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Journal of Hazardous Materials 171 (2009) 1178–1182

Contents lists available at ScienceDirect

Journal of Hazardous Materials

journa l homepage: www.e lsev ier .com/ locate / jhazmat

hort communication

queous phase partitioning of hexachlorocyclohexane (HCH) isomers byiosurfactant produced by Pseudomonas aeruginosa WH-2

uman Sharma b, Partapbir Singh b, Mayil Raj a, Bhupinder Singh Chadha b, Harvinder Singh Saini b,∗

MTCC, IMTECH, Sector 39-A, Chandigarh 160036, IndiaDepartment of Microbiology, Guru Nanak Dev University, Amritsar 143005, Punjab, India

r t i c l e i n f o

rticle history:eceived 31 December 2008eceived in revised form 22 April 2009ccepted 19 June 2009vailable online 27 June 2009

a b s t r a c t

The different isomers of technical-grade hexachlorocyclohexane (t-HCH) including the insecticidal �-isomer, commonly known as lindane, have been reported to be toxic, carcinogenic and endocrinedisrupters. The spatial arrangements of the chlorine atoms on different isomers and low aqueous phasesolubility contribute to their persistence in environment, �-HCH being the most resistance to transfor-mation. The biosurfactant preparation of Pseudomonas aeruginosa isolate WH-2 was evaluated for its

eywords:exachlorocyclohexane (HCH)CH-muckiosurfactant

ability to improve the aqueous phase partitioning of different isomers of HCH-muck. Further, the abilityof biosurfactant preparation to emulsify HCH and n-hexadecane was checked under different conditions,usually characteristic of sites contaminated with pollutants viz. wide range of pH, temperature, and salin-ity. The data obtained from this study will be helpful in designing suitable bioremediation strategies forhuge stock piles of HCH-muck and sites polluted by reckless use/disposal of HCH-isomers.

queous phase partitioning

CH-isomers. aeruginosa

. Introduction

Different formulations of hexachlorocyclohexane (HCH) haveeen the most extensively used broad-spectrum organochlorineesticides after ban of DDT, before being themselves banned foreneral use by 1990s. The technical-grade HCH (t-HCH) is composedf the following isomers: �-HCH (60–70%), �-HCH (5–12%), �-HCH10–15%), �-HCH (6–10%) and �-HCH (3–4%). Out of these �-HCH,ommonly known as lindane has a significantly higher insectici-al potential [1]. The extraction of lindane from t-HCH generatesCH-muck, almost six times of pure lindane produced. The muck

s usually stored in dumps and these over the years, are potentialources of pollution not only in the vicinity of production sites,ut also other pristine areas. There are reports that different HCH-

somers have been detected in trace amounts worldwide in air, soil,ater, oceans, food and even in cold drinks [2–4]. The residual lev-

ls of HCH near dumping sites in different regions of world, rangerom 17.02 mg kg−1 of soil in Germany to 1,25,280 mg kg−1 in India,hereby posing a serious toxicological problem [5]. According tone of the reports, various commercial brands of drinking water

amples available in Indian market contained almost 99–141-foldigher levels of HCH-isomers than the maximal permissible lim-

ts for drinking water i.e. 0.1 �g L−1 [6]. The isomers of HCH haveeen reported to have toxic, carcinogenic and endocrine disrupters

∗ Corresponding author. Tel.: +91 183 2450601 14x3318; fax: +91 183 2258820.E-mail address: sainihs@yahoo.com (H.S. Saini).

304-3894/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jhazmat.2009.06.116

© 2009 Elsevier B.V. All rights reserved.

leading to severe damage to reproductive and nervous systems inmammals [7,8]. Moreover, there are reports in the literature sup-porting isomerization of HCH-isomers and their persistence in theenvironment [7,9]. Thus, there is an urgent need to develop suitablebiological protocols for treatment of HCH residues to prevent theirbuild up in the environment.

The biotransformation of hydrophobic organic compounds(HOCs) is limited by low solubility of these compounds in aque-ous phase. Further their ageing over long term interaction withsoil particles significantly reduces their bioavailability to degrad-ing microorganisms [10]. The low aqueous phase solubility ofhydrophobic organic compounds (HOCs) can be improved by usingsurfactants [11,12]. The chemical surfactants usually have higherCMC (critical micelle concentration) values usually more than600 mg L−1 [13]. Thus, they are required at higher concentra-tions to get the desired results. The chemical surfactants, usuallyof petrochemical origin, are toxic to microbial flora at the con-centrations used and are themselves a source of pollution [14].Quintero et al. [15] reported use of three chemical surfactants,Triton X-100, Tween 80 and sodium dodecyl sulphate (SDS) toimprove sequestration of HCH-isomers from soil polluted withHCH. It was observed that with increase in ageing period, highamounts of surfactants were required to improve bioavailabil-

ity/biotransformation. However, Triton X-100 and SDS at the usedconcentrations were toxic to anaerobic microbes capable of trans-forming HCH.

Different microorganisms have been explored for their poten-tial to produce surface-active molecules with CMC values ranging

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rom 1 to 200 mg L−1 [16]. These surface-active preparations haveeen reported for their ability to improve aqueous phase sol-bility of HOC’s such as octadecane, phenanthrene, n-paraffin,tc. [17,18]. The advantage of biosurfactants as compared to syn-hetic surfactants is their ecological acceptance, owing to theirow toxicity and biodegradable nature [19]. However, field scalepplication of biosurfactants in bioremediation protocols is ham-ered due to their high cost, which is attributed to low yieldsnd poor recovery from production medium. However, with thesolation of efficient microbes and optimization of productionrotocols, it is possible to lower the cost of production of bio-urfactants; thereby making them cost effective as compared tohemical surfactants for use in bioremediation protocols [20,21].dditionally, there are reports that biosurfactants are quite effec-

ive even at their sub-CMC levels, thus lower amounts are requireds compared to chemical surfactants [22]. However, the efficacyf biosurfactant preparations under field conditions needs to bevaluated to ascertain their role in field scale applications. Therere few reports on the effect of factors such as pH, tempera-ure and salinity on the emulsifying activity of biosurfactants23,24].

In light of this, work was carried out to study the effect ofH, temperature and salts on the surface-active and emulsificationotential of the biosurfactant produced by Pseudomonas aeruginosaH-2 strain. The potential of the biosurfactant to partition different

somers of HCH to aqueous phase was also evaluated.

. Materials and methods

.1. Microorganism and growth medium

The bacterial isolate P. aeruginosa, designated WH-2, used in thistudy was isolated by enrichment of microbial populations in soilamples, collected from sites contaminated with petrochemicalsiz. petrol stations, etc. The soil samples were collected in plasticags from just below the soil surface, appropriately labeled andtored at 4 ◦C. The strain has been deposited in the Microbial Typeulture Collection (MTCC), IMTECH, Chandigarh with a catalogueumber MTCC 9303.

.2. Biosurfactant production and recovery

The biosurfactant production by WH-2 was carried out inhe mineral salts medium (MSM) [25] supplemented with 2.0%w/v) fructose and 0.25% (w/v) tryptone from their respective pre-terilized stock solutions. The flasks were inoculated with activatedells grown for 24 h in nutrient broth to achieve initial OD600 of.3 and were incubated at 30 ◦C and 100 rpm in an environmentalhaker (Scigenics, India). The surface-active components producedy WH-2 were recovered from the cell-free supernatant by lower-

ng the pH to 2.0 with 5N HCl followed by incubation at 4 ◦C for 48 h,o allow precipitation of surface-active molecules. The resultantxtract was further purified using silica gel column chromatography26].

.3. Critical micelle concentration (CMC)

The CMC of the biosurfactant preparation was determined byeasuring surface tension of different concentrations (0–1.0 g L−1)

f WH-2 biosurfactant prepared in alkaline water (pH 8.5). The sur-ace tension of respective solutions (20 mL) was measured usingSC-duNouy ring tensiometer (CSC, USA). The CMC (g L−1) wasetermined from the plot of surface tension and different biosur-

actant concentrations.

aterials 171 (2009) 1178–1182 1179

2.4. Determination of emulsification activity (EA)

The ability of biosurfactant to emulsify �-HCH was evaluatedby using method described by Appaiah and Karanth [27]. 20 mgof �-HCH dissolved in acetone (0.2 mL) was added to 5.0 mL ofWH-2 cell-free supernatant, vortexed for 1 min and was read at660 nm. For n-hexadecane, EA was studied by Cirigliano and Car-man method [28]. Briefly, the cell-free supernatant (800 �L) wasdiluted to 4 mL, to which 1 mL of n-hexadecane was added. The mix-ture was shaken vigorously on a vortex mixer for 2 min, incubatedfor 10 min and turbidity was measured at 540 nm.

2.5. Effect of different chemical conditions on biosurfactant

The effect of different conditions such as pH, temperature andsalinity on surface-active properties of biosurfactant preparation(0.2 g L−1) was evaluated. In these studies, the emulsification effi-ciency of biosurfactant from P. aeruginosa WH-2 was also comparedwith the commonly used anionic chemical surfactant, sodium dode-cyl sulphate (SDS). The effect of each treatment was assayed interms of percentage decrease in EA as compared to the respectivecontrols.

2.5.1. pH stabilityThe pH of biosurfactant solution and SDS was adjusted to var-

ious levels in the range of 2–12 at room temperature using 0.5NHCl/NaOH. The solutions were incubated at room temperature for24 h followed by evaluation of surface-active properties viz. surfacetension and emulsification activities for �-HCH/n-hexadecane asper methods described earlier.

2.5.2. Thermal stabilityThe effect of temperature on surface-active properties of bio-

surfactant preparation and SDS was determined by incubatingthe preparations at 100 ◦C for 4 h in a water bath. The sampleswithdrawn at regular intervals of 20 min were used to determinesurface-active properties as mentioned above.

2.5.3. Effect of different concentrations of saltThe effect of different salt concentrations present in WH-2 bio-

surfactant solution and SDS was studied by adding NaCl, CaCl2and MgSO4 in the range of 0–1.0 mol L−1. The effect of respectivesalt concentrations was assessed by measuring surface tension andemulsification activity of WH-2 biosurfactant solutions.

2.6. Aqueous phase partitioning of hexachlorocyclohexane (HCH)isomers

The ability of biosurfactant produced by WH-2 to solubi-lize HCH-muck isomers to aqueous phase was studied. For this,20 �g mL−1 of HCH-muck (dissolved in acetone) was added to thescrew cap vials (15 mL). The solvent was allowed to evaporate,followed by addition of 5 mL aliquot of MSM supplemented withdifferent concentrations equivalent to below CMC (0.02 g L−1), CMC(0.038 g L−1) and above CMC (0.06 g L−1) of filter-sterilized WH-2surfactant solution. The experiment was carried out in duplicatefor each concentration. The vials were placed on a rotary shaker at150 rpm and 30 ◦C for 24 h, to allow partitioning of HCH to aque-ous phase. The aqueous phase of P. aeruginosa WH-2 surfactantwas decanted to other vial and extracted thrice using equal vol-ume of solvent mix (acetone:hexane, 20:80) and the residual HCH

in the vials was extracted using same solvent mix. The sampleswere analyzed by gas chromatography (Model-5765 Nucon, India)fitted with electron capture detector (ECD) using fused silica cap-illary column BPX608 (Agilent) 25.0 m × 0.32 mm (i.d.) × 0.43 mm(o.d.) using split injection system. The injector, detector and oven

1 rdous Materials 171 (2009) 1178–1182

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ere maintained at 250, 270 and 240 ◦C, respectively. The flow ratef carrier gas (nitrogen) was 25 mL min−1.

. Results and discussion

.1. Production and characterization of biosurfactant

The strain WH-2 cells grown in MSM supplemented with fruc-ose and tryptone afforded production of surface-active moleculess evident by decrease in surface tension (28 mN m−1) and increasen total rhamnose content, estimated as per the protocol describedy Chandrasekaran and Bemiller [29]. There are reports in the litera-ure regarding production of rhamnolipids by P. aeruginosa [30]. Theharacteristic 1H NMR chemical shifts (data not presented) of puri-ed WH-2 biosurfactant preparation indicated that the sample hadolecular structure similar to rhamnolipid, produced by other P.

eruginosa species [31]. The CMC of purified biosurfactant of WH-2as equivalent to 0.038 g L−1, indicating to an efficient biosurfac-

ant preparation [32]. There are reports in the literature relating towide range of CMC values (0.055–0.163 g L−1) for rhamnolipids

roduced by different Pseudomonas spp. [33]. The biosurfactantreparation of WH-2, having low CMC would be a cost effectiveddition for bioremediation applications, as low amount of biosur-actant would achieve the desired results [30].

.2. Evaluation of surface-active properties of WH-2iosurfactant under different set of conditions

The chemical/environmental factors at the site of applicationuch as pH, temperature and salinity may affect micelle formationf biosurfactants, thus affecting its surface-active properties. There-ore, it is important to study the influence of these conditions, whenonsidering specific applications for these surface-active biologicalolecules [17]. The biosurfactant preparation of isolate WH-2 was

ubjected to extremes of chemical conditions viz. pH, temperaturend salinity in order to study their effect on surface tension andmulsification activity of biosurfactant.

.2.1. Effect of pHThe results presented in Fig. 1 show that no significant variation

as observed in surface tension of the biosurfactant preparationn the range of pH 2–12, indicating that the biosurfactant prepa-

ation retained micelle formation ability. However, the ability ofiosurfactant to emulsify �-HCH and n-hexadecane was affectedignificantly at different pH levels. The emulsification activity (EA)f biosurfactant preparation in the range of pH 4–6 and 9–11 for-HCH was maximum (100%). However, a low EA (40–60%) was

ig. 1. Effect of pH on surface tension (�) and emulsification activity for HCH (�)nd n-hexadecane (×) of the biosurfactant from P. aeruginosa WH-2.

Fig. 2. Effect of thermal stability on surface tension (�) and emulsification activityfor HCH (�) and n-hexadecane (×) of the biosurfactant from P. aeruginosa WH-2.

observed in the range of pH 6–8 and 12. The EA for n-hexadecanewas stable in the range of pH 4–7 with a steady decrease to 40%up to pH 12. Prieto et al. [34] studied the effect of pH (3–9) on EAof the biosurfactant produced by P. aeruginosa against soybean oiland reported the formation of stable emulsions in the pH rangefrom 6 to 9. The biosurfactant preparation of WH-2 maintains goodemulsion stability under acidic and alkaline environments.

3.2.2. Thermal stabilityIt was observed that biosurfactant of WH-2 was stable at 100 ◦C

even after 240 min exposure, as there was no effect on surface ten-sion of the biosurfactant solution (Fig. 2). Further, the biosurfactantsolution incubated to 100 ◦C was able to maintain up to 80% emul-sification activity for n-hexadecane and �-HCH after an exposurefor 240 and 200 min, respectively as compared to the control at30 ◦C. The biosurfactant preparation stored at 4 ◦C also retained thesurface-active properties. Such properties of WH-2 biosurfactantpreparation make it a potential candidate for use in the food, phar-maceutical and cosmetic industries, where emulsification activityis desirable, both at low and high temperatures.

3.2.3. Resistance to saltsThere are reports that the presence of salts results in disruption

ity of surfactants [35]. Thus, activity of biosurfactant preparationwas evaluated in presence of different concentrations of salts likeNaCl, CaCl2 and MgSO4 to determine its field application. As evidentfrom results presented in Fig. 3, it was observed that the surface ten-

Fig. 3. Effect of salinity on surface tension (�) and emulsification activity (EA) forHCH in the presence of NaCl (�), CaCl2 (+), and MgSO4 (�) of the biosurfactant fromP. aeruginosa WH-2.

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ion of WH-2 biosurfactant was not affected up to 1.0 mol L−1 of anyf these salts.

However, the 0.05 mol L−1 supplement of NaCl, CaCl2 and MgSO4esulted in decrease in �-HCH emulsification potential as ∼80%,5% and 15% of emulsification activity was observed, respectivelys compared to the control without any salt supplement. Furtherncrease in any of the salts did not significantly lower the emul-ification potential further. This also indicates that biosurfactantroduced by WH-2 is quite effective in presence of monovalent

ons like Na+, whereas divalent cations (Ca2+/Mg2+) may result inignificantly lowered the emulsification activity.

Prieto et al. [34] reported that the biosurfactant produced by P.eruginosa strain formed stable emulsions at low NaCl concentra-ions (below 0.05 mol L−1). However, it was capable of maintainingbout 80% of its original activity up to a salinity level of 0.3 mol L−1.bouseoud et al. [36] reported that little changes were observed

n the surface-active properties of P. fluorescens biosurfactant withddition of NaCl up to 2.0 mol L−1. There are a few reports in theiterature indicating that in saline soil degradation of parathionnd total crude oil was lowered, as compared to non-saline soils37,38]. Thus, there is a need for extensive studies in order to eval-ate the effectiveness of the bioremediation processes for pollutedites having different levels of salinity.

The emulsification potential of the biosurfactant preparation ofH-2 was also compared with a commonly used chemical sur-

actant, SDS. It was observed that as compared to biosurfactantroduced by WH-2, SDS supported significantly lower emulsifi-ation of �-HCH (data not presented). However, for n-hexadecaneoth SDS and WH-2 biosurfactant showed comparable emulsifi-ation properties. There are reports in the literature using SDSor comparing and evaluating the emulsification potential of bio-urfactants [39]. These preliminary observations indicate thatiosurfactants could prove to be environmental friendly optionsor bioremediation processes, where chemical surfactants are beingurrently used.

.3. Improved aqueous phase partitioning of HCH-muck by WH-2iosurfactant

The isomers of HCH have low aqueous phase solubility, rang-ng from 5 to 10 mg L−1 [40]. In order to improve aqueous phaseolubility of different HCH-isomers, there is a need to ensureioavailability of HCH to efficient degraders during bioremedi-tion applications. In light of this, the efficacy of biosurfactantroduced by WH-2 to improve the aqueous phase partitioning ofCH-isomers was studied at its different concentrations viz. 0.02,.038 and 0.06 g L−1, with 0.038 g L−1 being CMC of the prepa-ation. It is evident from the results presented in Fig. 4 that aignificant increase in the partitioning of the �- and �-isomersas observed with increasing concentration of biosurfactant. Theartitioning of �-isomer, major constituent of HCH-muck, to aque-us phase improved in the presence of biosurfactant at the ratef 0.038 and 0.06 g L−1, as compared to the control without sur-actant. There are many reports regarding improved bioavailabilityf oil-based hydrocarbons in the presence of biosurfactants [21];owever, there are few reports on improved partitioning of pes-icides by using biosurfactants [41,42]. Further, there is a need tonderstand the effect of improved aqueous phase availability ofhe pesticides on efficient degraders as the improved bioavailabil-

ty of such molecules may result in acute toxicity to microbialopulations [43]. Further studies to determine the effective con-entration of WH-2 biosurfactant under prevailing conditions, formproving the bioavailability of HCH-isomers to degrading strainsn bioreactors and microcosms, respectively are being carriedut.

Fig. 4. Solubility of various HCH-isomers in the aqueous phase of P. aeruginosa WH-2surfactant.

4. Conclusions

The strain P. aeruginosa WH-2, when grown on MSM affordedproduction of rhamnolipid-type biosurfactant with a CMC of0.038 g L−1. The effect of pH, temperature and salts on emulsifi-cation activity of biosurfactant preparation was studied and it wasobserved that biosurfactant retained its activity in the wide rangeof pH from 4 to 10 and can withstand temperature up to 100 ◦C for4 h. The presence of divalent salts (Ca2+/Mg2+) significantly affectedthe emulsification activity, whereas the WH-2 biosurfactant main-tain 50% emulsification potential in presence of 0.2 mol L−1 NaCl.The biosurfactant improved the aqueous phase solubility of all theisomers of HCH-muck, even at the levels below CMC. In light ofthis, biosurfactant produced by WH-2 strain could be an importantpart of the bioremediation protocols for degrading HCH-isomers inslurry bioreactors and solid phase treatment systems.

Acknowledgement

Financial support provided to Dr. Harvinder Singh Saini byDepartment of Biotechnology (DBT), Government of India, NewDelhi, India is gratefully acknowledged.

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