Observations of a seasonally shifting thermal optimum in peatland

carbon-cycling processes; implications for the global carbon cycle

and soil enzyme methodologies

N. Fenner*, C. Freeman, B. Reynolds

School of Biological Sciences, University of Wales, Memorial Building, Deiniol Road, Bangor, Gwynedd LL57 2UW, UK

Received 16 July 2004; received in revised form 21 January 2005; accepted 17 February 2005

Abstract

Thermal gradient apparatus has been used to study enzyme activity and carbon cycling in peat collected seasonally from a Northern upland

peatland. A thermal optimum was observed in the peat where maximum carbon-cycling enzyme activities (phenol oxidase and

b-glucosidase), phenolic compound concentrations, dissolved organic carbon (DOC) concentrations and microbial respiration (CO2 efflux)

were all found in a given season. The thermal optimum for these carbon-cycling processes coincided with the highest ambient soil

temperature recorded at the time of peat collection, suggesting microbial acclimation to the external conditions. Under the waterlogged

conditions of this experiment, phenol oxidase activites correlated positively with phenolic compounds (winter 0.96, P!0.01; spring 0.92,

P!0.001; summer 0.94, P!0.001; autumn 0.88, P!0.001) and b-glucosidase activities with DOC (winter 0.91, P!0.01; spring 0.85, P!0.01; summer 0.92, P!0.001; autumn 0.72, P!0.05). We propose, therefore, that the relative activities of these enzymes is crucial in

mobilising DOC from the peat matrix, with implications for carbon exports to the receiving waters (magnitude and molecular weight

distribution) and CO2 efflux to the atmosphere. The pronounced seasonality in carbon processing found here, must be taken into account

when modelling carbon flux in and from these systems, if responses to climate change are to be predicted satisfactorily. Furthermore, because

the optimum activity of these carbon-cycling enzymes shifted with seasonal changes in temperature, it is essential to perform enzyme assays

in soil ecological investigations at field temperatures (rather than standardised temperatures), when information on natural process rates is

required.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: Acclimation; b-Glucosidase; Carbon cycle; DOC; Enzyme; Mineralization; Peatland; Phenol oxidase; Thermal optimum; Water quality

1. Introduction

Peatland carbon cycling has attracted much attention

recently in relation to climate change, and phenol oxidase

activity, in particular, has been recognized as a major

regulator of carbon storage in these organic-rich northern

soils (Freeman et al., 2001a). The typically low oxygen

conditions inhibit phenol oxidase activity, which is thought

to allow phenolic compounds to accumulate. Phenolics

inhibit the action of other hydrolase enzymes, which are not

oxygen limited (Appel, 1993; Freeman et al., 1990, 2001a;

Wetzel, 1992), such as b-glucosidase, phosphatase

0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.soilbio.2005.02.032

* Corresponding author. Tel.: C44 1248 351151.

E-mail address: n.fenner@bangor.ac.uk (N. Fenner).

and sulphatase, leading to retarded rates of organic matter

decay (Freeman et al., 2001a).

Seasonal changes occur in many factors (e.g. temperature

and soil moisture levels) that affect microbial metabolism

which, in turn, is responsible for the production of most of

these enzymes (Kang and Freeman, 1999). Temperature has

a key role in controlling the rates of biogeochemical

processes in soils, including peats, with effects upon

dissolved organic carbon (DOC) release (Briones et al.,

1998; Freeman et al., 2001b; Ineson et al., 1995; Tipping

et al., 1999), CO2 (Moore and Dalva, 1993), CH4 (Wilson

et al., 1989) and N2O emissions (Bailey and Beauchamp,

1973; Lensi and Chalamet, 1982), as well as the availability

of nutrients (Koerselman et al., 1993; Ross, 1985). However,

little is known about the effect of seasonal temperature

changes on peatland carbon-cycling enzymes, despite the

fact that peatlands represent a vast store of carbon (ca. 455 Pg

Soil Biology & Biochemistry 37 (2005) 1814–1821

www.elsevier.com/locate/soilbio

Fig. 1. Thermal gradient bar used to incubate peat over a range of 2–20 8C.

N. Fenner et al. / Soil Biology & Biochemistry 37 (2005) 1814–1821 1815

or 1/3 of the world’s soil organic carbon), and such enzymes

may be critical in the regulation of carbon cycling within, and

exports from this globally important ecosystem.

Here a thermal gradient bar (Fig. 1) has been used to apply a

continuous thermal gradient of 2–20 8C to Northern peatland

soils, with the aim of determining whether carbon-cycling

enzymes show seasonality in relation to temperature. Phenol

oxidase activity was measured because of the aforementioned

properties and b-glucosidase activity (which specifically

hydrolyzes cellobiose as is present in cellulose (Killham,

1996)) was determined because it is an indicator of cellulose

decomposition (Sinsabaugh et al., 1991) and of carbon

mineralization rates (McLatchey and Reddy, 1998; Sinsa-

baugh et al., 1991). Typically, in mineral soil studies, enzyme

assays are done under optimal conditions (temperature and

pH) (e.g. Bandick and Dick, 1999; Saiya-Cork et al., 2002;

Tabatabai, 1982). This was avoided in the current study in

favour of maintaining conditions that are more representative

of those that occur naturally (Freeman et al., 1995). Such

optimal conditions are rarely found in the field and so

incubations were carried out at field temperature (in addition

to the higher and lower temperature ranges induced in the

thermal gradient bar) without pH buffer additions for the

incubation step. The concentration of potential phenol oxidase

and b-glucosidase substrates, i.e. pore water phenolic

compound and DOC concentrations, respectively, were also

determined. The DOC apparent molecular weight (AMW)

spectra were examined in order to ascertain whether seasonal

changes would induce altered DOC processing. CO2 flux from

the peat surface was measured to provide an indication of

microbial respiration.

2. Materials and methods

2.1. Operation

A thermal gradient (2–20 8C) was maintained along an

aluminium temperature bar (Fig. 1). The apparatus was

operated in a constant temperature environment in order to

ensure close temperature control.

2.2. Soil collection

Peat was collected in the winter (January), spring (April),

summer (August) and autumn (November) of 1999, to a

depth of 10 cm, from a peat-accumulating wetland in the

Upper Wye catchment on Plynlimon in Mid Wales (UK

NGR SN 820 866). Collections were made to allow two

thermal gradient bars to be run simultaneously per season,

each of which contained two replicates. The field site is

typical of many in the uplands of Wales (Hughes et al.,

1996), being characterized by Sphagnum and Juncus

communities with a pore water pH in the range of 3.9–4.8

(at a depth of 10 cm). A temperature probe was used to

measure the peat temperature at the surface before

collection.

2.3. Preparation and incubation of samples

Peat from each collection was treated separately; the

surface layer of vegetation was removed and the peat gently

homogenized by hand for 10 min in order to reduce spatial

heterogeneity. A 2 cm thick layer of this soil was placed on

the thermal gradient bar within two layers of clingfilm to

prevent water loss without inhibiting gas exchange (Gordon

et al., 1987). Soil water loss has been found to be negligible

(between 0.4 and 0.9% of the total water content of the peat

soil) using a single layer of clingfilm but a second layer was

used to ensure complete water retention (Dowrick, 1998).

Peat was incubated for 2 weeks.

2.4. Extracellular phenol oxidase activities

and b-glucosidase activities

Phenol oxidase activities were determined according to

Pind et al. (1994) and b-glucosidase activities using the

method of Freeman et al. (1995), at 2 8C intervals along the

thermal gradient bar. All solutions required for the assays

were incubated at the same temperature as that in the

thermal gradient bar from which the peat sample was taken.

2.5. Gas and hydrochemical analysis

Following 2 weeks of incubation, phenolic substances

and DOC concentrations were measured in peat pore waters

obtained by centrifugation (10,000g for 30 min) from 2 8C

intervals across the thermal gradient. The former using the

spectrophotometric method of Box (1983) and the latter a

Total Organic Carbon analyzer (Shimadzu 5000, Kyoto,

Japan).

A CECIL 1100 series (Cecil Instruments Limited,

Cambridge, UK) High Performance Liquid Chromatograph

(HPLC) with a gel filtration column (PL-GFC 8 mm, 300 A,

300!7.5 mm2 inner diameter, Polymer Laboratories Ltd,

N. Fenner et al. / Soil Biology & Biochemistry 37 (2005) 1814–18211816

Shropshire) was used to determine the AMW distribution of

DOC in the samples, with a CECIL 1200 variable

wavelength monitor detecting at 254 nm. The flow rate

used was 3 ml minK1 and the loop size 120 ml. Several

proteins were used to calibrate the column: albumin

(molecular mass, MMZ67,000), ovalbumin (MMZ43,000), chymotripsinogen A (MMZ25,000), ribonuclease

A (MMZ13,700), cytochrome C (MMZ12,400), and

vitamin B12 (MMZ3500) (Alarcon-Herrera et al., 1994)

along with a range of polystyrene standards (Chin et al.,

1998). Tris (Tris hydroxymethane aminomethane) hydro-

chloric acid (0.01 M, pH 7.5) was used as the eluent

(Dawson et al., 1989), after being degassed and filtered

using a 0.45 mm membrane filter (Whatman, Kent, UK). To

avoid confusion AMW spectra will be described in terms of

‘fractions’ rather than ‘peaks’, which are used to describe

maximum activities or concentrations across the thermal

gradient bar.

Microbial respiration (CO2 flux) along the thermal

gradient bar was measured using 90 mm by 13 mm petri-

dish bases as headspaces (placed upside down on the soil

surface). The dishes were pressed 1–2 mm into the peat at

2 8C temperature intervals. Tubing (PVC, 10 cm long, 3 mm

diameter, 1 mm bore), connected to a gas syringe and

running under the peat into the dishes, was used to facilitate

gas collection. Each dish was removed following every

sampling replicate run (of which four were carried out) and

flushed with ambient air. Five millilitres of gas was taken

from each chamber for analysis using a gas chromatograph

(Ai Cambridge, model 92, Analytical Measuring Systems,

Cambridge, UK). The increase in trace gas concentration

above the initial background concentration after 1 h was

used to estimate gaseous fluxes from the peat.

Microbial respiration was also measured in peat collected

from a second ombrotrophic bog site in the Nant Ffrancon

valley, North Wales (GR SH504 817).

2.6. Statistical considerations

Four replicate peat samples were used from each

temperature interval, for each of the four seasons. Data

was tested for normality using the Kolmogorov–Smirnov

test (Minitab version 13.32, Minitab, Inc.). ANOVA, with

temperature as the model and Tukey’s simultaneous test,

was used to determine whether differences within tempera-

ture treatments were significant. Significant correlations

(Pearson correlation coefficient) between variables within a

given season are also presented.

3. Results

In a given season, broad maxima (raised activities/con-

centrations) were present in all the measured variables at a

temperature (referred to as the ‘thermal optimum’) approxi-

mately predicted by the highest ambient peat temperature

recorded before peat collection. Maximum ambient peat

temperatures recorded were 2.5 8C in winter (January),

11 8C in spring (April), 15.8 8C in summer (August) and

5.2 8C in autumn (November) of 1999, respectively.

However, it should be noted that these maximum values

represent the highest temperature obtained during the

preceding 2 days only.

3.1. Winter peat

The winter peat showed the lowest activities/concentra-

tions in all measured variables compared to the other

seasons (Table 1). Peak values in all variables were

observed at 2 8C in the thermal gradient bar, with values

at 2 and 4 8C being significantly different to those at 6 8C in

all but the case of CO2 flux. These maxima coincided with

the highest temperature recorded in the field at the time of

peat collection (2.5 8C). Leachate collected from the winter

peat exhibited a DOC spectra with two distinct AMW

fractions, a primary fraction of O5000–!90,000 Da and an

O200,000 Da fraction, along with a certain amount of

lower molecular weight material (!5000 Da) in two

smaller fractions (Fig. 2). The primary fraction was most

prominent at 2 8C, while the other AMW fractions were

relatively similar over the temperatures studied.

3.2. Spring peat

The spring peat showed mid activities/concentrations in

all measured variables (Table 1). Peak values were found at

12 8C coinciding with the highest ambient temperature

recorded (11 8C), and values at 10–14 8C in the bar were not

statistically different.

3.3. Summer peat

The summer peat also produced leachate with a mid

range of enzyme activities and solute concentrations,

similar in magnitude to those in the spring peat (Table 1).

Peak values of all variables were found at approximately

20 8C, with the highest temperature recorded before peat

collection being somewhat lower (15.8 8C). However,

phenol oxidase and b-glucosidase activities showed a

broad temperature range where activities were raised

(14–20 and 16–20 8C, respectively) as did phenolic

compound concentrations (16–20 8C). The summer DOC

spectra showed a common AMW fraction to that of the

winter peat (O5000–!90,000), but the O200,000 Da

AMW fraction was not detected. The highest molecular

weight fraction instead corresponded to an AMW of

O90,000–!200,000 Da, and it was this fraction that

contained the largest amount of material at 18 and 20 8C.

The low AMW material was barely detectable in the

summer DOC spectra.

Table 1

Thermal optimum for enzyme activities, solute concentrations, CO2 emissions and dominant DOC molecular weight fraction in peat collected seasonally

Variable Winter mean

peak value

(2 8C)

Significantly

different

range (8C)

Spring mean

peak value

(12 8C)

Significantly

different

range (8C)

Summer

mean peak

value (20 8C)

Significantly

different

range (8C)

Autumn

mean peak

value (6 8C)

Significantly

different

range (8C)

Phenol oxidase

(nmol gK1 minK1)

1.14 (0.27) ns 1.42 (0.11) 10–14a,b 1.52 (0.15) 14–20c 313.52 (9.79) *6**

b-Glucosidase 0.178 (0.017) 2–4** 0.5 (0.037) *10–14b 0.31 (0.04) 16–20c 24.13 (1.7) *6**

Phenolic com-

pounds (mg lK1)

5.6 (0.52) 2–4* 6.2 (0.59) *10–14* 10.99 (0.47) 16–20** 1.3 (0.19) *6***

DOC (mg lK1) 80.65 (3.57) 2–4** 229.7 (5.96) *10–14** 152.44 (3.59) 18–20* 0.63 (0.062) **6***

AMW fraction (Da) O5000–

!90,000

2*** ND ND O90,000–

!200,000

18–20*** ND ND

CO2 (mg mK2 hK1) 15.7 (2.8) 2–6* ND ND 49.8 (2.57) 18–20* ND ND

The temperature (thermal optimum) at which peak enzyme activities, solute concentrations and CO2 effluxes were found, over the incubation range of 2–20 8C,

for peat collected during the four seasons (1999) is shown. The dominant AMW (apparent molecular weight) fraction of DOC (dissolved organic carbon) at the

thermal optimum is also given. Readings were taken at 2 8C intervals. Where the peak value was not significantly different to the flanking values the

temperature range is given. P values on the left of a temperature show the significance of the difference between values at this temperature and the next

consecutive lower temperature interval in the thermal gradient bar, similarly P values on the right refer to the next consecutive higher temperature, where ns

denotes non significant, *P!0.05, **P!0.01, ***P!0.001. Standard errors are given in parentheses, nZ4, ND denotes not determined.a Denotes that the peak was different to the value at 12 8C, P!0.05.b Denotes that the peak was different to the value at 16 8C, P!0.1 only.c Denotes that the peak was different to the value at 12 and 14 8C, respectively, P!0.1 only.

N. Fenner et al. / Soil Biology & Biochemistry 37 (2005) 1814–1821 1817

3.4. Autumn peat

The autumn peat showed the greatest enzyme activities

and solute concentrations (Table 1). Peaks in all variables

were found at 6 8C (with values at 4 and 8 8C being

significantly different) and maximum ambient temperatures

were 5.2 8C. A secondary maximum (lesser in magnitude)

was also evident at between approximately 14 and 20 8C

(Fig. 3).

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

mV

2C4C

6C8C

<5000>5000

<90 000>90 000<200 000

>200 000

Fig. 2. Pore water DOC apparent molecular weight spectra (Daltons) across

a thermal gradient (2–20 8C) for peat collected in winter 1999. Spectra at

2 8C (thick line), 4 8C (thin line), 6 8C (crossed line) and 8 8C (dots) are

shown.

3.5. Shifting thermal optimum for carbon processing

and site specificity

DOC concentrations in peat collected from all four

seasons are shown in Fig. 3, illustrating apparent temporal

shifts in the thermal optimum for carbon processing with

changes in ambient temperature. Similar patterns have been

observed in the peat from other sites, for example, peak CO2

emissions from the Nant Ffrancon valley peat

(258.6 mg mK2 hK1) were also found at 2 8C (P!0.001),

which corresponded to the highest ambient temperature

(2.6 8C).

0

50

100

150

200

250

300

350

0 2 4 6 8 10 12 14 16 18 20 22

winter spring summer autumn

DO

C (m

g l-1

)

Temperature (°C)

Fig. 3. Pore water DOC concentrations across a thermal gradient (2–20 8C)

in peat collected from all seasons (1999). Arrows show thermal optima

where peak DOC concentrations, enzyme activities and CO2 emissions

were found (winter dotted shading; spring unfilled; summer filled and

autumn hatched), error bars represent standard error of the mean, nZ4.

N. Fenner et al. / Soil Biology & Biochemistry 37 (2005) 1814–18211818

4. Discussion

Perhaps the most striking feature of these data was that a

thermal optimum for carbon-cycling processes was found to

occur in the peat. This can be seen as raised enzyme

activities along with DOC concentrations, phenolic com-

pounds and CO2 emissions (Table 1) above the trend line of

the response to rising temperature. These raised activities/

concentrations in a given season coincide approximately

with the highest ambient temperature recorded before peat

collection. This suggests that microbial respiration, along

with phenolic compound and more labile polysaccharide

degradation (indicated by phenol oxidase and b-glucosidase

activities, respectively), is acclimated to field temperature.

Phenol oxidase is involved in lignin degradation

(McLatchey and Reddy, 1998) and b-glucosidase is an

indicator of cellulase activity (Sinsabaugh et al., 1991).

However, few microbial species possess the entire suite of

enzymes required to completely degrade cellulose or lignin

to CO2 (Sinsabaugh et al., 1994). The thermal optimum of

phenol oxidase and b-glucosidase occurs at the same

temperature in a given season and corresponds to that of

microbial respiration (CO2), implying that community

degradation is occurring and seemingly the generation of

dissolved phenolic compounds and DOC from the peat

matrix as a result. However, in addition to temperature

control, co-limitation by changing pH may also contribute

to the results found here.

The presence of a thermal optimum for carbon proces-

sing in peat from two different ombrotrophic bog sites

indicates that it is not a site-specific phenomenon.

Furthermore, in a study of the bacterial communities present

in peat that has undergone self-heating, as a result of stock-

piling for horticultural purposes, growth rates were highest

when incubation temperatures matched those of the peat

from which the microbes had been extracted (Ranneklev

and Baath, 2001).

The organisms in the system studied here are likely to be

mesophiles (optimal growth temperatures between 20 and

50 8C) thus, the observed pattern of shifting thermal

optimum for carbon cycling with season (Fig. 3) probably

represents the proliferation of microorganisms and their

extracellular enzymes within this class, as environmental

temperatures change with season. A microorganism can

proliferate only when the environmental temperatures are

restricted to the thermal growth range of that organism, and

the ability of a microorganism to compete for survival in a

given system is increased when temperatures are close to its

optimal growth temperature (Atlas, 1988). The differences

in optimal growth temperatures and temperature growth

ranges result in spatial separation of these organisms in

nature, and since temperatures are changing through time,

temporal separation also. The current study shows major

shifts in carbon cycling can occur on a monthly time-scale

and there is evidence in the literature for more rapid

acclimation to changes in temperature; The self-heating

study carried out by Ranneklev and Baath (2001) shows that

acclimation to changes in temperature can occur within a

few days.

Some microbes can survive in a dormant state (through

production of endospores, for example) but do not grow at

temperatures outside their thermal growth range (Atlas,

1988), and this may be the mechanism which allows a

secondary maximum to occur in the autumn peat at 16 8C

and above (Fig. 3). Certain microbial species present in the

summer peat may survive as dormant endospores with the

decline in temperatures towards autumn and, in this case,

can proliferate again in the thermal gradient bar at the

artificially high temperatures. Peat may, therefore, possess a

‘microbial memory’, allowing optimal activities should

the prevailing conditions become favourable to growth.

Ranneklev and Baath (2001) found that an increased

temperature of incubation resulted in temperature relation

curves with only one optimum, however, a decrease in

incubation temperature produced curves with two optima.

This was true for the current study; peat collected in spring

and summer, exposed to increasing field temperatures

showed a single distinct thermal optimum for carbon-

cycling processes. Where field temperatures were changing

from warm to cool in the autumn, two optima were found.

Similarly, in a seasonal study of bacteria from a temperate

lake, temperature relationship curves with two optima were

frequent, where the lower optimum was closer to the actual

temperature of the lake (Simon and Wunsch, 1998), as

occurred in the autumn peat used in this study.

Conventionally, phenol oxidase activity is perceived to

be negatively correlated with phenolic compound concen-

tration, i.e. the higher the phenol oxidase activity the more

dissolved phenolic materials are removed from solution

(Freeman et al., 2001a; Pind et al., 1994). However, our

experiment suggests that the action of phenol oxidase can

generate phenolic substances under waterlogged conditions

by mobilizing the peat matrix, since phenolic compound

concentrations correlated strongly and positively with

phenol oxidase activities in all seasons (winter 0.96,

P!0.01; spring 0.92, P!0.001; summer 0.94, P!0.001;

autumn 0.88, P!0.001). While a positive correlation was

also reported by Freeman et al. (2001b), this is not proof of

cause and effect, and although phenol oxidase may make an

important contribution to DOC dynamics, numerous

enzymes will be involved. It is improbable though that

substrate induction (due to increased phenolic compound

concentrations already present at the thermal optimum) is

responsible for the peak phenol oxidase activities and

positive correlation observed in each season, because peaks

in phenolic compounds were not present before the thermal

gradient incubation began. Optimum phenol oxidase

activities occur at maximum ambient temperatures and if

the enzyme were only capable of degrading material

dissolved in the pore waters then a negative correlation

would be expected at such temperatures. The positive

correlation suggests that phenolic substances are being

N. Fenner et al. / Soil Biology & Biochemistry 37 (2005) 1814–1821 1819

cleaved from the vast peat resource faster than they can be

consumed/transformed by the microbial community, i.e. net

production over consumption is occurring. This is in line

with the role peatlands play as substantial aquatic carbon

sources (Freeman et al 2001b; Urban et al., 1989).

Temperature-dependent solubilities also seem unlikely to

be the cause of the observed maxima, given that all the

measured variables show thermal optima at the same

temperature in a given season (Table 1), including microbial

respiration, which suggests a biological cause.

The same was found in the case of b-glucosidase, with

activities correlating strongly and positively with DOC

concentrations (winter 0.91, P!0.01; spring 0.85, P!0.01;

summer 0.92, P!0.001; autumn 0.72, P!0.05). Care is

therefore required when dealing with soil enzyme activities,

because in experiments involving purely pore waters but

lacking any soil matrix, there would be a fixed amount of

dissolved material available for removal by the enzyme and

a negative correlation would be apparent. In contrast, the

presence of a matrix would allow an increased potential for

the release of materials due to enzymic mobilization,

leading to a positive correlation being produced. Indeed,

both positive and negative correlations between b-glucosi-

dase and DOC have been reported in the literature (e.g.

Freeman et al., 1997, 1998), although this may be due to

other factors such as end product inhibition, differing soil

moisture levels, oxygen status or substrate quality etc.,

illustrating a complexity that must be understood if we are

to accurately model carbon cycling within the peatland

system.

Phenolic compounds have (hydrolase) enzyme inhibiting

properties (Appel, 1993; Freeman et al., 2001a; Wetzel,

1992) and their concentration relative to that of DOC is

thought to be important in determining enzyme activities in

fresh water aquatic systems (Freeman et al., 1990). The

relative activities of phenol oxidase and b-glucosidase in

this experiment appear to be crucial in determining the

concentrations of dissolved phenolic compounds and DOC

available for export. Thus, these enzymes not only play a

major role in carbon cycling within the peatland itself, but

also may influence the concentrations of phenolic sub-

stances and DOC persisting in the aquatic systems that drain

such areas (Freeman et al., 2001b). This is an important

issue, with implications not only for stream ecology, but

also for drinking water quality (Alarcon-Herrera et al.,

1994; Worrall et al., 2003).

Generally, the magnitude of enzyme activities, solute

concentrations and CO2 effluxes increased throughout the

year from winter to autumn, probably reflecting plant and

microbial inputs during the growing season and subsequent

senescent period. The AMW spectra of pore water DOC

also apparently shifted with season, although only the

extremes of winter and summer were compared. In the

winter peat (Fig. 2), a primary fraction of mid AMW

material (O5000–!90,000 Da) was seen, present in the

greatest amount at the highest ambient temperature recorded

during peat collection. This fraction may represent a balance

of enzymic generation of high AMW material

(O200,000 Da) from the matrix (via the action of phenol

oxidase, for example) and community degradation of such

material to the mid fraction and lower AMW fractions

(!5000 Da), through enzymes such as b-glucosidase

(Burns, 1978; Meyer-Reil, 1991). Eventually a proportion

of these potentially more labile materials will be miner-

alized to produce CO2.

In the spectra produced by the summer peat (Table 1), a

somewhat different situation occurred in that it was the

(O90,000–!200,000 Da) AMW fraction, absent in the

winter peat, that appeared to be temperature sensitive. This

fraction became more dominant at 18 and 20 8C (along with

enzyme activities and microbial respiration), which is

somewhat higher than the highest ambient temperature

recorded. This may be because the microbial community

present has the potential to acclimate to higher temperatures

than are currently experienced in British summertime, or the

temperature recorded in the field was simply lower than that

reached in the field. The differing DOC spectra between

seasons may relate to changes in relative plant and microbial

inputs, but also to the presence of different active microbial

communities (inferred from the shifting thermal optimum of

enzyme activities) and thus catabolism of organic matter

through different biochemical pathways.

4.1. Methodological implications

The apparent sensitivity of peatlands to ambient seasonal

conditions, implies that soil warming (due to climate

change) could have a profound effect on carbon exports,

both to the aquatic system (quantity and quality) and to the

atmosphere. Clear seasonal differences in the biogeochem-

ical properties of the peat mean that its response to

environmental stimuli cannot be predicted by a single

sample, or even limited sample points in time, complicating

the modelling of such systems. Seemingly, the response of

peat not only differs between sites in its response to

temperature changes (Updegraff et al., 1998), but also

within the same site depending on season. Thus, without

knowing the thermal history of peat we may be unable to

predict its response to climate change.

A further methodological implication of our study

concerns the optimized or standardized conditions (such

as relatively high incubation temperatures and buffer

additions) that are typical in soil enzyme studies (Burns,

1978; Freeman et al., 1995; Tabatabai, 1982). While this

may be useful in determining potential enzyme activity, it is

clear that such results are totally unrepresentative of natural

process rates and major errors will occur unless assays are

performed at field temperature, because the thermal

optimum for an enzyme shifts according to season, in line

with ambient temperatures.

N. Fenner et al. / Soil Biology & Biochemistry 37 (2005) 1814–18211820

5. Conclusions

The presence of a thermal optimum for carbon proces-

sing in a given season (where enzyme activities, dissolved

product concentrations and microbial respiration reach their

peak at a temperature that coincides with maximum ambient

temperatures) suggests that the microbial community is

acclimated to the prevailing external conditions and is

dynamic, shifting with changing environmental tempera-

tures. Under the waterlogged conditions of this experiment,

phenol oxidase and b-glucosidase apparently generated

phenolic and DOC compounds, respectively, from the peat

matrix, rather than simply degrading those materials already

dissolved in the pore waters. The relative activities of these

enzymes may govern the nature and magnitude of DOC

exports to the receiving waters, as well as trace gas fluxes to

atmosphere, with considerable differences depending on

season. Such pronounced seasonality in carbon processing

must be taken into account when modelling carbon fluxes in,

and from peatland systems. Moreover, enzyme assays in soil

ecological analyses should be performed at field tempera-

tures, if information on natural process rates is required,

because the thermal optimum for carbon-cycling enzymes

shifts with seasonal changes in temperature.

Acknowledgements

The authors gratefully received funding from the Sir

William Roberts scholarship to NF (University of Wales,

Bangor), the Natural Environment Research Council,

Leverhulme Trust, UK and Royal Society. The authors

wish to thank R. Goode for supporting data and Professor K

Wieder for technical suggestions.

References

Alarcon-Herrera, M.T., Bewtra, J.K., Biswas, N., 1994. Seasonal variations

in humic substances and their reduction through water treatment

processes. Canadian Journal of Civil Engineering 21, 173–179.

Appel, H.M., 1993. Phenolics in ecological interactions: the importance of

oxidation. Journal of Chemical Ecology 19, 1521–1553.

Atlas, R.M., 1988. Microbiology: Fundamentals and Applications, second

ed. Collier Macmillan publishers, London.

Bailey, L.D., Beauchamp, E.G., 1973. Effects of temperature on NO3K and

NO2K reduction, nitrogenous gas production and redox potential in

saturated soil. Canadian Journal of Soil Science 53, 213–218.

Bandick, A.K., Dick, R.P., 1999. Field management effects on soil enzyme

activities. Soil Biology & Biochemistry 31, 1471–1479.

Box, J.D., 1983. Investigation of the Folin-Ciocalteau phenol reagent for

the determination of the polyphenolic substances in natural waters.

Water Research 17, 249–261.

Briones, M.J.I., Ineson, P., Poskitt, J., 1998. Climate change and Cognettia

sphagnetorum: effects on carbon dynamics in organic soils. Functional

Ecology 12, 528–535.

Burns, R.G., 1978. Soil Enzymes. 380 pp.

Chin, Y.P., Traina, S.J., Swank, C.R., Backus, D., 1998. Abundance and

properties of dissolved organic matter in pore waters of a freshwater

wetland. Limnology and Oceanography 43, 1287–1296.

Dawson, R.M.C., Elliott, D.C., Elliott, W.H., Jones, K.M., 1989. Data for

Biochemical Research, third ed. Oxford Science Publications.

Freeman, C., Lock, M.A., Marxsen, J., Jones, S.E., 1990. Inhibitory effects

of high molecular weight dissolved organic matter upon metabolic

processes of biofilms from contrasting rivers and streams. Freshwater

Biology 24, 159–166.

Freeman, C., Liska, G., Ostle, N.J., Jones, S.E., Lock, M.A., 1995. The use

of fluorogenic substrates for measuring enzyme activity in peatlands.

Plant and Soil 175, 147–152.

Freeman, C., Liska, G., Ostle, N., Lock, M.A., Hughes, S., Reynolds, B.,

Hudson, J., 1997. Enzymes and biogeochemical cycling in wetlands

during a simulated drought. Biogeochemistry 39, 177–187.

Freeman, C., Nevison, G.B., Hughes, S., Reynolds, B., Hudson, J., 1998.

Enzymic involvement in the biogeochemical responses of a Welsh

peatland to a rainfall enhancement manipulation. Biology and Fertility

of Soils 27, 173–178.

Freeman, C., Ostle, N., Kang, H., 2001a. An enzymic ‘latch’ on a global

carbon store. Nature 409, 149.

Freeman, C., Evans, C.D., Monteith, D.T., Reynolds, B., Fenner, N., 2001b.

Export of organic carbon from peat soils. Nature 412, 785.

Gordon, A.M., Tallas, M., Van Cleve, K., 1987. Soil incubations in

polythene bags: effect of bag thickness and temperature on nitrogen

transformations and CO2 permeability. Canadian Journal of Soil

Science 67, 65–75.

Hughes, S., Reynolds, B., Hudson, J., 1996. Release of bromide following

rewetting of a naturally drained acid gully mire. Soil Use and

Management 12, 62–66.

Ineson, P., Benham, D.G., Poskitt, J., Harrison, A.F., Taylor, K., 1995.

Climate change and UK hill grassland soils. Annual Report, Institute of

Terrestrial Ecology, 1994–5, pp. 78–81.

Kang, H.J., Freeman, C., 1999. Phosphatase and arylsulphatase activities in

wetland soils: annual variation and controlling factors. Soil Biology &

Biochemistry 31, 449–454.

Killham, K., 1996. Soil Ecology. Cambridge University Press, Cambridge.

Koerselman, W., Van Kerkhoven, M.B., Verhoeven, J.T.A., 1993. Release

of inorganic N, P and K in peat soils; effects of temperature, water

chemistry and water level. Biogeochemistry 20, 63–81.

Lensi, R., Chalamet, A., 1982. Denitrification in waterlogged soils: in situ

temperature-dependent variations. Soil Biology & Biochemistry 14,

51–55.

McLatchey, G.P., Reddy, K.R., 1998. Wetlands and aquatic processes.

Regulation of organic matter decomposition and nutrient release in a

wetland soil. Journal of Environmental Quality 27, 1268–1274.

Meyer-Reil, L.A., 1991. Ecological aspects of enzymatic activity in marine

sediments. In: Chrost, R.G. (Ed.), Microbial Enzymes in Aquatic

Environments. Springer, New York, pp. 84–95.

Moore, T.R., Dalva, M., 1993. The influence of temperature and water table

position on methane and carbon dioxide emissions from laboratory

columns of peatland soils. Journal of Soil Science 44, 651–664.

Pind, A., Freeman, C., Lock, M.A., 1994. Enzymic degradation of phenolic

materials in peatlands—measurement of phenol oxidase activity. Plant

and Soil 159, 227–231.

Ranneklev, S.B., Baath, E., 2001. Temperature-driven adaptation of the

bacterial community in peat measured by using thymidine and leucine

incorporation. Applied and Environmental Microbiology 67,

1116–1122.

Ross, S.M., 1985. Field and incubation temperature effects on mobilization

of nitrogen, phosphorus and potassium in peat. Soil Biology &

Biochemistry 17, 479–482.

Saiya-Cork, K.R., Sinsabaugh, R.L., Zak, D.R., 2002. The effects of long

term nitrogen deposition on extracellular enzyme activity in Acer

saccharum forest soil. Soil Biology & Biochemistry 34, 1309–1315.

N. Fenner et al. / Soil Biology & Biochemistry 37 (2005) 1814–1821 1821

Simon, M., Wunsch, C., 1998. Temperature control of bacterioplankton

growth in a temperate large lake. Aquatic Microbial Ecology 16,

119–130.

Sinsabaugh, R.L., Antibus, R.K., Linkins, A.E., 1991. An enzymic

approach to the analysis of microbial activity during plant litter

decomposition. Agriculture, Ecosystems and Environment 34, 43–54.

Sinsabaugh, R.L., Moorhead, D.L., Linkins, A.E., 1994. The enzymic basis

of plant litter decomposition: emergence of an ecological process.

Applied Soil Ecology 1, 97–111.

Tabatabai, M.A., 1982. Soil enzymes. In: Page, A.L. (Ed.), Methods of Soil

Analysis. Part 2 Chemical and Microbiological properties, second ed.

American Society of Agronomy, Soil Science Society of America,

Madison.

Tipping, E., Woof, C., Rigg, E., Harrison, A.F., Ineson, P., Taylor, K.,

Benham, D., Poskitt, J., Rowland, A.P., Bol, R., Harkness, D.D., 1999.

Climatic influences on the leaching of dissolved organic matter from

upland UK moorland soils, investigated by a field manipulation

experiment. Environment International 25, 83–95.

Updegraff, K., Bridgham, S.D., Pastor, J., Weishample, P., 1998. Hysteresis

in the temperature response of carbon dioxide and methane production

in peat soils. Biogeochemistry 43, 253–272.

Urban, N.R., Bayley, S.E., Eisenreich, S.J., 1989. Export of dissolved

organic carbon and acidity from peatlands. Water Resources Research

25, 1619–1628.

Wetzel, R.G., 1992. Gradient dominated ecosystems: sources and

regulatory functions of dissolved organic matter in freshwater

ecosystems. Hydrobiologia 229, 181–198.

Wilson, J.O., Crill, P.M., Bartlett, K.B., Sebacher, D.I., Harriss, R.C.,

Sass, R.L., 1989. Seasonal variation of methane emissions from a

temperate swamp. Biogeochemistry 8, 55–71.

Worrall, F., Burt, T., Adamson, J., 2003. Controls on the chemistry of runoff

from an upland peat catchment. Hydrological Processes 17, 2063–2083.