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Vol. 7(49), pp. 5587-5595, 11 December, 2013
DOI: 10.5897/AJMR2013.6080
ISSN 1996-0808 ©2013 Academic Journals
http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Full Length Research Paper
Isolation and characterization of glyphosate-degrading
bacteria from different soils of Algeria
Ouided Benslama and Abderrahmane Boulahrouf*
Laboratoire de Génie Microbiologique et Applications, Campus Chaâberssas, Faculté des Sciences de la Nature et de la
Vie, Université Constantine 1, Constantine, Algérie.
Accepted 18 November, 2013
Glyphosate (N-phosphonomethylglycine) is the most commonly used herbicide worldwide. Due to the
concern regarding its toxicity for non-targeted species in soil, finding glyphosate-degrading
microorganisms in soil is of interest. The success of this will depend on isolating bacteria with the
ability to grow in presence of glyphosate. Five bacterial strains were isolated from different untreated
soils of Algeria, the strains were able to grow in a medium containing glyphosate as sole carbon or
phosphorus source by enrichment cultures of these soils. Based on 16S rRNA gene sequence analysis,
MALDI-TOF MS and biochemical properties, the best strain amongst them (Arph1) was identified
as Pseudomonas putida. This isolate showed the highest growth level in the presence of glyphosate as
sole phosphorus source. Arph1 was therefore used for further studies for optimization of cultivation
conditions for an efficient glyphosate use. The best result of growth was on 1 g/L of glyphosate in
minimal medium supplemented with glutamate with initial pH 9.0 at 30°C at 150 rpm within 168 h.
Microbial growth during the study was monitored by measuring the optical density at 620 nm. Arph1
was able to tolerate up to 9 g/L of glyphosate. These results show that the bacterial strain may possess
potential to be used in bioremediation of glyphosate-contaminated environments.
Key words: Soil pollution, glyphosate-degrading bacteria, Pseudomonas putida, optimization, cultivation
conditions.
INTRODUCTION
The application of xenobiotic compounds generates
environmental concern by the potential of the unwanted
side effects, as large amounts of substances are
released into the environment. The degradation of xeno-
biotic compounds is an important indicator for healthy
ecosystems. Soil microorganisms can carry out pesticide
degradation and can use the xenobiotic as a source of
carbon, energy and other nutrients to promote microbial
growth (Durkin, 2003). The herbicide glyphosate is often
used to control weeds in grasslands. Despite its
extensive use in Algeria, detailed informations on glypho-
sate degrading-microorganisms are lacking.
Glyphosate (N-phosphonomethylglycine) is the most
commonly used herbicide worldwide (Franz et al., 1997);
it is a broad-spectrum, post-emergence, non-selective
herbicide, that inhibits the enzyme 5-enolpyruvylshikimic
acid-3-phosphate synthase (EPSPS), blocking the syn-
thesis of essential aromatic amino acids (Duke et al.,
2003). The importance of glyphosate degrading-bacteria
has been magnified by the biotechnology application.
Effectively, the bacterial genes encoding for glyphosate-
resistant EPSP synthase were cloned, endowed with
*Corresponding author. E-mail: boulahroufabderrahmane@yahoo.fr. Tel: (00 213) 555 136 100.
5588 Afr. J. Microbiol. Res.
chloroplast transit signals and used to transform plants
(Della-Cioppa et al., 1987) to enable them to survive
treatment following application of glyphosate. Use of this
herbicide in glyphosate-resistant crops has given farmers
cost-effective and broad-spectrum weed control options.
Several bacterial strains were isolated that were able to
degrade glyphosate; most of these bacteria were isolated
from sites already treated by the herbicide. However,
there are few reports of the isolation of bacteria from
untreated sites and no report of glyphosate degrading-
bacteria isolated from Saharian soils. Previous reports
were mainly focused on the screening of bacteria for their
ability to degrade glyphosate. However, the comprehensive
studies of the physiological regulation in bacterial cells
are rather few (Shushkova et al., 2012). Thus, the opti-
mization of cultivation conditions is important to appre-
ciate this physiological regulation, and the identification of
these conditions will make it possible to know which
factors can be applied for bacteria in soil during biore-
mediation.
The aim of the present work was to isolate and
characterize glyphosate-degrading bacteria by using
enrichment cultures for three different untreated Algerian
soils and the assessment of growth response of the
isolates, as well as the optimization of some abiotic para-
meters for the cultivation of isolated strains, providing
maximal effectiveness of the glyphosate degradation.
MATERIAL AND METHODS
Chemicals and media
The isopropylamine salt of glyphosate known as Roundup®
(containing 450 g active ingredient/L of glyphosate, Monsanto) was
purchased from a local store supplier of agricultural products in
Constantine, Algeria.
For the isolation of bacteria using glyphosate as sole source of
carbon and energy, mineral salt medium 1 (MSM1) was used. The
composition of the medium in gram per liter of distilled water, pH
(7.0 to 7.2) was: KH2PO4 (1.5), Na2HPO4 (0.6), NaCl (0.5), NH4SO4
(2), MgSO4 7H2O (0.2), CaCl2 (0.01) and FeSO4 7H2O (0.001).
Whereas, mineral salt medium 2 (MSM2) was used for the isolation
of bacteria using glyphosate as sole phosphorus source, its
composition in gram per liter of distilled water, pH (7.0 to 7.2) is:
Tris buffer (12), glucose (10), NaCl (0.5), KCl (0.5) NH4SO4 (2),
MgSO4 7H2O (0.2) CaCl2 (0.01) and FeSO4 7H2O (0.001). Both
media were supplemented with filter-st erilized (0.2 μm f ilter)
glyphosate and were used to enrich and isolate glyphosate-
degrading strains.
Experimental soil
Soil specimens were collected in April 2012 from three different
untreated soils. The first sample was an agricultural soil, taken from
the Institute of Field Crops in Constantine located between 7°35'
longitude and 36°23' latitude in the center of eastern Algeria. The
second sample was taken from the forest of Chaâberssas located
in the University of Constantine, Algeria. The third sample was
taken from a sandy field located in the region of Biskra located bet-
between 34°51'01" north latitude and 5°43'40" east longitude in the
north-eastern of Algeria on the northern edge of the Sahara,
Desert. Samples of about 1 kg were taken from the first 15 cm of
depth, pooled and sieved. Samples were air dried and stored in
sterile plastic bags at 4°C until use.
Enrichment and isolation of glyphosate-degrading strains
About 5.0 g of each soil were added to 95 mL of MSM1 or MSM2
medium in 250 mL flasks with the addition of glyphosate at a final
concentration of 0.5 g/L and incubated in the dark at 30°C under
shaking condition (150 rpm) for seven days. A 5 mL volume of
these suspensions were then transferred to fresh MSM1 or MSM2
containing 1g/L glyphosate and incubated for seven days. Three
additional successive transfers were made into media successively
containing 3, 6 and 12 g/L of glyphosate. The appropriate dilutions
of enriched samples were plated on plate count agar supplemented
with 1 g/L glyphosate. The plates were incubated at 30°C for 24 h.
Colonies were picked and purified. The strains Arph1, Arph2, Frglu,
Frph and Bisglu were isolated.
Conventional ident ification
For morphological and physiological studies, Arph1, Arph2, Frglu,
Frph and Bisglu isolates were grown in aerobically atmosphere at
30°C on Columbia Agar 5% sheep-blood media (Biomerieux, la
Balm-les-grottes, France). Apart from morphology, mobility,
catalase, oxidase and Gram reaction, physiological studies were
performed by using API 20E.
MALDI-TOF MS identification
The MALDI-TOF mass spectrometry protein analysis was carried-
out as previously described (Seng et al., 2009). Briefly, a pipette tip
was used to pick one isolated bacterial colony from a culture Agar
plate, and to spread it as a thin film on a MTP 384 MALDI-TOF
target plate (Bruker Daltonics, Leipzig, Germany). Twelve (12)
distinct deposits were done for each isolates from 12 different
colonies. Each smear was overlaid with 2 μL of matrix solution
(saturated solution of alpha-cyano-4-hydroxycinnamic acid) in 50%
acetonitrile, and 2.5% tri-fluoracetic-acid, and allowed to dry for five
minutes. Measurements were performed with a Microflex
spectrometer (Bruker).
Spectra were recorded in the positive linear mode for the mass
range of 2 to 20 kDa (parameter settings: ion source 1 (IS1), 20 kV;
IS2, 18.5 kV; lens, 7 kV). A spectrum was obtained after 675 shots
at a variable laser power. The time of acquisition was between 30
sand 1 minper spot. The 12 spectra of the different isolated strains
were imported into the MALDI BioTyper software (version 2.0,
Bruker) and analyzed by standard pattern matching (with default
parameter settings) against the main spectra of 6.213 bacteria in
the BioTyper database. For every spectrum, 100 peaks at most
were taken into account and compared with spectra in the
database.
16S rRNA gene amplification and sequencing
The 16S rRNA gene of the isolates was amplified using the primer
pair fD1-P2 (Weisburg et al., 1991). PCR amplifications were
carried-out in a 50 µL volume containing 5 µL template, 50 mM KCl,
1.5 mM MgCl2, 200 µM each dNTP, 0.2 µM each oligonucleotide
primers and 0.5 units of Taq DNApolymerase (EuroblueTaq,
Eurobio, Les Ulis, France). The thermal cycle consisted of an initial
5 min denaturation at 95°C followed by 35 cycles of 30 s
denaturation at 95°C, primer hybridization at 52°C for 30 s and
elongation at 72°C for 1 min and a final 5-min extension step at
72°C. PCR reactions were examined by electrophoresing 5 µL of
PCR product on a 1% agarose gel stained with ethidium bromide.
The gel was visualized using Gel Doc 1000 (Bio-Rad, California,
USA). Successful PCRs were transferred into PCR purification plate
(Macherey Nagel HOERDT, France) filtrated with vacuum manifolds
Millipore and agitated with a plate (Heidolph instrument Titramax
100). Purified PCR products were sequenced with the use of a
BigDye® Terminator v1.1 Cycle Sequencing Ready Reaction Kit
(Applied Biosystems), the Bvd1, 5X Sequencing Buffer and the
primers 536F, 536R, 800F, 800R, 1050F and 1050R. Sequencing
was then performed in ABI3700 automated capillary sequencer
(Applied Biosystems, Foster City, California, United States).
The nucleotide sequences were edited using ChromasPro 1.34
software (Copyright (c) 2003-2006 by Technelysium Pty Ltd).
Phylogenetic relationships of the genes were reconstructed using
neighbor-joining implemented in MEGA 5 software (Tamura et al.,
2011).
Glyphosate utilization patterns of the different isolates
Inoculums were prepared for each isolate by growing the strains in
50 mL of nutrient broth for three days at 30°C under shaking
condition (150 rpm) till the growth reached late exponential phase.
Cells were harvested by centrifugation at 4, 600 g for 5 min,
washed with 0.9% sterile saline and were re-suspended to a 0.5
McFarland nephelometer standard (Optical density of 0.18 at 625
nm) and this was then used as the inoculum.
Growth experiments with glyphosate as the sole source of carbon
or phosphorus were performed in 250 mL Erlenmeyer flasks
containing 100 mL sterile MSM 1 or MSM 2 with 1 g/L of
glyphosate. A 2 mL of each isolate was inoculated and triplicate
cultures were incubated on a rotary shaker at 150 rpm for 168 h at
30°C. Non-inoculated media served as control. Samples (2 mL)
were withdrawn periodically from the cultures to determine growth
by measurement of the turbidity at 625 nm using a spectro-
photometer.
Optimization of cultivation conditions
To optimize growth in glyphosate enriched media, some important
abiotic factors were chosen. The important factors and their
optimized ranges that were chosen in this experiment were
nutriments (yeast extract, glutamate and glycerol), temperature (30,
37 and 40°C), medium pH (5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) and
initial concentration of glyphosate (1, 3, 5, 7, 9, 12 and 15 g/L). All
experiments were performed in 250 mL flasks containing 100 mL of
MSM2 supplemented with an appropriate amount of glyphosate,
adjusted to an appropriate initial pH and inoculated with 2 mL of
Arph1 strain. The flasks were then incubated at the appropriate
temperature in the dark for seven days and stirred on a rotary
shaker at 150 rpm. Controls without inoculation were kept in similar
conditions. Bacterial growth was followed by taking a sample of 2
mL of cultures after every 24 h until 168 h of incubation and the
optical density was measured at 625 nm.
In the first step, the flasks containing the minimum media were
supplemented with 0.1%, w/v of various nutrients (yeast extract,
glutamate and glycerol). In the second step, minimum medium
supplemented with 0.1%, w/v of glutamate were incubated at
different temperatures (30, 37 and 40°C). In the third step, minimum
Benslama and Boulahrouf 5589
medium supplemented with 0.1%, w/v of glutamate were adjusted
to different initial pH (5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) and incubated
at 30°C. Finally, minimum medium supplemented with 0.1%, (w/v)
of glutamate with different concentrations of glyphosate (1, 3, 5, 7,
9, 12 and 15 g/L), adjusted to pH 9 were incubated at 30°C.
RESULTS
In the present study, five bacterial strains were isolated
from different untreated soils of Algeria; these isolates
have shown an ability to grow in a culture medium in the
presence of the herbicide glyphosate as sole source of
carbon or phosphorus. These isolates were named as
follows: Arph1 and Arph2 isolated from the agricultural
soil of Constantine in the medium containing glyphosate
as sole phosphorus source; Frglu isolated from the forest
soil of Constantine in the medium containing glyphosate
as sole C source; Frph isolated from the forest soil of
Constantine in the medium containing glyphosate as sole
P source and Bisglu isolated from the Saharan soil of
Biskra in the medium containing glyphosate as sole C
source.
Strains identification
For the MALDI-TOF analysis, the obtained score of the
isolate Arph1, was 2.5 close to the species
Pseudomonas putida. The sequence of 1500 bp of the
gene 16S rRNA was deposited in GenBank under
Accession number KC582298. The phylogenetic tree
showing the result of the 16S rRNA of Arph1 is
represented in Figure 1. The sequence of the 16S rRNA
gene of the isolate was 99.5% similar to the 16S rRNA
gene of P. putida (GenBank Accession No. gb
|EU439424.1|) and 99.4% similar to 16S rRNA gene of P.
putida (GenBank Accession No. gb |EU439425.1|). The
result of the analysis of 16S rRNA is consistent with that
of MALDI-TOF, morphological and biochemical properties
(Table 1). Therefore, the isolate was identified as P.
putida.
MALDI-TOF scores obtained for the isolates Arph2 and
Frglu were 2.4 and 2.3 near to Enterobacter cloacae,
respectively. For the analysis of 16S rRNA gene, the
isolate Arph2 showed a sequence similarity of 99.1% with
E. cloacae (GenBank Accession No. gb |JF772064.1|)
and 98.9% with E. cloacae (GenBank Accession No. gb
|CP003737.1|) and Pantoea agglomerans (GenBank
Accession No. gb |AY335552.1|) while the isolate Frglu
showed a similarity of 99.3% with E. cloacae (GenBank
Accession No. gb |JX307682.1|) and 98.8% with E.
cloacae (GenBank Accession No. gb |EF059833.1|). The
sequences of 1513 and 1288 bp of the gene 16S rRNA of
the isolates Arph2 and Frglu were deposited in GenBank
under the Accession numbers KC582299 and KC582300,
respectively. Phylogenetic analysis of 16S rRNA gene
5590 Afr. J. Microbiol. Res.
Figure 1. Neighbor joining tree based on 16S rRNA sequences for Enterobacteriacae. To examine the confidence
of NJ tree, 1000 bootstrap replicates were used.
clustered the two isolates with E. cloacae species as
shown in Figure 1. Based on the results of phylogenetic
and phenotypic tests, the isolates Arph2 and Frglu were
identified as Enterobacter cloacae.
The result of MALDI-TOF identified the isolate Arph3 as
Rahnella aquatilis with a high score of 2.5. The result of
the 16S rRNA gene analysis showed that the isolate Frph
shares a similarity of 99.2% with R. aquatilis (GenBank
Accession No. gb |FJ405361.1|) and 98.8% with R.
aquatilis (GenBank Accession No. emb |X79937.1|). The
sequence of 1337 bp of the gene 16S rRNA of the isolate
was deposited in GenBank under Accession number
KC582301. The result of the analysis of 16S rRNA gene
of the Frph strain is consistent with that of MALDI-TOF,
morphological and biochemical properties (Table 1).
Therefore, the isolate was identified as R. aquatilis.
MALDI-TOF identified the isolate Bisglu as Serratia
marcescens with a high score of 2.4. The result of the
16S rRNA gene analysis showed that the isolate Bisglu
shares a similarity of 99.3% with S. marcescens
(GenBank Accession No. gb |JQ308606.1|) and 98.9%
with S. marcescens (GenBank Accession No. dbj
|AB594756.1|). The sequence of 1233 bp of the gene
16S rRNA of the isolate was deposited in GenBank under
Accession number KC582302. Based on the results of
phylogenetic and phenotypic tests, the isolate Bisglu can
be identified as S. marcescens.
Glyphosate utilization patterns of the different
isolates
As shown in the Figure 2a, of the three isolates grown in
the media containing the glyphosate as sole phosphorus
source, and tested for the growth by measuring their
turbidimetry at 625 nm, P. pudida showed the highest
growth level (OD average = 0.129) suggesting extensive
use of glyphosate. This was followed by E. cloacae (OD
average = 0.100) and R. aquatilis (OD average = 0.084).
However, for the isolates grown in the media containing
the glyphosate as sole carbon source, the growth was
very low as showed in the Figure 2b. The ODs averages
Benslama and Boulahrouf 5591
Table 1. Morphological and biochemical properties of the different isolates. (+), strains positive; (-),
strains negative
Property
Arph1
Arph2
Frglu
Frph
Bisglu
Morphology
Rod-
shaped
Rod-
shaped
Rod-
shaped
Rod-
shaped
Rod-
shaped
Motility
+
+
+
+
+
Biochemical tests
Gram test
-
-
-
-
-
Oxidase/ catalase
+/+
-/+
-/+
-/+
-/+
β-galactosidase
-
+
+
+
+
Voges-Proskauer
-
+
+
+
+
Nitrate production
-
+
+
+
+
Lysine decarboxylase
-
+
+
-
+
Ornithine decarboxylase
-
+
+
-
+
H2S production
-
-
-
-
-
Urease
-
-
-
-
-
Tryptophan deaminase
-
-
-
-
-
Indole production
-
-
-
-
-
Gelatinase
-
-
-
-
+
Arginine dihydrolase
+
-
-
-
-
Citrate
+
+
+
-
+
Sugar use
Mannose
-
+
+
+
+
Glucose
-
+
+
+
+
Sorbitol
-
+
+
+
+
Rhamnose
-
+
+
+
-
Sucrose
-
+
+
+
+
Melibiose
-
+
+
+
+
Amygdalin
-
+
+
+
+
Arabinose
-
+
+
+
-
Inositol
-
-
-
-
+
were about 0.051 and 0.045 for S. marscecens and E.
cloacae, respectively.
Effect of abiotic factors
Figure 3 shows the effect of certain nutrients (yeast
extract, glycerol and glutamate) on bacterial growth. As
seen in Figure 3 in the presence of glutamate, growth
kinetic of P. putida strain shows a steady increase in
growth after 24 h of incubation and reached a maximum
of growth of 0.250 after 168 h of incubation, which
represent an increase of over 15% compared to its
growth in the medium without any nutriment. While in the
presence of glycerol and yeast extract, the growth
reached a maximum of 0.245 and 0.206 after 168 h of
incubation, respectively. Therefore, the glutamate was
selected as a carbon source for further studies.
Figure 4 shows the evolution of bacterial growth at
different temperatures (30, 37 and 40°C). A significant
increase in the growth of P. putida strain was noted at
30°C, where the growth of the strain reached its peak of
0.248 after 168 h of incubation. A less significant growth
was obtained at 37°C, reached a maximum of 0.145 after
168 h of incubation. At 40°C, the strain showed a slower
and weak growth and reached its maximum growth of
0.07 after 168 h of incubation. Therefore, the temperature
30°C was selected for further studies.
The effect of pH on the growth of P. putida is shown in
Figure 5. In general, the growth of the strain is greater in
alkaline pH ranging from 7 to 10 over 168 h. When the
initial pH is lower than 7, the growth of the strain
gradually decreased with the decrease of pH. At pH 9,
the growth peaked significantly and reached a maximum
of 0.261 within 168 h of incubation.
The growth kinetic at various initial concentrations of
glyphosate is shown in Figure 6. Increase in microbial
growth was observed till initial concentration of gly-
phosate was increased to 3 g/L. As the concentration of
5592 Afr. J. Microbiol. Res.
0
0.05
0.1
0.15
0.2
0.25
0.3
0
24
48
72
96
120
144
168
Absorbance (625 nm)
Time of incubation (hours)
P. putida
E. cloacae
R. aquatilis
Figure 2a. Growth kinetics of P.pudida, E. cloacae, R.aquatilis strains in
glyphosate as sole phosphorus source.
0
0.05
0.1
0.15
0.2
0.25
0.3
0
24
48
72
96
120
144
168
Absorbance (625 nm)
Time of incubation (hours)
S. marscecens
E.cloacae
Figure 2b. Growth kinetics of E. cloacae and S. marscecens strains in
glyphosate as sole carbon source.
0
0.05
0.1
0.15
0.2
0.25
0.3
0
24
48
72
96
120
144
168
Absorbance (625 nm)
Time of incubation (hours)
glutamate
glycerol
yeast extract
Figure 3. Growth kinetics of P. putida strain in glyphosate as sole
phosphorus source with different nutrients.
Benslama and Boulahrouf 5593
0
0.05
0.1
0.15
0.2
0.25
0.3
0
24
48
72
96
120
144
168
Absorbance (625 nm)
Time of incubation (hours)
30°C
37°C
40°C
Figure 4. Growth kinetics of P. putida strain in glyphosate as sole
phosphorus source supplemented with glutamate (0.1% w/v) in
different temperatures.
0
0.05
0.1
0.15
0.2
0.25
0.3
0
24
48
72
96
120
144
168
Absorbance (625 nm)
Time of incubation (hours)
pH 5
pH 6
pH 7
pH 8
pH 9
pH 10
Figure 5. Growth kinetics of P. putida strain in glyphosate as sole
phosphorus source supplemented with glutamate (0.1% w/v) with
different pH at 30°C.
Figure 6. Effect of the initial concentration of glyphosate on the growth
of P. putida strain.
5594 Afr. J. Microbiol. Res.
glyphosate increased, there was a decrease in the
growth of the isolate. The high concentrations of gly-
phosate severely inhibit bacterial growth.
The highest growth was observed at 1 g/L, which is the
least tested concentration of glyphosate. After 24 h of
incubation, the growth of P. putida in the medium
containing 1 g/L of glyphosate increased significantly and
reached a maximum of 0.265 after 186 h of incubation.
However, No inhibition of growth was observed when
initial gly-phosate concentration was increased further,
indicating that the isolate can tolerate up to 9 g/L of
glyphosate.
DISCUSSION
The limited number of strains isolated from the medium
containing glyphosate as sole carbon or phosphorus
source, is in agreement with the reports of Quinn et al.
(1988), Santos and Flores (1995) and Kryzsko-Lupicka
and Orlik (1997), which showed a significant reduction in
microbial population when glyphosate was added to the
medium culture. This result can be explained by the
toxicity of artificial media due to the mode of action of
glyphosate (the way of shikimic acid is ubiquitous in
microorganisms (Glyphosate makes the organism unable
to synthesize essential aromatic amino acids). In
addition, Liu et al. (1991), Dick and Quinn (1995) report
that when glyphosate is supplied as carbon source,
microbial growth is rare, but growth stimulation is more
apparent when applied in high concentrations. It was
found that the commonly isolated glyphosate-degraders
in the laboratory are the Pseudomonas spp. bacteria
(Jacob et al., 1988; Dick and Quinn, 1995). Five
pseudomonas species isolated that grew solely on
glyphosate were identified (P. maltophilia, P. putida, P.
aeruginosa and Pseudomonas sp.) that whose growth
were not inhibited due to a glyphosate-resistant EPSPS
(Schulz et al., 1985). As well, Bacillus megaterium (Quinn
et al., 1989), Alcaligenes sp. (Tolbot et al., 1984)
Flavobacterium sp. (Balthazor and Hallas, 1986),
Geobacillus caldoxylosilyticus (Obojska et al., 2002),
Rhizobium sp. and Agrobacterium sp. (Liu et al., 1991),
R. aquatilis (Peng et al., 2012) and Enterobacter cloacae
(Kryuchkova et al., 2013) have been reported as
degrading glyphosate. However, there is no report of the
isolation of Serratia marcescens as glyphosate degrading
bacteria. Thus, this finding adds to the list of glyphosate-
degrading bacteria a new degrading species that can be
used in further studies.
The use of microorganisms for bioremediation requires
an understanding of all physiological, microbiological,
ecological, biochemical and molecular aspects involved
in pollutant transformation (Iranzo et al., 2001). The effect
of abiotic factors on bacterial growth was used to
optimize the cultivation conditions that affect significantly
the glyphosate degradation and the bacterial growth. P.
putida was selected in this study because it showed the
highest growth potential in comparison with the other
species as showed in Figure 2a and b. The growth
kinetics of P. putida was monitored over time at 620 nm
using the MSM2 enriched with glyphosate as the sole
source of phosphorus, varying abiotic conditions of the
environment.
Growth rate was most important in the medium
supplemented with glutamate; this results is in agreement
with the report of Shushkova et al. (2012) who shows an
effective glyphosate degradation in the presence of
glutamate in the medium. Kumar and Philip (2006)
reported that the addition of auxiliary carbon to the
system having xenobiotic compounds increased the
biodegradation potential of bacterial culture which was
often because of increase in metabolic activity of the
microbes involved. As reported by Mallick et al. (1999)
and Guha et al. (1997), the co-metabolism appears to
occur commonly in nature. Microbial activity increases
with increasing temperature up to an optimum value. This
result can be attributed to the fact that, at low tem-
perature, the growth of the strain P. putida and the
reaction catalyzed by the enzyme degrading glyphosate
are increased. This indicates that the strain is psy-
chrophilic nature (Patel et al., 2012). Moorman (1994)
stated that within the range of temperature conditions
normally encountered in cultivated soils, the rate of
pesticide degradation generally increased with tem-
perature. Walker et al. (1992) considered soil tem-
perature to be the most important environmental factor
influencing pesticide degradation rate in soils. It has been
reported, that the incubation temperature does not only
affect the pesticide degradation rate but also affect the
growth of the strain.
Slightly alkaline pH is favorable for glyphosate
degradation by the strain, probably due to the increased
bioavailability and the decreased toxicity of glyphosate,
and to the optimal metabolic activity of the bacterial cells.
Singh et al. (2003a, b) report that in soils with higher pH a
higher copy numbers of organo-phosphate degrading
(opd) gene are found, suggesting that the activity of the
enzymes degrading organo-phosphate compounds is
more important at alkaline pH. Decrease in cell density at
high concentrations of glyphosate can be attributed to the
toxicity and the stress of glyphosate on strain. This can
also be explained by the fact that at high concentrations,
the appropriate catabolic enzymes may be repressed.
Another plausible explanation is that the strain may need
an acclimation period to induce the necessary degra-
dative path (Tang and You, 2011). A similar resultwas
found by Moneke et al. (2010) when testing different
initial concentrations of glyphosate on Acetobacter sp. and
and P. fluorescens. Tolerance to high pesticide concen-
trations is critical, since concentrations at contaminated
sites may be several orders of magnitude higher than the
recommended usage doses for these products.
Conclusion
This study is the first report of isolation and charac-
terization of a soil-borne bacterial strains (P. putida, E.
colacae, R. aquatilis and S. marcescens) from an
agricultural, Saharan and forest soil in Algeria that
possess the capacity to use glyphosate. The capacity of
these isolates to survive and grow in the presence of high
concentrations of the herbicide, show that these strains
may possess potential to be used in bioremediation of
glyphosate-contaminated environments or moreover, can
contribute on creating glyphosate-resistant crops.
In addition, this work adds to the list of glyphosate-
degrading bacteria a new degrading species that is S.
marcescens. This study provides also important infor-
mation on optimization of critical parameters of cultivation
conditions to enhance glyphosate degradation by P.
Putida strain.
ACKNOWLEDGEMENT
Authors gratefully acknowledge Professor Michel
Drancourt from Aix Marseille University, Unité des
Rickettsies, Faculté de Médecine, Marseille, France.
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