FEMS Microbiology Ecology 14 (1994) 221-232
© 1994 Federation of European Microbiological Societies 0168-6496/94/$07.00
Published by Elsevier
221
FEMSEC 00535
Thymidine, leucine and acetate incorporation into
soil bacterial assemblages at different temperatures
M. Diaz-Ravifia, A. F r o s t e g ~ r d a n d E. B ~ t h
*
Department of Microbial Ecology, Lund University, Helgonaviigen 5, S-223 62 Lund, Sweden
(Received 17 January 1994; revision received 9 March 1994; accepted 20 March 1994)
Abstract: Thymidine, leucine and acetate incorporation into soil bacterial communities extracted from two different soils using
homogenisation-centrifugation were measured at different temperatures (0-28"C). Similar effects of temperature were found for
both soils used. Optimum temperatures for incorporation of acetate into lipids were found between 20 and 24°C, while the
incorporation of thymidine and leucine into cold acid insoluble material increased with temperature. A good fit to the square root
model (Ratkowsky model) was found for all three methods, when only data below optimum was considered for the acetate
incorporation. The apparent Zmin calculated from this model was -8.4:i:0.77°C for thymidine incorporation. Tmi n for acetate
incorporation was slightly higher. Leucine incorporation had significantly higher Tmin (-6.0:1: 0.62°C), and the Q10 between 0 and
10°C was also higher than for the two other measurements. This resulted in a leucine/thymidine incorporation ratio which
increased from 0°C up to about 15°C, but remained constant at temperatures above 15°C. The amount of leucine incorporated into
hot acid insoluble material (protein) as a percentage of that incorporated into cold acid insoluble material (total macromolecules)
was also constant above 15°C (about 40%), but decreased at lower temperatures to less than 25%. No effects were found of
temperature on non-specific incorporation of thymidine into macromolecules other than DNA, or acetate incorporation into
different lipid fractions (neutral, glyco- and polar lipids). The fact that the temperature relationships for soil bacterial communities
appeared to follow the square root model will facilitate comparisons of such relationships between different soils, as well as
recalculation of data to actual field temperatures.
Key words: Thymidine; Leucine; Acetate; Incorporation; Soil bacteria; Temperature; Ratkowsky model
Introduction
Temperature is one of the most important
environmental factors affecting microbial activity
and must be considered when activity is measured in natural environments. In aquatic studies
the influence of temperature can be accounted
• Corresponding author. Fax: (46) 10 41 58.
SSD1 0 1 6 8 - 6 4 9 6 0 0 0 1 8 - R
for during actual in situ measurements. This is
not the case with soil studies, where in situ incasurements are rare. Instead, the procedure is
often to take soil samples destructively and perform the measurements at a standardised temperature in the laboratory. If the laboratory incubation temperature deviates from the in situ temperature, the measured values must be adjusted
to actual field temperatures, or to different temperatures if, for example, modelling on an annual
basis is an ultimate goal.
222
The effect of temperature is traditionally accounted for by using a Qm relationship or the
Arrhenius equation. Q10 values are usually between 2 and 3 at normal temperatures according
to the van't Hoffs rule (e.g. [1] for respiration),
but Qm usually increases with decreasing temperature and can be as high as 5 to 10 (see, e.g.
[2-4], for different soil microbial activites). Similarly, it is only within a specific interval below the
optimum temperature that the Arrhenius equation can be used for an approximation of a linear
relationship between the natural logarithm of the
activity and the inverse absolute temperature. If
the whole temperature range below the optimum
is considered, it has been shown that a curve
instead of a straight line is obtained [5], and that
the deviation from a simple straight line will
increase with decreasing temperature,
Different theoretical and empirical models
have been used to m o d e l / e x p l a i n temperature
relationships of microbial growth in pure culture
studies and within food microbiology [6-9]. In
many cases the best fit has been found by the
empirical square root model (Ratkowsky model, a
special case of the Belehrfidec model [5,10-12]),
which, up to or to just below optimum temperatures, implies a straight line relationship between
temperature and the square root of the growth
rate. Since the in situ temperature in natural
environments is usually well below the optimum
temperature, the model does not have to be extended to include supraoptimal temperatures in,
e.g., studies of soils. The square root model also
appears to provide a good estimate of temperature relationships not only in pure cultures, but
also in natural microbial communities. Li and
Dickie [13] compared different models for heterotrophic uptake of a mixture of 3H-labelled
amino acids by marine bacterial assemblages at
different temperatures, and found the square root
model to give the best fit. Bell and Ahlgren [14]
also reported this (using the Belehrfidek-model)
for 3H-thymidine uptake by bacteria in a lake
sediment. If the square root model was applicable
for soil bacterial assemblages, measurements of
temperature relationships would be facilitated,
since only two temperature points below the optimum are needed. The measurements of re-
sponses of different communities to temperatures
(seasonal changes)would thus be simplified.
Bacterial growth rates in natural environments
have been estimated using three different incorporation techniques; thymidine into DNA, leucine
into protein, and acetate into phospholipids [15].
Only the first method is specific for bacteria,
since fungi and algae apparently lack the ability
to incorporate thymidine into D N A with the experimental procedure used [16-18]. Incorporation of leucine and acetate therefore cannot be
applied to natural soil samples due to interference of fungi. B ~ t h [19,20] avoided this problem
by measuring incorporation of thymidine and
leucine in bacteria extracted from soil by homogenisation-centrifugation. This extraction
technique was used in the present study to cornpare relationships between temperature and activity of natural soil bacterial assemblages.
Thymidine incorporation into bacteria in a soil
slurry was also investigated, as was the effect of
temperature on unspecific labelling of macromolecules and on the extent of isotope dilution.
Materials and Methods
Soil
Two different soils were studied: an agricu!tural sandy loam with a p H ( H 2 0 ) of 7.8 and
4.4% loss on ignition, and a forest soil with
p H ( H 2 0 ) 5.8 and 12% loss on ignition. The soils
were collected at the beginning of October, sieved
the next day (2 mm mesh size) and stored at 4°C
until used (within 3 months).
Extraction of bacteria from soil
Bacteria were extracted from soil using the
method of Faegri et al. [21] with the modifications by B ~ t h [19]. Briefly, 10 g agricultural soil
or 2.5 g forest soil were homogenised in a Sorvall
Omnimixer for 1 min and centrifuged at 750 x g
for 10 min at 5°C. The supernatant was poured
through glass wool. This resulted in a bacterial
suspension with approximately 1-2 x 107 bacteria m1-1. The bacterial suspension was divided
into different portions, which were incubated in
water baths at different temperatures or on ice
223
for the 0°C treatment. The temperature was measured before and after incubation with the labelled substances. The bacterial suspensions were
kept a maximum of 30 min at the new temperature before addition of substrate,
Bacterial activity measurements
Thymidine and leucine incorporation was determined simultaneously on the same bacterial
suspension using the method of B ~ t h [ 2 0 ] .
Methyl[3H]thymidine (925 GBq mmo1-1, Amersham) and L-[U-14C]leucine (11.9 GBq mmo1-1)
were added to 2 ml bacterial suspension at 100
nM and 395 nM, respectively (final concentration). The concentrations used were chosen to
give 0 and about 50% isotope dilution, respectively, in the agricultural soil (see below). The
incubation was terminated by adding 1 ml 5%
formalin. The suspensions were then filtered
through glass fibre filters (Whatman G F / F ) , presoaked in 1% calgon (sodium polyphosphate) in
order to reduce unspecific binding of thymidine
to the filters. After washing with 3 x 5 ml ice cold
80% ethanol and 3 x 5 ml ice cold 5% TCA, the
rim of the filters was cut away. The filters were
then placed in scintillation vials with 1 ml 0.1 M
NaOH and heated for 2 1~ at 90°C to solubilise
macromolecules and thuslncrease the counting
efficiency. After cooling, 10 ml Ultima Gold
(Canberra-Packard) were added and radioactivity
was measured in a Beckman LS 1801 liquid scintillation spectrometer. Quenching was corrected
using Beckman's H#, an external standard
method. Duplicate or triplicate samples and one
zero-time control were used for each temperature. The coefficient of variation was typically
below 5%. Incorporation of leucine and thymidine at 0°C was found to be linear with time
between 2 and 24 h for the agricultural soil.
Linear incorporation over time for up to 4 h at
20°C has been found in previous experiments
[19,20].
The distribution of radioactivity in different
macromolecules and thus the degree of unspecific labelling was determined according to the
method of Riemann and Sondergaard [22]. The
cold acid treatment to precipitate total macromolecules was as above. The hot hydroxide treat-
ment to hydrolyse RNA consisted of heating for
30 min at 60°C in an equal amount of 1 M
NaOH, followed by neutralisation with 1 M HC1
and precipitation of solubilised DNA and protein
for 30 min at 0°C with 5% TCA. The hot acid
treatment consisted of heating with 20% TCA at
90°C for 30 min to hydrolyse DNA and RNA,
followed by 30 min at 0°C to precipitate solubilised protein. Isotope dilution was determined
according to the method of Pollard and Moriarty
[23]. Different amounts of non-radioactive substrate were added with the labelled substrate, and
the amount of exogenous and de novo synthesised substrate was determined using double reciproeal plots.
To measure incorporation of acetate into lipids,
bacterial suspensions were prepared as described
above. The bacteria were concentrated by centrifugation of the bacterial solution at 12000 X g
for 20 min. The bacterial pellet was suspended in
distilled water, and 1.2-ml portions of this suspension were put in water baths at different
temperatures. [1(2)-14C]NaAc (2.07 GBq mmol- 1,
Amersham) was added (30 /zM final concentration) and the samples incubated for varying times
according to temperature (see below). The concentration was chosen in order to give linear
ineoporation over time. The incorporation was
terminated by adding 1.5 ml CHCI 3 and 3.1 ml
MeOH. The lipids were then extracted for 2 h.
The different liquid phases were separated by
adding 1.5 ml CHC13 and 1.5 ml H 2 0 and the
samples were then left overnight for a complete
phase separation. The water phase containing
non-incorporated ~4C-acetate was carefully removed, and 1.5 ml of the lower phase taken out.
This lipid extract was fractionated into three different lipid classes on columns containing silicic
acid [24]. The lipid fractions were then added to
scintillation vials, the solvents were evaporated,
and a scintillation cocktail (Ultima Gold) was
added. Incorporation into total lipids was calculated by adding the incorporation into the three
different lipid fractions, neutral, glyco- and polar
lipids. Only one sample per temperature was
used. Incorporation at 20°C was found to be
linear with time for the 1 h tested.
The following approximate temperatures were
224
used: 0, 2, 5, 10, 15, 20, 24 and 28°C. The actual
temperature was measured each time. The incubation time with the labelled substrate was between 1 and 24 h, depending on temperature, for
thymidine and leucine incorporation measurements. The incubation time was adjusted to give
similar incorporation ml-1 bacterial suspension
irrespective of temperature. Similarly, for acetate
incorporation the incubation time was adjusted to
between 15 rain and 6 h. For thymidine and
leucine incorporation the whole temperature
range (0-28°C) was studied several times during
the 3 months storage. In additional experiments
incorporation at 0 and 20°C was compared. Acetate incorporation was only determined once for
the agricultural soil and twice for the forest soil.
Using the fresh soil, isotope dilution and unspecific labelling of macromolecules were determined for the agricultural soil at 0°C and 20°C
using both thymidine and leucine incorporation,
After storage, unspecific labelling was studied in
both soils and for all temperatures using only
leucine incorporation,
Thymidine incorporation into a soil slurry
Thymidine incorporation into the whole soil
(as a soil slurry) was studied in the agricultural
soil, using the method of B ~ t h [18] as modified
by B,~th [19]. To 1 g of soil in 2 ml of distilled
water, 0.2 nmol 3H-thymidine and 1.03 nmol
non-radioactive thymidine were added. The incubation time varied between 10 and 90 min depending on the temperature (1-30°C). Macromolecules were extracted with sodium hydroxide,
D N A and protein precipitated with cold TCA
and finally D N A solubilised using hot TCA and
proteins solubilised using hot NaOH. Values for
isotope dilution and D N A recovery given by B55th
[18] were used in the calculations. Four replicates
for each temprature and 4 zero-time controls
were used. The coefficient of variation varied
between 5 and 15%. The experiment was repeated twice,
Statistics
The data were fitted to the equation: ~ =
b(T - Tmin) [5], where A is the rate of incorpora-
tion of the substrate at the temperature T, Tmin is
the apparent minimum temperature for growth
and b is a slope parameter without any direct
biological meaning. Square root transformed data
were used to calculate apparent Tmin from linear
regression, since this stabilised the variance [25].
When the data for the highest temperatures deviated from the square root model (as revealed by
inspection of the residuals), they were not included in the calculations. The deviating data
suggest that the tested temperature was near or
above the bacterial optimum temperature. Ql0
was calculated for 2 temperature intervals (0 to
10°C, and 10 to 20°C) from the above equation by
dividing incorporation rate at the higher temperature with that at the lower temperature.
Only representative measurements are shown
in the graphs. The incorporation values per ml
suspension could differ from time to time due to
different amounts of bacteria being extracted.
However, since the same bacterial suspension was
used at all temperatures tested at each measuring
date, this would not affect the temperature relationships reported here, only the absolute values.
Results
Both leucine and thymidine incorporation into
total macromolecules increased with temperature
over the whole temperature range studied and in
both soils (Figs. 1 and 2). The data fitted the
square root model well, with r 2 values always
above 0.98 (Table 1). Only occasionally did incorporation at 28°C give slightly lower values than
expected. For acetate incorporation into lipids,
the optimum temperature in the agricultural soil
was around 24°C and that in the forest soil was
between 15 and 20°C, with decreasing incorporation rates found at higher temperatures (Fig. 3).
A similar optimum around 20°C was also found in
a preliminary experiment using the forest soil
(data not shown). A decreased incorporation with
decreasing temperature and a good fit to the
square root model was found below the optimum
temperature (Table 1).
There were no significant differences in apparent minimum temperature (Tmi n) and Q10 be-
225
500~"
2"
/x •
./~
Agricultural soil
400.
"
300-
~o
~ 2o0.~
10o-
o
-10
0
,
10
20
30
Temperature(°C)
Fig. 1. The effect of temperature on 3H-thymidine incorporation into cold acid insoluble material (total macromolecules)
of soil bacteria extracted by homogenisation-centrifugation,
Closed symbols denote fresh soil, open symbols denote soil
stored for two months at 4°C. Curves were fitted by the
square root model.
It is not meaningful to compare the slope
calculated from the square root model for the
three different techniques used, since this parameter will be scale-dependent [13], that is, it will
depend on the units used to express the incorporation data. Instead, Q10 was calculated for 2
t e m p e r a t u r e intervals in order to compare the
sensitivity of the different methods to temperature changes. For all three incorporation measurements, Q10 values for the 10-20°C interval
were between 2 and 3 (Table 1). Between 0 and
10°C Q10 was higher, with values between 3.7 and
6.7 (mean 5.1 _+ 0.54) for thymidine incorporation
and between 5.0 and 13.9 (mean 7.9 + 1.30) for
leucine incorporation. Qm for acetate incorporation was slightly higher than that for thymidine
incorporation.
The effect of t e m p e r a t u r e on thymidine incorporation into bacterial D N A in a soil slurry was
similar to that of the homogenisation-centrifugation extracted bacteria (Fig. 4). A good fit to the
square root model was found ( r 2 > 0.98), with
2500
tween the two different soil types studied (Table
1). T h e r e were also no indications of changing
values of Tmin and Q10 with time of storage.
However, there were differences between different measuring occasions (Table 1). Thus, when
comparing the different techniques, only data
from the same measuring occasion have been
used.
A p p a r e n t Tmin, calculated using the square
root model, differed between the three different
techniques (Table 1). Lowest values for Tmi n w e r e
found for thymidine incorporation (between - 6 . 3
and - 10.8°C, m e a n - 8.4 + 0.77°C), while consistently higher values were found for the leucine
incorporation (between - 3 . 7 and -8.1°C, m e a n
- 6.0 _+ 0.62°C). By comparing Tmin calculated at
the same measuring occasion, Tmin f o r l e u c i n e
incorporation was 2.4 -t- 0.47°C (n = 6) higher
than for thymidine incorporation. Tmi. for acetate
incorporation was slightly higher than for the
thymidine incorporation,
~"
"~
"
"~
2000-
A•
Agricultural
o=
Forestsoil
soil
/
,~
1500-
-~
og 1000.
.~
.~
~
.~
,¢'az="
500.
0
.
-10
/ /
.
0
.
.
10
20
30
Temperature (oc)
Fig. 2. The effect of temperature on 14C-leucine incorporation
into cold acid insoluble material (total macromolecules)of soil
bacteria extracted by homogenisation-centrifugation. Closed
symbols denote flesh soil, open symbols denote soil stored for
two months at 4°C. Curves were fitted by the square root
model.
226
Table 1
Tmin, r 2 and Qt0 calculated from the square root model for thymidine (TdR), leucine (Leu) and acetate (Ac) incorporation into
bacteria extracted from soils using homogenisation-centrifugation
Date
Soil
Method
Tmin
r2
Q0-x0
Q 10-2o
Oct 14
Agr
Oct 28
For
Nov 27
For
Dec 4
Agr
Dec 4
For
Dec 18
Agr
TdR
Leu
Ac
TdR
Leu
Ac
TdR
Leu
Ac
TdR
Leu
Ac
TdR
Leu
Ac
TdR
Leu
Ac
- 9.8
- 5.6
n.d.
- 7.7
- 6.3
n.d.
- 6.4
- 3.7
- 5.7
- 10.8
- 8.1
n.d.
- 9.4
- 7.1
n.d.
- 6.3
- 5.4
- 5.8
0.994
0.994
n.d.
0.998
0.996
n.d.
0.991
0.981
0.987
0.995
0.989
n.d.
0.995
0.997
n.d.
0.996
0.983
0.986
4.1
7.8
n.d.
5.3
6.7
n.d.
6.6
13.9
7.6
3.7
5.0
n.d.
3.7
5.8
n.d.
6.7
8.1
7.5
2.7
2.7
n.d.
2.4
2.6
n.d.
2.6
3.0
2.7
2.2
2.4
n.d.
2.2
2.5
n.d.
2.6
2.7
2.7
Agr = agricultural soil, For = forest soil. Q10 was calculated for two different temperature intervals, 0-10°C and 10-20°C. The soils
were collected at the beginning of October and stored at 4°C until used.
m0o0
/*
~"
_
8000-
/19/A/l
z~ • Agricultural soil
,x
"~
-~ 6000o
~
40oo-
20o0"~
a-
0
o •
-10
0
10
20
3O
Temperature (°C)
Fig. 3. The effect of temperature on 14C-acetate incorporation
into total !ipids of soil bacteria extracted by homogenisationcentrifugation from soils stored for two months at 4°C. Curves
were fitted by the square root model. Open symbols were not
included in the calculations,
c a l c u l a t e d Tmi n o f - - 6 . 9 a n d - 13.8°C. Q t 0 w a s
2.5 a n d 2.0 b e t w e e n 10 a n d 20°C, a n d 6.0 a n d 3.0
b e t w e e n 0 a n d 10°C, r e s p e c t i v e l y , f o r t h e t w o
measurements.
The incorporation into DNA/
(DNA + protein) was constant over the whole
t e m p e r a t u r e r a n g e t e s t e d . D N A w a s m e a s u r e d as
h o t T C A - s o l u b l e m a t e r i a l a n d p r o t e i n as c o l d
TCA-precipitated material left after the hot acid
treatment. The ratio DNA/(DNA
+ protein) was
0.39 + 0.03 f o r t h e f i r s t m e a s u r e m e n t a n d 0.38 +
0.01 f o r t h e s e c o n d .
The extent of dilution of the added isotope by
exogeneous and de novo synthesised precursor
m o l e c u l e s f o r b a c t e r i a e x t r a c t e d f r o m soil ( d e termined using the isotope dilution approach)
w a s d e t e r m i n e d u s i n g t h y m i d i n e a n d l e u c i n e incorporation at 0 and 20°C for the agricultural
soil. L i t t l e e f f e c t o f t e m p e r a t u r e o n i s o t o p e d i l u tion was found. The degree of participation of
the labelled substrate in the synthesis of macromolecules was almost 1 for thymidine, irrespective of temperature, indicating no isotope dilution. The corresponding values for leucine incorp o r a t i o n w e r e 0.48 ( 0 ° C ) a n d 0.58 (20°C).
227
The incorporation of thymidine into D N A was
11% of the incorporation into total macromolecules at 0°C, with no obvious difference at
20°C (12%, mean of two experiments). This was
only measured for the agricultural soil. Leucine
incorporation into protein as percentage of total
macromolecules, however, appeared to decrease
at lower temperatures, being 24% at 0°C, and
39% at 20°C (mean of two experiments). The
same difference was found for the forest soil
(23% at 0°C and 39% at 20°C). This relationship
was further studied using the whole temperature
range and both soil types (Fig. 5). Above approximately 15°C the incorporation into protein was
independent of the temperature, and was constantly around 40%. However, below 15°C, leucine
incorporation into protein decreased to 25%, irrespective of the soil type. This meant that calcu-
lating Tmin using leucine incorporation into protein only (hot acid insoluble) gave higher values,
than using incorporation into total macro-
50.
40'
~
~
-"
30~"
z 20
• Agricultural soil
• Forest so~
10-
o0
1'0
.....
2'0
30
Temperature(°C)
Fig. 5. Leucine incorporation into hot acid (HA) insoluble
material (protein) as a percentage of incorporation into cold
acid (CA) insoluble material (total macromolecules)at different temperatures for bacteria extracted by homogenisationcentrifugation from two different soils stored for two months
at 4°C.
6000.
~"
2-
5000.
4000-
-~
~o
30oo2000-
~
[
1000-
-10
0
1()
2()
30
40
Temperature(oc)
Fig. 4. The effect of temperature on 3H-thymidine incorporation into DNA of bacteria in a soil slurry using an agricultural
soil. Two different measurement occasions are denoted by
different symbols. Curves were fitted by the square root
model. Open symbolswere not included in the calculations,
molecules (cold acid insoluble material). The
mean difference was 2.3°C.
The ratio between leucine and thymidine incorporation into cold acid insoluble materials
(total macromolecules) varied with both temperature and soil type (Fig. 6). However, the soil type
only affected the level of this ratio, and the
temperature effect was thus similar for the two
soils. Above about 15°C, the ratio of leucine
incorporation to thymidine incorporation was unaffected by the incubation temperature. However,
below this temperature, the ratio decreased.
Incorporation of acetate into different lipid
fractions appeared to be unaffected by the incubation temperature and was similar in the two
soil types. For the agricultural soil the mean
value for incorporation into the chloroform fraction (neutral lipids) was 17% + 1.4, and corresponding values for the acetone fraction (glycolipids) and the methanol fraction (polar lipids)
were 15% + 1.6 and 67% + 3.0, respectively. The
228
40-
3
'~
0
-
~
z
x
20!,
i
10
A • Agrioultural soil
© • Forest soil
0
1'0
2'0
30
Temperature (oc)
Fig. 6. The effect of temperature on the ratio of leucine
incorporation to thymidine incorporation into cold acid insoluble material (total macromolecules) of soil bacteria extracted
by homogenisation-centrifugation. Closed symbols denote
fresh soil, open symbols denote soil stored for two months at
4°C. Expressed as tool incorporated without considering isotope dilution.
values for the forest soil were similar, 17% + 1.5,
21% + 1.1 and 62% + 1.1 in the three respective
fractions,
Discussion
The temperature relationships of soil bacterial
activities were well-explained by the square root
model below the optimum temperature. This was
true irrespective of whether the thymidine,
leucine or the acetate incorporation technique
was used with bacteria extracted from soil (Figs.
1-3), or whether thymidine incorporation was
measured in a soil slurry (Fig. 4). This result is in
accordance with those of Li and Dickie [13], who
studied amino acid uptake of bacterial assemblages in a marine habitat, and found that the
temperature relationships were well explained by
the square root model. This model therefore appears to be well-suited for bacterial communities
in diverse habitats. Li and Dickie [13] also found
that another model, the Schoolfield model [26],
gave a good fit to their data. This model was,
however, not tested in the present study.
Bacterial activity appears not to be the only
variable to follow the square root model in soil,
since the relationship appears to be valid for the
whole soil microbial community. For example,
recalculation of respiration data for a forest soil
[27] or from two samples of litter [2] showed a
good fit to the square root model (r 2 > 0.97), with
calculated Tmin of - 6 ° C for the forest soil and
- 8 and - 12°C for the two types of litter. Recalculation of other data on respiration, where fewer
temperatures have been studied, gave Tmi n between - 5 and - 10°C [4,28,29]. Lower values for
Tmi n have also been found [30-32]. One must
bear in mind that the calculated Tmi n is only a
hypothetical value, since the square root model is
valid only at temperatures where water activity is
not changing due to ice formation [5]. However,
the calculated Tmi n c a n be useful to compare
different treatments or techniques.
Although all the results in the present study
appeared to follow the square root model, there
were differences in the temperature response between the three techniques used. The most striking differences were for the acetate incorporation
into lipids compared to the two other metods. In
the former, optimum temperatures of about 20
and 24°C were found in the forest and agricultural soil, respectively (Fig. 3). In contrast, for the
thymidine and leucine incorporation, there were
only a few indications that the values measured at
28°C were lower than expected from the square
root model, indicating that the optimum temperature would be slightly above this temperature
(Figs. 1 and 2). Several indications, beside the
thymidine and leucine incorporation, also point
to optimum temperatures being above 25°C in
soil. For example, the respiration data quoted
above indicated a square root relationship between temperature and respiration up to 30°C.
Joergensen et al. [28] found that storage of a soil
at 25°C did not change the activity of the soil
microbial community. Storage at 35°C, on the
229
other hand, changed its capacity to degrade dissolved organic carbon. They concluded that at
this temperature the community had been drastically changed, presumably because 35°C, but not
25°C, was above both optimum and maximum
temperatures, and thus many soil organisms were
killed at this higher temperature. This all suggests
that the optimum temperatures found with the
acetate incorporation technique were too low.
The reason for the difference between acetate
incorporation and the other two measurements in
this respect is not known. Enzymes involved in
lipid metabolism may, however, differ in their
relationship to temperature from those of DNA
and protein synthesis. Since the incorporation of
acetate into different lipid fractions (neutral,
glyco-, and polar lipids) was constant, irrespective
of the temperature, the differences appear not to
be due to an altered partitioning of acetate incorporation into the different lipid fractions at different temperatures. Besides the difference in
optimum temperatures, both Tmin and Ql0 values
were similar for the acetate and thymidine incorporation. Similar Q10 values for these two techniques were also found by Vincent et al. [33] for
heterotrophic microorganisms in Antarctic waters.
There were also differences in the relationship
to temperature between the thymidine and
leucine incorporation techniques. The ratio of
leucine to thymidine incorporation into cold acid
insoluble molecules was constant above 15°C, but
decreased at lower temperatures (Fig. 6). This
was true, irrespective of the soil used, although
the absolute value of the ratio differed between
the different soils. This difference between the
soils was probably due to the fact that no compensation for isotope dilution was made. The
extent of isotope dilution differs between different soils [19] and could thus affect thymidine and
leucine incorporation differently ([20], B ~ t h , unpublished). The decrease in the ratio of leucine
to thymidine incorporation at lower temperatures
gave different apparent Tmin values for the two
techniques, being 2.4°C higher for leucine incorporation than for thymidine incorporation. This
difference was even more evident, if the nonspecific incorporation of labelled substrate into
different macromolecules was taken into account.
For thymidine incorporation, the percentage label incorporated into DNA was not affected by
the incubation temperature. Leucine incorporation into protein, on the other hand, decreased
with temperatures below 15°C (Fig. 5). Thus, it
appeared that protein synthesis of the soil bacterial community was more affected by low temperatures than the DNA synthesis. This can also be
seen when comparing Q10 values between 0 and
10°C, where Q10 for leucine incorporation was
always larger than for thymidine incorporation on
the corresponding date (Table 1).
The reason for these differences is unclear. It
is clearly not due to changing incorporation rates
during incubation at the different temperatures,
since the incubation times were always within the
linear part of incorporation. Thus, the ratio of
leucine to thymidine incorporation was, for exampie, constant during the 24 h incubation at 0°C.
Low temperatures might trigger an uncoupling of
protein and DNA synthesis and thus an unbalanced growth of the bacterial community. It was
recently reported that marine bacteria had an
increasing demand for organic nutrients at low
temperatures [34-36]. Thus, at low temperatures
the addition of more substrate increased the
growth rate of the bacteria, while at temperatures
nearer the optimum for the isolates, substrate
concentration did not affect the growth rates. It
might be that the bacteria extracted from soil had
higher demands for amino acids for protein synthesis at low temperatures, compared with the
demand for thymidine for DNA synthesis. Whatever the reason for the different effects of ternperature on Tramvalues for thymidine and leucine
incorporation, our results clearly show that the
temperature must be taken into account when
changes in the ratio of leucine to thymidine incorporation are to be interpreted in an ecological
context. Furthermore, when growth rate determination cannot be performed at field temperatures, temperature corrected values should be
calculated based on incorporation-temperature
relationships measured for the specific method
used.
There are indications that the different effects
of temperature on thymidine and leucine incor-
230
poration are of a more general nature, not only
because they are similar for bacteria from two
different soils, but also because similar effects are
found in water habitats. In a study of marine
bacteria, Kirchman et al. [37] measured Q10 for
thymidine and leucine incorporation on several
occasions. In all cases but one, Ql0 was higher for
leucine incorporation than for thymidine incorporation. The only exception also differed in that it
had Q10 values for thymidine incorporation more
than 5 times higher than in any of the other
samples. Excluding this sample gave Q10 values
for thymidine incorporation of 1.3 and 1.2 (mean
values of several samples for 2 different months),
while the corresponding values for leucine incorporation were 1.9 and 1.7. Li and Dickie [13]
studied 3H-amino acid incorporation into marine
bacterial assemblages during a year, and found
that the Wmin value for amino acid incorporation
into total macromolecules ( - 1.2°C) was significantly lower than the Tmin for amino acid incorporation into only protein (0.9°C). This difference
is similar to that we found for soil bacteria using
a single amino acid, leucine, where a smaller
fraction of the labelled amino acid was incorporated into protein at lower temperatures compared with incorporation into total macromolecules (Fig. 5). This resulted in Ymin b e i n g
higher for incorporation into proteins than for
incorporation into total macromolecules. The
mean difference was 2.3°C, which is similar to the
difference of 2.1°C found by Li and Dickie [13].
There a p p e a r e d to be little effect of temperat u r e o n the extent of isotope dilution o r degree of
non-specific incorporation into macromolecules
other than D N A when measuring thymidine incorporation by bacteria extracted from soil. Thus
it appears possible to study t e m p e r a t u r e relationships of bacterial D N A synthesis (growth rate)
using only thymidine incorporation into cold acid
insoluble material (total macromolecules). This
will facilitate the comparison of t e m p e r a t u r e relationships of different bacterial communities. The
fact that the t e m p e r a t u r e relationships appeared
to follow the square root model also will make
such comparisons easier, since only a few incubation temperatures have to be studied to be able
to calculate Ymi n.
Acknowledgements
This study was supported by a grant from
European Environmental Research Organisation
( E E R O ) to M.D.-R., and a grant from the
Swedish Natural Science Research Council to
E.B.
References
1 Koepf, H. (1953) Die Temperature/Zeit - Abh~ingigkeit
der Bodenatmung. Z. Pflanzenern. Diing. Bodenk. 61,
29-48.
2 Flanagan, P.W. and Veum, A.K. (1974) Relationships between respiration, weight loss, temperature and moisture
in organic residues on tundra. In: Soil Organisms and
Decomposition in Tundra (Holding, A.J, Heal, O.J.,
Maclean Jr., S.F. and Flanagan, P.W., Eds.), pp. 249-277.
Tundra Biome Steering Committee, Stockholm.
3 Donnelly, P.K., Entry, J.A., Crawford, D.L. and Cromack,
K., Jr. (1990) Cellulose and lignin degradation in forest
soils: Response to moisture, temperature, and acidity.
Mierob. Ecol. 20, 289-295.
4 Wilhelmi, V. and Rothe, G.M. (1990) The effect of acid
rain, soil temperature and humidity on C-mineralization
rates in organic soil layers under spruce. Plant Soil 121,
197-202.
5 Ratkowsky, D.A., Olley, J., McMeekin, T.A. and Ball, A.
(1982) Relationship between temperature and growth rate
of bacterial cultures. J. Bacteriol. 149, 1-5.
6 Heitzer, A., Kohler, H.-P.E., Reichert, P. and Hamer, G.
(1991) Utility of phenomenological models for describing
temperature dependence of bacterial growth. Appl. Environ. Microbiol. 57, 2656-2665.
7 Phillips, J.D. and Griffiths, M.W. (1987) The relation
between temperature and growth of bacteria in dairy
products. Food Microbiol. 4, 173-185.
8 Ratkowsky, D.A., Ross, T., McMeekin, T.A. and Olley, J.
(1991) Comparison of Arrhenius-type and Belehradek-type
models for prediction of bacterial growth in foods. J. Appl.
Bacteriol. 71, 452-459.
9 Zwietering, M.H., de Koos, J.T., Hasenack, B.E., de Wit,
J.C. and van't Riet, K. (1991) Modeling of bacterial growth
as a function of temperature. Appl. Environ. Microbiol.
57, 1094-1101.
10 Belehrfidec, J. (1926) Influence of temperature on biologi-
cal processes. Nature 118, 117-118.
11 Ratkowsky, D.A., Lowry, R.K., McMeekin, T.A., Stokes,
A.N. and Chandler, R.E. (1983) Model for bacterial culture growth rate throughout the entire biokinetic temperature range. J. Bacteriol. 154, 1222-1226.
12 McMeekin, T.A., Chandler, R.E., Doe, P.E., Garland,
C.D., Olley, J., Putro, S. and Ratkowsky, D.A. (1987)
231
13
14
15
16
17
18
19
20
21
22
23
24
25
Model for combined effect of temperature and salt concentrations/water actMty on the growth rate of Staphylococcus xylosus. J. Appl. Bacteriol. 62, 543-550.
Li, W.K.W. and Dickie, P.M. (1987) Temperature characteristics of photosynthetic and heterotrophic activities:
seasonal variations in temperate microbial plankton. Appl.
Environ. MicrobioL 53, 2282-2295.
Bell, R.T. and Ahlgren, I. (1987) Thymidine incorporation
and microbial respiration in the surface sediment of a
hypereutrophic lake. Limnol. Oceanogr. 32, 476-482.
Moriarty, D.J.W. (1990) Techniques for estimating bacterial growth rates and production of biomass in aquatic
environments. In: Methods in Microbiology (Grigorova, R.
and Norris, LR., Eds.), Vol. 22, pp. 211-234. Academic
Press, London.
Fallon, R.D. and Newell, S.Y. (1986) Thymidine incorporation by the microbial community of standing dead
Spartina alterniflora. Appl. Environ. Microbiol. 52, 12061208.
Moriarty, D.LW. (1986) Measurement of bacterial growth
rates in aquatic systems from rates of nucleic acid synthesis. Adv. Microb. Ecol. 9, 245-292.
B~t~th, E. (1990) Thymidine incorporation into soil bacteria. Soil Biol. Biochem. 22, 803-810.
B ~ t h , E. (1992) Thymidine incorporation into macromolecules of bacteria extracted from soil by homogenization-centrifugation. Soil Biol. Biochem. 24, 1157-1165.
B ~ t h , E. (1994) Measurement of protein synthesis of soil
bacterial assemblages using the leucine incorporation
technique. Biol. Fert. Soil. 17, 147-153.
Faegri, A., Lid Torsvik, V. and Goks6yr, J. (1977) Bacterial and fungal activities in soil: separation of bacteria and
fungi by a rapid fractionated centrifugation technique. Soil
Biol. Biochem. 9, 105-112.
Riemann, B. and Scndergaard, M. (1984) Measurements
of diel rates of bacterial secondary production in aquatic
environments. Appl. Environ. Microbiol. 47, 632-638.
Pollard, P.C. and Moriarty, D.J.W. (1984) Validity of the
tritiated thymidine method for estimating bacterial growth
rates: measurements of isotope dilution during DNA synthesis. Appl. Environ. Microbiol. 48, 1076-1083.
Frosteg~rd, ,~., Tunlid, A. and B ~ t h , E. (1991) Microbial
biomass measured as total lipid phosphate in soils of
different organic content. J. Microb. Methods 14, 151-163.
Alber, S.A. and Schaffner, D.W. (1992) Evaluation of data
transformations used with the square root and Schoolfield
26
27
28
29
30
31
32
33
34
35
36
37
models for predicting bacterial growth rate. Appl. Environ. Microbiol. 58, 3337-3342.
Schoolfield, R.M., Sharpe, P.J.H. and Magnuson, C.E.
(1981) Non-linear regression of biological temperature-dependent rate models based on absolute reaction-rate theory. J. Theor. Biol. 88, 719-731.
B ~ t h , E. and S6derstr6m, B. (1988) FDA-stained fnngal
mycelium and respiration rate in reinoculated sterilized
soil. Soil Biol. Biochem. 20, 403-404.
Joergensen, R.G., Brookes, P.C. and Jenkinson, D.S. (1990)
Survival of the soil microbial biomass at elevated temperatures. Soil Biol. Biochem. 22, 1129-1136.
Salonius, P.O. (1990) Respiration rates in forest soil organic horizon materials treated with simulated acid rain.
Can. J. For. Res. 20, 910-913.
Anderson, T.-H. and Domsch, K.H. (1986) Carbon assimilation and microbial activity in soil. Z. Pflanzenernaehr.
Bodenk. 149, 457-468.
Blet-Charaudeau, C., Muller, J. and Laudelout, H. (1990)
Kinetics of carbon dioxide evolution in relation to microbial biomass and temperature. Soil Sci. Soc. Am. J. 54,
1324-1328.
Howard, D.M. and Howard, P.J.A. (1993) Relationships
between CO 2 evolution, moisture content and temperature for a range of soil types. Soil Biol. Biochem. 25,
1537-1546.
Vincent, W.F. and Howard-Williams, A. (1989) Microbial
communities in southern Victoria Land streams: II. The
effect of low temperaure. Hydrobiol. 172, 39-49.
Pomeroy, L.R., Wiebe, W.J., Deibel, D., Thompson, R.J.,
Rowe, G.T. and Pakulski, J.D. (1991) Bacterial responses
to temperature and substrate concentration during the
Newfoundland spring bloom. Mar. Ecol. Prog. Ser. 75,
143-159.
Wiebe, W.J., Sheldon, W.M., Jr. and Pomeroy, L.R. (1992)
Bacterial growth in the cold: Evidence for an enhanced
substrate requirement. Appl. Environ. Microbiol. 58, 359364.
Wiebe, W.J., Sheldon, W.M., Jr. and Pomeroy, L.R. (1993)
Evidence for an enhanced substrate requirement by marine mesophilic bacterial isolates at minimal growth temperatures. Microb. Ecol. 25, 151-159.
Kirchman, D.L., Keil, R.G., Simon, M. and Welschmeyer,
N.A. (1993) Biomass and production of heterotrophic bacterioplankton in the oceanic subarctic Pacific. Deep-Sea
Res. 40, 967-988.