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Applied and Environmental Microbiology, August 1999, p. 3547-3554, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Community Analysis of Biofilters Using Fluorescence
In Situ Hybridization Including a New Probe for the
Xanthomonas Branch of the Class
Proteobacteria
Udo
Friedrich,
Michèle M.
Naismith,
Karlheinz
Altendorf, and
André
Lipski*
Abteilung Mikrobiologie, Fachbereich
Biologie/Chemie, Universität Osnabrück, 49069 Osnabrück, Germany
Received 15 March 1999/Accepted 25 May 1999
 |
ABSTRACT |
Domain-, class-, and subclass-specific rRNA-targeted probes were
applied to investigate the microbial communities of three industrial
and three laboratory-scale biofilters. The set of probes also included
a new probe (named XAN818) specific for the Xanthomonas branch of the class Proteobacteria; this probe is described
in this study. The members of the Xanthomonas branch do not
hybridize with previously developed rRNA-targeted oligonucleotide
probes for the 
-, 
-, and 
-Proteobacteria. Bacteria
of the Xanthomonas branch accounted for up to 4.5% of
total direct counts obtained with 4',6-diamidino-2-phenylindole. In
biofilter samples, the relative abundance of these bacteria was similar
to that of the -Proteobacteria. Actinobacteria
(gram-positive bacteria with a high G+C DNA content) and
-Proteobacteria were the most dominant groups. Detection
rates obtained with probe EUB338 varied between about 40 and 70%. For
samples with high contents of gram-positive bacteria, these percentages
were substantially improved when the calculations were corrected for
the reduced permeability of gram-positive bacteria when formaldehyde
was used as a fixative. The set of applied bacterial class- and
subclass-specific probes yielded, on average, 58.5% (± a standard
deviation of 23.0%) of the corrected eubacterial detection rates, thus
indicating the necessity of additional probes for studies of biofilter
communities. The Xanthomonas-specific probe presented here
may serve as an efficient tool for identifying potential
phytopathogens. In situ hybridization proved to be a practical tool for
microbiological studies of biofiltration systems.
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INTRODUCTION |
Many industrial processes produce
waste gases containing odorous organic or inorganic compounds, some of
which may be toxic to human health (26). While the principle
of filtering air with the help of microorganisms has been known for
some time, Pomeroy (27) seems to be the first who applied
biofiltration on a technical scale in the early 1950s. Biofiltration
usually exhibits high removal efficiencies and lower investment and
maintenance costs than physical and chemical treatment of waste gases.
Biofilters are thus applied increasingly in a variety of industrial settings.
Extensive efforts have been made to optimize the technical aspects of
biofilters. Comparably little is known about the biology of biofilters,
although detailed biological information is necessary for long-term
stability and further optimization of cleaning efficiencies. Most
microbial studies of biofilters have used isolation techniques, which
have led to the characterization of new species showing interesting
physiological capabilities (16, 29). Molecular biological
methods, in particular, are promising tools for studying the community
level in biofilters. A PCR-based approach (32) and
rRNA-targeted oligonucleotide probes (21) have been used to
study waste gas cleaning systems containing artificial carrier materials. To our knowledge, the present study is the first to apply
fluorescence in situ hybridization (FISH) to full-scale biofilters. In
this study, the bacterial communities of three industrial and three
laboratory-scale biofilters were characterized with oligonucleotide
probes targeting domain-, class-, and subclass-specific regions of the
16S and 23S rRNAs.
A significant portion of the bacteria previously isolated from
biofilters in our laboratory are members of the Xanthomonas branch of the class Proteobacteria, which forms a
monophyletic group close to the root of the
-Proteobacteria (22). Recently, the biofilter
strains isolated in our laboratory were identified as two new genera
and three new species of the Xanthomonas branch (9). Buchholz-Cleven et al. (7) isolated from a
freshwater sediment a strain belonging to the Xanthomonas
branch. They demonstrated that neither probe GAM42a, which is specific
for the -Proteobacteria (19), nor probe BET42a
hybridized with this strain. The present study confirms this finding on
a larger scale, as none of the 18 tested representatives of the
Xanthomonas branch hybridized with these probes. To allow in
situ identification and enumeration of these bacteria, we thus
designed, evaluated, and applied a new, 16S rRNA-targeted
oligonucleotide probe (XAN818) for the detection of all sequenced
members of the Xanthomonas branch.
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MATERIALS AND METHODS |
Cultivation and preparation of reference strains.
The
reference strains used are listed in Table
1. The strains were obtained from the
Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,
Braunschweig, Germany (DSM); Laboratorium voor Microbiologie,
Universiteit Gent, Ghent, Belgium (LMG); and our own collection
(strains with prefixes B, L, and R). Except for strains DSM
20300T, DSM 20140T, and LMG11114, all DSM and
LMG strains were cultivated according to the descriptions given by the
collections. Those three strains and our strains were cultivated in
nutrient broth (3 g of meat extract liter
1, 5 g of
peptone liter1, and 15 g of agar
liter1 were added for solid media) and harvested in the
exponential phase (optical density at 578 nm, between 0.5 and 0.8).
Pure cultures of members of the phylum Firmicutes
(gram-positive bacteria) (5 ml) were fixed with 5 ml of ethanol (100%)
and stored at 
18°C. All other pure cultures (5 ml) of the reference
strains (Table 1) were fixed with 15 ml of 4% paraformaldehyde (PFA)
solution (3) for 24 h and centrifuged at
11,200 × g for 20 min. Pellets were washed twice in 20 ml of phosphate-buffered saline (10 mM Na2HPO4 · 2H2O adjusted to
pH 7.4 with 10 mM Na2HPO4 · H2O; final concentration of NaCl, 130 mM) and centrifuged.
Cells were then resuspended in 1:1 phosphate-buffered saline-ethanol
and stored at 18°C.
Design of probes specific for the Xanthomonas
branch.
Specific target sequences for the 16S rRNA of bacteria of
the Xanthomonas branch were identified by use of the program
package ARB (35). The most recent data set available for ARB
(release 6pubmrz97) was supplemented with sequences obtained from the
EMBL database. The accession numbers of these sequences are as follows: Xanthomonas, X95917, X95918, X95919, X95920, X95921, X95922,
X99299, Y10754, Y10755, Y10756, Y10757, Y10758, Y10759, Y10760, Y10761,
Y10762, Y10763, Y10764, Y10765, and Y10766; Nevskia,
AJ001010 and AJ001011; Stenotrophomonas, AJ012229, AJ012230,
X95923, X95925, and U62646; Luteimonas, AJ012228;
Pseudoxanthomonas, AJ012231; denitrifying Fe(II)-oxidizing
bacterium BrG3, U51103; and iron-oxidizing lithotroph, AF012541. The
specificities of the target sequences obtained were also checked with
the PROBE MATCH tool of the Ribosomal Database Project (17).
Optimization of probe specificity and FISH tests.
For FISH,
3 µl from each fixed sample was spotted onto precleaned (washed in
1% HCl and rinsed with 70% ethanol) and gelatin-coated [0.075%
gelatin-0.01 CrK(SO4)2] slides. The slides
were then dried at 42°C for 10 min. Following dehydration in 50, 80, and 100% ethanol for 3 min each, the samples were covered with 8 µl
of hybridization buffer (19) and 1 µl of probe (50 ng/µl). Oligonucleotides were synthesized and fluorescently labeled
with Cy3 (Amersham) at the 5' end by Interactiva Biotechnologie GmbH
(Ulm, Germany). The rRNA-targeted oligonucleotides used are summarized
in Table 2.
A formamide gradient of 0 to 70% in hybridization buffer was used to
assess the optimal stringency for probe XAN818. The formamide
concentrations used for the other hybridizations are given in
Table
2.
Samples were hybridized at 46°C for 90 min in isotonically
equilibrated humid chambers. Samples were subsequently treated
with a
posthybridization wash as described by Manz et al. (
19)
at
48°C for 15 min. Sodium chloride concentrations in the washing
buffer
were adjusted by use of the formulae of Lathe (
14). Slides
were rinsed briefly with high-pressure liquid chromatography (HPLC)
water (Riedel), air dried, and mounted in Vectashield (Vector
Laboratories Inc., Burlingame, Calif.).
For optimization of the stringency for probe XAN818, fluorescence
intensities of reference strains hybridized with EUB338
and XAN818 were
detected by use of a Zeiss Axiovert 10 microscope
equipped with a 50-W
super-pressure mercury lamp and an HQ-Cy3
filter set (AF
Analysentechnik, Tübingen, Germany). Images were
recorded with a
charge-coupled device camera (SenSys; Photometrics
Ltd., Tucson, Ariz.)
and analyzed with the image analysis software
package IPlab
(Scanalytics, Fairfax, Va.). Camera parameters were
kept constant for
all measurements. Intensity threshold values
were adjusted to mark the
Cy3-labeled cells (
37). Probe-conferred
signal intensities
of the cells were divided by the cell area
to determine the intensities
independently of cell size (
25).
Values of background
fluorescence were subtracted from the fluorescence
intensity values
obtained. Mean values of signal intensities were
normalized to the
signal intensity obtained with probe EUB338
to correct for different
contents of ribosomes (
25). Between
200 and 400 cells were
examined per replicate, except for
Beggiatoa alba DSM 1416, for which 10 filaments were examined per
replicate.
For FISH tests of reference strains (Table
1), hybridized and washed
microorganisms were counterstained with 10 µl of
4',6-diamidino-2-phenylindole
(DAPI; 1 mg liter
1) at
4°C for 10 min in the dark and rinsed with HPLC water. DAPI-
and
probe-conferred fluorescence signals were examined by use
of a Zeiss
Axioskop epifluorescence microscope equipped with filter
set no. 01 (Zeiss) and the HQ-Cy3 filter
set.
Sample collection and physicochemical analyses.
Samples of
biofilter material were collected from three industrial and three
experimental biofilters. Samples Hamm A and B were collected from two
sites of a biofilter used for hexane waste gas treatment in an oil mill
(Hamm, Germany). Samples Sage A and B were collected from two sites of
a biofilter used for waste gas treatment in an industrially operated
chicken farm (Sage, Germany). Paderborn A and B were kindly provided by
H.-J. Warnecke, Universität-Gesamthochschule Paderborn,
Paderborn, Germany, from styrene-treated experimental biofilters.
Sample Alberta was derived from a hexane-treated experimental biofilter
and was kindly supplied by K. Budwill, Alberta Research Council,
Vegreville, Alberta, Canada. Sample Eversburg was collected
from a biofilter used for waste gas treatment in a municipal wastewater
treatment plant (Osnabrück-Eversburg, Germany). The biofilter
material consisted of crushed tree roots (Sage, Hamm, and Eversburg), a
mixture of crushed wood and bark compost (Paderborn A), peat (Alberta),
and porous clay (Paderborn B). Soil samples were taken just below the
litter layer of a forest consisting mainly of Fagus
sylvatica. The freshwater sample was collected from the upper 20 cm of a pond close to the University of Osnabrück. For pH
measurements, 10 g of fresh filter material and soil were stirred
in 25 ml of 1 M KCl for 30 min before being analyzed with a pH
electrode. Between 40 and 50 g of fresh filter material and soil
were dried at 80°C to assess the water content.
Direct enumeration and FISH of biofilter and environmental
samples.
For direct counts, 10 g of fresh filter material and
soil were stirred in 100 ml of Ringer solution (0.9% NaCl, 0.042%
KCl, 0.024% NaHCO3) for 30 min. Subsequently, 5 ml of the
solution was fixed with 15 ml of PFA solution (see above). Prior to
direct enumeration, fixed samples were diluted in sterile 0.9% NaCl
solution to obtain about 100 cells per microscopic field. After samples were stained with DAPI (final concentration, 2 mg liter1)
(28) for 5 min, 5-ml subsamples were filtered on black
polycarbonate membrane filters (pore size, 0.2 µm; Nuclepore) by
application of gentle vacuum pressure (
2 kPa). Filters were mounted
in melting-point bath oil (M-6884; Sigma). At least 400 DAPI-stained
cells were counted by examining at least 20 randomly chosen microscopic fields.
Biofilter and soil samples were hybridized, washed, and counterstained
with DAPI as described for the reference strains. Bacterial
detection
rates calculated from EUB338-positive counts relative
to DAPI counts
were corrected for different fixation procedures
when high proportions
(>10%) of members of the
Firmicutes (gram-positive
bacteria) were present. The differences between percentages of
ethanol-
and PFA-fixed samples for probes HGC69a, LGC354a, LGC354b,
and LGC354c
were thus added to the percentages of PFA-fixed samples
for probe
EUB338. When PFA-fixed samples yielded higher percentages
of LGC354a-,
LGC354b-, and LGC354c-labeled cells than ethanol-fixed
samples, these
(negative) differences were not taken into account
for the correction
of eubacterial counts. The freshwater sample
was directly enumerated as
described by Friedrich et al. (
10)
and hybridized as
described by Glöckner et al. (
12). Probe-positive
counts were determined relative to DAPI counts. The number of
DAPI-stained cells counted varied between 400 and 700 cells per
replicate. At least 20 randomly chosen fields were examined per
replicate. Between two and four replicates were counted per
sample.
Statistical analysis.
For the analysis of relationships
between variables, Pearson product-moment correlation coefficients were
calculated. When the assumptions of normality and equal variance did
not apply, the data were log10 transformed prior to
calculating regressions. Percentages were transformed by arcsine
x/100 before statistical analysis.
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RESULTS |
Design and optimization of probe XAN818.
The rRNA data set of
the software package ARB used for the probe design in the present study
contained 5,138 small-subunit rRNA sequences of at least 1,400 residues
(plus 2,778 sequences of shorter lengths). The alignment contained 34 nearly complete sequences of members of the Xanthomonas
branch. Potential target regions for a probe specific for the
Xanthomonas branch were positions 92 to 109, 818 to 835, 861 to 878, 1143 to 1160, and 1428 to 1445, based on Escherichia
coli 16S rRNA numbering (6). Only positions 861 to 878 proved to be inaccessible, as found by whole-cell hybridization, among
the targets that we tested (the latter four regions). Positions 818 to
835 were best suited for a probe specifically targeting bacteria of the
Xanthomonas branch, since all sequenced members have
retained this target sequence and nontarget organisms had a minimum of
two mismatches (Table 3).
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TABLE 3.
Difference alignment of the target sequence of probe
XAN818 and sequences of exemplary matching target organisms and
nontarget organisms
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The optimum hybridization stringency in order to discriminate between
target and nontarget organisms was evaluated by use
of a formamide
concentration gradient in the hybridization buffer
(
33). The
effect of increased formamide concentration on the
fluorescence
intensity conferred by Cy3-labeled probe XAN818 hybridized
with a
target organism,
Stenotrophomonas nitritireducens DSM
12575
T, and three nontarget organisms is shown in Fig.
1. Probe binding
to the target organism
decreased at formamide concentrations above
10 to 15%, attaining
background intensities at about 50% formamide.
Probe-conferred
fluorescence of the nontarget organisms was in
the background range,
even at 0% formamide. Nontarget organisms
can thus be discriminated
without the addition of formamide to
the hybridization buffer. However,
since there were no significant
intensity losses, a concentration of
10% formamide was used for
all subsequent hybridizations. FISH tests
with reference strains
confirmed a previous finding with an isolate of
the
Xanthomonas branch (
7) that representatives
of this lineage are detected
by neither probe GAM42a nor probe BET42a
(Table
1). Probe XAN818,
presented here, hybridized with all members of
the branch tested,
confirming the results of the database retrieval.
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FIG. 1.
Quantification of binding of probe XAN818 relative to
that of probe EUB338 measured as fluorescence intensity. Different
stringencies were obtained by varying the concentration of formamide in
the hybridization buffer. The target sequence of probe XAN818 and base
changes in nontarget organisms are shown in Table 3. Symbols:  ,
S. nitritireducens DSM 12575T;  , B. alba DSM 1416;  , A. acidophilum DSM
700T;  , G. oxydans subsp. oxydans
DSM 3503T. Error bars show standard deviations.
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Physicochemical and community analyses.
H+
concentrations varied by more than 3 orders of magnitude in the
different biofilter materials, with the pHs being 3.0 in sample Hamm A
and 7.5 in sample Paderborn B (Table 4).
The water content showed little variation among the organic materials,
with an average of 72.6% and a coefficient of variation of 5.8%. Cell concentrations, as determined by direct counts with DAPI, varied between 5.5 × 109 and 7.8 × 1010
per g of dry weight among the organic biofilter materials, whereas counts of only 2.2 × 109 were determined for
Paderborn B, which contained porous clay as the filter material (Table
4).
Treatment with Ringer solution removed considerable amounts of
particles from the biofilter material in the size range of
<10 µm to
a few hundred micrometers. Most of the bacteria present
in the
preparations were attached to these particles. This result
indicates
that varying affinities of different taxa for particulate
matter will
not affect the results obtained after the treatment
of biofilter
material with Ringer
solution.
Bacterial detection rates obtained with probe EUB338 varied between
39.9% in sample Eversburg and 68.2% in sample Sage B (Fig.
2) and were similar to the rates reported
for other systems (
12,
13,
25,
34,
39). Probe EUB338 is
usually applied in combination
with PFA-fixed samples. However, ethanol
is more suitable for
the fixation of members of the
Firmicutes (
30). In samples with
high proportions
of members of the
Firmicutes, bacterial detection
rates may
thus be underestimated when PFA is used as a fixative.
In the present
study, percentages of EUB338-positive bacteria
were therefore corrected
for samples with high proportions of
bacteria belonging to the
Firmicutes by hybridizing and enumerating
ethanol- and
PFA-fixed samples with probes LGC354a, LGC354b, LGC354c,
and HGC69a
(Table
5). Ethanol proved to be more
effective than
PFA for members of the
Actinobacteria. The
largest difference
between ethanol- and PFA-fixed samples was found for
the Alberta
sample. Detection rates (relative to those obtained with
DAPI)
obtained with probe HGC69a were 37.5% for the ethanol-fixed
sample
and merely 4.3% for the PFA-fixed sample. In comparison, there
were no consistent differences between the two fixatives for bacteria
belonging to the
Firmicutes with a low G+C content (Table
5).
Whereas ethanol was more effective than PFA for sample Sage B,
it
was less effective for samples Alberta and Sage A. The positive
differences between detection rates for ethanol- and PFA-fixed
samples
were then added to the proportions obtained with probe
EUB338. This
correction led to higher detection rates, particularly
for samples
Alberta and Sage B, for which 87.7 and 90.8% of all
DAPI-stained
cells, respectively, were thus identified as bacteria
with probe EUB338
(Fig.
2).
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FIG. 2.
Probe-specific counts, relative to direct enumeration
with DAPI (percentages) ( ). Percentages of EUB338
positive bacteria corrected for samples with high proportions of
members of the Firmicutes ( ). Percentages
of LGC obtained when PFA fixation was more efficient than ethanol
fixation () (see Materials and Methods and Table 5).
Percentages given are corrected for negative control NON338. Probe
names are abbreviations of those shown in Table 2. Error bars indicate
standard deviations for two to four replicates.
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TABLE 5.
Percentages of cells labeled with probes HGC69a, LGC354a,
LGC354b, and LGC354c after ethanol and PFA fixation, relative to
DAPI counts
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The two sampling sites for the Hamm biofilter showed similar bacterial
community patterns, with the
-
Proteobacteria (ALF)
being
the most dominant group, followed by the
-
Proteobacteria (BET) and the
Actinobacteria (HGC) (Fig.
2). Members of the
Cytophaga-Flavobacterium cluster (CF),
-
Proteobacteria (GAM),
Xanthomonas branch
(XAN),
Firmicutes with low G+C DNA content (LGC), and
Planctomycetes (PLA) varied between 0 and 2.3% (Fig.
2).
Interestingly, proportions
for members of the
Xanthomonas
branch were in the same range as
those for members of the
-
Proteobacteria. The Paderborn biofilters
used for
styrene degradation differed with respect to the filter
material used
(see Materials and Methods). They also differed
in terms of their
bacterial community patterns. Whereas in Paderborn
biofilter A ALF,
HGC, and LGC were of similar importance, ALF
dominated in biofilter B
(Fig.
2). In this biofilter, members
of the
Xanthomonas
branch were the second most dominant group,
accounting for 4.5% of all
DAPI-stained cells. The two sampling
sites for the Sage biofilter used
for waste gas treatment in an
industrially operated chicken farm showed
different detection
rates but similar community patterns. The bacterial
communities
of both sites were dominated by HGC, BET, ALF, and LGC
(Fig.
2).
Members of the
Xanthomonas group accounted for 2.6 and 0.9% at
sites A and B, respectively, and were again in the range
for members
of the
-
Proteobacteria. Members of the
Eucarya and
Archaea were
only of minor importance
in terms of abundance, representing 0.9
to 3.6%, relative to DAPI
counts. In the Alberta laboratory-scale
biofilter used for hexane
degradation, HGC was the most dominant
group, followed by ALF, BET, and
CF (Fig.
2). XAN and GAM accounted
for 2.0 and 1.8%, respectively
(Fig.
2).
The proportions of bacteria that were detected with probe EUB338 and
that could be assigned to taxa with the group-specific
probes used
varied greatly among biofilters and sites. Whereas
in sample Sage B
nearly all bacteria could be identified as members
of the taxa
examined, there were considerable proportions of unidentified
bacteria
among the other filter samples. These varied between
18% in Paderborn
B and nearly 46% in Hamm B, relative to DAPI
counts, indicating that
significant percentages of other, nontargeted
bacterial taxa were
present in these samples. The correction of
eubacterial detection rates
by use of the results shown in Table
5 had a significant influence on
the proportions of unidentified
taxa. Before correction, the sum of the
percentages derived from
bacterial class- and subclass-specific probes
was higher than
that derived from probe EUB338 for sample Sage B and
nearly equal
to the latter for sample Alberta. This situation changed
after
the correction was taken into account: whereas in the Sage B
sample
nearly all bacteria could be identified as members of the taxa
examined, 27.6% of bacteria belonged to unidentified taxa in the
Alberta sample. Hence, the correction of eubacterial detection
rates in
samples with high concentrations of members of the
Firmicutes,
as used in the present study, may be crucial to
derive meaningful
data. Percentages of
Planctomycetes in
counts obtained with probe
PLA46 were not subtracted from the counts
obtained with probe
EUB338, as these organisms generally do not
hybridize with probe
EUB338 (
24).
Probes EUB338, NON338, and XAN818 were also applied in terrestrial and
aquatic environments. Percentages of bacteria detected
with probe
EUB338 were 45.0% ± 0.2% and 50.4% ± 4.4% in soil samples
A and
B, respectively, and 55.9% ± 1.0% in the freshwater pond
sample.
Amounts of bacteria belonging to the
Xanthomonas branch
were
fairly low in these samples, being 1.5 and 1.1% in soil samples
A and
B, respectively, and close to the detection limit in the
freshwater
pond
sample.
There were no significant relationships between physicochemical (pH and
water content) and bacterial parameters (total cell
concentration,
probe-specific percentages) of the biofilter
data.
| |
DISCUSSION |
Design of probes specific for the Xanthomonas
branch.
Our alignment demonstrated that positions 818 to 835 (E. coli numbering) are most appropriate for a probe
targeting the 16S rRNA of members of the Xanthomonas branch.
Among the four potential target regions that we examined, only
positions 861 to 878 were inaccessible by whole-cell hybridization.
Interestingly, Fuchs et al. (11) recently found good
accessibility of positions 853 to 870 and 863 to 880 in cells of
E. coli DSM 30083T. Since both probes, i.e.,
that used by Fuchs et al. (11) to target positions 863 to
880 and the one that we used to target positions 861 to 878, were
self-complementary to the same extent, potential Watson-Crick pairings
within the probes may not be the reason for the observed differences in
accessibility. These results rather suggest that accessibility may vary
among different taxa. The fluorescence intensity conferred by probe
XAN818 was 97% that conferred by probe EUB338 at 5% formamide for
Stenotrophomonas nitritireducens DSM 12575T
(Fig. 1). This intensity was 5.7 times higher than that found for the
same region in cells of E. coli DSM 30083T,
which showed only 17% the fluorescence intensity obtained with probe
EUB338 (11). The hybridization stringency applied by Fuchs et al. (11) was similar to that obtained at 0% formamide in the present study (for fluorescence values, see Fig. 1). Thus, the
observed lower probe-conferred fluorescence in E. coli DSM 30083T may be due to probe-specific differences (e.g.,
probe quality, dissociation temperature, and so forth) and/or to
different accessibilities of the same target site among the taxa
examined. The latter could increase the taxonomic specificity of in
situ hybridization beyond the level obtained by sequence differences.
The results presented here stress the necessity to test every newly
developed probe individually.
In situ identification of members of the Xanthomonas
branch.
Methods for the in situ identification of bacteria of the
Xanthomonas branch are interesting for several applications.
Members of the genus Xanthomonas are sometimes difficult to
identify, especially if the origin of the isolates is uncommon
(5). In comparison to time-consuming methods based on
isolation techniques, in situ hybridization facilitates identification
procedures. Probe XAN818 may also be suited for the assessment of
Xylella- and Xanthomonas-caused plant diseases.
The identification of the causative microorganisms has been addressed
by a number of studies using different techniques (1, 2,
15). However, the methods used do not allow in situ
identification and enumeration, which are possible with FISH. Furthermore, probe XAN818 allows differentiation between bacteria of
the genera Pseudomonas and Xanthomonas, which is
sometimes a difficult task (5). Apart from its use for the
phytopathogenic genera, probe XAN818 may be useful in clinical studies
focusing on the identification of human pathogens such as
Stenotrophomonas maltophilia (8, 31).
Tanner et al. (
36) have recently shown that PCR may easily
amplify contaminant DNA, resulting in biased results obtained
by
PCR-based assessments of bacterial diversity. These problems
may arise
because of the difficulty in preparing genomic DNA absolutely
free from
contaminating DNA and the exquisite sensitivity of PCR.
Therefore, the
occurrence of an organism indicated by a cloned
rRNA sequence requires
explicit detection of that organism in
situ (
36). Probe
XAN818 may serve as a tool for such analyses
if cloned rRNA sequences
can be assigned to the
Xanthomonas group,
a contaminant of
which was found by Tanner et al. (
36) in their
negative
extraction
controls.
Community analysis of biofilter samples.
No significant
correlations were obtained from the statistical analysis of the
parameters pH and water content versus bacterial parameters of the
pooled data from the biofiltration systems. There were similar
community patterns between sampling sites for the industrial biofilters
Hamm and Sage. For samples from biofilter Hamm, detection rates also
were similar. These differed for the two Sage biofilter sites (Fig. 2).
The higher detection rates at Sage site B might be a result of the
nearly neutral pH at this site, because higher acidity, as found at
site A, might reduce overall bacterial activity, numbers of ribosomes
and, thus, detection rates with rRNA-targeted probes. It can be
expected that waste gas loading will be an important factor in
determining the microbial communities of biofilters. The rather complex
waste gas composition of a chicken farm (Sage) may thus result in a
different community pattern than artificial waste gas containing the
single compound hexane (Alberta) or styrene (Paderborn). Apart from
waste gas composition, the filter material itself also may have an
impact on the microbial community. The material of the Paderborn A
biofilter consisted of crushed wood and bark compost, whereas the
Paderborn B filter contained porous clay. Even though both biofilters
were used for styrene degradation, the community patterns differed substantially (Fig. 2). Also, the pHs differed, being 4.1 and 7.5 at
sites A and B, respectively. The different community patterns may be a
consequence of the differences in the physical and/or chemical environment.
The most commonly used biofiltration material, crushed tree roots,
poses some difficulties for investigations of the microorganisms
involved. The main reasons are the large number of different size
classes, the morphological heterogeneity of the material, and
the high
organic content of the material. The results presented
here demonstrate
that FISH is a valuable tool for studying biofiltration
systems, being
both practical and reproducible. Our results also
stress the necessity
to account for the different efficiencies
of formaldehyde and ethanol
in permeabilizing the cell wall of
bacteria of the phylum
Firmicutes, particularly in systems with
high concentrations
of these bacteria. Detection rates with bacterium-specific
probes can
be significantly improved if these differences are
corrected for. The
results of the present study also show the
presence of significant
proportions of bacteria that can be identified
with probe EUB338 but
not with probes targeting major groups within
the
Bacteria.
Therefore, identification and enumeration of the
remaining taxa of the
microbial community of biofilters should
be an interesting avenue for
future
research.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Abteilung
Mikrobiologie, Fachbereich Biologie/Chemie, Universität
Osnabrück, D-49069 Osnabrück, Germany. Phone:
49-541/969-2276. Fax: 49-541/969-2870. E-mail:
Lipski{at}biologie.uni-osnabrueck.DE.
| |
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Top
Abstract
Introduction
