Bulletin of Environmental and Scientific ResearchISSN 2278-5205, Available online at http://www.besr.org.inVol. 1, Issue(3-4),pp.1-3Received: 28 June 2012/ Accepted: 29 November 2012

Detoxification of Heavy Metals by Biosurfactants

1?Sankar Narayan Sinha and 1Dipak Paul1Environmental Microbiology Research Laboratory, Department of Botany

University of Kalyani,Kalyani- 741235, West Bengal, India

?Email corresponding author: sinhasn62@yahoo.co.in

AbstractIn recent times river pollution is a serious and grow-

ing problem in most developing countries. Industrialeffluents and sewage entering the water bodies are oneof the major sources of environmental toxicity, whichendangers aquatic biota and deteriorates water qual-ity. Biological methods for the removal of heavy metalsfrom industrial waste may provide an attractive alterna-tive to the physicochemical process. Biosurfactants areone of the compounds that help in alleviating the heavymetals. A large number of bacteria such as Bacillus sp.,Pseudomonas sp., Acinetobacter sp. and Arthrobactersp. are reported to produce biosurfactants. Comparedto synthetic compounds, biosurfactants provide the ad-vantages of little or no environmental impact and thepossibility of in-situ production. Studies in recent pasthave exhibited the successful use of biosurfactants forfacilitating the degradation of organic pollutants in soiland water. In the light of the above, the present studyis aimed to carry out the assessment of efficiency ofbiosurfactants producing bacteria isolated from heavymetal contaminated site of the river Ganga. The bacte-rial isolates (PGS1, PGS2 and PGS3) were screened be-longing to the genus Pseudomonas were found to re-move heavy metals from the medium. The isolate PGS1was found to be more effective in removing more than50% chromium and cadmium from the medium.

Keywords: Biosurfactants; Heavy metals; Detoxifica-tion; River Ganga; Pseudomonas

1 IntroductionSurfactants are amphoteric molecules which help in

alleviating the non-degradable pollutants that are themajor cause of pollution of reverine ecosystem for thepast few decades. Biosurfactants have the potential forremoval of metals from soil or sediment. Biosurfactantsare produced by plants, animals and many different mi-croorganisms (Zajic and Panchel, 1976). There are sev-eral apparent advantages to the use of biosurfactantsrather than synthetic ones, as for example, biosurfac-tants are biodegradable, cost-effective and it may bepossible to produce them in situ at contaminated sites.Some species of Pseudomonas are able to produce andexcrete a heterogeneous mixture of biosurfactants withglycolipid structure, known as rhamnolipids. Rhamno-lipids give good physico-chemical properties in term ofsurface activities, emulsification activities and stabili-ties. Moreover, these surface-active compounds show

antimicrobial activities against both pathogenic bacte-ria and fungi. Due to an increase in concerns regardingenvironmental protection and also the distinctive prop-erties of the rhamnolipids, it seems that rhamnolipidsfulfil the criteria for several industrial and environmen-tal applications, like environmental remediation and bi-ological control. Biosurfactants like rhamnolipids havealready been commercially manufactured, producingthem more economically competitive with other syn-thetic surfactants. So in the near future, rhamnolipidsmay be commercially successful biosurfactants. Bio-surfactants accelerate the solubilisation of hydropho-bic chemicals by forming micelles which contain hy-drophobic domains where the chemicals are incorpo-rated. Biosurfactants are produced by many differentbacterial genera. Though chemical structures of bio-surfactants vary widely, but more biosurfactants are an-ionic or non-ionic. The yield and composition of bio-surfactant are affected by growth conditions includecarbon sources, culture medium nutrients, tempera-ture, pH and agitation (Syldatk and Wagner, 1987; Hom-mel and Ratledge, 1993). Moreover, there are specieslevel differences in the chemical structure of biosurfac-tants. For example, the rhamnolipids produced by dif-ferent Pseudomonas species vary both in the numberof rhamnose molecules and the length of lipid moiety(Chandrasekaran and Bemiller 1980). Although it is wellknown that bacterial cells can complex metals from so-lution, there is little information in the literature con-cerning the use of biosurfactants to complex metals.With this perspective in mind, the present work is un-dertaken to isolate bacteria from a metal-contaminatedsite along the lower stretch of the river Ganga, to deter-mine the ability to produce biosurfactant and to assessthe efficacy of biosurfactant-producing bacteria to re-move heavy metals in a heavy-metal contaminated siteof the lower stretch of the river Ganga.

2 Methods

2.1 Sampling

Water sample was collected from the river Ganga nearthe effluent discharge point of Ichhapore Metal andSteel factory, West Bengal, India.

2.2 Isolation and characterization of bacte-ria

Bacteria were isolated by serial dilution method andspread plate technique (Sinha, 2006). The isolated bac-

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terial strain were subjected to staining and various mor-phological, physiological and biochemical characteri-zation such as size, shape, Gram staining, motility test,indole test, methyl red test, Voges Proskauer test, citrateutilization test, triple sugar iron test, catalase and oxi-dation test, mannitol fermentation, urea hydrolysis testand starch hydrolysis test (Benson, 2004; Sinha, 2006).

2.3 Screening for biosurfactant production

The production of biosurfactant by the isolated bac-teria was determined by the ability of the bacteria to lysethe erythrocytes. For this blood agar were prepared andpoured into the sterile Petri dish. The bacteria was theninoculated in the medium and incubated at 37oC for 24h and then the haemolyses was measured (Plaza et al,2006).

2.4 Biosurfactant production

The isolated bacterium was used for the productionof biosurfactant by growing the organism in a specificmedium. The yield and structure of bacterial surfac-tants depends on the choice of carbon source.

2.5 Production of rhamnolipid from bacte-rial isolates

In the present study, biosurfactant production wascarried out in water-insoluble medium containing1.5% (V/V) cooked vegetable as substrate along withMgSO4.7H2O, KH2PO4, NaNO3, yeast extract and pep-tone. The cultures were taken in 500 ml Erlenmeyerflasks with 100 ml of medium. Filter-sterilized traceelement solution was added to medium. Then wholemedium is autoclaved and allowed to cool. Then 2 ml ofthe culture was added to the medium and incubated at30oC for 48-72 h.

2.6 Extraction of rhamnolipid

During growth, the surfactants was produced and re-leased into the medium. This surfactant was extractedby the acid precipitation method. At first, the mediumwas centrifuged at 5000 rpm for 15 minutes. The cell-free broth containing surfactant was collected in a sep-arate tube. Then the surfactant in the broth was precip-itated at pH 2.0 by adding conc. HCl. The broth wascentrifuged again at 5000 rpm for 15 minutes. Now thesurfactant was extracted with dichloromethane. More-over, purification was achieved by recrystallization. Thedichloromethane extract was dissolved in distilled wa-ter containing sufficient NaOH to give pH 7.0. This so-lution was filtered with through Whatman No. 4 filterpaper and reduced to pH 2.0 with conc. HCl. After cen-trifugation, the white solid was collected as a pellet.

2.7 Effect of biosurfactant on the removal ofheavy metal

The extracted biosurfactants was used for the re-moval of metals such as chromium and cadmium.The nutrient broth medium containing the salts ofchromium sulphate and cadmium sulphate was pre-pared and sterilized. The salts of chromium and cad-mium were added to the medium at concentration of

10 mg, 20 mg, 30 mg, 40 mg and 50 mg per litres respec-tively. The pH of the medium was maintained to 7.0 to7.2 and sterilized. Then the extracted biosurfactant ( 50µl ml-1) was inoculated in the medium and incubatedat 30oC for 24 h. The medium with rhamnolipid waskept as treatment and medium without biosurfactantsand organism served as control. The tubes were thenanalysed for the concentration of metals present aftertreatment by atomic absorption spectrophotometer.

3 Results and discussionTable 1 indicated the biochemical characterization

of bacterial strains isolated from heavy-metal contam-inated site of the river sediment. Standard biochem-ical characterization indicated that all the isolates be-longed to the genus Pseudomonas. The biosurfactantsproduced by the isolates was confirmed as rhamno-lipid by observing dark blue halos around bacterialcolonies when cultured in blue agar. Rhamnolipids arethe group of the surfactants produced by Pseudomonasaeruginosa. Rhamnolipids are found to be efficient inbioremediation of heavy metal polluted sites (Mulligan,2005). The isolate PGS1 was indicated with various con-

Table 1: Characterization of isolates from metal-contaminated site

CharacteristicsBacterial isolates

PGS1 PGS2 PGS3Shape rod rod rodSize 0.5-0.8µm 0.4-0.7µm 0.5-0.8µmGram reaction -ve -ve -veMotility + + +Indole test - - -Methyl red - - -test -Voges Proskauer - - -testCitrate utilization + + +testTriple iron sugar + + +testCatalase activity + + +Oxidase activity + + +Urea hydrolysis - - -+ present or positive reaction, - absent or negative reaction.

centrations of chromium and cadmium. This strain re-moved more than 50% chromium as well as cadmiumfrom the medium (Figure 1-3). Chromium was removedmore efficiently than cadmium. Rhamnolipid was ableto reduce metal toxicity to microbial consortia in co-contaminated soils (Sandrin et al, 2003). Maslin andMaier (2000) studied the effect of rhamnolipids pro-duced by various Pseudomonas aeruginosa strains onthe phenanthrene degradation by indigenous popula-tions in two soils co-contaminated with phenanthreneand cadmium. Hazra et al (2010) noted that 50% and30% removal of lead and cadmium with 1% (V/V) rham-

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nolipid extract. The results indicated the activity of bio-surfactants in the removal of heavy metals. The presentstudy indicated the use of biosurfactants in the biore-mediation of metal-contaminated sediment from theriver Ganga.

Figure 1: Effect of rhamnolipid in removing heavy metalfrom media at different heavy metal concentration byisolate PGS1

Figure 2: Effect of rhamnolipid in removing heavy metalfrom media at different heavy metal concentration byisolate PGS2

4 Conclusion

The present study indicates that out of 3 isolatesPGS1 was found to be more effective in removing morethan 50% chromium and cadmium from the medium.On the other hand, isolates PGS2 and PGS3 exhibitedmore than 25% and 30% chromium removal from themedium respectively. Similarly isolates PGS2 and PGS3was found to be more efficient in removing 24% and30% cadmium respectively. The biosurfactants rham-nolipid might play a great role in the removal of thesetoxic heavy metals. The use of such biosurfactants pro-duced by the bacterial strain PGS1 may be used for thebioremediation of the heavy metal contaminated areaof river Ganga in future, though further research onstructural characterization, gene regulation of biosur-factant and cost of production is needed.

Figure 3: Effect of rhamnolipid in removing heavy metalfrom media at different heavy metal concentration byisolate PGS3

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