Degradation of Clodinafop Propargyl by Pseudomonas sp.Strain B2

Baljinder Singh

Received: 18 June 2013 / Accepted: 4 October 2013 / Published online: 12 October 2013

� Springer Science+Business Media New York 2013

Abstract Using clodinafop propargyl (CF) as a sole

carbon, nitrogen and energy source, a CF-degrading bac-

terial strain was isolated from crop soil field. This strain

was identified as Pseudomonas sp. strain B2 by 16S rRNA

gene sequence analysis. 87.14 % CF was degraded out of

initial provided 80 mg/L CF. Degradation of CF was

accompanied by release of chloride ion. The optimal pH

and temperature for the growth of B2 were 7.0 and 30�C,

respectively in the mineral salts medium supplemented

with CF. An actively growing culture of strain B2 degraded

CF to clodinafop acid and 4-(4-Chloro-2-fluoro-phenoxy)-

phenol within 9 h, as determined by GC–MS analysis. A

metabolic pathway for the degradation of CF by B2 has

been proposed.

Keywords Pseudomonas sp. strain B2 � Clodinafop

propargyl � Biodegradation � GC–MS

CF (prop-2-ynyl(R)-2-[4-(5-chloro-3-fluoro-2 pyridyloxy)

phenoxy]propionate), is an important aryloxyphenoxypro-

pionate herbicide. CF is used for post emergence control of

annual grasses in cereals, including Avena, Lolium, Setaria,

Phalaris and Alopecurus spp. (Tomloin 2006). CF is absor-

bed by the leaves and rapidly translocated to the growing

points of leaves and stems. It interferes with the production

of fatty acids needed for plant growth in susceptible grassy

weeds (Hammami et al. 2011). CF acts by targeting the

enzyme acetyl coenzyme-A-carboxylase, essential for lipid

biosynthesis (Devine and Shimabukuro 1994). The wide-

spread use of CF has resulted in the discharge of large amounts

of the compound into the environment, which eventually

reach the biosphere (Gherekhloo et al. 2010; Vazan et al.

2011). The half-lives of CF were 2.35–11.20 days in soil and it

rapidly degraded to the acid derivative clodinafop as major

metabolite in soil (Guan et al. 2013). Several studies have

demonstrated that CF and its derivatives are toxic and car-

cinogenic to humans and other living organisms (Kashanian

et al. 2008; Gui et al. 2011). Therefore, the degradation of CF

in the environment is of great concern. Only a single study

concerning the biodegradation of CF can be found in the lit-

erature. This might be because of its low persistence; the half-

life in soil was reported to be 5 days, dependent on the soil

type, pH, and microbial population (Roy and Singh 2006).

They had reported 97.9 % CF-degradation without identify-

ing its metabolites.

In this study, we successfully isolated a Pseudomonas

sp. That could use CF as the sole carbon, nitrogen and

energy source. We named this strain Pseudomonas sp.

strain B2. Degradation of CF by strain B2 in liquid culture

was studied. Importantly, this is the first report of degra-

dation of CF by genus Pseudomonas.

Materials and Methods

Soil samples were collected from crop field area with a

previous history of CF application, located in the city of

Patiala, Punjab, India. Soil samples were held in sterile

bottles and stored at 4�C until used. CF (99.4 % purity)

was purchased from Sigma Aldrich (PESTANAL, Fluka

analytical). All other chemicals used in this study were

analytical grade or higher purity.

B. Singh (&)

Department of Biotechnology, Panjab University,

Chandigarh 160014, India

e-mail: gilljwms2@gmail.com

123

Bull Environ Contam Toxicol (2013) 91:730–733

DOI 10.1007/s00128-013-1124-2

A selective minimal salt medium (MS) was prepared

containing 40 mg/L CF as a sole source of carbon, nitrogen

in addition to 4 g Na2HPO4.2H2O, 2 g KH2PO4 (0.025 %),

MgSO4.7H2O (0.05 %), and 1 mL of trace element solu-

tion (0.1 g of ZnSO4.7H2O, 0.03 g of MnCl2.7H2O, 0.3 g

of H3BO3, 0.2 g of CoCl2.6H2O, 0.01 g of CuCl2.2H2O,

0.02 g of NiCl2.6H2O, in 1 L of the solution). Five grams

of soil sample were inoculated into Erlenmeyer flask

(250 mL) containing 100 mL autoclaved water. Processed

soil sample (0.5 mL) was spread on MS media plates and

incubated at 30�C for 3 days until bacterial colonies

became visible. Colonies grown on these plates were

evaluated for their CF degrading capabilities. Single colony

types were separated and subcultured on fresh plates to

purity using identical growth conditions at each transfer,

except that the CF concentration was increased stepwise

from 40 to 120 mg/L. One strain, designated as B2, which

possessed the highest CF-degrading ability and could uti-

lise CF as the sole carbon source for growth, was purified

and selected for further investigation. Strain B2 was clas-

sified by Gram staining, starch hydrolysis test, gelatin test,

catalase test, NaCl, pH, temperature variation assay, and

16S rRNA analysis. Genomic DNA extraction from strain

B2 was performed using the method described by Sam-

brook et al. (1989). Partial fragment of 16S rRNA gene of

strain B2 was amplified by PCR with set of universal

primers 27F (50-AGAGTTTGATCCTGGCTCAG-30) and

1492R (50-TACGGYTACCTTGTTACGACTT-30) follow-

ing the PCR parameters as described by Singh et al. (2011).

Solutions of CF were freshly prepared in methanol at

concentration of 1 mg/mL.

A 1 mL aliquot of CF solution was transferred into

sterile 12 mL amber glass vials. The vials were left open

in a fume hood to allow the solvent to evaporate. A

100 lL pre-grown single bacterial clone (OD600 = 0.5)

was inoculated into each vial to give an initial CF con-

centration of 80 mg/L. The cultures were incubated at

30�C and 100 rpm on a rotary shaker for 12 h. In order

evaluate the effect of temperature and pH, MS media

containing 80 mg/L CF were incubated for 12 h at dif-

ferent temperatures (20, 25, 30, 35, 40, and 45�C) and

under different pH conditions (5.0–10.0, in increments of

1.0 pH units). Uninoculated MS media was used as con-

trol. Each treatment was performed in three replicates,

and the control experiment without microorganism was

carried out under the same conditions.

The chloride ion concentration was determined using

Mohr method (Korkmaz). Two hundred microliters of a

sample diluted so that the chloride concentration was up to

0.1 mM was added to 50 mL of 0.25 M potassium chro-

mate. The reaction mixture was titrated with 0.1 M silver

nitrate solution. Chloride ion concentrations were calcu-

lated by using volumetric analysis.

During degradation, 1 mL sample was removed from

each vial at regular intervals to measure the inoculants

growth of the cell and CF concentration. To analyze CF and

its metabolites sample aliquots (1 mL) were taken at regular

intervals. The culture broth was centrifuged (10,000 rpm,

4�C for 10 min) and supernatant was acidified with 1 M

H2SO4 to a pH \ 5. The solution was extracted with two

1 mL portions of hexane and derivatized by adding 0.1 mL

diazoethane (Sigma Aldrich). Diazoethane is an ethylating

agent that facilitates the simultaneous analysis of CF and

clodinafop acid. Excess diazoethane was removed under a

stream of cold nitrogen and the solution was dried over

anhydrous Na2SO4. A cold stream of nitrogen was used to

evaporate the hexane and to allow a solvent exchange to

toluene. The final volume was adjusted to 2 mL with toluene.

Samples were analysed by using high-pressure liquid chro-

matography (HPLC) on a reverse-phase C18 column at

258 nm. The mobile phase was acetonitrile/water (50:50

v/v) and flow rate was 1 mL/min. To calculate % degrada-

tion, peak areas were measured to quantify the CF. Extracts

were analyzed using GC–MS-QP2010 Plus system (Shima-

dzu Corporation, Japan). GC column oven temperature was

programmed for an initial hold of 1 min at 100�C; then

temperature was increased at 10�C/min to 200�C; then up to

260�C at the rate of 15�C/min; followed up to 300�C at the

rate of 3�C/min and then hold at 300�C for 2 min. The gas

flow rate was 1 mL/min in splitless mode with injection

temperature of 270�C. Conditions for MS measurements

were: MS ion source at 200�C, MS interface temperature

250�C, electron impact ionisation (EI) at 70 eV. Chro-

matographic data were collected and recorded by GC–MS

real time analysis software.

Results and Discussion

Standard isolation and enrichment techniques yielded a

bacterial isolate, designated as strain B2 capable of

degrading CF and selected for further studies. B2 could

degrade 87.14 % of 80 mg/L CF in 9 h. When grown on

LB agar, cells of this strain are non-spore-forming, gram

negative, motile, and globular- or globular-rod-shaped. The

nucleotide sequence coding for the 16S rRNA of strain B2

(1,081 bp) was deposited in the GenBank database with

accession number KF254765. BLASTN and phylogenetic

analysis of 16S rRNA gene sequence revealed that strain

B2 belonged to Pseudomonas sp. (99 % similarity).

HPLC chromatograms of control and test reactions were

recorded and CF peak was observed after retention time

2.779 min. Limits of detection (LODs) were calculated using

a peak-to peak height signal to noise ratio of 3:1, at the lowest

calibration concentration of analyte. LOD for CF was 2 ng/L.

The major metabolites, clodinafop acid and 4-(4-Chloro-2-

Bull Environ Contam Toxicol (2013) 91:730–733 731

123

fluoro-phenoxy)-phenol peaks were observed at 4.519 and

1.874 min respectively. In order to evaluate the effect of CF

concentration on microbial growth, strain B2 was cultivated in

MS at CF concentrations 40, 80 and 120 mg/L. The degra-

dation of CF by strain B2 could be affected by substrate

concentration (Fig. 1a). Substrate concentration is one of the

factors governing the diffusive mass transfer of pollutants to

microbes. Therefore, at a higher initial pollutant concentra-

tion, more of the pollutant is able to enter the cells as a result of

faster diffusion and up-take by the cells. Ma et al. (2013)

reported that higher intracellular pollutant concentration

would result in higher degradation rate, but this is not con-

sistent with our observation, which showed that higher CF

concentration resulted in slower degradation. The optimum

concentration at which strain B2 showed the maximum

growth was 80 mg/L (Fig. 1a). At a higher concentration of

CF (120 mg/L), it was observed that strain B2 could maintain

a gradual degradation rate to final 55 % degradation of the

initial amount of CF after 9 h of incubation (Fig. 1a). This

limited growth at higher concentrations of CF could result

from higher toxicity, meaning that CF uptake was limited. The

effects of pH and temperature on the biodegradation of CF

were also investigated. When the pH was between 7.0 and 8.0,

more than 80 % of 80 mg/L CF could be degraded by B2

within 9 h. The optimum temperature for the biodegradation

of CF was 30–35�C. However, CF biodegradation decreased

when the temperature dropped to 20�C or rose to 40�C, indi-

cating that lower and higher temperatures were not beneficial

for the biodegradation of CF by B2.

The growth of strain B2 on CF and its ability to degrade

CF is shown in Fig. 1b. With CF as the carbon, nitrogen

and energy source, strain B2 produced a typical sigmoidal

growth curve consisting of a relatively very short lag phase

and an exponential phase of approximately 9 h, followed

by a abrupt transition to the stationary phase (Fig. 1b).

The OD600 showed a steady increase in bacterial mass.

Simultaneously, HPLC analysis showed a substantial

reduction in the levels of CF. Results demonstrated an initial

CF degradation rate and the biomass formation were detec-

ted. After incubation of 9 h, 87.14 % CF (80 mg/L) initially

added to the MS medium was degraded by strain B2

(Fig. 1b). Further no further degradation was observed.

Uninoculated controls showed no change in CF concentra-

tion was observed in cultures that were not inoculated. Only

trace amounts of 4-(4-Chloro-2-fluoro-phenoxy)-phenol)

were detected during the early stages of growth (1–2 h), high

concentrations of this metabolite in the growth medium

during the log and stationary phases (14–30 h) suggested that

4-(4-Chloro-2-fluoro-phenoxy)-phenol was the major deg-

radation product. This was in agreement with previous

observations by Smith-Grenier and Adkins (1996). They

reported the degradation of diclofop-methyl by Chryseo-

monas luteola and Sphingomonas paucimobilis and forma-

tion 4-(2,4-dichlorophenoxy)phenol as metabolites. The

formation of phenol as metabolite during growth of strain B2

in MS medium provided an indication that it might be due to

esterase activity as reported previously (Hou et al. 2011).

However, on the basis of the structures of the metabolites, the

initial degradation of the compound is suggested to take

place via cleavage of the ester bond. The presence of

metabolite, [4-(4-chloro-2-fluorophenoxy) phenol], sup-

ported this suggestion. Other possible breakdown product,

including clodinafop acid was also observed. The release of

chloride ion from the ring of CF during the biodegradation

pathway was of the particular interest for this work. During

the reaction amounts of chloride ion (2.1 ± 0.3 mg/L) were

Fig. 1 Degradation of CF by strain B2. a Effect of concentration on

CF degradation. b A time course study of CF degradation in MS

medium supplemented with 80 mg/L CF. Data are presented as mean

and standard error of three independent observations. Some error bars

are not present because they are smaller than the diameter of the

symbol

732 Bull Environ Contam Toxicol (2013) 91:730–733

123

released from initial provided 80 mg/L CF in 9 h. Therefore,

it is possible that the chloride ion release leads to catabolism

of the pyridyl moiety in CF.

In summary, the results indicate that strain B2 is capable

of rapidly hydrolyzing the ester bond of CF to produce

clodinafop acid, which in turn may either be directly

hydrolyzed to form 4-(4-Chloro-2-fluoro-phenoxy)-phenol

(Fig. 2). Future experiments using 14C-labelled compounds

will assist in clarifying this point. At this time, it is difficult

to ascertain the full degradation potential of strain B2.

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Fig. 2 Proposed pathway of CF

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