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How-do-enzymes-catalysing-soil-nitrogen-transformations-respond-to-changing-temperatures 2012 ...

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Biol Fertil Soils
DOI 10.1007/s00374-012-0722-1
SHORT COMMUNICATION
How do enzymes catalysing soil nitrogen transformations
respond to changing temperatures?
Fiona C. Fraser
&
Paul D. Hallett
&
Philip A. Wookey
&
Iain P. Hartley
&
David W. Hopkins
Received: 4 March 2012 /Revised: 8 July 2012 /Accepted: 16 July 2012
#
Springer-Verlag 2012
Abstract Biological processes in soils are regulated in part
by soil temperature, and there is currently considerable
interest in obtaining robust information on the temperature
sensitivity of carbon cycling process. However, very little
comparable information exists on the temperature regulation
of specific nitrogen cycling processes. This paper addresses
this problem by measuring the temperature sensitivity of
nitrogen cycling enzymes in soil. A grassland soil was
incubated over a range of temperatures (
only being triggered when there was a supply of exogenous
carbon. Thus, carbon supply was a greater constraint to
amidase activity than temperature was in this particular soil.
Keywords Nitrogen mineralization
.
Temperature
response
.
Protease
.
Urease
.
Amidase
2 to 21 °C) reflect-
ing 99 % of the soil temperature range during the previous
50 years at the site. After 7 and 14 days of incubation,
potential activities of protease, amidase and urease were
determined. Activities of protease and urease were positive-
ly related to temperature (activation energy;
E
a
0
−
Introduction
Transformations of soil nitrogen (N), including N mineral-
ization, are well studied but, by contrast with carbon (C)
mineralization (decomposition) (see for example von
Lützow and Kögel-Knabner
2009
), the sensitivity of N
mineralization reactions to temperature is still relatively
poorly understood. This work addresses this issue and is
aimed at developing a more robust understanding on how N
transformations are regulated by temperature.
The effects of temperature on soil processes have often
been measured using atypical temperature ranges. Many
studies have considered kinetic properties of soil enzymes
over temperature ranges that rarely extend below 5 °C but
sometimes exceed 50 °C (e.g. Gould et al.
1973
; Lai and
Tabatabai
1992
;Moyoetal.
1989
; Trasar-Cepeda et al.
2007
). Such hot temperatures substantially exceed the max-
ima for many soils under field conditions (Qian et al.
2011
),
even though some of the enzymes apparently have temper-
ature optima as high as 50 °C (Bremner and Mulvaney
1978
; Ladd
1972
; Ladd and Butler
1972
). This limits the
value of data from such studies, except possibly for surface
soils which may reach 50
49.7 and
73.4 kJ mol
−
1
, respectively, and
Q
10
0
2.97 and 2.78, respec-
tively). By contrast, amidase activity was relatively insensi-
tive to temperature, but the activity was significantly
increased after the addition of glucose. This indicated that
there was a stoichiometric imbalance with amidase activity
:
P. D. Hallett
James Hutton Institute, Invergowrie,
Dundee DD2 5DA, UK
F. C. Fraser
P. A. Wookey
Biological and Environmental Sciences, University of Stirling,
Stirling FK9 4LA, UK
I. P. Hartley
College of Life and Environmental Sciences, University of Exeter,
Exeter EX4 4RJ, UK
D. W. Hopkins (
*
)
School of Life Sciences, Heriot-Watt University,
Edinburgh EH14 4AS, UK
e-mail: david.hopkins@hw.ac.uk
60 °C as a result of direct radia-
tive warming on recently tilled and sparsely vegetated soils
(Horton et al.
1996
). In this work, we have studied temper-
ature range that accounts for over 99 % of that experienced
at the site over the past 50 years, so that the data are relevant
to the current debate on climate change in recent decades
(Manabe et al.
2011
; Murphy et al.
2009
).
–
Present Address:
P. A. Wookey
Department of Geography, The University of Sheffield,
Sheffield S10 2TN, UK
 Biol Fertil Soils
We have measured the temperature responses of individ-
ual enzyme activities that catalyse three N transformations:
(1) protease, catalysing the hydrolysis of proteins to poly-
peptides, and oligopeptides to amino acids (Ladd and Butler
1972
); (2) amidase, catalysing the hydrolysis of amides to
NH
4
+
(Frankenberger and Tabatabai
1979
); and (3) urease,
catalysing the hydrolysis of urea to NH
4
+
(Kandeler and
Gerber
1988
). Our aim was to determine temperature re-
sponse functions over a range of temperatures regularly
experienced in the field below a depth of a few millimetres.
Therateconstants(
k
) for each enzyme activity were
calculated at each temperature, assuming zero-order kinet-
ics; these were then used to calculate the activation energy
(
E
a
) of the reaction using the Arrhenius equation:
E
a
¼
slope x ln
R
where
R
is the universal gas constant.
Q
10
values were calculated for different parts of the
temperature range (from 4 to 13, 11 to 21 and 4 to 21 °C
for protease, and from
2to9,11to21and
2 to 21 °C for
−
−
urease) using the equation below:
10
=
ð
T
2
T
1
Þ
Q
10
¼
ð
k
2
k
1
=
Þ
:
Materials and methods
These temperature ranges were selected to cover the full
range of temperatures available, and the upper and low
halves of the temperature range, within the resolution of
the temperature gradient block.
Paired
t
tests were used to compare rates of enzyme
activity observed on day 7 with those observed on day 14,
and exponential regression models fitted to each day's data.
The effects of glucose addition were also investigated using
paired
t
tests; all statistical tests were carried out using the R
statistical package version 2.11.1 (R Development Core
Team
2010
).
Over 50 years, soil temperature data have been collected at
the James Hutton Institute at Invergowrie, Dundee,
Scotland, UK (56.457° N,
3.073° E). Analysis of data
between 1959 and 2009 found that 99.7 % of the temper-
atures ranged from
−
2 to 21 °C at 10-cm depth.
Soil was collected from undisturbed grassland close to
the soil temperature monitoring location. The soil has a
sandy loam texture, a pH (water) of 5.7 and C and N
contents (gravimetric) of 3.5 and 0.25 %, respectively. It is
classified as a dystric cambisol (FAO classification).
Samples were taken from the 0
−
10-cm depth using a trowel
and immediately passed through a 2-mm sieve in the field-
moist state. It was then mixed and split into sub-samples for
incubation. The water content was maintained at the field-
moist state by water addition if necessary.
Soil was incubated at temperatures ranging from
–
Results and discussion
Protease and urease activities were detected at all incubation
temperatures and were positively related to temperature
(Fig.
1
). The temperature response of protease activity was
affected by the length of the incubation period (
p
−
2to
21 °C. Samples (8
12 g dry wt. equivalent) of the field-
moist soils were weighed into glass test tubes and incubated
for either 7 or 14 days in a temperature-gradient block, as
described by Isaksen et al. (
1994
), over the
–
0.0227),
0
2to21°C
range. Incubation temperatures were checked daily using
alcohol thermometers installed in the block and were stabi-
lised to ±0.2 °C. Assays were carried out at the temperature
at which the sample was incubated also using the tempera-
ture gradient block.
Enzyme activities were assayed using published proto-
cols (Frankenberger and Tabatabai
1979
; Kandeler and
Gerber
1988
; Ladd and Butler
1972
). Absorbances for all
assays were measured using a WPA Spectrawave S1000
spectrophotometer, and concentrations were determined by
reference to standard curves. For the amidase assays, NH
4
+
concentrations were determined using the salicylate method
(Anderson and Ingram
1993
).
During preliminary experiments, amidase was only active
in this soil after addition of a C-rich substrate (data not
shown); for this reason, glucose was added to supply 3.2 mg
of C per gram of soil prior to incubation, and these samples
were assayed after 24 and 72 h, rather than 7 and 14 days,
ensuring measurements were made prior to glucose depletion.
−
Protease 7 days
Protease 14 days
0
5
10
15
20
0
5
10
15
20
Urease 14 days
Urease 7 days
0
5
10
15
20
0
5
10
15
20
Temperature (°C)
Fig. 1 Exponential models (
solid lines
) fitted to enzyme activity data
on days 7 and 14 of incubation.
Dashed lines
equal to 95 % confidence
intervals
 Biol Fertil Soils
Table 1
Q
10
values calculated for different parts of the temperature
range
Firestone
1995
). The fact that only one other study could
be found for comparison suggests that substantial further
work is required to develop a more robust understanding on
how soil protease activity is regulated by temperature in
terms of
E
a
, especially in view of the significance for N
cycling in a warming world.
The
Q
10
values for protease and urease activities varied
with temperature range (Table
1
) with an increase of ap-
proximately 10 °C at the low end of the scale (
Enzyme activity
Temperature range (°C)
Q
10
value
Protease activity
4 to 13
2.969
11 to 21
1.56
4 to 21
2.558
Urease activity
−
2 to 9
2.765
11 to 21
1.541
2 to 13 °C),
resulting in considerably greater
Q
10
values (3.0 for both
protease and urease activities) than an increase of 10 °C
at the upper end of the temperature range between 11 and
21 °C (1.6 and 1.5 for protease and urease, respectively). By
using a selection of realistically low incubation tempera-
tures, this study gains a considerable amount of information
on the temperature responses of soil enzyme activities that
may have been missed in other studies using only higher
temperatures. As
Q
10
values are frequently used when build-
ing models simulating the effects of climate change on
biogeochemical cycles (Cox et al.
2000
; Dessureault-
Rompré et al.
2010
; Reichstein and Beer
2008
; Ryan and
Law
2005
), it is important that these give a full and accurate
picture of possible temperature effects. This also prompts
the question of whether carrying these assays out at field pH
rather than an optimum pH would provide more information
on how soil nutrient cycling works in acidic soil or is likely
to respond to acidification. The soil used here has a pH
(water) of 5.7 and, as such, pH 8.1 for protease and pH
8.5 for amidase are not ideal conditions.
Amidase activity was significantly greater in glucose-
amended soil than in unamended soil (
p
−
−
2 to 21
2.779
whereas urease was not (
p
0.1561). This may be due to a
growth response from the microbes synthesising protease
activity, whereas urease often operates as an extracellular
enzyme and, thus, the temperature response does not need to
be preceded by a growth or physiological response by soil
micro-organisms. The contrasting relationships between in-
cubation temperature and protease activity on day 14 com-
pared with day 7 suggest a possible up-regulation of enzyme
activity at lower temperatures, but further work would
be required to deconvolute this from substrate-mediated
effects or shifts in thermal optima (see Hartley et al.
2008
;
Bárcenas-Moreno et al.
2009
).
The activation energies were 49.7 kJ mol
−
1
for protease
activity and 73.4 kJ mol
−
1
for urease activity. Trasar-Cepeda
et al. (
2007
) reported that the
E
a
values for protease activity
in three grassland soils ranged between 33.9 and 44.9 kJ
mol
−
1
, which is within 10 % of our estimate. Estimates for
urease activation energy ranged from 16.3 to 96.3 kJ mol
−
1
(Dalal
1985
; Rachninskii and Pelttser
1967
), with a median
of 39.1 kJ mol
−
1
, spanning the estimate for our study. There
is a range of both biotic and abiotic factors that will result in
differences in
E
a
between soils, including root system char-
acteristics (Bandick and Dick
1999
); contrasting levels of
enzyme adsorption due to soil clay content (Burns
1982
;
Ladd and Butler
1972
); land management practices (Garcia-
Giletal.
2000
); and soil moisture content (Stark and
0
0.0049); amidase
activity was not detected consistently in soil that had not
been amended with glucose irrespective of temperature with
amidase activity being undetectable or not significantly
different from zero in soil that had not been amended with
glucose at the majority of temperatures (Fig.
2a
). By com-
parison for the glucose-amended treatments, where the am-
idase activity was detected at the majority of temperatures,
0
Fig. 2 Amidase activity
obtained from soil a without
glucose added (
light grey bars
)
and b with glucose added (
dark
grey bars
).
Error bars
±1
standard error
20
20
15
15
10
10
5
5
0
0
−5
−5
−10
−10
Temperature (°C)
 Biol Fertil Soils
there was no consistent trend in enzyme activity with tem-
perature (Fig
2b
). The lack of response of the soil to the
addition of formamide was probably because there is no
demand by the soil micro-organisms for N, so expression
of amidase was not energetically expedient in this soil. This
presumably occurred because the supply of N amino acids,
amino sugars, NH
4
+
and NO
3
−
to the soil micro-organisms
was already adequate without the need to hydrolyse large
peptides. However, when excess C is added to the soil, this
balance is shifted so that more N is needed to satisfy the
requirements for microbial growth (Hopkins and Dungait
2010
). This is supported by observations that substrate
addition can lead to both N and P limitation (Allison
and Vitousek
2005
; Hopkins et al.
2006
; Stotzky and
Norman
1961
), and more work is needed on the influence
of resource stoichiometry on organic matter transformations
(Mooshammer et al.
2012
) and how this might over-ride
regulation of biogeochemical processes by temperature. The
fact that there is an interaction between C availability and N
mineralization in regulating amidase activity in this soil is
supported by the observation of positive amidase activity for
a high molecular weight amide substrate (propionamide) but
not for a lower molecular weight amide substrate
(formamide) (results not shown), and the stimulatory effect
of added glucose on amidase activity. However, this does
not explain why only a poor relationship was found between
amidase activity and incubation temperature, compared with
the strong temperature responses of the two other enzymes.
Amidase activities observed in glucose-amended samples
were compared across sampling days (24 and 72 h after
incubation to catch the effect of glucose addition), and no
significant difference was found (
p
predictions in the UK for eastern Scotland by 2080; Murphy
et al. (
2009
)) falls well within the range examined.
In summary, soil temperature exerted a substantial influ-
ence on both urease and protease activity, but no amidase
activity was detected at any temperature unless glucose was
added to the soil, which relieved C restriction and created a
need for further N-containing molecules. However, once
these nutritional constraints were removed, no clear temper-
ature response was observed for amidase activity, suggest-
ing that factors other than temperature may be driving rates
of amidase activity in this soil.
Acknowledgments This work was financially supported by The
James Hutton Institute joint Studentship programme and the University
of Stirling. The James Hutton Institute receives funding from the
Scottish Government.
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