Changes to the structure of Sphingomonas spp. communities
associated with biodegradation of the herbicide isoproturon in soil
Shengjing Shi & Gary D. Bending
Warwick HRI, University of Warwick, Wellesbourne, Warwick, UK
Correspondence: Gary Bending, Warwick
HRI, University of Warwick, Wellesbourne,
Warwick CV35 9EF, UK. Tel.: 144 0 24
76575057; fax: 144 0 24 76574500;
e-mail: gary.bending@warwick.ac.uk
Received 25 September 2006; revised 4
December 2006; accepted 11 December 2006.
First published online 15 January 2007.
DOI:10.1111/j.1574-6968.2006.00621.x
Editor: Clive Edwards
Keywords
biodegradation; isoproturon; pH;
Sphingomonas spp.; diversity.
Abstract
The phenyl-urea herbicide isoproturon is a major contaminant of surface and
ground-water in agricultural catchments. Earlier work suggested that within-field
spatial variation of isoproturon degradation rate resulted from interactions
between catabolizing Sphingomonas spp. and pH. In the current study, changes to
the structure of Sphingomonas communities during isoproturon catabolism were
investigated using Sphingomonas-specific 16S rRNA gene primers. Growth-linked
catabolism at high-pH (4 7.5) sites was associated with the appearance of multiple
new denaturing gradient gel electrophoresis (DGGE) bands. At low-pH sites, there
was no change in DGGE banding at sites in which there was cometabolism, but at
sites in which there was growth-linked catabolism, degradation was associated with
the appearance of a new band not present at high pH sites. Sequencing of DGGE
bands indicated that a strain related to Sphingomonas mali proliferated at low pH
sites, while strain Sphingomonas sp. SRS2, a catabolic strain identified in earlier
work, together with several further Sphingomonas spp., proliferated at high-pH
sites. The data indicate that degradation was associated with complex changes to
the structure of Sphingomonas spp. communities, the precise nature of which was
spatially variable.
Introduction
Biodegradation is the principal process controlling pesticide
dissipation, and thereby pesticide persistence and susceptibility to leach from soil and contaminate surface and
ground water (Aislabie & Lloyd-Jones, 1995). Pesticide
biodegradation rates can show considerable within-field
spatial variability, with implications for patterns of leaching
losses (Walker et al., 2001a; Rodriguez-Cruz et al., 2006).
Spatial variation in pesticide biodegradation rates can
sometimes be correlated to variation in gross soil properties;
however, the mechanisms underlying such variability are
poorly understood (Walker et al., 2001a).
The phenyl-urea herbicides are particularly susceptible to
within-field spatial variability of biodegradation rates (e.g.
Beck et al., 1996; Cullington & Walker, 1999; Walker et al.,
2001a, b). Phenyl-urea compounds, including isoproturon
and diuron, are among the most widely used pesticides in
Europe, and their slow degradation and moderate motility
in soil results in these compounds being found widely as
contaminants of agricultural catchments (Srensen et al.,
2003).
At one site, Deep-Slade field in Warwickshire, UK, spatial
variability in isoproturon degradation rates has been shown
2007 Federation of European Microbiological Societies
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c
to arise from localization in the areas in which organisms
had adapted to use the compound as an energy source,
which were interspersed with areas in which degradation
showed first-order degradation. Such degradation kinetics
generally indicate that catabolic organisms do not proliferate (Walker et al., 2001a), and has been termed cometabolism, although this could actually reflect very slow
growth-linked metabolism masked as first-order degradation. Rapid degradation of isoproturon was associated with
high-pH areas, whereas areas with lower pH showed slow
degradation (Bending et al., 2001). Furthermore, eubacterial
community denaturing gradient gel electrophoresis (DGGE)
analysis showed proliferation of two Sphingomonas bands
during isoproturon degradation in rapid-degrading areas
(Bending et al., 2003). As the DGGE analysis used in the
Deep-Slade study profiled dominant eubacterial community
members only, it was not clear whether isoproturon degradation was associated with a wider group of sphingomonad
strains, as has previously been shown for polychlorophenol
degradation in contaminated groundwater (Tiirola et al.,
2002).
In slow-degrading sites within Deep-Slade, either isoproturon-degrading organisms proliferated slowly, or there was
no growth of catabolic organisms (Bending et al., 2001).
FEMS Microbiol Lett 269 (2007) 110–116
111
Changes to the structure of Sphingomonas spp.
DGGE analysis at slow-degrading sites showed no change in
banding (Bending et al., 2003), so it was not clear whether
degradation reflected slow growth of the same organisms
responsible for isoproturon degradation at fast-degrading
sites, or whether other strains were responsible.
Traditionally, enrichment techniques have been used to
isolate and identify organisms responsible for the degradation of pesticides and other xenobiotics in the environment
(Aislabie & Lloyd-Jones, 1995). However, degradation of
xenobiotics is commonly associated with mobile plasmids
that can be transferred between strains (Van der Meer et al.,
1992), particularly under the selective pressures associated
with enrichment (Newby et al., 2000). The extent to which
catabolic strains obtained by enrichment techniques accurately reflect in situ catabolic communities is therefore
unclear. In the Deep-Slade field studies, two different
Sphingomonas isolates were obtained from rapid-degrading
soil by enrichment (Srensen et al., 2001; Bending et al.,
2003). DGGE analysis suggested that one of these isolates
(SRS2) proliferated during isoproturon degradation (Bending et al., 2003), but it was unclear what role the other strain
(F35) played in degradation in situ.
The aim of the current study was to resolve questions
relating to the role and spatial diversity of Sphingomonas
communities associated with isoproturon catabolism, which
were raised in earlier studies in Deep-Slade field. Using
Sphingomonas selective rRNA gene primers, DGGE and
quantitative PCR were used to investigate changes in the
Sphingomonas community during isoproturon degradation
in fast- and slow-degrading areas of Deep-Slade field.
and degraded isoproturon rapidly, while transect 2 soil had
pH of 5.6–6.1.
Isoproturon addition and analysis
Description of the procedure for isoproturon addition and
characterization are given in Bending et al. (2003). Briefly,
an aqueous suspension of commercial formulation of isoproturon was added to soils to give 15 mg isoproturon kg 1
soil, and
33 kPa. Control samples were set up using
distilled H2O instead of formulation. Over 65 days, isoproturon residues were extracted from subsamples of soil using
acetonitrile, and concentrations measured by HPLC, as
described in Bending et al. (2006).
Using data presented in Bending et al. (2003), the
Gompertz model provided the best fit to the degradation
data and was used to derive time to 50% degradation
(DT50) and the length of the lag phase prior to exponential
degradation. Calculations were performed using GenStat
(7th edition, VSN International Ltd).
DNA extraction, PCR and DGGE analysis
At 90% (DT90) degradation of isoproturon, and after 9
months, DNA was extracted from control and isoproturontreated soil according to Cullen & Hirsch (1998). Sphingomonas community 16S rRNA genes were amplified using the
primers Sphingo108f/GC40-Sphingo420r (Leys et al., 2004).
PCR equipment and reagents were as described in Bending
et al. (2003).
Sphingomonas spp. 16S rRNA gene PCR products were
analysed by DGGE according to Muyzer et al. (1993), using a
denaturant gradient ranging from 20% to 60% [100%
denaturant contains 7 M urea with 40% (v/v) formamide],
a constant voltage of 70 V for 18 h and Ingeny PhorU
equipment (Amsterdam). Gel visualization and the cutting
and amplification of DNA from bands were as described in
Bending et al. (2003). Reamplified bands were run against
the original sample to check motility and purity. Where
bands were pure, they were sequenced directly. In the case of
a band cut from transect 2 (L1), the band was not pure, and
the PCR reaction was cloned using a pCR2.1-TOPO cloning
kit (Invitrogen Ltd, Paisley, UK) using chemically competent TOP 10 Escherichia coli (Invitrogen). Sphingomonas
spp. 16S rRNA gene was amplified from clones, and
Materials and methods
Soil samples
The soil samples described in Bending et al. (2003) were
used in this study. Briefly, samples of soil were taken from
two transects within Deep-Slade field, on the farm at
Warwick HRI, Wellesbourne, Warwickshire, UK. The soil is
a sandy-loam of the Wick series (Whitfield, 1974). The field
had received regular applications of isoproturon for 20 years
before the study. Soil samples were collected from five sites
at 20-m intervals (labelled B, C, D, E, F) along two transects
separated by 50 m. Transect 1 soil had pH of 7.1–7.4 (Table 1)
Table 1. pH and isoproturon degradation characteristics [calculated from data presented in Bending et al. (2003)] in soil from transects 1 and 2 across
Deep-Slade field
Transect 1
Transect 2
Site
B
C
D
E
F
B
C
D
E
F
pH
Lag phase (days)
DT50 (days)
7.4
5.0
6.1
7.4
5.6
6.7
7.3
5.2
6.2
7.2
4.9
6.1
7.1
5.2
6.2
6.0
4 65
25.7
5.6
4 65
25.1
5.9
11.1
18.0
6.1
6.3
8.4
5.8
38.8
21.3
FEMS Microbiol Lett 269 (2007) 110–116
2007 Federation of European Microbiological Societies
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c
112
migration was tested against the original sample using
DGGE. Clones containing the correct insert DNA were
sequenced using M13 forward and reverse primers (Invitrogen), while Sphingo108f/GC40Sphingo420r were used in
sequencing reactions directly from DGGE bands. The
PRISM BigDye Terminator Cycle Sequence reaction kit
(Applied Biosystems, Warrington, UK) was used for sequencing with products analysed on an Applied Biosystems 377
DNA sequencer.
Cloning and sequencing
Sphingo108f/GC40-Sphingo420r PCR products were generated using DNA from isoproturon-treated and untreated
samples of soil B from transect 1, at 90% isoproturon
degradation. The products were cloned and sequenced using
the methods described above. A total of 71 and 37 clones
were sequenced for the isoproturon-treated and control
samples, respectively.
The sequence data were edited and assembled using the
DNASTAR II sequence analysis package (Lasergene Inc., Wisconsin), and the sequences were compared with those on the
EMBL nucleotide database using the program BLAST. Using
the environmental sequences, and reference sequences from
the EMBL database, phylogenetic trees were constructed
using the PHYLIP (version 3.5c) packages SEQBOOT, DNADIST and
NEIGHBOR. The dendrogram was generated using neighbourjoining analysis, and the results were viewed using DRAWTREE. Clone sequences have been deposited in the EMBL
nucleotide accession database under the accession numbers
AM412676–AM412706.
Real-time PCR
Real-time PCR was performed on an ABI PRISMs 7900HT
Sequence Detection System (Applied Biosystems) using a
Platinums SYBRs Green qPCR SuperMix UDG Kit (Invitrogen). Amplification of Sphingomonas spp. 16S rRNA
gene was carried out using Sphingo108f/GC40Sphingo420r
and general eubacterial (Muyzer et al., 1993) primers. PCR
reactions contained 2 mL DNA template, 1 Platinums
SYBRs Green qPCR SuperMix-UDG, 0.4 mL ROX reference
Dye, 4.0 mM MgCl2 and 300 nM Sphingo420r primer or
200 nM eubacterial primer. The Sphingomonas spp. reaction
conditions were 2 min at 50 1C, 10 min at 95 1C, followed by
50 cycles of 95 1C for 15 s, 62 1C for 30 s and 74 1C for 30 s.
For eubacteria, the programme was 2 min at 50 1C, 10 min at
95 1C, followed by 50 cycles 92 1C for 45 s and 55 1C for 30 s,
68 1C for 45 s.
DNA from Sphingomonas strain SRS2 (Srensen et al.,
2001) was used to prepare standard curves. Reactions were
duplicated. Real-time PCR data were analysed by SDS 2.1
application software (Applied Biosystems). Copy numbers
of Sphingomonas spp. and eubacterial 16S rRNA genes were
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
S. Shi & G.D. Bending
calculated according to Park & Crowley (2005). The size and
specificity of the PCR product were confirmed using a 1%
agarose gel stained with ethidium bromide.
Results
Isoproturon degradation
Samples from transect 1 had a lag phase of 4.9 to 5.6 days
before exponential decay (Table 1). In transect 2, for two
samples (B and C), there was no phase of exponential decay.
For the remaining three samples, the lag phase ranged from
6.3 to 38.8 days. Time to 50% degradation (DT50) varied
between 6.1 and 6.7 days in transect 1, and between 8.4 and
25.7 days in transect 2.
DGGE analysis
Strain Sphingomonas SRS2 gave a single dominant DGGE
band, together with a number of faint minor bands (Fig. 1).
At DT90, new bands appeared in isoproturon-treated samples from transect 1, which were not present in control soils.
The major band that appeared matched migration of the
dominant band seen in SRS2 (S6), and the faint bands seen
in SRS2 also appeared (S1-5, S7). Three further bands (H1,
H2 and H3) appeared in all five samples, while H4 appeared
in samples C, D and E only, and band H5 and H6 appeared
only in samples C and F, respectively. None of these bands
matched banding of isolate F35 or SRS2.
At DT90 in transect 2, treatment with isoproturon had
induced no effect on banding in samples B and C. In
samples D and F, one new band (L1) appeared on treatment
with isoproturon, while the same band plus one matching
the position of the major band generated by SRS2 (L3),
together with one of the faint bands produced by SRS2 (L2),
appeared in sample E. After 9 months, the differences in
banding between isoproturon-treated and untreated samples in transect 1, but not transect 2, were still evident (data
not shown).
Phylogenetic analysis of 16S rRNA gene from
clones, isolates and DGGE bands
Of the 108 clones sequenced, 97 showed homology with
Sphingomonas spp., with the remainder showing the closest
homology to related Alphaproteobacteria, including Stella
sp., Skermanella sp. and Azospirillium sp. The clone libraries
generated 31 unique Sphingomonas spp. sequences. All
Sphingomonas spp. clones showed 95–100% similarity to
Sphingomonas sp. 16S rRNA gene sequences from the EMBL
database, with clones showing homology with Sphingomonas sensu stricto, Sphingobium, Novosphingobium and Sphingopyxis (Takeuchi et al., 2001). Relative to control soil,
isoproturon-treated soil showed increased numbers of
FEMS Microbiol Lett 269 (2007) 110–116
113
Changes to the structure of Sphingomonas spp.
(a) Transect 1
M 1
2
3
4
5
6
7
8
9
(b) Transect 2
1 2
3 4
10
5
6
7
8
9
10 M
H1
H2
H3
H4
1
2
3
4
5
6
7
]
L1
L2
L3
S
H5
H6
Fig. 1. Analysis of Sphingomonas 16S rRNA genes by DGGE at the point of 90% isoproturon degradation. Lanes: M, Sphingomonas sp. SRS2; 1–5,
control unamended soil; 6–10, isoproturon-treated soil. Bands appearing in isoproturon-treated soil are indicated. S1-7 reflect bands generated by
isolate Sphingomonas sp. SRS2. Samples in lanes 1–5 and 6–10 correspond to sites B to F in transects 1 and 2. (a) Transect 1. (b) Transect 2.
sequences related to Sphingomonas strain SRS2, which
represented 27% of the clone library in isoproturon-treated
soil, compared with 5% in the control soil library (Fig. 2).
DGGE band S6, which was the dominant band appearing
in all samples from transect 1 at DT90, showed 100%
similarity to Sphingomonas SRS2 (Table 2). DGGE band H4
showed 96% homology to Sphingomonas D12 (accession
number AB015809), and DGGE band H3 showed 99%
homology to Sphingomonas CFDS-1 (accession number
AY702969). DGGE band L1, which appeared in transect 2
only, showed 98% homology to Sphingomonas mali (accession number SM16SR).
Real-time PCR
The PCR efficiencies were 100% (R2 = 0.995) and 97%
(R2 = 0.985) for the Sphingomonas and eubacterial primer
sets, respectively. The Sphingomonas spp. 16S rRNA gene
copy numbers in the samples ranged from 6.41 109 to
2.30 1010 g 1 soil while the total eubacterial 16S rRNA
gene copy numbers ranged from 6.51 1012 to
1.47 1013 g 1 soil. The percentage of Sphingomonas spp.
within the bacterial community did not change significantly
during isoproturon degradation (data not shown).
FEMS Microbiol Lett 269 (2007) 110–116
Discussion
While some studies using general 16S rRNA gene primers
have shown a change in the structure of bacterial communities following the application of herbicides or other
pesticides to soil, others have reported no change (Engelen
et al., 1998; Sigler & Turco, 2002). The data in this study in
combination with that of an earlier study (Bending et al.,
2003), demonstrate that when group-specific primers are
used to provide a finer scale of resolution, the complexity of
changes to community structure can be far greater than
those indicated using general primers.
Strains of Sphingomonas spp. capable of degrading a wide
range of xenobiotics have been isolated, suggesting that this
genus is extremely effective at adapting to degrade new
compounds (Basta et al., 2004). The DGGE data show that
growth-linked degradation of isoproturon was associated
with proliferation of a variety of Sphingomonas spp. Strain
Sphingomonas SRS2, which was isolated from Deep-Slade
field using enrichment methods (Srensen et al., 2001),
clearly had a role in degradation in situ. However, strain
Sphingomonas F35, which was isolated from the isoproturon-amended soil described in this paper (Bending et al.,
2003), did not appear to proliferate in soil during isoproturon catabolism. This could suggest that the enrichment
2007 Federation of European Microbiological Societies
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114
S. Shi & G.D. Bending
Sphingomonas sp. MT1 (SSP303009)
C47(AM412680)
IPU102 (AM412704)
IPU49 (AM412694)
Sphingomonas sp. MN 18.3a (SPH555475)
IPU70 (AM412702)
Sphingomonas mali (SM16SR)
Sphingomonas koreensis (AF131296)
Sphingomonas sp. HPC838 (AY997008)
Sphingomonas sp. SIA181-1A1 (AF395032)
Sphingomonas sp. SAFR-028 (AY167833)
IPU34 (AM412698)
C28 (AM412676) (4)
IPU69 (AM412696)
IPU5 (AM412683) (12), C86 (9)
C7 (AM412678)
Sphingomonas sp. IW3 (AB076396)
IPU43 (AM412700)
Novosphingobium rhizogenes (D13945)
Sphingomonas sp. AV6C (AF434172)
Sphingomonas sp. MBIC1965 (AB025720)
IPU104 (AM412679)
C84 (AM412682) (3)
IPU47 (AM412693)
Sphingomonas adhaesiva (X72720)
IPU29 (AM412689)
IPU10 (AM412685) (2), C19 (2)
Sphingomonas sp. KIN163 (AY136093)
Sphingomonas aquatilis (AF13129)
Sphingomonas sp. JSS-7 (AY131295)
C79 (AM412681)
Sphingomonas sp. SRS2 (SSP251638)
IPU55 (AM412695) (19), C2 (2)
Sphingomonas wittichii DSM 6014 (AB02149)
Sphingomonas sanguinis (D13726)
Sphingomonas trueperi (X97776)
Novosphingobium sp. B.E4-1 (AB164692)
IPU35 (AM412699)
IPU6 (AM412684)
Novosphingomonas tardaugens (AB070237)
Sphingomonas sp. SKA59 (AY926494)
Sphingomonas sp. KIN150 (AY136092)
IPU50 (AM412701)
Sphingomonas sp. HTCC503 (AY584572)
IPU32 (AM412697)
Sphingomonas sp. KT-1 (AB022601)
IPU49a (AM412706)
Sphingopyxis terrae (D13727)
Sphingomonas sp. Ellin426 (AF432250)
IPU88 (AM412703)
IPU14 (AM412687), C76
IPU15 (AM412688)
Sphingomonas sp. S-2 (AY081166)
IPU28 (AM412705)
C5 (AM412677) (2)
Novosphingobium stygium (U20775)
Sphingomonas sp. D12 (AB105809)
Sphingomonas sp. AC83 (AJ717392)
IPU13 AM412686) (3)
Sphingobium yanoikuyae (D16145)
IPU38 (AM412691)
Sphingobium herbicidovorans FA3g (AY544996)
Sphingomonas sp. CFDS-1 (AY702969)
IPU44 (AM412692) (4), C25 (2)
IPU31 (AM412690) (6), C12 (4)
Sphingomonas sp. CFO6 (U52146)
0.01
Fig. 2. Distance tree constructed with partial (303 bp) 16S rRNA gene sequences, showing relationships of clones from isoproturon-treated and
untreated (C) soil with members of the genus Sphingomonas spp. Figures in italics show the number of clones matching the sequence with 4 98%
homology. Scale bar represents the number of changes per nucleotide position.
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FEMS Microbiol Lett 269 (2007) 110–116
115
Changes to the structure of Sphingomonas spp.
Table 2. Closest match of DNA sequenced from excised DGGE bands to sequences from the EMBL database
Band
EMBL accession number
Closest match and accession number
Similarity (%)
H3
H4
S6
L1
AM412812
AM412813
Sphingomonas sp. CFDS-1 AY702969
Sphingomonas sp. D12 AB105809
Sphingomonas sp. SRS2 SSP251638
Sphingomonas mali SM16SR
99
96
100
98
AM412814
process facilitated degradative gene transfer from pesticide
degraders to a background organism that was not involved
in degradation in situ (Newby et al., 2000).
The role of the other Sphingomonas spp. that appeared
during degradation of isoproturon at high-pH sites is
uncertain. Horizontal gene transfer can occur readily between Sphingomonas spp. (Basta et al., 2004). Tiirola et al.
(2002) showed evidence that natural horizontal transfer of
the pcpB gene between sphingomonads facilitated the evolution of polychlorophenol-degrading sphingomonads within
a contaminated groundwater. Given the potential for horizontal gene transfer between Sphingomonas spp., gene
transfer from SRS2 could provide a mechanism to explain
the proliferation of other Sphingomonas spp. during isoproturon degradation. However, it is possible that the other
strains could have proliferated via indirect mechanisms,
such as through altered competitive interactions resulting
from the activities of isoproturon-catabolizing communities. Further work using stable isotope probing (Mahmood et al., 2005) approaches would be required to provide
unequivocal evidence for the contribution of such Sphingomonas spp. strains to biodegradation.
There was spatial diversity in the response of the Sphingomonas spp. community during isoproturon degradation.
Within transect 1, several bands did not appear at all sites,
although this was not related to differences in degradation
rate or gross soil characteristics. In transect 2, SRS2 proliferated at only one of the sites. At sites in which SRS2 did not
proliferate, growth-linked degradation was associated with
the appearance of a strain related to S. mali, which did not
occur in transect 1. In sites B and C in transect 2, degradation followed first-order kinetics, suggesting that there had
been no proliferation of degraders. In these sites, there was
no change in the Sphingomonas spp. community DGGE
profile, providing some evidence that those strains which
did appear on the DGGE profile at locations at that there
was growth-linked metabolism could have been involved in
isoproturon degradation.
Most studies in which sphingomonad strains capable
of xenobiotic degradation have been identified have used
enrichment procedures from environmental samples
collected from a single sampling location (e.g. Schmidt
et al., 1992; Wittich et al., 1992; Tanghe et al., 1999). Clearly,
sphingomonad communities acting in situ may be
diverse. Single strains isolated using enrichment techniques
FEMS Microbiol Lett 269 (2007) 110–116
may not provide accurate models with which to understand degradation processes in the environment. The
presence of diverse communities of phylogentically
related catabolic strains, together with strict nutritional or
environmental requirements, such as pH, could also help to
explain why sphingomonad strains shown to degrade
xenobiotics in the laboratory may not show degradative
abilities when inoculated into environmental samples,
including systems from which they were isolated (Shi et al.,
2001).
Although the Sphingomonas spp. community profile
changed during isoproturon degradation, there was no
change in the number of Sphingomonas spp. or the proportion of Sphingomonas spp. within the bacterial community.
This could indicate that the change in Sphingomonas spp.
was small relative to the overall size of the Sphingomonas
spp. community. As all sequenced bands that appeared in
the DGGE profiles during isoproturon degradation belonged to Sphingomonas sensu stricto (Takeuchi et al.,
2001), the use of primers specific to this group may have
provided more useful data on population dynamics.
Sphingomonas sp. strains F35 and SRS2 were found to
yield multiple DGGE bands. Similarly, Leys et al. (2004)
showed that a number of pure Sphingomonas strains give
multiple banding patterns using the 16S rRNA gene primers
used in the current study. In prokaryotes, possession of
more than one 16S rRNA gene copies with sequence
divergence in the genome is a common phenomenon
(Klappenbach et al., 2001).
To conclude, strain SRS2 was required for rapid isoproturon catabolism, SRS2 was not involved in isoproturon
degradation at most low-pH sites, strain F35 had no
involvement in isoproturon degradation at high- or lowpH locations and isoproturon degradation was associated
with complex changes to Sphingomonas spp. community
structure that varied according to soil pH and whether
degradation occurred by growth-linked or cometabolic
processes.
Acknowledgements
The authors thank the Department for Environment, Food
and Rural Affairs and the Biotechnology and Biological
Sciences Research Council for funding.
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c
116
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