Previous Article | Next Article 
Applied and Environmental Microbiology, March 2000, p. 930-936, Vol. 66, No. 3
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Succession of Microbial Communities during Hot
Composting as Detected by PCR-Single-Strand-Conformation
Polymorphism-Based Genetic Profiles of Small-Subunit rRNA
Genes
Sabine
Peters,
Stefanie
Koschinsky,
Frank
Schwieger, and
Christoph C.
Tebbe*
Institut für Agrarökologie,
Bundesforschungsanstalt für Landwirtschaft, 38116 Braunschweig,
Germany
Received 30 September 1999/Accepted 14 December 1999
 |
ABSTRACT |
A cultivation-independent technique for genetic profiling of
PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to
characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR
amplifications were performed with DNA directly extracted from compost
samples and with primers targeting either (i) the V4-V5 region of
eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of
actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes.
Homologous PCR products were converted to single-stranded DNA molecules
by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and
increasing diversity of microbial populations with all primers. A total
of 19 single products were isolated from the profiles by PCR
reamplification and cloning. DNA sequencing of these molecular isolates
showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the
SSU rDNA of 
-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular
isolates could be attributed to different composting stages. The
diversity of cultivated bacteria isolated from samples taken at the end
of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also
detectable in the SSCP community profiles. Our study indicates that
community SSCP profiles can be highly useful for the monitoring of
bacterial diversity and community successions in a biotechnologically relevant process.
| |
INTRODUCTION |
Composting is the biological
conversion of solid organic material into usable end products such as
fertilizers, substrates for mushroom production, or biogas (methane).
Regardless of the product, the active component mediating the
biodegradation and conversion processes during composting is the
resident microbial community. Therefore, optimization of compost
quality is directly linked to the composition and succession of
microbial communities in the composting process. This means that tools
are required to monitor and characterize microbial communities during
the composting processes and to relate microbial communities to compost quality.
Cultivation of microorganisms extracted from compost samples allows one
to obtain pure cultures which can be used for further taxonomic or
physiological characterizations (2, 4, 11, 13, 14, 16, 47).
Rapid molecular PCR-based techniques, such as amplified ribosomal DNA
restriction analysis (ARDRA), are useful for comparison of a large
number of isolates at the phylogenetic level (48). This
technique allowed the characterization of Thermus strains
and Bacillus-related bacteria isolated from hot composting
material (5, 6). However, since any chosen cultivation
approach will inevitably favor the growth of some community members
while others are inhibited or not culturable at all, it is unlikely
that any cultivation method will allow a full description of the
microbial diversity. Therefore, cultivation-independent methods have
recently been used to characterize microbial-community successions
during composting. These include assessment of the diversity of
directly extracted phospholipids (9, 21, 25), measurement of
carbon source utilization by substrate-extracted microbial cell
consortia (9, 24, 42), and nucleic acid-based techniques
(29).
Several protocols have been used to directly extract total DNA from
compost material. This approach, in combination with PCR-mediated gene
detection, allowed the detection of specific genes of pathogenic microorganisms (37) or monitoring of the fate of recombinant DNA from decaying plant material (26) and the persistence of seeded microorganisms (31). In a recent study, bacterial 16S rRNA gene sequences were amplified from compost DNA and, after cloning
in Escherichia coli, were characterized by restriction enzyme analysis (7). The results indicated that some
molecular clones were identical to genes of cultivated species, but
others could not be matched. Also, the study showed repetitive
isolation of identical products. Such repetitive isolations can be
avoided if the community DNA-amplified PCR products are
electrophoretically separated to generate sequence-specific genetic
profiles. Denaturing gradient gel electrophoresis (DGGE) and
temperature gradient gel electrophoresis (TGGE) have become popular for
this purpose in biodiversity studies of microbial communities from a
variety of habitats (8, 12, 22, 32, 43). Kowalchuk and
coworkers recently used this technique to characterize
ammonia-oxidizing bacteria in composts (29). However, this
approach has not yet been used to study microbial-community succession
during composting at a broader phylogenetic level.
As an alternative to DGGE and TGGE, we recently developed a protocol
which allows the application of single-strand-conformation polymorphism
(SSCP) (18, 34) for the cultivation-independent assessment
of microbial-community diversity (41). In contrast to DGGE
and TGGE, no GC clamp or construction of gradient gels is required, and
thus the SSCP method has the potential to be more easily applied
(30). We have used the method to compare rhizosphere
microbial communities of different plants (41). Here we
report on the use of the SSCP approach to characterize microbial-community successions during composting. The method was used
to monitor the production of mushroom composts in an 18-day-long,
self-heating composting process.
| |
MATERIALS AND METHODS |
Composting, sampling, and chemical analyses.
Two separate
composting windrows were set up for this investigation. Each windrow
consisted of a wooden box (area, 1.6 m2; height, 2.0 m) filled with a mixture of field-grown shredded maize plants
(Zea mays) (750 kg), 0.5 m3 of wood chips, and
10% straw-bedded horse manure. This mixture was wetted before
composting for the initiation of the self-heating phase. The windrows
were turned every 2 to 3 days in order to enhance the composting
process and avoid the formation of anaerobic compartments. The
temperature was continuously measured at three depths (20, 30, and 50 cm) in the composting pile, using measuring lances to monitor the
process. Replicate samples were taken from a 50-cm depth. Chemical
properties of the compost samples (pH, organic carbon, total nitrogen,
ammonium, and nitrate) were analyzed by standard methods
(35). Samples designated for DNA extraction were stored
immediately at 
20°C.
Isolation and characterization of pure bacterial cultures from
compost.
In order to obtain pure cultures from composting
material, samples taken at the end of the composting process (18 days)
were suspended in sodium polyphosphate solution (0.2%, wt/vol).
Dilutions of this suspension were inoculated onto plate count agar
(PCA; Oxoid Unipath Ltd., Basingstoke, United Kingdom) supplemented with 50 mg of cycloheximide liter
1 for suppression of
fungal growth. The inoculated plates were incubated at 50°C for
16 h before single colonies were transferred to fresh agar and subcultured.
The ARDRA technique was used for the characterization of isolates
(48). Colonies of bacterial cells grown were suspended in 50 µl of lysis buffer (0.05 M NaOH-0.25% [wt/vol] sodium dodecyl sulfate) and incubated for 15 min at 95°C. The suspensions were diluted with 450 µl of water and centrifuged in a microcentrifuge at
the highest setting for 5 min at room temperature. An aliquot (1 to 5 µl) of the centrifuged solution was used as a template for PCR. A
1,060-bp product from the eubacterial 16S rRNA genes was amplified with
primers Ec41(f) and Ec1066(r) as described elsewhere (23). A
total of 5 µl of each PCR product was incubated with 5 U of a
restriction endonuclease (CfoI and RsaI,
separately; both from New England Biolabs, Schwalbach, Germany) and
buffer solution supplied by the manufacturer in a final volume of 20 µl for 1 h at 37°C. The restriction fragments were analyzed on 7% (wt/vol) polyacrylamide gels with 1× TBE (Tris-borate-EDTA buffer
[40]) and 7 M urea (Roth, Karlsruhe, Germany) using a Multiphor II apparatus (Pharmacia Amersham Biotech, Freiburg, Germany).
Products were stained with silver nitrate (3).
Sequencing of the PCR-amplified 16S rRNA genes (1,060 bp) obtained from
pure cultures was conducted by IIT Bioservice, Bielefeld,
Germany.
Alignments and data bank identifications were carried
out using BLASTN
2.0.4 (
1).
Extraction of DNA from compost material and generation of
PCR-SSCP genetic profiles.
Compost samples were ground in liquid
nitrogen, and total DNA was extracted from samples of 200 mg (wet
weight) using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany).
Compost DNA was eluted from DNeasy columns with 150 µl of
manufacturer-supplied AE buffer. A 102 dilution of the
eluate was used for PCR. The DNA concentration was measured
fluorometrically using Pico Green dye (Molecular Probes, Leiden, The
Netherlands) and a microtiter plate reader (Fluoroskan II; Labsystems,
Helsinki, Finland).
Three different primer systems were used to amplify 16S rRNA or 18S
rRNA genes from total community DNA of compost (Table
1). For SSCP, reverse primers were
phosphorylated at the 5' end.
Each PCR was performed in a total volume
of 100 µl in micro-test
tubes (Flat Cap Micro Tubes; MWG Biotech,
Ebersberg, Germany).
Reaction mixtures contained 1× PCR buffer with
1.5 mM MgCl
2, deoxynucleoside
triphosphates (200 µM each
dATP, dCPT, dGTP, and dTTP), 0.5 µM
each primer, 3.75 U of DNA
polymerase (Expand-Taq; Roche Diagnostics,
Mannheim, Germany), and 2 µl of the diluted DNA extract. To increase
amplification efficiencies
from compost DNA, T4 gene 32 protein
was added at a concentration of 5 µg per reaction (Roche Diagnostics)
(
46). Cycle conditions
for the reactions using Com and NS primers
(Table
1) were as follows:
initial denaturation at 94°C for 3
min; 35 cycles of 94°C for
60 s, 50°C for 60 s, and 72°C for 70
s; and a final
elongation for 5 min at 72°C. For PCR with primers
F243 and R531, the
annealing temperature was increased to 60°C.
PCR products were
purified (Qiaquick; Qiagen), and 15 µl of purified
product was
subsequently digested with 10 U of lambda exonuclease
(Pharmacia
Amersham Biotech) in a final volume of 50 µl to obtain
single-stranded DNA molecules (
41). The single-stranded DNA
molecules were purified and separated on a 0.6× MDE polyacrylamide
gel
(FMC Bioproducts, Rockland, Maine) according to our previously
described protocol (
41). To make the SSCP products visible,
gels were silver stained (
3).
Isolation and cloning of single products from SSCP community
profiles.
Selected products ("bands") identified in MDE
polyacrylamide gels after silver staining were excised with razor
blades, and single-stranded DNA was eluted from the gel by a "crush
and soak" procedure described by Sambrook et al. (40) and
modified by Schwieger and Tebbe (41). The single-strand DNA
molecules were reamplified by PCR using the same primers and conditions
as for the respective SSCP analysis. The resulting PCR products were analyzed by SSCP for purity and identity by comparing them with the
original fragments in the community profiles.
For cloning, the reamplified PCR products were subjected to another
PCR. Com primer-amplified products (including V4 and V5;
see Table
1)
were used in PCR with two phosphorylated primers
and Vent polymerase
(New England Biolabs). The amplicons generated,
which were blunt ended
and phosphorylated, were further purified
using a purification kit
(Qiaquick; Qiagen). These fragments were
ligated into the
dephosphorylated plasmid vector pUC18 and cut
with
SmaI
(Appligene Oncor, Heidelberg, Germany), and 5 µl of
the ligation mix
was used for the electroporation of competent
E. coli strain
JM 109 cells (Promega, Mannheim, Germany). For
subcloning of amplicons
generated with primer pair NS7-NS8 or
F243-R531, PCR-mediated
reamplifications were carried out under
the same conditions and with
the same primers as those used for
amplifications from compost DNA,
except that the reverse primer
was also dephosphorylated. The purified
PCR products were ligated
into the plasmid vector pGEM-T (Promega) in a
reaction volume
of 10 µl. Half of the ligation mix was used to
transform
E. coli JM 109 supercompetent cells by a protocol
provided by the
manufacturer.
The presence of inserts of the expected sizes was confirmed by PCR with
the flanking vector primers M13 forward and M13 reverse
(Promega). For
this purpose, ampicillin-resistant colonies were
treated as described
for ARDRA (
23), and 2 µl of lysate was
used as a template
in PCR with the same cycle conditions as those
described for the Com
primers. The sizes of these PCR products
were controlled on 1.2%
(wt/vol) agarose gels (
40). Amplicons
whose sizes
corresponded to those of the original fragments were
purified
(Qiaquick; Qiagen). These purified products were used
directly for
double-stranded sequence
analysis.
DNA sequencing of subcloned SSCP products.
For DNA
sequencing, infrared dye 800-labeled M13 sequencing primers (MWG
Biotech) were used in combination with the Thermosequenase sequencing
kit with 7-deaza GTP (Pharmacia Amersham Biotech). Primer and template
concentrations and PCR reagents were selected according to the
instructions of the manufacturer (Pharmacia Amersham Biotech).
Conditions for the cycle sequencing process, which was conducted in a
Primus 96 thermocycler (MWG Biotech), were as follows: initial
denaturation at 95°C for 2 min, followed by 30 cycles of 95°C for
30 s, 54°C for 20 s, and 72°C for 60 s. The
sequences were automatically analyzed on a 6% (wt/vol) polyacrylamide
gel (Rapid Gel XL; Pharmacia Amersham Biotech) using a LI-COR 4200 (MWG
Biotech). Alignments and database identifications of the consensus
sequences were carried out using BLASTN 2.0.4 (1).
Nucleotide sequence accession numbers.
The new DNA sequences
described in this study have been deposited in the GenBank sequence
database under accession numbers AF213262 to AF213286.
| |
RESULTS |
Conditions during the composting process.
The composting
process for the preparation of the mushroom compost lasted 18 days. The
initial temperature in the composting windrows was 20°C. Within 3 days of incubation the temperature increased to 40°C at a depth of 80 cm, 60°C at 50 cm, and 70°C at 30 cm. During further incubation,
interrupted by the turning of the material every 3 to 4 days, the
temperatures increased to a maximum of 80°C at all three depths after
10 days. From 10 days to 18 days, during the maturation phase,
temperatures decreased slowly but continuously to 65 to 70°C. The pH
decreased within the first 2 days from 5.6 to 4.5. Later, the pH
increased to 7.0. Nitrate was detectable only during the first 3 days
of incubation (threshold of detection, 1 mg kg1). Ammonia
levels increased during the process from 4 to 22 mg kg1.
The total nitrogen concentration decreased from 17 to 14 g
kg1 from day 0 to day 3. Subsequently, from day 3 to day
18, the concentration increased to 20 g kg1. In
summary, the parameters indicated a typical composting process of
organic agricultural material as desired for the production of mushroom
cultivation substrate.
SSCP genetic profiles of microbial communities during the
composting process.
Primers designed to amplify the eubacterial
16S rRNA gene sequences including the variable V4 and V5 regions
yielded complex SSCP patterns on polyacrylamide gels. The number of
bands increased during the composting process, and at the end of the
process (18 days) the profiles consisted of more than 30 different
products (bands) (Fig. 1). Specific
products (bands) occurred with similar intensities (yields) in profiles
obtained from two separate composting windrows. The successions of
patterns obtained from the two windrows were similar, which indicated
good reproducibility of the DNA extraction method and PCR
amplifications.
View larger version (142K):
[in this window]
[in a new window]
|
FIG. 1.
Succession of PCR-amplified products during a composting
process as detected by SSCP on a polyacrylamide gel. PCR primers were
designed to amplify the hypervariable regions V4 and V5 of eubacterial
16S rRNA genes from directly extracted compost DNA (S, standard DNA).
For each day (d), results obtained from two separate composting
windrows are shown.
|
|
With primers targeting the V3 region of actinomycete 16S rRNA genes,
the SSCP patterns consisted of fewer bands except for
the last sampling
date, when band intensities were too strong
to clearly differentiate
between products in some gel regions
(Fig.
2). The composting-stage-related increase
of individual
bands in the profiles indicated a succession of community
members
and suggested that the diversity of actinomycetes increased
during
compost maturation.
View larger version (96K):
[in this window]
[in a new window]
|
FIG. 2.
SSCP patterns of single-stranded PCR products obtained
from amplifications targeting the V3 region of actinomycetes. DNA
directly extracted from composting material was used as a template.
Days of sampling (d) are indicated for each lane.
|
|
Primers selected to amplify fungus-specific 18S rRNA gene sequences,
including the V8 and V9 regions, yielded products from
the organic
material even before composting (Fig.
3).
When these
SSCP products were compared with those of other samples, it
became
evident that one of the dominant products detected at day 0 was
identical to the product amplified from 18S rRNA genes of maize,
one of
the major sources of organic material in this composting
process (Fig.
3, lane M). Thus, the selected primers were not
exclusively specific
for fungal DNA. The disappearance of the
maize product after 4 days
suggested the degradation of plant
DNA during this early composting
stage. At this stage (4 days),
another dominant product was detected.
At the beginning of the
composting, this product was the only
detectable product. However,
during further composting, additional
products were detected,
first (6 and 10 days) only in one windrow and
later (12 and 15
days) in both windrows. Especially for the last two
sampling dates
shown in Fig.
3, another dominant product with
relatively low
electrophoretic mobility was detected. Both dominant
products
(indicated by arrowheads in Fig.
3), as well as other products
shown in the gels above (Fig.
1 and
2), were further identified
by DNA
sequencing.
View larger version (139K):
[in this window]
[in a new window]
|
FIG. 3.
Succession of SSCP patterns obtained from
single-stranded DNA products amplified by PCR from directly extracted
compost DNA. Primers targeted conserved regions to amplify the V8 and
V9 regions of the eukaryotic 18S rRNA gene sequence (M, products
obtained from DNA extracted from leaves of maize plants). Days of
sampling (d) are given above lanes. For each day, parallel samples were
obtained from two separate composting windrows. Arrowheads point to
products of the prospective clones CP-1 (4 d) and CP-2 (15 d) (see also
Table 2).
|
|
Identification of products from community SSCP profiles.
To
identify the predominant products by DNA sequencing, a total of 19 different DNA single strands ("bands") were excised from the three
SSCP profiles shown in Fig. 1 to 3. By PCR, the opposite strands were
regenerated and the products were reamplified. SSCP gel electrophoresis
was used to evaluate the purities and identities of the reamplified
products, as shown for products obtained from PCR targeting the
hypervariable regions V4 and V5 of eubacterial 16S rRNA genes (Fig.
4). In most cases, reamplification products corresponded to the expected positions in the community patterns and no additional products were observed. These products were
then directly used for cloning and DNA sequencing. Table 2 gives the period of detection and the
closest relatives found for each molecular isolate.
View larger version (117K):
[in this window]
[in a new window]
|
FIG. 4.
Comparison of PCR-SSCP community patterns with single
products isolated from profiles and reamplified by PCR. Patterns
obtained with primers amplifying the hypervariable regions V4 and V5 of
eubacterial 16S rRNA genes from directly extracted compost DNA are
shown in lanes A (sample taken after 2 days of composting), B (4 days),
C (6 days), D (10 days), and E (15 days). Amplified products of
prospective clones are shown in lanes 1 (clone CB-12), 2 (CB-2), 3 (CB-1), 4 (CB-3), 5 (CB-4), 6 (CB-5), 7 (CB-6), 8 (CB-7), 9 (CB-8), 10 (CB-9), 11 (CB-10), and 12 (CB-11). For identification of clones, see
Table 2.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Characterization of molecular isolates, cloned from
PCR-SSCP genetic profiles of 16S rRNA genes amplified from total DNA
extracted from compost
|
|
Most molecular isolates, 12 of 19, were obtained from PCR products
generated with primers targeting the eubacterial V4-V5
region (Table
2). Surprisingly, one of the sequenced products
was larger (556 bp
instead of approximately 400 bp), and this
product was, in fact,
closely related to a eukaryotic organism,
the yeast
Candida
krusei. Lactobacillus-related sequences were
detectable only
during the early stage of composting (2 to 6 days).
Five sequences were
related to
Bacillus species, among them one
with complete
sequence identity for this variable region found
in the 16S rRNA gene
of
Bacillus badius. Sequences related to
B. badius and
Bacillus thermocatenulatus were detectable
throughout
the whole composting process (2 to 18 days). One sequence
with
relatively low similarity was related to the anerobic
spore-forming
Clostridium thermolacticum. Two sequences were
related to
-
Proteobacteria and one sequence to an
actinomycete. The latter group was more
specifically targeted with
primers amplifying the V3 region. In
fact, except for one sequence
which was related to another
Bacillus species, the other
four sequences were related to actinomycetes.
These four sequences were
already detectable at the beginning
of the composting, but their
intensities, especially those of
CA-4, related to
Streptomyces
nodosus, and CA-5, related to
Streptomyces thermodiastaticus, increased continuously during the process (Fig.
2). The two dominant products detected in SSCP profiles of 18S
rRNA
gene-targeted PCR were related to two yeast sequences, both
members of
the genus
Candida. The same organism which was accidentally
detected with primers designed to amplify the V4-V5 16S rRNA gene
region after 6 days of incubation (CB-3) was also identified as
one of
the dominant fungal sequences (CP-1) throughout the composting
process.
Characterization of bacteria isolated by cultivation and comparison
to molecular isolates.
A collection of 290 strains was isolated
under aerobic conditions at 50°C from compost samples taken at the
end of the composting process. Incubation of inoculated agar plates
under aerobic conditions at 50°C resulted in fast growth of bacteria.
In order to avoid contamination of pure cultures, the incubation period
before subcultivation of isolates (single colonies) was only 16 h.
Using the combined results obtained with two restriction endonucleases
(CfoI and RsaI), we selected 22 isolates which
were further identified by sequencing of the uncut PCR product. These
isolates could be assigned to six different groups (Table
3). The 16S rRNA gene sequences of three
groups showed high similarity to those of Bacillus species. Two sequences were related to Pseudomonas species and one to
Xanthomonas campestris.
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Characterization of cultivated thermophilic and
heterotrophic bacteria isolated at the end of the hot-composting stage
|
|
From selected pure cultures of each group, PCR products generated with
Com primers (used for eubacterial-community analysis)
were loaded onto
SSCP gels with community profiles as a reference
(data not shown).
Similarities in electrophoretic mobility were
observed between isolate
Sko07 and clone CB-5, both related to
X. campestris.
Sequence alignments of both showed 295 matches
for 299 bp (98.7%).
Similar band positions in the profiles were
also observed for Sko17 and
CB-6 (related to
Pseudomonas stutzeri and to
Azotobacter salinestris). As judged by the almost-identical
DNA sequences of the selected 16S rRNA gene region (342 of 343
matches), these organisms were identical. Differences in identification
of their closest relatives can be explained by the different lengths
of
the sequences submitted to the databases. A third identity
in band
position was found for Sko08 and CB-12. The match in sequences
was 274 of 275 bp. Thus, it can be concluded that these organisms
too were the
same, or at least very closely related in their 16S
rRNA
genes.
| |
DISCUSSION |
The quickly changing physicochemical conditions in composting
processes, as described in this study, are likely to select for a
succession of different microbial communities. It can be expected that
temperature and the available substrates, including electron donors and
acceptors, are the main factors in this context (36). In
this study, we have shown that SSCP analysis of small-subunit rRNA
genes amplified from directly extracted compost DNA can be used to
visualize such community structures and successions. The SSCP-generated
genetic profiles consisted of more than 30 distinguishable products.
These products were different in electrophoretic mobility, probably due
to sequence differences causing different conformations under
nondenaturing conditions. However, the possibility that single products
may also have existed in more than one conformation under such
conditions and, thus, generated more than one band cannot be ruled out
(34). In contrast to another, previously applied protocol
for SSCP-based microbial-community analysis (30), our
protocol avoids the reannealing of DNA single strands and the formation
of heteroduplices by removal of one strand from each PCR product by
exonuclease digestion prior to nondenaturing electrophoresis
(41). As a further development of our protocol in this
study, we have isolated single products from community profiles and
identified them by DNA sequencing. With DGGE profiles the same purpose
was achieved in other studies (15, 39). In contrast to
direct cloning of PCR-amplified rRNA genes from environmental DNA, the
profiling technique has the advantage that single products can be
preselected before sequencing, and thus redundant information from
repetitive isolation of the same or highly similar sequences can be
avoided. This advantage of genetic profiling was shown in our study,
since each of 12 molecular isolates extracted from a genetic profile
generated with eubacterial primers was unique. However, in contrast to
directly cloned molecular isolates, where whole RNA operons can be
analyzed, the size of products available from genetic profiles is, to
our knowledge, still restricted to a maximum length of about 500 bp,
whether SSCP or DGGE-TGGE techniques are used for microbial-community
analysis. This can limit the accuracy of species identification but may
not be crucial for comparative assessment of microbial-community
structures in environmental samples (19).
For community profiling, as described in this study, PCR conditions and
primer selections are important factors which may influence the outcome
of an analysis. It is known that products amplified by PCR may not
accurately reflect the microbial diversity in the template mixture due
to different small-subunit rRNA gene copy numbers (17) or
biases during the amplification process (38, 45). However,
since the number of products which could be detected in the profiles
was limited to a maximum of approximately 40, it could be anticipated
that shifts in the community structure on the basis of eubacterial PCR
amplifications would be detected only when quantitatively dominant
organisms were affected (32). More-specific detections,
which may be suppressed with eubacterial primer amplifications, may be
possible if primers are used which select for specific groups of the
microbial community. Such primer systems have been designed to
characterize the diversity of actinomycetes in soil (22),
ammonia-oxidizing Proteobacteria in compost (29), or fungal genes in plant roots or a rhizosphere (28, 43). In
our study we used three primer systems in parallel with the same
template DNA, and it was therefore possible to characterize community
successions from the same samples with three different specificities.
In accordance, SSCP profiles generated with all three primer systems
demonstrated increasing microbial diversity during the composting
process. Sequence identification indicated the presence of several
species known to be involved in composting. Lactobacilli, which are the
typical dominant microorganisms in degrading plant material under
oxygen limitation, e.g., in silage (10), occurred at the
beginning of the composting process, when the substrate was relatively
wet. During the heating phase, 5 of 12 molecular isolates which were
closely related to the spore-forming aerobic genus Bacillus
and one which was related to the anaerobic genus Clostridium
were detected. Bacilli are typical microorganisms in hot composting
stages (6, 44). Two Bacillus-related isolates could be detected throughout the composting process. One of these isolates showed perfect homology to a known 16S rRNA gene sequence (B. badius), but the other (CB-9) had only 92.3% identity
to a known sequence. Possibly this dominant isolate was related to a
group of gram-positive bacteria with a low G+C DNA content which have
not been characterized yet.
Surprisingly, one sequence was related, not to bacteria, but to yeast.
This result can be explained by the fact that both the Com1 and the
Com2 primer have homologous regions in the 18S rRNA genes, each with
only one mismatch. The primers amplify between positions 556 and 1,117 (20, 33). The size of the sequenced product was in
accordance with this assumption. Only one actinomycete-related sequence
could be detected with eubacterial primers, but with primers targeting
this group more specifically, four of five isolates could be assigned
to this group. These results indicate the usefulness of the primers
suggested by Heuer et al. (22) for the analysis of
indigenous actinomycete populations in the environment. The increase in
pattern diversity observed with these primers is in accordance with the
general knowledge that actinomycete populations increase during compost
maturation (13, 36). Primers selected for monitoring of
fungal diversity also amplified plant DNA. By this means, we were able
to determine that plant DNA was degraded during the first 4 days of
composting. These results confirm the findings of a previous study in
which the stability of a recombinant marker gene was monitored under
similar composting conditions (27). Two different molecular
isolates, both related to the yeast genus Candida, were
identified from the SSCP profiles. The closest relatives of these
species, C. krusei and Candida tropicalis, are
known to be pathogenic for humans (20). DNA similarities do
not prove any pathogenic potential. It is, however, remarkable that
C. krusei was also accidentally identified as a close
relative of CB-3 with eubacterial primers targeting a different gene
region. Possibly the yeast originated from the fecal material (horse
dung) which was a component of the organic substrate composted in this study. For accurate identification of the molecular yeast isolates, other rRNA gene regions may be more appropriate, since the taxonomic resolution of small-subunit rRNA genes for fungi seems to be lower than
that for bacteria (33, 43). Other fungi may have been present in the composting process as well but may have been missed due
to inefficient DNA extraction or nonoptimal primer selection.
The diversity of the cultivated isolates was rather low; only 6 species
could be identified out of 290 isolates. It is likely that
more-appropriate cultivation media and more-sophisticated incubation
conditions would have yielded additional species. Cultivation of
bacteria was carried out only at the end of the composting process.
With pure-culture SSCP analysis, we found three products which had the
same electrophoretic mobilities as dominant products in the profiles.
As judged by base pair similarities in the selected 16S rRNA gene
region, it is highly probable that at least two of these isolates were
identical. One was B. badius, which could be detected
throughout the composting process. Since the 16S rRNA gene of this
organism was predominant and was also identified by cultivation, we
assume that it was quantitatively important. The other organisms with
high similarities belonged to the -Proteobacteria and
were related to P. stutzeri and A. salinestris.
In community profiles, this product was detectable from 4 days to 12 days, but by cultivation the related organism was found in 18-day-old samples.
The succession of products in combination with increasing and
decreasing band intensities during different composting stages, as
detected with SSCP profiles in this study, indicates the high potential
of this technique to monitor microbial communities and their variation
qualitatively and quantitatively. As more gene sequences become
available, PCR-SSCP-mediated monitoring of different subgroups or
microorganisms, due to optimized primer design, will become even more
attractive. The use of genetic profiles reduces the numbers of products
which need to be identified by DNA sequencing to those which are
assumed to be of specific importance. By this means and in combination
with highly efficient automated DNA sequencing, molecular analysis of
microbial communities gains new relevance for the monitoring of
biotechnological processes and applied microbial ecology.
| |
ACKNOWLEDGMENTS |
We thank Katja Lübke and Evelin Schummer for excellent
technical assistance and Klaus Grabbe for help in conducting the
composting process.
This work was supported by the Federal Environmental Agency of Germany
(grant 11201032).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Agrarökologie, Bundesforschungsanstalt für
Landwirtschaft (FAL), Bundesallee 50, 38116 Braunschweig, Germany.
Phone: 49 531-596 736. Fax: 49 531-596 366. E-mail:
christoph.tebbe{at}fal.de.
| |
REFERENCES |
| 1.
|
Altschul, S. F.,
T. L. Madden,
A. A. Schaffer,
J. Zhang,
Z. Zhang,
W. Miller, and D. J. Lipman.
1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res.
25:3389-3402[Abstract/Free Full Text].
|
| 2.
|
Andrews, S. A.,
H. Lee, and J. T. Trevors.
1994.
Bacterial species in raw and cured compost from a large-scale urban composter.
J. Ind. Microbiol.
13:177-182.
|
| 3.
|
Bassam, B. J.,
G. Caetano-Anolles, and P. M. Greshoff.
1991.
Fast and sensitive staining of DNA in polyacrylamide gels.
Anal. Biochem.
196:80-83[CrossRef][Medline].
|
| 4.
|
Beffa, T.,
M. Blanc, and M. Aragno.
1996.
Obligately and facultatively autotrophic, sulfur- and hydrogen-oxidizing thermophilic bacteria isolated from hot composts.
Arch. Microbiol.
165:34-40[CrossRef].
|
| 5.
|
Beffa, T.,
M. Blanc,
P. F. Lyon,
G. Vogt,
M. Marchiani,
J. L. Fischer, and M. Aragno.
1996.
Isolation of Thermus strains from hot composts (60 to 80°C).
Appl. Environ. Microbiol.
62:1723-1727[Abstract].
|
| 6.
|
Blanc, M.,
L. Marilley,
T. Beffa, and M. Aragno.
1997.
Rapid identification of heterotrophic, thermophilic, spore-forming bacteria isolated from hot composts.
Int. J. Syst. Bacteriol.
47:1246-1248[Abstract/Free Full Text].
|
| 7.
|
Blanc, M.,
L. Marilley,
T. Beffa, and M. Aragno.
1999.
Thermophilic bacterial communities in hot composts as revealed by most probable number counts and molecular (16S rDNA) methods.
FEMS Microbiol. Ecol.
28:141-149.
|
| 8.
|
Bruns, M. A.,
J. R. Stephen,
G. A. Kowalchuk,
J. I. Prosser, and E. A. Paul.
1999.
Comparative diversity of ammonia oxidizer 16S rRNA gene sequences in native, tilled, and successional soils.
Appl. Environ. Microbiol.
65:2994-3000[Abstract/Free Full Text].
|
| 9.
|
Carpenter-Boggs, L.,
A. C. Kennedy, and J. P. Reganold.
1998.
Use of phospholipid fatty acids and carbon source utilization patterns to track microbial community succession in developing compost.
Appl. Environ. Microbiol.
64:4062-4064[Abstract/Free Full Text].
|
| 10.
|
Cocconcelli, P. S.,
E. Triban,
M. Basso, and V. Botazzi.
1991.
Use of DNA probes in the study of silage colonization by Lactobacillus and Pediococcus strains.
J. Appl. Bacteriol.
71:296-301[Medline].
|
| 11.
|
Derikx, P. J. L.,
G. A. H. de Jong,
H. J. M. op den Camp,
C. van der Drift,
L. J. L. D. van Griensven, and G. D. Vogels.
1989.
Isolation and characterization of thermophilic methanogenic bacteria from mushroom compost.
FEMS Microbiol. Ecol.
62:251-258[CrossRef].
|
| 12.
|
Duineveld, B. M.,
A. S. Rosado,
J. D. van Elsas, and J. A. van Veen.
1998.
Analysis of the dynamics of bacterial communities in the rhizosphere of the chrysanthemum via denaturing gradient gel electrophoresis and substrate utilization patterns.
Appl. Environ. Microbiol.
64:4950-4957[Abstract/Free Full Text].
|
| 13.
|
Edwards, C.
1995.
Compost a natural substrate for studies of the diversity of thermophilic actinomycetes and monitoring the fate of genetically modified species, p. 229-233.
In
V. G. Debabov (ed.), The biology of the actinomycetes '94: proceedings of the Ninth Symposium on the biology of the actinomycetes. Biotechnologiya 7/8.
|
| 14.
|
Fermor, T. R.,
J. F. Smith, and D. M. Spencer.
1979.
The microflora of experimental mushroom composts.
J. Hortic. Sci.
54:137-147.
|
| 15.
|
Ferris, M. J.,
G. Muyzer, and D. M. Ward.
1996.
Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community.
Appl. Environ. Microbiol.
62:340-346[Abstract].
|
| 16.
|
Finstein, M. S., and M. L. Morris.
1975.
Microbiology of municipal solid waste management, p. 355-374.
In
R. Mitchell (ed.), Environmental microbiology. John Wiley & Sons, New York, N.Y.
|
| 17.
|
Fogel, G. B.,
C. R. Collins,
J. Li, and C. F. Brunk.
1999.
Prokaryotic genome size and SSU rDNA copy number: estimation of microbial relative abundance from a mixed population.
Microb. Ecol.
38:93-113[CrossRef][Medline].
|
| 18.
|
Hayashi, K.
1991.
PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA.
PCR Methods Appl.
1:34-38[Medline].
|
| 19.
|
Head, I. M.,
J. R. Saunders, and R. W. Pickup.
1998.
Microbial evolution, diversity, and ecology: a decade of ribosomal RNA analysis of uncultivated microorganisms.
Microb. Ecol.
35:1-21[CrossRef][Medline].
|
| 20.
|
Hendriks, L.,
A. Goris,
Y. van der Peer,
J. M. Neefs,
M. Vancanneyt,
M. Kersters,
G. L. Hennebert, and R. de Wachter.
1991.
Phylogenetic analysis of five medically important Candida species as deduced on the basis of small ribosomal subunit RNA sequences.
J. Gen. Microbiol.
137:1223-1230[Abstract/Free Full Text].
|
| 21.
|
Herrmann, R. F., and J. F. Shann.
1997.
Microbial community changes during composting of municipal soil waste.
Microb. Ecol.
33:78-85[CrossRef][Medline].
|
| 22.
|
Heuer, H.,
M. Krsek,
P. Baker,
K. Smalla, and E. M. H. Wellington.
1997.
Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients.
Appl. Environ. Microbiol.
63:3233-3241[Abstract].
|
| 23.
|
Hoffmann, A.,
T. Thimm,
M. Droge,
E. R. B. Moore,
J. C. Munch, and C. C. Tebbe.
1998.
Intergeneric transfer of conjugative and mobilizable plasmids harbored by Escherichia coli in the gut of the soil microarthropod Folsomia candida (Collembola).
Appl. Environ. Microbiol.
64:2652-2659[Abstract/Free Full Text].
|
| 24.
|
Insam, H.,
K. Amor,
M. Renner, and C. Crepaz.
1996.
Changes in functional abilities of the microbial community during composting of manure.
Microb. Ecol.
31:77-87.
|
| 25.
|
Klamer, M., and E. Baath.
1998.
Microbial community dynamics during composting of straw material studied using phospholipid fatty acid analysis.
FEMS Microbiol. Ecol.
27:9-20[CrossRef].
|
| 26.
|
Koschinsky, S.,
S. Peters,
F. Schwieger, and C. C. Tebbe.
2000.
Applying molecular techniques to monitor microbial communities in composting processes.
In
C. Bell (ed.), Progress in microbial ecology. Proceedings of the International Symposium on Microbial Ecology8, in press.
|
| 27.
|
Koschinsky, S.,
F. Schwieger,
S. Peters,
K. Grabbe, and C. C. Tebbe.
1998.
Characterizing microbial communities of composts at the DNA level.
Med. Fac. Landouww. Univ. Gent.
63/4b:1725-1732.
|
| 28.
|
Kowalchuk, G. A.,
S. Gerards, and J. W. Woldendorp.
1997.
Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis of specifically amplified 18S rDNA.
Appl. Environ. Microbiol.
63:3858-3865[Abstract].
|
| 29.
|
Kowalchuk, G. A.,
Z. S. Naoumenko,
P. J. L. Derikx,
A. Felske,
J. R. Stephen, and I. A. Arkhipchenko.
1999.
Molecular analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in compost and composted materials.
Appl. Environ. Microbiol.
65:396-403[Abstract/Free Full Text].
|
| 30.
|
Lee, D.-H.,
Y.-G. Zu, and S.-J. Kim.
1996.
Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single strand conformation polymorphism.
Appl. Environ. Microbiol.
62:3112-3120[Abstract].
|
| 31.
|
Malik, M.,
J. Kain,
C. Pettigrew, and A. Ogram.
1994.
Purification and molecular analysis of microbial DNA from compost.
J. Microbiol. Methods
20:183-196.
|
| 32.
|
Muyzer, G.,
E. C. de Waal, and A. G. Uitterlinden.
1993.
Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA.
Appl. Environ. Microbiol.
59:695-700[Abstract/Free Full Text].
|
| 33.
|
Neefs, J.-M.,
Y. Van der Peer,
P. De Rejk,
S. Chapelle, and R. De Wachter.
1993.
Compilation of small ribosomal subunit RNA structures.
Nucleic Acids Res.
21:3025-3049[Abstract/Free Full Text].
|
| 34.
|
Orita, M.,
H. Iwahana,
H. Kanazawa,
K. Hayashi, and T. Sekyia.
1989.
Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms.
Proc. Nat. Acad. Sci. USA
86:2766-2770[Abstract/Free Full Text].
|
| 35.
|
Page, A. L.,
R. H. Miller, and D. R. Keeney.
1982.
Methods of soil analysis. Part 2. Chemical and microbiological properties.
American Society of Agronomy, Inc. and Soil Science Society of America, Inc., Madison, Wis.
|
| 36.
|
Paul, E. A., and F. E. Clark.
1996.
Soil microbiology and biochemistry, 2nd ed.
Academic Press, San Diego, Calif.
|
| 37.
|
Pfaller, S. L.,
S. J. Vesper, and H. Moreno.
1994.
The use of PCR to detect a pathogen in compost.
Compost Sci. Utiliz.
2:48-54.
|
| 38.
|
Reysenbach, A.-L.,
L. J. Giver,
G. S. Wickham, and N. R. Pace.
1992.
Differential amplification of rRNA genes by polymerase chain reaction.
Appl. Environ. Microbiol.
58:3417-3418[Abstract/Free Full Text].
|
| 39.
|
Rölleke, S.,
G. Muyzer,
C. Wawer,
G. Wanner, and W. Lubitz.
1996.
Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.
Appl. Environ. Microbiol.
62:2059-2065[Abstract].
|
| 40.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 41.
|
Schwieger, F., and C. C. Tebbe.
1998.
A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis.
Appl. Environ. Microbiol.
64:4870-4876[Abstract/Free Full Text].
|
| 42.
|
Sharma, S.,
A. Rangger, and H. Insam.
1998.
Effects of decomposing maize litter on community level physiological profiles of soil bacteria.
Microb. Ecol.
35:301-310[CrossRef][Medline].
|
| 43.
|
Smit, E.,
P. Leeflang,
B. Glandorf,
J. D. van Elsas, and K. Wernars.
1999.
Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis.
Appl. Environ. Microbiol.
65:2614-2621[Abstract/Free Full Text].
|
| 44.
|
Strom, P. F.
1985.
Identification of thermophilic bacteria in solid-waste composting.
Appl. Environ. Microbiol.
50:906-913[Abstract/Free Full Text].
|
| 45.
|
Suzuki, M. T., and S. J. Giovannoni.
1996.
Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR.
Appl. Environ. Microbiol.
62:625-630[Abstract].
|
| 46.
|
Tebbe, C. C., and W. Vahjen.
1993.
Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.
Appl. Environ. Microbiol.
59:2657-2665[Abstract/Free Full Text].
|
| 47.
|
Vancanneyt, M.,
P. De Vos,
K. Kersters, and J. De Ley.
1987.
Isolation, characterization and identification of strictly anaerobic, thermophilic, ethanol producing bacteria from compost.
Syst. Appl. Microbiol.
9:293-298.
|
| 48.
|
Vaneechoutte, M.,
R. Roussau,
P. De Vos,
M. Gillis,
D. Janssens,
N. Paepe,
A. De Rouck,
T. Fiers,
G. Claeys, and K. Kesters.
1992.
Rapid identification of bacteria of the Comamonadaceae with amplified ribosomal DNA-restriction analysis (ARDRA).
FEMS Microbiol. Lett.
93:227-234[CrossRef].
|
| 49.
|
White, T. J.,
T. Burns,
S. Lee, and J. Taylor.
1990.
Amplification and direct sequencing of fungal ribosomal genes for phylogenetics, p. 315-322.
In
M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols. A guide to methods and applications. Academic Press, San Diego, Calif.
|
Applied and Environmental Microbiology, March 2000, p. 930-936, Vol. 66, No. 3
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Bomberg, M., Timonen, S.
(2009). Effect of Tree Species and Mycorrhizal Colonization on the Archaeal Population of Boreal Forest Rhizospheres. Appl. Environ. Microbiol.
75: 308-315
[Abstract]
[Full Text]
-
Klammer, S., Knapp, B., Insam, H., Dell'Abate, M. T., Ros, M.
(2008). Bacterial community patterns and thermal analyses of composts of various origins. Waste Manag Res
26: 173-187
[Abstract]
-
Briancesco, R., Coccia, A. M., Chiaretti, G., Della Libera, S., Semproni, M., Bonadonna, L.
(2008). Assessment of microbiological and parasitological quality of composted wastes: health implications and hygienic measures. Waste Manag Res
26: 196-202
[Abstract]
-
Di Giacomo, M., Paolino, M., Silvestro, D., Vigliotta, G., Imperi, F., Visca, P., Alifano, P., Parente, D.
(2007). Microbial Community Structure and Dynamics of Dark Fire-Cured Tobacco Fermentation. Appl. Environ. Microbiol.
73: 825-837
[Abstract]
[Full Text]
-
Peu, P., Brugere, H., Pourcher, A.-M., Kerouredan, M., Godon, J.-J., Delgenes, J.-P., Dabert, P.
(2006). Dynamics of a Pig Slurry Microbial Community during Anaerobic Storage and Management.. Appl. Environ. Microbiol.
72: 3578-3585
[Abstract]
[Full Text]
-
Nakasaki, K., Nag, K., Karita, S.
(2005). Microbial succession associated with organic matter decomposition during thermophilic composting of organic waste. Waste Manag Res
23: 48-56
[Abstract]
-
Anastasi, A., Varese, G. C., Filipello Marchisio, V.
(2005). Isolation and identification of fungal communities in compost and vermicompost. Mycologia
97: 33-44
[Abstract]
[Full Text]
-
Nakamura, K., Haruta, S., Ueno, S., Ishii, M., Yokota, A., Igarashi, Y.
(2004). Cerasibacillus quisquiliarum gen. nov., sp. nov., isolated from a semi-continuous decomposing system of kitchen refuse. Int. J. Syst. Evol. Microbiol.
54: 1063-1069
[Abstract]
[Full Text]
-
Nakamura, K., Haruta, S., Nguyen, H. L., Ishii, M., Igarashi, Y.
(2004). Enzyme Production-Based Approach for Determining the Functions of Microorganisms within a Community. Appl. Environ. Microbiol.
70: 3329-3337
[Abstract]
[Full Text]
-
Sliwinski, M. K., Goodman, R. M.
(2004). Spatial Heterogeneity of Crenarchaeal Assemblages within Mesophilic Soil Ecosystems as Revealed by PCR-Single-Stranded Conformation Polymorphism Profiling. Appl. Environ. Microbiol.
70: 1811-1820
[Abstract]
[Full Text]
-
Duthoit, F., Godon, J.-J., Montel, M.-C.
(2003). Bacterial Community Dynamics during Production of Registered Designation of Origin Salers Cheese as Evaluated by 16S rRNA Gene Single-Strand Conformation Polymorphism Analysis. Appl. Environ. Microbiol.
69: 3840-3848
[Abstract]
[Full Text]
-
Norris, T. B., Wraith, J. M., Castenholz, R. W., McDermott, T. R.
(2002). Soil Microbial Community Structure across a Thermal Gradient following a Geothermal Heating Event. Appl. Environ. Microbiol.
68: 6300-6309
[Abstract]
[Full Text]
-
Ranjard, L., Poly, F., Lata, J.-C., Mougel, C., Thioulouse, J., Nazaret, S.
(2001). Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability. Appl. Environ. Microbiol.
67: 4479-4487
[Abstract]
[Full Text]
-
Schmalenberger, A., Schwieger, F., Tebbe, C. C.
(2001). Effect of Primers Hybridizing to Different Evolutionarily Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based Microbial Community Analyses and Genetic Profiling. Appl. Environ. Microbiol.
67: 3557-3563
[Abstract]
[Full Text]
-
Yu, Z., Mohn, W. W.
(2001). Bacterial Diversity and Community Structure in an Aerated Lagoon Revealed by Ribosomal Intergenic Spacer Analyses and 16S Ribosomal DNA Sequencing. Appl. Environ. Microbiol.
67: 1565-1574
[Abstract]
[Full Text]
-
McOrist, A. L., Warhurst, M., McOrist, S., Bird, A. R.
(2001). Colonic Infection by Bilophila wadsworthia in Pigs. J. Clin. Microbiol.
39: 1577-1579
[Abstract]
[Full Text]
-
Schwieger, F., Tebbe, C. C.
(2000). Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicago sativa) and a Non-Target Plant (Chenopodium album)---Linking of 16S rRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the Diversity of Cultivated Bacteria. Appl. Environ. Microbiol.
66: 3556-3565
[Abstract]
[Full Text]
Top
Abstract
Introduction
