Responses of extracellular enzyme activi

Soil Biology & Biochemistry 56 (2013) 60e68

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Soil Biology & Biochemistry
journal homepage: www.elsevier.com/locate/soilbio

Responses of the extracellular enzyme activities in hardwood forest to soil
temperature and seasonality and the potential effects of climate change

ra Merhautová, Petra Dobiásová, Tomás Cajthaml, Vendula Valásková
Petr Baldrian*, Jaroslav Snajdr,
Ve
 ská 1083, 14220 Praha 4, Czech Republic
Institute of Microbiology of the ASCR, v.v.i., Víden

a r t i c l e i n f o

a b s t r a c t

Article history:

Received 30 August 2011
Received in revised form
20 January 2012
Accepted 23 January 2012
Available online 13 February 2012

The activities of extracellular enzymes that participate in the decomposition of litter and organic matter in
forest soils depend on, among other factors, temperature and soil moisture content and also reflect the
quality of litter, which changes dramatically after a short litterfall period. Here, we explored the effects of
soil temperature and seasonality on the sizes of extracellular enzyme pools and activities in a temperate
hardwood forest soil with dominant Quercus petraea (cambisol, mean annual temperature 9.3  C).
We hypothesized that the most significant variation of enzyme activity would occur in the litter, which
faces greater variations in temperature, moisture content and chemical quality during the season, which
decrease with soil depth. The site exhibited relatively large seasonal temperature differences and
moderate changes in soil moisture content. Enzyme activity, microbial biomass, soil moisture content,
temperature and pH were monitored for three years in the litter (L), organic horizon (O) and upper
mineral horizon (Ah). Enzyme activity in vitro strongly increased with temperature until 20e25  C, the
highest temperatures recorded in situ. While no significant differences in the pools of most extracellular
enzymes and in the content of microbial biomass were found among the seasons, enzyme activity typically increased during the warm period of the year, especially in the O and Ah horizons. Approximately
63%, 64%, and 69% of total annual activity was recorded during the warm period of the year in the L, O, and

Ah horizons, respectively. Significant positive correlations were observed between soil moisture content
and fungal biomass, but not bacterial biomass, indicating a decrease of the fungal/bacterial biomass ratio
under dry conditions. The effect of moisture on enzyme activities was not significant except for endoxylanase in the litter. If soil temperature rises as predicted due to global climate change, enzyme activity is
predicted to rise substantially in this ecosystem, especially in winter, when decomposition is not limited
by drought and fresh litter that can decompose rapidly is present.
Ó 2012 Elsevier Ltd. All rights reserved.

Keywords:
Extracellular enzymes
Forest soil
Lignocellulose
Litter
Microbial ecology
Quercus petraea
Seasonality
Climate change

1. Introduction
Substantial variations in temperature or moisture during the
year influence considerably the soil processes of biomes with

such climatic characteristics, including the decomposition of organic
matter. Thus, climatic factors have previously been identified as
major causes of the observed seasonal differences in decomposition
rates in such environments due to alterations in the pools of various
extracellular enzymes, including laccase, polysaccharide hydrolases,
phosphatase, urease, protease and others (Bastida et al., 2008;
Criquet et al., 2002; Prietzel, 2001; Wittmann et al., 2004). The
effects of temperature on respiration or on the activity of selected
enzymes has been repeatedly demonstrated (Ise and Moorcroft,
2006; Moore, 1986; Wallenstein et al., 2009). However, in some

* Corresponding author. Tel.: þ420723770570; fax: þ420241062384.
E-mail address: baldrian@biomed.cas.cz (P. Baldrian).
0038-0717/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2012.01.020

environments such as the Mediterranean zone, where periods
of high temperature are accompanied by temporary droughts,
the positive effect of temperature in the warm period of the year is
counteracted by the decrease of enzyme pools due to soil or litter

desiccation (Criquet et al., 2000, 2004; Sardans and Peñuelas, 2005).
Even in the temperate zone, soil moisture content was identified as
one of the most important factors affecting the spatial distribution of
microbial biomass and extracellular enzymes (Baldrian et al., 2010b).
In temperate forests, temperature variation during the year
can be considerable, whereas the effects of drought are usually less
pronounced than in the warmer zones. However, the seasonality
of the decomposition processes may be seriously affected by the
seasonal differences in belowground C flux via plant roots (Högberg
et al., 2010; Kaiser et al., 2010) and the fact that the quality of the
litter material on the soil surface changes abruptly during the litterfall season, typically restricted to autumn, when fresh litter with
a higher content of easily available nutrients and high C/N ratio
accumulates on the forest floor (Dilly and Munch, 1996; Fioretto

P. Baldrian et al. / Soil Biology & Biochemistry 56 (2013) 60e68


et al., 2000; Snajdr
et al., 2011). As a result of these phenomena,
spring and summer are characterized by a decrease of litter quality

due to its ongoing decomposition with the concomitant increase of
photosynthetic carbon allocation underground via the mycelia of
mycorrhizal fungi. This carbon flow ceases in autumn along with
leaf abscission, when it is replaced by the seasonal litterfall. Consequently, the relative proportion of decomposer to symbiotic life
strategies of fungi is predicted to increase during the cold period of
the year. In addition to changes in microbial community composition, the production of several hydrolases by symbiotic ectomycorrhizal fungi is also increased (Mosca et al., 2007).
Although this theoretical model seems to reasonably predict the
behavior of the temperate forest soils, the extent of seasonal differences in enzyme activities and the abundance of their microbial
producers have not been addressed sufficiently with the majority of
studies obtained in the boreal and tundra ecosystems. Interestingly,
these studies showed that considerable decomposition rates can also
be achieved during the cold period of the year and that the warmest
periods do not necessarily have the highest decomposition rates
(Kahkonen et al., 2001; Wallenstein et al., 2009; Wittmann et al.,
2004). Data derived from similar studies make it possible to predict
the direction and the potential extent of changes in decomposition
rates if temperatures increase as a consequence of global climate
change.
The aim of this work was to describe the seasonal variations
in enzyme pools (i.e., the amount of enzyme molecules) and the

biomass of soil microorganisms in hardwood forest soils with
dominant Quercus petraea and to quantify the seasonal variation of
enzyme activities calculated as enzyme pools multiplied by the
relative activity of the enzyme at the in situ temperature recorded.
Litter, organic soil horizon and mineral soil were separately analyzed
because they were previously demonstrated to differ substantially
in chemical quality, microbial biomass content and community

et al., 2008). The enzyme activity and climatic
composition (Snajdr
data were collected monthly for three years. We hypothesized that
the most significant variation of enzyme activity would occur in
the litter, which faces greater variations in temperature, moisture
content and chemical quality during the season, which decrease with
soil depth. The results obtained in this study were also used to predict
the potential increase of enzyme activity under the model scenarios
of the future climate change HadAM3H and ECHAM4/OPYC3 (Jacob
et al., 2007).
2. Materials and methods
2.1. Study site and sampling

Soil and litter samples were collected in a sessile oak (Q. petraea)
forest in the Xaverovský Háj Natural Reserve near Prague, Czech
Republic, a site where previous studies targeted the spatial vari
et al., 2008), the
ability of extracellular enzyme distribution (Snajdr
description of environmental and microbial factors affecting enzyme

et al., 2011) and the
production (Baldrian et al., 2010a,b; Snajdr
decomposition abilities of saprotrophic fungi (Baldrian et al., 2011;

Snajdr
et al., 2010; Valásková et al., 2007). The soil was an acidic
cambisol with litter (L), organic horizon (O), and the mineral horizons Ah and A Litter thickness was 0.5e1.5 cm, with average pH 4.3,
46.2% C,1.76% N; O horizon thickness was 1.5e2.5 cm, average pH 3.7,
21.5% C, 0.56% N; Ah horizon thickness was 6e8 cm, average pH 3.4,
3.0e14.3% C, 0.10e0.39% N.
For the study of seasonal variation of soil enzyme activities and
microbial biomass content, soil cores (45 mm in diameter) were
collected monthly from September 2005 to August 2008. At each

sampling date, a total of six cores were collected from the same
16 m2 sampling plot with a litter layer on the forest floor (no growth

61

of grasses). For each soil core, L horizon material (0.5e1.0 cm), O
horizon material and Ah horizon material were separated, and
the materials from all cores were combined to yield a composite
sample of each horizon. Samples of the L horizon were cut into
approximately 0.25-cm2 pieces, and the samples from the deeper
soil horizons were sieved using a 2-mm sieve. The resulting samples
were used for the enzyme assays and the ergosterol and PLFA
analyses. For the analysis of temperature effects on enzyme activity,
extracellular enzymes were extracted from twelve cores sampled in
late summer 2007. The composite sample combined the L, O, and Ah
material of all cores.
Soil pH was measured in soil water extract (1 g soil and 10 mL
deionized water were mixed and left to stand overnight at room
temperature), and the soil moisture content was assessed by drying
the soil at 85  C until a constant mass was reached.

The temperature was recorded hourly during the sampling
period at the soil surface and at interfaces between the L and O, O
and Ah, and Ah and A horizons. From these data, temperatures
in soil horizons were calculated as the averages of temperature
recorded immediately above and below the respective horizon.
2.2. Enzyme extraction and assays
Enzymes were extracted from samples on the day of sample

et al., 2008), and at least
collection as previously described (Snajdr
two independent extractions were performed from each sample.
Homogenized samples of soil or litter material were extracted at
4  C for 2 h on an orbital shaker (100 rpm) with 100 mM phosphate
buffer, pH 7 (16:1 w/v), filtered through Whatman # 5 filter paper
and desalted using PD-10 desalting columns (Pharmacia, Sweden),
according to the supplier’s protocol, to remove inhibitory smallmolecular-mass compounds. The desalted samples were immediately used for enzyme activity analysis. Enzymes for the determination of temperatureeactivity relationships were extracted
from combined samples of the whole L, O and Ah horizons with
a total mass >100 g. Three independent extractions were performed. Extracts were concentrated by ultrafiltration through
a 10-kDa nitrocellulose membrane (Amicon, Millipore) before
desalting.

Laccase (EC 1.10.3.2) activity was measured by monitoring the
oxidation of 2,20 -azinobis-3-ethylbenzothiazoline-6-sulfonic acid
(ABTS) in citrate-phosphate buffer (100 mM citrate and 200 mM
phosphate; pH 5.0) at 420 nm (Bourbonnais and Paice, 1990).
Manganese peroxidase (MnP, EC 1.11.1.13) activity was assayed
using a succinate-lactate buffer (100 mM, pH 4.5) according to
Bourbonnais and Paice (1990). 3-methyl-2-benzothiazolinone
hydrazone (MBTH) and 3,3-dimethylaminobenzoic acid (DMAB)
were oxidatively coupled by the enzymes, and the resulting purple
indamine dye was detected spectrophotometrically at 595 nm.
The results were corrected by the activities of the samples without
manganese (for MnP) e the addition of manganese sulfate was
substituted by an equimolar amount of ethylenediaminetetraacetate (EDTA). One unit of enzyme activity was defined as the amount
of enzyme forming 1 mmol of reaction product per min.
Endocellulase (EC 3.2.1.4) and endoxylanase (EC 3.2.1.8) activities were routinely measured with azo-dyed carbohydrate
substrates (carboxymethyl cellulose and birchwood xylan, respectively) using the protocol of the supplier (Megazyme, Ireland). The
reaction mixture contained 0.2 mL 2% dyed substrate in 200 mM
sodium acetate buffer (pH 5.0) and 0.2 mL sample. The reaction
mixture was incubated at 40  C for 60 min and the reaction was
stopped by adding 1 mL of ethanol, vortexing for 10 s and centrifuging at 10,000  g for 10 min (Baldrian, 2009). The amount of

released dye was measured at 595 nm, and the enzyme activity was
calculated according to standard curves correlating the dye release

62

P. Baldrian et al. / Soil Biology & Biochemistry 56 (2013) 60e68

with the release of reducing sugars. One unit of enzyme activity
was defined as the amount of enzyme releasing 1 mmol of reducing
sugars per min.
Cellobiohydrolase (EC 3.2.1.91) activity was assayed in microplates using p-nitrophenyl-b-D-cellobioside (PNPC). The reaction
mixture contained 0.16 mL 1.2 mM PNPC in 50 mM sodium acetate
buffer (pH 5.0) and 0.04 mL sample. Reaction mixtures were
incubated at 40  C for 60e120 min. The reaction was stopped by
adding 0.1 mL 0.5 M sodium carbonate, and the absorbance was
read at 400 nm. 1,4-b-glucosidase (EC 3.2.1.21), 1,4-b-xylosidase
(EC 3.2.1.37) and 1,4-b-N-acetylglucosaminidase (EC 3.2.1.52)
activities were assayed by the same method using p-nitrophenylb-D-glucoside, p-nitrophenyl-b-D-xyloside and p-nitrophenylN-acetyl-b-D-glucosaminide, respectively. Phosphomonoesterase
(EC 3.1.3.1) was assayed using 2 g L 1 p-nitrophenylphosphate in
50 mM sodium acetate buffer (pH 5.0), as previously described
(Baldrian, 2009). One unit of enzyme activity was defined as the
amount of enzyme releasing 1 mmol of p-nitrophenol per min.
Spectrophotometric measurements of seasonal samples were
performed in triplicate using a microplate reader (Sunrise, Tecan)
or a UVeVIS spectrophotometer (Lambda 11, PerkineElmer) and
expressed per g dry mass of the sample. These activities are
referred to as “enzyme pools” because they reflect the amount of
enzymes present in the samples. For the analysis of temperature
effects on enzyme activity, assays were performed at 5  C, 10  C,
15  C, 20  C, 25  C, and 40  C for each enzyme in triplicate
in a microplate incubator. To calculate enzyme activity, the enzyme
pool from each sampling was multiplied by the ratio of enzyme
activity of the corresponding enzyme at the actual temperature and
at the standard assay temperature (40  C). Actual temperatures
for each sampling date were defined as the mean temperatures
recorded over the period of seven days preceding soil sampling.
Enzyme activity was also calculated per mg total PLFA as a measure
of total microbial biomass in each sample, this value being termed
the specific enzyme activity.
2.3. Quantification of microbial biomass
The samples for phospholipid fatty acid (PLFA) analysis were
extracted by a mixture of chloroform-methanol-phosphate buffer
(1:2:0.8) according to Bligh and Dyer (1959). Phospholipids were
separated using solid-phase extraction cartridges (LiChrolut Si
60, Merck), and the samples were subjected to mild alkaline

et al., 2008). The free
methanolysis as described previously (Snajdr
methyl esters of phospholipid fatty acids were analyzed by gas
chromatographyemass spectrometry (Varian 3400; ITS-40, Finnigan). Fungal biomass was quantified based on 18:2u6,9 content
(PLFAF), and bacterial biomass was quantified as a sum of i14:0,
i15:0, a15:0, 16:1u7t, 16:1u9, 16:1u7, 10Me-16:0, i17:0, a17:0,
cy17:0, 17:0, 10Me-17:0, 10Me-18:0 and cy19:0 (PLFAB). The fatty
acids found in both bacteria and fungi, 15:0, 16:0 and 18:1u7, were
excluded from the analysis (Tornberg et al., 2003). The relative
content of individual PLFA molecules was also calculated. The total
content of all PLFA molecules (PLFAT) was used as a measure of total
microbial biomass. The fungal/bacterial biomass (F/B) ratio was
calculated as PLFAF/PLFAB.
Total ergosterol was extracted and analyzed as previously

described (Snajdr
et al., 2008). Samples (0.5 g) were sonicated with
3 mL 10% KOH in methanol at 70  C for 90 min. Distilled water
(1 mL) was added, and the samples were extracted three times
with 2 mL cyclohexane, evaporated under nitrogen, redissolved in
methanol and analyzed isocratically using a Waters Alliance HPLC
system (Waters, USA) with methanol as a mobile phase at a flow
rate of 1 mL min 1. Ergosterol was detected by UV detection at
282 nm.

2.4. Statistics
Statistical analyses were performed using the software package
Statistica 7 (StatSoft, USA). For statistical purposes, all measurements were grouped into seasons (spring: 22.3e23.6, summer:
22.6e23.9, autumn: 22.9e23.12, and winter: 22.12e23.3). Autumn
corresponded to the period of litterfall of Q. petraea, with leaf
abscission starting in late September, peaking in October/November
and continuing until mid-December. The year was also divided into
a warm period of 26 weeks (weeks 15e40), with mean weekly
temperatures above the mean annual temperature, and a cold
period of 26 weeks (1e14 and 41e52). Differences among soil
horizons were tested by the Wilcoxon pair test always comparing
the data from the same sampling time. Differences between seasons
were tested using one-way analysis of variance (ANOVA) followed
by the Fisher LSD post hoc test. The correlations between individual
variables were evaluated by linear regression analyses based on the
Pearson’s correlation coefficients. To study the effects of temperature and moisture content on enzyme activities and microbial
biomass, general linear regression models (GLM, Statistica 7, StatSoft
USA) were used. In these models, the percentage of variability
explained by individual factors (e.g., temperature and moisture) was
calculated as a ratio of the variability due to this individual factor
and the total variability among samples. The percentages of
explained variability are only given for these factors when the effect
of such factor was statistically significant. In all cases, differences of
P < 0.05 were regarded as statistically significant.
3. Results
3.1. Seasonal differences in soil temperature, enzyme production
and microbial biomass content
Mean daily air temperatures at the study site varied
between 6  C and 21  C, and the weekly averages ranged
from 1.5  C in the winter to 18.5  C in the summer (Fig. 1). The
mean annual temperature was 9.3  C. A mean weekly air temperature below freezing was typically recorded during 2e3 weeks of
the year, and mean daily temperatures typically dropped below
0  C for 6e8 weeks each year. The amplitude of soil temperatures
decreased from the L horizon to the Ah horizon, with weekly
minima and maxima of 0.2  C and 15.7  C in the L, 1.4  C and 14.4  C
in the O and 3.7  C and 11.1  C in the Ah horizons. Litter horizon
temperatures also showed high daily temperature variations,
especially in the warm months (data not shown). During the year,
the lowest mean temperatures were recorded in the winter (4.0  C
in L) and the highest in the summer (14.7  C). Spring was slightly,
but insignificantly, warmer than autumn (9.6  C vs. 8.1  C in the
L horizon; Fig. 2). In the L and O horizons, soil moisture significantly
correlated with temperature (P ¼ 0.049 and 0.041, respectively);
however, in the Ah horizon, no such correlation was found. Mean
moisture content was, however, not different among seasons in any
horizon (Table 1). There was a significant difference in soil moisture
among horizons, with Ah having the highest and the least variable
moisture content (Ah ¼ 0.25  0.08 g g 1, O ¼ 0.39  0.10 g g 1, and
L ¼ 0.50  0.13 g g 1).
Enzyme pools and enzyme activities generally decreased from
the L horizons to the Ah horizons (Table 1, Fig. 2). The differences
among horizons were significant during all seasons for most
enzymes, with the exception of Mn-peroxidase, whose activity in
the O and Ah horizons was not significantly different. Also, the
content of microbial biomass significantly decreased from the
L through the O to the Ah horizon. When expressed per unit
microbial biomass, specific enzyme activities of most enzymes were
significantly higher in the L horizon, but there were no significant

P. Baldrian et al. / Soil Biology & Biochemistry 56 (2013) 60e68

A

25
Winter

Spring

Summer

Autumn

Temperature (°C)

20
15
10
5
0
-5

1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
41
43
45
47
49
51

-10

B

25
L
O
Ah

Temperature (°C)

20
15
10

MAT 9.3°C

5
0
-5

1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
41
43
45
47
49
51

-10
Week
Fig. 1. Annual course of temperatures in a Quercus petraea forest. Panel A: Mean
weekly air temperature at soil surface (boxes) and lowest and highest mean daily
temperature (vertical bars). Panel B: Mean weekly temperatures in the middle of the L
horizon (full line), O horizon (dashed line) and Ah horizon (dotted line). The values
were calculated from hourly temperature records from September 2005eAugust 2008.

differences among the O and Ah horizons in this respect. As
exceptions, the specific activities of laccase and b-xylosidase did not
differ among horizons in spring and summer, and the specific Mnperoxidase activity was not different among horizons in all seasons.
Pools of enzymes showed high variation through-out the year,
but only Mn-peroxidase activity had significant seasonal differences
in pools among seasons, as it was most abundant in autumn in
all horizons (Table 1). In addition, pools of cellobiohydrolase,
N-acetylglucosaminidase and phosphomonoesterase activities had
seasonal maxima in autumn but only in the O horizon (Table 1). None
of the measurements of soil microbial biomass showed significant
seasonal effects, except that the PLFAB in the Ah horizon in winter was
half that in summer (820 and 1660 ng g 1, respectively; Table 1).
3.2. Climatic factors affecting seasonal variability in enzyme
activity and microbial biomass
The activity of all extracellular enzymes in the soil extract
increased with increasing temperature of the reaction mixture
(Fig. 3). Although the activity of most enzymes increased over
the whole range of tested temperatures, the highest activities of
cellobiohydrolase and phosphomonoesterase were measured at
25  C and not at 30  C. The Q10 values ranged from approximately
1.4e1.5 for cellobiohydrolase and both ligninolytic enzymes,
laccase and Mn-peroxidase, to more than 2.5 for endocellulase,
endoxylanase and N-acetylglucosaminidase (Table 2). The latter
enzymes and b-glucosidase also showed the lowest activity at
temperatures 5% (Taylor and Parkinson, 1988).
Our theoretical prediction shows that an increase of temperature
might increase enzyme activities in soils substantially, provided that
the enzyme pools remain the same and soil moisture is not limiting.
The increase of enzyme activity is predicted to be more pronounced
in the winter than in the summer. As a consequence, relatively more

% var.

39.6
27.4
28.0
28.4

ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
0.009
0.004

Model

Temperature

Moisture

% var.

P

P

% var.

P

0.016
ns
0.020
0.037
ns
0.004
0.007
0.037
ns
ns
ns
ns
ns

31.8

29.3
36.9

ns
ns
0.025
ns
ns
ns
ns
ns
ns
ns
ns
0.007
0.006

ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
ns
0.002
0.011

32.6
27.2
44.9
41.1
27.6

Model
% var.

P

42.5
26.6

0.030
ns
ns
ns
ns
0.014
0.022
ns
ns
ns
ns
0.005
0.005

organic matter is likely to be decomposed during the cold period of
the year than has occurred recently. Global warming can thus
ultimately lead to greater differentiation of the cold period, with
prevailing decomposition and dominance of soil saprotrophs, and
the summer, with an increased importance of carbon flow into soil
from plant photosynthates and the increased dominance of mycorrhizal fungi and other root-associated microorganisms.
These theoretical models, however, contrast with recent results
from experimentally warmed soils. In the organic and mineral soil
horizons of a temperate hardwood forest, microbial biomass and
substrate-induced respiration decreased after 12 years of heating by
5  C. Fungal biomass decreased more than bacterial biomass, and
PLFA analyses indicated changes in community structure (Frey et al.,
2008). In mountainous forests of the temperate zone, temperature
increases by 4e5  C resulted in no change or a decrease in soil
microbial biomass content, enzyme activity and carbon-use efficiency
of the microbial community (Arnold et al., 1999; Schindlbacher et al.,
2011). One of the probable causes is the reduction of soil moisture
content. In a boreal forest, a temperature increase by as little as 0.5  C
resulted in a 22% decrease of soil moisture content, accompanied
with the decrease of fungal and bacterial biomass content and
N-acetylglucosaminidase pools (Allison and Treseder, 2008).
This study shows that, temperate forests exhibit significant
seasonal variations in enzyme activities in litter and soil. These
changes, which might ultimately result in the changes in the rates
of decomposition, are not due to the changes in microbial abundance or enzyme pools but rather result from the effects of
temperature on enzyme activity. If the global temperature changes
in the future, the seasonal differences in decomposition may be less
significant due to the predicted higher increase of decomposition in
the cold period than in the warm period.
Acknowledgments
This work was supported by the Ministry of Education, Youth
and Sports of the Czech Republic (LA10001, ME10152), by the
Ministry of Agriculture of the Czech Republic (QH72216) and by the
Institutional Research Concept of the Institute of Microbiology of
the ASCR, v.v.i. (AV0Z50200510).
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