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Biosurfactant Facilitated Biodegradation Of QuinalphosAt High Concentrations By Pseudomonas aeruginosaQ10Ambilli M. Naira, Sharrel Rebelloa, Rishad K.Sa, Aju K. Asoka & Jisha M.S.a

a School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, IndiaAccepted author version posted online: 06 Dec 2014.

To cite this article: Ambilli M. Nair, Sharrel Rebello, Rishad K.S, Aju K. Asok & Jisha M.S. (2014): Biosurfactant FacilitatedBiodegradation Of Quinalphos At High Concentrations By Pseudomonas aeruginosa Q10, Soil and Sediment Contamination: AnInternational Journal, DOI: 10.1080/15320383.2015.988205

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Biosurfactant Facilitated Biodegradation Of Quinalphos At High

Concentrations By Pseudomonas aeruginosa Q10

Ambilli M. Nair; Sharrel Rebello; Rishad K.S; Aju K. Asok and Jisha M.S.*

School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala, India

Tel: +91-481-2731035, Fax: +91 481 2731002.Email: jishams@mgu.ac.in;

Abstract

Previous studies indicate that high concentration of pesticides and its associated toxic effects are

high at their point source of application. Use of pesticide degrading bacteria at point sources

could augment degradation and thereby reduce toxic effects associated with its persistence in

soil. Quinalphos, an organophosphorus insecticide, though ranked 'moderately hazardous' in

WHO’s acute hazard ranking still continues to be used extensively in developing countries. The

presence of a chloride radical usually makes this pesticide sparingly soluble in water and hence

difficult to degrade. The present study aimed to isolate autochthonous bacterial strains capable of

utilizing quinalphos as carbon source. Primary screening of pesticide contaminated soil by

enrichment culture and degradation analysis by UV-VIS spectrophotometry led to isolation of 12

different bacterial isolates of which 3 efficient isolates of Pseudomonas sp, Serratia sp and

Pseudomonas aeruginosa with degradation rate 86%, 82%, 94% respectively were selected. GC-

MS studies with P.aeruginosa confirmed formation of 2-hydroxy quinoxaline and

phosphorothioic acid as a result of biodegradation. The present study succeeded to isolate

autochthonous bacterial strains capable of utilizing high concentrations of quinalphos as carbon

source in a shorter incubation period. This strain also possessed biosurfactant production ability

which makes quinalphos available to cells at higher concentrations.

Key words: Biosurfactant; Quinalphos; Gas Chromatography; Mass Spectrometry.

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Introduction

The unprecedented population explosion and subsequent increased food demand, prompted man

to adopt various improved crop yielding strategies. The use of high breeding crop varieties,

better fertilizers and extensive pesticides helped man to achieve his goal of high crop yield, but it

also resulted in the massive disposal of xenobiotic pesticides into our environment. Apart from

its inhibitory role on various pests, pesticides often lead to several short-term and long-term

adverse effects on man. In developing countries like India, pesticides serves as a key pest control

mode, but they often result in the accumulation of this xenobiotic in our food products (Gupta,

2004),water resources (Sharma, 2007) and eventually sneak into the foetus (Whyatt and Barr,

2001) which is a matter of great concern. The rate of still births, spontaneous abortions,

premature births and birth defects are higher in those exposed to these pesticides.

Among the various pesticides, organophosphates such as quinalphos are widely used in Indian

agriculture over certain crops such as cotton, groundnut and rice (Jena et al., 1990; Reddy and

Ghewande, 1986).Quinalphos has contact and stomach action against a wide range of pests from

Lepidoptera, Diptera, Coleoptera and Hemiptera. Quinalphos acts by inhibiting

acetylcholinesterase, which in turn block the transmission of the nerve impulse at the nerve

endings. Among 323 reported events of pesticide toxicity in three villages of India, 83.6% was

associated with signs and symptoms of mild to severe poisoning, 10% of the pesticide

application sessions were associated with three or more neurotoxic/systemic signs and symptoms

typical of poisoning by organophosphates, which were used in 47% of the applications (Mancini

et al., 2005).Thus, studies on the degradation and toxicity of this pesticide gains much relevance.

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Efforts are currently under way to develop safe, convenient and economically feasible methods

for pesticide detoxification (Kulshrestha and Kumari, 2010).Isolation of indigenous bacteria

capable of metabolizing organophosphorus compounds has received considerable attention

because these bacteria provide an environmentally friendly method of in situ detoxification

(Mulchandani et al., 1999).However, isolation of better strains that can degrade higher

concentrations of quinalphos at a faster rate is great need with the increasing rates of pesticide

posing rates.

Materials and methods

Enrichment of soil samples and isolation of Quinalphos degrading bacteria

Quinalphos 25EC (emulsified concentrate) supplied by Sandoz (India) Limited was used in the

present study.All other chemicals used were of analytical reagent grade. A minimal media broth

(MMB) containing quinalphos as sole source of carbon was formulated to isolate quinalphos

degrading bacteria. The medium composition was as follows (g/l) (pH 7.0 ± 0.2): KH2PO4-1.0

g/l, K2HPO4 -1.0 g/l, NH4NO3-1.0 g/l, MgSO4.7H2O-0.2 g/l, CaCl2-0.02 g/l, NaCl-1 g/l.The

mineral salt medium was also supplemented with 2ml/l of stock trace elements to provide

bacteria with essential trace elements.Trace element solution stock was prepared in distilled

water with composition FeSO4.7H2O (0.1 g/l), MnCl4.4H2O (0.1 g/l) and ZnSO4.7H2O (0.1 g/l).

Bacteria capable of utilizing quinalphos as sole source of carbon was isolated from pesticide

contaminated soils by enrichment in MMB. Five grams of soil sample was added to 100 ml of

minimal medium containing 0.001 g/l (w/v) quinalphos and incubated at 28 ± 2°C for 7-8 days

at 120 rpm. After eight days, 10 ml of supernatant was transferred to 90 ml each of fresh medium

with 0.005 g/l and 10 g/l quinalphos each and incubated. Subculturing was done six times from

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each concentration of quinalphos to fresh minimal medium of the same quinalphos

concentration. After six serial transfers, 1ml of the supernatant from the final enrichment flask

were taken; plated directly on minimal media agar with 10 g/l quinalphos by spread plate

technique and incubated at 30°C for 3-4 days. Single colonies obtained were maintained for

further analysis.

Primary screening of quinalphos degrading bacteria

The ability of the bacterial isolates to utilize quinalphos was assessed by checking the quinalphos

level left over in the culture medium after incubation period. One ml of 1 Optical Density (OD)

culture of isolated bacterial strains were inoculated into the flasks with 10g/l quinalphos

supplemented MMB and incubated at 28 ± 2°C for 8 days on a shaker at 120 rpm. After

incubation, the cell free supernatant was extracted three times with an equal volume of

chloroform and absorbance was measured at 350 nm in Hitachi U-2800 UV-VIS

Spectrophotometer (Chiranjeevi et al., 1997).The percentage of quinalphos leftover was

calculated and the percentage of degradation was computed. Since quinalphos was the sole

carbon source in the medium, the decrease in the amount of quinalphos left over in the medium

after incubation indicated a greater level of degradation.

Identification of the selected cultures

The selected bacterial isolates were identified based on their morphological, physiological and

biochemical characteristics according to Bergey’s Manual of Determinative Bacteriology (Holt

et al., 1994).The 16S rDNA typing of the most efficient isolate Q10 was done using universal

eubacterial primers Fd1 and Rd1 primers amplifying the 16S rDNA gene in most of bacteria

(Weisburg et al., 1991).Chromosomal DNA was isolated from 5 ml of nutrient broth culture of

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Q10 incubated at 37 ˚C for 16 hours (Wilson, 2001).The amplification was performed using

Personal Thermal cycler (Biorad, USA).The 1.5 kb amplicon was purified with PCR clean up

kit- 100 (Chromus biotech, Bengaluru).The purified amplicon was sequenced using big dye

terminator v3.2 cycle sequencing chemistry for ABI Bioprism (Applied Biosystems).The

sequence was analyzed using the BLAST (www.ncbi.nlm.nih.gov) search algorithm and aligned

to their nearest neighbors. The sequence was deposited in the NCBI Gene Bank database under

accession number JX256192.

Optimization of quinalphos degradation

The quinalphos biodegradation process was optimized at different operational conditions such as

pH (5.0 to 9.0), incubation temperature (25–37°C) and nitrogen sources in MMB media

supplemented with 10g/l quinalphos. In place of (NH4NO3), equivalent quantity of other nitrogen

compounds (on nitrogen basis) were used viz, NaNO3 (2.124 g/l) or KNO3 (2.527 g/l) or L-

Asparagine (1.65 g/l) or Urea (0.75 g/l). The isolates were inoculated at different operational

conditions in 100 ml of MMB media in 500 ml Erlenmeyer flasks for 8 days and the degradation

rates were assessed spectrophotometrically as described earlier.

The growth profile of quinalphos degrading organism in optimized conditions of quinalphos

degradation were analysed by constructing growth curves. The growth profile was analysed at 8

hour intervals in MMB with 10g/l quinalphos for 8 days.

GC-MS Analysis of Quinalphos Degradation

The various products of quinalphos degradation were extracted with hexane and analysed by Gas

Chromatography (Shimadzu GC-2010) using Capillary column DB-1 (30 M X 0.25 mm X 0.25

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mm) and ECD detector (Agilent Technologies, USA). The operating conditions were as follows:

nitrogen (carrier gas) flow rate 0.79 ml min-1, injector temperature 250°C, column temperature

350°C and detector temperature 300°C. The degradation of quinalphos by the strain Q10 was

measured at 0th hour, 20th hour and after 8 days.

The metabolites formed as a result of degradation of quinalphos by Q10 after 8 days of

incubation were identified using GC-MS analysis. The sample preparation was similar to that

used for GC-analysis. The sample was analyzed using Shimadzu GC-MS QP 2010 Plus. The

conditions used in the MS analysis were mass scan 40-400 m/z, ion source temperature 200°C,

interface temperature 280 °C and solvent cut time of 2.50 min.

Biosurfactants in degradation

Siegmund-Wagner Medium previously developed for the screening of anionic biosurfactant

rhamnolipid from Pseudomonas sp was used (Siegmund and Wagner, 1991). The biosurfactant

production was detected by the formation of dark blue halo around the colony after 24 – 48 hours

of incubation. Rhamnolipid concentration of cell free broths were assessed by quantification of

L-rhamnose by the 6-deoxy-hexose method after 8 days of incubation (Chandrasekaran and

Bemiller, 1980). Extracellular glycolipids concentration was evaluated in triplicate by measuring

the concentration of rhamnose. Briefly, 333 µl of the culture supernatant was extracted twice

with 1ml diethyl ether, the ether fractions were evaporated to dryness and 0.5 ml of H2O was

added. To 100 µl of each sample 900 µl of 0.19 % orcinol reagent was added, heated for 30 min

at 80°C, cooled to room temperature and the OD421 was measured. The rhamnolipid

concentrations were calculated from a standard curve prepared with L-rhamnose and expressed

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as rhamnose equivalents. All experiments were conducted in triplicates and controls were kept

under similar conditions.

Production of biosurfactant and calculation of CMC concentration

After adjusting pH to 2.0 using 6 N HCl, the culture supernatant was incubated at 4 C overnight

to precipitate the surfactant. The precipitated surfactant was isolated by centrifugation at 10,000

rpm for 20 min at 4 C. The precipitate was washed twice with aqueous HCl (pH 2.0), dissolved

in 1N NaOH and adjusted to pH 7. The biosurfactant mixture was extracted with ethyl acetate

(100 %) in a separating funnel and the organic phase was collected and concentrated using a

rotary evaporator at temperature of 40 C to yield the crude biosurfactant. The crude

biosurfactant was dissolved in 0.05 M NaHCO3, filtered and the pH was adjusted to 2.0 using 6

N HCl. The solution was kept at 4 C for 24 hours. The precipitate was finally collected by

centrifugation at 12,500 rpm for 15 minutes. The freeze dried sample was considered as the pure

form of biosurfactant (Yin et al., 2009).

The surface tension of the biosurfactant was measured with a KSV Sigma 701 tensiometer using

the Du Nouy ring method. The density of each sample was calculated using Hares apparatus

(Harkins et al., 1959).The critical micelle concentration (CMC) was determined by measuring

the surface tensions of dilutions of isolated biosurfactant in distilled water up to a constant value

of surface tension. Each result was the average of 10 determinations after stabilization. The value

of CMC was obtained from the plot of surface tension against surfactant concentration.

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Statistical Analysis

The data represents the arithmetical averages of at least three replicates, and the error bars

indicate the standard deviations. Comparison between groups was performed by a one-way

analysis of variance with post hoc analysis by Tukey’s test. p < 0.05 was considered statistically

significant.

Results and Discussion

Isolation, screening and identification of quinalphos degrading bacteria

Saprophytic microorganism plays a vital role in the degradation and transformation of pesticides

in soil. During this study, 12 different bacterial strains capable of utilizing quinalphos as the sole

carbon source were isolated from soil. Spectrophotometric analysis of cell free broths indicated

that isolates Q3, Q7 and Q10 were capable of degrading quinalphos to 57.47 %, 65.97 % and

74.14 % respectively (table 1). A visible decrease in colour intensity of pesticide in the medium

was observed on the microbial growth as shown in fig 1. No decrease in pesticide concentration

was observed in uninoculated control flasks, thus proving the microbial role in pesticide

reduction.

The selected bacterial isolates Q3, Q7, Q10 were gram negative, aerobic, motile, nonsporing and

catalase positive rods. Q3 and Q10 showed fluorescence in King’s B medium. On the basis of

cultural and biochemical characteristics, the isolates Q3, Q7 and Q10 were identified as

Pseudomonas aeruginosa, Serratia sp. and Pseudomonas sp. respectively. The 16S rDNA

sequence of the isolate Q10 was deposited in the NCBI gene bank under accession number

JX256192 and it showed 99% similarity to Pseudomonas aeruginosa on NCBI BLAST. The

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nearest neighbors of the isolate Pseudomonas aeruginosa (Q10) were P.aeruginosa

(HQ271084) and P.nitroreducens (HQ271083) (fig 2).

Previous research reveals that microorganisms produce various hydrolases to degrade pesticides

and thus they can be effectively used to overcome pesticide associated pollution problems

(Bhadbhade et al., 2002). Pseudomonas sp. has been reported as a good candidate with

versatile bioremediatory potential capable of degrading wide array of xenobiotics such as

hydrocarbons, surfactants, phenols etc. The role of Pseudomonas sp. in the degradation of

malathion, methamidophos, cartap and cypermethrin (Jilani and Altaf Khan, 2004), difocol

(Sarkar et al., 2009) and 4-chloro benzoic acid were reported. This paper presents another

example of bioremediatory potential of Pseudomonas in quinalphos remediation.

Optimization of Quinalphos degradation & Microbial growth

The rate of quinalphos degradation varied at different pH, temperature and nitrogen sources as

depicted in fig 3. During this study it was observed that a pH between 7 and 8 favored pesticide

degradation, which was similar to previous results (Devi S.G., 2006). This indicated that

quinalphos is biodegraded by an alkaline catalyzed hydrolysis reaction. The isolates Q3 and Q10

showed a greater growth and pesticide degradation at 37°C whereas Q7 showed better

degradation at 30°C. The nitrogen source of the medium greatly influenced the bacterial

degradation of quinalphos. The supplementation of sodium nitrate instead of ammonium nitrate

resulted in 10- 20 % increase in rate of pesticide degradation than pH optimization step in the

case of Q7 & Q10 (fig 3). This increase in bacterial growth and pesticide degradation could be

attributed to the increased biosurfactant production in presence of NaNO3 as nitrogen source (fig

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4). The influence of nitrogen sources on rhamnolipid production is previously reported (Rebello

et al., 2013).

The isolates Q3, Q7 and Q10 were capable of degrading quinalphos to 85.6 %, 87.55 % and

93.87 % respectively after 8 day, under optimal degradation conditions. Extent of quinalphos

degradation and growth profile of the efficient bacterial isolate Q10 in MMB was plotted (fig 5).

Studies on biodegradation of quinalphos are meager compared to reports on its toxicity. Previous

studies reported that 83.71% degradation of 12 mg/l quinalphos was degraded by mixed cultures

of Pseudomonas and Bacillus sp in 10 days (Devi S.G., 2006). Bioremediation of quinalphos by

combined incubation with medicinal plants and Trichoderma leave back only traces of the

pesticide, but the process requires a long incubation time of 60 days (Subashini et al., 2007). The

current paper describes isolation of bacterial isolates capable of degrading of 85-94 % of 2.5 g/l

of quinalphos within 8 days of incubation. A comparison of the above studies reveals that the

strains used in this study were superior to the former in the time extent and quantity of pesticide

it degraded, implying a better optimization of quinalphos degradation. Thus, Q10 could serve as

an ideal candidate for quinalphos degradation capable of degrading even a high concentration of

pesticide in a shorter incubation time.

Degradation studies of Quinalphos

A 92.58% decrease in peak areas of quinalphos was observed in Q10 inoculated flasks on Gas

Chromatographic analysis (fig 6). The gas chromatographic analysis and the optical density

measurements confirmed substantial removal of quinalphos with simultaneous increase in

bacterial growth. Two new peaks were formed in the chromatogram of Q10 on 8th day (retention

times 3.90, 9.53), which were due to metabolites formed as a result of degradation (fig 7). The

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additional peak at 9.53 min gave fraction with a molecular weight of 146 and was identified

as 2-hydroxyquinoxaline (fig. 7a). The peak at 3.9 min gave fraction with a molecular weight

of 298 and was identified as phosphorothioic acid (fig. 7b). Degradation or detoxification of

organophosphorus pesticides by the action of microorganisms is generally through the hydrolysis

of P–O alkyl and P–O aryl bonds. Such degradation makes the compound more vulnerable to

further metabolism (Ortiz-Hernadez and Sanchez-Salinas, 2010).

Biosurfactants in pesticide degradation

The three bacterial isolates Q3, Q7 & Q10 possessed biosurfactant production potential as per

halo on Siegmund Wagner agar plates. Quantization of rhamnolipids by rhamnose assay

indicated that the isolates produced rhamnolipids in quantities such that Q3< Q7<Q10 (table2).

The CMC values of the purified biosurfactants were found to range between 10- 22 mg/l during

our study (table 2).This agreed with former studies that suggested CMC values of rhamnolipids

could range between 10- 230 mg/l (Nitschke et al., 2005). The increased quinalphos degradation

ability by Q10 than other isolates could be due to its efficiency to produce more amounts of

rhamnolipids.Since the solubility of quinalphos is low in water, it is possible to claim that the

activity of biosurfactants is promising in the degradation of quinalphos. Quinalphos exhibits less

water solubility (20 mg/l) compared to other organophosphates such as monocrotophos (100%

solubility in water), which suggests the chance of its persistence in environment (Kaur et al.,

2013). But in the presence of biosurfactants, the solubility of quinalphos in water is increased.

During our study it was observed that the use of rhamnolipid at concentrations above CMC value

enhanced quinalphos solubility in water at greater rate than at concentrations below its CMC.

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Previous studies indicate that addition of synthetic surfactants enhanced the solubility and

degradation of the pesticide-endosulfan by 94% (Jayashree and Vasudevan, 2007). The inherent

biosurfactant production ability of the isolates will help to solubilise insoluble compounds,

thereby increasing their bioavailability (Shreve et al., 1995).

Conclusion

Degradation of organophosphorus compounds has attracted considerable attention because of

their widespread use as pesticides and their high mammalian toxicity. The present study has

analyzed the bioremediation of pesticides using bacterial isolates, which seems to be a safe and

effective means of remediation. Considering the use, toxicity and demand of organophosphorous

pesticides in agriculture, studies on biodegradation of these pesticides are of immediate concern.

The studies carried out so far suggests that microorganisms endowed with the ability to degrade

toxic pollutants are a boon to humanity. Microbial degradation process to detoxify pesticides

contaminants can be effectively used to overcome the pollution problems. The effective

utilization of pesticide degrading isolates in immobilized forms in industrial treatment plants

could further increase its prospects in pesticide bioremediation. Construction of recombinant

strains for degradation of organophosphorous pesticides and their metabolites would be a

challenge. The current work successfully led to the isolation of Pseudomonas aeruginosa strain

unique in its ability to degrade large concentration of quinalphos in a shorter incubation time,

which is noteworthy based on its bioremediatory potential.

Acknowledgements

The authors are thankful to College of Agriculture, Vellayani for GC-MS analysis.

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Table 1. Screening of quinalphos degrading bacteria

*Values are averages of three replications

Sl. No. Isolates Percentage of quinalphos

degradation (Mean ± SD)

1 Q1 2.63± 0.01

2 Q2 8.02 ±0.02

3 Q3 57.47 ± 0.01

4 Q4 23.97 ± 0.11

5 Q5 32.24 ±0.11

6 Q6 32.99 ± 0.1

7 Q7 62.97 ± 0.02

8 Q8 28.17 ±0.01

9 Q9 50.83 ± 0.01

10 Q10 74.14 ±0.12

11 Q11 34.74 ± 0.12

12 Q12 56.08 ± 0.12

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Table 2: Analysis of biosurfactant extracts of Q3, Q7, and Q10

Organism Concentration of

Biosurfactant By

Rhamnose Assay

(mg/l)

CMC concentration of

Biosurfactants

(mg/l)

Emulsification Index

(E24 %)

Q3 20 ± 0.01 22 ± 0.08 39.0625 ± 0.12

Q7 28 ± 0.03 15 ± 0.17 50.625 ± 0.26

Q10 37 ± 0.12 9.8 ± 0.39 81.25 ± 0.30

*Results are averages of three replications

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Fig 1: Comparison of medium colour of Q3 & Q10 during 1st and 8th day of incubation with

respect to control

Fig 2: Phylogenetic tree of the isolates generated using neighbour joining method

Fig 3: Optimisation of quinalphos degradation by Q3, Q7 & Q10 at various temperature, pH and

nitrogen sources (p value < 0.05)

Fig 4: Rhamnolipid yield using different nitrogen sources during quinalphos degradation

Fig 5: Comparison of growth and quinalphos degradation by the isolate Q10

Fig 6: Gas chromatography analysis of the extract of Q10 inoculated mineral medium

containing quinalphos at retention time 11.485 min A) after 20 hours of incubation B) after

8 days of incubation.

Fig 7:Mass spectroscopic analysis of the gas chromatogram of biodegraded quinalphos

residues A) additional peak at 9.53 min showing 2- hydroxyquinoxaline (Molecular formula

C8H6N2O m/Z 146) formation B) additional peak at 3.9 min showing phosphorothioic acid

(Molecular formula C12H15N2O3 m/Z 298) formation.

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Fig 1

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Fig 2

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Fig 3

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Fig 4

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Fig5:

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Fig 6

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Fig 7

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