Peat porewater dissolved organic carbon concentrationand lability increase with warming: a field temperaturemanipulation experiment in a poor-fen

Evan S. Kane • Lynn R. Mazzoleni • Carley J. Kratz • John A. Hribljan •

Christopher P. Johnson • Thomas G. Pypker • Rodney Chimner

Received: 8 October 2013 / Accepted: 9 January 2014

� Springer International Publishing Switzerland 2014

Abstract Studies conducted across northern Europe

and North America have shown increases in dissolved

organic carbon (DOC) in aquatic systems in recent

decades. While there is little consensus as to the exact

mechanisms for the increases in DOC, hypotheses

converge on such climate change factors as warming,

increased precipitation variability, and changes in

atmospheric deposition. In this study, we tested the

effects of warming on peat porewater composition by

actively warming a peatland with infrared lamps

mounted 1.24 m above the peat surface for 3 years.

Mean growing season peat temperatures in the warmed

plots (n = 5) were 1.9 ± 0.4 �C warmer than the

control plots at 5 cm depth (t statistic = 5.03,

p = 0.007). Mean porewater DOC concentrations

measured throughout the growing season were 15 %

higher in the warmed plots (73.4 ± 3.2 mg L-1) than in

the control plots (63.7 ± 2.1 mg L-1) at 25 cm

(t = 4.69, p \ 0.001). Furthermore, DOC from the

warmed plots decayed nearly twice as fast as control plot

DOC in laboratory incubations, and exhibited lower

aromaticity than control plot porewater (reduction in

SUVA254 in heated plots compared with control plots).

Dissolved organic nitrogen (DON) concentrations

Responsible Editor: Jan Mulder

E. S. Kane

U.S. Forest Service, Northern Research Station,

Houghton, MI 49931, USA

E. S. Kane (&) � C. J. Kratz � J. A. Hribljan �C. P. Johnson � T. G. Pypker � R. Chimner

School of Forest Resources and Environmental Science,

Michigan Technological University, Houghton,

MI 49931, USA

e-mail: eskane@mtu.edu

C. J. Kratz

e-mail: cjkratz@mtu.edu

J. A. Hribljan

e-mail: jahriblj@mtu.edu

C. P. Johnson

e-mail: cpjohnson8@wisc.edu

T. G. Pypker

e-mail: tgpypker@mtu.edu

R. Chimner

e-mail: rchimner@mtu.edu

L. R. Mazzoleni

Department of Chemistry, Michigan Technological

University, Houghton, MI 49931, USA

e-mail: lrmazzol@mtu.edu

Present Address:

C. P. Johnson

Department of Forest and Wildlife Ecology, University of

Wisconsin-Madison, 1630 Linden Drive, Madison,

WI 53706, USA

Present Address:

T. G. Pypker

Department of Natural Resources, Thompson Rivers

University, Kamloops, BC V2C OC8, Canada

123

Biogeochemistry

DOI 10.1007/s10533-014-9955-4

tracked DOC patterns as expected, but the amount of

dissolved N per unit C decreased with warming.

Previous work has shown that warming increased net

primary production at this site, and together with

measured increases in the activities of chitinases and

glucosidases we suggest that the increased DOC

concentrations observed with warming were derived

in part from microbial-plant interactions in the rhizo-

sphere. We also detected more nitrogen containing

compounds with higher double bond equivalents (DBE)

unique to the warmed plots, within the pool of

biomolecules able to deprotonate (16 % of all com-

pounds identified using ultrahigh resolution ion elec-

trospray mass spectrometry); we suggest these

compounds could be the products of increased plant,

microbial, and enzyme activity occurring with warming.

With continued warming in peatlands, an increase in

relatively labile DOC concentrations could contribute to

dissolved exports of DOC in runoff, and would likely

contribute to the pool of efficient electron donors (and

acceptors) in the production of CO2 and CH4 in

terrestrial and aquatic environments.

Keywords Peat � Climate change �Extracellular enzyme � Dissolved organic

nitrogen � Dissolved organic carbon � Ericaceae �Sedge

Introduction

Long-term studies of northern peatlands and brown-

water fed freshwater bodies of Europe and North

America have revealed increasing concentrations of

dissolved organic carbon (DOC) over the last two

decades (Freeman et al. 2001a; Driscoll et al. 2003;

Worrall et al. 2003; Freeman et al. 2004; Evans et al.

2005; Yallop et al. 2010; Urban et al. 2011). In carbon

rich ecosystems such as northern peatlands, DOC

pools are significant (Battin et al. 2009) and fluxes of

DOC can be similar in magnitude to that of long-term

C accrual in dead organic matter (Moore 2003; cf.

Rapalee et al. 1998). Not only are DOC pools and

fluxes important to consider with respect to carbon

cycling, but changes in DOC concentrations affect

critical ecosystem properties in aquatic environments,

such as pH (Kullberg et al. 1993), light attenuation

(Bukaveckas and Robbins-Forbes 2000), mineral

complexation and the mobility of heavy metals (Urban

et al. 1990; Kolka et al. 2001), peat porewater

electrochemistry and microbial activity (Heitmann

et al. 2007; Alewell et al. 2008; Keller et al. 2009) and

foodweb structure (Hessen 1992). It is therefore

important to understand the direct mechanisms driving

the observed increases in DOC concentrations.

To date there is still uncertainty associated with

identifying the primary drivers of observed increases

in DOC concentrations in a wide range of ecosystems

(Sucker and Krause 2010; Clark et al. 2010; Preston

et al. 2011). Debate surrounding the isolation of single

mechanisms responsible has focused on factors related

to climate change, notably increasing temperatures

(Freeman et al. 2001a), changes in atmospheric

deposition (Monteith et al. 2007; Clark et al. 2010;

Evans et al. 2012), altered precipitation or drainage

patterns (Strack et al. 2008; Holl et al. 2009; Kane

et al. 2010), and increased atmospheric CO2 concen-

tration (Freeman et al. 2004; Fenner et al. 2007;

Johansson et al. 2009). At the heart of uncertainty

concerning a direct mechanism for increasing DOC

concentrations in streams (considered as an export

from the terrestrial system) is distinguishing between

changes in soil DOC concentrations versus changes in

runoff patterns and hydrologic connectivity (Tranvik

and Jansson 2002; Roulet and Moore 2006). As such,

studies involving landscape temperature gradients

(Kane et al. 2006; Borken et al. 2011) or mesocosms

(Judd and Kling 2002; Blodau et al. 2004) report

increased soil DOC concentrations with warming, but

consensus as to the direct effects of temperature on

DOC concentrations in runoff are mixed (Pastor et al.

2003; Porcal et al. 2009; Clark et al. 2009; Preston

et al. 2011). Notwithstanding, peatlands (or Histosols)

in particular have been shown to be strong sources of

DOC export in comparisons across diverse catchment

types (Aitkenhead and McDowell 2000; Alvarez-

Cobelas et al. 2012). Improved understanding of the

underlying mechanisms of changes in DOC concen-

trations in peatlands is necessary to understand

observed changes in DOC export and to predict future

changes in DOC.

The concentration of peat porewater DOC is largely

dependent on the relativity between DOC production

and consumption in the soil matrix, and both of these

processes are likely to increase with temperature in

northern organic soils. DOC production increases with

temperature-mediated increases in plant production,

owing to root exudates (Kuzyakov 2002; Freeman

Biogeochemistry

123

et al. 2004) and changes in the types and activities of

extracellular enzymes exuded (Fenner et al. 2007) and

increased inputs and leaching of plant litter and soils

(McDowell and Likens 1988; Moore and Dalva 2001;

Park et al. 2002). DOC production also increases as the

turnover of dead organic matter increases (which

increases with temperature), as the water-soluble

products of decomposition accumulate (Kalbitz et al.

2000; Michalzik et al. 2001). DOC mineralization can

also increase with temperature (Moore et al. 2008),

with rates of change being dependent on structural

characteristics of the DOC (Christ and David 1996;

Michaelson et al. 1998; Wickland et al. 2007). Since

DOC is produced by microbes while also being a labile

substrate for microbial activity (McKnight and Aiken

1998), production and consumption relationships of

DOC are hard to disentangle (Muller-Wegener 1988;

Neff and Hooper 2002; Kalbitz et al. 2003). Field

manipulation studies with controls over variables

which co-vary with temperature are necessary to test

specific mechanisms for the observed increases in

DOC concentrations with warming (cf. Jassey et al.

2013).

Accumulation of the water-soluble products of

decaying organic matter in response to warmer tem-

peratures is also affected by enzyme production and

biophysical limitations to the microbial decay of DOC.

Microbial acquisition of nutrients is primarily exe-

cuted through the release of extracellular enzymes.

These enzymes depolymerize organic matter hydro-

lytically (Sinsabaugh et al. 2008) and oxidatively

(Sinsabaugh 2010) to release soluble components

which can be assimilated by microorganisms (see

reviews by Munster and de Haan 1998; Nannipieri

et al. 2002; Caldwell 2005; Burns et al. 2013). Exactly

how sensitive enzyme activities are to changes in

temperatures can be described using the first principles

of thermodynamics, with the sensitivity of organic

matter degradation being directly related to the acti-

vation energy of a particular enzyme (Wallenstein et al.

2011). This is an important additional consideration in

northern organic soils because the temperature sensi-

tivities of enzyme activity are generally higher in

colder environments (Koch et al. 2007). For example,

Wallenstein et al. (2009) found that temperature

explained 72 % of the variation in modeled hydrolytic

enzyme activity (b-glucosidase) in tundra soil. More-

over, Jassey et al. (2012) recently demonstrated that

oxidative enzyme activity (peroxidase) increased by

over 30 % with passive warming in a Sphagnum

peatland. To date the direct effects of enzyme activity

on DOC concentrations in peatland ecosystems have

largely assumed DOC accumulates when enzyme

function is inhibited, such as by complexation with

organic molecules (Joanisse et al. 2007) or with the

presence of oxygen (Freeman et al. 2001b, 2004).

Relationships between DOC and extracellular enzyme

activities are not straightforward, and there may be

positive feedbacks wherein labile DOC promotes

hydrolytic enzyme production, which in turn increases

the quantity of DOC (Shackle et al. 2000). However,

there is almost no work investigating in situ temper-

ature effects on hydrolytic and oxidative enzyme

function, and how these factors in turn effect DOC

concentrations in northern peatlands.

To examine the direct effects of warming on peat

porewater composition, we actively heated the soil

surface of a poor fen peatland ecosystem in northern

Michigan, USA for three growing seasons (Johnson

et al. 2013). We used a combination of spectral

indexes, porewater incubation studies (for mineraliza-

tion and enzymatic activities), ion chromatography,

and ultra-high resolution electrospray ionization mass

spectrometry to test our hypotheses. We hypothesized

that DOC inputs to porewater derived in part from

microbial/plant interactions in the rhizosphere would

outstrip losses from DOC mineralization in the heated

plots, leading to an increase in porewater DOC quantity

and quality (or lability) with warmer temperatures. We

expected higher activities of both hydrolytic and

oxidative enzymes with warming, which would also

increase DOC concentrations through the cycling of

phenolic compounds. Alternatively, warming could

cause a reduction in the solubility of oxygen, which

would lead to a decrease in phenol-oxidase activity.

We hypothesized that microtopography (hummock vs.

hollow or lawns) would be a significant arbiter of

temperature effects on DOC concentrations, owing to

changes in thermal conductivity and relative water

table position with change in peat microtopography.

Methods and materials

Study site and instrumentation

The study site is located in a poor fen peatland on the coast

of Lake Superior in the Upper Peninsula of Michigan,

Biogeochemistry

123

USA near the town of Pequaming (46.85�N 88.37�W,

Elev. 183 m). The peatland initiated *2,225 years ago

on a tombolo in Lake Superior and has 290 cm of sedge

and Sphagnum peat overlying sandy mineral soil (Bois-

vert 2009). The peatland is categorized as a barrier beach

lagoon-tombolo (Albert et al. 2005). Hummock and lawn

microtopography is prevalent, with vegetation typical of

poor fens in the region (trees: Picea mariana and Larix

laricina; shrubs: Chamaedaphne calyculata, Ledum

groenlandicum, Kalmia polifolia, Andromeda glauco-

phylla, Myrica gale, Vaccinium oxycoccos; herbs: Dro-

sera rotundifolia, Sarracenia purpurea; sedges: Carex

oligosperma, C. exilis, C. utriculata; mosses: Sphagnum

fuscum, S. rubellum, S. magellanicum, S. papillosum).

The average annual temperature and total precipitation

for the region are 4.5 �C and 833 mm, respectively.

Our study consisted of 10, 2 m by 1 m plots that

were divided equally between hummocks and lawns.

Plot locations were chosen based on available micro-

topography and were randomly subdivided into heated

or control treatments. The heating treatments consisted

of infrared lamps (MRM-1215, Kalglo Electronics

Company Ltd., Bethlehem, PA, USA; 1,500 W,

165 cm long) positioned 124 cm above the peat

surface on steel conduit that was anchored into the

underlying sandy substrate, as described in detail by

Johnson et al. (2013). Elevated boardwalks and planks

were built to allow access to all plots without damaging

vegetation. Warming occurred in three different peri-

ods: 2008 from June to November (initiation phase,

data not presented); 2009 from May to December; and

2010 from April to November.

The study area was instrumented with an air

temperature/relative humidity sensor (CS-215, Camp-

bell-Scientific Inc., Logan, UT, USA; height =

183 cm) and a tipping bucket rain gauge (TE525WS,

Texas Instruments, Dallas, TX, USA; height =

100 cm) wired to a datalogger (CR1000, Campbell-

Scientific Inc.). Data were collected every minute, and

were binned into 20 min averages. Within each plot, a

thermocouple (Type-T copper constantan, Campbell-

Scientific Inc.) was installed 5 cm below the peat

surface and wired to a multiplexor (AM25T, Camp-

bell-Scientific Inc.) and a datalogger. Water table

levels were measured at 1-h intervals in a 1.5 m long,

10 cm diameter PVC well with a water table logger

(Levellogger Junior, Solinst, Georgetown, Ontario,

Canada) and a barometric pressure logger (Barrolog-

ger Gold, Solinst, Georgetown, Ontario). Volumetric

water content at 0–6 and 0–12 cm vertically beneath

the Sphagnum moss surface at each plot was measured

manually with a HydroSense� Water Content Sensor

(Campbell Scientific Inc., Australia) in July–October

of 2010. Temperature data from the nearby MTU Ford

Center Research Forest in Alberta, MI were used to

compare mean growing season temperatures and

summed degree-days (SDD; sum of mean daily

air temperatures [10 �C) from June–September,

2009–2011.

Porewater collection and analysis

Porewater was harvested from piezometers approxi-

mately bi-weekly throughout the growing seasons of

2009–2010, and approximately monthly following the

cessation of the heating treatments in 2011. Piezom-

eters were constructed of 2.54 cm diameter polyvinyl-

chloride pipe with 10 cm slotted regions covered with

40 lm Nitex nylon mesh. Slotted regions were

centered at depths of 25 and 50 cm beneath the peat

surface. Prior to sampling for chemical analyses, the

piezometers were pumped dry and allowed to

recharge. A small amount of pore water was pumped

into the collection flask as a pre-rinse for sample

collection in high density polyethylene (HDPE) Nal-

gene bottles. Approximately 150 mL of sample was

collected, and then samples were kept on ice packs in a

cooler and brought back to the laboratory where they

could be filtered through 0.45 lm nylon membrane

filters (less than a day from time of collection). After

filtration, samples were split into three parts: (1)

50 mL was acidified (pH 2) and refrigerated prior to

DOC and TDN analysis, (2) 50 mL was kept frozen

prior to anion and organic acid analysis, and (3) 10 mL

was diluted (1:10) and its ultraviolet absorbance at

k = 254 nm was immediately determined on a spec-

trophotometer using a 1 cm quartz cuvette (Spectra-

Max M2 multimode microplate reader; Molecular

Devices Corporation, Sunnyvale, CA, USA). Blanks

of deionized water (with and without acidification)

were run with all analyses (approximately one for

every 15 samples). DON and TDN were run with a

TOCV Analyzer with TDN module, (Shimadzu Sci-

entific Instruments, Columbia, MD, USA), and nitrate

(NO3-), acetate, formate, and oxalate were deter-

mined with an ICS-2000 ion chromatograph with an

IonPac AS11 separator column (Dionex Corporation,

Bannockburn, IL, USA). Ammonium (NH4–N) was

Biogeochemistry

123

determined spectrophotometrically using salicylate

and cyanurate reagents (Hach Co., Loveland, CO,

USA) following Sinsabaugh et al. (2000). Dissolved

organic N (DON) was determined as the difference

between TDN and dissolved inorganic N (DIN;

NH4–N plus NO3–N). We calculated specific ultravi-

olet absorbance (SUVA) by dividing absorbance at

k = 254 nm by DOC concentration, and reported

SUVA254 of DOC in units of L mg C-1 m-1. Total

dissolved iron concentrations were quantified spec-

trophotometrically (ammonium thioglycolate reagent,

Martini instruments MI 408 Iron High Range Meter,

Rocky Mount, NC, USA) throughout the summer of

2010 to determine possible interferences with

SUVA254 (cf. Weishaar et al. 2003). Dissolved oxygen

(DO) and specific conductivity were measured at 25

and 50 cm depths throughout the growing season.

Measurements were made within a slotted PVC well

(capped when not being measured) with Hach probes

(LDO101 and CDC401 probes with a HQ40d18 meter;

Hach Co., Loveland, CO, USA) which were calibrated

monthly with water-saturated air for DO and at

1,000 lS cm-1 (as NaCl) for conductivity.

Ultrahigh-resolution ESI-FT-ICR MS analysis

A 10 mL aliquot was prepared for Electrospray

Ionization Fourier Transform Ion Cyclotron Reso-

nance Mass Spectrometry (ESI-FT-ICR MS) analysis

using solid phase extraction (Strata-X; Phenomenex

Inc., Torrence, CA, USA). For this purpose, 10 mL of

filtered and frozen porewater from two warmed plots

and two control plots in 2009, when the DOC

concentration differences were the greatest between

treatments at 25 cm (day of year 249). These samples

were applied to Strata-X solid phase extraction

cartridges (pre-conditioned with methanol and aceto-

nitrile), the cartridges were rinsed with high purity

water and dried, and DOC was washed out with

1.5 mL methanol (CHROMSOLV for HPLC). Sam-

ples were stored at -5 �C until further analysis.

The ultrahigh resolution mass spectrometric ana-

lysis was performed on a 7 tesla FT-ICR MS (LTQ FT

Ultra, Thermo Scientific) equipped with an ESI

source. For the analysis, the signal was optimized

with methanol dilution. The solution was directly

infused at 4 lL min-1 into the ESI interface. The ESI

probe was placed in position ‘‘B’’ and the needle

voltage was set between -3.7 and -3.8 kV (blanks

were run at -4.0 kV). The sample delivery apparatus

was flushed after each run with a minimum of 500 lL

of methanol/water (50/50) and 100 % methanol until

background noise level was reached. Negative ion

mass spectra were collected for the mass range of

100 \ m/z \ 1,000. The mass resolving power was set

at 400,000 (defined at m/z 400). Automatic gain

control was used to consistently fill the instrument

with the same number of ions (n = 1 9 106) for each

acquisition to avoid space charge effects from over

filling the mass analyzer. The instrument was exter-

nally calibrated in negative ion mode with a standard

solution of sodium dodecyl sulfate and taurocholic

acid, and the resulting mass accuracy was better than

2 ppm. Over 200 individual mass spectra were

collected and stored as transients by use of Thermo

Xcalibur software (Thermo Scientific, USA). Repli-

cate mass analysis was performed. Variances in the

analyte intensity and the reproducibility of low

relative abundance (RA) signals were calculated from

the replicate analyses.

Ultrahigh-resolution FT-ICR MS data were pro-

cessed using Sierra Analytics Composer software

previously described (Mazzoleni et al. 2010; Putman

et al. 2012). Briefly, *200 transients recorded in the

time domain were co-added to improve analyte detec-

tion (Kujawinski 2002; Stenson et al. 2003). The

molecular formula calculator was set to allow up to 70

carbon (C), 140 hydrogen (H), 25 oxygen (O), and 4

nitrogen (N) atoms per molecular formula composition.

A higher number of N was tested during the method

development. However, a higher number of chemically

unreasonable formula assignments without a significant

increase to the proportion of the assigned total ion

current was observed. Thus, the more conservative

method (with N B 4) was selected. The molecular

formula calculator, based on the PREDATOR algorithm

(Blakney et al. 2005), uses a Kendrick mass analysis

(Hughey et al. 2001) to sort ions into CH2 ‘‘homologous

series’’ and then assigns the de novo molecular formulas

B500 Da. In other words, all of the ions[500 Da either

belong to a CH2 series with de novo formulas B500 Da

or were not assigned. A threshold of 6 times the root

mean square values between 950\ m/z \ 1,000

(RA C 0.4 %) was applied to the data. Formulas

containing 13C corresponding to the monoisotopic

masses were also assigned and evaluated. Blank samples

were analyzed using the same method. The molecular

formulas resulting in measurement errors[3 ppm were

Biogeochemistry

123

discarded. Additional data filtering of the assigned

formulas was done by applying rules and assumptions as

described by Koch et al. (2005).

Enzyme activity assays

Enzyme assays were conducted on porewater samples

harvested prior to and following the senescence of

vegetation (early September and late October, respec-

tively). The enzymes assays were conducted using a

96-well plate format according to Sinsabaugh et al.

(2005). There were 16 technical replicates per sample.

Each replicate contained 200 lL of porewater and 50 lL

of substrate. Hydrolytic enzyme activity was estimated

using 200 lM fluorometric substrates including acid

phosphatase, b-glucosidase (glucosidase), cellobiohy-

drolase (cellulase) and N-acetyl-glucosaminidase (chiti-

nase). The hydrolytic enzyme assays included a blank

with 250 lL of reverse osmosis water, a substrate

correcting blank containing 200 lL of water and 50 lL

of substrate, a quenching blank containing 200 lL of

water and 50 lL of methylumbelliferone. Each of these

had 8 technical replicates per plate. Additionally, there

were 8 technical replicates containing 50 lL of meth-

ylumbelliferone and 200 lL of porewater for each

sample to calculate the quenching coefficient. Oxidative

enzyme activity was estimated using the L-dihydroxy-

phenylalanine (L-DOPA) substrate method to measure

phenol oxidase and peroxidase activity. There were 16

technical replicatesper sample, and 8 technical replicates

for blanks including 250 lL of water, 200 lL of buffer

and 50 lL of 5 mM L-DOPA substrate, and 200 lL of

porewater with 50 lL of water. The peroxidase assays

also had 10 lL of H2O2 added to all wells. All samples

were incubated at room temperature. Hydrolytic enzyme

samples incubated for 2 h, and then 10 lL of 1.0 M

NaOH was added to each well in order to stop the

reaction. Fluorescence was measured at an excitation of

364 nm and an emission wavelength of 450 nm using a

SpectraMax M2 plate reader (Molecular Devices Cor-

poration, Sunnyvale, CA, USA). Oxidative enzyme

samples incubated for 24 h and the absorbance was

measured at 450 nm. Enzyme activities were calculated

according to German et al. 2011.

Porewater incubation

In May of 2010, 150 mL of porewater was harvested

from three heated and three control plots at 25 cm

depth. Samples from each plot were filtered as

previously described and 30 mL was put into five

60 mL precleaned and sample-rinsed HDPE bottles,

for a total of 30 bottles. 1 mL of common inoculum of

pore water filtered through a 1.6 mm glass fiber filter

(sequentially diluted to 10-3) was pipetted into each

bottle, following methods described by Wickland et al.

(2007). Bottles were then capped, gently shaken, and

incubated at 20 �C. A subset of six bottles (three

heated and three control plots) was acidified to

pH = 2 and measured for DOC and TDN concentra-

tions on the day the incubation was set up and 7, 14,

21, and 56 days later.

Statistical analyses

A repeated measures analysis of variance was con-

ducted using PROC MIXED to test for differences in

pore water DOC, DON, and SUVA254 between heated

and control treatments (SAS Institute Inc., Cary, NC,

USA, version 9.2). Warming treatment, depth, micro-

topography (hummock or hollow/lawn), day of year,

year, and depth and warming interactions were treated

as fixed effects, and day of year was treated as a

repeated measures with plot number acting as the

subject. We used variance components symmetry

covariance structure for repeated measures analysis as

determined by looking at the fit statistics and the

Kenward and Roger’s correction for degrees of

freedom (Littell et al. 2006). Type-3 tests of fixed

effects were considered significant at p \ 0.05. Ana-

lysis of covariance was used to determine the signif-

icance of changes in the relationship between DOC

and DON as a function of heating (Cody and Smith

1997). Least-squared mean value comparisons by

heating treatment and depth employed the Tukey–

Kramer adjustment. For comparisons of seasonal

mean values between heated and control plots we

conducted two-tailed t tests (a = 0.05).

Results

Temperature effects on DOC concentrations

Mean monthly peat temperatures measured at five cm

were 1.9 �C warmer in the heated plots (mean ± stan-

dard error; 18.4 ± 0.2 �C) than in the control plots

Biogeochemistry

123

(16.5 ± 0.2 �C) measured from June–August, 2009–

2010 (t = 5.03, p = 0.007). There was no significant

difference in mean monthly temperatures across

hummock or lawn microtopographies across all sam-

pling times at 5 cm (t = 0.18, p = 0.86), but some

opposing trends in temperatures between hummocks

and lawns were observed in June and July (Fig. 1e, f).

Mean temperatures measured at 42 cm throughout the

growing season of 2010 were not significantly differ-

ent between heated (16.6 ± 0.2 �C) and control plots

(16.3 ± 0.1 �C). Mean seasonal dissolved O2 concen-

tration at 50 cm was 1.1 ± 0.8 mg L-1 and mean

seasonal conductivity was 53.1 ± 4.7 lS s-1. Dis-

solved O2 declined as temperature increased in con-

current measurements at 50 cm throughout the growing

season (r = -0.69, F1,14 = 11.98, p = 0.004).

Growing season precipitation (May–September)

was 343 mm in 2009 and 483 in 2010, but mean

monthly water table depths (below peat surface) were

not different between 2009 (13.7 ± 3.2 cm) and 2010

(16.4 ± 3.1 cm) (t = 1.90, p = 0.15). The plots

received 40 % more precipitation in July of 2010

(77 mm) than in July of 2009 (47 mm), but mean July

water table depths were similar for these 2 months

(15.2 ± 2.5 vs. 16.8 ± 1.4 cm, respectively). Mean

peat moisture at 6 cm (6.0 ± 0.6 g g-1) and at 12 cm

(15.1 ± 2.1 g g-1) measured throughout the growing

season were not significantly different between heated

and control plots (t = 0.45, p = 0.66; and t = 0.34,

p = 0.74, respectively).

Mean peat porewater DOC concentrations at 25 cm

were 15 % higher in the heated plots (73.4 ±

3.2 mg L-1) than in the control plots (63.6 ± 2.1

mg L-1) across all sampling times in 2009–2010

(t = 4.69, p \0.001) (Fig. 2). Mean monthly porewater

DOC concentrations at 50 cm were not significantly

different between heated and control plots in 2009

(t = 1.01, p = 0.35; mean of 69.9 ± 2.0 mg L-1), but

were significantly different in 2010 (t = 4.47,

p = 0.001). There was a significant warming treatment

effect on DOC concentrations after accounting for

seasonal changes (Table 1). In fact, the increases in

mean monthly DOC concentrations in heated plots

relative to control plots generally corresponded with

mean increases in peat temperatures (Fig. 3). The largest

increases in DOC and peat temperature in heated plots

relative to control plots occurred later in the growing

season (August and September). There were no changes

in DOC concentrations by year (Fig. 2), depth, or

microtopography (hummock or lawn terrain)

(Table 1A). In 2011 there was no power being supplied

to the infrared lamps, and there were no differences in

mean DOC concentrations between the previously

heated (83.2 ± 3.4 mg L-1) and control (84.7 ±

5.3 mg L-1) plots measured at 25 cm throughout

June-October (n = 6 sampling dates, t = 0.33,

p = 0.76).

Fig. 1 Seasonal differences between the mean hourly peat temperatures (5 cm) of plots heated with infrared lamps and unheated

control plots. Negative values in pane (a) indicate periods when the heated plots were actually cooler than the control plots

Biogeochemistry

123

2009 2010

40

50

60

70

80

90

100

110

DO

C a

t 25

cm

(mg

L -1 )

control

heated

40

50

60

70

80

90

172

183

197

207

224

249

338

113

121

136

160

177

199

216

233

247

274

286

295

DO

C a

t 50

cm

(mg

L -1 )

Fig. 2 Mean daily DOC concentrations for heated and control plots at 25 cm (top pane) and 50 cm (bottom pane) throughout

2009–2010. Error bars are standard errors of the mean values

Table 1 Mixed-effects model of predictors of (A) dissolved

organic carbon and (B) dissolved organic nitrogen

Term Fixed

effects

F value

Degrees of

freedom

(num, den)

p

A: DOC

Depth 1.7 1, 276 0.1931

DOY 10.32 18, 276 \.0001

Heated or control 15.7 1, 276 \.0001

Hummock or lawn 1.69 1, 276 0.1944

DOY 9 heated 1.16 18, 276 0.291

Depth 9 heated 8.82 1, 276 0.0032

B: DON

Depth 0.89 1, 204 0.3460

DOY 5.64 16, 204 \.0001

Heated or control 0.15 1, 204 0.7030

Hummock or lawn 1.32 1, 204 0.2526

DOY 9 heated 0.71 16, 204 0.7868

Depthv 9 heated 5.56 1, 204 0.0193

Jun

Jul

Aug

Sep

*Jul

R2 = 0.99p = 0.004

R2 = 0.83p = 0.09

-5

0

5

10

15

20

25

30

0.5 1 1.5 2 2.5 3

Mea

n in

crea

se in

DO

C

rela

tive

to c

on

tro

l plo

ts (2

5 cm

, mg

L -1

)

Mean increase in peat temperature relative to control plots (5 cm, °C)

2009

2010

Fig. 3 Mean monthly increases in peat temperature are compared

to mean monthly increases in porewater DOC concentrations at

25 cm. Asterisk denotes a point not included in the regression

owing to a period of relatively heavy rainfall (standardized residual

error = 1.32). Error bars are standard errors of the mean values

Biogeochemistry

123

Temperature effects on porewater composition

and quality

A significant change in DOC with heating, with no

significant change in DON, would result in a different

DOC:DON ratio in response to heating. While DOC and

DON both exhibited significant seasonal changes, there

was no direct effect of heating on porewater DON

concentrations across all samples (Table 1B). There was

an interaction between heating and depth in explaining

variation in DON concentrations (Table 1B), owing to

the increase in DON at 25 cm in heated plots

(1.31 ± 0.06 mg N L-1) compared to control plots

(1.20 ± 0.06 mg N L-1) across all dates (t = 2.37,

p = 0.03); this difference was not found at 50 cm

(t = 1.14, p = 0.27). As expected, DON increased with

DOC, and there was a marginal interaction between

heating and DOC in explaining variation in DON

(F1,240 = 2.90, p = 0.09; Fig. 4). When the correlation

between DOC and DON was accounted for in

ANCOVA, heating had a weak but significant effect

on DON concentrations across all sampling depths

(F1,240 = 4.19, p = 0.04). As such, the amount of DON

per unit DOC declined in heated plots relative to control

plots (Fig. 4). Mean DIN concentrations were small

across all sampling times (0.08 ± 0.02 mg N L-1), and

there were no significant differences between heated and

control plots (t = 1.06, p = 0.29).

Heating significantly reduced porewater aromatic-

ity (Fig. 5). Porewater aromaticity changed with depth

and season as could be expected, but also changed

with heating and was a significant covariate with the

DOC:DON ratio (Table 2). Mean total dissolved Fe

concentrations were not different between heated

(2.1 ± 0.4 mg L-1) or control plots (1.6 ±

0.2 mg L-1; t = 1.55, p = 0.16), and therefore any

potential Fe interference of treatment effects on

SUVA254 were minimal. In addition, DOC from the

heated plots decayed twice as fast as control plot DOC

control

R2 = 0.36

heated

R2 = 0.49

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

2.1

2.3

2.5

30 60 90 120

DOC (mg L -1)

DO

N (m

g L

-1)

control

heated

Fig. 4 The slope of the relationship between DON and DOC

declined with heating in analysis of covariance (F1,240 = 4.19,

p = 0.04). Coefficients describing the trends are b0 = 0.38 ±

0.09, b1 = 0.013 ± 0.001 for heated plots and b0 = 0.18 ±

0.13, b1 = 0.017 ± 0.002 for control plots

1:1

2.5

3.5

4.5

2.5 3.5 4.5

Mean SUVA254

in control plots

Mea

n S

UV

A25

4

in h

eate

d p

lots

25 cm

50 cm

Fig. 5 Mean daily porewater Specific Ultraviolet Absorbance

at 254 nm (SUVA254; L mg C-1 m-1) measurements were

lower in heated plots than in control plots across all depths and

sampling days (t = 2.76, p = 0.011). Error bars are standard

errors of the mean values

Table 2 Mixed-effects model of predictors of specific ultra-

violet absorbance at 254 nm (SUVA254; L mg C-1 m-1)

Term Fixed

effects

F value

Degrees of

freedom

(num, den)

p

Depth 5.23 1, 143 0.0236

DOY 2.61 12, 143 0.0036

DOC:DON 29.18 1, 143 \.0001

Heated or control 5.05 1, 143 0.0261

Hummock or lawn 0 1, 143 0.9811

DOC:DON 9 DOY 2.71 12, 143 0.0025

DOC:DON 9 heated 4.47 1, 143 0.0362

Depth 9 heated 3.45 1, 143 0.0654

Biogeochemistry

123

within the first 3 weeks of a 2 month laboratory

incubation experiment, with exponential decay coef-

ficients for heated plot DOC being -1.8 9 10-3

± 8.4 9 10-4 compared to -8.0 9 10-4 ± 3.7 9

10-4 for the control plot DOC (Fig. 6). The exponen-

tial decay coefficients for DOC incubated for 56 days

were not different for porewater collected from the

heated (-8.6 9 10-4 ± 3.2 9 10-4) and control

plots (-9.2 9 10-4 ± 1.1 9 10-4).

Perhaps surprisingly, SUVA254 was negatively

related to the dissolved organic C:N ratio across all

samples (r = -0.47, p \ 0.001; see also Table 2).

Collectively, these results suggest that the higher DOC

concentrations present in the warming treatments were

relatively aliphatic in nature, while also being asso-

ciated with a higher dissolved C:N ratio.

Ultrahigh resolution mass spectrometry revealed

that, within the pool of relatively small compounds

(*200–800 Da) that are able to deprotonate, there

were small differences in peat porewater composition

with heating. Compounds unique to the heated plots

had lower mean H:C ratios and higher DBE than

compounds unique to the control plots (Fig. 7).

Compounds unique to the heated plots (289 identified)

were generally more condensed and less oxidized than

compounds unique to the control plots (Fig. 8). 17 %

of the 1,545 biomolecular assignments made for the

warmed plot DOC were in the range of protein and

carbohydrate containing compounds (H:C [ 1.5,

O:C [ 0.3), whereas only 8 % of the 1,516 biomo-

lecular assignments fell within the range of amino-

sugars in the control plot DOC (Fig. 8). Porewater

y = 56.7e-0.0018x

R2 = 0.69

y = 49.6e-0.0008x

R2 = 0.70

47

50

53

56

59

0 7 14 21 28 35 42 49 56 63

day of incubation

DO

C o

f in

cub

ated

sam

ple

s (m

g L

-1 )

Fig. 6 Reduction in dissolved organic carbon over a 56 day

laboratory incubation. Each point is the mean DOC concentra-

tion of porewater from three heated (opaque diamonds) or three

control (squares) plots. Negative exponential relationships are

plotted for the first 21 days only. Error bars are standard errors

of the mean values (n = 3 plots)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

O:C H:C N:C DBE/10

Wei

ghte

d R

elat

ive

Abun

danc

e

Control, all Heated, allControl, unique Heated, unique

Fig. 7 Mean weighted relative abundances of O:C, H:C, N:C and

the DBE of all molecular formulas present in the sample, and of the

molecular formulas unique to a given sample. Data represent the

assigned molecular formulas of samples from two control plots

and two heated plots collected on the same day at 25 cm depth.

Error bars are standard errors of the mean values (n = 2)

Fig. 8 Van Krevelen diagram of porewater harvested from two

heated and two control plots. Points represent the composition of

molecular assignments unique to heated (gray) or control

(white) plots. Points depicted by a times symbol are molecular

assignments common in all pore water

Biogeochemistry

123

from the heated plots exhibited greater biomolecular

assignments associated with aromatic compounds,

with 35 % of the biomolecular assignments occurring

above the threshold for AI [ 0.5 (Koch and Dittmar

2006).

The three organic acids measured changed in

different ways with the heating treatment, though

responses were variable and not statistically significant.

Least-squares mean acetate concentrations decreased

from 0.32 to 0.16 mg L-1 with heating, but were not

significantly different (F1,244 = 1.80, p = 0.18). For-

mate (mean concentrations ranging from 0.10 mg L-1

in control plots to 0.12 mg L-1 in the heated plots) and

oxalate (mean concentrations ranging from 0.20 to

0.26 mg L-1, respectively) concentrations were also

unaffected by the heating treatment (F1,244 = 2.04,

p = 0.16, and F1,244 = 2.25, p = 0.14, respectively).

Least-squared mean acetate concentrations increased

with depth from 25 to 50 cm (0.06–0.42 mg L-1

respectively; F1,244 = 7.90, p = 0.005).

Enzyme activities

Porewater extracellular enzyme activities were

affected by heating treatment and also changed

seasonally (Table 3). Chitinase and glucosidase activ-

ities were significantly affected by warming, with

mean activities increasing by 58 and 138 %, respec-

tively (Table 3, p = 0.005 and p = 0.03, respec-

tively). Cellulase, acid phosphatase, and phenol

oxidase activities exhibited seasonal changes as might

be expected, but did not change in response to warming

(Table 3). Cellulase activity increased during leaf

senescence in both the heated and control treatments

(p = 0.002). Phosphatase and phenol oxidase activity

were higher during leaf out versus leaf senescence

in both treatments (p = 0.004 and p = 0.002,

respectively).

Discussion

Temperature effects on DOC concentrations

and composition

The increase in DOC concentrations with heating

observed in this experiment was expected because the

water-soluble products of decomposition and biogenic

DOC should both increase with warming, because

decomposition and microbial activity are temperature

dependent. We observed a strong seasonal effect on

DOC concentrations, which is consistent with many

studies reporting patterns of peat DOC concentrations

that track temperatures and plant and microbial activity

throughout the growing season (Dalva and Moore

1991; Heikkinen 1994; Tegen and Dorr 1996; Scott

et al. 1998; Tipping et al. 1999; Kalbitz et al. 2000).

The growing season of 2011 was considerably warmer

(1962, SDD) than 2010 (1919 SDD) or 2009 (1814

SDD); this was coincident with mean porewater DOC

concentrations that were*22 % higher across all plots

in 2011 than was measured in 2009–2010. This

seasonal increase in mean DOC concentrations

observed in the warm year is similar in magnitude to

the increase in DOC measured with active heating at

the peat surface. A unique aspect of this study was the

increase in DOC concentrations measured in response

to warming which was independent of temporal

variability. This effect is confirmed by the fact that

there were no differences in DOC concentrations

Table 3 Changes in porewater enzyme activities (25 cm depth) in late summer and following senescence in both control and heated

plots (±standard error of the mean)

Treatment Season Chitinase Glucosidase Cellulase Phosphatase Phenol oxidase Peroxidase

Control Leaf out 0.118 (0.009) 0.287 (0.087) 0.066 (0.001) 1.475 (0.270) 0.030 (0.002) 0.013 (0.003)

Senescent 0.140 (0.002) 0.123 (\ .001) 0.208 (0.011) 0.539 (0.131) 0.002 (0.001) 0.011 (0.002)

Heated Leaf out 0.186 (0.005) 0.593 (0.003) 0.079 (0.002) 1.879 (0.480) 0.017 (0.002) 0.018 (0.009)

Senescent 0.182 (0.025) 0.380 (0.137) 0.319 (0.044) 0.432 (0.087) 0.002 (0.001) 0.001 (0.001)

Differences (t, p; n = 8)

By treatment 4.28, 0.005 2.76, 0.03 0.76, 0.48 0.27, 0.80 0.73, 0.50 0.35, 0.74

By season 0.34, 0.75 1.42, 0.21 5.12, 0.002 4.53, 0.004 5.44, 0.002 1.90, 0.11

Units are in nmol h-1, except phenol oxidase and peroxidase, which are in lmol h-1. Significant differences by treatment or season

(a = 0.05) are in bold text

Biogeochemistry

123

between previously heated and control plots in 2011,

when there was no power being supplied to the infrared

lamps. Moreover, we began to see separation in deeper

porewater DOC concentrations with warming in the

second year of the heating treatment, even though

temperatures at 50 cm were not significantly altered by

the experiment. This, too, likely reflects an influence of

heating on vegetation, and resulting interactions with

root-associated microorganisms and root activity in

addition to the downward diffusion of porewater. In a

3 year passive warming experiment in an alpine fen

peatland, a structural equation modeling effort esti-

mated a 23 % increase in the weight of litter-layer

microbial community effects on water chemistry

(Jassey et al. 2012). Jassey et al. (2012) suggested that

by inhibiting higher trophic levels within the microbial

community, interactions between Sphagnum-derived

polyphenols and warmer temperatures could actually

increase nutrient levels and dissolved C release. We

note here that passive warming effects on DOC and

TDN concentrations in the alpine fen study were

variable, with warming only significantly increasing

DOC concentrations in the third year of temperature

manipulation, and with no effect on TDN. Neverthe-

less, these results generally agree with those presented

herein, and demonstrate that modest warming at the

peat surface can have significant direct effects on DOC

concentrations and complex indirect effects on micro-

bial activity and porewater character.

Humic substances released by plant roots and

microorganisms are of a relatively lower molecular

weight (more aliphatic) than are the water-soluble

products of decomposition, which are more comprised

of high molecular-weight (aromatic) compounds (Thur-

man 1985; McKnight and Aiken 1998). In this study,

the relative aromaticity of peat porewater declined in

the warmed plots relative to control (Table 2; Fig. 5),

but the character of the pool of biomolecules that were

able to deprotonate (*200–800 Da) did not change

appreciably with modest warming at the peat surface

(Fig. 7). Compounds unique to the heating treatment

had narrower H:C and O:C ratios than control plots

(Fig. 8), suggesting a more aromatic or condense

structured DOM (Hedges 1990). While these results

may appear to be at odds with the SUVA254 data, they

suggest that most of the change in bulk porewater

composition with heating occurred in the fraction of

compounds not well quantified with the ultra-high

resolution mass spectrometry in this study. Increases in

the concentrations of relatively labile low molecular

weight DOC are consistent with increased microbial

activity (as observed with hydrophilic acids; Bourbon-

niere 1989; Guggenberger et al. 1994). It is possible that

the changes in novel compounds present in the heated

plots relative to control reflect increasing amounts of

microbially derived humic acids or the diagenesis of

polysaccharides (oxygen-rich biochemicals)-these pro-

cesses would result in narrower H:C and O:C ratios

within the pool of relatively large molecular weight

DOM (see, for example, Visser 1983). Interpretation of

these data are not straightforward, however, because

larger biomolecules initially released from peatland

vegetation and mosses can be degraded into smaller

components fairly rapidly (Gonet and Debska 1998;

Strack et al. 2011; cf. Wickland et al. 2007); this is a

transformation which would likely be facilitated at

warmer temperatures. In this study, novel compounds in

the heated and control plots both generally had lower

DBE and there was a decrease in the appearance of

unsaturated and/or oxygenated compounds, which

could indicate that the unique compounds in both

treatments were more labile (Tfaily et al. 2013). One

consistency between the FT-ICR MS data and analysis

of mean porewater constituent concentrations within

the much larger dataset was that an increase in N:C ratio

in novel compounds generally corresponded with

higher DBE (Fig. 7). This finding is consistent with a

higher N:C generally occurring in the unheated control

plots (Fig. 4), which also had higher SUVA254. The

increase in N:C while also having higher aromaticity

could be indicative of amine complexation with

carboxylic groups or protein complexation with phe-

nolic OH groups (McManus et al. 1985; Spencer et al.

1988; Munster and de Haan 1998). It could be that with

increased breakdown of larger humic substances with

warming, or with an increase in the abundance of lower

molecular weight compounds, there is less complexa-

tion of proteinaceous compounds with DOM (lower

dissolved N:C). Future studies employing emerging

technologies in liquid chromatography and mass spec-

trometry (cf. Liu et al. 2011; Remucal et al. 2012), in

conjunction with field manipulation experiments, are

needed to fully appreciate exactly how the character of

DOM is likely to change in a warmer climate.

Notwithstanding, these findings suggest different mech-

anisms of DOM production and consumption within

different biomolecular size classes in response to

warming at the peat surface.

Biogeochemistry

123

In this study, warming resulted in an increase in

relatively aliphatic DOC, with no statistical increases

in DON (Table 1B; Fig. 4). In contrast, studies have

shown that the ratio of aliphatic to aromatic DOC is

generally higher in aquatic systems with more DON

relative to DOC (McKnight and Aiken 1998). Aro-

matic structures present in DOC would largely come

from the degradation of plant-derived lignin (and

possibly the products of burning), whereas aliphatic

DOC would largely come from microbes, algal

exudates, and root exudates in the rhizosphere

(Hatcher and Spiker 1988; McKnight and Aiken

1998; Benner 2003). The observed declines in aro-

matic DOC relative to DON with warming could

suggest that N-containing biomolecules were not

generally exuded within the rhizosphere, or that

soluble N was rapidly immobilized. Because of the

absence of lignin in microorganisms and the fact that

SUVA254 decreased with warming, these results

suggest that the increase in DOC with warming was

largely derived from plants and plant–microbe inter-

actions within the rhizosphere, and not from the

breakdown of organic matter. This is consistent with

previous research in boreal peatlands showing that

porewater DOC is quite reactive and is composed of

relatively modern C affected by rhizosphere pro-

cesses, particularly when sedges are abundant (Chan-

ton et al. 2008). Complementary research at this site

has shown increased gross ecosystem productivity

with warming on the hummock-dominated plots, with

no differences in ecosystem respiration (Johnson et al.

2013). Together, these findings suggest that the

increased DOC concentrations observed with warm-

ing were derived more from plants and plant–microbe

interactions within the rhizosphere than from the

accumulation of products from decomposition of

organic material.

Temperature effects on porewater enzyme activity

Extracellular enzyme activities of the porewater dif-

fered by warming treatment for chitinase and glucosi-

dase, and differed seasonally for cellulase, phosphatase

and phenol oxidase activities. This suggests that the

controls on enzymatic metabolism are dependent on

environmental conditions. Chitinase, a carbon and

nitrogen-acquiring enzyme, may have higher activity

in heated treatments due to a higher rate of nitrogen

cycling in heated plots. This has been demonstrated in

upland temperate forests (Melillo et al. 2011, Butler

et al. 2012). In Arctic tundra soils, temperature has been

shown to be the strongest driver of hydrolytic enzyme

activities, though low activities have been observed in

the summer- possibly owing to a tight nitrogen cycle

and nitrogen limitations for enzyme production (Wal-

lenstein et al. 2009). More chitinase enzymes may be

produced, or the efficiency of chitinase enzymes may

increase with warming. Fungi produce the majority of

chitinase enzymes, and it has been demonstrated that

fungal abundance increases with warming in tundra

ecosystems (Clemmensen et al. 2006). Allison and

Treseder (2008) found that warming with closed-toped

greenhouses in a boreal forest lead to a decline in fungal

abundance and chitinase activity, however their study

might have been confounded with drying of the organic

horizon caused by the impediment of precipitation due

to the structure of the greenhouses. This study used

infrared lamps, which did not block moisture inputs

from precipitation. In a survey of 32 diverse wetlands

from around the world, Kang et al. (2005) demonstrated

that chitinase (N-acetylglucosaminidase) activities

associated with solid-phase peat increased with mean

annual temperatures measured in the field. While the

increasing potential chitinase activities measured with

increasing temperatures presented by Kang et al. (2005)

were over a magnitude greater than those measured in

this study (confer with Table 3), the results indicate that

chitinase activities associated with both solid and

dissolved phases are likely to increase with modest

warming in peat soil.

Glucosidase activity was also higher in heated

treatments. This may be due to an increase in the

amount of substrate available in heated treatments, or

to changes in the amount or efficiency of glucosidase

enzymes in the porewater. Since relatively aliphatic

compounds and DOC concentrations both increased

with warming, it seems likely that the concentration of

polysaccharides broken down by glucosidase has

increased with warming. Moreover, there was no

measured increase in phenol oxidase activity with

warming, which could allow for an accumulation of

phenolics with warmer temperatures, barring any co-

occurring changes in water table position and

increased aeration (Freeman et al. 2001b). Taken

together, these data suggest increased hydrolytic

enzyme activity could contribute in part to higher

concentrations of DOC with a warmer climate, which

fits nicely with reports suggesting a temperature (and

Biogeochemistry

123

enzyme) mediated rising trend in DOC concentrations

in runoff is underway (Freeman et al. 2001a).

We observed seasonal changes in hydrolytic and

oxidative enzyme activity potentials. Phosphatase

activity was higher in the leaf out period versus the

senescent period, indicating that phosphorus-acquiring

enzymes either have more substrate available, or have

a higher abundance or specific activity earlier in the

season. Phenol oxidase activity was also higher during

leaf-out than at the end of the growing season. This

suggests that there is either higher abundance or

efficiency of phenol oxidase enzymes or that there is a

higher polyphenol concentration early versus late in

the growing season. Although it is unclear which

mechanism may be involved, it is possible that the type

of phenol oxidase enzymes changes seasonally, due to

the wide variety of enzymes that depolymerize poly-

phenols and the relative instability of oxidative

enzymes versus hydrolytic enzymes (Sinsabaugh

2010). A previous study in an alpine peatland has

shown increased peroxidase activity with passive

warming, which was three orders of magnitude higher

than phenol oxidase activity (Jassey et al. 2012). While

we did observe higher oxidative enzyme activity in

September than in October, we did not observe large

differences between phenol oxidase or peroxidase

enzyme activity potentials, and there was no signifi-

cant effect of heating on oxidative enzyme activity

potentials. We measured enzyme activity potentials in

peat pore water, and it is possible that oxidative

enzymes associated with the solid phase (particle-

associated extracellular enzymes, or humus-enzyme

complexes) are more sensitive to changes in environ-

mental conditions than are free floating extracellular

enzymes associated with dissolved organic matter

(Hoppe 1991; Munster and de Haan 1998). Cellulase

activity increased during leaf senescence versus leaf

out. This may be due to an increase in concentrations of

more complex polysaccharides as leaf litter accumu-

lates. It is also possible that there is a decrease in the

relative concentration of simple polysaccharides dur-

ing this period, as evidenced by a non-significant

decrease in glucosidase activity from leaf out to

senescence. More data is needed on the relative

abundances of the chemical species present in the

two seasons to allow us to draw further conclusions

about the impact of substrate concentrations on

porewater enzyme activities.

Conclusions

We observed increases in porewater DOC concen-

trations and a slight decline in porewater aromaticity

with modest warming at the peat surface over

2 years. We also observed increases in hydrolytic

enzyme activity potentials in response to warming.

Earlier work at this study site has shown increased

gross primary production, with little change in

ecosystem respiration with active warming at the

peat surface (Johnson et al. 2013) and the observed

changes in porewater character are consistent with

increases in vegetation production and increased

microbial activity in shallow peat. Moreover, we

observed increases in DON:DOC generally corre-

sponding with higher aromaticity in the unheated

control plots. This was also observed within the suite

of novel compounds measured in the heated plots,

which had higher N:C and DBE than the control

plots. However, all novel compounds had lower DBE

than did compounds common to both heated and

control plots. These data suggest a tight coupling of

climate effects on the peatland biosphere and pore-

water chemistry, with different consequences for the

production and diagenesis of relatively large and

small biomolecules. Collectively, these data support

other studies which indicate DOC concentrations are

likely to increase in organic soils with the predicted

warming likely to occur in the coming decades. With

continued warming in peatlands, an increase in

relatively labile DOC concentrations could contrib-

ute to dissolved exports of DOC in runoff, and would

likely contribute to the pool of efficient electron

donors (and acceptors) in the production of CO2 and

CH4 in terrestrial and aquatic environments. Future

work examining how DOC export is likely to change

in a warmer climate, either by mineralization or in

runoff, is necessary to understand the consequences

of any temperature mediated increases in DOC

concentrations in soil.

Acknowledgments This research was supported by the U.S.

Department of Energy’s Office of Science (BER) through the

Midwestern Regional Center of the National Institute for Climatic

Change Research at Michigan Technological University as well

as the Michigan Technological University Ecosystem Science

Center. We would like to thank Justina Silva and Jennifer

Eikenberry for help with laboratory analysis, and Arvo Aljaste

and Kristen Schmitt for help with field work. We appreciate the

constructive comments from two anonymous reviewers.

Biogeochemistry

123

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