Nitrous Oxide Release in Salt Marsh Ecosystems
Jennifer Reeve (Haverford College, 2014)
With guidance from: Anne Giblin (The Ecosystems Center, Marine Biological Laboratory), Julie
Huber (The Josephine Bay-Paul Center, Marine Biological Laboratory) and Victor Schmidt
(Brown-MBL Joint Program)
oxide, nitrate, methionine
2
Abstract
Denitrification and associated nitrous oxide fluxes were measured for sediments from 3
sites in the Plum Island Estuary and 2 sites in Great Sippewissett Marsh. The fluxes were
measured using whole-core incubations. The percentage of denitrification being lost as nitrous
oxide was lowest in the cores from sites with high nitrate levels. 8 additional cores were set up in
the lab with 4 treatments: a control, a nitrate treatment, a nitrate and methionine treatment, and a
nitrous oxide treatment. These cores were treated for 3 weeks before their consumption of
nitrous oxide was measured. The nitrate and methionine treatment consumed nitrous oxide faster
than the other three treatments, which consumed nitrous oxide at similar rates.
Introduction
Salt Marshes—Salt marshes are important sites of biogeochemical activity. They are
known to be highly productive ecosystems, and their roles as carbon sinks have been researched
extensively (Teal 1962, Kirwan 2012). Associated with these high levels of productivity are high
levels of nitrogen uptake. Salt marshes have been shown to remove upwards of half the terrestrial
inorganic nitrogen inputs (Seitzinger 1988). It is important to note that these marshes are known
to be highly influenced by the tides, and the chemical and physical changes brought with tides
can have large effects on elemental cycling in salt marshes (Dausse 2011).
Denitrification—Denitrification is one of the key microbial processes that take place in
salt marshes. Denitrification results in the creation of several intermediate nitrogen compounds.
This study looks specifically at nitrous oxide and its reduction. Nitrous oxide is of special
importance as a subject of study as it is a powerful greenhouse gas, approximately 300 times
more potent than carbon dioxide (IPCC 2001). Additionally, because nitrous oxide is a gas and
fairly insoluble in water, it is possible for nitrous oxide to be leaked to the atmosphere during
denitrification (Canfield et al. 2010). Understanding what influences the release of nitrous oxide
during denitrification is important due to the role of salt marshes in nitrogen processing.
The factors which increase the leakage of nitrous oxide during denitrification are not well
known. Eutrophication has been shown to increase nitrous oxide release in both ecosystem and
microcosm experiments (Seitzinger 1988). In general, it appears that higher nitrous oxide release
occurs when conditions are rich in nitrate and organic matter and low in oxygen (Moseman-
Valtierra 2011). This study looks more deeply into the controls on nitrous oxide release to gain a
better understanding of salt marshes as sinks or sources of nitrous oxide.
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different microbes within the environment. The first step in microbial denitrification is the
reduction of nitrate to nitrite. This step is carried out by dissimilatory nitrate reductases. The
nitrite is converted to nitric oxide by nitrite reductase (Nir). The nitric oxide is then reduced to
nitrous oxide by nitric oxide reductase (Nor). The final step in denitrification is to reduce nitrous
oxide to nitrogen gas. This is carried out by nitrous oxide reductase (Nos) (Canfield 2010).
The genes, nirK and nirS, usually used to analyze the occurrence of denitrification in an
environment encode for two different Nir enzymes, one containing a heme cytochrome and one
containing a copper cytochrome (Schreiber 2012). These genes are used because essentially all
denitrifiers have either nirK or nirS, however, only one-third have genes encoding for the Nos
enzyme, noted by the genetic subunit nosZ (Schreiber 2012). While this has the negative
consequence of generating nitrous oxide in the process of denitrification, it may be useful for
estimating nitrous oxide fluxes from environments by looking at the ratio of nosZ to (nirK +
nirS).
Another key aspect of the reduction of nitrous oxide is the effect that reduced sulfur
compounds have on the Nos enzyme. Studies conducted by C. Magalhães, et al. (2011 and 2012)
have shown that dimethylsulfoniopropionate (DMSP) and its breakdown products have
inhibitory effects on denitrification, specifically the reduction of nitrous oxide to nitrogen gas.
Sulfur compounds are very prevalent in salt marshes and this inhibitory effect could have a large
impact on the role of salt marshes as sinks or sources of nitrous oxide.
Hypotheses—More microbes are expected to be involved in nitrous oxide reduction at
sites with high levels of nitrogen, specifically nitrate and nitrous oxide, than at sites low in
nitrogen due to exposure to higher levels of nitrous oxide and nitrate. The ratio of nitrous oxide
reducing genes to nitrite reducing genes is expected to be higher in high nitrous oxide sites due
to this increased exposure to nitrous oxide. Sites high in sulfur compounds are expected to have
lower ratios of nitrous oxide reducing genes to nitrite reducing genes. Higher percentages of
denitrification are expected to be lost as nitrous oxide at sites with lower levels of nitrate. In-lab
treatments are expected to follow similar trends with treatments containing methionine, a
precursor to methanethiol, having relatively low abundances of nosZ genes and low rates of
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nitrous oxide consumption, and treatments containing nitrous oxide to have relatively high
abundances of nosZ and high rates of nitrous oxide consumption.
Methods
Sites—This study looked at 3 sites in the Plum Island Estuary (PIE) and two sites in the
Great Sippewissett Marsh. Both marshes are dominated by Spartina alterniflora and Spartina
patens. At PIE, one of the streams, Greenwood, has sewage inputs, while the other site, near the
Rowley Field House, lacks direct nitrogen inputs. Greenwood Site 2 is closer to the sewage
inputs than Greenwood Site 3. Near Greenwood Site 2 Phragmites australis is becoming the
dominant species. The Sippewissett sites are two of the plots involved in Ivan Valiela’s long-
term salt marsh fertilization experiment (Valiela et al 1976). One site, Sippewissett Unfertilized,
has seen no added nitrogen whereas the other, Sippewissett Fertilized, has had high levels of
organic nitrogen added to it for over 40 years.
Sampling—For the nutrient flux measurements from Sippewissett and PIE, cores were
collected from creek bottom sediment at each site. 6 cores were collected at Sippewissett, 3 from
Sippewissett Unfertilized and 3 from Sippewissett Fertilized (Fig. 1). 6 cores were collected at
PIE: 2 each from Greenwood Sites 2 and 3 and 2 from Rowley House (Fig. 2). Sediment samples
were taken for later molecular analysis from each site and frozen at -80°C. At both of the
Greenwood creek sites two 30mL syringes were filled with water from the creek. At the Rowley
House site, one 30mL syringe was filled with water. At both the Sippewissett sites one 30 mL
syringe was filled. In the lab these samples were used for three measurements: 10 mL was frozen
for nitrate analysis, 10 mL was acidified with 10 µL 5N HCl for ammonium analysis and 10 mL
was combined with 20 mL nitrogen gas for nitrous oxide analysis.
Core Incubations—In lab, the cores had the water on top of them removed and replaced
with filtered seawater. Each core was equipped with a stir bar and incubations were run in the
dark. For the nutrient flux measurements, the water was once again removed and replaced. For
the Sippewissett cores, two of the cores from each site received water spiked with nitrate to a
concentration of 100 µM and one core received normal filtered seawater. For the PIE cores, one
core received spiked water and one received normal filtered seawater. At the beginning of the
flux measurement time series the cores were sealed and all samples were removed via ports in
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the lid of the core. As water was being removed for sampling, water was added via a second port
in the lid. At each time point, each core had 10 mL removed for nitrous oxide measurements and
20 mL removed that was filtered through a GF/F filter into two scintillation vials. One
scintillation vial was frozen for nitrate measurements and the other had 10 µL of 5N HCL added
to fix the sample for ammonium measurements.
Treatment Setup—For the lab treatments eight cores were collected in the bottom
sediment of an undisturbed creek in Great Sippewissett Marsh. Sediment samples were also
taken and frozen at -80°C for DNA extraction. In lab, the water in the cores was removed and
replaced with filtered seawater. For the control cores the seawater was added without any
additional nutrients. For the nitrate treatment the seawater was spiked with nitrate to reach a
concentration of 200 µM. The nitrate and methionine treatments had seawater spiked with nitrate
to 200 µM into which a gram of methionine was dissolved. The nitrous oxide treatments had
filtered seawater added before they were sealed and 60 mL of nitrous oxide was injected into
them. The water for all of the cores was changed a week later with the same treatments. The
nitrous oxide treatments additionally had 60 ml nitrous oxide injected into them 4 days after they
were originally set up, on the day the water was changed and 5 days after the water was changed.
Treatment Incubations—15 days after the treatments began half of the cores (one from
each treatment) had the water drained off of them, microbial samples were taken and then the
water replaced, incubations were run on these cores, but nitrous oxide measurements could not
be taken due to technical problems. 25 days after the treatments began microbial samples were
taken from the other half of the cores. These cores then had water added to them, and they were
sealed with only water in the headspace. After each core was sealed, 5 mL of nitrous oxide
saturated water was added. The cores were left to stir for approximately 30 minutes before
sampling began. At each time point, the dissolved oxygen and temperature of each core was
measured with a DO probe. Approximately 5 mL of water was removed into a 30 mL syringe,
and closed off with a stopcock. These samples were then measured for nitrous oxide via the
method described above.
Measurements of Nitrate—The nitrate samples were measured using the protocol laid out
in Lachat Flow Injection Analyzer (FIA) For Measuring Nitrate (2012).
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Measurements of Ammonium—Ammonium samples were measured using a modification
of the phenol-hypochlorite method laid out by Solórzano (1969).
Measurements of Nitrous Oxide—For the nitrous oxide measurements, the samples were
kept in a tub of seawater until needed for analysis. To prepare the samples, the water level in
each syringe was lowered to 10 mL, and then 20 mL of nitrogen gas was added to the syringe to
a final volume of 30 mL. The syringe was shaken for 1 minute to equilibrate nitrous oxide
between the water and gas. The gas was then injected into a Shimadzu GC-14A. The area of the
peak of nitrous oxide was noted, and a standard curve was used to convert the area into
concentration. The concentration of nitrous oxide in the sample was calculated using Henry’s
Gas Law. The KH of nitrous oxide in seawater used was 0.02318 mol/L/atm.
Molecular Analyses—Microbial samples were extracted with a MoBio UltraClean Soil
DNA Extraction Kit. After extraction they were kept frozen. Concentrations of DNA in each
extract were measured using a Nanodrop. Polymerase chain reactions (PCR) were attempted
using primers for nosZ and GoTaq Mastermix, but no products were ever observed in subsequent
gel electrophoresis. Several attempts were made to obtain PCR products using bacterial 16S
primers, but these attempts were also unsuccessful.
Results
Environmental Nitrogen Concentrations—The two sites in Greenwood had the highest
concentrations of all three forms of inorganic nitrogen (Table 1). The nitrate concentrations at
both Greenwood sites were nearly 30 times the concentrations at the Sippewissett sites, and 200
times the concentration at Rowley House. The ammonium concentrations at the Greenwood sites
were approximately 7 times the concentrations at the Sippewissett sites, and 60 times the
concentration at Rowley House. The nitrous oxide concentrations at Greenwood are again 7
times those at the Sippewissett sites, but only 20 times the concentration at Rowley.
Control Core Fluxes—The rates of the fluxes of nitrate, ammonium and nitrous oxide
were calculated from the slopes of concentrations over time using linear regressions; the R²
values for the regressions are shown in Table 2. Maximum rates of denitrification were
calculated from the nitrate flux of each core. All of the PIE sites had their last time point
removed from the regression for nitrous oxide as the cores were anoxic at this point. Sippewissett
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Unfertilized consumed nitrate the fastest, slightly more than twice as fast as Greenwood Site 2,
the second fastest consumer, and 20 times faster than Rowley House, the slowest consumer
(Table 3). Only Sippewissett Unfertilized consumed ammonium. The Greenwood sites produced
ammonium faster than Rowley House and Sippewissett Fertilized. Neither of the sites in
Sippewissett showed no measureable nitrous oxide production. Greenwood Site 2 consumed
nitrous oxide at approximately the same rate as Site 3 produced it. Rowley House produced
nitrous oxide at approximately the same rate as Greenwood Site 3. Rowley House and
Greenwood Site 3 both lost significant portions of their denitrification potentials as nitrous oxide.
Nitrate Added Core Fluxes—Fluxes for nitrate, ammonium and nitrous oxide in the
nitrate added cores were calculated in the same manner as the control cores; the R² values for the
regressions are shown in Table 4. The PIE cores again had their last time point removed. The
replicates of Sippewissett Unfertilized and Sippewissett Fertilized are shown in Table 5 in
addition to the specific data from each replicate (Table 5). Sippewissett Fertilized had the fastest
consumption of nitrate, two and half times faster than the consumption of Rowley House, the
slowest consumer. The two Greenwood sites and Sippewissett Fertilized produced ammonium
much faster than either Rowley House or Sippewissett Unfertilized, varying between nearly two
times the rate and almost 7 times the rate. Sippewissett Unfertilized and Sippewissett Fertilized
had the highest rates of nitrous oxide consumption, more than twice the rates of any of the other
sites. At every site except Sippewissett Unfertilized the rate of denitrification was at least 10
times higher than in the control cores.
Treatment Core Fluxes—Only nitrous oxide was measured in the treatment cores. The
nitrous oxide treatment had its first time point removed for the linear regression as it appears
some of the nitrous oxide from the treatment was trapped in the sediment. The nitrate and
methionine treatment had its last time point removed as it had run out of nitrous oxide at the third
time point. The slopes of the linear regressions were used to calculate rates of consumption. The
nitrate and methionine core was the only one to have a significantly different rate of
consumption (Table 6).
8
Environmental Nitrogen Loading—The nitrate, ammonium and nitrous oxide
concentrations taken from all sites for measurements of nitrogen loading showed approximately
the levels expected. Sippewissett Fertilized was expected to have higher levels of nitrogen than
Sippewissett Unfertilized; however, the reverse was true. The reason for this discrepancy may be
that the fertilization had stopped for the winter prior to the water sample being taken. This could
have affected the nitrogen concentrations measured due to the sediments consuming all the
nitrogen added onto Sippewissett Fertilized. The cores however were all taken prior to the end of
fertilization for the year.
Percent Potential Denitrification Lost as Nitrous Oxide—Sites with higher long-term
nitrate loading did not have higher rates of denitrification occurring in the nitrate added cores.
However, these sites did show a greater ability to convert the nitrous oxide produced during
denitrification into nitrogen gas. The two Greenwood sites both had the lowest percentages of
potential denitrification being lost as nitrous oxide (Table 5). This is implies that while the
nitrous oxide loss is greater in marshes with nitrogen loading, it is actually a smaller term
relative to the total denitrification taking place. This contradicts the results of several other
studies (LaMontagne 2003, Magalhães 2005, Moseman-Valtierra 2011) and thus merits further
investigation and additional study. It is possible that with long-term nitrogen loading, the
marshes initially lose nitrous oxide at a faster rate, and then the microbial community adapts and
becomes more capable of reducing most of the nitrous oxide to nitrogen gas. If this is the case,
marshes would initially release nitrous oxide at a higher rate, and then slowly become less of a
source of nitrous oxide. It is possible that after nitrogen loading stopped, a marsh could, at least
temporarily, be a nitrous oxide sink.
The percentage of potential denitrification lost as nitrous oxide at Rowley suggests that
while the measured nitrogen loading at Rowley was very low, Rowley may see much higher
levels of nitrogen, specifically nitrate, at times. This additional nitrogen may be coming from
nearby fertilized sections of marsh.
Treatments—The nitrous oxide treatment did not stimulate nitrous oxide consumption.
More significantly, the nitrate and methionine treatment did not inhibit nitrous oxide
consumption and seems to have stimulated it instead. This is in direct opposition with the results
of previous studies (Magalhães 2011, Magalhães 2012). Unfortunately, the lack of replicates due
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to technical problems lessens the reliability of these results. Further investigation of the
microbial community in these cores will allow for greater understanding of the cause of these
results.
This study suggests quite a few further areas of research that would elaborate and
potentially explain the results seen here. The most obvious and potentially the most helpful
would be to complete the analysis of the genetic samples taken at each site and from each of the
treatments. While merely comparing the ratios of nosZ to (nirK + nirS) would provide insights
into the molecular basis for the differences in fluxes, sequencing may allow for a greater
understanding of the diversity of the genes involved at each site.
Along similar lines, it would be interesting to test different sulfur compounds to see
which show inhibitory effects and which do not. If, as it appears, methionine and its breakdown
products in fact stimulate nitrous oxide reduction in at least some microbes, this is of great
importance as it means that the sulfur compounds in salt marshes may not be inhibiting nitrous
oxide reduction as suggested previously but rather stimulating it. An important part of this
process would be to test the effects of many of the sulfur compounds frequently found in salt
marsh sediments to see which interact with nitrous oxide reduction and in what manner. It may
be that there are variations of nosZ that are inhibited by sulfur compounds and others that are not.
The ability to distinguish between these would improve the prediction of nitrous oxide fluxes
occurring in sediments containing high levels of sulfur compounds.
Figures and Tables
Figure 2: Sampling Sites at Plum Island Estuary (PIE)
Table 1: Environmental Inorganic Nitrogen Concentrations
Table 2: Coefficients of Determination for Control Cores
Table 3: Control Core Fluxes
Table 4: Coefficients of Determination for Nitrate Added Cores
Table 5: Nitrate Added Core Fluxes
Table 6: Rates of Nitrous Oxide Consumption in Treatment Cores
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Appendix
Figure 2: Sampling Sites at Plum Island Estuary (PIE)
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Site Nitrate (µM) Ammonium (µM) Nitrous Oxide (µM)
Sippewissett Unfertilized 26.3 11.0 0.0832
Sippewissett Fertilized 2.41 6.82 0.0798
Rowley House 2.27 1.11 0.0329
Greenwood Site 2 404 65.3 0.843
Greenwood Site 3 348 55.6 0.269
Table 2: Coefficients of Determination for Control Cores
Table 3: Control Core Fluxes
Site Change
in Nitrate
% of Potential
Sippewissett
Rowley
Greenwood
Greenwood
Site R² of linear
regression for
for Ammonium
for Nitrous Oxide
Greenwood Site 2 0.846 0.9898 0.7175
Greenwood Site 3 0.1403 0.9925 0.9217
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Table 5: Nitrate Added Core Fluxes
Site Change in
Change in
Change
in
Nitrous
Oxide
Maximum
Potential
Denitrification
% of Potential
Sippewissett
Unfertilized
Sippewissett
Unfertilized
Sippewissett
Fertilized
Sippewissett
Fertilized
Rowley
Greenwood
Site R² of linear
regression for
for Ammonium
for Nitrous Oxide
Greenwood Site 2 0.9882 0.9682 0.8571
Greenwood Site 3 0.9811 0.9692 0.6163
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Greenwood
Table 6: Rates of Nitrous Oxide Consumption in Treatment Cores
Treatment Nitrous Oxide Consumption (µM m² hr¹) R² of linear regression
Control 37.6 0.6048
Nitrate 29.2 0.9798
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