FEMS Microbiology Ecology 86 (1992) 213-219
0 1992 Federation of European Microbiological Societies 0168-6496/92/$05.00
Published by Elsevier
213
FEMSEC 00366
Microbiological response to Ca(OH) treatments in a forest soil
Rosa Nodar, Maria J. Acea and Tarsy Carballas
Instituto de Inuestigaciones Agrobioldgicas de Galicia (CSIC), Santiago de Compostela, Spain
Received 23 April 1991
Revision received 16 August 1991
Accepted 27 September 1991
Key words: Liming; Bacterial population; Fungal population; Nitrobacter; Denitrifiers
1. SUMMARY
2. INTRODUCTION
Lime was added to a forest acid soil rich in
organic matter. During five weeks of initial incubation at room temperature (until the various
limed soil samples reached stabilized pHs of 5.5,
6.0 or 6.51, there were rapid increases in the
bacterial population, denitrifiers, and fungal
mycelia, particularly in the heaviest limed sample.
Conversely, nitrite oxidizers decreased to undetectable numbers regardless of the lime dose applied. At this time soil samples were amended
with 5% of fresh soil. During 12 weeks of subsequent incubation at 28"C, the bacterial population was favoured by increasing soil pH; nevertheless, at the end of the incubation the positive
effect was only significant at pH 6.0 and 6.5. By
contrast fungi were depressed by raising the pH.
Nitrifiers and denitrifiers were more numerous in
the limed than in the unlimed soils but only in
samples at pH 6.5 were the differences significant
throughout the incubation period.
For many acid soils, liming is a basic soil
management practice. The treatment of acid soils
with lime accelerates the decay of plant tissues,
simple carbonaceous compounds or native soil
organic matter [ 11. Much research has involved
the study of the effects of lime on nutrient availability and on soil chemical properties [2,31. In
contrast, research on the effect of liming on microbial populations is scarce. Microbiota play an
essential role in the energy circulation through
ecosystems and in the cycles of C and N, hence
knowledge of their response to soil lime amendments is needed.
The composition of the soil microbial community is highly dependent on soil pH [ 1,431. Generally soil bacteria are less tolerant of acid conditions than fungi [6]. Most soil nitrification appears to be limited to a restricted number of
autotrophic bacteria [4]. The activity of the nitrifying bacteria is markedly influenced by certain
environmental conditions, chief among which is
soil pH [7-lo], and Nitrobacter densities are very
low in acid soils [10,11]. However, this genus
carries out most of the nitrate production in soils
[12]. Denitrification removes nitrogen forms that
Correspondence to: M.J. Acea, lnstituto de Investigaciones
Agrobiol6gicas de Galicia (CSIC), Apartado 122, 15080 Santiago de Compostela, Spain.
214
can be assimilated by non-nitrogen-fixing organisms, and microbial denitrification is highly sensitive to pH [1,9,13,14].
This laboratory study was performed to evaluate the effects of different doses of lime, and the
subsequent increase in pH, on total bacterial and
fungal populations and on the microorganisms
that carry out nitrite oxidation and denitrification
in soil.
3. MATERIALS AND METHODS
3.1. Soil treatment
Samples were collected from the top 5-15 cm
of the umbric horizon of a humid temperate zone
soil classified as an Andic Haplumbrept [15] developed over gabbros under oakwood. This soil
had an initial pH of 5.0, an organic C content of
11% (determined by combustion) and a N content of 0.82% (determined by digestion and steam
distillation). The soil was air-dried just sufficiently for the moist soil to be sieved ( < 4 mm)
and hornogeneized prior to use. Lime was added
to separated soil samples at rates of 0, 4.0, 8.1
and 12.2 mg of reagent grade Ca(OH)2 g - ' soil
to reach pHs of 5.0 (Lo), 5.5 ( L , ) , 6.0 (L,) and
6.5 (L3), respectively, according to the lime-requirement method described by McLean [161.
Changes in microbiota before the predetermined
pH was attained were studied by incubating limed
Table 1
pH change in limed ( L I - L , ) and unlimed Lo soil samples.
Day
0
1
2
4
7
14
35
42
49
63
77
98
119
Soil sample
L,,
L,
L2
L3
5.0
5.0
4.9
4.7
4.6
4.6
4.6
4.5
4.3
4.3
4.3
4.3
4.3
5.0
6.0
6.5
6. I
5.9
5.8
5.5
5.4
5.4
5.3
5.3
5.3
5.3
5.0
6.7
6.8
6.5
6.4
6.3
6.0
6.0
6.0
5.9
5.Y
5.9
5 .Y
5.0
7.5
7.3
7.2
7.0
6.9
6.5
6.5
6.5
6.5
6.5
6.5
6.5
and unlimed soil samples for 35 days at room
temperature and 80%- of field capacity. Counts of
viable bacteria, fungal hyphae. Nitrobacter SPP.
and denitrifiers were made at 0, 2, 4, 7, 14 and 35
days of incubation (see 3.2.). Once the desired
pH was attained, all soil samples were Supplemented with 5% of fresh soil to assure microbial
diversity and then 50 g portions of each sample
were incubated in 500-ml Erlenmeyer flasks for
12 weeks at 28°C and 80% of field capacity.
Optimal conditions of humidity and aeration w e r e
maintained by daily passage of a stream of humidified COz-free air. Microbial counts w e r e
made at 0, 1, 2, 4, 6, 9 and 12 weeks of incubation. Three replicates were used for each count.
3.2. Microbiological analyses
20-g subsamples of soil were diluted in IO-fold
series in sterile water. Five Petri dishes containing solid media or five test-tubes containing liquid media were inoculated from each dilution
and incubated at 28°C. Viable bacterial numbers
were determined by colony counts after 7-10
days incubation on yeast extract agar plates. PJitrite-calcium carbonate medium was used to culture chemoautotrophic nitrite oxidizers (Nitrobacter spp.) and the diphenylamine-sulphuric acid
reagent was utilized to detect their growth. T h e
method and media described by Alexander [ 171
were used to culture and count denitrifying populations. Nitrifiers and denitrifiers were incubated
in test tubes for 4 weeks and their number w a s
estimated by the most-probable-number method
[18]. For estimation of length of fungal hyphae,
the membrane filter technique [191 combined with
the mathematical formula of Newman [20] was
followed. The density of microorganisms was related to dry weight (110°C) of soil. The least
significant difference (LSD) test [211 was applied
to the results.
4. RESULTS
Upon liming, the soil pH rose abruptly from
5.0 to 6.5, 6.8 and 7.5 in L , , L, and L,, respectively (Table 1); thereafter it declined to reach
the desired value of 5.5, 6.0 and 6.5 five weeks
215
after liming. In the unlimed sample (Lo)pH
declined slightly. There were only slight pH
changes in unlimed and limed samples after this
period. The microbial population during the initial period when pH increased rapidly differed
from that during the second period when pH
stabilized.
4.1. Microbial counts during oariable p H period
Microbial population changes during the 35day incubation period, with and without added
Ca(OH), are represented graphically in Fig. 1.
Initially the bacterial population density was
about lo6 CFU g - ' soil. Bacterial counts increased in limed and unlimed samples. However,
the rate and extent of change was significantly
related to the dose of hydroxide applied. Thus,
although a slight increase was observed in L,, and
L , samples, this was not significant ( P < 0.03,
whereas in L, and L, samples counts reached,
respectively, three and two orders of magnitude
higher than the initial value, the maximum bacte-
FUNGAL MYCELIUM
9
6
0
7
14
2 1
28
0
3 5
7
days
1 4
21
28
35
days
a
I
DENlTRlFlERS
.0
f n 1
Y
I
z
a
IJ)
z
n.
I
I
m
0
0
0
4
0
0
7
1 4
21
days
2 8
35
0
7
1 4
2 1
days
28
35
-
Fig. 1. Microbial population in soil samples following Ca(OH), addition. Treatments: Lo, no lime; L,, L, and L, limed at rates of
4.0.8.2 and 12.2 mg Ca(OH), g-' soil, respectively. LSD ( P < 0.05) shown as bars at all sampling times.
216
rial numbers being reached during the first week
of incubation.
Total fungal hyphal length was initially 32.5 m
g- soil. Fungi showed two different patterns of
behaviour according to the dose of lime applied
(Fig. 1). During the first week of incubation, the
fungal hyphae were reduced 50% and 30% in L,,
and L I , respectively, and thereafter they increased. In L, and L, the opposite occurred,
'
increasing 30% and 50% respectively, and decreasing thereafter. As with bacterial counts,
there were no significant differences between Lo
and L,. Although L, and L, soil samples showed
significantly longer total mycelium than Lo a n d
L,, total mycelial length in all samples tended to
approach original values after 35 days.
Counts of chemoautotrophic nitrite oxidizerwere initially low (Fig. 11, whereas, the addition
*"
-
I
FUNGAL MYCELIUM
Q
40
0
u)
.I
UI
E
30
20
-
6
0
2
4
6
8
1 0 1 2
0
2
4
6
8
1 0 1 2
weeks
weeks
6r
DENlTRlFlERS
Nitrobacter
spp.
- 0
0
2
4
6
8
1 0 1 2
weeks
Fig. 2. Microbial population in unlimed (pH 5) and limed (pH 5.5, 6.0 and 6.5) soil samples once pk1 became stable (i.e. after 35
days of liming). LSD ( P< 0.05) shown as bars at all sampling times.
217
of Ca(OH), resulted in a Nitrobacter population
size of close to zero regardless of the dose. Immediately before liming the soil denitrifier population was about lo3 microorganisms g-' soil; their
density was essentially constant during the experiment in Lo and L,, while their number rose
abruptly and significantly in L, and L,, reaching
densities 4 orders of magnitude higher than the
initial value two weeks after liming and declining
two orders of magnitude afterwards.
However in the pH 6.0 sample the positive effect
of liming was only apparent for 4 weeks, after
which period this sample showed numbers similar
to pH 5.5 soil. By contrast, the increase of denitrifiers in the pH 6.5 sample was significant ( P <
0.05) throughout the incubation period.
4.2. Microbial counts during stable pH period
During the 12 weeks following amendment
with 5% of fresh soil, pH remained essentially
constant in the limed samples and decreased
slightly in the unlimed soil (Table 1). Bacterial
counts and trends were generally similar in the
limed and unlimed soil (Fig. 2). However, during
the first week following amendment, bacterial
counts increased to their maximum values in the
unlimed and limed-to-pH-5.5 soil samples and
then declined until 6 weeks of incubation, but
counts continuously declined in pH 6.0 and 6.5
samples. Population sizes were related to the
lime dose applied, thus throughout the incubation period the samples at pH 6.0 and 6.5 had
significantly higher bacterial population than the
unlimed soil while the positive effect of liming
was not significant in pH 5.5 samples at the end
of the incubation. By contrast, fungi were negatively affected by lime. Hyphae increased in pH
5.0 and 5.5 samples and decreased in pH 6.0 and
6.5 samples. Therefore the fungal length was
inversely related to pH. Nevertheless, at the end
of the incubation the negative effect of increasing
pH was similar in the limed samples regardless of
the dose applied, pH 5 soil showing about 60%
more mycelium than the limed samples.
Ca(OH),-amended soils to Ph 5.5 and 6.0 had
slightly more chemoautotrophic nitrite oxidizers
than the unlimed soil. However, only in pH 6.5
samples was the Nitrobacter population significantly (P<O.O5) higher than in the unlimed
treatment. Denitrifiers showed similar numbers
in pH 5.0 and 5.5 samples, both increasing over 9
weeks and then decreasing. Initially, numbers of
denitrifiers decreased in pH 6.0 and 6.5 samples,
but were significantly greater than at lower pHs.
The effect of liming on the microbial community can be divided into two well-differentiated
periods. In the short-term, bacterial density and
fungal length increased significantly, particularly
in L2 and W.This fact agrees with the increase
found by some authors in soil microbial activity
[1,22-271 in the short-term after liming. The initial rapid multiplication of bacteria and fungi in
soil samples limed to pH 6.0 and 6.5 was interpreted as due to the utilization of dead microbial
tissue by living cells and/or to the multiplication
of acid-intolerant bacteria [24] and of fungi
favoured by basic pH. The possibility that microbial death occurred in response to addition of
high doses of Ca(OH), was supported by the
finding that microorganisms highly sensitive to
environmental stress, as autotrophic nitrite oxidizers, practically disappeared after liming. This
is in accordance with the lack of stimulation of
nitrification found by some authors [24,27] in the
first weeks following liming. Therefore, it is probable that microbial diversity had been initially
reduced by liming. MacDonald [28] observed that,
immediately after liming, a relatively small number of morphological types of microorganisms
predominated. The positive effect of liming on
denitrifier populations was only significant at pH
6.0 and 6.5. Given the fact that many heterotrophic and autotrophic bacteria bring about
denitrification [l] it is likely that the increase in
denitrifying microorganisms could be attributed
to the increase in bacterial population observed
at the same pHs. In the long-term, bacteria were
favoured by the increase in pH. However, after
12 weeks of incubation, significant differences
with the unlimed soil were only found at pH 6
and 6.5, which is in agreement with the fact that
5. DISCUSSION
218
the optimum pH for most bacteria is near neutrality. By contrast, fungi are generally.,more tolerant of acid conditions than bacteria 16,291;
therefore it is not surprising that fungi were depressed as pH increased. Nevertheless at the end
of the incubation, similar counts were found in
the samples at pH 5.5, 6.0 and 6.5 while the
unlimed soil showed a significantly higher mycelial
length.
Innoculation of limed samples with fresh soil
restored the population of Nitrobacter and other
microorganisms which were favoured by increasing pH. Morril and Dawson [30] showed that
generation times of Nitrobacter in soil were pH
dependent and varied from 58 h at pH 6.2 to 21 h
at pH 6.6 and above. Further, Richards [lo] reported that nitrate production in soil falls off
rapidly below pH 6.0 and generally is negligible
below pH 5.0. On the other hand, nitrification is
favoured at neutral or slightly alkaline pH [311.
However, in the soil studies the decrease in acidity less than pH 6.5 may not be sufficient to
stimulate the activity of autotrophic nitrifiers,
which are favoured by a more neutral environment [321. This could explain the rapid growth of
these microorganisms in pH 6.5 samples, the only
pH at which the positive effect was highly significant at all the sampling times. Denitrifiers were
also favoured by increasing pH, but as in the case
of nitrite oxidizers, only at pH 6.5 were the differences consistently significant. Sprent [ 121 reported a neutral or slightly alkaline pH as optimum for overall denitrification. Therefore, it is
suggested that liming at pH lower than 6.5 does
not significantly alter microbial nitrification and
denitrification capacities of this soil. Likewise,
neither does it effect P mineralization [33]. On
the other hand, liming to pH 6.0 and above had a
beneficial effect on bacterial numbers while increasing the pH of this acid soil had a detrimental effect on fungal biomass.
'
ACKNOWLEDGEMENTS
The authors thank Mrs. Blanca Arnaiz for
technical assistance.
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