IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Using o f Some Bacterial Species to Treat Polluted Soils with Hyd rocarbons. I.K. AL-Mayaly Departme nt of Biology, College of science , Unive rsity of Baghdad Abstract Three bacterial sp ecies were isolated from three p olluted soils with gasolin e which leaks from electricity generators that used in different regions in Baghdad; the regions choices to collect the polluted soils were (Al-Shaab, Al- Jadry ia and Al-Saydiya). The bacterial sp ecies were identified according to international b iochemical methods. It was found t hat these sp ecies were Escherichia coli, Enterobacter aerogenes and serratia marcesens. It was found that the optimum temp erature and pH were 37C°and 9 these were to cultivate E.coli and S.marcesens, while for E.aerogenes were 25 C° and 9. FT IR (Fluori Transmission Infra Red) sp ectrum technique was depended to test the ability of isolated bacteria to biodegrade the gasoline in order to use these bacteria in bioremed iation for polluted soils with hy drocarbons. Introduction Hy drocarbons are one of the p ollutants which are harmful for ecosy st ems if it increased more than the accep table level in water, soil, as well as air [1]. Biological cleaning p rocedures (bioremediation) depend on the fact that most organic chemicals are subject to enzy matic att ack of living microorganisms. These activities are summarized under the term biodegradation. However, the end p roducts of these enzy matic p rocesses might differ drastically. For instance, an organic substance might be mineralized (i.e. transformed to carbon dioxide and water). It might also be converted to a p roduct that binds to natural materials in the soil, or to a toxic substance[ 1] . Essentially , there are three major categories of sites with p olluted soils. (a) Sites that have been p olluted by either sp illage or leakage during p roduction, handling, or use of industrial material Also the activities t o gain raw materials, such as mining and oil drilling; (b) locations that have been used as disp osal sites for diverse wast e; (c) farmlands that have been excessively exp osed to p esticides.(d) in Iraq (st udy region) from generators which used to generate electricity . Contaminated land sites are health hazards for human beings and thus are unsuitable for housing or agriculture. The downward migration of p ollutants from the soil into the groundwater is esp ecially p roblematic in developing countries where groundwater is often directly used for drinking without any p rior treatment [ 2 ]. Leticia (2006) discuses the p otential of some actinomy ces to degrade p olycyclic aromatic hy drocarbons (PAH) and the effect of co-subst rates, p lants and other additives on their degradation and bioavailability [ 3]. M icrobial degradation app ears to be the most environmentally friendly method for removing of oil p ollutant since other methods such as surfactant washing and incineration lead to t he introduction of more toxic comp ounds t o the environment. Hy drocarbon-degrading microorganisms are widely distributed in marine, freshwater, and soil ecosy st ems [ 4].T he IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 ability to isolate high numbers of certain oil-degrading microorganisms from an environment is commonly taken as an evidence that those organisms are the active degraders of that environment [5]. Although, hy drocarbon degraders may be exp ected to be readily isolated from a p etroleum-p olluted environment, the same degree of exp ectation may be anticip ated for microorganisms isolated from a total unrelated environment. There is an extensive body of knowledge on mineralization or degradation of hy drocarbons by microorganisms [ 6&7] Materials and Methods - S oils poll ute d with hydrocarbons: Native hy drocarbonoclast bacteria were isolated from p olluted soils with gasoline obtained from three p olluted soils with generators gasoline, which were taken from (Al- Shaab; Al-Jadriyia and Al- Saydiyia) regions in Baghdad. - Isol ation of strains from pollute d soi ls: 10 gm of p olluted soil was humidified with 250 ml of mineral medium (modified mineral salts medium –M M SM -) which comp osed of: 4 gm NH4NO3; 4 gm KH2PO4; 0.2 gm M gSO4.7H2O and 0.01 gm CaCl.2H2O p er litre [ 8 ] . The bacteria was sep arated from the soil p articles by gentle shaking of 1 gm soil dry weight with 10 ml of st erile water for 30 minutes. Aft er sedimentation, the sup ernatant susp ension was used to p repare app rop riate dilutions (from 1x10 -1 to 1x10 -5 ) with st erile water. Aliquots of 0.2 ml were sp read –p lated on the nutrient agar medium. The p lates were incubated at 25 C° for 5 days. T he bacteria were allowed to sp read until p urification and were then conserved in a refrigerator ready for use in the p roduction of inocula for characterization and the hy drocarbon degradation exp eriments [ 9]. The morphology of the bacterial st rains was determined by gram st aining, and they were then st reaked in Petri dishes. The isolated bacterial cultures were identified according to [ 10&11] - The Optimum Conditi ons of Cultivation: In order to know the op timum conditions for the three isolated bacteria; they were cultivated with different degrees of temp erature which include :in 25 C° ; 37 C° and 40 C° and pH 5,7 and 9. - Hydrocarbons De gradation i n Liqui d Culture: To test the ability of isolated bacteria to biodegrade the gasoline, about 1 gm sample of p olluted soil was t aken and dissolved in (1 acetone: 3 hexane) and added to a flask of 250 ml, after the evap oration of solvents at 25° C, about 50 ml of st erile medium and 1 ml of the isolated bacteria was inoculated into each flask and then incubated at 35 C° and 150 rp m for 7 days [ 9] The ability of the isolated bacteria to biodegrade gasoline was determinate by using FT IR (Fluori Transmission Infra Red) sp ectrum technique. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Re sults and Discussion: According to [ 10&11] and the biochemical tests which done and exp lained in table (1) , the most p redominant bacteria sp ecies in p olluted soils that t aken from Al-shaab, Al-jadriyia and Al-saydiya were E.coli; Enterobacter aerognosa and Serratia marcesens, resp ectively. Usually, the tested soil samples were filled with gasoline fuel which used in generators of electricity So the saturated with gasoline may have effect to limit the kind of microbial content [ 12] .The difference in microbial p op ulation is a reflection of many factors such as nutrients and oxy gen levels, temp erature and availability of minerals [ 13]. Usually, the temp erature, p H and other factors like available energy source, moist ure availability and the ap p rop riate depend to know the optimum conditions of cultivation [ 14], So, it was found that t he optimum conditions to cultivate E.coli and Serratia marcesense were 37C° and p H9 but 25C°and p H9 were op timum conditions to cultivate Enterobacter aerogenes (Tables 2, 3& 4). The results of Flouri Transmission Infra- red (FT IR) sp ectra exp lain the effect of bacteria to biodegrade the gasoline, as following: Figure (1-A) represents the sp ectrum of control samp le , the important bands absorbency of infra-red in 2931.80 ; 2897.08 ; 2877.79 and 2850.79 cm -1 , these wave numbers represents a strong and wide absorbency for blended frequency for NH3 + group in primary amino acids. The frequency in the wave number 1720.50 cm -1 related to amine salts.also1458.18 cm -1 represents as st rong absorbency for the sy mmetrical blended frequency of the amine salts. T he nitrop arfins ap p ear two bands in 1377.17and 1357.89 cm -1 which related to asy mmetrical and sy mmetrical frequency for NO2. There is a weak absorbency for the alip hatic amines in 1215.15; 1064.71 cm -1 . Three p eaks of blended frequency in 887.26; 794.67 and 725.23 cm -1 are for C-H group . Also, there is absorbency for disulphides in 528.50 and 455.20 cm -1 . Figures (1, B-E) exp lain that t he sy mmetrical and a sy mmetrical frequency of 2974.23 and 2931.80 cm -1 related to alkanes group [15] while 2357.01 cm -1 represents the identical absorbency of st retch frequencies for OH and NH3 + group s. The wave number 1361.74 cm -1 represents the blended frequency was related to carboxy l group while 1134.14 cm -1 was related to asy mmetrical blended frequency for C-O group . The disap p ear of 2357.01; 1064.71; 794.67 cm -1 is the main notice in the fifth day of the exp eriment. Also the app earance of other wave numbers in 1292.31; 1033.85; 786.96 cm -1 is very clear. Usually these changes of hy drocarbon group s give good evidence that E.coli has good effect on biodegrade the gasoline [16] where in the seventh day 786.96 and 759.95 cm -1 were disapp eared completely , also a clear ap p earance for 2966.52 ; 2977.94 and 2862.36 cm -1 can be recognized . In the tenth day of the exp eriment (last day), many changes in absorbency of the hy drocarbon group s would occur like app earance of peak in 1716.65 cm -1 as a blended IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 frequency of N-H group , and there is a st rong absorbency in 1219.01 cm -1 as result of the blended frequency for carboxy l group . The sy mmetrical blended frequency for the aromatic ethers group is in 1056.99 cm -1 while the number wave 891.11 cm -1 represents the frequency for C-C group in alkaline. The p eak app ears in 725.23 cm -1 is related to C-N group absorbency for isobuty ramide.also, T here is a bromine compounds frequency in 528.50 cm -1 and frequency of disulphides in 447.49 cm -1 . Figures (2, A-D) represent t he treat of Enterobacter aerogenosa with gasoline during ten days. It was found t here are weak absorbency bands of C≡C, C ≡N and S-H group s in 2357.01 cm -1 and a blended frequency of C =O group of aliphatic Easters in 1716.56 cm -1 , also there is absorbency for C-H group in 786.96 and 763.81 cm -1 . Simp le change would occur in the fifth day of the exp eriment , some absorp tion would app ear other disap p ears but all of them in the same region of the absorbency would app ear in p revious day s; but in the seventh day , more than one p eak of new absorbency regions would app ear, esp ecially in 3421.72 and 3309.85 cm -1 which represent acute st rong absorbency of O-H group in phenols compounds. M any changes in absorbency p eaks would be clear in tenth of the exp eriment, these are usually related to the effect of bacteria activity with biodegradation p rocess which has sure effect in initial new absorbency p eaks [ 15&16]. Figures (3, A-D) represent the treat of Serratia merscens with 1m /50 ml of gasoline. It can by found that t he absorbency p eak in 794.67 cm -1 is sharp er in first day comp arative with control sample. This wave number rep resents a blended absorbency of C-H group for benzene comp ounds. T he long chain of these comp ounds which led to p revent or reduce the bacterial enzy me activity [ 17] but its sharp shap e is be more reduced in the fifth day and may disapp ear till the end of the exp eriment as a result of bacteria effect. M any absorbency p eaks would disapp ear in the fifth day which give a good sight to the change that occur in the chemical comp ound of gasoline. Also p eaks disapp ear and other app ears in the seventh day. In the tenth day, there is absorbency frequency of C=O group of aliphatic Easter app ear in 17166.66 cm -1 , a frequency of C-C group in 1465.90 cm -1 , other frequency of C-H group in 786.96 and 759.95 cm -1 [ 17]. Re ferences 1- Pizzul, L., Castillo, M .D.P.and Stenstrom, J.(2006).Characterization of selection actinomy cetes degrading p olyaromatic hy drocarbons in liquid culture and sp ecies. World Journal of M icrobiology and Biotechnology .10.s11.9100-6. 2- Lehmann, V.(1998).Bioremedation: A solution for p olluted soils in the sout h? J.Biotechnology and Development M onitor,.34, p .12-17. 3- Leticia, P.(2006).Degradation of p olycyclic aromatic hy drocarbons by actinomy cetes. SLU.Acta Universitatis agriculture sueciae..2006:50. 4- At las, R.M . and Bartha, R.(1973).Abundance, distribution and oil degradation p otential of microorganism in Raritan Bay. Environ .p ollut.4:291-300. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 5- Okerntugba, P.O.and Ezerony e, O.U.(2003).Petroleum degradation p otentials of single and mixed microbial cultures isolated from river and refinery effluent in Nigeria .Afr.J.Biotechnol.2:288-292. 6- Ojumu, T.V.; Bella, O.Oand; Sonibare, J.A.(2005).Evolution of microbial sy st em for bioremediation of p etroleum refinery effluents in Nigeria.Afr.J.Biotechnol.4 (1):31- 35. 7- Adenipekun, C.O. and Fasidi, I.O.(2005).Bioremediation of oil-p olluted soil by Lentinus subnudus, a Nigerian white- rot fungus.Afr.J.Biotechnol; 4(8):796-798. 8- Herman ,D.C.;Zhang ,Y.and M iller,R.M .( 1997).Rhamnolip id (Biosurfactant) Effect On Cell Agreement and Biodegradation Of Residual hexadecane Under Saturated Flow Conditions.Ap p l.Envi Ron.M icrobiol.,63:3622-3627. 9- Lopez, M .E. ; Rodriguez, M .T.;Rios ,E.L. ; Esp arza ,F.G.;Chavez,,G.; Rodriguez, V.and Barrera, J.C.(2007).Fungi and Bacteria isolated from two highly p olluted soils for hy drocarbon degradation.Acta Chim.Slov.(technical p aper) , 54,201-209. 10- Johnson, M .T.(2007).Identification of the Enterobacteriaceae.Indiana University . India. 11- (NHS) National Health Service .(2001).Introduction to the Preliminary Identification of M edically Important Bacteria. 12- Akp or, O.B.; Lgbinosa, O.E. and Lgbinosa, O.O.(2007).Studies on the effect of p etroleum hy drocarbon on the microbial and p hy sico-chemicals characteristics of soil. African journal of biotechnology .6 (16):1939-1943. 13- Haris, J.O.(1962).M icrobiological st udies of oil and gas p ipelines. Technology bulletin.p p .135. 14- Englert, C.J.; Kenzie, E.J.and Dragun, J. (1993). Bioremediation of Petroleum Products in Soil. In Princip les and Practices for Petroleum Contaminated Soils.Ed.E.J.Calabreseand P.T .Kostechi.Lewis Publishers, Chelsea, M I.PP.112- 115,121. 15- At wo, A.T .; Abedulkader, A;and Abdul kader, K.(1989).T he sp ectral and organic diagnosis .M inist ry of Higher Education and Scientific Research. College of Education. Ibn Al-Haitham. Dar Al-Kitab for p ublishing. Baghdad. 16- Jacobucci,D.F.C.;Vasconcelos,C.K.;M atsuura,A.B.;Falconi,F.A.and Durrant,L.R.(2001)Degradation Of Diesel Oil by Biosurfactant -Producing Bacterial Strains.J.of Soil Sediment &Water (The magazine of Environmental Assessment &Remediation.htt p ://www.Aehsmag.com/issues/2001. 17-Fritsche, W. and Hofrichter, M . (2006). Aerobic Degradation by M icroorganism.http ://www.engg.ksu.edu/HSRC/JHSR/V3. Tabl e (1) Biochemical tes ts to i den tifi cation the i sol ate d bacte ri a Test Strain 1 Strain 2 Strain 3 Oxidase _ _ _ Growth on Mac Conky ag ar P ink colony P ink colony Whit e colony Indol reaction + _ * Urease * _ H2S * * _ Cit rate * * + Gram st rain _ _ _ Morphology Bacilli -cocci Sho rt bacilli bacilli IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Table (2): Bacterial densi ty in 25C ° and pH 5, 7and 9. ( cel lx10 5 ). Bacteria Time ( day s ) p H 0 3 5 7 10 E.coli 0.005 0.015 0.025 1 5 5 0.005 0.015 1.5 5.5 7.5 7 0.005 0.007 0.055 0. 175 25 9 E.aerogenes 0.005 0.006 1.5 15 20 5 0.005 0.015 16 18 20 7 0.005 0.2 0.5 15 50 9 S.marcesen s 0.005 1 1.2 4 5 5 0.001 2.4 5 7.5 25 7 0.005 0.7 5 5.5 0.195 9 Table (3): Bacterial densi ty in 37C ° and pH 5 7and 9. ( cel lx10 5 ). Bacteria Time ( day s) p H 0 3 5 7 10 E.coli 0.005 100 15 15 12 5 0.005 200 350 150 50 7 0.005 140 300 350 400 9 E.aerogenes 0.005 20 30 35 12 5 0.005 20 10 3 3 7 0.005 100 100 30 20 9 S.marcesen s 0.005 3 1 zero zero 5 0.005 250 300 500 500 7 0.005 125 150 400 500 9 Table (4): Bacterial densi ty in 40C ° and pH 5 7and 9. ( cel lx10 5 ). Bacteria Time (day s) p H 0 3 5 7 10 E.coli 0.005 0.03 6.5 10 6.6 5 0.005 0.02 55 30 15 7 0.005 0.12 45 33 20 9 E.aerogenes 0.005 0.007 2 2.5 3.5 5 0.005 0.03 25 8 4.6 7 0.005 100 100 30 25 9 S.marcesen s 0.005 3.5 4.1 1.6 0.3 5 0.005 0.5 2 5 5.5 7 0.005 5.5 20 50 55 9 IHJPAS B C D A IHJPAS E A B C D Fig. (2, A-D): FT IR S pectrum for gasoline with Enterobacter aerogenes where: A= FT IR spectrum of gasoline after 3 days of the treat with E. aerogenes B= F T IR spectrum of gasoline after 5 days of the treat with E. aerogenes C= F T IR spectrum of gasoline after 7 days of the treat with E aerogenes D= FT IR spectrum of gasoline afte r 10 days of the treat with E aerogenes. Fig. (1, A-E): FT IR S pectrum for gasoline with E.coli.where: A= FT IR spectrum of gasoline without treat with E.coli. (Control sample) B= F T IR spectrum of gasoline after 3 days of the treat with E.coli. C= FT IR sp ectrum of gasoline after 5 day s of t he treat with E.coli D= FT IR sp ectrum of gasoline after 7 days of the treat with E.coli. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 A B C D Fig. (3, A-D): FT IR S pectrum for gasoline with S.marcesens where: A= FT IR spectrum of gasoline afte r 3 days of the treat with S.marcesens B= FT IR spectrum of gasoline after 5 days of the treat with S.marcesens C= FT IR spectrum of gasoline after 7 days of the treat with S.marcesens D= FT IR spectrum of gasoline after 10 days of the treat with S.marcesens. IHJPAS 2010) 3( 23مجلة ابن الهیثم للعلوم الصرفة والتطبیقیة المجلد ة الترب الملوثة بالھیدروكاربوناتاستخدام بعض األنواع البكتیریة لمعالج ـالـــــــــــي ــمیــــ ـــل الــ ـــــار كـــــامـــ إیـــث جامعة بغداد،كلیة العلوم ، قسم علوم الحیاة الخالصة دات المسـتخدم ولینأنواع بكتیریة من ثالث تـرب ملوثـة بالكـاز ةثالث عزلت ن الموـل د الطا ةالمتسـرب ـم قـة عـادة لتولـی ــاد ) والسـیدیة،والجادریـة ،الشـعب( الكھربائیـة فـي منـاطق مختلفـة مـن بغـداد واع البكتیریـة المعزولـة باعتم ، شخصـت األـن :، وقد وجد إن ھذه األنواع كانت " ق البایوكیمیائیة المعتمدة عالمیاائالطر ــا ــد . S.marcesens وE.aerogenes ,و E.coliبكتری ــل أووج ــدروجیني األمث ــرقم الھی ــى و ال ــرارة المثل ــة الح ن درج . 9و °م 25كانت E.aerogenesبكتریا ل بینما 9 و ،°م37ھما S.marcesensو ،.coli Eلتنمیة FT( تم اعتماد تقنیة طیف األشعة تحت الحمراء IR( ك الكـاز ،ولین الختبار قدرة األنواع البكتیریـة المعزولـة علـى تفكـی .في المعالجة الحیویة للترب الملوثة بالھیدروكاربوناتھذه البكتریا استعمال لغرض IHJPAS