi.org/10.5194/bg-16-167-2019-supplement.
NGB, RVS, CMD, and NM designed the study. NGB and RVS performed the fieldwork
and sample and data analysis. NGB, RVS, CMD, and NM interpreted the
results. NGB wrote the first draft of the paper. All authors contributed
substantially to the final paper.
The authors declare that they have no conflict of
interest.
Acknowledgements
This work was funded by the Spanish Ministry of Economy, Industry and
Competitiveness (Medshift project, CGL2015-71809-P) with baseline funding
allocated by the King Abdullah University of Science and Technology to CMD. We
thank Carlos Alex Morell Lujan Williams for field assistance and Maria
Trinidad García Barceló for lab support. RVS was supported by a
Juan de la Cierva incorporación contract (ref. IJCI-2015-23163).
Edited by: Clare Woulds
Reviewed by: Carmen B. de los Santos and one anonymous referee
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