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Biodegradation of glyphosate herbicide in vitro using bacterial isolates from four rice fields

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Glyphosate is a compound used as herbicide in the control and/or killing of grasses and herbaceous plants. It can be used in no-till agriculture, to prepare fields before planting, during crop development and after crop harvest. Because of its toxicity to non-target organisms, there is need to decontaminate glyphosate contaminated soils and bioremediation is a very useful alternative to conventional cleanup methods. The success of this will depend on isolating bacteria with the ability to degrade glyphosate in a changing environment. The abilities of five bacterial species (Escherichia sp, Azotobacter sp., Alcaligenes sp., Acetobacter sp. and Pseudomonas fluorescens) to degrade glyphosate herbicide under varying environmental conditions were evaluated in this study. The isolates were screened for glyphosate utilization using mineral salt medium containing glyphosate as carbon and/or phosphorus source. Of the five bacterial isolates, P. fluorescens and Acetobacter sp. showed the capacity to utilize glyphosate efficiently and were therefore used for further biodegradation studies. Time course of growth of the isolates on mineral salt medium containing glyphosate showed that both grew significantly (P < 0.05). Microbial growth during the study was monitored by measuring the optical density at 660 nm. The comparative effects of glyphosate as carbon and/or phosphorus source on the growth of the isolates showed that there was significant (P < 0.05) growth in the medium containing glucose and glyphosate. The effects of different concentrations of glyphosate on the growth of the isolates (P. fluorescens and Acetobacter sp) were evaluated. Significant (P < 0.05) growth was observed at lower concentrations (7.2 -25 mg/ml) of glyphosate. No inhibition of growth was observed at high concentrations (100 -250 mg/ml), indicating that the isolated bacteria can tolerate up to 250 mg/ml of glyphosate. However, there was subsequent decrease in growth of both isolates as the concentration of glyphosate increased. This study showed that P. fluorescens and Acetobacter sp. exhibited a high capacity to efficiently degrade glyphosate under the environmental conditions studied. Thus, the organisms can be exploited for biodegradation of glyphosate and should be studied for their ability to degrade other organophosphates.
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African Journal of Biotechnology Vol. 9 (26), pp. 4067-4074, 28 June, 2010
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2010 Academic Journals
Full Length Research Paper
Biodegradation of glyphosate herbicide in vitro using
bacterial isolates from four rice fields
A. N. Moneke*, G. N. Okpala and C. U. Anyanwu
Department of Microbiology, University of Nigeria, Nsukka, Enugu State, Nigeria.
Accepted 18 May, 2010
Glyphosate is a compound used as herbicide in the control and/or killing of grasses and herbaceous
plants. It can be used in no-till agriculture, to prepare fields before planting, during crop development and
after crop harvest. Because of its toxicity to non-target organisms, there is need to decontaminate
glyphosate contaminated soils and bioremediation is a very useful alternative to conventional cleanup
methods. The success of this will depend on isolating bacteria with the ability to degrade glyphosate in a
changing environment. The abilities of five bacterial species (Escherichia sp, Azotobacter sp.,
Alcaligenes sp., Acetobacter sp. and Pseudomonas fluorescens) to degrade glyphosate herbicide under
varying environmental conditions were evaluated in this study. The isolates were screened for
glyphosate utilization using mineral salt medium containing glyphosate as carbon and/or phosphorus source.
Of the five bacterial isolates, P. fluorescens and Acetobacter sp. showed the capacity to utilize glyphosate
efficiently and were therefore used for further biodegradation studies. Time course of growth of the
isolates on mineral salt medium containing glyphosate showed that both grew significantly (P < 0.05).
Microbial growth during the study was monitored by measuring the optical density at 660 nm. The
comparative effects of glyphosate as carbon and/or phosphorus source on the growth of the isolates
showed that there was significant (P < 0.05) growth in the medium containing glucose and glyphosate.
The effects of different concentrations of glyphosate on the growth of the isolates (P. fluorescens and
Acetobacter sp) were evaluated. Significant (P < 0.05) growth was observed at lower concentrations (7.2 -
25 mg/ml) of glyphosate. No inhibition of growth was observed at high concentrations (100 - 250 mg/ml),
indicating that the isolated bacteria can tolerate up to 250 mg/ml of glyphosate. However, there was
subsequent decrease in growth of both isolates as the concentration of glyphosate increased. This study
showed that P. fluorescens and Acetobacter sp. exhibited a high capacity to efficiently degrade
glyphosate under the environmental conditions studied. Thus, the organisms can be exploited for
biodegradation of glyphosate and should be studied for their ability to degrade other organophosphates.
Key words: Glyphosphate herbicide, degradation process.
INTRODUCTION
The intensive use of herbicides in rice-based cropping
systems is a general practice and thus a matter of
environmental concern. This is as a result of the potential
hazardous effects of these chemicals on soil biological
processes, non-target organisms and pollution of streams
*Corresponding author. E-mail: annymoneke@yahoo.com. Tel:
08033357734.
and rivers through runoffs. The common herbicides in
use include 2,4-dicholorophenoxyacetic acid (2,4-D) and
Roundup® (isoproplyamine salt of glyphosate).
Glyphosate on its own may be relatively harmless to
humans. It is however formulated with surfactants such
as polyoxyethylene amine (POEA) which is more toxic than
glyphosate alone (Atkinson, 1985). Also, 2,4-
dichlorophenoxy-acetic acid which is used by most
farmers to spike glyphosate in order to boost its efficacy
increases the toxicity of glyphosate.
4068 Afr. J. Biotechnol.
Table 1. State and location from where the soil
samples were collected.
State Location Code
Anambra Omor OMR
Anambra Omasi OMS
Enugu Adani ADA
Ebonyi Abakaliki AKL
Organophosphates, including glyphosate, account for
half of the pesticides used worldwide with glyphosate
based formulations such as Roundup®, Accord® and
Touchdown® consisting the commonest types used for
agricultural purposes (Franz et al., 1997). Glyphosate is a
broad spectrum, non-selective herbicide used in the
control and/or killing of grasses, herbaceous plants,
including deep rooted perennial weeds, brush, some
broad-leaf trees and some shrubs (United States
Department of Agriculture (USDA), 2000; Cox, 2000). It
can be used in no-till agriculture, to prepare fields before
planting, during crop development and after crop harvest
(USDA, 2000). Its mode of action is the
inhibition of 5-
enolpyruvylshikimate-3-phosphate synthase, resulting
in
the depletion of essential aromatic amino acids needed
for
plant survival (Arhens, 1994; Zablotowicz and Reddy,
2004). Although most living organisms lack this metabolic
route such that they would not be potentially affected by
this herbicide, the environmental consequences of the
widespread use of glyphosate have been reported (Cox,
2000; Santillo et al., 1989). On application, glyphosate
remains unchanged in the soil for varying lengths of time,
as a result of its adsorption on clay particles and organic
matter present in the soil (Penaloza-Vazquez et al.,
1995). The removal of glyphosate from the environment
is usually by microbiological processes as chemical
process of degradation is ineffective because of the
presence of highly stable bonds (carbon-phosphorus
bond) present in the compound (Gimsing et al., 2004).
Glyphosate is microbially degraded in soil and
water and
has a reported field half-life of 47 days and a laboratory
half-life of < 25 days (Ahrens, 1994). Studies of
glyphosate degrading bacteria have involved selection
for, and isolation of pure bacterial strains with enhanced or
novel detoxification capabilities for potential uses in
biotechnology industry and biodegradation of polluted
soils and water. Microorganisms known for their ability to
degrade glyphosate in soil and water include
Pseudomonas sp strain LBr (Jacob et al., 1988),
Pseudomonas fluorescens (Zboinska et al., 1992),
Arthrobacter atrocyaneus (Pipke et al., 1988) and
Flavobacterium sp. (Balthazor and Hallas, 1986). Bacteria
degrade glyphosate via two general pathways leading to
the intermediate production of either glycine or
aminomethylphosphonate (AMPA). The use of glyphosate
based formulations in rice farming is a common practice
in south eastern Nigeria and in oil palm (Elaeis
guineensis) plantation in the rainforest area of Nigeria.
No work has been done to identify glyphosate degrading
bacteria from rice farms in Nigeria and the conditions that
enhance the degradation process. The main objectives
of the present study are the isolation and identification of
glyphosate utilizing bacteria from rice farms using an
enrichment culture technique, assessment of the growth
response of the isolates in liquid medium containing
different concentrations of glyphosate and analysis of the
comparative effects of glyphosate as carbon or
phosphorus source on the isolates.
MATERIALS AND METHODS
Chemicals used
The isopropylamine
salt of glyphosate known as Roundup®
(containing 360 g active ingredient/L of glyphosate, Monsanto) was
purchased from a local dealer’s store in Nsukka, Enugu state,
Nigeria. All other chemicals were of the highest purity commercially
available.
Collection of soil samples
Soil samples were obtained from four rice fields located at Adani in
Enugu State, Nigeria, Omor and Omasi in Anambra State, Nigeria
and Abakaliki in Ebonyi State, Nigeria, all in south eastern Nigeria.
These rice fields are known to have been previously exposed to
glyphosate-based formulation (Roundup®) for long periods of time.
Soil samples were collected from depths of 0 - 15 cm from three
different sites in each of the four locations. Soil samples from each
site were thoroughly mixed. They were designated as shown in
Table 1. All samples were placed in sterile polyethylene bags and
taken immediately to the laboratory and stored at C before use
within 24 h (Nannipieri, 1994).
Isolation medium
A modified mineral salts medium (MSM) of Dworkin and Foster
(1958) consisting of (g/l) (NH
4
)
2
SO
4
,
0.375;
MgSO
4
, 0.075; CaC0
3
,
0.03;
FeSO
4
.7H
2
O, 0.001; H
3
BO
3
, 0.000001, MnSO
4
, 0.000001 and
yeast extract, 0.0053 was used. Phosphate buffer was replaced by
tris buffer 6.05 g/L and pH was adjusted to 7.0. All glasswares were
washed with 1 N HCl and thoroughly rinsed with deionized water to
remove contaminating phosphate before use. The medium was
autoclaved at 121°C and 15 psi for 15 min prior to the addition of
the filter sterilized Roundup® (isopropylamine salt of glyphosate)
and glucose (1.0) autoclaved at 110°C and 10 psi as carbon
source.
Isolation of glyphosate utilizing bacteria
The soil samples were air-dried and sieved using a 2 mm mesh. 5 g
of each soil sample was suspended in 250-ml Erlenmeyer flasks
containing a mixture of 50 ml of mineral salts medium and 1 ml of
Roundup® (7.2 mg/ml of glyphosate). This concentration was used
because it is equivalent to the field application rate. The flasks were
incubated on a rotary shaker (Gallenkamp, England) at 120 rpm for
7 days at 30°C. The above steps were repeated by taking 1.0 ml of
sample from each broth culture and transferred to fresh enrichment
medium followed by incubation as described for 7 days. Isolation
was done using the spread plate method on the solid mineral salts
medium described above with added glyphosate. The plates were
incubated at 30°C for 5 days. Morphologically distinct colonies were
re-isolated and were repeatedly sub-cultured on nutrient agar
(Fluka). Identity of the isolates was affirmed after characterization
by standard bacteriological methods (Holt et al., 1994; Cheesbrough,
1984). Stock cultures were maintained in nutrient agar slants at
C.
Inoculum preparation and standardization
Inocula used for the study were prepared by inoculating isolates
into nutrient broth and incubated at 30°C for 24 h using sterile
normal saline; the cells from the above cultures were resuspended
to a 0.5 McFarland nephelometer standard (Optical density of 0.17
at 660 nm).
Glyphosate utilization patterns of the different isolates
A 1.0 ml portion of each isolate was inoculated into 150 ml of the
screening medium (contained in a 500-ml flask) which is the
isolation medium without yeast extract. It contained 3 ml of roundup
(7.2 mg/ml of glyphosate). The flasks were incubated on a rotary
shaker (Gallenkamp, England) at 120 rpm for 180 h at 30°C. The
ability of each isolate to utilize glyphosate was measured based on
the turbidity of the medium at 660 nm using a spectrophotometer
(Spectronic 20, USA).
Determination of time course of the growth of P. fluorescens
and Acetobacter sp.
500-ml Erlenmeyer flasks containing 150 ml of the sterile screening
medium was prepared and 3 ml of roundup (containing 7.2 mg/ml of
glyphosate) was added to each flask. 1 ml of the inoculum (0.5
Macfarlane standards) of each selected isolate was used to
inoculate each flask (experiments were carried out in 3 replicates).
The two isolates used were selected based on their utilization
patterns. The medium was then incubated at 30°C for 192 h on a
shaker at 120 rpm. 5 ml of the culture medium was collected from
each flask at 12 h intervals and assayed for growth by measuring
the optical density at 660 nm using a spectrometer.
Comparative role of glyphosate as carbon or phosphorus
source
The screening medium (150 ml) was prepared as earlier described
and 3.0 ml filter-sterilized roundup (containing 7.2 mg/ml of
glyphosate) was added as phosphorus or carbon source. When
used as carbon source, denoted by Gly and Pi, the medium
consisted of the following (g/L): (NH
4
)
2
SO
4
,
0.375; MgSO
4,
0.075;
CaC0
3
,
0.03;
FeSO
4
.7H
2
O, 0.001; H
3
BO
3
, 0.000001; MnSO
4
,
0.000001; NaHPO
4
, 6.0
and KH
2
PO
4
, 2.0. When used as carbon
and phosphorus source, denoted by (Glyphosate), the medium
consisted of the following (g/L): (NH
4
)
2
SO
4
,
0.375;
MgSO
4,
0.075;
CaC0
3
,
0.03;
FeSO
4
.7H
2
O, 0.001; H
3
BO
3
, 0.000001; MnSO
4
,
0.000001; tris buffer 6.05 g. When used as phosphorus source
denoted by (Gly and Glu), the medium consisted of the following
(g/L): (NH
4
)
2
SO
4
,
0.375;
MgSO
4,
0.075; CaC0
3
,
0.03;
FeSO
4
.7H
2
O,
Moneke et al 4069
0.001; H
3
BO
3
, 0.000001; MnSO
4
, 0.000001; glucose, 1.0; tris buffer
6.05 g. The media was incubated at 3C for 120 h on a shaker at
120 rpm. 5 ml of the culture medium was collected from each flask
at 12 h intervals and assayed for growth by measuring the optical
density at 660 nm using a spectrometer.
Effects of different concentrations of glyphosate on the growth
of the isolates
Aliquots (1.0 ml) of 24 h old bacterial cultures (0.5 MacFarland
standard) grown in nutrient broth were inoculated into 500-ml
Erlenmeyer flasks containing 150 ml of MSM supplemented with
various concentrations of glyphosate (25, 50, 100 and 250 mg/ml).
A control was maintained with MSM supplemented with 7.2 mg/ml
of glyphosate. Bacterial growth was monitored by withdrawal of 5.0
ml of culture sample immediately after inoculation for the 0 h and
every 12 h up to 108 h of incubation and optical density was measured
at 660 nm.
Treatment effects on the growth of the bacterial isolates at the
different time periods were analysed using 2-way ANOVA.
RESULTS
Isolation of glyphosate utilizing bacteria
The preliminary studies with glyphosate as phosphorus
source showed that a total of twelve bacterial isolates
were able to grow in the presence of glyphosate as sole
phosphorus source. On further sub-culturing on solid
medium, five isolates consistently grew on the MSM
enriched with glyphosate as phosphorus source. They
are: Acetobacter sp. - G ADA3, Escherichia sp. - G AKL2,
P. fluorescens - G AKL5, Azotobacter sp. - G OMR1 and
Alcaligenes sp. - G OMS1.
Glyphosate utilization patterns of the different
isolates
The five bacterial isolates were screened for glyphosate
utilization by measuring their growth turbidimetrically at
660 nm. Of the five bacterial isolates grown on the
medium containing glyphosate as sole phosphorus
source, P. fluorescens significantly (P < 0.05) utilized
glyphosate (mean OD 0.1268). This was followed by
Acetobacter sp., Azotobacter sp. and Alcaligenes sp.
(mean OD 0.1069, 0.0858 and 0.0841, respectively).
Escherichia sp. did not show any appreciable growth as
shown in Figure 1.
Growth kinetics of P. fluorescens and Acetobacter
sp. in glyphosate
The growth kinetics of P. fluorescens and Acetobacter sp.
were further monitored over time at 660 nm, using the
MSM enriched with glyphosate as sole phosphorus source.
Their growths were both significant (P < 0.001), but that
4070 Afr. J. Biotechnol.
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180
Time (Hours)
A b s o rb a n c e (6 6 0 n m )
Acetobacter sp Escherichia sp P seudomo nas fluo rescens Azo to bacter s p Alcaligenes sp
Figure 1. Screening of the isolates for glyphosate utilization.
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192
Time (Hours)
Ab so rb an ce (660 n m )
Acetobacter sp Pseudomonas fluorescens
Figure 2. Growth kinetics of Acetobacter sp. and P. fluorescens on glyphosate.
but that of P. fluorescens was significantly (P < 0.05)
higher than that of Acetobacter sp. as shown in Figure 2.
Comparative role of glyphosate as carbon or
phosphorus source
The ability of glyphosate to serve as carbon source,
phosphorus source, carbon and phosphorus source was
monitored. Optical density measurement at 660 nm was
used to monitor increase in cell numbers. The growth of
Acetobacter sp. was non-significantly (P < 0.05) higher in
the Glucose and Glyphosate (Gly and Glu) medium
(mean OD value = 0.0907) when compared to P.
fluorescens (mean OD value = 0.09003) (Figure 3). On
the medium with glyphosate as carbon and phosphorus
source, the growth of P. fluorescens was significantly
higher when compared to Acetobacter sp. The growth
kinetics of the isolates in the different carbon sources
showed that there was progressive increase in growth of
the isolates when glyphosate was used as a phosphorus
source and glucose as carbon source. The growth of
Acetobacter sp. after 24 h incubation on Glu and Gly
medium was more significant (P < 0.05) with a mean OD
value of 0.0933 when compared with the growth on the
GPi, glyphosate and the control (Figure 4). Also, the
growth of Acetobacter sp. on the GPi medium peaked
after 36 h with mean OD value of 0.02733, which was
significantly (P < 0.05) higher than growth on the medium
containing glyphosate and the control. After 36 h of
incubation, the growth on the Glu and Gly medium
consistently increased till the end of the monitoring (120
Moneke et al 4071
0.00000
0.01000
0.02000
0.03000
0.04000
0.05000
0.06000
0.07000
0.08000
0.09000
0.10000
Glucose Gly and Glu Glyphosate Gly and Pi
Carbon source
Optical Density (660nm )
Acetob acter sp Pseudomonas fluorescens
Figure 3. Comparative effect of glyphosate as carbon or phosphorus source on Acetobacter sp. and P
fluorescens.
0.00000
0.05000
0.10000
0.15000
0 12 24 36 48 60 72 84 96 108 120
Time (Hrs)
Absorbance (660nm)
Glucose Glyphosate Gly and Glu Gly and Pi
Figure 4. Growth kinetics of Acetobacter sp. in glyphosate as carbon or phosphorus source.
h). The growth of P. fluorescens in the Glu and Gly
medium followed a similar pattern as that of Acetobacter sp.
as shown in Figure 5.
Effects of different glyphosate concentration on the
growth of Acetobacter sp. and P. fluorescens
The growth of Acetobacter sp. and P. fluorescens in
different concentrations of glyphosate gave an inverse
result as shown in Figure 6. As the concentration of
glyphosate increased there was a corresponding
decrease in the growth of the isolates. The highest
growth was observed in the control (7.2 mg/ml) which
contained the least concentration of glyphosate. The
growth kinetics of both isolates in increasing
concentrations of glyphosate followed a similar pattern
with a lag phase of about 12 h and steady increase in
growth as seen in Figures 7 and 8. After 84 h of
incubation the growth of P. fluorescens in medium
containing 25 mg/ml of glyphosate increased significantly
(P < 0.05) when compared with its growth in the medium
with 7.2 mg/ml of glyphosate till the end of the monitoring
at 108 h.
DISCUSSION
Twelve bacterial isolates were initially isolated from rice
field soil samples. On further sub-culturing on solid media
enriched with glyphosate, only five showed the capacity
to grow in the presence of the herbicide. The five
bacterial isolates were identified as Acetobacter sp.,
Escherichia sp., P. fluorescens, Azotobacter sp., and
4072 Afr. J. Biotechnol.
0.00000
0.05000
0.10000
0.15000
0 12 24 36 48 60 72 84 96 108 120
Time (Hrs)
Glucose Glyphosate Gly and Glu Gly and Pi
Figure 5. Growth kinetics of P. fluorescens in glyphosate as carbon or phosphorus source.
0.0000
0.0200
0.0400
0.0600
0.0800
0.1000
0.1200
0.1400
0.1600
Optical density (660 nm )
7.2mg/ml 25mg/ml 50mg/ml 100mg/ml 250mg/ml
Acetobacter sp
Pseudomonas fluorescens
Figure 6. Effects of the different concentrations of glyphosate on the growth of Acetobacter sp.
and P. fluorescens.
0.000
0.050
0.100
0.150
0.200
0.250
0 12 24 36 48 60 72 84 96 108
Time (hours)
Ab so rb a n ce (660n m )
7.2mg/ml 25mg/ml 50mg/ml 100mg/ml 250mg/ml
Figure 7. Growth kinetics of Acetobacter sp. on the different concentrations of glyphosate.
Moneke et al 4073
0.0000
0.0500
0.1000
0.1500
0.2000
0 12 24 36 48 60 72 84 96 108
Time (Hours)
A b s o rb a n c e (6 6 0 n m )
7.2mg/ml 25mg/ml 50mg/ml 100mg/ml 250mg/ml
Figure 8. Growth ksinetics of P. fluorescens on the different concentrations of glyphosate.
Alcaligenes sp. The results of this study, which showed a
reduction in the number of bacterial species grown on
glyphosate solid medium, are consistent with the report of
Busse et al. (2001) who showed that culturable bacteria
and fungi are usually reduced in number or eliminated
when extracted from soil or grown on solid media
containing glyphosate. Other studies (Quinn et al., 1988;
Santos and Flores, 1995; Kryzsko-Lupicka and Orlik,
1997) had found similar reductions in population counts
when glyphosate was added to culture media. Toxicity of
the artificial media is expected to be based on the mode
of action of glyphosate (inability of the organism to
synthesize the needed aromatic amino acids). Unlike the
response in artificial media, no toxicity was expressed
when glyphosate was added to soil in laboratory
bioassays (Busse et al., 2001).
The isolated bacterial species had previously been
obtained from other soil samples (Zboinska et al., 1992,
Franz et al., 1997). Of the seven identified bacterial species,
two (Acetobacter sp. and P. fluorescens) were selected
for further biodegradation studies based on their short lag
phase and rapid utilization of glyphosate. Many
Pseudomonas species have been used extensively in the
degradation and/or metabolism of glyphosate (Jacob et
al., 1988; Shinabarger et al., 1984; Kishore and Jacob,
1987; Talbot et al., 1984. However, Zboinska et al. (1992)
reported that P. fluorescens could not utilize glyphosate.
The strain of P. fluorescens used in this study was not
only able to utilize glyphosate but was also able to thrive
at high concentrations of the herbicide. The use of
Acetobacter in the degradation/metabolism of glyphosate
has not been reported.
Both organisms used in this study showed appreciable
growth in the culture medium containing glyphosate as
sole phosphorus source. The differences observed in the
growth of the isolates in the medium are indicative of the
differences between the organisms in tolerating the
herbicide. The short lag phase, coupled with rapid growth
of the two isolates, showed effective utilization of
glyphosate by the selected isolates. P. fluorescens attained
maximum growth and peaked at 132 h incubation, while
Acetobacter sp. achieved maximum growth and peaked
at 72 h incubation. There have been several reports on
the ability of microorganisms, including some Pseudomonas
sp., to effectively utilize glyphosate by naturally
synthesizing appropriate enzymes or as a result of genetic
mutation (Jacob et al., 1988; Shinabarger et al., 1984;
Kishore and Jacob, 1987). So far, there has been no
report on the ability of P. fluorescens to utilize glyphosate
as sole phosphorus or carbon source. The high capacity
of these two organisms to utilize this herbicide in vitro
could be attributed to their previous contact with the
herbicide in the soil (rice fields) from where they were
isolated. It is also possible that the organisms have
undergone genetic mutation leading to the adaptability of
the organisms to their microenvironment.
The results of the comparative role of glyphosate as
carbon or phosphorus source for the two isolates showed
that glyphosate serves as a better phosphorous source
than as a carbon source. Many bacterial isolates have
been reported to utilize glyphosate as a phosphorus
source (Liu et al., 1991; Balthazor and Hallas, 1986; Dick
and Quinn, 1995). Of the two isolates used in the present
study, P. fluorescens demonstrated a better capacity to
utilize glyphosate as carbon and phosphorus source.
Glyphosate served as a better phosphorus source than
carbon source for the Acetobacter sp. The presence of
inorganic phosphate in the growth medium affected the
effective uptake of glyphosate in both organisms. This is
because inorganic phosphate has been reported to sup-
4074 Afr. J. Biotechnol.
press the genes coding for the C-P lyase system and
thus make them unable to metabolise glyphosate (Liu et
al., 1991). Our results in this study agree with this report.
The results of this study showed that increase in
glyphosate concentration led to a concomitant decrease
in the growth of the isolates. This is in contrast with the
report of Amoros et al. (2007), who, while studying the
effects of roundup (glyphosate) at different concentrations
(50 and 100 mg/l) observed an increase in Aeromonas
counts in contrast with the control which contained no
glyphosate. However, Carlisle and Trevors (1988)
observed that glyphosate can either stimulate or inhibit
soil microorganisms depending on the soil type or
herbicide concentration. In our study, high cell density
was recorded for both isolates at glyphosate
concentrations of 7.2 - 50 mg/ml after 24 h at 3C.
However, at higher concentrations (100 and 250 mg/ml),
the cell density was very low compared with the control
(7.2 mg/ml). Even though a severe decline in growth of
the organisms occurred at high concentrations (100 and
250 mg/ml), the isolates were still able to tolerate 250
mg/ml of glyphosate. A possible explanation may be the
presence of novel degradative systems in the organisms.
Conclusion
Herbicides are extensively used in farming practices.
Some of the farmers, due to illiteracy and impatience,
apply more than the stipulated quantity of herbicide per
application. These chemicals may persist for long periods
of time in the environment, whereby they may affect non-
target organisms. This study reports the isolation and
identification of two bacterial species, P. fluorescens and
Acetobacter sp. that possess the capacity to utilize
glyphosate. The ability of these isolates to utilize
glyphosate effectively provides a means of removing this
compound from the environment. Thus the capacity of
the isolates to survive and grow in the presence of high
concentrations of the herbicide marks them out as good
candidates for the bioremediation of glyphosate-polluted
environments.
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... Исследования проведены при содержании глифосата в среде 3,0 мг/мл. Следующий этап скрининга выполнен в жидкой минеральной среде (MSM) Дворкина-Фостера [36]. В ходе экспериментов в конические колбы объемом 1000 мл, содержащие 450 мл жидкой питательной среды Дворкина-Фостера (без источника углерода), вносили 5 мл инокулюма исследуемой двухсуточной бактериальной культуры. ...
... На завершающем этапе проведен количественный скрининг способности ризобактерий метаболизировать глифосат как единственный источник углерода. Для этого бактерии культивировали в жидкой питательной среде Дворкина-Фостера [36]. Скрининг в жидких питательных средах позволяет количественно оценивать рост по плотности популяции микроорганизмов. ...
... По литературным данным общей физиологической особенностью процессов биодеградации фосфорорганических соединений является наличие латентного периода, то есть требуется определенный временной промежуток для адаптации микробных клеток к гербициду [20]. Замедление роста и более длинная lag-фаза были отмечены в работах многих исследователей -Moneke A. N., Okpala G. N., Anyanwu C. U. [36], Quinn J. P., Peden J. M, Dick R. E. [37]. ...
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... [15], Achromobacter sp. Strain kg16, Ochrobactrumenthropi strain GPK3 [16], Arthrobacteratrocyaneus [17] and Acetobactersp [5]. ...
... Firdous [24] reported the presence of Alcaligenes in glyposate contaminated soil and this also agreed with the findings of the present study. Mussali-Gadante eLebeau, [21] also reported the presence of Sinorhizobium which corresponds with the findings in this study.Acinetobacter sp has been reported by Olawale et al, (2011) [21] as a glyphosate degrader while Moneke et al, (2010) [5] reported the presence of Alcaligenes sp. in glyphosate contaminated soil. Mussali et al. [22] also reported the presence of Sinorhizobium sp. ...
... These bacteria have particularly shown tolerance to endosulfan, Graphical Abstract phorate, carbendazim, chlorpyrifos (CP), pendimethalin, among others (Castillo et al. 2011;Chennappa et al. 2014a;Gurikar et al. 2016;Rani and Kumar et al. 2017), without showing growth inhibition . Also, there are reports describing the degradation of lindane (Anupama and Paul 2009), phorate (Moneke et al. 2010), endosulfan (Castillo et al. 2011), pendimethalin (Chennappa et al. 2018a), glyphosate (Mousa et al. 2021), and CP by Azotobacter isolates (Chennappa et al. 2019). ...
... (Askar and Khudhur 2013;Chennappa et al. 2013;Walvekar et al. 2017;Kumar et al. 2019); e.g. reduced growth rate in the presence of CP (Menon et al. 2004), glyphosate (Moneke et al. 2010) and endosulfan (Castillo et al. 2011), inhibition of diazotrophic activity (Menon et al. 2004;Chennappa et al. 2019), reduced respiration rate with glyphosate, pendimethalin and fomesafen (Chennappa et al. 2013(Chennappa et al. , 2014bWu et al. 2014), cell damage and loss of viability after exposure to different concentrations of glyphosate and atrazine (Shahid et al. 2019). ...
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... These bacteria have particularly shown tolerance to endosulfan, phorate, carbendazim, chlorpyrifos (CP), pendimethalin, among others (Castillo et al. 2011;Chennappa et al. 2014a;Gurikar et al.2016;Rani and Kumar et al. 2017), without showing growth inhibition . Also, there are reports describing the degradation of lindane (Anupama and Paul, 2009), phorate (Moneke et al. 2010), endosulfan (Castillo et al. 2011), pendimethalin (Chennappa et al.2018a), glyphosate (Mousa et al. 2021), and CP by Azotobacter isolates (Chennappa et al. 2019). ...
... (Askar and Khudhur 2013;Chennappa et al. 2013;Walvekar et al. 2017;Kumar et al. 2019); e.g. reduced growth rate in the presence of CP (Menon et al. 2004), glyphosate (Moneke et al. 2010) and endosulfan (Castillo et al. 2011), inhibition of diazotrophic activity (Menon et al. 2004;Chennappa et al. 2019), reduced respiration rate with glyphosate, pendimethalin and fomesafen (Chennappa et al. 2013, Wu et al. 2014Chennappa et al. 2014b), cell damage and loss of viability after exposure to different concentrations of glyphosate and atrazine (Shahid et al. 2019) The genus Azotobacter can exhibit varied behaviors depending on the species and strains, growth conditions, type of pesticide, and contaminant concentrations; therefore, it is useful to evaluate the effect of these factors on model organisms such as Azotobacter vinelandii (Noar and Bruno-Bárcena, 2018); A. vinelandii is a strictly aerobic free-living bacterium with growth and metabolite production, both in vitro and in soil, closely related to physicochemical parameters (Lenart 2012;Plunkett et al. 2020), nutrient concentration and availability (essentially carbon and nitrogen sources) (Tejera et al. 2005;Then et al. 2016), microbial interactions (Bhosale et al. 2013), exposure to toxic substances (Chennappa et al. 2019) and oxygenation levels Castillo et al. 2013), the latter being one of the critical parameters because of the high oxygen rate consumption of Azotobacter spp. On this regard, some aspects of the respiration in A. vinelandii have been evaluated widely concerning its growth and polymers synthesis (Lozano et al. 2011;Castillo et al. 2020). ...
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This study aimed to evaluate the growth, respiratory activity, and biodegradation of chlorpyrifos in cultures of Azotobacter vinelandii ATCC 12837. A strategy based on the modification of culture media and aeration conditions was carried out to increase the cell concentration of A. vinelandii , in order to favor and determine its tolerance to chlorpyrifos and its degradation ability. The culture in shaken flasks, using sucrose as a carbon source, significantly improved the growth compared to media with mannitol. When the strain was cultivated under oxygen-limited (5.5, 11.25 mmol L- 1 h- 1 ) and no-oxygen-limited conditions (22 mmol L -1 h -1 ), the growth parameters were not affected. In cultures in a liquid medium with chlorpyrifos, the bacteria tolerated a high pesticide concentration (500 ppm) and the growth parameters were improved even under conditions with a reduced carbon source (sucrose 2 g L -1 ). The strain degraded 99.6 % of chlorpyrifos at 60 h of cultivation, in co-metabolism with sucrose; notably, A. vinelandii ATCC 12837 reduced by 50% the initial pesticide concentration in only 6 h (DT 50 ).
... The intensive use ofherbicide is a general practice and thus a matter of environmental concern. This is as a result of the potential hazardous effects of the chemicals on soil biological processes, non-target organisms and pollution of streams and rivers through runoffs [2]. Pesticides have significant chronic human health effects including cancer, neurological effects, diabetes, respiratory diseases, fetal disease and genetic disorder [3,4]. ...
... The MIC values of the extracts were found to have various ranges, thus indicating that evaluation of MIC is sufficient for measuring bacterial activity [10]. ...
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... Pseudomonas species have been found to grow on diesel and jet fuel where they are regarded as hydrocarbon utilizing micro-organism (Biopharma, 2007) and in glyphosate treated soil by utilizing inorganic phosphate as a source of nutrient (Moneke et al., 2010). Studies of glyphosate degrading bacteria have involved selection for and isolation of pure bacterial strains with enhanced or novel detoxification capabilities for potential uses in biotechnology industry and biodegradation of polluted soils and water (Gimsing et al., 2004). ...
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Glyphosate, as one of the broad-spectrum herbicides for controlling annual and perennial weeds, is widely distributed in various environments and seriously threatens the safety of human beings and ecology. Glyphosate is currently degraded by abiotic and biotic methods, such as adsorption, photolysis, ozone oxidation, and microbial degradation. Of these, microbial degradation has become the most promising method to treat glyphosate because of its high efficiency and environmental protection. Microorganisms are capable of using glyphosate as a phosphorus, nitrogen, or carbon source and subsequently degrade glyphosate into harmless products by cleaving C–N and C–P bonds, in which enzymes and functional genes related to glyphosate degradation play an indispensable role. There have been many studies on the abiotic and biotic treatment technologies, microbial degradation pathways and intermediate products of glyphosate, but the related enzymes and functional genes involved in the glyphosate degradation pathways have not been further discussed. There is little information on the resistance mechanisms of bacteria and fungi to glyphosate, and previous investigations of resistance mechanisms have mainly focused on how bacteria resist glyphosate damage. Therefore, this review explores the microorganisms, enzymes and functional genes related to the microbial degradation of glyphosate and discusses the pathways of microbial degradation and the resistance mechanisms of microorganisms to glyphosate. This review is expected to provide reference for the application and improvement of the microbial degradation of glyphosate in microbial remediation.
Chapter
The usage of pesticides in agricultural practices contributes to an improvement in food production through monitoring insects, weeds, and crop diseases, with the aim of ensuring food sustainability to meet the needs of increasing population. However, the widespread application of these substances has numerous harmful consequences on the soil and on both environmental and human health. Moreover, in developed nations, the nonrational usage of chemicals, as well as banned forms, presents a major danger and increases contaminated agricultural lands at alarming rates, as well as polluting surface and groundwater. Glyphosate [N-(phosphonomethyl) glycine] is a broad-spectrum systemic herbicide that blocks the enzyme required by plants to produce amino acids and proteins. At present, in all fields (environment, agriculture, toxicology), it has become important to talk about paraquat (1,1′-dimethyl-4,4′-bipyridium) when it comes to glyphosate. Similarly, in recent years, these two herbicides have been the focus of many toxicities and biodegradation studies. Bioremediation by microbial biotechnology is also one of the most recommended solutions to alleviate the impact of these contaminants and is known to be an environmentally safe soil and water remediation technology. The principle of bioremediation is the modification and removal of pesticides in the form of nontoxic compounds used as nutrients for plants. Several approaches are commonly used, such as biostimulation and bioaugmentation. The discovery of potent microbial strains and the screening of degradation genes are currently a challenge for scientific researchers. In this context, this chapter highlights and summarizes contaminants, their environmental implications, and the biotechnological use of bacteria that may be used for bioremediation in order to remediate polluted areas.
Chapter
Among all the microbial genera, plant growth-promoting rhizobacteria (PGPR) proved that they stimulate the growth and development of almost all plants and conserving essential properties of soil ecosystem. PGPR is the alternate method in replacing the synthetic fertilizer for crop improvement and better cultivation of different food crops by fixing the nitrogen in the atmosphere and by also producing different growth promoting elements (indole acetic acid, gibberellic acid). Among PGPR genera, species of Azotobacter plays a vital role and is known to produce a wide variety of plant growth-stimulating secondary metabolites and directly influences complete vigor of the plant. Further, Azotobacter species produces certain antimicrobial compounds, which helps in maintaining the plant diseases caused by different group of phytopathogens. Azotobacter species are known to tolerate and degrade synthetic pesticides and are potential bio-agents for sustainable agriculture and maintains the ecological imbalance in the environment.
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The wild-type strain of Pseudomonas fluorescens was found to utilize a range of structurally diverse organophosphonates as its sole carbon or nitrogen sources. Representative compounds included aminoalkylphosphonates, hydroxyalkylphosphonates, oxoalkylphosphonates, and phosphono dipeptides. Among them, amino(phenyl)methylphosphonate,2-aminoethylphosphonate, aminomethylphosphonate, diisopropyl 9-aminofluoren-9-ylphosphonate, and 2-oxoalkylphosphonates were used by P. fluorescens as its sole sources of phosphorus. Only slight growth was observed on the herbicide glyphosate (N-phosphonomethylglycine), which was metabolized to aminomethylphosphonate. Neither phosphinothricin nor its dialanyl tripeptide, bialaphos, supported growth of P. fluorescens. The possible mechanisms of organophosphonate degradation by this strain are discussed.
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We investigated effects of herbicide-induced habitat changes on small mammals in clearcuts in northcentral Maine. Fewer small mammals were captured on glyphosate (nitrogen-phosphonomethyl glycine) (Roundup, Monsanto, St. Louis, Mo.)-treated clearcuts 1-3 years post-treatment compared to untreated clearcuts. Insectivores (Soricidae) comprised 72% of small mammal captures and were less abundant (P < 0.001) for all 3 years post-treatment. Herbivores (Microtinae) were less abundant 1 (P < 0.01) and 2 years (P < 0.001) post-treatment. Omnivores (Cricetinae and Zapodidae) were equally abundant on treated and untreated clearcuts. Differences in small mammal abundance paralleled herbicide-induced reductions in invertebrates and plant food and cover. Patches of untreated vegetation within herbicide-treated clearcuts provided a source of invertebrates and plant food and cover.
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The effect of the herbicide glyphosate (N-(phosphonomethyl)glycine) on the growth, respiration and nitrogen fixation of Azotobacter chroococcum and A. vinelandii was studied. Azotobacter vinelandii was more sensitive to glyphosate toxicity than A. chroococcum. Recommended dosages of glyphosate did not affect growth rates. More than 4 kg ha-1 is needed to find some inhibitory effect. Specific respiration rates were 19.17 mmol O2 h-1 g-1 dry weight for A. chroococcum and 12.09 mmol h-1 g-1 for A. vinelandii. When 20 kg ha-1 was used with A. vinelandii, respiration rates were inhibited 60%, the similar percentage inhibition A. chroococcum showed at 28 kg ha-1. Nitrogen fixation dropped drastically 80% with 20 kg ha-1 in A. vinelandii and 98% with 28 kg ha-1 in A. chroococcum. Cell size as determined by electron microscopy decreased in the presence of glyphosate, probably because glyphosate induces amino acid depletion and reduces or stops protein synthesis.
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The herbicide glyphosate was used as a selection agent for isolation of fungal strains capable to degrade phosphorus-to-carbon bond from standard sandy-clay soil. The studies have shown that the herbicide used in Martin medium as a sole source of phosphorus br carbon caused the decrease of the fungal population and substantially changed strain composition, thus selecting those which are able to degrade glyphosate.
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We assessed the direct and indirect effect of the herbicide glyphosate on soil microbial communities from ponderosa pine (Pinus ponderosa) plantations of varying site quality. Direct, toxic effects were tested using culture media and soil bioassays at glyphosate concentrations up to 100-fold greater than expected following a single field application. Indirect effects on microbial biomass, respiration, and metabolic diversity (Biolog and catabolic response profile) were compared seasonally after 9–13 years of vegetation control using repeated glyphosate applications in a replicated field study. Three pine plantations were selected to provide a range of soil characteristics associated with glyphosate binding (clay, Fe and Al oxide content) and site growing potential from the lowest to the highest in northern California. Glyphosate was toxic to bacteria and fungi from each plantation when grown in soil-free media. Culturable populations were reduced, as was the growth rate and metabolic diversity of surviving bacteria, by increasing concentrations of glyphosate. This toxicity was not expressed when glyphosate was added directly to soil, however. Microbial respiration was unchanged at expected field concentrations (5–50 μg g−1), regardless of soil, and was stimulated by concentrations up to 100-fold greater. Increased microbial activity resulted from utilization of glyphosate as an available carbon substrate. Estimated N and P inputs from glyphosate were inconsequential to microbial activity. Long-term, repeated applications of glyphosate had minimal affect on seasonal microbial characteristics despite substantial changes in vegetation composition and growth. Instead, variation in microbial characteristics was a function of time of year and site quality. Community size, activity, and metabolic diversity generally were greatest in the spring and increased as site quality improved, regardless of herbicide treatment. Our findings suggest that artificial media assays are of limited relevance in predicting glyphosate toxicity to soil organisms and that field rate applications of glyphosate should have little or no affect on soil microbial communities in ponderosa pine plantations.