Appl Microbiol Biotechnol (2010) 88:585–594
DOI 10.1007/s00253-010-2775-0
ENVIRONMENTAL BIOTECHNOLOGY
Bioremediation of glyphosate-contaminated soils
Inna T. Ermakova & Nina I. Kiseleva &
Tatyana Shushkova & Mikhail Zharikov &
Gennady A. Zharikov & Alexey A. Leontievsky
Received: 31 March 2010 / Revised: 9 July 2010 / Accepted: 10 July 2010 / Published online: 31 July 2010
# Springer-Verlag 2010
Abstract Based on the results of laboratory and field experi-
ments, we performed a comprehensive assessment of the
bioremediation efficiency of glyphosate-contaminated soddy-
podzol soil. The selected bacterial strains Achromobacter sp.
Kg 16 (VKM B-2534D) and Ochrobactrum anthropi GPK 3
(VKM B-2554D) were used for the aerobic degradation of
glyphosate. They demonstrated high viability in soil with the
tenfold higher content of glyphosate than the recommended
dose for the single in situ treatment of weeds. The strains
provided a two- to threefold higher rate of glyphosate
degradation as compared to indigenous soil microbial
community. Within 1–2 weeks after the strain introduction,
the glyphosate content of the treated soil decreased and
integral toxicity and phytotoxicity diminished to values of
non-contaminated soil. The decrease in the glyphosate
content restored soil biological activity, as is evident from a
more than twofold increase in the dehydrogenase activity of
indigenous soil microorganisms and their biomass (1.2-fold
and 1.6-fold for saprotrophic bacteria and fungi, respective-
ly). The glyphosate-degrading strains used in this study are
not pathogenic for mammals and do not exhibit integral
toxicity and phytotoxicity. Therefore, these strains are
I. T. Ermakova (*) : T. Shushkova : A. A. Leontievsky
G. K. Skryabin Institute of Biochemistry and Physiology
of Microorganisms, Russian Academy of Sciences,
142290 Pushchino, Moscow Region, Russia
e-mail: ermakova@ibpm.pushchino.ru
A. A. Leontievsky
Pushchino State University,
142290 Pushchino, Moscow Region, Russia
N. I. Kiseleva : M. Zharikov : G. A. Zharikov
State Federal Enterprise for Science “Research Center
for Toxicology and Hygienic Regulation of Biopreparations”,
142253 Serpukhov, Moscow Region, Russia
suitable for the efficient, ecologically safe, and rapid
bioremediation of glyphosate-contaminated soils.
Keywords Glyphosate . Bioremediation . Herbicide .
Biodegradation . Microorganism degraders . Toxicology
Introduction
Glyphosate (GP) is an organophosphonate compound that has
a very stable carbon–phosphorus (C–P) bond in its molecule.
It is an active ingredient in herbicides (Roundup, Roundup
Ultra, Rodeo, Glycel, Ground Bio) widely used in agriculture
for the elimination of annual and perennial weeds. The
application of GP results in the yellowing and decay of leaves
within 5–10 days (sometimes 30 days) caused by the
breakdown of aromatic amino acids synthesis.
The studies conducted at the initial stage of Roundup
production by Monsanto Chemical Co. (USA) showed a
relatively high GP degradation in the contaminated soils
with a half-life period of degradation about 20 days
(Rueppel et al. 1977). However, the 30-year application of
the herbicide under different ecological conditions showed
that GP persists in the contaminated soil for a longer time
depending on soil type, processing technique, climatic
conditions, and other factors (Cox 1998). The occurrence
of GP in the contaminated soils 2 years after application
shows its ability to accumulate in soils and cause negative
effects on human health and the environment (Eberbach
1998). It has been reported that the GP exposure may cause
the degradation of liver cells, gene mutations, structural
changes in chromosomes, etc. (Rank et al. 1993).
GP can alter natural ecosystem by affecting different
components of soil microbial community. This compound
inhibits the growth and decreases the content of different
586
soil microorganisms—bacteria, fungi, actinomyces, and
yeasts (Carlise and Trevors 1988). The GP impact on
plants makes them amenable to the infection by phytopath-
ogenic microorganisms (Levesque and Rahe 1992). After
7 years of GP application, some researchers observed the
adaptation of weeds to this herbicide, as well as an
alternation of the species composition and the displacement
of perennial species from the cenosis (Protasova et al.
2008). The GP transported to aqueous systems via surface
runoff displayed toxic effects to some aquatic organisms
(Alberdi et al. 1996).
The main way of GP elimination in soils is its biodegra-
dation by the enzyme systems of some microorganisms
(Rueppel et al. 1977; Strange-Hansen et al. 2004). The
intensity of herbicide mineralization in soils depends on
its bioavailability and on the size and the activity of
microbial population containing indigenous GP-degrading
microorganisms (Sorensen et al. 2006).
The inactivation of GP is facilitated by its sorption on
soil matrix. On the other hand, GP sorption makes the
herbicide more persistent because it decreases its bioavail-
ability (Schnurer et al. 2006). The efficiency of GP
degradation can be altered because of its vertical mobility
in soils as a result of strong watering or atmospheric
precipitations. This was confirmed by the detection of GP
in the subsurface, groundwater, and in deeper soil
horizons, where it is not subject to biodegradation (Veiga
et al. 2001).
The accurate assessment of remediation efficiency is
impossible without reliable information on changes in the
ecotoxicological characteristics of soil (Casable et al.
2007). The reaction of test organisms may serve as a
criterion for the assessment of the actual exposure and
environment risks posed by the contaminant. There are no
universal test systems for the reliable detection of all
possible toxicants. That is why various combinations of
different test organisms are usually used in the environ-
mental monitoring practice. In our research, we used
Daphnia as the most sensitive test organism for the short-
term bioassays of soil integral toxicity (ISO 2000) and oat
as a phytotest for the direct evaluation of soil purity after
the application of broad-host herbicide (ISO 2005).
In order to prevent the accumulation of GP in soils, it is
important to develop a method for its rapid elimination after
the weed treatment. Moreover, bioremediation strategy
should be developed to remove this herbicide in case of
spills or leakage during the GP manufacture, storage,
transportation, and application.
The present paper describes a complex study of the
bioremediation efficiency of GP-contaminated soils using
the introduced GP-degrading bacterial strains under labora-
tory (in the microcosm) and field (in situ) experiment
conditions.
Appl Microbiol Biotechnol (2010) 88:585–594
Materials and methods
Soil
Soddy-podzol (SP) loamy soil (clay 39.8%, sand 16.1%,
silt 25.4%, organic matter 1%, exchangeable phosphorus
45 μg Р2О5 g−1, mobile potassium 80 μg K2O g−1, pH =
7.1) was used. The site intended for our bioremediation
studies was a part of a special soil territory free of any
pollutant. The absence of pollution was confirmed by the
analysis of integral toxicity (Daphnia and earthworms
mortality) and phytotoxicity assessment (inhibition of
seed germs and roots of oat, pea, and radish) (data not
shown).
Bacterial strains, cultivation conditions
Bacterial GP degraders were isolated by the method of
enrichment cultures (Ermakova et al. 2008) from soils
contaminated with organophosphonates. Initially, mineral
salt medium (MS1) (Shushkova et al. 2010) supplemented
with 0.5 g l−1 GP and 10 g l−1 sodium glutamate (Difco,
USA) was used for the selection of most active strains in
batch cultures. The isopropylamine ammonium salt of GP
as a component of commercial Ground Bio herbicide
(Technoexport, Russia) was used for the cultivation of
microorganisms only as a source of phosphorus rather than
carbon or nitrogen. The composition of Ground Bio was
similar to that of the Roundup herbicide with a GP content
of 360 g l−1.
At the next stage, the strains selected in batch cultures
were tested in open microcosms. Two of them, Kg 16 and
GPK 3, with the maximal efficiency of GP degradation
were found to be non-toxic for the test organisms. They
were chosen for the complex assessment of soil remediation
in laboratory and field experiments. Based on the analysis
of the primary nucleotide sequences of 16S rRNA genes
(Ermakova et al. 2008), they were identified as Achromo-
bacter sp. Kg 16 (VKM B-2534D) and Ochrobactrum
anthropi GPK 3 (VKM B-2554D).
To be introduced to soil, the strains were grown in batch
cultures in the MS1 medium with 250 mg l−1 of GP and
10 g l−1 of sodium glutamate. Cells were harvested in the
logarithmic growth phase by centrifugation (5,000×g for
20 min), twice washed with MS1 medium without
phosphorus source, suspended in the same medium supple-
mented with 10 g l−1 of sodium glutamate and incubated for
48 h to provide phosphorus starvation (Ermakova et al.
2008). All procedures were carried out under sterile
conditions. Then the cells were centrifuged again, washed,
and suspended in the MS1 medium lacking phosphorus and
carbon sources. The cell density of the introduced microbial
suspensions was ∼108 CFU ml−1.
Appl Microbiol Biotechnol (2010) 88:585–594
Soil bioremediation in the open microcosm
The soil sample free of roots and plant debris was grinded,
sieved (2-mm mesh), placed into plastic cups (h=15 cm,
d=10 cm, 0.5 kg of soil in each), and moistened. Then 12.5
or 25 ml of 0.33% (v/v) Ground Bio water solution (14.85
and 29.7 mg of GP, respectively) was applied to the soil
surface. These doses (equivalent to 50 and 100 l of
herbicide ha−1) were fivefold and tenfold higher than the
recommended dose for single field application. Carbon
sources were not added.
Microbial suspension in a volume of 25 ml (106 CFU g−1
of soil) was evenly introduced into the soil by drip irrigation
72 h after the GP application. In control 1 (non-inoculated
soil), the same volume of MS1 medium was added instead of
the microbial suspension. In control 2 (non-contaminated
and non-inoculated soil), distilled water (25 ml) was added.
Soil was incubated at the room temperature (22–24оС); its
humidity was maintained at 14–19% of total moisture
capacity. Soil samples were taken at the beginning and at
the end of the experiments. For analysis, total soil from
every cup was thoroughly mixed. The averaged soil samples
were immediately analyzed for the total content of GP, as
well as separately dissolved and adsorbed GP, the density of
introduced and indigenous GP-degrading microorganisms,
the biomass of saprotrophic bacteria and fungi, integral
toxicity, phytotoxicity, and the dehydrogenase (DH) activity
of soil microbial community. The experiments were done
in triplicate. The data were re-calculated for the weight of
air-dried soil.
In situ soil bioremediation
The experiments were carried out on 1×2 m plots. The plots
were dug and thoroughly loosened to the depth of 25 cm;
roots, plant debris, and other foreign inclusions were removed.
Two blank soil plots with indigenous microorganisms served
as control 1 (GP-contaminated soil) and control 2 (native non-
contaminated soil). The herbicide was applied in the amount
equivalent to 100 l of Ground Bio preparation ha−1. The
experiment was carried out in July–August with night
temperatures of 12–17°C and day temperatures of 18–28°C.
Soil humidity varied from 7–9% to 18–21% after rain or
watering. The procedures of GP application to soil, the
preparation and introduction of microbial suspensions, as
well as all analytical procedures were analogous to those
used in the laboratory experiments. Soil samples were taken
weekly through 28 days from the soil horizon of 0–20 cm by
using a core auger tool with an inner diameter of 4.5 cm
(AMS, Inc., USA). The soil samples taken from ten different
points of each plot were pooled and thoroughly mixed. Each
averaged sample was analyzed at least in triplicate. At the
end of the experiment, soil samples were also taken from the
587
horizon of 20–30 cm to estimate the herbicide mobility
through the vertical soil profile.
Microbiological analysis
For counting viable cells of GP-degrading microorganisms
in the soil population, serial dilutions of soil samples were
inoculated on MS1 agar medium with 500 mg l−1 of GP
and 10 g l−1 of sodium glutamate. CFU were determined
after the incubation of agar plates at 28°С for 48 h. In every
test, the total amount of GP-degrading cells was deter-
mined. The CFU of the introduced strain was calculated as
the difference between the total amount GP-degrading cells
and the amount of indigenous GP-degrading cells (the latter
was determined in the control experiment 1).
Growth of microorganisms in liquid media was monitored
by measuring OD560 on a Specol 210 spectrophotometer
(Carl Zeiss, Jena, Germany). The OD data were converted to
the dry biomass weight by assuming that one OD unit
corresponds to 0.5 g of biomass. This value for the bacteria
studied was determined experimentally.
To assess the biological activity of soil, indigenous
bacterial cells were counted in soil suspensions stained with
an aqueous solution of acridine orange. The length of
fungal mycelium was measured in the soil preparations
stained with calcofluor white. The samples were examined
under a luminescent microscope. Biomass was calculated
by assuming that the weight of one dry bacterial cell is 2×
10−14 g and the weight of 1 m of dry fungal mycelium is
3.9×10−6 g (Zvyagintsev 1991).
Analytical determinations
GP in the culture liquid and soil extracts was determined by
HPLC using an LKB 2150 UV-detector (204 nm) and
Repro-Gel H column, 9 μm (250×8 mm). The column was
kept at 65°С, the mobile phase was 0.01 N sulphuric acid.
The alkaline extraction of soil samples was used for the
evaluation of the total GP content. The soluble fraction of
GP was determined after the aqueous extraction of soil
samples. Then, the GP adsorbed on soil particles was
extracted by alkali (extractable GP). The non-extractable
GP (irreversibly bound GP) was calculated as the difference
between the total, soluble, and extractable GP (Shushkova
et al. 2010). Aminomethylphosphonic acid and sarcosine
were determined in the culture liquid by HPLC as acyl
derivatives (Zelenkova and Vinokurova 2008).
The DH activity of indigenous soil microbial community
was measured under anaerobic conditions with glucose as
the metabolized substrate and 2,3,5-triphenyltetrazolium
chloride as the hydrogen acceptor. The DH activity was
calculated from the amount of triphenylformazan produced.
The latter was measured spectrophotometrically at 540 nm.
588
Non-contaminated soil served as the standard of 100% DH
activity (Zvyagintsev 1991).
Toxicological analysis
The integral toxicity of the selected strains and GP-
contaminated soil was evaluated through the vital
activity of synchronized laboratory Daphnia magna
culture (ISO 2000). The culture liquid of the GP-
degrading strains grown in Luria–Bertani (LB) nutrient
broth was taken in the stationary growth phase, diluted
to 106, 107, 108 cells ml−1 and assessed for Daphnia
survivability. After 96 h, the number of survived Daphnia
was counted, and percentage of their death was calculated
as the difference with the control (LB medium instead of
the culture liquid). The integral toxicity of soil was
evaluated in the same way using aqueous soil extracts
instead of the culture liquid. D. magna loss was calculated
as the difference with control 2 (non-contaminated soil
with a toxicity of 0%).
The phytotoxicity of strains and soil was assessed on oat
sprouts (Berestetsky 1971). Oat seeds were soaked for 24 h
in the diluted culture liquid prepared as for assessment of
integral toxicity (from 106 to 108 cells ml−1). Then the
seeds were kept for 5 days at the room temperature on
moistened filter paper under a lid. Morphometric indices
(root and germ lengths) were determined in comparison
with the same parameters in the control variants with LB
medium and sterile tap water instead of the culture liquid.
To assess soil phytotoxicity, oat seeds were kept for 5 days
on soil covered with moistened filter paper under the same
conditions as during the phytotoxicity assessment of the
strains. To calculate the level of phytotoxicity, the charac-
teristics of non-contaminated soil with a phytotoxicity of
0% were used.
The pathogenicity for mammals of GP-degrading strains
was determined according to the recommendations of the
Word Health Organization (WHO 1981) on laboratory
outbred white mice and rats. Virulence (LD50), toxicity,
toxigenicity, and dissemination in the internal organs of
animals were evaluated. The strain exhibiting even one
unfavorable feature was considered to be pathogenic. The
microorganisms were cultivated in liquid LB medium and
sampled in the stationary growth phase. For the determina-
tion of LD50, 1 ml of suspensions containing 106–108 and
108–1010 bacterial cells, respectively, was i/p and i/g
administered in animals. Control groups were treated with
1 ml of physiological solution. Each group contained 12
animals. The toxicity of the strains was assessed in mice by
the i/p administration of 1-day microbial suspension inacti-
vated at 70°С. The doses were 108, 109, and 1010 cells per
animal (four animals per dose). Toxigenicity was assessed in
mice by the i/p and i/g administration of sterile cultural liquid
Appl Microbiol Biotechnol (2010) 88:585–594
filtrate of 3- and 7-day cultures in doses of 0.5 and 1.0 ml,
respectively (four animals per dose). Control animals
received sterile liquid nutrient medium in the same doses.
To assess the dissemination of microorganisms in the internal
organs of animals, 30 days after the injection of microbial
culture into the animals, they were euthanized by CO2, and
samples of the blood and internal organs (lungs, liver,
kidneys, and spleen) were plated by replica method on MS1
agar medium supplemented with GP.
Results
Selection of active GP-degrading bacterial strains
At the initial stage of the study in batch culture, the criteria of
selection were: (1) biomass in the stationary phase not lower
than 0.5 g l−1 and (2) the biodegradation efficiency within
20–50 mg of GP g−1 of dry biomass. Among the 25 bacterial
soil isolates tested, 13 active strains were selected. Six
cultures actively utilized the herbicide immediately after
isolation from soil, whereas seven other strains could utilize
GP only after adaptation via several re-inoculations in the
liquid MS1 medium supplemented with GP and glutamate
(data not shown).
In order to assess the potential of these strains for the
bioremediation of GP-contaminated soils, we evaluated
their integral toxicity on D. magna. The results of this assay
have demonstrated that these microorganisms at concentra-
tion up to 108 cells ml−1 (commonly used for microbial
introduction to soils) were not toxic for this test organism.
None of the 13 isolates displayed phytotoxic effect to oat
sprouts.
At the next stage assessment of the degrading activity of
the selected strains, they were introduced into the SP soil in
the microcosm (Table 1). The initial GP content was 30–
35 mg kg−1, the duration of the experiment was14 days.
Assuming that the intensity of GP adsorption in soil samples
is the same, the decrease in its content served as a criterion of
the degrading activity of the strains. For ten cultures, the
decrease in the toxicant content was higher in comparison
with the control containing only indigenous GP degraders.
The study of their pathogenicity showed that only six strains
(MPK 7, MPS 10, MPS 11, GPK 1, Kg 16, and GPK 3) met
the requirements to hazard class 4 (WHO 2004) and could be
used for introduction into contaminated soils. The decrease
in the GP content of soils due to the degrading activity of the
introduced microorganisms diminished the integral toxicity
of soil samples to 3–9%, as compared to the toxicity of 24%
in the control samples and 30% immediately after the
application of GP.
As a result of the screening, two strains were selected:
Achromobacter sp. Kg 16 (VKM B-2534D) and O. anthropi
Appl Microbiol Biotechnol (2010) 88:585–594 589

Table 1 Selection of GP degraders in open microcosm In control 1 without the introduced strains, the total GP
Strain degraders GP decrease, Strains Integral soil content decreased only by 23.3% (14.3 mg kg−1). The
mg kg−1 of soil pathogenicity toxicity, percent largest portion of the utilized herbicide (20%) was in the
for mammals of Daphnia death liquid soil fraction, and only about 3% was desorbed from
the soil matrix.
Indigenous microbial <6 nd 24±0.4
community, control The elimination of GP via its biodegradation by the
R1 <6 nd nd introduced strains decreased the integral toxicity of soil
P 15 <6 nd nd to the values close to those of non-contaminated soil
MPK 7 12±3 − 5±0.2 (1.3% and 5.3%), while in control 1 with indigenous
MPK 8 <6 nd nd microorganisms, this value decreased only from 36.7% to
KM 11 10±3 + nd 16.7%.
GPK 1 15±3 − 3±0.3 The phytotoxicity assessment of GP-contaminated soil
GPK 3 19±4 − 9±0.4 demonstrated that, in control 1, the inhibition of the oat root
Kg 16 23±5 − 7±0.3 length had slightly decreased (from 44% to 37%). At the
B5 11±3 + nd same time, in the presence of Achromobacter sp. Kg 16 and
G4 10±3 + nd O. anthropi GPK 3, this value decreased to 14% and 24%,
SM 51 10±3 + nd respectively. The germ lengths changed in the same
MPS 10 12±3 − 5±0.2 proportions.
MPS 11 16±4 − 8±0.3 GP contamination leads to a threefold reduction of the
Values are means ± standard error
DH activity of soil microbial community. At the end of the
nd not determined; − negative result; + positive result
experiment, this activity in control 1 remained almost
unchanged (34%), while the introduction of Achromobacter
sp. Kg 16 and O. anthropi GPK 3 caused a twofold increase
GPK 3 (VKM B-2554D). These strains possessed maximal in the enzyme activity (from 30% to 62–64%).
degrading activity towards GP (23 and 19 mg GP kg−1 of Following the addition of GP, the biomass of saprotro-
soil). Possible GP degradation products (aminomethylphos- phic soil bacteria and fungi decreased by 42% and 62%,
phonic acid and sarcosine) were not detected in the growth respectively. Due to the herbicide elimination by the
media of both strains (data not shown). introduced GP degraders, the biomass of indigenous soil

Soil bioremediation in the open microcosm

%
80
In laboratory-scale experiments, the efficiency of GP biodeg-
radation by the introduced Achromobacter sp. Kg 16 and O. 70
anthropi GPK 3 strains was estimated in comparison with
60
the indigenous microbial community (control 1). The
duration of the experiment was 21 days under controlled 50
conditions (temperature and soil humidity).
40
The evaluation of GP adsorption demonstrated that up to
80% of the initial herbicide dose was adsorbed by the soil 30
matrix within 72 h and only 20% remained in the soluble
20
phase. The adsorbed GP could be fully extracted with
NaOH (extractable GP). It was available for bioutilization 10
by microorganisms as a phosphorus source. At the end of
0
the experiment, soluble GP was utilized in all the 1 2 3 4 5 6
experimental variants. The residual GP was extractable
fraction. The non-extractable fraction (irreversible bound Fig. 1 Change of toxicological and biological characteristics of GP-
contaminated SP soil during the bioremediation in open microcosm: 1—
toxicant) was absent. GP decrease of initial content (100%), 2—integral toxicity, 3—
The results are shown in Fig. 1. At the initial GP content phytotoxicity (2, 3 of control 2 with non-contaminated soil, 0%), 4—
of 58–61 mg kg−1 of soil, the degree of elimination reached DH activity, 5—saprotrophic soil bacteria biomass, 6—soil fungi
65.8% (38.6 mg kg−1) with Achromobacter sp. Kg 16 and biomass (4–6 of control 2 with non-contaminated soil, 100%). Error
bars indicate the standard deviation. All values at the beginning of
49.5% (29.5 mg kg−1) with O. anthropi GPK 3. This means experiment after 72 h following GP adding. The values at the end of
that 20% of the soluble and 45.8% and 29% of the adsorbed experiment: control 1, indigenous microbial community; with
GP were bioutilized by the GP-degrading microorganisms. Achromobacter sp. Kg 16; with Ochrobactrum anthropi GPK 3
590 Appl Microbiol Biotechnol (2010) 88:585–594

microorganisms during the experiment increased by 20– a 80

26% and 56–60%, respectively, as compared to control 1.
70

GP content, mg kg-1 of soil

In situ soil bioremediation 60

Soil bioremediation under field conditions was studied in 50

dynamics for 28 days. All parameters were evaluated for 40

the arable soil horizon (0–20 cm from the surface).
30
Within 72 h after GP application, 88.8% of the herbicide
was adsorbed by the soil matrix (extractable GP), and 20
11.2% remained in the liquid fraction (soluble GP).
10
Residual GP at the end of the experiment was found only
in the extractable fraction. No movement of the herbicide 0
down soil profile to the depth of 20–30 cm with
atmospheric precipitations was observed. b 60
The experimental results shown in Fig. 2 demonstrate that
the GP biodegradation rate was maximal during the first week 50

Integral toxicity, %
after the introduction of the GP-degrading strains. The GP
content decreased by 33.3 mg kg−1 (56.3%) in the case of 40
Achromobacter sp. Kg 16, and by 21.6 mg kg−1 (35%) in the
30
case of O. anthropi GPK 3 (Fig. 2a). Cell density in this
period slightly changed; namely, from 3.5±0.2×106 to 3.0±
20
0.2×105 for Achromobacter sp. and from 12±0.9×106 to 15±
0.7×105 CFU g−1 for O. anthropi. Taking into account the 10
insignificant decrease in the GP content in control 1, we
evaluated the biodegradation efficiency by the introduced 0
strains. The efficiency was equal to 18±3 μg GP 106 cells−1
for Achromobacter sp. and 3.0±0.8 μg GP 106 cells−1 for O. c
80
anthropi. In batch cultures, the degrading activity of Achro-
mobacter sp. Kg 16 was equal to 10±2 μg GP 106 cells−1 70

during the growth phase (96 h) and that of O. anthropi GPK 3 60

DH activity, %

was equal to 20±5 μg GP 106 cells−1 (72 h). 50

The degradation of GP by the introduced strains sharply
40
decreased by day 14, when cell density was 2–3 orders
lower (103–104 CFU g−1). By that time, soluble GP had 30

been completely utilized. By the end of the experiment, the 20

total GP content decreased by 44.5 mg kg−1 (75.2%) in the
case of Achromobacter sp. Kg 16 and by 37.9 mg kg−1
(61.5%) in the case of O. anthropi GPK 3 (Fig. 2a).
At a lower density and activity of indigenous GP degraders,
the GP content decreased gradually to 17.9 mg kg−1 (29.3%)
during the experimental period (28 days).
The integral toxicity of soil decreased to the level of
non-contaminated soil 2 weeks after the introduction of the
degrading strains. In control 1, the integral toxicity of soil
decreased twofold by the end of the experiment (Fig. 2b).
As a result of the GP contamination of soil, the
morphometric parameters of oat sprouts decreased 1.5 times
as compared to non-contaminated soil. By the end of the
experiment, the phytotoxicity of soil could not be detected
probably due to the herbicide degradation by the GP-
degrading strains. In control 1, the phytotoxicity of soil
decreased to a level of 14%.
10
0
0 5 10 15 20 25 30
Time, days
Fig. 2 Dynamics of GP content (a), integral toxicity (b), and DH
activity (c) during the bioremediation of GP-contaminated SP soil in
field conditions: (filled circle)—control 1 (with indigenous GP
degraders); (filled square)—with introduction of Achromobacter sp.
Kg 16; (filled triangle)—with introduction of O. anthropi GPK 3.
Error bars indicate the standard deviation
Similar to the data obtained in the microcosm, the DH
activity of soil microbial community increased from 30%
to 64–66% during the experiment (28 days) in the case
of the introduced strains, whereas in control 1, this
activity declined to 38% (Fig. 2c). Changes in the
Appl Microbiol Biotechnol (2010) 88:585–594
biomass of saprotrophic bacteria and fungi in the field
experiments were similar to those observed in the
laboratory experiments.
Discussion
GP degraders, belonging mostly to the genera Pseudomonas,
Arthrobacter, and Alcaligenes, were isolated from different
sources, including soil, water, as well as GP-containing
household and industrial wastes. Their physiological proper-
ties were studied in batch cultures (Balthazor and Hallas
1986; Pipke et al. 1987; Dick and Quinn 1995). However, no
data is available on the degrading activity of such bacteria in
natural soil ecosystems contaminated with GP-containing
herbicides.
In earlier studies, the biodegradation of GP in soil was
quantitatively assessed only for indigenous microorganisms
with different levels of adaptation to GP. The biodegradation
of GP was found to be low and dependent on the conditions
and duration of the process. The maximum value of GP
biodegradation did not exceed 11% for a period of 50 days (in
that experiment, GP dose was 100 mg kg−1 of soil) (Getenga
and Kengara 2004). In clay soils, the GP biodegradation rate
varied from 9.3% to 14.7% for a period of 3 months
(Sorensen et al. 2006). In sand soil contaminated with GP at
a dose 1 mg kg−1 of soil, GP was mineralized by only 2% for
a month (Strange-Hansen et al. 2004).
The mobility of GP down the soil profile and its
sorption by soil matrix promotes the accumulation of GP
in the studied SP soil. When applied in a dose of
50 mg kg−1 soil, the GP content in the 0–10 cm soil
horizon decreased during 21 days by 29% in the case of
indigenous GP degraders and by 56% in the case of O.
anthropi GPK 3. In these experiments, some amount of
GP (18% and 14%, respectively) was revealed in the soil
horizon 10–20 cm, where cells of O. anthropi GPK 3 were
absent and the cell density of indigenous microorganisms
was very low. The total biodegradation of GP made up
11% in the control and 42% in the case of the introduced
strain. Intense watering (an imitation of rainfall) washed
out 26–28% of GP down to depths of 10–30 cm, and,
consequently, reduced the amount of bioavailable toxicant
in the upper soil horizons (Shushkova et al. 2010). Similar
results were obtained with other types of loamy soils:
intense irrigation or precipitations washed out GP down to
the sub-arable layer (30 cm deep), where it could not be
attacked by GP degraders for a long time (Veiga et al.
2001; Barrett and McBride 2007).
The distribution of GP between liquid (soluble GP) and
solid (adsorbed GP) soil phases depends on its sorption
capacity. In soil with high levels of gravel, where the
degree of GP sorption was the lowest, GP mineralization by
591
the native microbial community reached 30% in compari-
son with 2% in the case of a sandy soil with high sorption
capacity (Newton et al. 1994; Strange-Hansen et al. 2004).
The bioavailability of GP depends on its degree of sorption.
As soluble GP is consumed, extractable GP desorbs from
the soil matrix. The adsorbed GP is degraded considerably
slower than soluble GP. This effect enhances the persistence
of GP in contaminated soils. The irreversibly bound (non-
extractable) GP is not accessible to microbial degradation
and accumulates in contaminated soils (Eberbach 1999;
Zablotowicz et al. 2009; Shushkova et al. 2009). Under
some conditions, extractable GP can gradually convert to
non-extractable GP (Al-Rajab et al. 2008; Getenga and
Kengara 2004).
In our studies, the screening of bacterial strains isolated
by the method of enrichment cultures from soils contam-
inated with organophosphonates allowed us to select some
cultures with a high survivability in the environment and
maximal degrading activity towards GP. Two most active
GP-degrading strains Achromobacter sp. Kg 16 (VKM B-
2534D) and O. anthropi GPK 3 (VKM B-2554D) (Table 1)
did not exhibit pathogenicity, integral toxicity, and phyto-
toxicity, and according to safety requirements, can be used
for the bioremediation of GP-contaminated soils. The
bioremediation potential of these isolates was studied in
the laboratory microcosm and under field conditions with a
Ground Bio dose of 100 l ha−1. The recommended dose of
this herbicide applied singly in the field is no more than
10 l ha−1 (3.6 kg of GP). In our experiments, we increased
this dose tenfold in view of its possible accumulation in soil
as a result of multiple treatments of weeds and due to GP
sorption and mobility.
O. anthropi has already been known capable of GP
degradation (Gard et al., 1997). Among bacteria of the
Achromobacter genus, a unique strain Achromobacter sp.
LW9 was described, which utilized GP in batch cultures
as a carbon source in the presence of orthophosphate.
This strain transformed GP into aminomethylphosphonic
acid without cleavage of the С–Р bond; therefore, it did
not use GP as a phosphorus source (McAuliffe et al.
1990).
We evaluated the bioremediation efficiency of GP-
contaminated soil not only through the decrease in the GP
content of soil but also through changes in the toxicological
and biological characteristics (integral toxicity and phyto-
toxicity), as well as in the DH activity and the biomass of
saprotrophic bacteria and fungi.
SP soil is known to have high sorption capacity. When
Ground Bio was applied to this soil at a dose of 100 l ha−1, 80–
89% of GP was adsorbed by the soil matrix and only 20–
11% remained in the liquid phase 72 h after the GP
application. The irreversible binding of the toxicant to the
soil matrix did not take place. It means that all adsorbed GP
592
can be used as a phosphorus source by the introduced
microorganisms.
In the microcosm (Fig. 1), indigenous microorganisms
degraded GP (control 1) by 23.3% (20% was soluble GP
and only 3.3% was extractable GP). More than 75% of GP
remained adsorbed to the soil matrix. Within the same time
period, the biodegradation efficiency of O. anthropi GPK 3
and Achromobacter sp. Kg 16 was 2–3 times higher (49.5%
and 65.8%, respectively). In this case, the degradation of
adsorbed GP by these two strains comprised 29.5% and
45.8%, respectively.
In control 1, the integral toxicity of soil decreased from
36.7% to 16.7%, and its phytotoxicity from 44% to 37%
(control 1). Following the introduction of O. anthropi GPK
3 and Achromobacter sp. Kg 16, the integral toxicity of soil
decreased to levels close to those of non-contaminated soil,
and the inhibition of oat roots and germs decreased by 2–3
times. Generally, the decrease in the integral toxicity and
phytotoxicity of soil correlated with the intensity of GP
elimination from soil due to the degrading activity of the
two GP degraders.
GP was found to suppress the development of indigenous
microbial community in the contaminated soil. Indeed, the
DH activity (as a characteristic of the metabolic activity of the
microbial complex), as well as the biomass of saprotrophic
bacteria and fungi, considerably decreased after the applica-
tion of GP. It should be noted that soil fungi were more
strongly inhibited by GP than soil bacteria. Due to the intense
degradation of GP by the introduced strains, the biological
activity of soil was restored. By the end of the experiment,
total DH activity, bacterial biomass, and fungal biomass
increased by 2, 1.2, and 1.6 times, respectively, in comparison
with the contaminated soil in control 1. The high residual
content of GP (over 70%) in this control soil was still
inhibitory to indigenous soil microorganisms, as is evident
from the following results: by the end of the experiment, the
DH activity was almost unchanged, bacterial biomass
increased only by 6% and fungal biomass by 20%.
In the field experiments (Fig. 2), changes in the basic
bioremediation parameters were similar to those in the
microcosm experiments. The maximum rate of GP biodeg-
radation in the contaminated soil was observed 1–2 weeks
after the introduction of Achromobacter sp. Kg 16 and O.
anthropi GPK 3. These strains utilized soluble GP by
11.2% and a considerable part of adsorbed GP (61.4% and
37.8%, respectively). In the following period, the degrada-
tion of GP drastically fell.
The two-phase dynamics of GP degradation in soils by
indigenous microorganisms was described by a number of
authors. The initial fast phase of degradation was due to the
bioutilization of soluble GP. The decrease in the soluble GP
content promotes the desorption of GP from the soil matrix.
The slower second phase of GP degradation is explained by
Appl Microbiol Biotechnol (2010) 88:585–594
microbial attack to the desorbed GP. The availability of GP
to biodegradation depends on the rate of its desorption
(Eberbach 1998; Getenga and Kengara 2004; Sorensen et
al. 2006). The fast phase of degradation lasts from 2–5
(Zablotowicz et al. 2009) to 40 days (Eberbach 1998),
depending on the activity of soil microbial community and
environmental conditions.
The intensity GP biodegradation by O. anthropi GPK 3
in batch culture increased 1.5 times when bacterial cells
starved of phosphorus and contained no more 2% of this
compound were used for inoculation (Ermakova et al.
2008). Taking into account these data, the high rate of GP
consumption in the first phase of soil remediation can be
due to the introduction of microbial cells starved of
phosphorus.
The decrease in the intensity of GP biodegradation in the
field experiments 2 weeks after introduction can be
explained by the decrease of the number of introduced
cells by 2–3 orders of magnitude. Cell lysis may result from
the exhaustion of some nutrition sources, in particular,
carbon source in soil, as well from unfavorable temperature
and humidity conditions. As was shown in experiments
with batch cultures, the growth of O. anthropi GPK 3 and
the consumption of GP was arrested as the result of
glutamate exhaustion as carbon source. In this case, the
number of viable cells in the presence of GP drastically
decreased because of cell lysis (Ermakova et al. 2008).
Similar effects were observed in the case of phosphorus
limitation and the excess of carbon source (data not shown).
In SP soil, the biomass of O. anthropi GPK 3 cells
introduced to soil without additional carbon source
increased twofold within the first week after introduction
and then decreased. In this case, the content of GP in soil
remained at a rather high level. (Shushkova et al. 2010). In
water suspensions of the same soil, the initial increase in
the O. anthropi biomass was even higher, probably due to
extraction of available carbon source(s) from the soil
(Shushkova et al. 2009).
It is noteworthy that the amount of adsorbed GP utilized
by the introduced bacteria was 4–6 times higher than that of
soluble GP. This can be explained by the fact that the GP
degraders isolated from organophosphonate-contaminated
soils acquire ability to utilize directly the GP adsorbed on
the soil matrix (that is, without the stage of GP desorption
into the soluble phase). Such a situation was described for
phenanthrene-degrading microorganisms (Tang et al. 1998).
In control 1, the GP degradation rate by indigenous soil
microorganisms (unlike the introduced strains) did not rise
through the whole experiment. Presumably, they need quite
a long period of adaptation to GP as the phosphorus source.
The intensity of GP biodegradation with the indigenous
microbial community is low in comparison with the
introduced strains. This fact makes the natural remediation
Appl Microbiol Biotechnol (2010) 88:585–594
of GP-contaminated soils quite long and increases the risk
of herbicide migration down to deeper soil horizons.
The data presented provide evidence that the biological
properties of GP-contaminated soils can be restored. These
data can be used to intensify the process of soil cleanup
after weed treatment to prevent the accumulation of GP in
the soil and the elimination of soil toxicity. This problem
can be solved by the introduction of the selected GP-
degrading strains and the maintenance of their high activity
through the optimization of habitat conditions.
The advantages of the bacterial degraders we selected are
as follows: high degradation capacity without the accumula-
tion of any metabolite, in particular, aminomethylphosphonic
acid; the ability to maintain rather high levels of biomass in the
presence of necessary nutrient components; and the rapid
elimination of introduced cells from soil after exhaustion of
the phosphorus source. The strains meet the toxicological
requirements and are suitable for reducing the GP content in
contaminated soils to ecologically safe levels in the course of
soil remediation.
Acknowledgements The work was supported by the International
Science & Technology Center, project #1892.2, Ministry of Education
and Science of the Russian Federation, project “Development of
Scientific Potential of the Higher School” (2.1.1.9227) and Russian
Foundation of Basic Research, project # 09-04-00320.
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