Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol33.Issue1.Jan2001:
Soil Biology & Biochemistry 33 (2001) 75±81
www.elsevier.com/locate/soilbio
Dynamics of carbofuran-degrading microbial communities in soil during
three successive annual applications of carbofuran
S.L. Trabue, A.V. Ogram, L.-T. Ou*
Soil and Water Science Department, P.O. Box 110290, University of Florida, Gainesville, FL 32611-0290, USA
Received 4 October 1999; received in revised form 3 May 2000; accepted 29 May 2000
Abstract
The in¯uence of repeated annual ®eld applications of the insecticide±nematicide carbofuran on carbofuran-degrading microbial communities was studied at a site in Florida that exhibited enhanced degradation toward the chemical. Three successive annual applications of
carbofuran at 4.5 kg ha 21 y 21 did not result in an increase in the size of the microbial community capable of mineralizing uniformly ringlabeled 14C-carbofuran (carbofuran-ring degraders) in surface soil (0±15 cm depth). After the second annual application, however, the
community capable of mineralizing carbonyl-labeled 14C-carbofuran (carbofuran hydrolyzers) in the treated surface soil was signi®cantly
larger than that after the ®rst annual application. Communities of methylamine utilizers in treated and untreated soils were much larger than
the communities of carbofuran phenol degraders, but not statistically different from the sizes of carbofuran hydrolyzers. In addition to no
increase in the number of carbofuran ring degraders in the treated site during three consecutive annual applications of carbofuran, laboratory
addition of 10 mg carbofuran g 21 to treated and untreated soils collected in 1995 did not result in an increase in the number of carbofuran ring
degraders. This suggests that degradation of the ring structure of carbofuran in soil is a cometabolic process. q 2001 Elsevier Science Ltd.
All rights reserved.
Keywords: Carbofuran; Most-probable-number; Carbofuran ring degraders; Carbofuran hydrolyzers; Enhanced soil
1. Introduction
It has been known since 1981 that repeated ®eld applications of the insecticide±nematicide carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) cause
enhanced degradation of the chemical in the soil (Felsot et
al., 1981; Read, 1983; Camper et al., 1987; Turco and
Konopka, 1990). As a consequence of enhanced degradation, there may be a loss in the ef®cacy of the pesticide to
control target pests, resulting in crop failure (Felsot et al.,
1981; Racke and Coats, 1990). Enhanced degradation of
pesticides in soils is a microbial process (Racke and
Coats, 1990). There are two schools of thought on the
mechanism by which microorganisms in soil develop
enhanced degradation. One is as a consequence of repeated
applications of a pesticide, where an increase in the number
of microorganisms capable of degrading the chemical
results in more rapid degradation of the chemical compared
to untreated soil. Evidence supporting this hypothesis is
based on the observations that after addition of the phenoxy
herbicide 2,4-D (Ou, 1984; Holben et al., 1992; Ka et al.,
* Corresponding author. Tel.: 11-352-392-1951; fax: 11-352-392-3902.
E-mail address: [email protected]¯.edu (L.-T. Ou).
1994) or the organophosphate insecticide isofenphos (Racke
and Coats, 1987), the numbers of soil microorganisms
capable of degrading the chemicals increased substantially.
The other hypothesis is that repeated applications of a pesticide result in an increase in the enzyme activity, but not
community size, speci®cally toward the degradation of the
pesticide. Evidence for the second hypothesis is supported
by the ®ndings that the sizes of EPTC-degrading communities in soils with or without a history of ®eld applications
of the herbicide were similar (Moorman, 1988).
At present, published information on the changes of
carbofuran-degrading community sizes in soils after development of enhanced degradation toward carbofuran is
mixed. Hendry and Richardson (1988) and Dzantor and
Felsot (1989, 1990) pointed to an increase in the number
of carbofuran degraders in soils with enhanced degradation
compared to the number of the indigenous carbofuran
degraders. Racke and Coats (1988), Merica and Alexander
(1990), Scow et al. (1990) and Robertson and Alexander
(1994) failed to link an increase in the number of carbofuran
degraders with the number of carbofuran applications in
enhanced soils.
There are likely to be three groups of microorganisms that
may be involved in the degradation of carbofuran in soils
0038-0717/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0038-071 7(00)00116-4
76
S.L. Trabue et al. / Soil Biology & Biochemistry 33 (2001) 75±81
(Chaudhry and Ali, 1988). Groups 1 and 2 are capable of
hydrolyzing carbofuran and utilizing methylamine, one of
the hydrolysis products, as a sole source of N, and as sole
source of C and N for growth, respectively. Group 3 utilizes
the aromatic ring as a sole source of carbon. It is likely that
group 3 also has the capacity to hydrolyze carbofuran and
then utilize the aromatic ring of carbofuran phenol, one of
the hydrolysis products, for growth. Thus, all three groups
are likely involved in hydrolysis of the carbamate linkage.
The majority of carbofuran-degrading bacteria that have
been isolated from soils belong to group 2. Topp et al.
(1993) isolated a methylotrophic bacterium from an agricultural soil that hydrolyzed carbofuran. If some carbofuran
hydrolyzers are methylotrophic bacteria, the number of
the microorganisms should increase after carbofuran hydrolysis. Hendry and Richardson (1988) reported a signi®cant
increase in the size of carbofuran hydrolyzers from 1:6 £
103 to 3:1 £ 105 cells g21 after one laboratory treatment of
soil with carbofuran. However, Merica and Alexander
(1990) and Robertson and Alexander (1994) failed to link
carbofuran degradation with the growth of carbofuran
hydrolyzers as well as ring degraders.
We located a ®eld site in Florida that had been treated
with carbofuran annually two consecutive times, enhanced
degradation of carbofuran in soil at this site was observed
(Ou, unpublished observation). This site provided an unique
opportunity to ®nd out whether enhanced degradation of
carbofuran was due to an increase in microbial communities
capable of degrading carbofuran or due to an increase in
enzyme activity per cell (cometabolism). We determined
changes of microbial communities capable of degrading
carbofuran (carbofuran ring degraders and carbofuran hydrolyzers) in soil from this site after two to three successive
annual ®eld applications of carbofuran. In addition, microbial
community sizes capable of degrading carbofuran phenol or
methylamine were determined as part of this study.
2. Materials and methods
2.1. Chemicals
Technical grade carbofuran (99% purity), analytical
grade carbofuran phenol (99.3% purity), carbonyl-labeled
(CBL) 14C-carbofuran (speci®c activity, 131 MBq mmol 21),
and uniformly ring-labeled (URL) 14C-carbofuran (speci®c
activity, 229 MBq mmol21) were provided by FMC Corporation (Princeton, NJ). Prior to use, the two 14C-labeled
compounds were puri®ed to .98% radiopurity by thin-layer
chromatography (TLC) using preparative silica gel G TLC
plates (Ou et al., 1982, 1985). URL 14C-carbofuran phenol
was obtained by alkaline hydrolysis of URL 14C-carbofuran
overnight at 708C, and was puri®ed by preparative TLC to
.98% radiopurity. Analytical grade methylamine hydrochloride (.99% purity) was purchased from Aldrich
(Milwaukee, WI).
2.2. Field plots, carbofuran treatments and soil sampling
Field plots at an experimental site located in northeast of
Florida (60 km northeast of Gainesville, FL) were treated
with carbofuran annually in March or April from 1992 to
1996 at a rate of 4.5 kg ha 21. This site had been under
continuous potato cultivation for more than 10 y. Control
(untreated) plots at this site had never been treated with
carbofuran or any structurally similar pesticides, but
received fertilizers at rates identical to the treated plots.
Soil samples were collected from three plots at two depths
(0±15 and 45±60 cm) 4 weeks after annual applications of
carbofuran in three consecutive years (third, fourth and ®fth
annual applications in 1994, 1995 and 1996, respectively).
Soil samples from each depth were combined and mixed.
Soil samples were stored in plastic bags in the dark at 48C
and used within 3 months after collection. All soil samples
were classi®ed as Ellzey ®ne sand (sandy, siliceous,
hyperthermic Arenic Ochraqualfs). Key soil properties of
the samples have been reported by Trabue et al. (1997).
2.3. 14C-most-probable-number (MPN) assay
A 14C-MPN technique similar to that used by Ou (1984)
to determine 2,4-D-degrading microbial community sizes in
soils was employed to determine the sizes of microbial
communities capable of degrading carbofuran or carbofuran
phenol. URL or CBL 14C-carbofuran were used to determine community sizes capable of mineralizing the aromatic
ring structure or hydrolyzing the carbamate linkage of
carbofuran. URL 14C-carbofuran phenol was used to estimate the numbers of microorganisms capable of mineralizing the phenolic structure of carbofuran phenol.
One gram of soil (oven-dry weight basis) was transferred
to a sterile capped MPN tube containing 9 ml of a minimal
mineral medium, including 10 mg tryptone ml 21 (Ou,
1984). The medium also contained 10 mg ml 21 of technical
grade carbofuran and 16.7 Bq ml 21 of URL 14C-carbofuran,
CBL 14C-carbofuran or URL 14C-carbofuran phenol. Five
replicates of successive 5- to 10-fold dilutions were made.
All tubes were incubated in the dark at 288C for 28 d.
Control MPN tubes were treated as described above, except
that soil was not added to the tubes. At the end of the
incubation, 0.1 ml of concentrated HCl was added to each
tube, and after mixing, they were kept in a fume hood for 4±
6 h. Afterwards, 0.5 ml of the MPN media was assayed for
14
C-activity by liquid scintillation counting (LSC). Tubes
were scored as positive when # 70% of the initial 14Cactivity remained in solution. 14C-activity in all control
14
C-MPN tubes was unchanged after 28 d.
To determine community size changes after laboratory
treatment of carbofuran, 100 g of soil (oven-dry weight
basis) were added to a 250 ml Erlenmeyer glass ¯ask with
a cotton plug and mixed with 1.0 mg of technical grade
carbofuran to give a concentration of 10 mg g 21. The ¯asks
were incubated in the dark at ambient temperature
S.L. Trabue et al. / Soil Biology & Biochemistry 33 (2001) 75±81
Table 1
MPN (cells g 21) of carbofuran ring degraders in treated and untreated
surface soils collected in three consecutive years (1994±1996); CI Ð
con®dence interval
Depth (cm)
MPN
Lower 95% CI
Upper 95% CI
Treated soil (1994)
0±15
45±60
21.6abc a
2.0d
6.5
0.6
71.4
6.2
Untreated soil (1994)
0±15
45±60
32.9abc
0
10.0
0
108.7
0
43.0
30.5
464
225
Treated soil (1995)
0±15
45±60
Untreated soil (1995)
0±15
45±60
Treated soil (1996)
0±15
45±60
Untreated soil (1996)
0±15
45±60
141a
82.7ab
12.6bcd
0
4.9
0
34.1
0
183a
8.8cd
70.8
3.4
471
22.7
0
0
0
0
0
0
a
Values followed by the same letter are not signi®cantly different
P , 0:05:
(23 ^ 28C), and once a week their weights were checked,
sterile deionized water was added to compensate for any
water loss. Periodically, 1 g of soil was removed and placed
in a sterile MPN tube containing 9 ml of the MPN medium
(including 10 mg tryptone ml 21), technical grade carbofuran
(10 mg ml 21), and either URL 14C-carbofuran, CBL 14Ccarbofuran or URL 14C-carbofuran phenol. Community
sizes were estimated as above.
Table 2
MPN (cells g 21) of carbofuran hydrolyzers in treated and untreated surface
soils collected in 1994 and 1995; CI Ð con®dence interval
Depth (cm)
MPN
Lower 95% CI Upper 95% CI
Treated soil (1994)
0±15
45±60
1:61 £ 105 bc a
2:78 £ 104 c
4:87 £ 104
8:42 £ 103
5:31 £ 105
9:18 £ 104
Untreated soil (1994)
0±15
45±60
1:61 £ 105 bc
3:45 £ 104 bc
4:87 £ 104
1:05 £ 104
5:31 £ 105
1:14 £ 105
Treated soil (1995)
0±15
1:23 £ 107 a
2:27 £ 106
6:68 £ 107
Untreated soil (1995)
0±15
4:99 £ 105 ab
9:21 £ 104
2:70 £ 106
a
Values followed by the same letter are not signi®cantly different
P , 0:05:
77
2.4. MPN for methylamine utilizers
An MPN technique that determined the community size
capable of utilizing an organic substrate for growth was
used for determination of the number of microorganisms
capable of utilizing methylamine for growth (Alexander,
1982). The MPN method was similar to the 14C-MPN
assay, except that non-labeled methylamine hydrochloride
equivalent to 100 mg methylamine ml 21 was added to the
MPN tubes as a sole source of carbon and no tryptone was
added. Five replicates of successive 5-fold dilutions were
made. Tubes were scored positive if turbidity developed in
the MPN solutions in the tubes after 28 d at 288C. Control
tubes, which contained the MPN medium and methylamine,
were all devoid of turbidity after 28 d.
2.5. Statistical analysis
Numbers of microorganisms per gram soil based on the
MPN assays were obtained using the Eureka software
(Borland International, Scotts Valley, CA). The MPN and
the 95% con®dence limits were estimated by the software.
3. Results
3.1. Carbofuran degrading communities in ®eld soils
The numbers of microorganisms capable of mineralizing
the aromatic ring of carbofuran (ring degraders) in treated
surface soils (0±15 cm depth) collected 4 weeks after three
annual ®eld applications of carbofuran were not statistically
different (Table 1). The MPN in treated subsurface soils
(45±60 cm depth) were also not statistically indistinguishable, with the exception of the subsurface soil collected in
1995. The MPN in this soil was signi®cantly larger than the
MPN in the other two subsurface soils. The MPN in the
treated surface soils were statistically larger than the MPN
in the corresponding subsurface soils, with the exception of
the surface and subsurface soils collected in 1995. The MPN
in the two soils were not statistically different. The MPN in
untreated surface soils collected in 1994 and 1995 were not
signi®cantly different from each other, nor were they significantly different from the treated surface soils, with the
exception of the untreated surface soil collected in 1995.
The MPN in this soil was statistically smaller than the
MPN in the treated surface soil collected in the same year.
The MPN in untreated subsurface soils collected in three
consecutive years as well as in untreated surface soils
collected in 1996 were zero.
The MPN of microorganisms capable of hydrolyzing the
carbamate linkage of carbofuran (carbofuran hydrolyzers)
in the treated and untreated soils collected in 1994 and 1995
(Table 2) were three to ®ve orders of magnitude larger than
the MPN of carbofuran ring degraders. The MPN of carbofuran hydrolyzers in the treated surface soil collected in
1995 was signi®cantly larger than the MPN in the treated
78
S.L. Trabue et al. / Soil Biology & Biochemistry 33 (2001) 75±81
Table 3
MPN (cells g 21) of methylamine utilizers and carbofuran phenol ring
degraders in treated and untreated surface soils collected in 1995; CI Ð
con®dence interval
Degraders
MPN
95% CI
Upper 95% CI
Treated soil
Methylamine
Carbofuran phenol
7:93 £ 106 a a
16.0b
2:40 £ 106
6.2
2:62 £ 107
41.2
Untreated soil
Methylamine
Carbofuran phenol
4:93 £ 106 a
8.5b
1:49 £ 106
3.3
1:62 £ 107
21.9
a
Values followed by the same letter are not signi®cantly different
P , 0:05:
and untreated surface and subsurface soils collected in 1994,
but not signi®cantly different from the MPN in the untreated
surface soil collected in 1995. However, the MPN in the
untreated surface soil collected in 1995 was not statistically
different from the MPN in the treated surface soil, and
untreated surface and subsurface soils collected in 1994,
but signi®cantly larger than the MPN in the treated subsurface soil collected in 1994. Due to larger than usual precipitation in late winter and early spring in 1995, the water
table at the site at the time of sampling was fairly shallow;
occurring at 50±55 cm from the soil surface.
Fig. 1. Most probable numbers (MPN) of carbofuran ring degraders (A) and
carbofuran hydrolyzers (B) in ®eld treated and untreated soils after receiving 10 mg carbofuran g 21 in the laboratory during 14 or 28 d of incubation.
Soil samples were collected from treated and untreated plots in 1995.
3.2. Carbofuran phenol- and methylamine-degrading
communities in ®eld soils
The MPN of microorganisms capable of utilizing methylamine in the treated and untreated surface soils collected in
1995 were much larger 5 £ 105 times) than the MPN of
microorganisms capable of mineralizing the aromatic ring
of carbofuran phenol (Table 3). The MPN of methylamine
utilizers in the treated and untreated surface soils were not
statistically different from each other. It is interesting to
point out that the MPN of methylamine utilizers and carbofuran hydrolyzers in the two soils were not statistically
different (Tables 2 and 3).
The numbers of carbofuran phenol ring degraders in the
treated and untreated soils averaged 16 and 9 cells g 21 soil,
respectively, and were statistically indistinguishable (Table
3). The MPN of carbofuran ring degraders and carbofuran
phenol ring degraders in treated and untreated soils were all
small (Tables 1 and 3). They were not statistically different
in the untreated soil (Tables 1 and 3). But the MPN of
carbofuran ring degraders in the treated soil was statistically
larger than the MPN of carbofuran phenol ring degraders.
3.3. Growth of carbofuran degraders after a laboratory
treatment of carbofuran
Laboratory treatment of carbofuran at 10 mg g 21 to the
®eld treated surface and subsurface soils collected in 1995
did not result in a statistically signi®cant increase in the
MPN of carbofuran ring degraders after 28 d of incubation
(Fig. 1A). After 14 d, the MPN in the ®eld treated surface
soil was signi®cantly smaller than before the laboratory
treatment. The MPN of carbofuran ring degraders in the
®eld untreated surface soil did not change after laboratory
treatment with carbofuran, with the exception that 14 d after
the treatment, the MPN was signi®cantly smaller than
before the treatment. In the untreated subsurface soil, carbofuran ring degraders were not detected until 28 d after the
laboratory treatment of carbofuran, when 4.5 cells g 21 were
detected in this soil.
The MPN of carbofuran-hydrolyzers in the ®eld treated
surface soil collected in 1995 was initially very large 1:23 £
107 cells g21 soil), and after laboratory treatment with carbofuran at a rate of 10 mg g 21 soil, the MPN remained stable
during the ®rst 7 d of incubation (Fig. 1B). After 14 d, the
MPN declined signi®cantly. The MPN of carbofuran hydrolyzers in the ®eld untreated surface soil did not change during
the ®rst 3 d after the laboratory application of carbofuran;
however, between days 3 and 7, the MPN increased signi®cantly. After 14 d, it declined to the level statistically similar
to that before the treatment.
3.4. Growth of carbofuran phenol degraders and
methylamine utilizers after laboratory treatment with
carbofuran phenol and methylamine
After laboratory treatment of URL 14C-carbofuran phenol
S.L. Trabue et al. / Soil Biology & Biochemistry 33 (2001) 75±81
79
laboratory treatment, although they were not signi®cantly
different.
4. Discussion
Fig. 2. Most probable numbers (MPN) of carbofuran phenol ring degraders
(A) and methylamine utilizers (B) in ®eld treated and untreated surface soils
after receiving 10 mg carbofuran phenol g 21 and 10 mg methylamine g 21,
respectively, in the laboratory during 14 or 28 d of incubation. Soil samples
were collected from treated and untreated plots in 1995.
at 10 mg g 21 to ®eld treated and untreated surface soils
collected in 1995, the MPN of carbofuran phenol ring degraders in these two soils were statistically indistinguishable
during the entire 28 d of incubation (Fig. 2A). Even though
the average MPN of carbofuran phenol degraders in the
surface soil after 28 d was 6.8 times larger than before the
laboratory treatment, due to large errors associated with
the MPN determination, the two numbers were not statistically different. Results similar to the MPN of carbofuran
phenol degraders were obtained for the MPN of methylamine utilizers in treated and untreated surface soils (Fig.
2B). The average MPN in the treated soil 7 d after the
laboratory treatment was 10 times larger than before the
In this study, we chose MPN assays over other enumeration techniques. Dilution-plate count methods were inadequate. Although small semi-transparent watery colonies
developed on basal mineral-carbofuran agar plates after
5±7 d of incubation, no carbofuran degradation and no
growth were observed when biomass of individual colonies
was inoculated into the liquid basal mineral-carbofuran
medium (Ou, unpublished observations). Methods such as
gene probing are not available for those strains. Only one
gene involved in carbofuran degradation has been cloned.
This gene, the methyl carbamate degrading (mcd) gene, was
cloned from Achromobacter sp. WM111 and encodes for a
carbofuran hydrolase (Tomasek and Karns, 1989). mcd
appears to be not widely distributed among carbofuran
degraders in the US. DNA isolated from many soils that
have been exposed to carbofuran did not hybridize with
this gene (Derk et al., 1993). 14C-MPN assays at present
appear to be the only means for determination of carbofuran-degrading communities in soils.
The sequence of carbofuran degradation in soil is ®rst via
hydrolysis to carbofuran phenol and methylamine. Carbofuran phenol is degraded further through aromatic ring cleavage, and eventually to CO2 and H2O (Ou et al., 1982;
Trabue et al., 1997). Therefore, the same microorganisms
that mineralized the aromatic ring of carbofuran were likely
also to be responsible for carrying out mineralization of the
aromatic ring of carbofuran phenol. It was not clear why the
MPN for carbofuran ring degraders was statistically larger
than the MPN for carbofuran phenol ring degraders in treated soil collected in 1995 where there was enhanced degradation of carbofuran (Trabue et al., 1997). They were all
small in numbers, however.
Carbonyl-labeled 14C-carbofuran was used to determine
the number of carbofuran hydrolyzers in soil based on the
14
C-MPN technique. When the carbamate linkage of carbofuran is broken by hydrolysis, 14C-carbonyl is immediately
converted to 14CO2, carbofuran phenol and methylamine
(Fig. 3). Due to unavailability of furanyl ring-labeled
Fig. 3. Hydrolysis of carbonyl-labeled 14C-carbofuran.
80
14
S.L. Trabue et al. / Soil Biology & Biochemistry 33 (2001) 75±81
C-carbofuran, the number of microorganisms capable of
degrading the furanyl ring could not be determined.
However, it is conceivable that carbofuran ring degraders
may also have the capacity to mineralize the furanyl ring.
We found that URL 14C-carbofuran in treated and untreated
surface and subsurface soils collected in 1995 from the same
site was rapidly mineralized to 14CO2 (Trabue et al., 1997).
Prior to this study, the treated site had been treated with
carbofuran annually for two consecutive years and soil of
this site was found to exhibit enhanced degradation toward
carbofuran after the second annual treatment (1993) (Ou,
unpublished observation). Enhanced degradation was continually observed from 1994 to 1996 (Trabue, Unpublished
PhD thesis, University of Florida, 1997; Trabue et al.,
1997). Despite the fact that carbofuran was applied to the
treated site annually at a rate of 4.5 kg ha 21 for ®ve consecutive years, the MPN of carbofuran ring degraders in the
surface soils during the last three annual applications were
small and not statistically different. This suggests that
mineralization of the ring structure of carbofuran was
mainly by cometabolism.
The fact that the MPN of carbofuran hydrolyzers and
methylamine utilizers were not statistically different may
be fortuitous. However, some carbofuran-degrading
bacteria are methylotrophic organisms (Chaudhry and Ali,
1988; Singh et al., 1993; Topp et al., 1993). They were
capable of hydrolyzing carbofuran and utilizing methylamine as a sole source of C for growth. It is known that
methylotrophic bacteria utilize 1 C compounds, including
methane and methylamine, as a sole source of C for growth
(Hanson and Hanson, 1996). Large communities of methanotrophs ranging from 10 6 to 10 8 cells g 21 soil exists in oxic
soils (Hanson and Hanson, 1996). Since methanotrophs are
a subgroup of methylotrophs, the sizes of methylotrophic
communities in soils should be at least equal or larger than
the sizes of methanotrophic communities. In our study,
community sizes of methylamine utilizers in the two soils
were similar to the reported values for methanotrophs.
Our ®ndings suggest that some carbofuran hydrolyzers
may also be methylamine utilizers. Hydrolysis of 4.5 kg
carbofuran ha 21 (equivalent to 2 mg g 21) will result in the
formation of 280 ng methylamine g 21 soil. Thus, the methylamine will provide 110 ng C and 130 ng N g 21 for serving
as C and N sources for methylotrophic bacteria. A typical
bacterial cell has a dry mass of 280 fg and consists of 50% C
and 14% N (Neidhardt et al., 1990). Thus, 280 ng methylamine g 21 could support from 7:9 £ 105 to 3:3 £
106 cells g21 soil, if N and C are not limiting factors, respectively. Due to the large difference between the lower and
upper con®dence intervals (Table 3), even an increase of
3:3 £ 106 cells g21 would not result in a statistically significant increase in the community size of methylamine utilizers. In this study, ®ve replicates for each dilution were
employed for all MPN experiments, an increase to 10 replicates may provide smaller difference between lower and
upper con®dence intervals. As a consequence, release of
280 ng methylamine g 21 from hydrolysis of carbofuran
may result in a statistical increase in MPN for methylamine
utilizers.
Communities of carbofuran ring degraders in the ®eld
treated and untreated soils after receiving 10 mg carbofuran g 21 in laboratory did not increase numerically, suggesting that the ring structure of carbofuran did not provide a
carbon source for the growth of microorganisms, even
though the structure was microbially degraded. This ®nding
was supported by the results of MPN estimations of carbofuran ring degraders in soil from the treated plots during
three consecutive annual ®eld applications of carbofuran
(Table 1). Similarly, the MPN of carbofuran phenol ring
degraders in the treated and untreated soils after receiving
10 mg carbofuran phenol g 21 did not increase numerically
as well. Therefore, enhanced degradation of carbofuran in
the treated soil (Trabue et al., 1997) may have been due to
an increase in enzyme activity responsible for carbofuran
ring degradation. Merica and Alexander (1990) also found
that the MPN of carbofuran ring degraders did not increase
in enhanced soil in 2 and 12 d after receiving 10 mg URL
14
C-carbofuran g 21.
In conclusion, our results indicated that communities of
microorganisms capable of degrading the ring structure of
carbofuran during three consecutive years of carbofuran
treatments remained small and stable, and hence the degradation is a cometabolic process.
Acknowledgements
We thank John E. Thomas for technical assistance.
Sincere appreciation is extended to David P. Weingartner
for ®eld treatments of carbofuran. This study was partially
supported by a grant from USDA. Florida Agricultural
Experimental Station journal series No. R-06805.
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Ou, L.-T., Gancarz, D.H., Wheeler, W.B., Rao, P.S.C., Davidson, J.M.,
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biodegradation. Pesticide Science 41, 311±318.
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carbofuran. Biodegradation 4, 115±123.
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Microbiology 59, 3339±3349.
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www.elsevier.com/locate/soilbio
Dynamics of carbofuran-degrading microbial communities in soil during
three successive annual applications of carbofuran
S.L. Trabue, A.V. Ogram, L.-T. Ou*
Soil and Water Science Department, P.O. Box 110290, University of Florida, Gainesville, FL 32611-0290, USA
Received 4 October 1999; received in revised form 3 May 2000; accepted 29 May 2000
Abstract
The in¯uence of repeated annual ®eld applications of the insecticide±nematicide carbofuran on carbofuran-degrading microbial communities was studied at a site in Florida that exhibited enhanced degradation toward the chemical. Three successive annual applications of
carbofuran at 4.5 kg ha 21 y 21 did not result in an increase in the size of the microbial community capable of mineralizing uniformly ringlabeled 14C-carbofuran (carbofuran-ring degraders) in surface soil (0±15 cm depth). After the second annual application, however, the
community capable of mineralizing carbonyl-labeled 14C-carbofuran (carbofuran hydrolyzers) in the treated surface soil was signi®cantly
larger than that after the ®rst annual application. Communities of methylamine utilizers in treated and untreated soils were much larger than
the communities of carbofuran phenol degraders, but not statistically different from the sizes of carbofuran hydrolyzers. In addition to no
increase in the number of carbofuran ring degraders in the treated site during three consecutive annual applications of carbofuran, laboratory
addition of 10 mg carbofuran g 21 to treated and untreated soils collected in 1995 did not result in an increase in the number of carbofuran ring
degraders. This suggests that degradation of the ring structure of carbofuran in soil is a cometabolic process. q 2001 Elsevier Science Ltd.
All rights reserved.
Keywords: Carbofuran; Most-probable-number; Carbofuran ring degraders; Carbofuran hydrolyzers; Enhanced soil
1. Introduction
It has been known since 1981 that repeated ®eld applications of the insecticide±nematicide carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) cause
enhanced degradation of the chemical in the soil (Felsot et
al., 1981; Read, 1983; Camper et al., 1987; Turco and
Konopka, 1990). As a consequence of enhanced degradation, there may be a loss in the ef®cacy of the pesticide to
control target pests, resulting in crop failure (Felsot et al.,
1981; Racke and Coats, 1990). Enhanced degradation of
pesticides in soils is a microbial process (Racke and
Coats, 1990). There are two schools of thought on the
mechanism by which microorganisms in soil develop
enhanced degradation. One is as a consequence of repeated
applications of a pesticide, where an increase in the number
of microorganisms capable of degrading the chemical
results in more rapid degradation of the chemical compared
to untreated soil. Evidence supporting this hypothesis is
based on the observations that after addition of the phenoxy
herbicide 2,4-D (Ou, 1984; Holben et al., 1992; Ka et al.,
* Corresponding author. Tel.: 11-352-392-1951; fax: 11-352-392-3902.
E-mail address: [email protected]¯.edu (L.-T. Ou).
1994) or the organophosphate insecticide isofenphos (Racke
and Coats, 1987), the numbers of soil microorganisms
capable of degrading the chemicals increased substantially.
The other hypothesis is that repeated applications of a pesticide result in an increase in the enzyme activity, but not
community size, speci®cally toward the degradation of the
pesticide. Evidence for the second hypothesis is supported
by the ®ndings that the sizes of EPTC-degrading communities in soils with or without a history of ®eld applications
of the herbicide were similar (Moorman, 1988).
At present, published information on the changes of
carbofuran-degrading community sizes in soils after development of enhanced degradation toward carbofuran is
mixed. Hendry and Richardson (1988) and Dzantor and
Felsot (1989, 1990) pointed to an increase in the number
of carbofuran degraders in soils with enhanced degradation
compared to the number of the indigenous carbofuran
degraders. Racke and Coats (1988), Merica and Alexander
(1990), Scow et al. (1990) and Robertson and Alexander
(1994) failed to link an increase in the number of carbofuran
degraders with the number of carbofuran applications in
enhanced soils.
There are likely to be three groups of microorganisms that
may be involved in the degradation of carbofuran in soils
0038-0717/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0038-071 7(00)00116-4
76
S.L. Trabue et al. / Soil Biology & Biochemistry 33 (2001) 75±81
(Chaudhry and Ali, 1988). Groups 1 and 2 are capable of
hydrolyzing carbofuran and utilizing methylamine, one of
the hydrolysis products, as a sole source of N, and as sole
source of C and N for growth, respectively. Group 3 utilizes
the aromatic ring as a sole source of carbon. It is likely that
group 3 also has the capacity to hydrolyze carbofuran and
then utilize the aromatic ring of carbofuran phenol, one of
the hydrolysis products, for growth. Thus, all three groups
are likely involved in hydrolysis of the carbamate linkage.
The majority of carbofuran-degrading bacteria that have
been isolated from soils belong to group 2. Topp et al.
(1993) isolated a methylotrophic bacterium from an agricultural soil that hydrolyzed carbofuran. If some carbofuran
hydrolyzers are methylotrophic bacteria, the number of
the microorganisms should increase after carbofuran hydrolysis. Hendry and Richardson (1988) reported a signi®cant
increase in the size of carbofuran hydrolyzers from 1:6 £
103 to 3:1 £ 105 cells g21 after one laboratory treatment of
soil with carbofuran. However, Merica and Alexander
(1990) and Robertson and Alexander (1994) failed to link
carbofuran degradation with the growth of carbofuran
hydrolyzers as well as ring degraders.
We located a ®eld site in Florida that had been treated
with carbofuran annually two consecutive times, enhanced
degradation of carbofuran in soil at this site was observed
(Ou, unpublished observation). This site provided an unique
opportunity to ®nd out whether enhanced degradation of
carbofuran was due to an increase in microbial communities
capable of degrading carbofuran or due to an increase in
enzyme activity per cell (cometabolism). We determined
changes of microbial communities capable of degrading
carbofuran (carbofuran ring degraders and carbofuran hydrolyzers) in soil from this site after two to three successive
annual ®eld applications of carbofuran. In addition, microbial
community sizes capable of degrading carbofuran phenol or
methylamine were determined as part of this study.
2. Materials and methods
2.1. Chemicals
Technical grade carbofuran (99% purity), analytical
grade carbofuran phenol (99.3% purity), carbonyl-labeled
(CBL) 14C-carbofuran (speci®c activity, 131 MBq mmol 21),
and uniformly ring-labeled (URL) 14C-carbofuran (speci®c
activity, 229 MBq mmol21) were provided by FMC Corporation (Princeton, NJ). Prior to use, the two 14C-labeled
compounds were puri®ed to .98% radiopurity by thin-layer
chromatography (TLC) using preparative silica gel G TLC
plates (Ou et al., 1982, 1985). URL 14C-carbofuran phenol
was obtained by alkaline hydrolysis of URL 14C-carbofuran
overnight at 708C, and was puri®ed by preparative TLC to
.98% radiopurity. Analytical grade methylamine hydrochloride (.99% purity) was purchased from Aldrich
(Milwaukee, WI).
2.2. Field plots, carbofuran treatments and soil sampling
Field plots at an experimental site located in northeast of
Florida (60 km northeast of Gainesville, FL) were treated
with carbofuran annually in March or April from 1992 to
1996 at a rate of 4.5 kg ha 21. This site had been under
continuous potato cultivation for more than 10 y. Control
(untreated) plots at this site had never been treated with
carbofuran or any structurally similar pesticides, but
received fertilizers at rates identical to the treated plots.
Soil samples were collected from three plots at two depths
(0±15 and 45±60 cm) 4 weeks after annual applications of
carbofuran in three consecutive years (third, fourth and ®fth
annual applications in 1994, 1995 and 1996, respectively).
Soil samples from each depth were combined and mixed.
Soil samples were stored in plastic bags in the dark at 48C
and used within 3 months after collection. All soil samples
were classi®ed as Ellzey ®ne sand (sandy, siliceous,
hyperthermic Arenic Ochraqualfs). Key soil properties of
the samples have been reported by Trabue et al. (1997).
2.3. 14C-most-probable-number (MPN) assay
A 14C-MPN technique similar to that used by Ou (1984)
to determine 2,4-D-degrading microbial community sizes in
soils was employed to determine the sizes of microbial
communities capable of degrading carbofuran or carbofuran
phenol. URL or CBL 14C-carbofuran were used to determine community sizes capable of mineralizing the aromatic
ring structure or hydrolyzing the carbamate linkage of
carbofuran. URL 14C-carbofuran phenol was used to estimate the numbers of microorganisms capable of mineralizing the phenolic structure of carbofuran phenol.
One gram of soil (oven-dry weight basis) was transferred
to a sterile capped MPN tube containing 9 ml of a minimal
mineral medium, including 10 mg tryptone ml 21 (Ou,
1984). The medium also contained 10 mg ml 21 of technical
grade carbofuran and 16.7 Bq ml 21 of URL 14C-carbofuran,
CBL 14C-carbofuran or URL 14C-carbofuran phenol. Five
replicates of successive 5- to 10-fold dilutions were made.
All tubes were incubated in the dark at 288C for 28 d.
Control MPN tubes were treated as described above, except
that soil was not added to the tubes. At the end of the
incubation, 0.1 ml of concentrated HCl was added to each
tube, and after mixing, they were kept in a fume hood for 4±
6 h. Afterwards, 0.5 ml of the MPN media was assayed for
14
C-activity by liquid scintillation counting (LSC). Tubes
were scored as positive when # 70% of the initial 14Cactivity remained in solution. 14C-activity in all control
14
C-MPN tubes was unchanged after 28 d.
To determine community size changes after laboratory
treatment of carbofuran, 100 g of soil (oven-dry weight
basis) were added to a 250 ml Erlenmeyer glass ¯ask with
a cotton plug and mixed with 1.0 mg of technical grade
carbofuran to give a concentration of 10 mg g 21. The ¯asks
were incubated in the dark at ambient temperature
S.L. Trabue et al. / Soil Biology & Biochemistry 33 (2001) 75±81
Table 1
MPN (cells g 21) of carbofuran ring degraders in treated and untreated
surface soils collected in three consecutive years (1994±1996); CI Ð
con®dence interval
Depth (cm)
MPN
Lower 95% CI
Upper 95% CI
Treated soil (1994)
0±15
45±60
21.6abc a
2.0d
6.5
0.6
71.4
6.2
Untreated soil (1994)
0±15
45±60
32.9abc
0
10.0
0
108.7
0
43.0
30.5
464
225
Treated soil (1995)
0±15
45±60
Untreated soil (1995)
0±15
45±60
Treated soil (1996)
0±15
45±60
Untreated soil (1996)
0±15
45±60
141a
82.7ab
12.6bcd
0
4.9
0
34.1
0
183a
8.8cd
70.8
3.4
471
22.7
0
0
0
0
0
0
a
Values followed by the same letter are not signi®cantly different
P , 0:05:
(23 ^ 28C), and once a week their weights were checked,
sterile deionized water was added to compensate for any
water loss. Periodically, 1 g of soil was removed and placed
in a sterile MPN tube containing 9 ml of the MPN medium
(including 10 mg tryptone ml 21), technical grade carbofuran
(10 mg ml 21), and either URL 14C-carbofuran, CBL 14Ccarbofuran or URL 14C-carbofuran phenol. Community
sizes were estimated as above.
Table 2
MPN (cells g 21) of carbofuran hydrolyzers in treated and untreated surface
soils collected in 1994 and 1995; CI Ð con®dence interval
Depth (cm)
MPN
Lower 95% CI Upper 95% CI
Treated soil (1994)
0±15
45±60
1:61 £ 105 bc a
2:78 £ 104 c
4:87 £ 104
8:42 £ 103
5:31 £ 105
9:18 £ 104
Untreated soil (1994)
0±15
45±60
1:61 £ 105 bc
3:45 £ 104 bc
4:87 £ 104
1:05 £ 104
5:31 £ 105
1:14 £ 105
Treated soil (1995)
0±15
1:23 £ 107 a
2:27 £ 106
6:68 £ 107
Untreated soil (1995)
0±15
4:99 £ 105 ab
9:21 £ 104
2:70 £ 106
a
Values followed by the same letter are not signi®cantly different
P , 0:05:
77
2.4. MPN for methylamine utilizers
An MPN technique that determined the community size
capable of utilizing an organic substrate for growth was
used for determination of the number of microorganisms
capable of utilizing methylamine for growth (Alexander,
1982). The MPN method was similar to the 14C-MPN
assay, except that non-labeled methylamine hydrochloride
equivalent to 100 mg methylamine ml 21 was added to the
MPN tubes as a sole source of carbon and no tryptone was
added. Five replicates of successive 5-fold dilutions were
made. Tubes were scored positive if turbidity developed in
the MPN solutions in the tubes after 28 d at 288C. Control
tubes, which contained the MPN medium and methylamine,
were all devoid of turbidity after 28 d.
2.5. Statistical analysis
Numbers of microorganisms per gram soil based on the
MPN assays were obtained using the Eureka software
(Borland International, Scotts Valley, CA). The MPN and
the 95% con®dence limits were estimated by the software.
3. Results
3.1. Carbofuran degrading communities in ®eld soils
The numbers of microorganisms capable of mineralizing
the aromatic ring of carbofuran (ring degraders) in treated
surface soils (0±15 cm depth) collected 4 weeks after three
annual ®eld applications of carbofuran were not statistically
different (Table 1). The MPN in treated subsurface soils
(45±60 cm depth) were also not statistically indistinguishable, with the exception of the subsurface soil collected in
1995. The MPN in this soil was signi®cantly larger than the
MPN in the other two subsurface soils. The MPN in the
treated surface soils were statistically larger than the MPN
in the corresponding subsurface soils, with the exception of
the surface and subsurface soils collected in 1995. The MPN
in the two soils were not statistically different. The MPN in
untreated surface soils collected in 1994 and 1995 were not
signi®cantly different from each other, nor were they significantly different from the treated surface soils, with the
exception of the untreated surface soil collected in 1995.
The MPN in this soil was statistically smaller than the
MPN in the treated surface soil collected in the same year.
The MPN in untreated subsurface soils collected in three
consecutive years as well as in untreated surface soils
collected in 1996 were zero.
The MPN of microorganisms capable of hydrolyzing the
carbamate linkage of carbofuran (carbofuran hydrolyzers)
in the treated and untreated soils collected in 1994 and 1995
(Table 2) were three to ®ve orders of magnitude larger than
the MPN of carbofuran ring degraders. The MPN of carbofuran hydrolyzers in the treated surface soil collected in
1995 was signi®cantly larger than the MPN in the treated
78
S.L. Trabue et al. / Soil Biology & Biochemistry 33 (2001) 75±81
Table 3
MPN (cells g 21) of methylamine utilizers and carbofuran phenol ring
degraders in treated and untreated surface soils collected in 1995; CI Ð
con®dence interval
Degraders
MPN
95% CI
Upper 95% CI
Treated soil
Methylamine
Carbofuran phenol
7:93 £ 106 a a
16.0b
2:40 £ 106
6.2
2:62 £ 107
41.2
Untreated soil
Methylamine
Carbofuran phenol
4:93 £ 106 a
8.5b
1:49 £ 106
3.3
1:62 £ 107
21.9
a
Values followed by the same letter are not signi®cantly different
P , 0:05:
and untreated surface and subsurface soils collected in 1994,
but not signi®cantly different from the MPN in the untreated
surface soil collected in 1995. However, the MPN in the
untreated surface soil collected in 1995 was not statistically
different from the MPN in the treated surface soil, and
untreated surface and subsurface soils collected in 1994,
but signi®cantly larger than the MPN in the treated subsurface soil collected in 1994. Due to larger than usual precipitation in late winter and early spring in 1995, the water
table at the site at the time of sampling was fairly shallow;
occurring at 50±55 cm from the soil surface.
Fig. 1. Most probable numbers (MPN) of carbofuran ring degraders (A) and
carbofuran hydrolyzers (B) in ®eld treated and untreated soils after receiving 10 mg carbofuran g 21 in the laboratory during 14 or 28 d of incubation.
Soil samples were collected from treated and untreated plots in 1995.
3.2. Carbofuran phenol- and methylamine-degrading
communities in ®eld soils
The MPN of microorganisms capable of utilizing methylamine in the treated and untreated surface soils collected in
1995 were much larger 5 £ 105 times) than the MPN of
microorganisms capable of mineralizing the aromatic ring
of carbofuran phenol (Table 3). The MPN of methylamine
utilizers in the treated and untreated surface soils were not
statistically different from each other. It is interesting to
point out that the MPN of methylamine utilizers and carbofuran hydrolyzers in the two soils were not statistically
different (Tables 2 and 3).
The numbers of carbofuran phenol ring degraders in the
treated and untreated soils averaged 16 and 9 cells g 21 soil,
respectively, and were statistically indistinguishable (Table
3). The MPN of carbofuran ring degraders and carbofuran
phenol ring degraders in treated and untreated soils were all
small (Tables 1 and 3). They were not statistically different
in the untreated soil (Tables 1 and 3). But the MPN of
carbofuran ring degraders in the treated soil was statistically
larger than the MPN of carbofuran phenol ring degraders.
3.3. Growth of carbofuran degraders after a laboratory
treatment of carbofuran
Laboratory treatment of carbofuran at 10 mg g 21 to the
®eld treated surface and subsurface soils collected in 1995
did not result in a statistically signi®cant increase in the
MPN of carbofuran ring degraders after 28 d of incubation
(Fig. 1A). After 14 d, the MPN in the ®eld treated surface
soil was signi®cantly smaller than before the laboratory
treatment. The MPN of carbofuran ring degraders in the
®eld untreated surface soil did not change after laboratory
treatment with carbofuran, with the exception that 14 d after
the treatment, the MPN was signi®cantly smaller than
before the treatment. In the untreated subsurface soil, carbofuran ring degraders were not detected until 28 d after the
laboratory treatment of carbofuran, when 4.5 cells g 21 were
detected in this soil.
The MPN of carbofuran-hydrolyzers in the ®eld treated
surface soil collected in 1995 was initially very large 1:23 £
107 cells g21 soil), and after laboratory treatment with carbofuran at a rate of 10 mg g 21 soil, the MPN remained stable
during the ®rst 7 d of incubation (Fig. 1B). After 14 d, the
MPN declined signi®cantly. The MPN of carbofuran hydrolyzers in the ®eld untreated surface soil did not change during
the ®rst 3 d after the laboratory application of carbofuran;
however, between days 3 and 7, the MPN increased signi®cantly. After 14 d, it declined to the level statistically similar
to that before the treatment.
3.4. Growth of carbofuran phenol degraders and
methylamine utilizers after laboratory treatment with
carbofuran phenol and methylamine
After laboratory treatment of URL 14C-carbofuran phenol
S.L. Trabue et al. / Soil Biology & Biochemistry 33 (2001) 75±81
79
laboratory treatment, although they were not signi®cantly
different.
4. Discussion
Fig. 2. Most probable numbers (MPN) of carbofuran phenol ring degraders
(A) and methylamine utilizers (B) in ®eld treated and untreated surface soils
after receiving 10 mg carbofuran phenol g 21 and 10 mg methylamine g 21,
respectively, in the laboratory during 14 or 28 d of incubation. Soil samples
were collected from treated and untreated plots in 1995.
at 10 mg g 21 to ®eld treated and untreated surface soils
collected in 1995, the MPN of carbofuran phenol ring degraders in these two soils were statistically indistinguishable
during the entire 28 d of incubation (Fig. 2A). Even though
the average MPN of carbofuran phenol degraders in the
surface soil after 28 d was 6.8 times larger than before the
laboratory treatment, due to large errors associated with
the MPN determination, the two numbers were not statistically different. Results similar to the MPN of carbofuran
phenol degraders were obtained for the MPN of methylamine utilizers in treated and untreated surface soils (Fig.
2B). The average MPN in the treated soil 7 d after the
laboratory treatment was 10 times larger than before the
In this study, we chose MPN assays over other enumeration techniques. Dilution-plate count methods were inadequate. Although small semi-transparent watery colonies
developed on basal mineral-carbofuran agar plates after
5±7 d of incubation, no carbofuran degradation and no
growth were observed when biomass of individual colonies
was inoculated into the liquid basal mineral-carbofuran
medium (Ou, unpublished observations). Methods such as
gene probing are not available for those strains. Only one
gene involved in carbofuran degradation has been cloned.
This gene, the methyl carbamate degrading (mcd) gene, was
cloned from Achromobacter sp. WM111 and encodes for a
carbofuran hydrolase (Tomasek and Karns, 1989). mcd
appears to be not widely distributed among carbofuran
degraders in the US. DNA isolated from many soils that
have been exposed to carbofuran did not hybridize with
this gene (Derk et al., 1993). 14C-MPN assays at present
appear to be the only means for determination of carbofuran-degrading communities in soils.
The sequence of carbofuran degradation in soil is ®rst via
hydrolysis to carbofuran phenol and methylamine. Carbofuran phenol is degraded further through aromatic ring cleavage, and eventually to CO2 and H2O (Ou et al., 1982;
Trabue et al., 1997). Therefore, the same microorganisms
that mineralized the aromatic ring of carbofuran were likely
also to be responsible for carrying out mineralization of the
aromatic ring of carbofuran phenol. It was not clear why the
MPN for carbofuran ring degraders was statistically larger
than the MPN for carbofuran phenol ring degraders in treated soil collected in 1995 where there was enhanced degradation of carbofuran (Trabue et al., 1997). They were all
small in numbers, however.
Carbonyl-labeled 14C-carbofuran was used to determine
the number of carbofuran hydrolyzers in soil based on the
14
C-MPN technique. When the carbamate linkage of carbofuran is broken by hydrolysis, 14C-carbonyl is immediately
converted to 14CO2, carbofuran phenol and methylamine
(Fig. 3). Due to unavailability of furanyl ring-labeled
Fig. 3. Hydrolysis of carbonyl-labeled 14C-carbofuran.
80
14
S.L. Trabue et al. / Soil Biology & Biochemistry 33 (2001) 75±81
C-carbofuran, the number of microorganisms capable of
degrading the furanyl ring could not be determined.
However, it is conceivable that carbofuran ring degraders
may also have the capacity to mineralize the furanyl ring.
We found that URL 14C-carbofuran in treated and untreated
surface and subsurface soils collected in 1995 from the same
site was rapidly mineralized to 14CO2 (Trabue et al., 1997).
Prior to this study, the treated site had been treated with
carbofuran annually for two consecutive years and soil of
this site was found to exhibit enhanced degradation toward
carbofuran after the second annual treatment (1993) (Ou,
unpublished observation). Enhanced degradation was continually observed from 1994 to 1996 (Trabue, Unpublished
PhD thesis, University of Florida, 1997; Trabue et al.,
1997). Despite the fact that carbofuran was applied to the
treated site annually at a rate of 4.5 kg ha 21 for ®ve consecutive years, the MPN of carbofuran ring degraders in the
surface soils during the last three annual applications were
small and not statistically different. This suggests that
mineralization of the ring structure of carbofuran was
mainly by cometabolism.
The fact that the MPN of carbofuran hydrolyzers and
methylamine utilizers were not statistically different may
be fortuitous. However, some carbofuran-degrading
bacteria are methylotrophic organisms (Chaudhry and Ali,
1988; Singh et al., 1993; Topp et al., 1993). They were
capable of hydrolyzing carbofuran and utilizing methylamine as a sole source of C for growth. It is known that
methylotrophic bacteria utilize 1 C compounds, including
methane and methylamine, as a sole source of C for growth
(Hanson and Hanson, 1996). Large communities of methanotrophs ranging from 10 6 to 10 8 cells g 21 soil exists in oxic
soils (Hanson and Hanson, 1996). Since methanotrophs are
a subgroup of methylotrophs, the sizes of methylotrophic
communities in soils should be at least equal or larger than
the sizes of methanotrophic communities. In our study,
community sizes of methylamine utilizers in the two soils
were similar to the reported values for methanotrophs.
Our ®ndings suggest that some carbofuran hydrolyzers
may also be methylamine utilizers. Hydrolysis of 4.5 kg
carbofuran ha 21 (equivalent to 2 mg g 21) will result in the
formation of 280 ng methylamine g 21 soil. Thus, the methylamine will provide 110 ng C and 130 ng N g 21 for serving
as C and N sources for methylotrophic bacteria. A typical
bacterial cell has a dry mass of 280 fg and consists of 50% C
and 14% N (Neidhardt et al., 1990). Thus, 280 ng methylamine g 21 could support from 7:9 £ 105 to 3:3 £
106 cells g21 soil, if N and C are not limiting factors, respectively. Due to the large difference between the lower and
upper con®dence intervals (Table 3), even an increase of
3:3 £ 106 cells g21 would not result in a statistically significant increase in the community size of methylamine utilizers. In this study, ®ve replicates for each dilution were
employed for all MPN experiments, an increase to 10 replicates may provide smaller difference between lower and
upper con®dence intervals. As a consequence, release of
280 ng methylamine g 21 from hydrolysis of carbofuran
may result in a statistical increase in MPN for methylamine
utilizers.
Communities of carbofuran ring degraders in the ®eld
treated and untreated soils after receiving 10 mg carbofuran g 21 in laboratory did not increase numerically, suggesting that the ring structure of carbofuran did not provide a
carbon source for the growth of microorganisms, even
though the structure was microbially degraded. This ®nding
was supported by the results of MPN estimations of carbofuran ring degraders in soil from the treated plots during
three consecutive annual ®eld applications of carbofuran
(Table 1). Similarly, the MPN of carbofuran phenol ring
degraders in the treated and untreated soils after receiving
10 mg carbofuran phenol g 21 did not increase numerically
as well. Therefore, enhanced degradation of carbofuran in
the treated soil (Trabue et al., 1997) may have been due to
an increase in enzyme activity responsible for carbofuran
ring degradation. Merica and Alexander (1990) also found
that the MPN of carbofuran ring degraders did not increase
in enhanced soil in 2 and 12 d after receiving 10 mg URL
14
C-carbofuran g 21.
In conclusion, our results indicated that communities of
microorganisms capable of degrading the ring structure of
carbofuran during three consecutive years of carbofuran
treatments remained small and stable, and hence the degradation is a cometabolic process.
Acknowledgements
We thank John E. Thomas for technical assistance.
Sincere appreciation is extended to David P. Weingartner
for ®eld treatments of carbofuran. This study was partially
supported by a grant from USDA. Florida Agricultural
Experimental Station journal series No. R-06805.
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