1. ARE SALTMARSHES SUSCEPTIBLE TO CARBON LOSS FOLLOWING N
LOADING?
Ulrich Kakou, Sanderman J.
Abstract
There has been a growing interest in salt marsh protection and restoration for its economical,
recreational and, more recently, its value as a carbon sink. Salt marshes and many other coastal
wetlands have the capacity to store large amounts of carbon in the soil but that carbon may be in
danger of being lost to eutrophication. This project focuses on evaluating soil quality and microbial
activity following eutrophication using diffuse reflectance Fourier­transform infrared spectroscopy
(DRIFTS) and bioavailability assay. Using DRIFTS revealed that the litter quality has improved as the
fertilized plots (XF) show more decomposition than the control plots (C). The amount of decomposition
can also be demonstrated using C:N ratio where a low ratio correlated with higher decomposition ratios
(index I values). Comparing C­plots bulk samples to XF indicated that XF plots were more decomposed
than the C­plots which is a testament to an overall change in OM composition following eutrophication.
The bioavailability assay revealed that the XF soil was on average less decomposable than soil from
the C­plots and that additional nitrogen loading feedbacks positively in soils deeper than 10 cm.
However, top soils did not respond favorably to additional nitrogen loading. The data provides some
important insights that suggest belowground carbon cycle has been substantially altered.

2. INTRODUCTION
Salt marshes are among the most productive and most important vegetated coastland ecosystems on
the planet. Their most notable roles include quenching storm surges, keeping up with sea level rise
(SLR), sequestering carbon (​McLeod et al. 2011​ ), and because of their incredible ability to retain
nitrogen (N) and other nutrients (​Valiela I 2015​ ), efficiently filter excess nutrient which prevents
eutrophication and pollution of other marine ecosystems. In the past few years, there has been an
increasing interest in salt marsh protection and restoration because of its economical and recreational
value (​McLeod et al. 2011, Chmura et al. 2012​ ). Despite their relatively low greenhouse gas emission
(​Chmura et al. 2003), there is new evidence that suggests that salt marshes are susceptible to emitting
greenhouse gases, such as methane, nitrous oxide, and carbon dioxide, at a higher rate following
eutrophication (​Moseman­Valtierra et al. 2011​ ). This potential for increased greenhouse gas
emissions with high N loading may result in salt marshes switching from being carbon sinks to carbon
sources. Unfortunately, pertinent data on eutrophication effects on the carbon balance of salt marshes
is lacking thus this project’s goal is to provide a more conclusive insight on this relationship based on a
40+ year study of artificial eutrophication via fertilization on the Great Sippewissett salt marsh (GSM) in
Falmouth, Massachusetts.
Most natural ecosystems’ biomass are nutrient limited which prevents optimal growth for some species.
This limitation is not inherently detrimental to the ecosystem. However, for the salt marsh ecosystems
alleviating this limitation can potentially destabilize the natural balance between plant species, microbial
activity, and carbon sequestration (​Deegan et al. 2007, Morris & Bradley 1999​ ). Salt marshes can
succumb to nitrogen enrichment via agricultural, urban and suburban runoff because pollution
anywhere in the upstream catchment can reach the marsh (​Moseman­Valtierra et al. 2011​ ). To further
understand this balance, parts of the Great Sippewissett marsh has been nitrogen, phosphorus, and
potassium enriched since 1971 (​Valiela I 2015). As expected, the control (C) plots have continued to
grow at the same rate maintaining the same plant species. However, the plots fertilized at an extra­high
(XF) rate have seen a significant increase in aboveground biomass and a species shift. In contrast, the
belowground biomass in the XF­plots has experienced a dramatic decrease compared to C­plots
(​Valiela I 2015). This has many implications for carbon storage and accumulation because one of the
greatest sources of the carbon for the marsh is from the root material that plants develop to forage for
necessary nutrients. Following nutrient enrichment, plants may spend less resources building their
roots deeper in the soil as nutrients are more readily available (​Levine et al. 1998​ ). Additionally, plants
may invest fewer resources into building defence structures resulting in litter that can be more easily
decomposed. Based on these observations, we believe that salt marshes might be at risk of becoming
carbon sources rather than carbon sinks under eutrophication
We hypothesize that under eutrophication:
(1) The quality of the litter will increase with chronic fertilization.
(2) The increased litter quality (H1) in conjunction with greater nutrient availability to the microbial
community will feedback positively on decay rates.

3. Under the anoxic conditions of the marsh (​Young and Frazer 1987, Morris & Bradley 1999​ ), lignin is
much less decomposable (​Benner et al. 1984) than other organic matter such as proteins and
carbohydrates. If fertilization results in the root litter having less lignin and more protein, then this would
result in more decomposable organic matter for microbes. Additionally, in the fertilized plots there are
more nutrients available to the microbial community itself which will likely stimulate decay rates. These
greater losses of organic matter coupled with the observed decreases in root biomass (i.e. Valiela I
2015) can the act together to reduce or even reverse carbon accumulation in marshes receiving high N
loads.
We tested these hypotheses by evaluating soil quality and microbial activity following eutrophication
using diffuse reflectance infrared spectroscopy (DRIFTS), elemental (C and N) analysis and a
bioavailability assay. Using DRIFTS will allow testing of hypothesis I by detecting any shift in overall soil
organic matter composition that usually occurs in the mid­range (4000­400 cm­1) infrared region of soil
samples. The bioavailability assay will help elucidate the effect that nitrogen has on the decay rates by
capturing, quantifying, and comparing CO2 emissions (H2).
METHODS
Site description
The GSM has many sites (habitats) that have been sectionalized by plots which received various
fertilizer treatments. The plots are 10 m in radius and each plot is separated by a creek. Some plots
have received high fertilizer treatment (HF), some extra fertilizer (XF), and some no treatment (C). The
plots have been chosen to represent the main habitats on the marsh and their height from mean sea
level (MSL), i.e., low marsh (LM, 50­70 cm above MSL), high marsh (HM, >70 cm above MSL), and
creek bank (CB, 30­50 cm above MSL), which may have different vegetative species. Each habitat,
divided in plots, received the various fertilizer treatments of a mixed fertilizer (10% N, 6% P2O4, 4%
K2O), i.e., 2.52 for HF, 7.56 g N m​­2
week​­1
for XF. These treatments are 30 and 90 times the US
Department of Agriculture recommended annual dosage for oats (​Valiela et al. 1973, Fox et al. 2012,​ ).
Each core sampled was 30 cm deep and split by 2 cm increments. For this project, we focused on the
low marsh habitat C, HF, and XF whose dominant vegetative species is ​Spartina alterniflora because
this is the most common vegetative assemblage found in salt marshes of southern New England.
Preparing samples
Core segments were oven dried and half of each increment had live and dead root/rhizome material
separated from the bulk sediments. Bulk density had already been determined by measuring the
volume and weighing the half of each core segment that was not used for root biomass. Prior to
analysis, all samples were first broken down using a coffee grinder then ground to a fine powder using
the ​D8000 Mixer/Mill grinder. Upon completion, each sample was placed in small vials and labelled to
be prepared for the FTIR run, which consisted of filling each sample cup with finely ground GSM
samples and carefully place them on the FTIR tray.

4.
DRIFTS (FTIR)
The bioavailability assay soil samples and soil samples cored from the various plots on GSM were ran
on a Bruker Vertex 70 FTIR spectrometer with a Pike Autodiff diffuse reflectance accessory. For each
sample, 60 scans were collected at a 2 cm­1 resolution. Background subtracted spectra were then
baseline corrected and peaks attributed to major functional groups were identified as laid out in the
Margenot et al. (2015). Amines show up around 3400 cm­1, amides closer to 1575 cm­1, phenols at
1270 cm­1, polysaccharides at 1110 and 1080 cm­1, aromatics at 920 and 840 cm­1, and aliphatic
groups (most abundant) at 2924, 2850, 1470, 1405, and 1390 cm­1 (​Margenot et al. 2015). To
determine the relative amount of decomposition, the ratio of aromatic to aliphatic functional groups was
calculated as Index I. Index II ratio is the ratio of C to O­functional groups which determines relative
recalcitrance of soil organic matter (​Veum et al. 2014). These shifts in organic matter composition
detected by DRIFTS are attributed to the dipole moments of the aforementioned functional groups
(​Margenot et al. 2015).
LECO
The LECO employs a ​high temperature oxidative combustion followed by infrared detection to
determine the carbon and nitrogen content of organic matter. Each sample was weighed, placed in an
aluminum foil, and combusted to determine its elemental composition. A series of certified standards
were used to produce calibration curves that spanned the C and N concentrations in our unknown
samples.
BIOAVAILABILITY ASSAY
To test whether microbial activity, or carbon decay rates may increase following eutrophication, an
incubation experiment was designed. Core samples from 3 plots (C­plot, high fertilizer­HF, extra high
fertilizer­XF) on the GSM of various depths and treatments, were grounded, weighed, and placed in 50
mL vials. After a 10 day preincubation period to restore microbial activity to the previously dried
samples, soil respiration rates were measured over a 3 week period. Accumulated CO2 was measured
in the headspace of the incubation vials using an infrared gas analyser (LICOR Li­820) at 5 time
points over the 3 week period. About ~2L of creek water was sampled from GSM and used as
inoculum. For the incubation, the low marsh (LM) habitat alone had samples from 3 depths,
categorized as top, mid, and deep. Each depth had 6 repetitions: 3 of which were treated with only the
inoculum and the remaining 3 were treated with the inoculum and urea applied at a rate of
approximately 200 kg N ha­1. To isolate the CO2 produced by microbes in the soil, 6 vials that
contained no soil received the same amount of inoculum and inoculum+nutrient treatment to serve as
control. Along with 3 empty vials to account for the CO2 already present in the air, making a total of 63
vials.
RESULTS AND DISCUSSION

5. Have carbon accumulation rates changed?
We compiled some data on depth to amount of carbon which shows that the XF­plots have a lower %C
but greater bulk density, resulting in greater overall carbon content in XF plots (F​igure 1​). While the
lower C concentration is suggestive of a shift in belowground carbon cycling, to test whether
eutrophication has curtailed or increased the amount of carbon that is coming into the marsh, it would
be advantageous to know the carbon accumulation rate on the various plots. But because we lack data
to accurately age the soil, the accumulation rates cannot be determined for the span of this project.
Figure 1: downcore profile of C to XF plots. %C is lower in XF but XF bulk density is higher thus higher
C density
Has litter quality increased?
Looking at the results from the FTIR and C:N ratio data we see that there is in fact an increase in litter
quality. Because of the protein and carbohydrate content, we expect to see the C:N ratio of live roots to
be greater than both the dead roots and bulk material. If the C:N ratio is 22 then for every unit of N
there are 22 units of C. Higher index I values are consistent with a more decomposed organic matter
and higher index II values are consistent with more recalcitrant organic matter (​Veum et al. 2014). The
data supports our initial expectation that C­plots would demonstrate less decomposition than
XF­plots.The lower index I data in conjunction with the higher C:N data of C­plots live and dead roots
suggest that there is more protein in the litter. The XF plots, however, show a great shift in overall
composition and lower C:N ratio (see ​Figure 2&3​). These findings support our hypothesis that nitrogen
loading will make more labile organic matter available to microbes to be decomposed.

6.
Figure 2: Live and Dead roots index I ratio. Higher index I indicate more decomposition
Figure 3: C:N ratio of live and dead roots. Higher C:N indicates less decomposition
Has bulk OM composition shifted?

7.
Figure 4: Bulk samples index I and C:N ratio. Higher index I values indicate more decomposition.
Higher C:N ratio indicate less decomposition
The data shows that the bulk material in XF are more decomposed than the C plots (see ​Figure 4​).
This trend can be explained when we consider that the bulk material in C­plots have more recalcitrant
OM, specifically lignin with the pronounced root system observed in C­plots, compared to XF (​Valiela
2015). The lower bulk C:N ratio results typically indicate more decomposed material since this ratio will
decrease towards the C:N ratio of microbial biomass (6­8) as decomposition of plant litter progresses.
In addition, coupling this data with the increased CO2 emission related to microbial decay (see next
section) offers a solid perspective for the difference in decomposition. With this evidence, we can
conclude that bulk OM composition has shifted following eutrophication.
Have decomposition rates changed in response to chronic loading and short term nutrient addition?
CO2 accumulation is expected to be greater in N(urea) treated vials in both C­plots and XF­plots. The
data shows that the amount of CO2 that accumulated in the ambient treated incubation vials is greater
in the C­plots (Figure 5) likely due to the lower observed decomposition state in the C­plots relative to
the HF and XF plots (Figure 4). The natural log of the ratio of CO2 accumulated in vials treated with
urea versus vials that received no urea treatment helps us evaluate the response to nitrogen loading,
where a positive number indicates that more CO2 was formed in response to urea and vice versa. The
data shows that deeper soils (10­30 cm) responded positively to the urea treatment but the top 10 cm
responded negatively (see ​Figure 6)​. The positive response to the urea treatment indicates that
decomposition rates were nutrient limited and this limitation has been alleviated and consequently
increased microbial activity. The negative response to urea treatment in both C­plots and XF­plots in
the top soils is not expected because top soils show less decomposition than deeper soils (see ​Figure
5​). Further testing is required to validate or refute this response and identify the possible causes.

8.
Figure 5: CO2 accumulated per gC against treatment in ambient treated incubation vials
Figure 6: Response to the urea treatment in incubation vials. Top soils produced less CO2 than mid to
deep.
IMPACT
The data gathered for this project highlights some of the effects of eutrophication on soil quality and the
microbial activity. Our data suggests that carbon concentrations have decreased but because bulk
density has increased, the carbon storage in the XF­plots is greater than the C­plots. This difference
may be closely linked to the increase in aboveground biomass following nutrient loading. However, we
don’t know whether the accumulation rates are similar in C­plots and XF­plots. Learning about the rates
will safely answer the question about salt marshes ability to store carbon under eutrophication.
The FTIR and C:N data suggest that litter quality is better. This means that there is in fact more labile
carbon and less recalcitrant carbon, which predicts more microbial activity. When coupling litter quality

9. results to the bioavailability assay results, we find that the data is consistent with hypothesis II, i.e.,
microbial activity will deplete labile organic matter faster and produce more CO2. However, this trend is
only observed in mid to deep soils as the top soils respond negatively to the urea treatment. This
seems to suggest that microbial activity might actually be stifled in top soils. While not consistent with
soil quality data, further testing will prove advantageous in that we will learn of other factors that may
prevent or promote microbial decay.
Given the size of the data on this project, we cannot draw definite conclusions or make generalized
statements about the fate of salt marshes at large. But the data does provide some important insights
that suggest belowground carbon cycle has been substantially altered.
ACKNOWLEDGEMENTS
I’d like to thank Jonathan Sanderman for helping make this project an absolute success by providing a
means of transport to and from work, for teaching all the necessary lab techniques, and skills, and
assisting with presenting my mid summer report and final poster, and writing this paper. Elizabeth
Elmstrom and Ivan Valiela for providing the dried, segments cores for this project as well as bulk
density data and giving an onsite detailed tour of the Great Sippewissett marsh. Michael Ernst for
helping me print my poster. SSF for organizing this entire internship program. Kama Thieler for her
support and being our main resource for everyday things, including providing a bike helmet, free rides,
and safekeeping mail. The WHRC for letting me use its facility to accomplish this project. NSF for
providing the funding, and WHOI for providing the housing.

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