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Identification of Hydrocarbon-Degrading
Bacteria in Soil by Reverse Sample Genome
Probing
Yin Shen, Lester G. Stehmeier and Gerrit Voordouw
Appl. Environ. Microbiol. 1998, 64(2):637.
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1998, p. 637–645
0099-2240/98/$04.0010
Copyright © 1998, American Society for Microbiology
Vol. 64, No. 2
Identification of Hydrocarbon-Degrading Bacteria in Soil
by Reverse Sample Genome Probing
YIN SHEN,1 LESTER G. STEHMEIER,1,2
AND
GERRIT VOORDOUW1*
Department of Biological Sciences, The University of Calgary, Calgary, Alberta, Canada T2N 1N4,1
and NOVA Research and Technology Centre, Calgary, Alberta, Canada T2E 7K72
Received 29 May 1997/Accepted 19 November 1997
tion of the success of a remediation effort at sites where C51
spills have occurred is therefore determined largely by the
concentration of residual DCPD.
The objective of this study was to determine whether reverse
sample genome probing (RSGP), a technique that is suitable
for monitoring the response of environmental microbial communities to chemical changes (19), can be used to identify the
response of a soil microbial community to the introduction of
unsaturated hydrocarbons. Bacteria enriched in the presence
of a metabolizable compound (toluene) or a recalcitrant compound (DCPD) were tested for their ability to oxidize these
substrates.
Unsaturated hydrocarbons are obtained as by-products in
the pyrolysis of ethane to ethylene. These higher-molecularweight products collect at the bottom of the quench tower of
an ethane pyrolysis plant and are referred to as the C51
stream. C51 contains benzene, cyclopentadiene, dicyclopentadiene (DCPD), ethylbenzene, styrene, toluene, and xylenes as
major components and many other hydrocarbons as minor
components. It is further processed to obtain the pure components; e.g., pure DCPD is used for polymer production. As a
result of transport and handling, accidental releases of C51
occur at pyrolysis plant sites. It was shown recently (18) that
the bioremediation of C51-contaminated soil is characterized
by rapid removal of more easily degradable BTEX (benzene,
toluene, ethylbenzene, and xylene) components followed by
the much slower removal of cyclopentadiene, DCPD, and higher-molecular-weight hydrocarbons (C111).
Bacteria capable of mineralizing the BTEX components of
the C51 stream are readily isolated from contaminated soil,
but microorganisms capable of DCPD mineralization have not
been found (17). Similarly, the pathways by which BTEX components are metabolized are generally known (5, 30) whereas
the pathway for DCPD degradation is not. Laboratory experiments have shown that soil microbial communities convert
some [14C]DCPD into 14CO2 while forming larger amounts of
oxygenated derivatives as identified by gas chromatographymass spectrometry (GC-MS) (17). van Breemen et al. (23)
found two monooxygenated DCPD derivatives in contaminated groundwater in which oxygen was incorporated at position 8 of the DCPD carbon skeleton (Fig. 1E and F), while
Stehmeier et al. (17) demonstrated incorporation at position 3
(Fig. 1G and H). All of these are distinct from the epoxides
(Fig. 1C and D) formed by rabbit liver cytochromes P-450 (23).
The recalcitrance of DCPD in the environment is of concern
primarily because of its pungent smell (2). The human percep-
MATERIALS AND METHODS
Biochemical reagents. Radioisotopes a-35S-dATP (10 mCi/ml; 400 Ci/mmol
[Amersham]) and [a-32P]dCTP (10 mCi/ml; 3,000 Ci/mmol [ICN]) were used.
Reagent-grade chemicals were from BDH, Fisher, or Sigma, and enzymes and
bacteriophage l DNA (0.5 mg/ml) were obtained from Pharmacia. Polyvinylpolypyrrolidone (PVPP) was from Sigma, and the Hybond-N hybridization transfer membrane was from Amersham.
Culture media. Hydrocarbon degradation medium (HDM) and tryptone yeast
extract (TY) medium were as described previously (17, 29). Minimal salts medium for studies of hydrocarbon degradation in soil contained 4 g of NaNO3,
1.5 g of KH2PO4, 0.5 g of Na2HPO4, 0.0011 g of FeSO4 z 7H2O, 0.2 g of MgSO4 z
7H2O, and 0.01 g of CaCl2 per liter of water at pH 7.0. PTYG medium contained
1 g of tryptone, 2 g of yeast extract, 2 g of glucose, 0.6 g of MgSO4 z 7H2O, and
0.07 g of CaCl2 z 2H2O per liter of water. Liquid medium C and plating medium
E for the growth of sulfate reducers were as formulated by Postgate (13).
Isolation and characterization of soil bacteria. Members of the microbial
community in C51-contaminated soil were obtained by being grown at room
temperature (22°C) on the media listed in Table 1. The set in Table 1 had limited
cross-hybridization of genomic DNAs. Species with little or no genomic crosshybridization under stringent conditions have been referred to as standards in
earlier work (19, 20, 25–28). Higher degrees of cross-hybridization (up to 30%)
were found in the present study for genomes from Pseudomonas species. Standards 1 to 20 and 22 to 24 were isolated from C51-contaminated soil under
aerobic conditions by streaking for single colonies on PTYG medium. Restreaked, isolated colonies were grown in 5 ml of PTYG medium, which was used
to inoculate 300 ml of medium in 500-ml Erlenmeyer flasks shaken at 150 rpm.
Following growth to stationary phase, cells were harvested by centrifugation,
frozen at 270°C, and used for DNA preparation. Glycerol stocks of all cultures
were also kept at 270°C.
Standards 21, 25, and 26 (Table 1) were isolated on aerobic HDM plates
incubated in an atmosphere of benzene, naphthalene, or styrene, respectively, by
M. M. Francis, NOVA Research & Technology Corp., Calgary, Canada. Cells for
* Corresponding author. Mailing address: Department of Biological
Sciences, The University of Calgary, 2500 University Dr. NW, Calgary,
Alberta, T2N 1N4, Canada. Phone: (403) 220-6388. Fax: (403) 2899311. E-mail: voordouw@acs.ucalgary.ca.
637
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Bacteria with limited genomic cross-hybridization were isolated from soil contaminated with C51, a mixture
of hydrocarbons, and identified by partial 16S rRNA sequencing. Filters containing denatured genomic DNAs
were used in a reverse sample genome probe (RSGP) procedure for analysis of the effect of an easily degradable
compound (toluene) and a highly recalcitrant compound (dicyclopentadiene [DCPD]) on community composition. Hybridization with labeled total-community DNA isolated from soil exposed to toluene indicated
enrichment of several Pseudomonas spp., which were subsequently found to be capable of toluene mineralization. Hybridization with labeled total-community DNA isolated from soil exposed to DCPD indicated enrichment of a Pseudomonas sp. or a Sphingomonas sp. These two bacteria appeared capable of producing oxygenated
DCPD derivatives in the soil environment, but mineralization could not be shown. These results demonstrate
that bacteria, which metabolize degradable or recalcitrant hydrocarbons, can be identified by the RSGP
procedure.
638
SHEN ET AL.
DNA isolation were obtained from 1-liter cultures in HDM in which these
hydrocarbons served as the sole carbon and energy source. Standards 27 to 29
were isolated on aerobic TY plates, while standards 30 to 33 were obtained on
TY plates under anoxic conditions in a 5% H2–10% CO2–85% N2 gas atmosphere. Two Desulfovibrio species (Table 1, standards 34 and 35) were obtained
on medium E plates (13), and single colonies were grown on liquid medium C (13).
The colony morphology and cellular morphology of isolated bacteria were
recorded for future reference by using descriptors and microscopy procedures
defined elsewhere (6).
DNA isolation. DNA was extracted from cells by the Marmur method (11)
modified as described elsewhere (25) and also including three cycles of freezing
and thawing (22) for better cell lysis. Final preparations were dissolved in TE (10
mM Tris-HCl, 0.1 mM EDTA [pH 8]).
DNA was isolated from soil by a modification of the technique described by
Bakken (3). Soil samples (5 to 20 g) were combined with acid-washed PVPP,
suspended in 0.1% (wt/vol) sodium pyrophosphate, and homogenized by stirring
for 20 min. The soil particles and acid-washed PVPP were removed by centrifugation at 1,000 3 g for 10 min at 4°C. The soil extraction was repeated twice,
and the combined supernatants were centrifuged at 15,000 3 g for 20 min at 4°C
to collect the bacteria. The pellet was resuspended in 0.15 M NaCl–0.1 M EDTA
(pH 8.0) and used for DNA isolation by the modified Marmur method. Agarose
gel electrophoresis was used as an additional, final purification step to obtain
DNA free from humic acids.
RSGP. The concentrations of selected DNA preparations were adjusted to ca.
70 ng/ml by a fluorimetric method (27), and 2 ml of each denatured DNA
preparation was spotted on Hybond-N hybridization membrane filters. The exact
amounts of DNAs on the filter are listed in Table 1. Denatured bacteriophage l
DNA was spotted at 10, 20, 40, 60, 80, 100, 200, and 400 ng in the bottom row
of each filter. The filters were dried and baked for 10 min at 80°C in a vacuum
oven, after which the DNAs were further cross-linked to the filter by UV irradiation as described elsewhere (26, 27). For probe preparation, 100 ng of purified
chromosomal DNA (e.g., as obtained from a single standard or from a soil
sample), 0.1 ng of l DNA, 6 ml of primer extension mix containing random
hexadeoxyoligonucleotides (26), 2 ml of Klenow polymerase (2 U/ml), and 2 ml of
[a-32P]dCTP were combined in a total volume of 30 ml. Following reaction at
room temperature for at least 3 h, during which all of the label was incorporated,
the probes were boiled and then hybridized to the filters at 68°C under highly
stringent conditions (29). Following washing and drying, the dot blots were
exposed to BAS-III imaging plates, which were scanned with a Fuji BAS1000
bioimaging analyzer. Net hybridization intensities for all dots (Ix and Il) were
determined in units of photostimulable luminescence (DPSL) by subtracting a
local background. The fractions fx of all genomes were calculated from the
hybridization data as described previously (19). Relative hybridization constants
(kl/kx) were determined for all standards by hybridizing labeled, single genomic
DNAs in duplicate (19), and the average values derived for each genome (Table
1) were used for calculation of fx. The degree of cross-hybridization between the
chromosomal DNAs of all 35 standards was also derived from these experiments.
As in the previous study (19), the hybridization intensities observed for the
internal standard bacteriophage l DNA (Il) increased linearly with cl, the
amount of denatured l DNA spotted on the filter, for low concentrations only.
Il/cl values obtained for the range from 10 to 60 ng were averaged for all
calculations. The calculated fx values can be subject to systematic errors (19), but
this tends to affect all values equally. The general appearance of bar diagrams
(plots of fx against standard number) was reproducible in duplicate incubations.
Identification by 16S rDNA sequencing. A partial 16S rRNA gene sequence
was determined for all of the standards listed in Table 1. The 16S rRNA genes
were amplified by PCR with primers f8 (12) and r1406 (9), as explained elsewhere (19). The PCR products were sequenced directly with the Promega fmol
cycle-sequencing system, with EUB388 (1) or primer P76 (GCCAGC[A/C]GC
CGCGGT) targeting conserved regions of the 16S rRNA (positions 338 to 356
and 517 to 531, respectively [Escherichia coli numbering]). The best-matching
sequence in the Ribosomal Database Project (RDP) database was then identified
with the program SIMILARITY_RANK (10).
Soil incubations. Soil was obtained from either the northwestern (NW) or the
northeastern (NE) end of a soil pile constructed for a C51 bioremediation
project at an ethane pyrolysis plant. This soil had ca. 70 mg of DCPD/g and 70 mg
of BTEX/g at the start and 30 mg of DCPD/g and 0 mg of BTEX/g at the
conclusion of the bioremediation project (18). The NW side of the pile received
nutrients and bulking agents, while the NE side was an unamended control. The
soils were stored at 4°C in the dark. Soil samples (10 g) were placed in sterile
100-ml glass beakers loosely covered with aluminum foil. After the addition of 10
ml of sterile minimal salts, the beakers were placed in glass desiccators containing a saturated atmosphere of either DCPD and H2O, toluene and H2O, or H2O
only. The desiccators were incubated at room temperature in the dark for 4 to 8
weeks. The soil-medium mixture was then centrifuged for 10 min at 10,000 3 g.
The soil-cell pellet was extracted with 0.1% (wt/vol) sodium pyrophosphate
containing acid-washed PVPP for DNA isolation.
For studying DCPD degradation by specific strains, steam-sterilized soil (10 g)
and 10 ml of minimal salts were inoculated with 10 to 20 ml of a culture grown
to saturation in TY medium. Following incubation in a DCPD- and H2Osaturated atmosphere for 4 to 8 weeks and centrifugation, the soil-cell pellet was
extracted for DNA isolation. The supernatant was saturated with sodium chloride and extracted three times with a total volume of 45 ml of ethyl acetate.
Extracts were combined and concentrated in a rotary evaporator. The yield of
these extractions was in excess of 80%. For quantitative analysis of oxygenated
DCPD derivatives, the concentrated ethyl acetate extracts (ca. 0.5 ml) were dried
with a stream of nitrogen and redissolved in 0.2 ml of dichloromethane containing 20 mg of p-dichlorobenzene as the internal standard.
GC and GC-MS analysis. GC-MS and capillary GC analyses were performed
with a Hewlett Packard 5890 gas chromatograph/mass spectrometer (Hewlett
Packard 5971A mass selective detector) equipped with either a liquid-phase
DB-1701 fused-silica capillary column (30 m by 0.25 mm) for GC-MS or an OV-1
fused-methyl-silica column (15 m by 0.32 mm) for capillary GC. The injector
temperature was 220°C, and the gas chromatograph oven temperature was programmed for 2 min at 60°C and then run from 60 to 250°C at 10°C/min. The
flame ionization detector temperature was 250°C. For each run, 1 ml of concentrated sample was injected directly into the gas chromatograph/mass spectrometer. The MS spectra were compared with those published previously (17, 23, 24).
Mineralization of [14C]DCPD and [14C]toluene. Mineralization studies were
carried out essentially as described by Bazylinski et al. (4). Steam-sterilized soil
(1 g), 5 ml of mineral salts medium, 10 ml of uniformly labeled [14C]DCPD (2 ml
of 0.15 mCi/ml diluted with 8 ml of cold DCPD), and 10 ml of a culture grown to
saturation in TY medium were combined in a 20-ml ampoule, which was then
sealed. Following incubation for 4 weeks, the ampoule was connected in series to
two test tubes containing 10 ml of 0.6 M KOH each. The ampoule seal was then
broken, and 1 ml of 1 M HCl was added. [14C]CO2 was transferred to the KOH
trap for 30 min via a gentle stream of nitrogen. The contents of the test tubes
were placed in two scintillation vials, mixed with 5 ml of EcoLite scintillation
fluid (ICN), and counted with an LKB 1215 RACKBETA liquid scintillation
counter for 2 min. The values observed in control experiments without inoculum
were subtracted from the counts obtained. [14C]toluene (2 ml of 0.06 mCi/ml,
diluted with 8 ml of cold toluene) was used for toluene mineralization studies.
Nonsterilized soils without an added inoculum were also used in some experiments.
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FIG. 1. Structures of DCPD and several oxidized derivatives. (A and B)
DCPD (B) can be formed from cyclopentadiene (A) by a reversible reaction at
room temperature. Incubation at higher temperature results in further polymerization. (C to H) Structures of six mono-oxygenated DCPD derivatives. (I)
Structure of a dioxygenated DCPD derivative.
APPL. ENVIRON. MICROBIOL.
VOL. 64, 1998
RSGP OF SOIL BACTERIA
639
TABLE 1. Composition of the soil master filter
Nameb
Mediumc
Sabd
Nearest homologe
cf
kl/kxg
sh
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
LQ1
LQ5
LQ6
LQ10
LQ11
LQ14
LQ15
LQ16
LQ17
LQ19
LQ20
LQ21
LQ26
LQ27
LQ29
LQ30
LQ33
LQ34
LQ35
LQ36
Q1
Q2
Q3
Q4
Q5
Q6
Q7
Q8
Q9
Q10
Q11
Q12
Q13
Q14
Q15
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
PTYG
HDM, benzene
PTYG
PTYG
PTYG
HDM, naphthalene
HDM, styrene
TY
TY
TY
TY, anoxic
TY, anoxic
TY, anoxic
TY, anoxic
Medium C, anoxic
Medium C, anoxic
0.802
0.375
0.396
0.598
0.769
0.615
0.580
0.861
0.931
0.349
0.816
0.791
0.865
Bordetella bronchiseptica
Pseudomonas syringae
Azospirillum sp.
Sphingomonas parapaucimobilis
Bacillus macroides
Xanthomonas campestris
Bacillus pseudomegaterium
Pseudomonas syringae
Agrobacterium rubi
“Flavobacterium” lutescens
Pseudomonas syringae
Bordetella parapertussis
Bordetella parapertussis
(Bordetella sp.)i
Bordetella parapertussis
Sphingomonas yanoikuyae
NDj
Pseudomonas flavescens
Pseudomonas flavescens
Pseudomonas flavescens
Rhodococcus sp.
Bacillus cereus/thuringiensis
Nocardioides luteus
Flavobacterium ferrugineum
Pseudomonas syringae
Rhodococcus globerulus
Pseudomonas syringae
Bacillus benzoevorans
Bacillus polymyxa
Bacteroides distasonis
Bacteroides heparinolyticus
Clostridium xylanolyticum
Clostridium sp.
Desulfovibrio longus
Desulfovibrio desulfuricans
164
156
200
110
148
132
116
176
152
152
156
148
130
130
152
140
180
138
148
136
172
148
184
160
160
116
172
148
146
146
240
152
128
172
146
238
180
283
112
46
61
68
95
112
157
210
294
166
149
208
200
249
170
160
109
574
60
330
93
142
202
151
51
73
72
80
65
61
127
94
20
10
4
11
2
12
24
1
5
14
30
0
25
36
32
5
14
13
6
6
98
6
32
10
4
5
12
7
7
10
8
6
17
6
8
0.843
0.935
0.969
0.972
0.965
0.851
0.879
0.763
0.683
0.779
0.836
0.741
0.879
0.809
0.504
0.498
0.846
0.337
0.582
0.571
a
Position of denatured chromosomal DNA on master filter.
Name assigned at time of isolation.
c
Medium used for isolation. Standards 1 to 29 were isolated under aerobic conditions.
d
Similarity coefficient for query and matching sequences (10).
e
Nearest homolog in the RDP database as determined by the program SIMILARITY_RANK (10).
f
Amount of denatured chromosomal DNA (ng) spotted on the filter.
g
Ratio of hybridization constants for bacteriophage l and genomic DNA (x) from equation 2 (19).
h
Average deviation between duplicate measurements of kl/kx.
i
Inferred from cross-hybridization with other Bordetella sp. genomes on the filter.
j
ND, not determined.
b
RESULTS
Characterization of isolated bacterial standards. Bacteria
were isolated from contaminated soil with the media indicated
in Table 1. A minimal set was obtained by eliminating species
with strong genomic cross-hybridization in dot blots. The 35
selected standards are listed in Table 1 in the order in which
they were spotted on the master filter. Partial sequencing of
PCR-amplified rRNA genes and comparison of the sequences
obtained with those in the RDP database allowed identification of 33 standards (Table 1). Identifications with low values
for the similarity coefficient Sab (10) are unlikely to be significant beyond the genus level. The genus Pseudomonas was
most prevalent among the aerobes. Five standards had Pseudomonas syringae and three had Pseudomonas flavescens as the
closest RDP homolog (Table 1). The genera Bacillus and Bordetella were also well represented, with five and four standards,
respectively. Six anaerobic isolates included two Bacteroides
spp., two Clostridium spp., and two Desulfovibrio spp. (Table
1).
Two hundred master filters were made by spotting 2-ml vol-
umes of denatured genomic DNA for all 35 standards. The
amounts of DNA applied to the filters are listed in Table 1.
Seventy of these were used for duplicate hybridizations with
labeled chromosomal DNA (spiked with l) from each of the 35
represented standards. The results for five of the six P. syringae
homologs and for the five Bacillus homologs are shown in Fig.
2. These experiments allowed the cross-hybridization as well as
the ratio kl/kx to be evaluated. The average kl/kx values are
listed in Table 1. The P. syringae genomes had substantial
degrees of cross-hybridization of up to 30% (Fig. 2A, standards
2, 8, 11, 25, and 27). These also cross-hybridized at a level of 5
to 10% with three genomes that had P. flavescens as the closest
RDP homolog (Fig. 2A, standards 18, 19, and 20). Crosshybridization with standards assigned to other genera by 16S
rRNA sequencing was generally below 2%. The five Bacillus
genomes displayed lower (,5%) degrees of cross-hybridization with each other (Fig. 2B).
Cross-hybridization data for all 35 genomes are displayed in
a matrix in Fig. 3, which has been rearranged to group phylogenetically related genomes. Strong cross-hybridizations occur
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Positiona
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FIG. 2. Hybridization of soil community master filters with single genomic DNAs. The hybridization intensity (corrected for background) relative to that observed
for the genome used as a probe (100%) is plotted against standard number in the same order as in Table 1. The patterns shown are means of duplicate hybridizations.
(A) Patterns for standards 2, 8, 11, 25, and 28, which all have P. syringae as the nearest RDP homolog. (B) Patterns for standards 5, 7, 22, 28, and 29, which all have
a Bacillus sp. as the nearest RDP homolog.
640
VOL. 64, 1998
RSGP OF SOIL BACTERIA
641
only within the drawn squares, indicating a correlation between
16S rRNA-derived phylogeny and the degree of cross-hybridization. Low degrees of cross-hybridization for species within
the same genus were observed for the genera Sphingomonas,
Bacteroides, and Clostridium, in addition to the genus Bacillus
(Fig. 2). The cross-hybridization data can be used to correct
RSGP hybridization patterns of synthetic microcosms, as discussed elsewhere (19).
Extraction of DNA from soil. Soils obtained from either the
NW or the NE end of a C51-contaminated pile were incu-
FIG. 4. Agarose gel electrophoresis of community DNA extracted from soil.
Soil was incubated for 4 weeks at room temperature with 10 ml of minimal salts
medium in an atmosphere saturated with water (lane 2), water and DCPD (lane
3), or water and toluene (lane 4). Extracted DNAs were electrophoresed through
agarose. Lane 1 represents DNA extracted from soil prior to incubation; lane l
represents size markers (bacteriophage l DNA restricted with HindIII; from left
to right, 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb).
bated with DCPD or toluene, two significant components of
the C51 mixture. An agarose gel of DNAs extracted from NW
soil samples is shown in Fig. 4. Incubation in mineral salts
increased the amount of extracted DNA (Fig. 4, lanes 1 and 2)
from 0.06 to 0.12 mg/g of soil. Incubation in a DCPD or toluene
atmosphere further increased the extracted amounts of DNA
(lanes 3 and 4) from 0.25 to 0.5 mg/g. The extraction efficiency
was estimated to be 20% by measuring the amount of DNA
obtained from sterilized soil to which a known volume of a
bacterial culture was added. Importantly, if subsequently extracted DNA fractions were analyzed by RSGP, identical community profiles were obtained, indicating that these results
were not affected by the extraction yield.
Effects of toluene. The community profiles for NE and NW
soil samples incubated with minimal salts medium only were
very similar. The averaged profile in Fig. 5A shows a broad
population distribution with calculated fx values from 0.001 to
0.015. Incubation in the presence of toluene led to a significant
shift in the community profile. A limited number of standards
became enriched by an order of magnitude, as shown for the
NW soil sample in Fig. 5B. Standard 11 (Pseudomonas strain
LQ20) was the dominant community member. Standards 2, 8,
25, and 27 have the same RDP homolog as standard 11 (Table
1, P. syringae). The peaks for these standards in Fig. 5B are
therefore caused in part by cross-hybridization with the LQ20
genome, as may be seen by comparison with the hybridization
pattern for pure LQ20 in Fig. 2A. To establish whether LQ20
and the other four P. syringae homologs could metabolize toluene, the formation of [14C]CO2 from uniformly labeled
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FIG. 3. Cross-hybridization matrix. Hybridization data, as in Fig. 2, were arranged as columns in a matrix. The order of columns and corresponding rows was
changed to bring genomes with similar 16S rRNA phylogeny in close proximity. The enclosed squares represent Pseudomonas spp. (P. flavescens inside and P. syringae
outside the smaller square) (A), Bordetella spp. (B), Sphingomonas spp. (C), various (D), Rhodococcus spp. (E), various (element Q3/Q4 is anomalously high) (F),
Bacillus spp. (G), Bacteroides spp. (H), Clostridium spp. (I), and Desulfovibrio spp. (J).
642
SHEN ET AL.
APPL. ENVIRON. MICROBIOL.
[14C]toluene was investigated. When [14C]toluene was incubated with the NW soil sample and minimal salts for 4 weeks,
4 to 10% of the label was recovered from the alkali traps as
[14C]CO2. The mineralization activity of individual standards,
inoculated into sterilized soil and minimal salts medium containing [14C]toluene, relative to the soil consortium is indicated
in Fig. 5C. Pseudomonas strain LQ20 was the most active of
five standards tested (LQ5, LQ16, LQ20, Q5, and Q7).
Effects of DCPD. Exposure to DCPD also led to significant
changes in the community profile, which were distinctly different from those observed for toluene. Standard 16 (LQ30, with
Sphingomonas yanoikuyae as the nearest RDP homolog) was
the most abundant after 4 weeks of incubation of both NW and
NE soil samples (Fig. 5D and E). Continued liquid culture
incubations, obtained by inoculating the supernatant of the
NW soil-minimal salts medium culture into minimal salts medium and incubating it in a DCPD atmosphere, gave limited
growth. The absorbance at 600 nm of the culture typically
increased from 0.02 to 0.08 over a 2-week period and declined
subsequently. The cultures showed a different RSGP profile in
which standard 15 (Pseudomonas strain Q5) was dominant
(hybridization pattern similar to that shown for Pseudomonas
strain Q5 in Fig. 2A). Plating of these cultures on TY medium
gave colonies with uniform morphology. RSGP testing of two
of these indicated both to be standard 15.
Inocula of LQ30, Q5, or LQ30 plus Q5 were added to sterilized soil (10 g) and minimal salts medium (10 ml) and incubated in a DCPD atmosphere for 7 weeks. A sample of nonsterilized soil was similarly incubated. GC patterns of ethyl
acetate-extracted organics were similar for all five incubations.
Those for the nonsterilized and sterilized soil incubations are
shown in Fig. 6A and B, respectively. More oxidized DCPD
derivatives were formed in the nonsterile soil incubation (Table 2). Comparison with known MS spectra for oxygenated
DCPD derivatives (17, 23, 24) allowed the tentative identification of three mono-oxygenated derivatives (Fig. 1C through
E) and one dioxygenated derivative (Fig. 1I). The addition of
LQ30, Q5, or LQ30-plus-Q5 inocula also appeared to result in
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FIG. 5. RSGP of community DNAs from soils at the NE or NW side of a contaminated soil pile. (A) Soils were incubated with minimal salts medium only. The
pattern shown is an average for NE and NW soils. (B) NW soil sample incubated with minimal salts medium in a toluene atmosphere. (C) Percent toluene
mineralization by individual species relative to mineralization by the NW soil community. Data are plotted for Pseudomonas sp. standards 2, 8, 11, 25, and 27. (D and
E) NW and NE soils, respectively, were incubated with minimal salts medium in a DCPD atmosphere. The fraction of each standard (fx) is plotted against standard
number in panels A, B, D, and E.
VOL. 64, 1998
RSGP OF SOIL BACTERIA
larger yields of oxygenated DCPD derivatives (Table 2). Extraction of DNA from these incubations and RSGP assays of
the extracted DNAs confirmed that the inoculated bacteria
were present (Fig. 7). However, mineralization experiments in
which sterilized soil, minimal salts medium, uniformly labeled
[14C]DCPD, and inocula of LQ30, Q5, or LQ30 plus Q5 were
combined gave negligible amounts of [14C]CO2 in the alkali
traps after 4 weeks of incubation (0.1 to 0.5% of added label).
DISCUSSION
Bioremediation often involves the degradation of complex
mixtures of contaminants by undefined mixed populations of
microorganisms (16). The degradation of C51 by a soil microbial community certainly falls into this category. The changes
TABLE 2. Extraction of oxidized DCPD derivatives from soil
minimal salts medium incubations
NW soila
Inoculumb
Amt (mg) of oxidized
derivativesc
Sterile
Sterile
Sterile
Sterile
Nonsterile
None
Q5
LQ30
Q5 1 LQ30
None
37
103
58
88
162
a
Sterilized or nonsterilized NW soil (10 g) was combined with 10 ml of
minimal salts medium and incubated in a DCPD atmosphere for 7 weeks.
b
Saturated cultures of Q5 (10 ml), LQ30 (20 ml), or both (10 and 20 ml) were
added.
c
Peak areas of oxidized DCPD derivatives (masses indicated in Fig. 6) were
combined and converted into micrograms by comparison with the internal standard, p-dichlorobenzene. The results of duplicate experiments agreed within
10%.
in community composition upon introduction of a single or
mixed hydrocarbon substrate have been characterized by plating and by the use of probes for hydrocarbon degradation
genes. Sayler et al. (14) used the TOL and NAH plasmids to
track the fraction of the population capable of growing with
either toluene or naphthalene as the sole carbon and energy
source. It was found that only a fraction of the colonies appearing on selective media reacted with these probes. In the
case of toluene degradation, the unreactive bacteria may have
degraded toluene through a pathway other than TOL, e.g., by
the dioxygenase-initiated pathway catalyzed by Pseudomonas
putida F1. In the case of naphthalene degradation, genes with
no homology to NAH have recently been described (7). Thus,
even the analysis of a single degradative function may require
the application of multiple probes. Greer et al. (8) have therefore proposed a biotreatability protocol, which involves the use
of multiple probes to assess the presence of hydrocarbondegrading bacteria as well as physical and chemical analysis of
the contaminated soil and assessment of pollutant mineralization and respiratory capacity of the resident community.
We have shown here that the effects of the introduction of
hydrocarbon or xenobiotic compounds on a soil microbial community can also be monitored by RSGP, a technique used
previously to characterize the dynamics of microbial communities in oil fields (19, 25–27). Whole genome probes can easily
distinguish species from different genera (Fig. 3) and can, to a
degree, distinguish species within the same genus (Fig. 2 and
3). The RSGP format allows rapid tracking of the abundance
of multiple microbial genomes. The present collection of 35
genomes is modest relative to the microbial diversity that is
thought be present in soil environments. Torsvik et al. (21)
have estimated from analysis of Cot curves that soil communities contain 103 to 104 genome equivalents. If this number
also applies to the community present in pyrolysis plant soils,
the master filters created in this study cover only a small fraction of the resident community and are clearly not representative. The experimental data obtained in this study on the
effect of toluene suggest that the actual situation in our target
environment may be more favorable. Most of the species represented on the filter were cultured on rich media (Table 1).
Only three were isolated in minimal media with a hydrocarbon
as the sole source of carbon and energy, and toluene was not
used in these isolations. However, when the soil community
was exposed to toluene in the presence of minimal salts medium, one of the standard genomes on the filter was estimated
to represent 40% of the extracted community DNA (Fig. 5B).
Standard 11 (Pseudomonas strain LQ20), harboring this genome, was originally isolated on rich medium but was subsequently shown to be indeed capable of mineralizing toluene to
a similar extent to the entire soil community (Fig. 5C).
The toxicity of individual unsaturated hydrocarbons to microbial cells (15) and the large number of components that can
be present may preclude the isolation of all specific degraders.
However, assuming that many of these can also be cultured on
regular plating media, a general strategy to identify these species could be to (i) isolate an extensive set on rich plating
media, (ii) generate a master filter of genomes with limited
cross-hybridization, and (iii) identify possible degraders of specific components by determining the response of the community to introduction of the chemical. This approach appears to
work in the case of toluene degradation by the community
present in C51-contaminated soil (Fig. 5B), but identification
of degraders of the extremely recalcitrant petrochemical
DCPD is more difficult.
Evidence for a role of microorganisms in the conversion of
DCPD to oxidized derivatives was obtained by Stehmeier et al.
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FIG. 6. GC patterns of organic compounds extracted from soil incubated
with minimal salts medium in a DCPD atmosphere. Incubation was carried out
with nonsterilized soil (A) and sterilized soil (B). The masses of peaks corresponding to oxidized DCPD derivatives are indicated.
643
644
SHEN ET AL.
APPL. ENVIRON. MICROBIOL.
(17), who showed that this conversion is absent in sterilized
media. van Breemen and Tsou (24) suggested that oxidized
DCPD derivatives are formed by nonmicrobial, especially photochemical, mechanisms, although the existence of specialized
microorganisms with DCPD-oxidizing ability in soil was not
ruled out by these experiments.
Our data support a role for microorganisms in the generation of oxidized DCPD derivatives (Fig. 6; Table 2). However,
even in the absence of light and microbes, some oxidized derivatives appeared (Table 2), perhaps because of the different
mode of delivery of the chemical (through an aerobic, saturated vapor phase) compared to earlier studies (adsorbed to
charcoal [17, 24]). Sphingomonas strain LQ30 was implicated
in DCPD oxidation by RSGP assays (Fig. 5D and E). The
calculated fractions of the total population of this organism
(0.04 to 0.08) are much lower than in the case of enrichment of
Pseudomonas strain LQ20 by toluene (0.40), indicating that
none of the microorganisms currently represented on the filter
can derive significant energy from DCPD oxidation. These
results confirm the recalcitrant nature of this chemical and
suggest that in the soil environment DCPD oxidation may
result solely from cometabolic reactions. Cometabolic degradation of DCPD will be explored in future studies by determining the change in the community composition by RSGP
upon exposure to mixtures of DCPD and degradable BTEX
compounds. The possibility that Pseudomonas strain Q5, which
can use naphthalene as the sole source of carbon and energy
(Table 1) and which was identified by RSGP as the dominant
component of continued liquid culture incubations in the presence of DCPD, degrades DCPD cometabolically when growing
on naphthalene will also be investigated.
ACKNOWLEDGMENTS
This work was supported by a Strategic Grant from the Natural
Science and Engineering Research Council of Canada to G.V. Partial
salary support for L.G.S. was provided by Novacor Research and
Technology Corp., Calgary, Canada.
We thank F. Sun for help in the acquisition of GC-MS spectra.
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