Influence of biosurfactant on the diesel oil remediation in soil-water system

ISSN 101--07427Journad of Environmental SciencesVol. 18, No.3, pp.587- -590, 2006CN 11- 2629/XAricle ID: 1001-0742(2006)03-0587-04CLC number: X703Document code: AInfluence of biosurfactant on the diesel oil remediation in soil-watersystemLI Yu-ying]2", ZHENG Xi-lai, LI Bing'(1. Department ofChermistry and Environmental Engincring, Wuyi University, Jiangmen 529020, China. E-mail: liyuying2002@yahoo com.cn;2. Institute of Environmental Science and Engineering. Ocean University of China, Qingdao 266003, China)Abstract: There werc six high diesel oil degrading bacteria strainsCity. The strain Y1 was able to produce biosurfactant rhamnolipid when cultivated on diesel oil as carbon source. The critical micelleconcentations (CMC) of rhamnolipid in water and in thc soil were measured respectively according to the crrelation between thesurface tension of the mecdium and the added rhamnolipid concentration. The results showed that the CMC of rhamnolipid in walerwas 65 mg/L, and was 185 mg/L in soil. The testsdiesel oil biodegradation were conducted with the addition of diferentconcentrations of rhamnolipid in water and in soil respectively. When 0.01% hamnolipid was added to water, the diesel oildegradation was enhanced. On the contrary, when the same concentration of rmamnolipid was added to the soil, the degradation ofdiesel oil was inhibited. The results suggested that the thamnolipid could enhance the diesel oil biodegnadation, indicating that theconcentration of rhamnolipid was higher than the corrcsponding CMC in the medium. Kinetics parameters for the diescl oilbiodegradation paramcters such as biodegradation constant (入), coefficient of correlation (r) and half lifc (tu2) in both lests werenumerically analyzed in this paper, indicating that the moderate concentration of rhamnolipid in the medium could not only enhancethe extent of diesel oil biodegradation but also shorten the time for oil remnediation.Keywords: biosurfactant; diescl oil; remediation; bacteriaon the influence of biosurfactants on the microbialIntroductiondegradation of hydrocarbons have been carried out onHuman activities in the production， trans-pure compound such as phenantheren (Barkay et ul,portation and storage of petroleum are routes of oil1999) or fluoranthene (Willunsen, 2001), and fewpollution since such activities inevitably involve thestudies are done on complex mixtures. At the samerisk of accidental spills that can be only minimized buttime the reported data indicated that biosurfactantsnot eliminated entirely. Petroleum releases to thecould stimulate， inhibit or have no effect onenvironment can cause safety hazards, ecologicalbiodegradation of bydrocarbons (Kanga et al., 1997;harm, and adverse human health effects. In recentBruheim, 1997).years, leakage of diesel fuel at automobile serviceIn this study, diesel oil was used as a modelstations and from pipelines has been experienced at ancontaminant because it is a common oil pollutant inalarming rate due to their increasing usage in ourthe terrestrial environment. This paper describes dieselsociety. With the increasing attention towards theoil degrading microorganisms, of which one isolateprotection of the environment, the applied cleanupcould produce a biosurfactant. The biosufactanttechnologies gained increasing interest.isolated from that microorganism was further used inThe capacity of microorganisms for trans-the test on diesel oil biodegradation. And theformation of hydrocarbons had been recognized verybiodegradation tests of diesel oil were conducted withearly (Alexander et al., 1982; Swanell and Head,the addition of different biosurfactant concentrations1994). But the remediation rate of oil contaminantseither in water or in soil, in order to find out the effctwas slow duc to their hydrophobicity and low waterof biosurfactant in different medium.solubility. Recently, a signifcant interest has been1 Materials and methodsfocused on the application of surfactants in oilbioremediation due to its ability of enhancing1.1 Materialshydrocarbons solubility (Kanga et al, 1997; ZhangThe uncontaminated subsoil were collected inand Miller, 1994). Biosurfactants have specialLinzi City, Shangdong, China in this study. The soiladvantages over synthetic surfactants, because of theirpH (measured in 0.01 mo!/L CaCl2, the ratio of watersmall size， lower toxicity, structural diversity,to soil is 2.5:1]) was 7.2. The particle size distributionbiodegradable nature, effectivencss at extreme中国煤化宁，29.7 % si (.02-temperatures, pH, salinity, easc of synthesis, or0.00: mm).perhaps even stimulation of in situ productionYHC N M H Gstudy was obtained(Kosaric, 1993; Desai and Banat, 1997). Most studiesfrom Qilu Petrochemical Corporation, China, and theFoundation item: The National Natural Science Foundation ofChina (No. 40272108) and the Doclor Subject Foundation of the Ministry of Educationof China (No. 20040423016); Corresponding authorLI Yu-ying et al.Vol.18properties of diesel oil were: density 0.8392 mg/cm;,the same time. The critical micelle concentrationssurface tension 26.8 mN/m, and viscosity 2.87 mPa.s.(CMC) of rthamnolipid in water and in the soil were1.2 Isolation and identification of bacteriameasured respectively according to the correlationDiesel oil degrading bacterial strains werebetween the surface tension of the medium and theobtained from the soil samples at the sequencing 0added rhamnolipid concentration.enrichment, isolation and purification with the diescl1.5 Tests on dicescl oil biodegradationoil as the sole carbon source. And 6 strains wereThe tests on diesel oil biodegradation weresclected based on the high diesel oil degradation rate,conducted with the addition of diffcrent concen-the bacteria identification was conducted according totrations (here 0.01% and 0.02% were selectedthe Manual of Determinative Systematic Bacteriologyaccording the experience in Li (2005)) of rhamnolipid(Dong and Cai, 2001; Zhou, 1983).to the water and the soil with the pH of 7.2The 6 strains of bacteria were inoculated in therespectively. Blank tests on diesel oil biodegradationmineral medium with the 1% of diesel oil as the solewithout the addition of rhamnolipid in water and incarbon source for 60 h at 28C respectively. And thenthe soil werc conducted at the same time. The dieselthe diesel oil biodegradation rate for 6 strains andoil concentration was 1% (the appropriate concen-surface tension in culture solution were determined,tration for bioremediation) in both biodegradationrespectively.tests. During the test, the diesel oil concentrations1.3 Recovery and measurement of the biosur-were measured by infrared spectrometry (Li et al,factant2004).for isolation and purification of biosurfactant2 Results and discussionfrom bacterial culture, the culture suspension wascentrifuged at the centrifugal force of 5000 g for 202.1 Isolation and selection of biosurfactantnin to remove cells. The supernatant was removedproducing strainsfrom the centrifuge tube, and then concentrated HCIThere were 14 strains of bacteria isolated fromwas added to obtain a final pH of 2.0 in order tothe soils. The 6 strains of bacteria that have higherpromote the precipitation of rhamnolipid, and thenthan 15% diesel oil biodegradation rate werewas centrifuged at the centrifugal force of 8000 g foridentified based on the Manual of Determinative20 min. The precipitate was dissolved in 0.05 mol/LSystermatic Bacteriology (Table 1). After 60 hsodium bicarbonate, reacidified, and recentrifuged atincubation, the surface tensions of culture solutionthe centrifugal force of 8000 g for another 20 min. Thewere determined for 6 strains of bacteria respectivelyprecipitate was extracted with chloroform-ethanol(2:1)(Table 1). The strain of Y1 was able to producein a separator funnel three times. The equivalentsurface -active compounds when cultivated on dicsclvolume of distilled water was also introduced into theoil as the carbon source. The results are listed in Tablefunnel to separate the chloroform layer (containing the1. The strain Y1 was selected for further studies sincelipids) from the water- soluble phase. The organicit showed a higher reduction on the culture brothsolvent was removed with the aid of a rotarysurface tension to 35.2 mN/m. The previous result ofevaporator, and the residue was dissolved in 0.05recovery and determination of biosurfactant producedmol/L sodium bicarbonate (pH 8.6). And theby strain Y1 has indicated that the biosurfactant wasbiosurfactant produced in this research was identifiedrhamnolipid (Li, 2005).as rhamnolipid (Li, 2005).Table 1 Characteristics of the 6 isolatesThe rhamnolipid content of biosurfactant wasDiesel oilSurface tension,determined by orcinol method. Briefly, samples wereStrain number Bacteria strainremoval, %mN/mair dried and re-dissolved in same volume of waterYlPsendamonas35.2(pH adjusted to 7.5). And 0.9 ml of a solutionY:Sphingomonas35.365.3containing 0.19% orcinol (in 53% H2SO.) was addedYMicrocoecus21.467.8to 0.1 ml of sample. After heating for 30 min at 80C,Arthrobacter20.167.4the sample was cooled for I5 min at room temperatureAcinetobacter25.159.8and the absorbancc at 421 nm was measured with aγNocerda19.868.5721 spectrophotometer. The concentration of therhamnolipids was calculated in terms of rhamnose2.2 CMC in water and in soilcontent by comparing the data with those of rhamnose; an indicator tostandard solutions.deter中国煤化工d solutions in water1.4 CMC of rhamnolipid in different mediumtensiou of uc vuin luaivuipiu solution and those ofandMHCNMH Ggearch. The surfaccThe different concentrations of rthamnolipid wereadded to the water and the soil respectively, and thehe supermatants from soil-rhamnolipid solutions atsurface tensions of different media were measured atdifferent concentrations were determined. Two seriesNo.3Influence of biosurfactant on the diesel oil remediation in soil-water system589of measured data were plotted on one graph at theTable 2 Impacts of difTerent rhamnolipids on diesel oilsame scale, and then the CMC in water and in soildegradationwere determined respectively.Theresults arTest TestDicesel oilAddedDiesel oilpresented in Fig.l. In water, the CMC of rhamnolipidlo.mediumconcentration, rhamnolipidremoval, %concentration, %was 65 mg/L(0.0065%). But the CMC of rhamnolipidoil061.4in the soil was as high as 185 mg/L (0.0185%). At theCMC values, the concentration of the bulk solutionsSoil0.0147.6will be saturated with rhamnolipid monomers. The0.0284.9diference between the CMC of rhamnolipid in soilWater76.1and in water indicated the amount of the rhamnolipid8.7loss in the soil. Therefore there was 72.2% of89.1rhamnolipid sorption by soil.8contaminants. So the diesel oil removal rate was lower- In water70女m soilthan that in the test No.1 where without the addition of自650rhamnolipid. But when 0.02% of rhamnolipid (which40 [was higher than the CMC in soil) was added into theg 30soil (test No.3), even there was some rhamnolipid虽2adsorbed by the soil surface, the rested rhamnolipidenhanced the water-solubility of hydrocarbons and)一200reducc the surface tension of soil water, resulting in aRhamnolipid concentration, mg/Lhigher improvement on the removal of diesel oil.When 0.01% of rhamnolipid (which was higherFig.1 CMC of the rhamnolipid in water and in soilthan the CMC in water) was added into the water, the2.3 Tests on diesel oil biodegradationsurface tension of water was reduced and there wasresults from the tests on diesel oilthe larger oil-water surface area which is in favor ofbiodegradation under different conditions are pre-the contact of cell with the oil. And as a result, thesented in Table 2. Without the addition ofdiesel oil removal rate was higher than that in the testrhamnolipid, the diesel oil removal in soil (test No.l)No.4 without the addition of rhamnolipid. But whenwas lower than in water (test No.4) at the same testing0.02% of thamnolipid was added, the amount and theconditions of diesel oil content. There is lowersize of rhamnolipid micelle increased while thebioavailablity of diesel oil in soil than in water as theoil-water surface was without the same correspondinghydrophobic diesel oil tended to adsorb onto soilincrease, and as a result, the diesel oil removal rate insurface, limiting the biodegradation extent, althoughtest No. 6 has a lttle enhance than the test No. 5.one research indicated that the addition of biosur-Whereas there were different results aboutfactant could enhance the desorption of hydrocarbonsbiosurfactant in hydrocarbons biodegradation (Bliorafrom the soil surface and then enhance theand Eugene, 2002; Vipulanandan and Ren, 2000;biodegradation extent (Rahman el al, 2003). ButKanga el al, 1997; Bruheim, 1997), tbe result in thiswhen 0.01% of rhamnolipid was added into the soilexperiment showed that the effects of rhamnolipid on(test No.2) in this research, the removal of diesel oilhydrocarbons biodegradation depended on both thewas even lower than that without rhamnolipid; whileadded rhamnolipid concentration and environmental0.02% of rhamnolipid was added into the soil (test No.medium.3), the removal of diesel oil has a distinct enhance toDuring the diescl oil biodegradation tests, the84.9%. But when adding 0.01% of rhamnolipid intodiesel oil contents both in the water and in the soilthe watcr (test No.5), the removal of diesel oil has adecreased with the time(Fig.2). The equation could belarge improvement than that without rhamnolipid; andexpressed as follows: C = Co e-xi, where Co denotes thewhen adding 0.02% of rhamnolipid into the water (testinitial diesel oil contcnt in environment medium (inNo.6), the removal of diesel oil has no distinetsoil or in water), C denotes the diesel oil content at theimprovement (Table 2).ime t, λ denotes biodegradation constant. So theWhen 0.01% of the rhamnolipid (which wasdiesel oil biodegradation rate accorded with thelower than the CMC in soil) was added into the soilfirst-rate kinetics, eauation: dC/dt = AC, and the half(test No.2), the rhamnolipid form a layer of hemi-life(t中国煤化工n could derivate asmicelles on the soil surface into which thefollovbasis of numericalhydrophobic hydrocarbons could partition， andconduce to the lower concentration of hydrocarbons inbiodegradation such as biodegradation constant (A),soil-water and lead to a lower bioavalability of oilcoefficient of correlation (r) and half life (12) in tests590LI Yu-ying et al.VoL.18are listed in Table 3. In both tests, there was a fastsame concentration of rhamnolipid! was added to thebiodegradation of diesel oil during the initialsoil, the degradation of hydrocarbons was inhibited.experimental period, but the biodegradation rateThe addition of rhamnolipid to the medium must beslowed down with the time especially in soil medium.higher than corresponding CMC in that medium.When 0.02% of rbamnolipid was added in soil, theThe further research also indicated that thehalf-life of diesel oil biodegradation decreased fromappropriate addition of rhamnolipid could enhance the23 d (without rhamnolipid) to 11.7 d; and when 0.01%degradation of hydrocarbons, and shorten the time forrhamnolipid was added in water, the half-life of diescloil remediation.oil biodegradation decreased from 14.9 d (withoutReferences:added thamnolipid) to 10.1 d (Table 3)。This indicatedthat the moderate content of rhamnolipid in mediumAlexander s K, Schropp s J, Schwars J R, 1982. 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Microorganism experiment manual [M]. Shaunghai:soil, respectively.Shanghai Science and Technology Press.When 0.01% rhamnolipid was added to water,the hydrocarbon degradation enhanced, but when the(Received for review August 8, 2005. Accepted October 27.2005)中国煤化工MYHCNMHG