ELSEVIER
FEMS Microbiology
Ecology
I9
(I 996) 227-237
Adaptation of soil bacterial communities to prevailing pH
in different soils
Erland B&h
Received 3 I May 1995: revised 20 January
*
1996; accepted 6 February
1996
Abstract
The bacterial community response to pH was studied for 16 soils with pH(H,O) ranging between 4 and 8 by measuring
thymidine incorporation into bacteria extracted from the soil into a solution using homogenization-centrifugation.
The pH of
the bacterial solution was altered to six different values with dilute sulfuric acid or different buffers before measuring
incorporation. The resulting pH response curve for thymidine incorporation was used to compare bacterial communities from
the different soils. There was a correlation between optimum pH for thymidine incorporation and the soil pH(H,O). Even
bacterial communities from acid soils had optima corresponding to the soil pH, indicating that they were adapted to these
conditions. Thymidine incorporation was also compared with leucine incorporation for some soils. The leucine to thymidine
incorporation ratio was constant over the tested pH interval when incorporation values were adjusted for isotope dilution. A
good correlation was found between the scores along the first component (explaining 80% of the variation) and soil pH
( r2 = 0.85). if principal component analysis of the pH response curves for thymidine incorporation was used. The pH
response curves differed most for the extreme pH values used, and a linear relationship was found between the logarithm of
the ratio of thymidine incorporation at pH 4.3 to incorporation at pH 8.2 and the soil pH (r’ = 0.86). Thus, a simplified
technique using only two pH values, when measuring the thymidine incorporation. could be used to compare the response to
pH of bacterial communities.
Kqvnrds:
Soil; pH response;
Thymidine
incorporation:
Bacterial
community
1. Introduction
It is well known that bacteria usually are less
numerous in relation to fungi in acid forest soils
compared to arable soils with a higher pH [1,2]. One
explanation for this phenomenon has been that bacteria as a group have a higher pH optimum compared
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to fungi, and thus are less adapted to the more acidic
conditions in forest soils (e.g. [ 1,3-61). For example.
bacteria that have higher pH optima than the prevailing soil pH are frequently isolated from acid soils
[7,8], and bacterial counts from such soils can be
equal or even higher on media with a neutral pH
than on media adjusted to low pH [9,10]. On the
other hand, in several studies in forest soils, where
the pH has been increased by up to 2 pH units due to
liming. ash treatment or alkaline pollution, there was
little effect on the bacterial biomass estimated using
Societies.
All rights reserved
acridine orange direct counts [IO- 121. This casts
doubt on the conventional
explanation
that fewer
bacteria are found in acid soils due to lack of tolerance to low pH.
Until now one has been forced to rely on agar
plate techniques. when testing the pH response of a
bacterial community, either after isolating and testing
single isolates, or by performing plate counts on agar
medium with different pH. However, due to the
selectivity of the agar media, only a minor part of the
soil bacterial community can be studied in this way.
Furthermore, it is difficult to maintain an exact pH
during prolonged incubation of the agar plates. The
technique is also time consuming, since an incubation time of several weeks is needed to maximize
colony counts [13].
Recently a new technique to measure the response
of the soil bacterial community to pH was described
[lo]. It used short-term incubations of bacteria extracted directly from soil by homogenization-centrifugation and measured bacterial community growth
rates (estimated
using incorporation
of labelled
thymidine) at different pH values. This technique has
been used to detect shifts in the pH response of
bacterial communities
after treatments such as liming, ash fertilization, prescribed burning and alkaline
Table I
pH and organic
pollution, which raise soil pH [ 10,12.15]. However,
the method has so far only been applied to forest
soils with a limited range of pH values.
In the present study the bacterial community response to pH was studied for different soils ranging
between pH 4 and pH 8. One aim was to study
whether the bacterial community response to pH was
directly related to soil pH over the whole pH interval. Special emphasis was placed on acid soils. The
second aim was to compare different ways of expressing the effect of soil pH on the bacterial community response to pH. This might enable us to use
the pH response as a way of elucidating actual pH
values in microhabitats.
where pH is difficult to
measure using normal procedures.
2. Materials
and methods
The soils were chosen in order to cover a wide
range of pH. Initially it was tried to obtain soils for
which no correlation existed between organic matter
content and pH, but this was not possible for the
soils with the lowest pH. Forest soils were sampled
matter contenr in the 16 soils studied
Number
Soil
pH(H,O)
pH(KCI)
PH.,,,
Organic matter
I
Deciduous forest
Coniferous forest
Coniferous forest
Coniferous forest
Arable (gas>)
Arable (grass)
Garden
Arable
Deciduous forest
Garden
Garden
Arable (grass)
Deciduous forest
Garden
Deciduous forest
Agricultural
3.90
3.21
4.39
1.58
3.90
5.10
6.00
6.23
6.24
6.75
6.88
6.91
7.21
7.69
7.83
8.10
3.35
3.52
3.67
3.82
4.09
4.13
5.28
5.52
5.57
6.12
6.62
7.00
6.61
7.31
7.58
7.99
4.58
1.55
4.73
1.89
5.09
5.83
6.39
6.55
6.36
6.82
6.91
7.02
6.67
7.63
7.09
7.88
51.1
25.8
3.5
8.9
Il.3
I I.2
13.4
10.4
5.1
4.7
2.9
0.8
S.6
8.8
9.6
3.3
2
3
4
5
6
7
8
9
IO
II
I2
13
I4
I5
16
pH(H,OI
was measured in distilled water and pH(KCl) in 0.2 M KCI. pH,,,,
homogenization-centrifufarion.
These values were means of two determinations unless otherwise stated.
‘I Only determined once.
,’
a
rl
d
was the pH of the bacterial
(57-j
solution
extracted
by
bacterial community was measured on bacteria extracted from soil by homogenization-centrifugation
using thymidine incorporation into cold acid-insoluble material [ 151. In short, 2.5 to 10 g soil (wet
weight), depending on the organic matter content,
was homogenized with 200 ml distilled water in a
Sorvall Omnimixer,
centrifuged at 750 X g for 10
min. and the supernatant collected. To the resulting
bacterial
suspension
[’ H-methyllthymidine
was
added (200 nM final concentration,
925 GBq
mmol-‘, Amersham. UK), and the incorporation (at
20°C) was stopped after 2 h with formalin. A zerotime control (formalin
added before the labelled
thymidine) was always included. Filtration, washing
and measurement of incorporated radioactivity were
as described by BEith [16].
Before adding thymidine, the pH of the bacterial
suspensions
was altered to different values using
different buffers or dilute H, SO,. On both measur-
just beneath the litter layer, while O-5 cm was taken
for the other soils. The soils were sampled, brought
to the laboratory and sieved (mesh size 2 mm) the
same day, and then stored at 4°C.
Soil pH was measured in distilled water or KC1
(0.2 M) using 5 g wet weight of soil and 50 ml liquid
after shaking on a rotary shaker for 1 h. pH was also
measured in all bacterial suspensions (see below) at
each measuring
occasion. Organic matter content
was approximated
from loss on ignition measured
after 4 h at 600°C. Background data for the soils are
presented in Table 1.
2.2. pH respome
measurements
The bacterial community
response to pH was
measured twice for each soil. the first time within a
week of sampling and the second time after about
two months of storage. The pH response of the
4
5
PH
Fig. I, The effect of pH on thymidine incorporation into macromolecules
(cold acid insoluble material) of bacteria extracted from a forest
soil (soil 2 in Table I) by homogenization-centrifugation.
0. pH was altered with diluted sulphuric acid. where the sample with the highest
pH was used without any acid (distilled water); 0, pH was altered with citrate-phosphate
buffer; 0. pH was altered with phosphate buffer.
Bars denote SE. (n = 3) from separate experiments. Soil pH(H,O) was 4.21 and pH(KCI) was 3.51.
ing occasions. pH was altered to approx. 5.5. 6.2, 7.2
and 8.2 using a potassium phosphate buffer (6.6
mM. final concentration).
On the first measuring
occasion a citrate-potassium
phosphate buffer (I .65
mM citric acid and 3.3 mM K,HPO,.
final concentration) was used to give pH values of approx. 3.5
and 4.3. This was only done for I2 soils. For the
second measurement occasion 0.025 M H,SO, was
added to give these low pH values. The actual pH
was always measured immediately before the thymidine addition. Two replicate measurements
were
made for each pH level and a distilled water control.
The data are expressed as percentages of the thymidine incorporation
values obtained when only distilled water was used, that is. when the natural pH of
the soil prevailed. Values for the zero-time control
was always subtracted before these calculations.
The bacteria1 community
from soil 2 was also
studied more specifically by altering the pH by small
additions of dilute sulfuric acid in order to detect the
optimum pH for thymidine incorporation with more
precision.
Leucine incorporation
was performed simultaneously with the thymidine
incorporation
measurements by adding L-[U-‘JC]leucine
(775 nM final
concentration.
1 1.9 GBq mmol-‘,
Amersham, UK)
together with the radioactive thymidine. This was
only made for 4 soils.
The degree of participation
(DP) of the added
labelled substrates in the incorporation was measured
for two soils by the isotope dilution approach [ 171.
Different amounts of non-radioactive
substrate were
added with the labelled substrates. If the reciprocal
of disintegrations
min-’
are plotted against the
amount of added thymidine or leucine. the intercept
with the .r-axis will be the amount of exogenous and
PH
Fig. 2. The effect of pH on thymidine incorporation into macromolecules (cold acid insoluble material) of bacteria extracted from soils by
homogenization-centrifugation.
Data were expressed as percentages of distilled water controls. pH was altered with dilute sulphuric acid for
the two lowest pH values and with phosphate buffer for the four other values. Soil numbers refer to Table I.
0.
soil 7; a.
soil 13: A. hoi1 14: ~1. soil 16.
0.
soil I: n
, soil
7: 0.
soil 6;
E. BBBth / FEMS Microbiology
de novo synthesised substrate
macromolecular
synthesis.
participating
Ecolo,~y IY (1996) 227-237
231
increased the thymidine incorporation up to an optimum slightly above pH 4. Decreasing the pH even
more resulted in decreased thymidine incorporation
of the bacterial community. The optimum pH of the
thymidine incorporation thus corresponded well with
the pH(H,O) of the soil (4.21), but was higher than
pH(KC1) (3.52).
The addition of the citrate-phosphate buffer to set
the two lowest pH values appeared to inhibit thymidine incorporation. This can be seen in Fig. 1, where
the thymidine incorporation using this buffer is compared with the use of dilute sulfuric acid to alter pH.
The use of phosphate buffer. however, appeared not
to inhibit bacterial incorporation of thymidine. Judging from Fig. 1 the use of this buffer for pH around 5
gave similar values as would have been expected
from a pH response curve extended to this pH value.
Thus, when not otherwise stated only data using
phosphate buffer and dilute sulfuric acid from the
second measurement occasion were used to compare
the pH response of bacterial communities from different soils.
Large differences were found, when the pH re-
in the
2.3. Statistics
Thymidine
incorporation
at different pH values
was subjected to principal component (PC) analysis,
after standardization of the data by dividing with the
distilled water controls. In order to separate the soils
according to the pH response of their bacterial communities, each soil sample was considered one object. The multivariate
calculations
were performed
using the computer programme SIRIUS [ 181.
3. Results
The pH response of the bacterial community of
soil 2 (an acid soil from a coniferous forest) was
initially determined by decreasing pH using small
increments of added dilute sulfuric acid (Fig. 1). The
distilled water control of the extracted bacterial solution had a pH of around 4.8. Decreasing the pH
5011
9
5
;
8
&
E
s
-a
.a
s
‘i:
a
25-
3
14
6
16
O12
7
::!__’
-100
-50
0
Principal
component
'ts
50
100
1
Fig. 3. Principal component analysis of pH response curves for bacterial communities extracted from differents soils using homogenizationcentrifugation.
pH was altered with dilute sulphuric acid for the two lowest pH values and with phosphate buffer for the four other values.
Numbers refer to soils in Table I.
pal component explained about 80% of the variation
in the data. The low pH soils (Nos. I. 2 and 3) were
all situated to the left in the PC plot. while the high
pH soils (Nos. I3 to 16) were found to the right.
Thus. the soils were ordered according to their pH
along the first PC axis. This relationship was further
underlined by correlating pH(H,0)
and the scores
for each soil for the first principal component (Fig.
4). A strong linear relationship was found (1.’ = 0.85,
17 =
16, P < 0.001).
Although the citrate-phosphate
buffer appeared to
inhibit thymidine incorporation (Fig. I), the use of
this buffer to set the lower pH values could also be
used to differentiate between the pH response of the
bacterial communities of the different soils (data not
shown). Thus. when a PC analysis was performed on
the pH response curves for the 12 soils tested in this
way. a picture similar to that in Fig. 3 was seen. The
first principal component explained 867~ of the variation in the data, and when the scores were correlated against soil pH(H,O), a linear relationship was
found (Y’ = 0.85, 12= 12. P < 0.001).
sponse curves from different soils were compared.
This is seen in Fig. 2. where some examples of such
curves are shown. The optimum pH for the low pH
soils I and 2 was around pH 4. while for the soils
with higher pH, the optimum incorporation rate was
gradually shifted to a higher pH. Thus, soil 16 with
the highest pH (8.10) had a response curve with the
highest pH optimum. Similar results. with the optima
of the pH response curves correlated with the soil
pH(H,O). were found for the other soils tested (data
not shown). A significant linear correlation (P <
0.001)
was found, when the optimum pH for thymidine incorporation
was correlated to soil pH(H?O)
(Y’ = 0.62. 17 = 16).
It was. however, difficult to exactly determine
optimum pH values, since only six different pH
values were used for the thymidine incorporation.
Instead, in order to compare the different bacterial
communities.
a principal component (PC) analysis
was performed, in which each soil was considered as
an object and each pH used for the thymidine incorporation were the variables (Fig. 3). The first princi100
y = 35.1x
-100
- 213.9
12 = 0.85
!
I
I
I
I
I
3
4
5
6
7
8
Soil pH(H20)
Fig. 4. Correlation between scores for the first principal
Numbers refer to soils in Table I.
component
from the analysis
of the pH response
curves and the soil pH(HIO).
E. Biiiith / FEMS Microbiology
Inspection
of Fig. 2 revealed that the largest
differences
between
bacterial
communities
were
found when thymidine incorporation
was measured
at extreme pH values. Thus, a good separation between the different bacterial communities should be
possible, if the ratio between thymidine incorporation at those extreme pH values are compared. This
also appeared to be the case, since the logarithm of
the ratio of thymidine incorporation at pH 4.3 to that
at pH 8.2 (Fig. 5) or thymidine incorporation at pH
3.5 to that at pH 8.2 (not shown) were negatively
and linearly related to soil pH(H,O) (r2 = 0.86 and
0.84, respectively).
The negative slope was greater
for the latter ratio and would thus be better for
separating the different bacterial communities. However, since very low values of thymidine incorporation were found at pH 3.5 for bacterial communities
from the high pH soils, and thus the errors in the
measurements
were large, the variation was higher
using the ratio incorporation at pH 3.5 to that at 8.2
=-0.37x
+ 2.25
Ecology
19 (1996) 227-237
333
Table 2
The effect of pH on degree of participation (DP) of the added
labelled substance in the incorporation
into cold acid insoluble
substances of bacteria extracted from soil by homogenizationcentrifugation
Soil 7
Soil I6
pH
DP,,
(T/c)
DP,,,
(c/c)
Leu/TdR
(mol/mol)
without DP
Leu/Tdr
(mol/mol)
with DP
3.98
5.90 d
8.01
4.08
7.10
7.91 a
50.5
49.1
58.7
100
100
100
17.6
38.6
51.9
34.4
59.6
58.1
1.9
Il.6
14.4
15.8
19.2
26.1
14.4
12.0
14.0
45.9
49.0
45.0
The leucine (Leu) to thymidine (TdR) ratio was either calculated
directly, without considering DP, or after taking account of DP.
’ pH in the bacteria1 solution directly extracted from the soil.
than using the ratio incorporation
8.2.
The pH dependent thymidine
at pH 4.3 to that at
incorporation
was
ra = 0.06
3
8
Soil pH(H20)
Fig. 5. Correlation between the logarithm of the ratio of thymidine incorporation into macromolecules
(cold acid insoluble material) at pH
4.3 and 8.2 of bacteria extracted from soils by homogenization-centrifugation
and pH(H?O) of the soils. pH was altered with dilute
sulphuric acid for the lowest pH value and with phosphate buffer for the highest. Numbers refer to soils in Table I.
233
25-
20a
E
%
.s
15-
f
_)
lo-
U
Soil 3
Soil 5
Soil 15
5Soil 16
1
9
PH
Fig. 6. The effect of pH on the leucine to thymidine
incorporation
compared with leucine incorporation for several soils.
If these two methods were affected in a similar way
by pH, the ratio leucine to thymidine incorporation
should be constant over the whole pH interval tested.
However, especially at pH values below 5 the leucine
to thymidine incorporation
ratio decreased (Fig. 6).
This appeared to be due to high isotope dilution and
thus low degree of participation of the added leucine
in the incorporation at low pH values, while isotope
dilution for thymidine incorporation appeared not to
be affected by pH (Table 2). The leucine to thymidine incorporation
ratio was not affected by pH
when the values were adjusted for isotope dilution.
4. Discussion
The crucial question, that needs to be answered in
order to be able to use thymidine incorporation
to
determine the pH response of a bacterial community.
is if thymidine incorporation
at a certain pH only
ratio calculated
withou
compensating
for isotope dilution.
reflects the activity (growth rate) of the community
or if pH per se affects the uptake and incorporation
of thymidine irrespective of the origin of the bacterial community. The latter could for example be due
to pH affecting the chemical form of thymidine, thus
affecting the uptake rate. This could not be answered
definitely
using the present data. However, both
thymidine and leucine incorporation gave the same
results, when isotope dilution was considered (Table
2). indicating that the result for thymidine incorporation was not due to a direct pH effect. Furthermore,
any physical/chemical
effects would be the same at
one particular pH irrespective of the origin of the
bacterial community. Thus, the fact that thymidine
incorporation was relatively high at pH 3.5 for bacterial communities
from acid soils and low for communities from soils with high pH (Fig. 2) could only
be due to properties of the bacteria.
The bacterial community
appeared to be well
adapted to the prevailing pH of the soil, irrespective
of the soil being acid or having a more neutral pH
E. B&?th / FEMS Micmbiolog~
(Figs. 1 and 2). This was also found for the denitrifying populations of two soils with different pH [19].
where the denitrifying
potential of the population
from the low pH soil was adapted to the acidic pH
conditions prevailing in that soil.
In a study of 15 soils different from the ones used
here, 0.7 X lo’“-7.2 X 10” acridine orange stained
bacteria g- ’ organic matter and a strong negative
exponential relationship between bacterial counts and
organic matter content of the soil were found ([2],
recalculated from their Table I >. A negative correlation, but less strong, was also found with the soil
pH(H,O).
Although it is thus clear that, on an
organic matter basis. bacteria are less abundant in
low pH soils high in organic matter than in high pH
soils with little organic matter, we cannot deduce
which factor is the most important, pH or organic
matter, since they are often correlated. However,
since the optimum activity for the bacterial communities was found around the actual pH(H,O) of the
soils (Figs. 1 and 2). it might indicate that pH per se
was not the main factor causing low bacterial counts
in acid soils. Thus, earlier explanations
(see Introduction) of low bacterial numbers in acidic soils
being due to the bacterial community
not being
adapted to the pH conditions might be to simplistic.
One can hypothesize that the bacterial community
response curves might indicate under what pH conditions the soil organisms are actually growing. It has,
for example, been argued that pH in soil will be
lowest at particle surfaces. Thus, soil pH measured
in a water suspension will overestimate the actual pH
for surface
inhabiting
organisms
like bacteria.
pH(KCl), which should reflect the approximate ionic
strength normally present in the soil solution, should
therefore be a more appropriate pH indicating the
conditions
encountered
by soil bacteria. A closer
inspection
of Fig. 1 indicates, however, that the
optimum thymidine
incorporation
of the bacterial
community was slightly below pH(Hz0) of this acid
soil, but well above pH(KC1). This might indicate,
that pH(H20) may better reflect [H+] encountered
by soil bacteria than pH(KC1). A direct technique to
measure pH in soil is to extract pore water directly
from soil using centrifugation.
Extracted pore water
had about 0.3 units lower pH than pH(H,O),
but
about 0.5 units higher than the pH determined after
extraction
with a salt solution in a forest with
Ecology
19 C1996) 227-237
235
pH(H,O) between 4 and 5.5 [20]. Thus, the fact that
the optimum thymidine incorporation of the bacterial
community is slightly lower than pH(H,O) in Fig. 1
might indicate that the pH of a directly extracted
pore water is the most correct one. at least when it
comes to the environment encountered by the bacteria.
There are different ways of comparing the pH
response curves for the different bacterial communities. The most obvious would be to use the optimum
pH directly. However, although the optimum pH was
significantly correlated to the soil pH, the degree of
explanation
was rather low. This was not unexpected. since the use of only six pH values in the
response curve gave low precision in the estimated
optimum pH, and also that this approach did not
consider the whole pH response curve. One could
also fit a mathematical model to the data and compare the different cardinal pH values calculated using
this model. This has been used to compare pure
culture bacterial isolates [21.22]. However, to get a
good fit one would need data with a higher resolution than the six different pH values used in the
present study, and the models therefore had low
precision when tried. Furthermore, the models used
for pure cultures do not necessarily hold for communities.
An objective way of comparing different curves
without fitting a specific equation but still using all
the data points is the use of principal component
(PC) analysis. This was, for example, a better way to
compare temperature effects on growth of fungal
isolates than fitting a model equation [23]. In the
present study a very good separation along the first
PC axis was found (Fig. 3) and the scores along this
axis were well correlated to the soil pH (Fig. 4).
An even easier way of comparing the pH relations
of different bacterial communities is to calculate the
logarithm of the ratio of thymidine incorporation at
two extreme pH values, as illustrated for pH 4.3 and
8.2 in Fig. 5. This will make the measurements very
rapid and less expensive than using the full range of
pH values. Furthermore, there is no need to have a
distilled water control to standardize the incorporation values. Thus, only two replicates for two pH
values are needed for each soil sample. Compared to
plate counting techniques the use of the thymidine
incorporation
method in this way will be very cost
efficient when measuring community
responses to
pH. Furthermore,
since usually no more than 100
colonies can be counted on an agar plate, while in
the thymidine incorporation technique more than IO’
bacteria are used in the measurements,
the former
technique will probably also be less reproducible.
Earlier attempts to use the thymidine incorporation technique to study the bacterial community pH
response used citrate-phosphate
buffer to set the
lower pH levels [ 10.141. This buffer inhibits thymidine incorporation
at the concentrations
used (Fig.
I), and this explains why the optimum pH for incorporation was not found at the low pH values expected in the acid soils used in these studies.
On the other hand, B%th et al. [I21 used dilute
sulfuric acid to set the lower pH values when measuring the impact of different pH increasing measures in two different forest soils on the bacterial
community pH response. Their data can therefore be
directly compared with the ones presented here, and
can be used as a validation data set. Recalculating
their data and using Fig. 5 to predict soil pH showed.
for example, that the two non-treated control soils
with pH(H,O) of 3.93 and 5.2-5.4 had a predicted
pH of 3.95 and 5.35. respectively,
using the pH
response curves for the bacterial communities.
By
using all 8 treatments studied by B%%thet al. [ 121, a
linear regression between predicted soil pH from the
bacterial community response and measured soil pH
explained a large part of the variation (r’ = 0.79).
Thus, a calibration curve, either using a multivariate
statistical technique. like PC analysis, as in Fig. 4, or
the ratio of two values as in Fig. 5. could be
constructed once. Bacterial communities
extracted
from other soils or environments could then be fitted
into this correlation to indicate the pH conditions
that had prevailed in their environment.
A similar extraction technique as the one used
here for estimating the pH response of a bacterial
community have been used to study metal tolerance
of soil bacteria [24]. In a later study [25] it was
shown that leucine incorporation was as sensitive as
thymidine incorporation
to measure bacterial tolerance to metals. This was also the case for pH
measurements,
when isotope dilution for leucine incorporation was considered (Table 2). However. the
measurements
will be both more time consuming
and less precise, if isotope dilution has to be esti-
mated for each pH tested. Thus, thymidine incorporation, where isotope dilution was not affected by
pH, appeared to be better suited for estimating pH
response curves of bacterial communities than leucine
incorporation.
Acknowledgements
This study was supported by a grant from the
Swedish Natural Science Research Council.
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141Gray, T.R.G. and Williams, S.T. (1971) Soil Microorganisms. Longman, London.
[Sl Alexander, M. (1977) Introduction to Soil Microbiology.
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