Soil Biology & Biochemistry 55 (2012) 85e92

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Soil Biology & Biochemistry

journal homepage: www.elsevier.com/locate/soilbio

Modeling the effects of temperature and moisture on soil enzyme activity: Linking
laboratory assays to continuous eld data
J. Megan Steinweg a, b, *, Jeffrey S. Dukes c, d, Matthew D. Wallenstein a, b, e
a

Natural Resource Ecology Laboratory, Colorado State University, Fort Collins, CO 80523-1499, USA
Graduate Degree Program in Ecology, Colorado State University, Fort Collins, CO 80523-1878, USA
c
Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN 47907, USA
d
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
e
Department of Ecosystem Science and Sustainability, Colorado State University, Fort Collins, CO 80523, USA
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 29 February 2012
Received in revised form
14 June 2012
Accepted 19 June 2012
Available online 4 July 2012

Although potential enzyme activity measurements have a long history of use as an indicator of microbial
activity, current methods do not provide accurate estimates of in situ activity. In the eld, diffusion rates
typically limit the rate at which enzymes can pair with substrates. However, the common laboratory
practice of creating soil slurries removes all diffusion constraints. In addition, temperature strongly
affects in situ enzyme activities, but is rarely considered in enzyme assays. To address these limitations,
we developed a new protocol to measure the moisture and temperature sensitivity of enzyme activities.
We incorporated sensitivity data obtained using this protocol into a model to estimate the effects of
temperature and moisture on in situ b-glucosidase enzyme activity, recognizing that other factors such as
substrate concentrations and diffusion constraints also affect in situ enzyme activities.
Soil samples were collected from the Boston-Area Climate Experiment every two weeks over a 10week period to track enzyme dynamics as eld temperature and moisture changed. Precipitation
inputs to an old-eld were manipulated to produce drought (50% ambient precipitation), ambient, and
wet (150% ambient precipitation) treatments. Temperature sensitivity of b-glucosidase was determined
by assaying for the enzyme in soil slurries at three different temperatures (15, 25 and 35
C). Moisture
sensitivity was determined by exposing soils to different moisture levels in the lab and adding substrate
to homogenized dry or moist soils instead of slurries. Temperature sensitivity was calculated as Q10 and
moisture sensitivity was calculated using a linear regression for each eld treatment at each sample
collection date.
Moisture sensitivity varied signicantly among the ve sample dates and treatments, whereas
temperature sensitivity remained stable. At almost every time point, b-glucosidase activity responded
more strongly to increased moisture in soils of drought plots than in soils of ambient and wet plots. We
estimated in situ b-glucosidase activity in the fall using the temperature and moisture sensitivities.
Estimates that used only temperature or only moisture sensitivity suggested that ambient plots had the
highest activity, followed by wet and then drought plots. Estimates based on both temperature and
moisture suggested that b-glucosidase activity responded primarily to changes in temperature, except
when soils were dry, with water potentials below 1 MPa. These results demonstrate that low soil
moisture can strongly limit in situ enzyme activity in soils, negating any positive effect of warming. This
study provides a template for parsing out the role of specic abiotic drivers on in situ enzyme activities,
which could lead to the explicit incorporation of enzymes in biogeochemical models, improving upon the
ability of current models to predict rates of biogeochemical processes in dynamic environments.
2012 Elsevier Ltd. All rights reserved.

Keywords:
b-glucosidase
Moisture threshold
Temperature sensitivity
In situ conditions
Drought
Enzyme assay methods

1. Introduction
* Corresponding author. Present address: Oak Ridge National Laboratory, PO Box
2008, MS 6038, Oak Ridge, TN 37831-6038, USA. Tel.: 1 865 241 3776; fax: 1 865
576 8646.
E-mail addresses: steinwegjm@ornl.gov (J.M. Steinweg), jsdukes@purdue.edu
(J.S. Dukes), matthew.wallenstein@colostate.edu (M.D. Wallenstein).
0038-0717/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.soilbio.2012.06.015

Current soil organic matter (SOM) models (e.g. DAYCENT, RothC)

are able to reproduce large-scale changes in carbon dynamics
under most conditions (Coleman and Jenkinson, 1999; Parton et al.,
1998, 1987; Schimel et al., 1997) without explicitly modeling

86

J.M. Steinweg et al. / Soil Biology & Biochemistry 55 (2012) 85e92

microbial community dynamics (Andren and Balandreau, 1999).

However, this is often not the case in highly variable environments,
which may require more mechanistic models that include enzyme
catalysis and substrate utilization efciency parameters (Allison
et al., 2010; Lawrence et al., 2009; Li et al., 2010). These parameters may be important to include in models because microbes often
respond to changing environmental conditions through altered
resource allocation and changes in community composition, which
can affect enzyme kinetics and the aggregate physiology of
microbial communities (Collins et al., 2008; Fierer et al., 2003;
Stone et al., 2012). Enzymatic depolymerization of biopolymers is
the rate-limiting step in decomposition of unprotected SOM
(Bengtson and Bengtsson, 2007; Conant et al., 2011; Schimel and
Bennett, 2004). A few studies have explicitly incorporated
enzymes into models and have demonstrated the potential to
improve predictions of litter decomposition rates and microbial
responses in dynamic systems when enzyme pools and kinetics are
explicitly modeled (Davidson et al., 2012; Lawrence et al., 2009;
Moorhead and Sinsabaugh, 2000; Schimel and Weintraub, 2003).
However, these models have also exposed a fundamental gap in our
knowledge of abiotic controls on the rates of enzyme activities
under eld conditions, and a disconnect between what is being
measured in laboratory assays and model needs (Wallenstein and
Weintraub, 2008).
Enzyme activity has been assayed in soils for over sixty years
(Skujins, 1976) and used as a descriptor of soil quality, an indicator
of substrate use and nutrient cycling, and to provide a mechanistic
understanding of decomposition in natural and disturbed systems
(Bandick and Dick, 1999; Dilly and Nannipieri, 1998; Nannipieri,
1994; Sinsabaugh et al., 2009). There have been signicant
changes in the methodology used to assess activity, but current
methods still only provide estimates of potential enzyme activity
and do not measure parameters that drive in situ activity (Drobnk,
1961; Skujins, 1976; Wallenstein and Weintraub, 2008). Two
characteristics of current enzyme assays that hamper our ability to
better estimate in situ activity are the lack of diffusion limitations
and the use of a single assay temperature (German et al., 2011b;
Wallenstein and Weintraub, 2008).
Almost all contemporary enzyme assays are performed in slurry
(Dick, 2011; Saiya-Cork et al., 2002) to ensure adequate homogenization of added substrates and consistent estimation of activity rates
over time. However, most upland-soils are rarely water saturated, and
even when saturated, substrates are not well mixed. The slurry
decreases the substrate diffusion limitation observed in most soils
resulting in higher activities than would be expected in the eld
(Geisseler and Horwath, 2009; Geisseler et al., 2011). In addition,
substrates are not homogenously distributed throughout soil (Ettema
and Wardle, 2002) as in the slurry, again leading to an overestimation
of activity. Diffusion constraints on substrates, enzymes, or both can
have a large impact on in situ activity, especially under drought
conditions where diffusion is limited (Koch, 1990).
In early soil enzyme protocols, assay temperatures were very
high (e.g. 40
C) to optimize activity, but were outside the
temperature range experienced in most soils (Skujins, 1976). More
recently, many studies have measured activity at temperatures
closer to those experienced by soils in the eld. However, enzyme
activity is typically measured only at one temperature. Enzymatic
reactions, like all other chemical reactions, are sensitive to
temperature (Trasar-Cepeda et al., 2007; Wallenstein et al., 2011,
2009). The temperature sensitivity of enzymes has previously been
found to vary seasonally in several ecosystems (Brzostek and Finzi,
2012; Wallenstein et al., 2009). Field soil temperatures can change
drastically over the course of a day or year, and the use of one assay
temperature does not provide enough information on the
temperature sensitivity of soil enzyme activities to predict in situ

rates (McClaugherty and Linkins, 1990). Temperature sensitivity of

enzyme reactions can be tempered by diffusion limitations in the
eld, resulting in a lack of enzyme temperature dependence
(Brzostek and Finzi, 2012; Davidson and Janssens, 2006). Thus
temperature and moisture need to be considered together when
trying to understand eld activity.
We evaluated the independent and combined effects of
temperature and moisture on b-glucosidase activity at the BostonArea Climate Experiment (BACE). Utilizing data generated from two
different enzyme assay methods in combination with eld soil
moisture and temperature data, we modeled the joint effects of
temperature and moisture on b-glucosidase activity, to predict how
these factors affect in situ activity. b-glucosidase was chosen as the
model enzyme because it is involved in the nal step of cellulose
breakdown and is produced by a variety of microorganisms.
Enzyme assays were performed at three different temperatures and
multiple soil moistures to assess changes in temperature and
moisture sensitivity of b-glucosidase activity in soils collected every
two weeks during Fall 2009 from the BACE soil moisture manipulation plots. We hypothesized that soil moisture was the dominant
control on enzyme activity, and that the activity of existing
enzymes would increase as diffusion limitations were alleviated.
2. Methods
2.1. Study site
Soils were obtained from the BACE, which is located in an oldeld ecosystem in Waltham, Massachusetts, at the University of
Massachusetts Suburban Experiment Station (42
230 300 N, 71
120
5200 W). Mean annual temperature and precipitation in the area is
9.5
C and 1194 mm yr1 (Hoeppner and Dukes, 2012). Soils were
classied as mesic Typic Dystrudepts; the upper 30 cm consists of
loam soils (45% sand, 46% silt, and 9% clay), with a pH of 5.5. The site
was previously an apple orchard, and has harbored old-eld
vegetation for over 40 years. Current vegetation includes 42
species of grasses and forbs, which are primarily non-native
(Hoeppner and Dukes, 2012).
2.2. Precipitation manipulation
We used soil from nine plots in the BACE; all plots experienced
ambient temperatures, but plots were divided among three precipitation treatments, with three replicates per treatment. Precipitation
treatments were designated as ambient, wet (150% of ambient
precipitation during the growing season), and drought (50% of
ambient precipitation during the growing season). Precipitation was
controlled by clear partial roofs in the drought plots, with half of the
incoming precipitation diverted to wet plots during the growing
season (MayeOctober). During the colder months of the year,
NovembereApril, drought plots were maintained, but additional
water was not added to the wet plots. Drought treatments began in
January 2007 and wet treatments in June 2008. Thus, by the fall of
2009, soils had been exposed to drought treatments for 2.6 years and
to wet treatments for 1.1 years. During the non-freezing months, soil
moisture was measured weekly using time-domain reectometry
(waveguides were installed across 0e10 and 0e30 cm depths).
Dataloggers recorded soil temperature near the center of each plot
every 30 min throughout the year, as measured by linear temperature sensors positioned at 2 and 10 cm below the surface.
2.3. Soil sampling and processing
Soils were collected every two weeks from August 2009 through
October 2009 (Fig. 1). Three cores (5 cm diameter) were collected

J.M. Steinweg et al. / Soil Biology & Biochemistry 55 (2012) 85e92

in situ % volumetric soil moisture

a
35
30
25
20
15
10
5
0
230

240

250

260

270

280

290

300

30

in situ average soil temperature ( C)

drought
ambient
wet

25
20
15
10

87

used to create a standard curve for each sample at 25
C. The

reference standard for b-glucosidase activity was 4methylumbelliferone (MUB). The standard curve plates had
a column for each soil slurry sample and a different concentration
of the MUB standard in each of the slurry wells (0, 2.5, 5, 10, 25, 50
and 100 mM).
A 2.75 g subsample from each soil core was warmed to room
temperature (w20
C) and homogenized with 50 mM sodium
acetate buffer (pH 5.5) for 1 min in a Waring laboratory grade
blender on high to make a soil slurry. Each column on the 96-well
deep-well plates corresponded to one sample. After homogenization, 800 mL of soil slurry was aliquoted into each of the eight wells of
one column on all four plates, one plate for each of the three assay
temperatures and one for the standard curve at 25
C. Following the
addition of twelve samples into their respective columns, 200 mL of
200 mM MUB-b-D glucopyranoside substrate was added.
The plates were incubated for different lengths of time depending on incubation temperature, 1.5 h at 35
C, 3 h at 25
C, and 6 h at
15
C. Three temperatures were used to estimate two Q10 values in
case there was a signicant difference between Q10s at 15e25
C and
25e35
C. Following incubation, the plates were centrifuged for
3 min at 350 g. Afterwards, 250 mL of supernatant from each well was
placed into the corresponding well on a 96-well black plate. A Tecan
Innite M500 spectrouorometer was used to measure uorescence
with wavelengths set at 365 nm and 450 nm for excitation and
emission, respectively. The plates with the standards were used to
calculate a linear standard curve and determine b-glucosidase
activity for each sample as nmol g1 dry soil h1.

5
0
230

240

250

260

270

280

290

300

day of year, 2009

Fig. 1. In situ (a) volumetric soil moisture and (b) daily average temperature from
August 2009 through October 2009 for precipitation manipulation plots. Lines indicate
soil sample collection dates.

from each plot at 0e5 cm depths. Cores were packaged on ice and
shipped to the laboratory overnight, where the soils from each plot
were sieved (2 mm), separated from rocks and roots, homogenized,
and frozen at 10
C until analysis.
Soil moisture was measured on all soil samples by weighing 5 g
of eld-moist soil, drying the soils for 24 h at 60
C, and then
reweighing. Soil water potential was measured on soil samples
using a Decagon WP-4 potentiometer. Water potential was
measured on eld-moist and air-dried samples. In addition, 0, 100,
200 or 300 mL of water was added to dried samples and 0, 100, or
200 mL was added to the moist samples to measure the range of
water potentials that were used in the enzyme moisture sensitivity
assay.
2.4. Enzyme assays
2.4.1. Temperature sensitivity assays
Enzyme assays were performed on samples from all plots at
each collection date, using two different methods to assess
temperature and moisture sensitivity. Temperature sensitivity of
samples was assayed using a protocol modied from Saiya-Cork
et al. (2002). The temperature sensitivity of b-glucosidase was
assessed by performing assays in deep-well 96-well plates at three
temperatures: 15, 25 and 35
C. Each column of 8 wells on each
plate corresponded to one soil sample. One additional plate was

2.4.2. Moisture sensitivity assays

The moisture sensitivity assays were performed using soils from
the eld precipitation treatment plots with additional soil moisture
manipulations in the laboratory. Two subsamples, each w20 g
when eld-moist, were taken from each frozen soil sample. One
subsample was kept at eld moisture (hereafter called moist)
overnight, while the other was allowed to dry overnight (hereafter
called dried) at room temperature, both about 20
C. The following
day 2 g of soil from each group, moist and dried, were weighed into
seven different scintillation vials. Next, between 0 and 300 mL DI
water was added to alter the soil moisture in dried samples and
0e200 mL DI water was added to alter the soil moisture in moist
samples. Water was added in small drops and then the sample was
stirred with a small spatula for 5e10 s to distribute water more
evenly throughout the sample. Immediately following water
addition, 250 mL of 591 mM MUB-b-D glucopyranoside substrate
was added to samples at 20
C and stirred for 5 s. A greater
concentration of substrate was added than in the temperature
sensitivity assays because the lack of diffusion limited enzyme
activity resulting in very rapid loss of activity in preliminary
experiments. Lower concentrations of substrate made it difcult
to measure the maximum rate of enzyme activity on the
microplate reader. Eight minutes after substrate addition, 31 mL
of 50 mM sodium acetate (pH 5.5) was added and the sample
was vortexed for about 5 s. After vortexing, 800 mL of slurry from
each sample was added to three wells in a deep-well plate and
centrifuged for 3 min at 350 g. Following centrifugation, 250 mL of
each sample was transferred to a black 96-well plate and uorescence was measured as previously described. Standard curves were
also created using MUB standard at 0, 5, 25, 50 and 100 mM.
Preliminary trials indicated that soils dried down and then rewetted to their original eld moisture content produced activity similar
to eld moist samples that had not been dried. In addition,
preliminary assays indicated that 8 min provided an adequate
incubation period to assess enzyme activity before substrate limitation occurred in the moisture sensitivity assays.

J.M. Steinweg et al. / Soil Biology & Biochemistry 55 (2012) 85e92

2.5. Calculations and statistics

The temperature sensitivity of b-glucosidase activity was
assessed on samples incubated in the lab at 15, 25 and 35
C and
calculated as Q10:


Q10

10
T2 T1

activity at T2
activity at T1

(1)

where T2 is the incubation temperature 10
C greater than T1. The

Q10 values were calculated from the 15 and 25
C assays. There were
ve dates resulting in 15 Q10 values, one for each of the three eld
precipitation treatments. For the predictive model, Q10 values were
interpolated between dates to obtain a Q10 for each day.
Moisture sensitivity for b-glucosidase activity was estimated
using separate linear regressions of enzyme activity measured in the
lab for each date and treatment. PROC GLM (SAS Institute, Cary, NC)
was used to determine if there were signicant differences among
treatments and dates in moisture sensitivity slope and intercept.
Precipitation treatments signicantly affected slope and intercept at
each date, so we used different slopes and intercepts to estimate
activity at each date for each precipitation treatment. Only ve dates
were assayed, so the slopes and intercepts were interpolated for the
days between sample dates for each eld precipitation treatment for
the model. In addition, we interpolated between weekly eld soil
moisture measurements to obtain daily soil moisture values from
August 24, 2009 to October 22, 2009. PROC CORR was used to estimate the correlation coefcient and signicance of the correlation
between enzyme activity and soil moisture.
The effect of temperature on predicted in situ b-glucosidase
activity was estimated using the Q10 values estimated from lab
temperature incubations in conjunction with eld soil temperature.
We estimated temperature-based in situ activity using the
following equation from Wallenstein et al. (2009):

in situ activity from field temperature

t25
10
R25 *Q10


(2)

where R25 is the b-glucosidase activity at 25
C, Q10 is derived from

the 15 and 25
C assays, and t is the in situ temperature. Q10 did not
signicantly vary by treatment (PROC GLM, SAS Institute, Cary, NC),
but instead of averaging over treatments we chose to use the
estimated Q10 from each treatment.
We also modeled the effect of soil moisture on in situ enzyme
activity using a linear regression:

in situ activity from field moisture msm xsm bsm

(3)

where m and b are the slope and intercept, respectively, determined from enzyme assays conducted at a range of soil moistures
and x is the eld soil moisture. A linear regression model was used
because the response of enzyme activity to moisture in the lab was
linear across the range of soil moistures we used.
To estimate the effects of both temperature and moisture on in
situ b-glucosidase activity we combined the Equations (2) and (3).
Equation (2) was modied to estimate activity using both eld
variables, by replacing R25 in Equation (2) with y mx b from
Equation (3), as shown in Equation (4):

where the variables are the same as above. Multiple comparisons

ANOVAs using PROC GLM were made for model predictions to
determine if average predicted activity from each precipitation
treatment over all dates was signicantly different based on the model
used, i.e. the temperature, moisture, and temperature  moisture
models.
3. Results
Precipitation treatments altered soil moisture substantially,
with soil moisture in drought treatments being on average 50% of
ambient moisture at 0e10 cm in 2009 (Fig. 1a). There was no effect
of additional water on the soil moisture of wet plots compared to
ambient. Volumetric soil moisture increased from the end of
August 2009 to the end of October 2009, while soil temperature
declined by about 17
C during the same period (Fig. 1b). Drought
plots tended to be slightly warmer than ambient and wet plots but
the difference was minimal; about 0.25
C higher on average.
Temperature sensitivity of b-glucosidase activity did not differ
signicantly among precipitation treatments or sample dates. The
temperature sensitivity of b-glucosidase activity (Q10) in all plots on
average was about 3 throughout the sampling period (Fig. 2).
Laboratory manipulation of soil moisture resulted in a wide
range of soil water potentials, from 4 MPa in the dried samples
to 0.10 MPa in eld-moist soils amended with 200 mL water
(Fig. 3). At every sampling period, except September 10 and October
22, 2009, b-glucosidase activity in the drought treatment was more
sensitive to soil moisture than activity in ambient or wet plots, with
slope values 2e5 times higher (Table 1; Fig. 4). Drought plots
demonstrated a signicant positive correlation between soil
moisture and enzyme activity for every sample date except
September 10, 2009. Enzyme activity in ambient soils was negatively correlated with soil moisture on the rst two sample dates,
but the correlation became positive for the two October sample
dates. In wet plots, enzyme activity was positively correlated with
soil moisture except on September 25, 2009. There were differences
(P < 0.05) in slope and intercept values between drought and other
treatments at each date except October 22, 2009, when ambient
and drought plots had similar responses.
Temperature-based in situ b-glucosidase activity estimates
declined as eld temperatures dropped from August to October
(Fig. 5a). Drought plots had the lowest estimated activity despite

5.0

temperature sensitivity (Q )

88

4.5
4.0
3.5
3.0
2.5
2.0
230

t20
10

240

250

260

270

280

290

300

day of year, 2009

in situ activity from field temperature and moisture



mSM *xSM bSM *Q10

drought
ambient
wet

(4)

Fig. 2. Temperature sensitivity of b-glucosidase activity during Fall 2009 calculated

from lab activity at 15
C and 25
C enzyme assays. Points are the average Q10 collected
from sample dates with standard error bars, n 3.

J.M. Steinweg et al. / Soil Biology & Biochemistry 55 (2012) 85e92

lab moisture manipulation

driest

dried+200uL

field moist

wettest

soil water potential (MPa)

-2

drought field treatment

ambient field treatment
wet field treatment

-4

Fig. 3. Water potential in soils from drought, ambient and wet treated plots on
September 10, 2009, DOY 253 following laboratory moisture manipulations via drying
and water additions, n 1. The term driest on the x-axis refers to the air dried
subsample, with no water or substrate solution addition. Moving from left to right, the
next three points are the air dried sample with additions of 100, 200 and 300 mL water,
respectively. The eld moist point is the thawed soil sample with no moisture
manipulation. The two points following the eld moist label have 100 and 200 mL
water added, with the 200 mL water addition termed wettest.

having, on average, slightly higher plot temperatures because of

low potential activity measured in the lab. Moisture-based in situ
activity estimates suggested increasing enzyme activity as soil
moisture increased over the course of the season (Fig. 5b). There
was a dry down in all plots around day 250, and estimated enzyme
activity immediately began to decline in drought and ambient
treatments. Soil moisture increased immediately following the dry
down event, but enzyme activity did not increase in ambient- or
drought-treated plots until after day 270. In situ activity estimates
in the wet plots increased over the course of the sample period and
was not as sensitive to changes in soil moisture.
For ambient and wet precipitation treatments, estimated
activity averaged over the whole season was highest in the
moisture-based model, followed by the combined temperaturemoisture model and then the temperature-based model (Table 2).
In drought treatments the moisture-based and temperature-based
models had similar average estimated activity over the whole

Table 1
Slope and intercept values determined from linear regressions of moisture sensitivity assays for each eld treatment at each date. Bold lettering indicates signicant
regression, P < 0.05.
Date

Field treatment

Slope

Intercept

Pearsonss R

24-Aug-09

Drought
Ambient
Wet

15.43
2.571
3.193

L91.41
263.07
41.32

0.73
0.22
0.27

10-Sep-09

Drought
Ambient
Wet

3.472
2.069
6.702

119.6
209.3
19.53

0.09
0.17
0.48

25-Sep-09

Drought
Ambient
Wet

10.37
2.488
0.2223

4.406
76.50
181.9

0.55
0.25
0.01

6-Oct-09

Drought
Ambient
Wet

14.32
5.998
0.3521

L8.926
173.1
234.8

0.52
0.57
0.04

22-Oct-09

Drought
Ambient
Wet

5.988
6.386
2.410

53.68
37.60
107.94

0.46
0.75
0.35

89

season, but had very different trends in activity over the course of
the season (Fig. 5c). Using both eld soil moisture and temperature
to estimate in situ activity resulted in average estimated activities
signicantly different from the temperature-based and moisturebased models in all precipitation treatments (P < 0.05; Table 2).
In the drought plots, the predicted activity trend in the combined
model followed the moisture-based model throughout the season.
In ambient and wet plots, estimated activity trends from the
combined model were similar to those from the temperature-based
model throughout the sampling period.
4. Discussion
Given the critical role of extracellular enzymes in decomposition and nutrient cycling, our limited ability to predict enzyme
activities is an important constraint in understanding feedbacks
between climate change and biogeochemical cycles. Predicting
soil enzyme activities in dynamic environments is challenging due
to our limited understanding of enzyme production, residence
time and turnover (Burns, 1982; Wallenstein and Weintraub,
2008). In this study, we demonstrated the importance of considering the sensitivity of enzyme activity to both temperature and
moisture. The lack of a consistent effect of experimental climate
change on enzyme activity at the BACE (unpublished data) masked
important changes in the moisture sensitivity of enzyme activity
that appear to control changes in enzyme activities under eld
conditions.
By measuring b-glucosidase activity in soils at different soil
moistures, we observed a strong positive effect of moisture on
activity in the drought soils and occasionally in the ambient and
wet soils. These assays were very short, and therefore detected the
activity of the extant enzyme pool rather than that of newly
produced enzymes. As lab soil moisture increased, the extant pool
of functional enzymes was able to interact more freely with
substrates as diffusion constraints were alleviated. The maintenance of an enzyme pool during drought at the beginning of the
Fall could have been due to either increased production (Harder
and Dijkhuizen, 1983) or reduced enzyme turnover due to
enzyme stabilization (George et al., 2007). If substrate limited
enzyme activity, due to either diffusion constrains or low
substrate quantity (German et al., 2011a, 2011b), the nutrient
requirements to produce enzymes could have exceeded the net
increase in nutrient availability due to enzyme activities (Allison
and Vitousek, 2005). Increased enzyme production would not
have been favored in this scenario, because nutrient mineralization would have declined at low moistures and nutrient redistribution would have been diffusion-limited, but a decline in enzyme
turnover could have accounted for the relatively large pool of
enzymes (indicated by potential activities) detected under dry
conditions.
As soils dried, enzymes may have become adsorbed to clays and
tannins, rendering them less susceptible to proteolytic breakdown
but able to maintain reactivity (Ensminger and Gieseking, 1942;
Kandeler, 1990). The results of this study indicate that low soil
moisture can limit b-glucosidase activity, particularly when soil
water potential falls below 1 MPa, indicated by reduced estimated
enzyme activity in the drought plots following a dry down event
around September 10, 2009. Our results contrast with those of
Lawrence et al. (2009) who suggested that enzyme activity is
insensitive to moisture because activity can continue in water lms
during drought. The steep increase in activity as diffusion limitations were eliminated in the lab indicated that soil moisture in the
drought plots constrained the activity of the available enzyme pool.
In ambient and wet plots, eld soil moisture stayed above 15% for
the duration of the study and diffusion limitations may not have

90

J.M. Steinweg et al. / Soil Biology & Biochemistry 55 (2012) 85e92

b
DOY 236

500

drought
ambient
wet

400

DOY 253

400

-1

nmol activity g dry soil h

-1

500

300

300

200

200

100

100

0
0

10

20

30

40

50

60

10

20

30

40

50

60

800

DOY 268

DOY 279

800
700

600

600

500

500

400

400

300

300

200

200

-1

nmol activity g dry soil h

-1

700

100

100

0
0

10

20

30

40

50

60

500

-1

10

20

30

40

50

60

% volumetric soil moisture

DOY 295
400

-1

nmol activity g dry soil h

300

200

100

0
0

10

20

30

40

50

60

% volumetric soil moisture

Fig. 4. Potential b-glucosidase activity and respective signicant (P < 0.05) linear regressions for each precipitation treatment at different lab induced soil moistures at each sample
date, (a) Day of year (DOY) 236: August 24, 2009, (b) DOY 253: September 10, 2009, (c) DOY268: September 25, 2009, (d) DOY 279: October 6, 2009 and (e) DOY 295: October 22,
2009.

been imposed. As a result, predicted activity in ambient and wet

plots did not consistently show a strong positive correlation with
increasing eld moisture. Despite the presence of an available
enzyme pool in drought plots, a concurrent decline in soil respiration at BACE (Suseela et al., 2012) indicated a moisture limitation
control on enzyme activity and/or diffusion of soluble substrates for
microbial assimilation and metabolism.
Enzyme assays conducted at multiple temperatures conrmed
that temperature is a strong driver of enzyme activity rates, as has
been previously demonstrated in other systems (German et al., 2012;

Stone et al., 2012; Trasar-Cepeda et al., 2007; Wallenstein et al., 2009).

Despite the positive response of enzymes to increasing temperature
in the lab assays, there was no measurable change in the enzyme
temperature sensitivity over the course of the sample dates as others
have measured (Wallenstein et al., 2009), which was unexpected due
to a 17
C decline in soil temperature over the sample period. Changes
in temperature sensitivity could have been due to production of
isoenzymes with temperature sensitivities that differ from those of
the isoenzymes they are replacing within the enzyme pool
(Wallenstein and Weintraub, 2008). The replacement of an enzyme

J.M. Steinweg et al. / Soil Biology & Biochemistry 55 (2012) 85e92

estimated activity by in situ soil temperature

-1

-1

(nmol activity g dry soil h )

300

200

100

0
230

240

250

260

270

280

290

300

240

250

260

270

280

290

300

400

-1

-1

(nmol activity g dry soil h )

estimated activity by in situ soil moisture

drought
ambient
wet

400

300

200

100

0
230

-1

estimated in situ activity

-1

(nmol activity g dry soil h )

400

200

100

240

250

260

270

280

290

300

day of year, 2009

Fig. 5. Modeled effects of (a) eld soil temperature and lab measured temperature
sensitivity, (b) eld soil moisture and moisture sensitivity, (c) combined temperature
and moisture sensitivity on b-glucosidase activity.

Table 2
Average estimated in situ b-glucosidase activity during Fall 2009 by eld treatment
for each model. Letters indicate whether estimated in situ activity estimate averages
are signicantly different between models for each precipitation treatment,
P < 0.05.

Drought
Ambient
Wet

pool with enzymes that had different temperature optima may have
taken longer than the 60-day period of observation.
The temperature-based model estimated similar enzyme
activity trends to the temperature  moisture model in ambient
and wet plots, indicating that temperature was an important
controller of activity when moisture was not limiting. The estimated activity from the temperature model in the ambient plots
was lower than the moisture model because temperature was
constant at 20
C in the moisture model, whereas in the temperature model it was often below 20
C. Also, despite the lack of
a signicant difference in temperature sensitivity across moisture
treatments, the ambient plots had lower temperature sensitivities
for three of ve dates resulting in lower estimated activity. A
strong effect of moisture on estimated activity in the drought plots
was apparent in the rst half of fall, when drought plots were
extremely dry. The lack of moisture overwhelmed any enzyme
temperature response in the temperature  moisture model, so
that the moisture  temperature model resulted in lower estimated activity than the temperature-based or moisture-based
models individually. Later in the season when soil water potential increased, temperature did inuence estimated activity in the
drought plots, but was not as dominant a control as it was on
ambient and wet plot enzyme activity.
Predicted enzyme activities averaged over the whole season for
the moisture- and temperature-based models individually were
quite different in the ambient and wet plots. The moisture-based
model estimated higher activity than the temperature-based
model, likely due to the higher substrate concentration in the
assay and the assumption of a constant 20
C temperature in the
moisture-based model. In general, the methodology used to
ascertain moisture and temperature sensitivities likely overestimated in situ enzyme activity because of high substrate
concentrations compared to in situ conditions. Despite these limitations, the combined models provided a more comprehensive and
realistic view of soil enzyme dynamics, because moisture and
temperature can both strongly affect these dynamics.
5. Conclusion

300

230

91

Moisture

Temperature

Moisture  temperature

97.72  4.66a
211.1  7.46a
167.6  5.67a

96.59  3.77a
129.2  7.33b
123.0  3.58b

84.10  3.18b
173.7  3.41c
142.0  2.79c

The three models developed here were the rst attempts to

estimate enzyme activity using eld soil moisture and temperature data. Current enzyme methodology provides information on
potential activity, but relationships between enzymes and
ecosystem processes have remained poorly understood due to the
constraints of laboratory methodology (German et al., 2011b). As
climate change increases the variability of precipitation and the
frequency of droughts, diffusion constraints on enzyme activities
may emerge as an important control on soil C cycling in more
ecosystems. Temperature sensitivity of enzyme activity has
received increasing attention as of late (McClaugherty and Linkins,
1990; Trasar-Cepeda et al., 2007; Wallenstein et al., 2009), but
there is still a lack of understanding regarding moisture sensitivity
(Wallenstein and Weintraub, 2008). Our study demonstrates that
both moisture and temperature can strongly control enzyme
activity; we found temperature was the dominant control when
soil moisture was not limiting. Since both of these variables
inuence in situ enzyme activities, and warming can often induce
soil drying in the eld, it is important to understand the interactive effects of these factors. Our results suggest that soil drying
could mitigate the positive effect of warming on enzyme activity.
This research provides new insights into climate controls on in situ
enzyme activity, adding to an increasingly sophisticated understanding of how microbial responses to climate will affect soil C
and nutrient cycling (Wallenstein and Hall, 2012; Wallenstein
et al., 2012).

92

J.M. Steinweg et al. / Soil Biology & Biochemistry 55 (2012) 85e92

Acknowledgments
We thank Carol Goranson for sampling and shipping soils and
maintaining the BACE. This research was supported by grants to
MDW and JSD from the U.S. Department of Energys Ofce of
Science (BER), through the Northeastern Regional Center of the
National Institute for Climate Change Research, and from NSF (DEB
to JSD).
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