Biosurfactant production by Pseudomonas sp. and its role in aqueous phase partitioning and biodegradation of chlorpyrifos

Authors


Harvinder Singh Saini, Department of Microbiology, Guru Nanak Dev University, Amritsar-143005, Punjab, India. E-mail: sainihs@gmail.com

Abstract

Aim:  To study the effect of biosurfactant on aqueous phase solubility and biodegradation of chlorpyrifos.

Methods and Results:  A Pseudomonas sp. (ChlD), isolated from agricultural soil by enrichment culture technique in the presence of chlorpyrifos, was capable of producing biosurfactant (rhamnolipids) and degrading chlorpyrifos (0·01 g l−1). The partially purified rhamnolipid biosurfactant preparation, having a CMC of 0·2 g l−1, was evaluated for its ability to enhance aqueous phase partitioning and degradation of chlorpyrifos (0·01 g l−1) by ChlD strain. The best degradation efficiency was observed at 0·1 g l−1 supplement of biosurfactant, as validated by GC and HPLC studies.

Conclusion:  The addition of biosurfactant at 0·1 g l−1 resulted in more than 98% degradation of chlorpyrifos when compared to 84% in the absence of biosurfactant after 120-h incubation.

Significance and Impact of the Study:  This first report, to the best of our knowledge, on enhanced degradation of chlorpyrifos in the presence of biosurfactant(s), would help in developing bioremediation protocols to counter accumulation of organophosphates to toxic/carcinogenic levels in environment.

Introduction

The organophosphate pesticides (OPs) first developed and used in 1937 emerged as less toxic and safe alternative to highly toxic chlorinated pesticides. However, over the years, because of their indiscriminate use, the OPs poisoning has emerged as a worldwide health problem with around three million poisonings and 200 000 deaths annually (Karalliedde and Senanayak 1999; Sogorb et al. 2004). Chlorpyrifos [O,O-diethyl O-(3,5,6- trichloro-2- pyridyl) phosphorothioate] is an organophosphate pesticide used worldwide having half-life of 60–120 days in soil. However, under different pH levels, concentrations and repeatability of application, the rate of degradation of chlorpyrifos in diverse type of soil varies from 50 days to 570 days (Singh et al. 2003). Further, its low aqueous solubility (2 ppm) and higher adsorption affinity to organic matter/soil lower its bioavailability for microbial conversion hence limiting the rate of degradation in aqueous and soil system (Volkering et al. 1998 and Singh et al. 2003). The problem is further compounded by formation of 3,5,6-trichlorpyridinol (TCP), an intermediate of chlorpyrifos degradation (Fig. S1), reported to have antimicrobial activity preventing the proliferation of chlorpyrifos degrading micro-organisms (Racke et al. 1990).

Thus, it is desirable to isolate chlorpyrifos degraders that metabolize chlorpyrifos without the accumulation of TCP to avoid build-up of their toxic levels in environment.

Surfactants are surface-active amphipathic molecules that have potential to improve partitioning of hydrophobic compounds to aqueous phase by forming emulsions at and above their critical micellar concentration (CMC), which may improve bioavailability of those compounds to their potential degraders (Noordman and Janssen 2002). There are reports regarding the use of biosurfactants as alternate options to chemical surfactants in bioremediation studies because of their being highly effective at low concentrations, stability at wide range of pH, salinity and temperature, biocompatibility and environmentally friendly nature. However, antagonistic properties of the biosurfactant preparations and micellerization of pollutants, resulting in lower bioavailability, at higher concentration of biosurfactants (above their CMC level) may inhibit degradation (Itoh and Suzuki 1972; Bellingsley et al. 1999). Thus, it is desirable to develop a compatible combination of biosurfactant and degrading microbes and to optimize the concentration of biosurfactant to be used in biodegradation studies so as to strike a balance between transformation potential of degrader and toxicity of the target pollutant.

In this regard, we report isolation of a Pseudomonas sp. (ChlD) capable of producing biosurfactant (rhamnolipid) and degrade chlorpyrifos. The use of partially purified biosurfactant significantly improved the degradation of chlorpyrifos by isolate ChlD.

Materials and methods

Analytical (technical) grade chlorpyrifos and 3,5,6-trichloropyridinol (97% purity) generously provided by Gharda Chemical Ltd. Ratnagiri, Maharashtra, India, were used in this study. All the media components/solvents/standards were of analytical/HPLC grade. Soil samples, for enrichment of microbial populations, were collected from agricultural fields located on outskirts of Amritsar having a history of spray of OP pesticides viz monocrotophos, chlorpyrifos and malathion since last 6 years.

Media

The isolation of microbial populations present in the soil samples was carried out in nutrient broth (NB) medium of following composition in g l−1: peptone (5 g), beef extract (3 g) and sodium chloride (5 g). The mineral salt medium (MSM) and M-9 medium were used for biosurfactant production and chlorpyrifos degradation respectively by selected isolates unless specified otherwise. Mineral salt medium (MSM) used was of the composition as given by Khehra et al. (2006). The MSM was supplemented with 0·25% (w/v) of yeast extract and 2% (w/v) glucose from their filter-sterilized stock solutions. M-9 medium used was of the following composition (g l−1): Na2HPO4 (12·8), KH2PO4 (0·9), MgSO4 (0·0096), NaCl (0·5), CaCl2.2H2O (0·00146), yeast extract (0·005) and ZnSO4 (0·0287). The final pH of the medium was adjusted to 8·0.

Isolation of bacterial strains

The isolation of microbial populations present in soil samples was carried out using 1/10 and 1/100 times diluted nutrient broth (50 ml in 250-Erlenmeyer flask) supplemented with 0·1 g l−1 of chlorpyrifos. The isolates were further screened for biosurfactant production and chlorpyrifos degradation as per the following protocols.

Screening of biosurfactant producers

The morphologically distinct strains were screened for biosurfactant production by growing them on MSM supplemented with glucose 2% (w/v) and yeast extract 0·25% (w/v). The reduction in surface tension of cell-free supernatant was monitored by Du Nouy ring method using CSC-Du Nouy tensiometer (CSC, Fairfax, VA, USA). The biosurfactant producers were further screened on CTAB-methylene blue medium (Siegmund and Wagner 1991).

Screening of chlorpyrifos degraders

The isolated strains were screened for their ability to degrade chlorpyrifos on M-9 medium (50 ml in 250-ml Erlenmeyer flask) supplemented with 0·01 g l−1 chlorpyrifos from its 10% (w/v) stock solution in acetone. The flasks were incubated at 28°C at 150 rev min–1 on orbital shaker. Flasks with uninoculated medium were kept as controls to rule out abiotic loss of chlorpyrifos. The medium in the flasks was extracted thrice with equal volume of hexane, and the pooled extract was concentrated using Buchi Rota-vapour R-114. The concentrated samples were further analysed as per analytical procedures described.

Biosurfactant recovery and analysis

The cells of ChlD were grown on MSM medium supplemented with 2% (w/v) glucose and 0·25% (w/v) yeast extract for 96 h under shaking conditions (150 rev min–1) at 30°C. The cell-free supernatant was acidified to pH 2·0 with concentrated HCl and kept at 4°C for 24 h to precipitate the anionic surface-active molecules. The pellet collected after centrifugation at 6000 g was dissolved in 2 volumes of 0·0005 mol l−1 NaHCO3, and the solution was reprecipitated with concentrated HCl. The process was repeated twice to ensure the removal of pigments and other molecules of microbial origin. The precipitates thus obtained were redissolved in 0·0005 mol l−1 NaHCO3 (pH 8·2) to obtain a stock solution of 10% (w/v). The stock solution was sterilized by passing through 0·22-μm filter and stored at 4°C for further use. The CMC of the biosurfactant was determined by standard methods as reported earlier (Haba et al. 2000). The type of sugar polar head was determined by saponification of partially purified biosurfactant preparation. To saponify the biosurfactant, 0·2 g acid precipitates were dissolved in 10 ml of diethyl ether followed by treatment with 15 ml of 0·5 mol l−1 NaOH (in 90% ethanol) for 12 h at 60°C to hydrolyse the ester bonds. After hydrolysis, 5 ml water was added and extracted twice with equal volume of diethyl ether to remove lipid portion of biosurfactant. The aqueous phase was dried and dissolved in methanol to spot on TLC plate along with the pure standard of rhamnose. The spots were developed in chloroform : methanol : water in the ratio of 65 : 25 : 4. The plates were sprayed with 2% (w/v) anthrone solution in concentrated sulphuric acid and developed by mild and even heating on the hot plate to notice the characteristic colour change from pale to greenish yellow for spots of rhamnose-containing biosurfactants.

Enhanced chlorpyrifos bioavailability in aqueous phase by partially purified biosurfactant

The ability of the biosurfactant solution at different concentrations to partition chlorpyrifos (0·1 g l−1) to aqueous phase was evaluated. Chlorpyrifos from its 10% (w/v) stock solution in acetone was added to glass vials (15 ml), and acetone was allowed to evaporate completely. This was followed by the addition of 5 ml of M-9 medium supplemented with different concentrations of partially purified biosurfactant ranging from 0·02 g l−1 to 0·4 g l−1 from its filter-sterilized stock solution. The vials were kept on shaker at 100 rev min–1 for 24 h. The aqueous phase was transferred to the fresh vials for extraction with hexane, and the unpartitioned chlorpyrifos was extracted directly with hexane. The chlorpyrifos content of the samples was determined by GC/HPLC analysis.

Degradation of chlorpyrifos by ChlD in the absence and presence of biosurfactant

The ChlD cells were activated in M-9 medium (pH 8·0) containing 2% (w/v) glucose, 0·25% (w/v) yeast extract, ZnSO4 (0·0001 mol l−1) and chlorpyrifos (0·01 g l−1) at 30°C under shaking conditions at 150 rev min–1 for 16–18 h. The activation was carried out twice, using 2% (v/v) inoculum from first stage, to obtain desired biomass with optimum degradation potential. The cells were harvested by centrifugation at 4500 g for 10 min and washed twice with the basic M-9 buffer without supplements to remove unused nutrients and residual chlorpyrifos. The activated cells were used to inoculate M-9 medium (50 ml in 250-ml Erlenmeyer flask) supplemented with yeast extract 0·0005% (w/v), ZnSO4 (0·0001 mol l−1) and chlorpyrifos (0·01 g l−1) to achieve initial OD540 ≈ 1·0. The ability of isolate ChlD to grow on M-9 medium supplemented with chlorpyrifos (0·01–0·075 g l−1) as sole source of carbon and energy was evaluated by inoculating 9·2 × 107 cells ml−1 of M-9 medium without yeast extract supplement. The CFU ml−1 at regular interval of 24 h was monitored up to 120 h. The M-9 medium without chlorpyrifos inoculated with same inoculum level was kept as control.

The effect of biosurfactant addition to M-9 medium on chlorpyrifos degradation was monitored by supplementing medium with different concentrations of biosurfactant ranging from 0·02 g l−1 to 0·4 g l−1. The abiotic control flasks without and with different biosurfactant concentrations in M-9 medium were kept to determine the abiotic loss of chlorpyrifos and to evaluate the effect of biosurfactant on extraction efficiency of chlorpyrifos. The experiments were performed in triplicates, and the data presented relate to three independent experimental observations.

Analytical procedures

The pooled hexane extracts of M-9 medium for chlorpyrifos degradation was analysed by gas chromatography using 5765 Nucon Gas Chromatograph with split injector, equipped with BPX608 capillary column of 25 m × 0·32 mm i.d. (SGE) using electron capture detector (ECD). The operating parameters were as follows: column temperature set at 240°C, injector 250°C and detector at 270°C. Nitrogen was used as a carrier gas at the flow rate of 25 ml min−1 with split ratio of 1 : 1. High performance liquid chromatography (HPLC) of the extracts was carried out using Dionex P680 HPLC pump, TCC-100 thermostat column compartment equipped with C-18 column (250 × 4·6 mm i.d., Varian) set at 30°C and UVD340U UV–visible detector at 300 nm. Mobile phase used was acetonitrile and water in the ratio of (80 : 20) with flow rate set at 1 ml min−1.

Results

Isolation and screening of bacterial isolates

The enrichment of microbial populations present in (five) different soil samples grown on N/10 and N/100 diluted nutrient broth supplemented with 0·01 g l−1 of chlorpyrifos yielded more than 90 morphologically distinct isolates. Nine morphologically distinct strains were selected on the basis of their ability to degrade chlorpyrifos and/or to produce biosurfactant. All the nine potential isolated strains were identified by biochemical tests and partial 16S rRNA sequencing using universal primers (27 F and 1492 R).

The biosurfactant-producing strains ChlD (Pseudomonas sp.), MC-1 (Aerococcus viridans), C-4 (Pseudomonas sp.), M-2 (Ochrobactrum sp.) and an unidentified isolate M-3 showed blue colouration with halo around their colonies on CTAB-methylene blue medium, indicating formation of anionic biosurfactant. The isolate ChlD was efficient biosurfactant producer resulting in decrease of surface tension from initial 65 N m−1 to final 28 N m−1.

The screening of the isolates for their ability to grow in the presence of chlorpyrifos (0·1 g l−1) showed that five isolates including ChlD viz F-3 (Klebsiella pneumonia), CH-y (Stenotrophomonas maltophilia), 13·9 (Ochrobactrum sp.) and C-2 (Bacillus subtilis) were able to degrade 43% to 78% chlorpyrifos. On the basis of these observations, the isolate ChlD, a Gram-negative, aerobic, motile, catalase and oxidase positive, capable of utilizing citrate and hydrolysing lipid was selected for further studies.

Biosurfactant production and recovery

The surface-active molecules produced by ChlD were recovered by acid precipitation, washed and reprecipitated to remove impurities. The partially purified biosurfactant preparation was dissolved in 0·0005 mol l−1 NaHCO3 to make a working stock solution of 10% (w/v). The stock solution was filter sterilized and stored at 4°C. The biosurfactant precipitates were saponified to determine the nature of sugar polar head. TLC of the methanol-extracted aqueous phase of the saponified biosurfactant preparation was developed by spraying with anthrone reagent, which showed single spot with Rf values of 0·32 in comparison with the pure standard of rhamnose (Fig. 1).

Figure 1.

 TLC of aqueous phase of the saponified biosurfactant preparation (2) and pure rhamnose (1) developed by spraying with 2% (w/v) anthrone in concentrated sulfuric acid.

Enhanced aqueous phase partitioning of chlorpyrifos

The ability of different concentrations of biosurfactant ranging from 0·02 g l−1 to 0·4 g l−1 in M-9 medium to improve partitioning of chlorpyrifos to aqueous phase was evaluated. As evident from the results presented in (Fig. 2), the chlorpyrifos solubility in aqueous phase increased from 2·5% in control to >87% in medium supplemented with 0·4 g l−1 of biosurfactant preparation. The amount of chlorpyrifos in the vials decreased concomitantly with increase in biosurfactant concentration from 0·02 g l−1 to 0·4 g l−1, indicating significant increase in aqueous phase partitioning of chlorpyrifos.

Figure 2.

 Effect of different concentrations of biosurfactant (g l−1) on aqueous phase partitioning of chlorpyrifos (0. 1 g l−1). Chlorpyrifos concentration in aqueous phase (bsl00001), chlorpyrifos concentration in vials (□).

Degradation of chlorpyrifos by ChlD in the absence and presence of partially purified biosurfactant

The isolate ChlD under optimized conditions (M-9 medium supplemented with 0·0005% (w/v) yeast extract and 0·0001 mol l−1 ZnSO4 at pH 8·0) at initial OD560 ≈ 1 without any additional carbon source supported 84% degradation of chlorpyrifos (0·01 g l−1) in 120 h of incubation at 28°C without the accumulation of TCP at any stage (Fig. 3). Further, the CFU count increased with the increase in chlorpyrifos concentration from 0·01 g l−1 to 0·05 g l−1 in medium without yeast extract supplement, indicating the ability of ChlD to use chlorpyrifos as a sole source of carbon and energy (Fig. S2).

Figure 3.

 Degradation profile of chlorpyrifos (0·01 g l−1) using different biosurfactant concentrations (0·02–0·4 g l−1) at 120-h incubation.

The effect of addition of partially purified biosurfactant on degradation of chlorpyrifos was studied, and the results show that it took 168 h to degrade 91·7% of chlorpyrifos in the absence of biosurfactant (data not shown). While the results presented in Fig. 3 indicate that with increase in biosurfactant concentration >98% of chlorpyrifos degradation was achieved at 0·1 g l−1 biosurfactant supplement in 120 h. However, further increase in biosurfactant concentration resulted in steady decrease in overall degradation rate.

The M-9 supplemented with different concentrations of biosurfactant and 0·1% (v/v) acetone respectively with same level of inoculum was incubated under similar set of conditions. It was observed that there was no change in CFU ml−1 when compared to the control flasks without biosurfactant and acetone supplements. Further, it was observed that the addition of different concentrations of biosurfactant (0·01–0·4 g l−1) did not affect the extraction efficiency of chlorpyrifos (0·01 g l−1) by hexane from the M-9 medium (see Fig. S3). The HPLC chromatograph presented in Fig. 4 indicated >98% of chlorpyrifos degradation within 5 days of incubation at 28°C in comparison with 84% of degradation of chlorpyrifos by ChlD in the absence of biosurfactant.

Figure 4.

 Degradation of chlorpyrifos (0·01 g l−1) by ChlD in the absence of biosurfactant (♦) and in the presence of 0·1 g l−1 biosurfactant (□) (I) and HPLC profile of chlorpyrifos (0·01 g l−1) degradation by ChlD. (a) Control, (b) in the absence of biosurfactant and (c) in the presence of 0·1 g l−1 biosurfactant (II).

Discussion

The organophosphate pesticide, chlorpyrifos, extensively used for pest control has low solubility (2 ppm) in aqueous phase thus limiting its bioavailability for transformation. The strain ChlD, isolated from soil collected from agricultural fields with a history of organophosphate pesticide application was able to metabolize 91% of the added chlorpyrifos (0·01 g l−1) in 168 h without accumulation of TCP. The majority of the microbial isolates reported till date are co-metabolizer of the OPs with the exception of Enterobacter sp. reported to be capable of catabolically metabolizing chlorpyrifos using as a carbon and phosphorous source (Singh et al. 2003, 2004). The isolate ChlD was observed to be capable of utilizing chlorpyrifos, up to 0·05 g l−1, as a sole carbon source. Further increase in chlorpyrifos did not support comparable increase in biomass, which might be because of toxicity by higher concentrations of chlorpyrifos. Similarly, increase in CFU ml−1 of bacterial isolates had been reported by Lakshmi et al. 2008 and Zeinat et al. 2008, indicating the ability of respective bacterial cultures to use chlorpyrifos and malathion as sole source of carbon and energy.

The partially purified biosurfactant preparation of ChlD consisted of mono- and di-rhamnolipids as evident from characteristic spots of Rf value 0·72 and 0·42 (data not shown). The saponified biosurfactant preparation showed the presence of rhamnose when compared to the pure standard of rhamnose, indicating the presence of rhamnose–based, surface-active glycolipid. Different Pseudomonas sp. have been reported to produce similar surface-active compounds (Siegmund and Wagner 1991 & Zhang and Miller 1994). The biosurfactant preparation, having CMC of 0·2 g l−1, was evaluated for its role in improving the aqueous phase solubility of chlorpyrifos. The strain ChlD achieved almost 100% degradation of chlorpyrifos (0·01 g l−1) in 120 h, in medium supplemented with 0·1 g l−1 of partially purified biosurfactant preparation when compared to 84% degradation that achieved in the absence of biosurfactant. Further, it was observed that biosurfactant supplement of more than 0·1 g l−1 lowered the degradation efficiency of ChlD. This is in line with earlier observations that higher concentrations of the surfactants caused micellerization of PCBs, leading to inhibition of their biodegradation (Bellingsley et al. 1999).

This first time report regarding the potential of isolate ChlD, to efficiently degrade chlorpyrifos without accumulation of TCP and to produce biosurfactant which can improve its chlorpyrifos transformation potential, will provide insights into designing bioremediation protocol where the dual character of the strain can be exploited to achieve desired efficiency. There are reports regarding the use of biosurfactant in bioremediation of polychlorinated biphenyls (PCB) and polycyclic aromatic hydrocarbons (PAHs), including petroleum hydrocarbons (Noordman et al. 2002), but only a few reports are available related to role of biosurfactants in improved degradation of pesticides. Awasthi et al. (1999) observed enhanced biodegradation of endosulfan in both flask and soil systems using biosurfactant produced by B. subtilis MTCC 1427.

Further efforts are required to make the field application of biosurfactant cost competitive with chemical surfactants. However, the application of crude extract/ partially purified biosurfactant effective at sub-CMC levels, thus saving the cost of processing and application, can be a potential solution to reclamation of polluted sites.

Ancillary