The effects of salinity and nutrient limitation on microbial

processes in coastal sediments

Emily Waters

Hampshire College

December 2011

Mentor: Anne Giblin

Abstract

In order to better understand the relationship between salinity, nutrient limitation, and

microbial activity, I collected high and low salinity sediments and overlying water

samples from Little Sippewissett Marsh (Falmoth, MA). I analyzed the overlying water

for NO3-, NH4

+ and PO4

3- concentrations and the sediments for percent carbon and total

phosphate. I gave the homogenized sediments one of four treatments (control, nitrogen,

nitrogen and phosphorus, and phosphorus) and incubated them for three weeks. At each

of four time points, I measured respiration and conducted endopeptidase and phosphatase

assays. Results showed higher phosphate concentrations in the low salinity water and

sediment samples, however significantly higher phosphatase activity than endopeptidase

activity (p<0.0002 for high and low salinity). This suggests that the microbial community

in both high and low salinities is phosphorus limited. Low salinity sediments had an

average percent carbon of 9.48%, while the high salinity sediments had an average

percent carbon of 1.97%, potentially accounting for the significantly higher respiration in

the low salinity samples compared to the high salinity samples (p< 3.5 x10-5

). Both

phosphatase and respiration did not appear to be much affected by the nutrient treatments,

however trends in the endopeptidase activity suggest that endopeptidase activity was

lower in treatments where nitrogen was added. I recommend a further study with a

longer incubation time that accounts for the loss in available carbon over the incubation

period.

Key Phrases: nutrient limitation, extracellular enzymes, coastal sediments, microbial

activity

Key Words: salinity, enzymes, respiration, sediments, phosphatase, endopeptidase,

nitrogen, phosphorus

Introduction

Not only are coastal wetlands extremely productive systems, but they provide many

ecosystem services, contributing to storm protection for coastal communities, buffering

nutrient loads from land to the ocean, and supplying habitat and nursing ground for fish

and birds (Kostka et al., 2002). The key to these environmental and economic services is

the wetlands’ ability to accumulate organic matter and peat. It is likely that this service

will only become more important, because as sea levels rise these marshes will

accumulate sediment, counteracting the potential harm from sea level elevation (Morris

and Bradley, 1999). Peat accumulation in marshes is the result of saturated, and therefore

anoxic sediment. This anoxia limits organic matter oxidation, leading to carbon

accumulating more rapidly than it is decomposed (Amador and Jones, 1993).

As a result of this carbon accumulation, wetlands represent a large sink of global

carbon. (Morris and Bradley, 1999). Therefore, if wetland carbon mineralization were to

accelerate, the consequence of this potentially large CO2 flux could further contribute to

changes in the global climate. One factor that has the potential to increase wetland

carbon mineralization is anthropogenic nutrient loading, which has been shown to alter

the soil carbon storage due to increased decomposition, in some settings (Morris and

Bradley, 1999). Coastal wetlands are especially threatened by nutrient loading because

they receive inputs from surface water runoff and groundwater discharge (Morris and

Bradley, 1999). Besides increased decomposition, a potential consequence of

eutrophication includes increased microbial activity and biomass (Penton and Newman,

2007).

These effects of nutrient loading may result from either nitrogen or phosphorus

inputs, however the responses of different aquatic systems to either nutrient may vary.

The degree of a system’s response may be determined by what nutrient is limiting

production, as well as the vegetation, microbial community, and sediment chemical

quality (Paludan et al., 1999). The typical trends in nutrient limitations of aquatic systems

suggest that freshwater is more often phosphorus limited, while salt water is more often

nitrogen limited. If so, we can presume that over a salinity gradient of an estuary, the

corresponding gradient of limited nutrients would follow. Freshwater may be phosphorus

limited because phosphorus becomes bound in the sediments and is not biologically

available. In salt water, iron is trapped in the sediments in place of phosphorus, making

phosphorus more available. While the scientific community generally accepts these

limitation patterns, there are many examples of systems where this salinity-nutrient trend

does not hold. Additionally, while these patterns may be typical of pristine systems, most

aquatic systems now receive high nutrient loading, which has the potential to shift

nutrient limitation.

Arguably the most important step in the marsh’s food chain is the microbial loop,

which cycles through most of the nutrients and energy in the system (Hill et al., 2006).

Microbial activity is often controlled by their access to nutrients, acquired through the

synthesis and activity of extracellular enzymes (Penton and Newman, 2007). If nutrient

loading causes increased organic carbon cycling and nutrient limitation shifts in wetlands,

the microbial activity controlling these processes would be further driven by the

microbes’ enzyme production and activity (Penton and Newman, 2007).

Microbes produce enzymes in order to access large polymeric substrates in

organic matter that they would otherwise not be able to use (Karner and Rassoukadegan,

1995). In this way, enzymes can speed up decomposition and mineralization (Allison

and Vitousek, 2005). However enzyme synthesis is energetically and nitrogen-

expensive, so microbes will usually only synthesize enzymes when there is a limiting

nutrient (Allison and Vitousek, 2005). In a study by Penton and Newman (2007), N and P

additions resulted in higher carbon mineralization while enzyme activity was lowered.

Allison and Vitousek (2005) found that carbon and nitrogen availability, which is needed

for enzyme synthesis, may also control enzyme production. Temperature, pH, oxygen

conditions, hydrogen sulfide, humic substances, and heavy metals may also regulate

enzyme activities (Nausch and Nausch, 2000).

The measurement of phosphatase and endopeptidase activity is useful in

determining the nutrient limitations of microbial communities. Phosphatases hydrolyze

the esters and anhydrides of organic phosphorus to make inorganic phosphorus available

(Huang and Morris, 2003). Phosphatases are produced by plant roots, fungi, and bacteria

(Olander et al., 2000) and have been correlated to phosphorus stress and plant growth

(Makoi, 2008). Huang and Morris (2003) found phosphatase activity was positively

correlated with with plant biomass and negatively correlated with phosphorous in

porewater, sediments and total phosphorus. There is a well-documented negative

feedback, where phosphatase activity will increase the bioavailability of P, repressing

phosphatase activity and synthesis, decreasing P mineralization, and making P limited

again, back to where there more phosphatase is needed. In the same vein, it has been

suggested that high phosphatase activity may occur when an ecosystem is in a P-

accumulation phase, while low phosphatase activity signals that the ecosystem is P-

neutral (Huang and Morris, 2003).

Endopeptidase is a type of protease, which catalyzes the degradation of proteins,

supplying primarily nitrogen, but also carbon in low molecular weight compounds that

bacteria are able to assimilate (Nausch and Nausch, 2000). They are limited by the

presence of proteins and peptide substrates (Jankiewicz, 2007), and inorganic nitrogen

has been shown to suppress their activity (Nausch, 2000).

In order to better understand the relationships between salinity, nutrient limitation,

and microbial activity, I sampled sediments in high salinity and low salinity areas of a

coastal marsh in Cape Cod, MA. I incubated the sediments with nitrogen and phosphorus

treatments and measured phosphatase and endopeptidase activity at four time points

during the incubation. At each time point, I also measured sediment respiration, as an

additional measurement of microbial activity and carbon mineralization. The relationship

between salinity and nutrient limitation in aquatic systems has been studied extensively,

but the focus has been on limitation in the water column. This study will emphasize the

sediments. I hypothesize that the low salinity site will be more phosphorus limited and

the high salinity site will be more nitrogen limited. Further, I expect higher phosphatase

activity where the sediments are phosphorus limited, and higher endopeptidase activity in

nitrogen limited sediments. I expect these patterns to become more extreme over time, as

the nutrients are being consumed, and to additionally increase as sediment respiration

increases. Cape Cod coasts receive a substantial anthropogenic nutrient load, which

poses a threat to these marshes. For this reason, understanding how nutrient additions

influence the microbial communities is essential to the management of these wetlands.

Methods

Field Sampling and Experimental Design

I collected overlying water samples and three replicate sediment samples from a high

salinity (34 ppt) and low salinity (4 ppt) site at Little Sippewissett Marsh in Falmouth,

MA (Figure 1). Sediment samples were taken from the top 5 cm, however not at the

same distance from water. I filtered the water samples and analyzed them for NO3-, NH4

+

and PO43-

concentrations. I homogenized the sediments and sub-sampled them into 24

samples per replicate (total of 96, 48 high salinity and 48 low salinity). Each subsample

was about 15-20 g wet weight (WW), however after the first time point, an additional 10

g WW was added in order to increase respiration fluxes. For each set of 24 subsamples,

there were 4 control (HSC=high salinity control, LSC= low salinity control), 4 nitrogen

treatments (HSN=high salinity nitrogen, LSN- low salinity nitrogen), 4 phosphorus

treatments (HSP= high salinity phosphorus, LSP= low salinity phosphorus), and 4

nitrogen and phosphorus treatments (HSNP= high salinity nitrogen and phosphorus,

LSNP= low salinity nitrogen and phosphorus). I added nitrogen in the form of 1 mL 200

mM NH4+ and phosphorus as 1 mL of 12.5 mM PO4

3-. I set aside 20 g WW from each

sediment sample for initial respiration and enzyme assays. For each time point during the

22 day incubation, I harvested a set of 24 samples, took measurements, and added an

additional nutrient treatment to the remaining samples. The time points occurred on day

5, 9, 18, and 22. Each set of measurements consisted of respiration measurements and an

endopeptidase and phosphatase assay. I incubated the samples at room temperature and

added DI water each day to bring the samples back to their initial WW to regulate for

evaporation.

Overlying water and Sediment Analysis

I followed the phosphate protocol adapted from Murphy and Riley (1962) and used

the Shimadzu UV-1800 UV spectrophotometer (Shimadzu Coroporation; Kyoto, Japan).

For ammonium analysis, I followed the ammonium protocol modified from Strickland

and Parsons (1969) and used the Shimadzu UV-1601 UV spectrophotometer (Shimadzu

Coroporation; Kyoto, Japan). To determine nitrate concentrations, I followed the Lachat

flow injection analyzer (FIA) for measuring nitrate (adapted from Wood, Armstrong, and

Richards, 1967). I performed CHN and Total Phosphorus (adapted from Asplia et al.,

1976) analysis on the sediment samples using the Perkin Elmer Series II CHNS/O

Analyzer 2400 (Shelton, CT) and the Shimadzu UV-1800 UV spectrophotometer

(Shimadzu Coroporation; Kyoto, Japan) respectively.

Respiration

I measured respiration on both the Li-Cor 6200 and Li-Cor 6400 Portable

Photosynthesis System (Li-Cor Biosciences, Lincoln, Nebraska). I used the Li-Cor 6200

for the initial measurements and the first time point and the Li-Cor 6400 on the remainder

of the measurements. The machine measured CO2 every 15 seconds for five minutes per

sample.

Enzymes

I placed 1 gram of fresh sediment in a 15 mL falcon tube and added 8 mL of acetate

buffer (0.1 M) and 400 uM substrate (1 mM, 4-Methylumbelliferyl phosphate and L-

Leucine-7-amido-4-methylcoumarin hydrochloride). At room temperature, I tumbled the

falcon tubes during the incubation to keep the sediment mixing. At 10 min, 30 min, and

60 min, I centrifuged the tubes, withdrew 2.5 mL of supernatant and combined it with 2.5

mL pH 10 glycine buffer (200 mM), which kills the activity due to its high pH. I

analyzed the supernatant and glycine buffer fluorometrically on 10-AU Fluorometer

(Sunnyvale, California) at 445 nm.

Calculations

I averaged the final 8 respiration measurements of the five minutes recorded for each

sediment sample. To calculate enzyme activity, I plotted MUF concentration versus time

and used a linear regression to get activity in nmole l-1 h-1. All data analysis was

completed on Microsoft Excel 2008.

Results

Nutrient analysis of the overlying waters from the low salinity site showed higher

concentrations of NH4+ and PO4

3- than the high salinity site (Table 1). The average

ammonium concentration was prominently greater, with 5.29 uM NH4+ -in the low

salinity water and 0.47 uM NH4+ in the high salinity water. The average phosphate

concentration in the low salinity overlying waters was 2.11 uM PO43-

, while just 1.03 uM

PO43-

in the high salinity water. NO3- concentrations were fairly similar in high and low

salinity waters (0.73 and 0.61 uM NO3-, respectively). Sediment analysis was consistent

with the phosphate concentrations in the water samples. Total phosphate in the low

salinity sediment samples (6.09 mmol/g) was three times as much as that in high salinity

sediments (2.17 mM/g). Additionally, low salinity sediments had a C:N ratio of 21 with

an average percent carbon of 9.48%, while the high salinity sediments had a C:N ratio of

only 15.38 with an average percent carbon of 1.97%. Initial enzyme assays showed that

the low salinity sediments had higher phosphatase and endopeptidase activities as well as

higher respiration compared to the high salinity sediments. It is worth noting that prior to

respiration measurements the sediments were homogenized. This disturbance likely

altered respiration measurements, as the rates are about an order of magnitude higher than

the respiration rates we measured throughout the incubation.

Over the course of the incubation, respiration measurements in all nutrient treatments

and in both salinities initially increased (Figure 2). At the second measurement,

respiration slightly decreased and the congruence deteriorates. HSN, HSP, and LSP drop

off, HSC and HS N+P increase, and LSN and LSN+P show no change. Respiration was

significantly higher in the low salinity sediments compared to the high salinity sediments

(p< 3.5 x10-5

) with an average respiration rate across the time points of 0.104 ug CO2/g

dry sediment/min in high salinity sediments and .351 ug CO2/g dry sediment/min in low

salinity sediments.

At the beginning of the incubation, endopeptidase was highest in the phosphorus-

treated samples regardless of salinity. However, by day 5 these activities decreased and

we saw the highest activity in the control samples (Figure 3). Activity tended to decrease

until the last time point, where activity was highest in the phosphorus-treated and control

samples. Activity in the samples treated with nitrogen or both nitrogen and phosphorus

were close to zero throughout the incubation. The patterns of the high and low salinities

were fairly similar, although measurements from the first time point showed higher

endopeptidase activity in the low salinity P-treated sediments compared to the P-treated

high salinity sediments.

Phosphatase activity was significantly higher than endopeptidase activity in both high

salinity and low salinities (p<0.0002 for high and low salinity). There was a positive

correlation between the phosphatase activity in high and low salinity (r2= 0.79), where

phosphatase activity decreased over the course of the incubation, regardless of the

sediment salinity. Within this trend, activity varied among the nutrient treatments within

a given salinity (Figure 4). However, there were no significant differences between

treatments, except that the high salinity control was significantly higher (p<0.02) than the

high salinity nitrogen treatment. At the first time point in the high salinity sediments, the

highest phosphatase activity was in the samples treated with both N and P and lowest in

the sediments treated with N. However, at day 22, the phosphorous treatment and the

control had the highest activity. The phosphorus-treated and control sediments had the

highest phosphatase throughout the incubation in the low salinity sediments. All low

salinity treatments showed decreasing activity, although the control samples had

increased phosphatase activity. Overall, there was much higher and more frequent

phosphatase activity than endopeptidase activity.

Until now, we have only considered the patterns of a single measurement over the

incubation period. However, in some treatments, emergent patterns suggest that these

microbial processes may be working in conjunction with each other. For example, the in

the low salinity control sediments, all three measures increased between day five and day

nine (Figure 5). Between day nine and eighteen, endopeptidase activity stopped, while

phosphatase activity continued to increase and respiration declined slightly. Between the

final time points, respiration and phosphatase decreased dramatically, while

endopeptidase activity revived itself.

Just as in the low salinity control samples, the high salinity controls showed microbial

activity increases in all three processes between the first and second time point (Figure

6). Opposite to the low salinity, for the remainder of the incubation, endopeptidase and

phosphatase activities declined while respiration increased. These considerations may

help determine what nutrients are limiting the microbial communities in these sediments.

Discussion

The results of the initial enzyme assays and respiration measurements showed higher

microbial activity in the low salinity sediments compared to the high salinity sediments.

Initial respiration measurements are higher than those throughout the incubation, likely

due to homogenizing the sample. Kostka et al. (2002) found that homogenizing the

sediments resulted in increased respiration by a factor of two to six, however the

measurements recovered to typical levels after two days. If our sediments followed this

recovery, then the respiration measurements throughout the incubation are likely a good

reflection of typical fluxes and were not hampered by the initial homogenization.

Additionally, there were higher ammonium and phosphate concentrations in the

overlying waters of the low salinity site, and the nitrate concentration was only slightly

lower than the overlying waters of the high salinity site. Sediment total phosphate was

about three times as high in the low salinity site. While we would expect higher enzyme

activity where nutrients are limited, this suggests that the microbial community in the

high salinity sediments was fairly nutrient limited at the start of the experiment, perhaps

limiting its ability to synthesize enzymes. On the other hand, the low salinity sediments

had a higher C:N ratio than the high salinity site (21.00 versus 15.38). With more

available carbon, the microbial community in the low salinity site may be more active,

which is reflected in the higher respiration. Despite the site being more nutrient rich, the

microbes may require more nutrients in order to sustain this level of activity, which is

reflected in the higher enzyme activity. High soil organic matter often suggests high soil

fertility and productivity (Bolton Jr. et al., 1985). Additionally, the high salinity

sediments had a comparatively lower C:N ratio and percent carbon, which suggests that

there is less available carbon for mineralization and may explain the lower respiration.

The microbes in the high salinity sediments also had more nitrogen available to them

compared to the low salinity microbes. Therefore, they may not need to be producing

those enzymes. In considering these initial enzyme activities, it is necessary to recall that

plants as well as microbes may produce enzymes. The sediments were taken within the

root zone of the sediments and it is likely that the sediments contained previously

synthesized enzymes from the surrounding vegetation. While both sites were in

vegetated areas, the vegetation in the low salinity site was denser, although this was not

measured. Huang and Morris (2003) showed that phosphatase activity had a positive

correlation to aboveground biomass.

Respiration increased in all treatments between the first two time points, and then

decreased over the remainder of the incubation, except in HSC, HSN+P, and LSN. This

general decrease over the time may be contributed to the fact that no new carbon was

added to the sediment. While studies have shown an increase in microbial activity and

carbon mineralization with greater availability of inorganic nutrients (Amador and Jones,

1993), we likely didn’t see this pattern, despite the nutrient additions because the

microbial community may have become carbon limited. Further, the initial high

respiration and microbial activity may have reflected the addition of nutrients. At the

second time point, in the high salinity sediments, the two highest measurements are in

sediments with nutrient treatments. In the low salinity sediments, the highest respiration

at the second time point is in the LSP and LSN+P treatments, which may reflect the high

phosphatase activity in the initial measurements. This is consistent with Sundareshwar et

al. (2003), who found increased soil respiration and carbon turnover in plots treated with

N+P. White and Reddy (2000) found a positive correlation between microbial biomass

and high P treatments, suggesting that wetlands are phosphorus limited. Further, they

found that the addition of P made nitrogen more available because the microbial

community was more active and microbes drive the nitrogen cycle. Sundareshwar et al

(2003) additionally found that although the plants in the studied salt marsh were nitrogen

limited, which follows the typical pattern of nitrogen limitation in high salinity pristine

aquatic systems, the microbes were P-limited. Consistent with this study, he found that

the microbial community had a secondary carbon limitation when phosphorus was added.

The enzyme activity of both the high salinity and low salinity sediments supports

the microbial community being phosphorus limited, as phosphatase was significantly

higher than endopeptidase activity. The initial increase in phosphatase activity may be a

result of established active phosphatase that had been synthesized before sampling, as

opposed to new phosphatase synthesis as a result of nutrient treatments (Spier et al.,

1978). However, it has been suggested that the microbial community will respond to

changes in environmental conditions readily, the first response being enzyme synthesis

and activity on the short-term (Karner and Rassoukadegan, 1995).

Nutrient treatments did not follow the expected pattern of inhibiting enzyme activity.

In both high salinity and low salinity sediments, phosphorus additions appeared to

stimulate phosphatase activity, as the highest activities were in the phosphorus addition in

the low salinity sediments and in the nitrogen and phosphorus treatment in the high

salinity sediments. It is possible that the sediments had enough of a phosphorus

limitation that the treatments did not pass a threshold where phosphatase synthesis would

be inhibited. In fact, it is possible that a small enough phosphorus addition may stimulate

phosphatase activity in the microbial community. Penton and Newman (2007) suggested

that until a certain threshold, small shifts in phosphorus concentrations will not change

enzyme-based resource allocation. Another explanation for the lack of responsiveness to

nutrient treatments is that because the sediments were phosphorus limited, the microbes

may be accustomed to producing phosphatase regularly, regardless of the availability of

phosphate. Microbes have been shown to constituently produce enzymes that target the

nutrient that is typically limiting of them, regardless of its availability (Allison and

Vitousek, 2005). While phosphatase activities dropped off by the end of the incubation,

there were not significant differences between the nutrient treatments and the controls,

suggesting that this is probably not a result of the nutrient additions, and possibly a

carbon limitation. The coupling of phosphatase activity and respiration further support

this possibility. In most treatments, there is an initial increase in all activities, followed

by a crash in respiration after the second time point, and a crash in phosphatase activity

after the third time point. If the sediments became carbon limited, this would stop

respiration and carbon mineralization, followed by a shift in the microbial community to

focus its resources on acquiring more recalcitrant carbon. Allison and Vitousek (2005)

suggest that the two most significant regulators of enzyme production are microbial

demand and access to C and N, which are both needed for enzyme synthesis. If carbon

became limiting, which is reflected in the respiration measurements, then the microbial

community would not have carbon to spare for enzyme synthesis.

Endopeptidase activity seemed to be more responsive to nitrogen additions than

phosphatase to phosphorus additions. The controls in both low and high salinity

sediments followed the respiration and phosphatase activity pattern, with initial increases

and then dropping off after the second time point. Where phosphorus alone was added,

endopeptidase activity was initially higher than the other treatments, then dropped off

after the first time point, and increased slightly on day 22, the fourth time point. In the

nitrogen and phosphorus as well as nitrogen only treatment, endopeptidase activity was

lowest, especially in the high salinity sediments. These results contrasted with other

studies, which found that endopeptidase activity was less responsive to increased nitrogen

concentrations than phosphatase activity was to phosphorus additions. The explanation

to this trend is that phosphatase directly makes phosphate biologically available, while

endopeptidase indirectly makes nitrogen available from proteins. Moreover, inorganic

nitrogen may be accessed in multiple ways outside of enzyme acquisition, while

phosphatase is a primary pathway for microbes to access phosphate. Accessing nitrogen

from proteins requires a suite of enzymes in addition to endopeptidase, and this process is

often incomplete and does not result in the complete mineralization to ammonium

(Olander et al., 2000). While the treatments did seem to control the endopeptidase

activity, the overall higher activity in phosphatase activity may be contributed to the more

easily-accessed phosphate, as well as the phosphorous limitation, Additionally, the

increase in endopeptidase activity in many treatments towards the end of the incubation

may be contributed to the carbon limitation. Because endopeptidase goes after proteins,

it helps free up carbon, as well as nitrogen. Olander et al. (2000) found that

endopeptidase activity increased when carbon was limiting. Endopeptidase is a type of

protease, which often are excreted in order to cope with starvation (Karner and

Rassoukadegan, 1995).

There are many methodological problems associated with the short term, small scale

nature of this study. The small samples (about 30 g WW) resulted in small CO2 fluxes,

and it is recommended that future studies use larger sediment samples to measure

respiration. While I added water to the sediments daily to bring them back to their

original wet weight, keeping moisture constant, as well as salinity, was difficult. An

additional problem is that these coastal sediments receive new detrital carbon, face daily

tides, and fluctuations between aerobic and anaerobic conditions. These conditions were

not replicated in the lab and the samples were essentially treated like terrestrial samples.

The degree to which this would impact the results is unknown. Finally, in order to better

understand the dynamics between nutrient limitation, salinity, and microbial activity, a

longer incubation would be preferable. Future studies should be done, adjusting for these

problems. Additional studies could assay for enzymes that target carbon, as a way to

determine the carbon limitation as well as measure total N mineralization, in order to

determine the degree to which N acquisition is through enzyme activity.

Acknowledgements

I would like to thank Anne Giblin, my mentor, for her support and guidance through

this project, and inspiring my interest in this topic. I would like to thank Stefanie Strebel

for helping me develop an enzyme assay protocol, as well as Laura van der Polt for being

patient with the Li-Cor, when I couldn’t be. I also need to thank Rich McHorney and

Carrie Harris for their daily help in lab. I couldn’t have done it without you.

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Figures and Tables:

Figure 1: Map of field site, Little Sippewissett Marsh (Falmouth, MA).

Table 1: A comparison of initial and background measurements between a high and low salinity marsh site.

Figure 2: A comparison of sediment respiration in high and low salinity

sediments and across nutrient treatments over the incubation period.

Figure 3: A comparison of endopeptidase activity in high and low salinity

sediments and across nutrient treatments over the incubation period.

0

1000

2000

3000

4000

5000

6000 HIGH SALINITY

control

N+P

N

P

0

1000

2000

3000

4000

5000

6000

5 8 19 22

LOW SALINITY

control

N+P

N

P

Time (days)

Figure 4: A comparison of phosphatase activity in high and low salinity

sediments and across nutrient treatments over the incubation period

Figure 2: How endopeptidase activity, phosphatase activity, and respiration change over time in low salinity control

sediments.

Figure 6: How endopeptidase activity, phosphatase activity, and respiration change over time in high salinity

control sediments.