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Appl Environ Microbiol, July 1998, p. 2739-2742, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Microbial Community Changes in a Perturbed
Agricultural Soil Investigated by Molecular and Physiological
Approaches
Lise
Øvreås,1
Sigmund
Jensen,2,*
Frida
Lise
Daae,1 and
Vigdis
Torsvik1
Department of Microbiology, University of
Bergen, N-5020 Bergen,1 and
Department
of Biotechnological Sciences, Agricultural University of Norway,
N-1432 Ås,2 Norway
Received 12 December 1997/Accepted 14 April 1998
 |
ABSTRACT |
Changes in soil microbial activity and diversity after incubation
either with nitrogen or with a mixture of methane and air were
examined. The perturbation by methane and air were characterized in
detail and led to reduced diversity and enrichment of methanotrophs which were identified by denaturing gradient gel electrophoresis and
16S rRNA sequencing.
| |
TEXT |
Biologically mediated processes in
soils are central to the ecological function of soils. Soil methane
oxidation is caused by methanotrophs and a heterogeneous collection of
cooxidizing bacteria (3-6, 9, 10, 13, 25). Environmental
perturbation such as soil management introduces poorly known changes in
microbial communities (7, 19, 26). The response of
bacterial communities to environmental changes can be assessed by
analyzing in situ activity in combination with molecular analysis of
the community DNA. In this study the response of the soil bacterial
community as well as methanotrophic and methylotrophic activities to
anaerobic and methane-enriched atmosphere was investigated. The aim was to investigate whether shifts in microbial activity were reflected in
shifts in the community structure. Bacterial community DNA was analyzed
to assess the total diversity and generate a profile of the
community. Changed profiles indicated altered community structure.
Broad resolution approaches such as DNA reannealing kinetics
(28), base composition profiles (24, 28), and
denaturing gradient gel electrophoresis (DGGE) (20) were
applied.
Soil collection and perturbation.
Samples (depth, 0 to 5 cm)
were taken from an organic agricultural soil (Krohnestykket, Stend,
south of Bergen, Norway). Methods of sampling, characterization, and
storage are described elsewhere (15). In laboratory
experiments the soil was incubated in two different types of atmosphere
at 15°C for 3 weeks. One set was incubated with N2
gas (N2 perturbation) and the other was incubated with an
atmosphere of air with 17% methane (CH4 perturbation). Twenty grams of soil (wet weight) was incubated for 3 weeks in 120-ml
sterile serum bottles capped with butyl rubber stoppers. The headspace
atmosphere was created by flushing to obtain a final concentration of
99.8% nitrogen (flushed once) or 17% methane in air (flushed twice a
week).
Physiological measurements.
Methanol and methane were measured
by gas chromatography as described by Lindahl et al. (18)
and Jensen and Olsen (14), respectively. Methanol oxidation
measurements were modified as follows: bottles containing 5 g (wet
weight) of soil, 20 ml of autoclaved distilled water, and 5.0 µl of
methanol (100% high-performance liquid chromatography grade) were
incubated in a shaking water bath at 15°C at 150 rpm. Rate constants
of atmospheric methane oxidation and methanol oxidation were calculated
(2, 13).
Genetic analyses.
Soil bacteria were extracted from nine 20-g
(wet weight) soil samples, and DNA from each soil bacterial fraction
was isolated and purified as described by Torsvik et al.
(27). Thermal denaturation and reassociation were determined
spectrophotometrically in a Cary 4E, UV-visible light spectrophotometer
with temperature holder (Varian Instruments) as previously described
(27). The DNA complexity was calculated as described by
Torsvik et al. (27) with a data acquisition program
developed by Svein Norland (University of Bergen, Norway).
The V3 regions of 16S rRNA genes from community DNA were PCR amplified
and analyzed by DGGE as described by Øvreås et al. (23).
DGGE bands selected for sequencing were reamplified and sequenced as
previously described (1, 23).
Physiological changes of soil bacterial communities.
Both
perturbations changed the activity of the methane oxidizers and
the methanol oxidizers (Table 1). Methane
caused the most dramatic changes with highly increased oxidation
rates. Nitrogen under practically anaerobic conditions had the opposite
effect, reducing both oxidation rates. The control soil revealed
methanol oxidation to be biologic and perturbation to increase methane production (Table 1).
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TABLE 1.
Physiological changes in the community of methane
and methanol oxidizers in agricultural soil after 3 weeks of
perturbation at 15°Ca
|
|
Genetic changes of soil microbial communities.
Community DNA
extracted from the bacterial fraction from the control soil had a
steeper melting profile than that of perturbed soils (Fig.
1A). The difference in mean melting
temperatures (Tm) between that for DNA from
N2-perturbed soil (78.4°C) and that for DNA from
CH4-perturbed soil (79.2°C) was only 0.8°C. The
Tm of the control soil DNA was 79.0°C. This
could idicate minor differences between the communities. The DNA
melting range (25 to 75% melting), however, revealed greater
community differences. The control soil DNA melted over a smaller
temperature range (4.3°C) than those from the perturbed soils (6.3 and 7.6°C for N2- and CH4-perturbed soils,
respectively). The base composition of DNA from the control soil and
the N2-perturbed soil ranged from 45 to 70 and 40 to 70 mol% G+C, respectively. The DNA from CH4-perturbed soil
ranged from 35 to 77 mol% G+C. The base composition profile had two
peaks, indicating that the community consisted of two fractions; one ranging from 35 to 60 and the other from 62 to 77 mol% G+C (Fig. 1B).
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FIG. 1.
(A) Thermal denaturation for DNA from control soil
( ), N2-perturbed soil ( ), CH4-perturbed
soil ( ), and E. coli ( ). (B) DNA melting profiles (1st
derivative of the melting curves). Melting profiles were converted to
moles percent G+C (mol % G+C) profiles by using the equation mol% G+C = [(Tm/50.2) 0.99] × 100 (18a). The moles
percent G+C of DNA from control soil (), N2-perturbed
soil (), CH4-perturbed soil (), and E. coli () are shown.
|
|
Reassociation showed that perturbation caused a significant change in
community diversity (Fig.
2). The
reassociation rate
of DNA from the control soil was low and showed the
highest
C0t1/2 value (6,300 mol
s
1 liter
1), corresponding to approximately
8,000 different
Escherichia coli genomes. N
2
perturbation decreased the
C0t1/2
value to 2,500
mol s
1 liter
1, which
corresponds to 3,200 different
E. coli genomes. The lowest
C0t1/2 value (300 mol
s
1 liter
1) was found in DNA from the
CH
4-perturbed soil, corresponding
to 380 different genomes.
The results show that perturbations
caused a significant reduction in
bacterial diversity, but the
data provided no information on species
composition.
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FIG. 2.
Reassociation (C0t
plots) of DNA from bacterial fractions of control soil (),
N2-perturbed soil (), CH4-perturbed soil
(), and E. coli (). The DNA was sheared to about
420,000 Da and reassociated at 49°C.
|
|
Separation of PCR-amplified 16S ribosomal DNA by DGGE
showed a complex community structure with more than 100 different
bands
covering the entire gradient (Fig.
3). N
2 perturbation resulted
in minor differences in the community profile with two bands becoming
slightly more intense. In the CH
4-perturbed soil some
intensified
bands were seen on top of the community profile, and we
were able
to purify and reamplify three of these. Sequences of the
purified
fragments revealed that they showed phylogenetic affiliation
to
Methylomicrobium album (band 1),
Methylobacter
sp. strain BB5.1
(band 2), and
Methylomonas rubra (band 3)
with sequence homology
of >90%. These are all type I methanotrophs
belonging to the
subclass of the class
Proteobacteria.
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FIG. 3.
DGGE analysis of PCR-amplified 16S ribosomal DNA
fragments from soil bacterial communities. DNA was derived from the
control soil, a high-organic pastureland (lane A), the same soil
incubated for 3 weeks at room temperature with methane (lane B), and
the same soil incubated for 3 weeks with nitrogen (lane C). The
positions and numbering of bands discussed in the text are indicated
with arrows.
|
|
We observed gas perturbation to change both the structure and the
physiology of microbial communities in an agricultural soil.
All
changes were found after only 3 weeks and incubation at 15°C.
Substrate addition (CH
4) produced greater changes than
oxygen
removal (N
2). The atmospheric methane oxidation
increased after
methane and decreased after nitrogen perturbation.
Methanol oxidation
changed in parallel, indicating that methanol and
atmospheric
methane were oxidized by methanotrophs. Fast-growing
methanotrophs
are likely to have leaked exudates such as methanol or
formate
(
11,
12), which can be substrates for, e.g.,
Methanosarcina spp. or
Methylobacterium spp.
(
2,
8). Hence, perturbation
with a substrate
(CH
4) specific for the methanotrophs may cause
secondary
effects which change other microbial populations in
the soil as well.
Shifts in the bacterial community structure were observed after both
perturbations, as reflected in the broader DNA melting
range, skewed
base distribution towards lower moles percent G+C,
and reduced DNA
complexity. The most profound changes were found
in
CH
4-perturbed soil. The molecular analyses strongly
indicated
growth of methanotrophic bacteria during the methane
perturbation.
During the N
2 perturbation the moles
percent G+C was lowered by
5%, indicating growth of bacterial types
not abundant under aerobic
conditions.
DNA from the control soil had an extremely slow reassociation rate,
indicating high bacterial diversity prior to perturbation.
In this soil
the genetic heterogeneity was more than twice the
diversity in the
N
2-perturbed soil. In the CH
4-perturbed soil
the diversity was 1/20 of that in the control soil. The diversity
reflects the number of genetically different bacterial types responding
to the added substrate and indicates an outgrowth of a few dominant
bacterial species. Complex microbial communities like soil are
difficult to analyze using the DGGE technique (
22). More
than
100 amplified fragments were seen in all the samples. Use of this
method indicated that anaerobic conditions (N
2) caused
growth
of bacteria which were suppressed under aerobic conditions or
were stimulated by substrates becoming available, e.g., from cells
lysed due to oxygen deficiency. In the CH
4-perturbed soil,
growth
of methane oxidizers was demonstrated by the DGGE analysis.
Sequence
analysis suggested that the dominant community members in this
soil were related to cultivable methanotrophs, all of which are
found
to have low affinity to methane (
16,
17).
The parallel increase in methanol and methane oxidation indicated that
methanotrophs consume both methane and methanol in
the soil. In the
methane-perturbed soil, DGGE analyses revealed
an increase in numbers
of known methanotrophs, and an increased
oxidation rate at 1 part per
million per volume (ppmv) methane
was found. Our data support the view
that in the presence of atmospheric
methane concentrations the known
methanotrophs oxidize atmospheric
methane by the concomitant
consumption of methanol (
6,
13).
We conclude that apparent changes in microbial activity and community
structure were observed after perturbation. The bacterial
communities
from perturbed soils showed a significant decrease
in diversity
compared to that from the control soil. The methane-perturbed
soil was
dominated by a few community members showing phylogenetic
affiliation
to known methanotrophic bacteria. Our investigation
indicated that
these methanotrophs, although they were related
to cultured,
low-affinity methanotrophs, were responsible for
oxidation of
atmospheric concentrations of methane. Our investigation
demonstrates
that the polyphasic approach can elucidate the relationship
between
microbial activity and community structure and identify
the bacteria
which have a key role in a process.
Nucleotide sequence accession numbers. The sequences
obtained in this study are available from GenBank (band 1,
AF067423;
band 2,
AF067424; band 3,
AF067425).
| |
ACKNOWLEDGMENTS |
We thank Tonje Castberg for technical assistance on the isolation
of total DNA and on the DGGE analyses. We also thank Anders Priemé for helpful discussions on methanol uptake in soil.
Finally, we thank Jostein Goksøyr and Larry Forney for valuable
comments on the manuscript.
This work was funded by the Norwegian Research Council.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biotechnological Sciences, Agricultural University of Norway, Box 5040, N-1432 Ås, Norway. Phone: 47 64 947726. Fax: 47 64 947750. E-mail: sigmund.jensen{at}ibf.nlh.no.
| |
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Appl Environ Microbiol, July 1998, p. 2739-2742, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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