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2018 Effects of Nutrient Addition and Two Invasive Plants on Wetland Methane Production Jaymes Dempsey

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Recommended Citation Dempsey, Jaymes, "Effects of Nutrient Addition and Two Invasive Plants on Wetland Methane Production" (2018). Senior Honors Theses. 605. https://commons.emich.edu/honors/605

This Open Access Senior Honors Thesis is brought to you for free and open access by the Honors College at DigitalCommons@EMU. It has been accepted for inclusion in Senior Honors Theses by an authorized administrator of DigitalCommons@EMU. For more information, please contact lib- [email protected]. Effects of Nutrient Addition and Two Invasive Plants on Wetland Methane Production

Abstract Wetlands provide a number of important ecosystem services, but their anoxic conditions also favor the production of several greenhouse gases. Wetlands are an ideal environment for methanogenesis, the process by which carbon dioxide is reduced to methane by wetland microbes (methanogens). As wetlands are the largest natural contributor to the atmospheric methane pool, it is important to understand variables that control wetland methane production. Nitrogen availability is one variable that likely affects methanogen communities. Nitrate drains from agricultural areas, where wetlands act as a nitrogen sink, preventing nitrate from contaminating aquatic systems. Another factor that may affect wetland methanogenesis is vegetation type. In recent years, invasives such as exotic cattail (Typha angustifolia and Typha x glauca} and Phragmites have spread through wetlands, negatively impacting ecosystem processes. To determine the influence of nitrate availability and vegetation type on wetland methanogenesis, we incubated sediment from Phragmites and exotic cattail dominated stands, added increasing concentrations of nitrate, and measured methane production. We hypothesized that if nitrate allows methanogens to be outcompeted by another group of wetland microbes, denitrifiers, then we would observe decreased methane production with high nitrate concentrations.

Degree Type Open Access Senior Honors Thesis

Department Biology

First Advisor Kristin Judd

Second Advisor Aaron Liepman

Third Advisor Marianne Laporte

Keywords Ecology, Wetlands, Methane, Emissions, Global Warming

Subject Categories Biology

This open access senior honors thesis is available at DigitalCommons@EMU: https://commons.emich.edu/honors/605 EFFECTS OF NUTRIENT ADDITION AND TWO INVASIVEPLANTS ON WETLAND

METHANEPRODUCTION

By

Jayznes Dempsey

A Senior Thesis Submitted to the

EasternMichigan University

Honors College

In Partial Fulfillmentof the Requirements forGraduation

With Honors in Biology

SupervisingInstructor (Prin Name and Sign)

Department Head (PrintName and Sign)

1 TABLEOF CONTENTS

ABSTRACT...... 3

INTRODUCTION...... 4

MATERIALS& METHODS...... 16

RESULTS ...... 20

DISCUSSION...... 24

REFERENCES ...... 29

2 ABSTRACT

Wetlands provide a number of important ecosystem services, but their anoxic conditions also favorthe production of several greenhousegases. Wetlands are an ideal environment for methanogenesis, the process by which carbon dioxide is reduced to methane by wetland microbes (methanogens). As wetlands are the largest natural contributorto the atmospheric methane pool, it is important to understand variables that controlwetland methane production.

Nitrogenavailability is one variable that likely affects methanogen communities. Nitratedrains fromagricultural areas, where wetlands act as a nitrogen sink, preventing nitrate from contaminating aquatic systems. Another factorthat may affectwetland methanogenesis is vegetation type. In recent years, invasives such as exotic cattail (Typha angustifoliaand Typha x glauca} and Phragmites have spread through wetlands, negatively impacting ecosystem processes. To determine the influence of nitrateavailability and vegetation type on wetland methanogenesis, we incubated sediment fromPhragmiles and exotic cattail dominated stands, added increasing concentrations of nitrate,and measured methane production. We hypothesized that if nitrateallows methanogens to be outcompeted by another groupof wetland microbes, denitrifiers,then we would observe decreased methane production with high nitrate concentrations. Increased nitratelowered methane production (p=0.03) and heightened denitrificationpotential (p<0.005). Exotic cattail increased methane production (p<0.0 1 ). Our fmdings suggest that nitrate bolsters denitrifiers, causing methanogens to be outcompeted. Our fmdings also suggest that exotic cattail facilitates methane production. Nitrate availability and vegetation type likely influence sediment methane production. It is importantto integratethese conclusions into our understanding of invasive vegetation, water contaminants, and methane production, to consider the best possible methods of mitigation and prevention.

3 INTRODUCTION Wetlands provide myriad ecosystem services. They supply habitat fororganisms and offerrecreational value (Silvius 2000, Millenium Ecosystem Assessment 2005). As a primary contributorto flood mitigation, millions of people depend upon wetlands {Silvius 2000,

Millenium Ecosystem Assessment 2005). Additionally, their unique biogeochemical processes situate wetlands at the center of several significantenvironmental issues. Wetlands are nitrogen sinks, decreasing agriculturalrun-off from contaminating downstreamwaterways {Huang and

Pant 2009). However, wetlands also contributeto global warming {Segers 1998). Wetlands emit methane, a potent greenhousegas, which is produced by sediment microbes (methanogens)

(Segers 1998, Huang and Pant 2009). Methanogen communities are affected by soil properties such as nitrate availability (Kim et al. 2015), and by direct interactions with vegetation (Wolfe and Klironomos 2005). Different plant species offerroot exudates, leachates, and leaf litter that shapes soil microbial communities (Wolfe and Klironomos 2005, Weidenhamer and Callaway

2010). The invasion of exotic cattail to wetlands in the Great Lakes region, followedby the more recent invasion of Phragmitesgenotypes, has resulted in a need to understand the effectsthese plants have on wetland ecosystems.

Wetlands and Methane

Wetlands negatively impact the environment by releasing methane and other greenhouse gases into the atmosphere. Wetlands are the single largest natural methane source, contributing approximately 78% of methane produced in natural systems (Figure l; Bosquet et al. 2006).

Wetlands also account for32-47% of total methane emissions (Denman et al. 2007).

4 Natural Sources of Methane Emissions

■ Wetlands ■ Termites ■ Oceans

Figure 1 Wetlands are the largest natural contributorsto the global methane pool, followedby termites and oceans (based on figure fromBosquet et al. 2006).

This is due to the anaerobic conditions in water-logged wetland sediments, which are ideal for methane-producing microbial processes called methanogenesis (Huang and Pant 2009). As methane is a potent greenhouse gas (28 times stronger than CO2) (Segers 1998), the release of methane fromecosystems can substantially increase global warming and alter natural systems.

Wetland methanogenesis is part of the natural decomposition process. During methanogenesis, the breakdown of organic matter under anaerobic conditions results in accumulation of carbon dioxide, before it is reduced by microbes to methane (Figure 2). These carbon dioxidereducing microbes are members of the domain Archaea, known as methanogens (Segers 1998, Denman et al. 2007). While the atmospheric methane pool has been rising fordecades, in recent years it has experienced significant unexplained growth (Kirschke et al. 2013). Possible explanations include increased anthropogenic combustion of fossil fuels, and increased wetland methanogenesis

(Kirschke et al. 2013; Figure 3). s Methane oxidation: Methanogenesls·

CH4 + 2 OJ ➔ COJ + 2 HiO co,+ 4 H1 ➔ 2 H,o + cH,. CH1 COOH ➔ CO2 + CH,.

Figure 2 Methanogens talce up CO2, and convert it to CH.. This methane may then be released into the atmosphere directly fromthe sediment; alternatively,it may escape through the leaves of wetland plants (Diagram modifiedfrom Brevik 2012).

6 20 - NOAA l.800 - AGAGE -uc1 15 CSIRO 1.750 "' 10 > !;. .r, 0 ..s-C. ., 1,700 .. � �· 5 ,, n l # e ?/ 3 0 ·; // .z: 0- iil C. /I 1,650 a. / r E e I .,.... ,. "0 :r. ., "0 u -5 ,, e I I 1,600 -10

-15 1,550 I 1980 1985 1990 1995 2000 2005 2010 Year Figure 3 Atmosphericmethane levels since 1980 (Modifiedfrom Kirschke et al. 2013). Methane

concentrationsleveled offfrom 2000-2005, then began increasing in 2006. Dotted lines

correspond to methane mole fractions,and solid lines correspond to growth rate.

7 Nutrient Enrichment and Methane

Nutrient enrichment (eutrophication) negatively impacts ecosystems (Smith et al. 1999).

Excess nitrogen and phosphorus in aquatic systems cause increased algae growth, decreased biodiversity, and reduced ecosystem services (Smith et al. 1999). Nitrate, as a mobile formof nitrogen, often drains from agricultural areas into wateiways (Randall and Mulla 2001 , Mitsch et

al. 2005). However, wetlands are oftengeographically situated to intercept this nitrate runoff before it can contaminate aquatic systems (Mitsch et al. 2005). Plant uptake and denitrification then remove this excess nitrogen fromthe wetland ecosystem (Mitsch et al. 2005}. Additionally,

increased nitrate availability has been implicated in the spread of exotic Typha spp. (a US invasive} (Morton 1975), and Phalaris arondinacea, another aggressive wetland species (Craft et al. 2006). Eutrophication also results in fishkills and contaminates water sources (Carpenter et al. 1998). These have become especially significant problem in recent years, due to increasing

nitrogenand phosphorus inputs from agriculture and industry (Carpenter et al. 1998).

Nitrateavailability also affectsmicrobial communities. However, the influence of nitrate

on methanogens is unclear. While some studies have found that nitrate increases methane

production (Liu and Greaver 2009), others have suggested that nitratesuppresses methanogen

growth (Kim et al. 2015). Because both methanogens and denitrifiers(another group of wetland

microbes) require organic carbon (Vymazal 2013), competition may occur between the two

groups. Nitrateaddition potentially favorsdenitrifier growth, bolstering the ability of denitrifiers

to compete with methanogens. This competition may result in the inhibition of methanogen

activity (Figure 4 and 5), causing reduced methane production (Kim et al. 2015}.

Denitrifiers are a groupof microbes that are also active in anaerobic sediments, where

they carry out an ecologically valuable process: denitrification(Knowles 1982). Denitrifying

8 bacteria reduce nitrate(NO,) to nitrogengas (N2), removing nitratecontaminants fromthe ecosystem (Knowles 1982). Therefore,denitrification is a vital process, mitigating the threat that nitrateposes to the environment. Like methanogenesis, denitrificationmust take place under anaerobic conditions (Chen and Lin 1993). However, because denitrificationis carriedout at a higher redox potential than methanogenesis, the addition of nitrate,which elevates redox potential (Chen and Lin 1993), results in conditions more favorableto denitrifiers.Thus, increased nitrate availability likely bolsters denitrifiergrowth. Over the last century, use of nitrogenin fertilizershas led to increased nitratelevels in lakes, rivers, and oceans (Galloway and Cowling 2002). These denitrifier-methanogeninteractions may lead to an environmental trade-offconcerning nitrogen: nitrate potentially suppresses methane production,but is also linked with many environmental problems (Galloway and Cowling 2002, Mcisaac et al. 200 I,

Mitsch and Reeder 1991 ).

9 Figure 4 Interactions between denitrifiers and methanogens in low nitrateconditions.

Methanogens successfully obtain sediment carbon, and produce higher methane quantities.

Arrowsindicate size of interaction.

Figure 5 Interactions between denitrifiers and methogens in high nitrateconditions. If nitrate increases denitrifierpopulations, denitrifierswould potentially compete with methanogens for sediment carbon, reducing methanogen growth,and thus reducing methane production. Arrows indicate size of interaction.

10 Vegetation Type and Methane

Vegetation type is another factorthat influencesmany microbial processes, including methanogen activity (Bubier 1995, Wolfeand Klironomos 2005, Audet et al. 2013). Specific wetland plant species (e.g., Scheuchzeria palustris) have been linked to high methane output

(Bubier 1995), potentially due to ways in which these wetland plants alter soil conditions. Plant species affectsoil hydrology, soil structure, and soil biogeochemistry(Weidenhamer and

Callaway 2010). This, in turn,shapes soil microbial communities (Wolfe and Klironomos 2005).

For instance, various plant species differentiallyinfluence soil nutrient availability by taking up nutrientsand by producing leaf litter (Weidenhamer and Callaway 2010). In addition, some plants release root exudates into the sediment (Caillo-Quiroz et al. 2009, Zhai et al. 2013).

Exudates can be conducive to microbial growth, especially when rich in nutrients(Caillo-Quiroz et al. 2009). These exudates differin quantity and quality depending on plant species and environmental conditions (Wolfeand Klironomos 2005, Zhai et al. 2013). By offeringexudates, leachates, and leaf litter of differingquantity and quality, certain species have the potential to facilitate the growth of certain microbes (Wolfe and Klironomos 2005): belowgrowid microbial commwiities differdepending on abovegroundvegetation (Callaway et al. 2004). Furthermore, increased microbial growth is linked with the production of substratesby plants ( e.g., acetate) that can be used by methanogens (Jespersen et al. 1998). Plants also release oxygen from roots, furthermodifying the surrounding environment in a way that can potentially favorthe growth of one microbial species over another (Jespersen et al. 1998).

Typha is a genus of wetland plants that has been negatively impacting Great Lakes ecosystems (Tuchman et al. 2009). While Typha latifolia(broad leaf cattail) is native to North

America, Typha angustifoliais a non-native species (Grace and Harrison 1986). Typha /atifolia

11 and Typha angustifolia hybridize, resulting in Typha x glauca, a second invasive Typha genotype

(Grace and Harrison 1986; Figure 6). Typha angustifo/ia and Typha x glauca (hereafterreferred to as ..exotic cattail") have colonized much of the United States, including the Great Lakes area

(Morton 1975, Grace and Harrison 1986). Exotic cattail decreases wetland biodiversity

(Tuchman et al. 2009), in part due to the large amounts ofleaf litter that it produces (Mitchell et al. 2011), which reduces seed growth of native plants (Vaccaro et al. 2009). Exotic cattail has also been observedto decrease wetland function; forexample, Typha x glauca may reduce the capacity of wetlands to remove nutrients fromwaterways (Angeloni et al. 2006).

Phragmites australis is a wetland plant that is native to N. America, but a non-native genotype has recently spread extensively thought wetlands in the United States, oftenreplacing exotic cattail in freshwaterwetlands (Chambers et al. 1999; Figure 7). The Eurasian variety of

Phragmiles australis(hereafter referred to as Phragmites) that has recently invaded the Great

Lakes region (Saltonstall 2001, Tulbure et al. 2007) has proven to be especially aggressive

(Saltonstall 2002). The spread of Phragmites is thought to have been facilitatedby many factors, including wetland habitat alteration and wetland eutrophication( Chambers et al. 1999, Tulbure et al. 2007). Concernscenter around the loss of biodiversity resulting fromwetland invasion by

Phragmites, as well as the potential forPhragmites to modify trophic structures or disturbance regimes (Chambers et al. 1999).

12 ......

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Alt.,-,.olib...... �, -�111iA..,_•aw 1•�L-� ....ft,...... - ,t'&'....-a,w,, 1>tlllll...... _1"'...... tlfft1ik,._._1w-,. ..w..._,.,.._..._.._..�.,u ...... 1.-.....­ 'WIS(l)IISII tti.-c> f1__...... ,,..,_..,.�. srm HUIIAKIUM cw,tl

Figure 6 Typha x glauca has invaded Great Lakes wetlands, with detrimentaleffects.

Image Credit: Wisconsin State Herbarium-University of Wisconsin, Madison.

13 ASU 2350i2

a&.1"AODPUJflA DI' oUIIZDIIA ta•,- fll Artiana S•••• � --.T­ IUAOA&

Figure 7 Phragmitesaustralis is a Great Lakes invasive that bas been causing much concernin recent years. Image credit: http://swbiodiversity.org.

14 Experimental Approach To understand factors affecting methane release fromwetlands (Kirschke et al. 2013), in addition to the effects of increased nitratedraining from agricultural systems (Galloway and

Cowling 2002}, we focusedon two broad questions. First, what are the effectsof elevated nitrate concentrationson methanogen activity? Second, what are the effectsof vegetation type (i.e., exotic cattail and Phragmites)on methanogen activity? We hypothesized that if nitrate bolstered the growthof competitors of methanogens, higher nitrateconcentrations would result in reduced methane production. We also hypothesized that if exotic cattail facilitatedmethanogen growth, sediment fromexotic cattail dominated stands would exhibit higher methane production. We

sampled wetland sediment from stands dominated by exotic cattail or Phragmites, and incubated

the sediment in jars. We added various levels of nitrate, beforemeasuring methane production.

To furtherexamine whether competition occurs between methanogens and denitrifiers, we also

measured sediment denitrification potential. We predicted that if nitrate bolsters denitrifier

growth, then the sediment with greater nitrateavailability would exhibit increased denitrification

potential.

1S MATERIALSAND METHODS

Study Site

Sampling was conducted at Paint Creek Wetland, Ypsilanti, Michigan (42°12'N,

83°37W) in both fall of2015 and fallof 2016. Paint Creek's waters come fromthe Paint Creek watershed. Paint Creek is a mitigated wetland, with vegetation of two main types: Phragmites australis, exotic cattail (Typha angustifoliaand Typha x glauca). Paint Creek has swathes ofland dominated by each species, with little overlap.

Sediment sampling forthe firstexperiment was conducted in November201 5; sediment sampling forthe second experiment was conducted in November 2016. A soil corer was used to obtain sediment fromexotic cattail dominated stands (referredto as exotic cattail sediment) and

Phragmites dominated stands (referredto as Phragmites sediment) fromthe topmost 15 cm of sediment. In2015, the sediment was frozenuntil early December, when it was thawed, homogenized, and placed in jars. In2016, homogenization and jar set-up occurred the day after sediment sampling.

Experimental Set-Up

To examine the influenceof nitrate on methane production from wetland sediments, nitratewas added to jar incubations of exotic cattail and Phragmites sediment, and sediment methane production was measured over the course of one week. Three nutrientconcentrations and two sediment types were used, fora total of six treatments, with five replicates per treatment.

In the2015 experiment, 15 g of sediment was placed in each jar ( either Phragmites sediment or exotic cattail sediment}, and 13 mL of nutrient solution was added. Three nutrient

16 concentrations were used: distilled water, 2.5 mM ofKNO3, and 5.0 mM KNO3. To create anoxic conditions, the jars were bubbled with nitrogengas fortwo minutes. The lids were secured, then the jar headspace was flushed out forfour minutes with nitrogengas, by way of rubbersepta embedded in the lids. The jars were incubated in darknessat 25°C forfive days.

The same experimental set-up was used in 2016, except with 30 g of sediment, 40 mL of nutrientsolution (same concentrations),and bubbling and flushing with N2 gas forthree and five minutes, respectively. We also modifiedthe technique to ensure sediments were at atmospheric pressure following flushing by inserting a water-filled syringe in the jar septum and allowing bubbling to occur. The jars were incubated in darknessat 18°C forsix days.

Methane Measurements:

To measure methane production, we used a Gas Chromatograph (Shimadzu 2014, equipped with a flameionizing detector). A syringe and needle were used to sample the jar headspace through the septa in their lids; immediately aftersampling, the gas sample was injected into a Gas Chromatograph to measure the methane concentration.

Sediment Carbon Content

To measure organic carbon content in sediment samples, Phragmilesand exotic cattail sediment was dried in a drying oven at 65°C. The dry weights were recorded. To remove all organic carbon content, the samples were placed in a combustion oven. The final (mineral) weights were recorded, and the difference between the dry weights and mineral weights was used to calculate exotic cattail and Phragmitessediment carbon content.

17 DenitrificationMeasurements:

To furtherassess the effectsof nitrate on microbial processes, we measured sediment denitrificationpotential using the acetylene-inhibition method (as described by Groffinan et al.

2006). This experiment was conducted only in 2016, using the sediment fromthe methane experiment.

Sediment was removed fromthe jars, and approximately 7 g of each replicate was added into an Erlenmeyer flask. Flasks were capped with rubbersepta. A needle was used to inject 6 mL of acetylene through the septa to prevent the conversion ofN2O to N2. The flaskswere shaken on a shaker-table for30 minutes, beforean additional 3 mL of acetylene was injected.

The flaskswere shaken foran additional 60 minutes, then the flaskheadspace was sampled with a syringe. A set of vials was flushed with nitrogen to ensure anoxic conditions, and the samples were injected into the vials.

Approximately one month aftergas sampling, the samples were run on a gas chromatograph (equipped with an electroncapture detector) to measure N2O accumulation. The standards forthe GC machine were collected on the same day as the samples, and stored in. evacuated vials.

Data Analysis

Methane standards of known concentration were used to produce a standard curve and calculate jar methane concentration. To better compare data between years, methane measurements were converted to micrograms of carbon per day per dry gramof sediment using the following equation:

18 A two-way ANOVA was runon both the methane and denitrification data, and a two­ tailed t-test on the exotic cattail /P hragmites denitrificationcontrol. A two-tailed t-test was run on the sediment carbon content data, and on the methane production data for2015 and 2016.

19 RESULTS

Methane Emissions

To test the hypothesis that nitrate additions inhibit methanogen activity, we measured methane production of Phragmites and exotic cattail sediments supplied with varying nitrate additions. In fall 2015, methane production in sediment with nitrate addition was many times lower than methane production sediment with no nitrateaddition (p<0.01; Figure 8). The same experiment examined whether exotic cattail facilitated methane production. In fall2015, methane production in exotic cattail sediments was over twice that of Phragmites sediments

(p<0.01; Figure 8).

0.09

bij0.08

� 0.07 r -� 0.06 C O 0.05

-6 0.04

a.. 0.03 C � 0.02

� 0.01

0 0 .. 2.5 .5 ..... Nitrate Concentration (mM) ■ Exotic Cattail ■ Phragmites

Figure 8: Effects of nitrate concentration on methane production in sediments from exotic cattail and Pliragmites sediment with standard error. Fall, 2015. N=5.

20 0.003 I 0.003 0 +-' - 0.002

0 � '-- ct1 0.002 � "'C QJ L3' 0.001

+-' 0.001 0 -- 0 2.5 5 Nitrate Concentration (mM)

■ Exotic Cattail ■ Phragmites

Figure 9: Effectsof nitrateconcentration on methane production in sediments fromexotic cattail

and Phragmites sediment with standard error. Winter 2016. N=5.

In fall 2016 samples, methane concentrationin controljars aftersix days was notably

higher than methane concentrations in the low and high nutrient treatments, especially in the

exotic cattail sediment (p=0.15; Figure 9). Samples fromexotic cattail sediment produced

approximately double the methane than those fromPhragmites sediment aftersix days (p<0.01;

Figure 9). The data suggest that nitrate does inhibit methanogen activity, and that exotic cattail

bolsters methanogen growth.

Differencebetween 2015 and 2016 methane production was significant, with 2015

methane production nearly an order of magnitude higher than that of2016 methane (t=2.51;

p=0.01). This suggests that there are other important factors that affectmethane production in

wetlands.

21 Sediment Carbon Content

To examine sediment carbon content, we combusted exotic cattail and Phragmites sediment in a muffle furnace, and calculated the percent carbon present in the sediment. In 2015, exotic cattail sediment had significantly higher carbon content as compared to Phragmites

{t=3.72; p<0.005; Table 1). This trend was even more notable in 2016, when exotic cattail sediment had nearly 50% higher carbon content as compared to Phragmites sediment (t=8.74; p<0.005; Table I). The data suggest that exotic cattail alters soil properties by increasing sediment carbon content.

Table 1: Relationship between vegetation (Exotic cattail and Phragmites) and percent carbon content ± standard error. N=15 and N=5 for 2015 and 2016, respectively.

% Carbon 2015 2016

Exotic 31.7±0.01 34.9±0.01 Cattail 21.S Phragmites 26.7±0.008 ±0.007

DentrificationPotential

To furtherexamine whether nitrateadditions increase competition between methanogens and denitrifiers, denitrification potential of 2016 sediments was measured.. Nitrate addition nearly doubled denitrification potential (p<0.005; Figure 10). In the control,exotic cattail sediments had significantlylower denitrificationpotential as compared to Phragmites {t=7.35; p<0.0 1; Figure 10), though the effectof vegetation type on denitrificationwas not significant

22 overall (F0.202; Figure 10). This suggests that nitrate addition does bolster denitrifier

communities, increasing denitrifier-methanogen competition. This also suggests that Phragmites

may facilitatedenitrification.

co 400 � C Q,J 350

C 300 0 :P � 250 � "C ..:: ...... ·-...... ti.o 200 -� E � §: 150 0- 100 50 0 5 Nitrate Concentration (mM)

■ Phragmites ■ Exotic Cattail

Figure 10: Effects of nitrate concentration and vegetation on denitrificationpotential with

standard error. Winter 2016. N=S.

23 DISCUSSION

To examine the relationship between nitrate, vegetation type, and wetland methane

production, we incubated sediment in jars with differentlevels of nitrateavailability and

measured methane production. We hypothesized that if nitratebolstered the growth of methanogen competitors, higher nitrateconcentrations would result in reduced methane

production. We also hypothesized that if exotic cattail facilitatedmethanogen growth, exotic

cattail sediment would have higher methane production. Our results support these hypotheses.

Nitrateavailability was negatively correlatedwith decreased methane production and positively

correlatedwith denitrification potential. These findingssuggest that denitrifierscompete with

methanogens, inhibiting methane production. Sediments from exotic cattail stands produced

more methane than Phragmites sediments.

Effectsof Nitrate Addition on Sediment Methane Production

Our findings support the competition hypothesis, which suggests that the increased

denitrification and inhibited methanogenesis is caused by denitrifiers{Kim et al. 2015), which

are bolstered by higher nitrateavailability (Huang and Pant 2009). Given the competition over

carbon substratebetween the microbes discussed, this denitrifiergrowth appears to be at the

expense of the methanogen population (Fang and Zhou 1999). Previous studies have foundthat

denitrifiers outcompete methanogens in certain decomposition processes (Fang and Zhou 1999).

A second possibility is that nitrateincreases denitrification, and that toxic intermediates,such as

N02, are produced during this process, which inhibit methanogen activity (Banihani et al. 2009).

Even small increases in nitratehave been observed to inhibit methanogenesis (Yuan and Lu

2009). These mechanisms are not mutually exclusive; methanogens and denitrifiers may be

impacted by both. To determine the extent to which each of these factorsaffects methane

24 production, furtherstudies could be conducted in which methane production is measured after the sediment has been exposed to denitrification intermediates. If methane production decreases, this would offerevidence that methanogens are inhibited by these gases. The extent to which they are inhibited could then be investigated with a more careful quantitative analysis in which methanogens are exposed to differingconcentrations of denitrificationintermediates.

Our results are generally consistent with those fromother studies. Several studies founda similar relationship between nitrateand methane production (Kim et al. 2015, Yuan and Lu

2009, Banihani et al. 2009). One study had results notably differentfrom ours, indicating increased methane production with increased nitrate (Liu and Greaver 2009). However, this study did not specifically isolate production fromsediment, but instead conducted a meta­ analysis that considered overall production fromwetlands (Liu and Greaver 2009). Our results account only for production directly fromsediments, and do not include other potential contributorsto the atmosphericmethane pool, such as methane transportedthrough plant aerenchyma systems (Ding et al. 2005). In addition, methane production fromwetlands may be context-dependent, depending on factors such as water level and climate. Future research could consider various factorsthat influence the effectsof nitrate addition on methane production by conducting similar jar incubations with varyingwater levels or temperature.

Our findings indicate significant year-to-year variation in methane production. This may

have been caused, in part, by the higher incubation temperature in 2015 as compared to 2016.

Ideally, incubation would have been conducted at the same temperatureboth years. However, it

is unlikely that this can account completely forthe observed variation. Another possible cause

could be water level differences at the sampling site. Lower water levels allow forgreater

sediment oxygen availability, which would raise the redox potential and favordenitrifiers. This,

25 in turn,may suppress methanogen communities and alter methanogen community composition.

Future studies should investigate annual and seasonal differences in methane production, and consider such additional variables that might be correlated with these differences. Another study on the relationship between nitrate and denitrification revealed a relationship parallel to our results, with increased nitratecorrelated with increased denitrification(Hanson et al. 1994).

There is also evidence that the relationship between nitrate, denitrification, and methanogenesis is not as straightforward as it appears, with other nutrients,such as carbon and phosphorus, altering the influence of nitrate {Kim et al. 2015). For example, phosphorus was observed to decrease methane production when added alongside nitrate; phosphorus was also observedto increase denitrificationpotential when added in conjunction with carbon and nitrate {Kim et al.

2015). This is another avenue forfurther study: methane production and denitrificationpotential could be measured in samples containing various combinations of nitrate, phosphorus, and carbon.

Nitrate is a waterwaycontaminant; over the last century, the use of nitrogenin fertilizers has led to increased nitratelevels in lakes, rivers, and oceans with detrimental effects {Galloway and Cowling 2002). Increases in nitrateavailability have also been implicated in eutrophication

{Mcisaac et al. 2001), adverse health effectson both humans and animals, and water acidification{Camargo and Alonso 2006). Yet nitrateappears to provide some benefit: it decreases methane production. Therefore, the impacts of nitrateaddition into waterways are not simple, but multifaceted.To what extent does this suppression of methane production counterbalance the negative effects caused by nitrate pollution? Further studies could attempt to quantify the extent to which nitrateinfluences methanogenesis by conducting jar incubations with more incremental differences in nitrate concentration.

26 Effectsof Vegetation on Sediment Methane Production

Our results support the hypothesis that exotic cattail facilitate methanogen growth. In both years, exotic cattail sediments bad higher methane production than Phragmites sediments.

The results also suggest that Phragmitesaustralis facilitates denitrifier growth, as denitrification in the Phragmites sediment controlwas significantly higher than denitrificationin exotic cattail sediment control.

One possible mechanism forincreased methane production in exotic cattail sediment is that exotic cattail raises the carbon content of sediment, bolstering methanogen communities.

Plants produce substrates that have been correlatedwith increased microbial growth {Jesperson et al. 1998), and carbon is required by methanogens (Vymazal 2013). If Typha spp. offer these carbon substrates, this is a potential source of methanogen growth. Furthermore, a hypothesis

that focuses on carbon content is supported by our results, which found increased carbon content

in exotic cattail as compared to Phragmites sediment (Table 1). Future studies could examine

potential correlations between sediment carbon content and methane production, and at the

effectsof exotic cattail leaf litter on methane production by conducting jar incubations with leaf

litter additions.

Another possible mechanism for increased methane production in exotic cattail sediments

is that Phragmites may aerate sediments at an increased rate. Sediment aeration, in which oxygen

is provided to sediment through plant roots, raises redox potentials (Jespersen et al. 1998). This,

in turn,favors denitrifiers, as denitrification talces place under a higher redox potential than

methanogenesis (Chen and Lin 1993 ). If exotic cattail aerates sediment at a greater rate than

Phragmites australis, this could be the mechanism behind our observedfacilitation of

methanogenesis. This would also explain why Phragmites appeared to facilitatedenitrification

27 (Figure 10). However, there appears to be significantvariation in terms of the effects that

Phragmites spp. and exotic cattail spp. have on methane production, with one study observing no clear link between either Phragmites or exotic cattail stands and methane production (K.aki et al.

2001). Another study observedlittle difference between Typha angustifoliaand Phragmites australis microbial biomass (Findlay et al. 2002). A third study, however, found that Phragmites australis reduced methane production (Grunfeld and Brix 1999). A useful next step would be to compare the microbial communities around the roots of these plants directly, by sampling for

DNA to identify the microbes present. If vegetation type affects methanogen community size and community composition, then we expect significant differences in methanogen populations around exotic cattail roots as compared to roots of Phragmites australis.

Invasive plants, such as Phragmitesaustralis and exotic cattail, have been observed to colonize wetlands, alter habitat structure, and reduce biodiversity (Chambers et al. 1999, Zedler and Kercher 2004). The ability of Phragmites australis to dominate ecosystems is of particular concern,which has led to attempts to remove these invaders fromwetlands (Ailstock et al.

2001). However, it appears that Phragmites australis may offer an ecosystem service: the reduction of methane production fromwetlands. Therefore,it is important to consider all factors before attempting to exterminatePhragmites australis and restore native wetland vegetation.

Further studies could also examine the difference between methane production in native versus restored wetlands.

Conclusion

Given the increasing threat of global climate change as the result of rising levels of greenhouse gasses in the atmosphere, in part due to methane production (Seger 1998), and the current high levels of nitraterunoff (Naslas 1994), it is necessary to consider mitigation and

28 prevention of methane production and nitraterunoff . As wetlands play a significantrole in the methane cycle and in nitratecycling (Seger 1998, Denman 2007), it is essential that we consider the type and extent of their involvement. We must unravel the complex web of interacting factors in order to recommend avenues for change. Our results indicate that nitratedecreases methane production; this is likely due, at least in part, to the beneficial effects that nitrate has on denitrifiergrowth, bolstering the ability of these microbes to compete with methanogens. Our results also indicate that exotic cattail bolsters methane production, potentially due to effects of exotic cattail on sediment carbon content. Future studiesshould continue to look at the links between these variables, and especially assess the relationship between methanogenesis and nitrate, to better comprehend the intersection between these potentially harmfulfactors.

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