e ISSN:2582 5208
InternationalResearchJournal of Modernization in Engineering TechnologyandScience ( Peer Reviewed, Open Access, Fully Refereed International Journal )
ISOLATION, IDENTIFICATION AND OPTIMIZATION OF DIESEL DEGRADING BACTERIA FROM DIESEL CONTAMINATED SOIL
*1DepartmentOfAgriculturalChemistryAndBiochemistry,TheUniversityOfAgriculture, Peshawar,25130,Pakistan
*2,3,4,5,6InstituteOfBiologicalSciencesFacultyOfLifeSciences,SarhadUniversityOfScienceAnd InformationTechnology,Peshawar,Pakistan
ABSTRACT
Environmentalpollutionwithpetroleumhasbeenrecognizedamongsttheseriousthreatingproblemsglobally. Someofthemostexistingpetroleumhydrocarbonspollutantsincludediesel,gasolineandfueloils. Intherecent times, bioremediation/biodegradation techniques are used to remove environmental pollutants. A list of microorganisms have the ability to use petroleum hydrocarbons as the sole source of carbon for energy in order to carry out their metabolic activities. It involves the use of microbes to purify and humiliate environmental contaminants. The present study is carried out during 2019 20 by conducting isolation, identification and optimization of diesel degrading microbes from diesel oil polluted in selected sites of Peshawar, Pakistan. Bacteria like Acinetobacter baumani, Acinetobacter pitti, Acinetobacter calcoactius, and Pseudomanasauroginosawereisolatedonthebasisofmorphologicalandphysiologicalcharacteristics.Further the optimization was carried out for various culture parameters i.e., Hydrocarbon concentration and pH. The present study suggests that Acinetobacter pitti has the degrading ability at pH 7 when hydrocarbon concentrationiskeptat2%.
Keywords: Bioremediation,EnvironmentalPollutant,DieselOil,Bacteria,Optimization
I. INTRODUCTION
In the recent times, one of the most serious threating problem the world is facing is Environmentalpollution containingpetroleum.Ithasbeenknownasthemostseriousissueduetoitstoxiceffectsonanimals,plantsand humans [1]. Petroleum oil spill is amongst the top contributors observed daily which eventually results in environmental pollution. Petroleum iscomposed of a hydrocarbon (HC) along with other organic compounds. Someofthemajorsourcesofsoilandaquifercontaminationincludedieseloilleakages,distributionfacilitiesandvarious industrialoperations[2].Oilspillcanoccurduringthedrillingprocess,accidentorleakagefromoil tankersor ships during transportation, seepage from storage tank and from natural process [3]. These contamination sourcesleadtopollutebothterrestrialandaquaticecosystems[4].Dieseloil(apetroleumproduct)contaminants affectsthesoilassoilabsorbitinitssurfaceduetoitspropertieslikelowemulsifyingpotentialanddensitywhile highlevelofviscosity.Thisleadtoaffecttheabsorbencyandsoilporosity[5],[6].PetroleumHCconsistsofCarbon(C) alongwithNitrogen(N), resultinginthe chemical changeof soilorganic matter (SOM). Thisalsodisturbsthesoil salinityalongwithpHandelectricalconductivity(EC)[7].AliphaticHCshowmorepersistentinthesoildueto largechain(C20 C40).Thisleadstodifficultyinvolatilizationanddegradationduetotheirlowwatersolubility and structure [8], [9]. Petroleum HCs are toxic when present in low quantity and can be carcinogenic or mutagenictowildlifeandhumans[10].
In the recent times, sustainable techniques are applied in order to tackle this problem. One of the techniques include bioremediation which is an accepted environmental friendly technology. Bioremediation refers to the use of microbes, such as bacteria and fungi, in order to transform the environmental contaminants into less harmful substances. This technique engages the microbes in order to degrade pollutants effectively and efficiently [11]. This technique has numerous advantages if compared with conventional remediation technologies,i.e.,easytobeinstalled,eco friendly,massapplicationinlargeareas,lowcostandcanleadtothe complete destruction of different contaminants [12]. Bacteria are usually used for the remediation of soil and watercontainments affected with petroleumproducts. Bacteria are foundthroughoutthe environment dueto
Volume:04/Issue:06/June-2022 Impact Factor- 6.752 www.irjmets.com www.irjmets.com
@InternationalResearchJournalofModernizationinEngineering,TechnologyandScience [5682]
Muhammad Baseer Us Salam*1, Nihal*2, Adnan Khan*3, Zahoor Ahmad*4 , Sania Iqbal*5 , Muhammad Mubasser Ahsan*6
e ISSN:2582 5208
InternationalResearchJournal of Modernization in Engineering TechnologyandScience ( Peer Reviewed, Open Access, Fully Refereed International Journal )
their adoptability. They can be grown in an extensive variety of environmental conditions. The nutritional abilityofadaptabilityofbacteriacanalsobemanipulatedforbiodegradation[13].Severalbacteriastrainshave been isolated and studied for their potential to degrade various petroleum products [14]. The microbial transformations are dependent upon certain factors like pH, temperature, Salinity, Electrical Conductivity, Moisture level (for soil). It is also dependent on the chemical structure and composition along with physical state and concentration of the contaminant hydrocarbons. Mostly, Mesophilic bacteria have the tendency to degradepetroleumhydrocarbons Theypossesstheiroptimumtemperaturewhich rangesfrom25ºCto 45ºC [15] StudieshaveshownthattherateofbiodegradationisatpeakwhenpHisnearneutrality[16],[17],[18]. StudieshaveshownanacceptablepHrangeforbiodegradationofpetroleumhydrocarbonsi.e.,pH6topH8while thebestgrowthrateisobservedatpH7(neutral)[19].PetroleumBiodegradationHCscanundergobothaerobicand anaerobicenvironments.Aerobicbiodegradationisconsideredasmoreefficientandeffectivebecauseitresults incompletemineralizationoftheHCs.Whiletheanaerobicbiodegradationprocessisslowanddonotresultin complete mineralization of HCs [20]. This research aims to isolate and identify some of the diesel degrading bacteria from diesel contaminated soil and further evaluate the best growth condition for those isolated bacteria.
II. MATERIALS AND METHODS
Sample collection:
Oilpolluted soil was collected fromShellStationlocated at Kohat Road,Peshawar and Oil Depot in Tarujabba, Nowshera.Sampleswerecollectedat2 5cmdepthusingastainless steelsampler.Thesampleswerestoredin specializedsoilcans.
Serial dilution:
Seven fold serial dilution of each soil sample were done separately. For serial dilution seven pre labeled test tubes, each containing 9ml distilled water, were taken and then autoclaved at 121℃ for 15 minutes. Then 1 gramofsoilsamplewastransferredtothefirsttesttubeandmixedvigorously.1mlofsuspensionfromfirsttest tubewastransferredtothesecondtesttubeandwasshakenwell.Thiswasrepeatedtillthe lasttesttube.Asa result,followingdilutionwereachieved10 1,10 2,10 3,10 4 ,10 5,10 6and10 7 .
Isolation of hydrocarbon degrading bacteria:
Stock solution comprising of Bushnell Hass agar media was prepared for each component of the media. The mediawaspreparedforthe isolationofhydrocarbonsdegrading bacteria. The recipe of media (1 L) is given in Table1. Themediawaspreparedandautoclavedat121℃for15 minutes.
Pure culture preparation:
Nutrient agar media was prepared for pure culturing. The media was prepared in flask and sterilized in autoclaveat121℃for15minutes.Thesterilizedmediumwaspouredintopetriplatesandleftforsolidification. Aftersolidification,thebacterialcoloniesfromBushnellHassagarmediaplateswerestreakedintheneutralagar media.
Addition of carbon source (Diesel):
Dieselwasusedasacarbonsourceindifferentconcentrationsi.e.,2%,4%,6%.Apartfromthis,noothersource ofcarbon/energywasaddedintothemedia.
Biochemical Identification and Chemical Tests of isolated bacteria
Gram staining:
StandardprocedureforGramStainingwasfollowed.Theprocedureinvolvedtransferringofaloopfull offresh culturefollowedbynormalsmearonthecenteroftheslide.Thesmearwasdried intheair.Acleanglassslide was taken along with a sterilized inoculating loop on flame of Bunsen burner. The dry smear was fixed via passingovertheflame.Then,theslidesweresubjected togramstaining. Slideswas subjectedto crystal violet stainforaminutefollowedbywashing.Then,Gram’sIodinesolutionwasaddedtotheslides.Itwasthendrained off by washing the slides. The smear was then flooded with the decolorizing agent (Ethyl alcohol) for 30 seconds. The decolorizing agent was added drop wise to the slide till decolorization. The slides were later washed with distilled water. In last, the smearwas covered with safranin for about 1 minute. The slides were
Volume:04/Issue:06/June-2022 Impact Factor- 6.752 www.irjmets.com www.irjmets.com
@InternationalResearchJournalofModernizationinEngineering,TechnologyandScience [5683]
e ISSN:2582 5208
InternationalResearchJournal of Modernization in Engineering TechnologyandScience ( Peer Reviewed, Open Access, Fully Refereed International Journal )
againcleaned with distilled wateruntilpinkorpurplecolorappears.Finally,theslideswereobservedunderthe lightmicroscope
Indole test:
Freshculturewastakenfromnutrientagarplateswiththe helpofsterilizedloopandtransferredto testtubes containing tryptophan media.The test tubes were then kept in incubator at 37 ºC for 24 hours. Two drops of Kovac’sreagentwereaddedtothetesttubesafterincubation Ringformation(Pink)within5minutesindicated positiveresultsforindole.
Coagulase test:
Twodropsofdistilledwaterwerepouredoncleanglassslidefollowedbysterilizationoftheloop.Freshculture was taken with the help of sterilized loop and inoculated on the slides to which 2 drop of distilled water was added. Then blood plasma was added to it kept for 5 min to form clot. Formation of clot on the slides were indicatepositiveresultwhilenoformationofclotindicatesnegativeresult.
Catalase Test:
A small amount of fresh culture was taken from nutrient agar plates and transferred to a sterile slide. Two drops of3%hydrogenperoxide(H2O2)wereaddedandmixed.Formationof bubblesafter30secondsshows thepresencewhilenobubblesformationindicatesnegativeresult.
Triple Sugar Iron (TSI)
AnisolatedcolonywerepickedthroughsterileinoculatingneedleandtransferredtotesttubescontainingTSI agarmedia.TSIwaslaterinoculatedandthenstreakedonthesurfaceoftheslant.Capswereleftlooseandthe tubes were further incubated at 37℃ for 24 hrs. Results were noted by analyzing the color of the butts and slants.
Motility test
Pureculturewaspickedthroughsterileneedleandtransferredtotesttubescontainingsemisolidmedia(SIM). Theneedlewasthenstreakedonthesurfaceoftheslants.Thetesttubeswerekeptinincubatorat37℃for24 hrs. A turbid area that extends away from the inoculation line indicated positive result, while a growth along theinoculationlinebutdoesnotextendindicatednegativeresult.
Urease test
Freshculture fromnutrient agar plates were picked througha sterile needle and transferred to the test tube containingurea agarslant bystreaking ina zigzag manner.Incubation ofthe slantswere made at37℃for 24 hrs. The results were interrupted by analyzing the color of the butt and slants. Pink color indicated positive resultwhileyellowcolorformationindicatednegativeresults.
Optimization of Cultures:
The optimization of cultures was subjected to pH and concentration of diesel oil. The analysis was made to checkthegrowthrateanddegradationuponthesetwofactors.BushnellHassmediumwithdifferentdieseloil concentration(2%,4%,and6%)waspreparedatdifferentpH(6.0,7.0,8.0)andsterilizedinautoclaveat120ºC for15mins.1NHCland1N NaOHwasusedtoadjustthepHofmedia. Aftertheinoculationofbacteria,itwas incubatedat37ºCfor72hours.Thegrowthwasmeasuredat600nmbyusingspectrophotometer.
III. RESULTS AND DISCUSSIONS
Atotalof10bacterialspeciesisolateswereobtainedbyusingBHmedia.Onthebasisofsize,shape,andcolors, fourcolonieswereselectedandstreakedonnutrientagar plates.Itwasfurtherfollowedbygramstainingand biochemical tests. Biochemical tests such as catalase, coagulase, indole, motility, TSI and urease tests were carried out. The isolated strains were identified as Acinetobacter baumani (A), Acinetobacter pitti (B) Acinetobactercalcoaceticus(C), andPseudomanasauroginosa(D).Gramstraintestshowsthatalltheisolates’ strainsweregramnegative.FurthertestresultsareshowninTable2.
Bacteria is the mostly used organism for the biodegradation of petroleum oils. Several studies are presently focusing on the isolation of bacterial strains which contains the ability to degrade/ remediate petroleum oils efficientlyandeffectively[21],[22],[23].Thereareseverala bioticfactorswhichcanaffectthebio degradation of diesel oil. This research has evaluated the effect of substrate concentration and pH on growthof bacterial
Volume:04/Issue:06/June-2022 Impact Factor- 6.752 www.irjmets.com www.irjmets.com
@InternationalResearchJournalofModernizationinEngineering,TechnologyandScience [5684]
e ISSN:2582 5208
InternationalResearchJournal of Modernization in Engineering TechnologyandScience ( Peer Reviewed, Open Access, Fully Refereed International Journal )
Volume:04/Issue:06/June-2022 Impact Factor- 6.752 www.irjmets.com
strains.Itwasevidentfromthestudythatbacterialgrowthissubjectedtothesevariationsinoilcontaminated media.ResultshaveshownthatneutralpHand2%dieseloil concentrationisthebestfitmediaforthegrowth ofallthebacterialstrainsisolatedduringthisresearchwork.Whileacidicandbasicmediaalongwith4%and 6% substrate concentration resulted in a decline growth rate. According to [24] diesel oil offers a healthier carbon source for the growth of bacteria. The current research work is in line with [25] which reports an optimumgrowthinin2%dieseloilcontaining media.Higherconcentrationofdieseloilnegativelyaffectedthe growthofbacteriainminimalmedia;suggestinghighconcentrationofsomecomponentsofdieseloilmayhave toxiceffectonthebacterialstrains[26].ResultofpHforgrowth of Acinetobacterbaumani,Acinetobacterpitti, Acinetobacter calcoactius, and Pseudomanas auroginosa with diesel oil was evaluated further. All microbes resulted in optimum growth in pH 7. Hence, it is evident from the study that neutral pH is vital for the best growth of bacteria. Compared to this, acidic and basic condition don’t provide the best growth condition to these microbes. The optimization of pH is an essential step while selecting the bioremediation approach as it shows the best fit growth condition for the microbes The conducted research is in line with [27] which has reported thattheoptimum pHforthe degradationof diesel oil by individual bacterial strainsand anassorted bacterialconsortiumwasinitiatedtobe7
Table 1: RecipeofBushnellHassAgar Component g/liter MgSO4, 0.2 CaCl2 0.02 KH2PO4 1.0 K2HPO4 1.0 NH4NO3 1.0 FeCl3 0.05 Agar 15.0
Table 2: TestsresultsofBacterialIsolates
@InternationalResearchJournalofModernizationinEngineering,TechnologyandScience [5685]
e ISSN:2582 5208
InternationalResearchJournal of Modernization
Engineering TechnologyandScience ( Peer Reviewed, Open Access, Fully Refereed International Journal )
in
Volume:04/Issue:06/June-2022 Impact Factor- 6.752
www.irjmets.com
e ISSN:2582 5208
InternationalResearchJournal of Modernization in Engineering TechnologyandScience ( Peer Reviewed, Open Access, Fully Refereed International Journal )
Volume:04/Issue:06/June-2022 Impact Factor- 6.752 www.irjmets.com
e ISSN:2582 5208
InternationalResearchJournal of Modernization in Engineering TechnologyandScience ( Peer Reviewed, Open Access, Fully Refereed International Journal )
Volume:04/Issue:06/June-2022 Impact Factor- 6.752 www.irjmets.com
Optimization of growth conditions
Effect of pH on growth
The effect pH on growth of Acinetobacter baumani, Acinetobacter pitti, Acinetobacter calcoactius, and Pseudomanas auroginosa is given in figure 6. The fig 1 shows that growth of the all the isolated strains were optimum at neutral pH (i.e., 7). Decrease in growth was observed when the pH was either increased or decreasedfromtheoptimumlevel.
Photospectrometric absorbtion
0.8 pH 6 pH 7 pH 8 0.7 0.6 0.5 0.4 0.3
Acinetobacter baumani Acinetobacter pitti Acinetobacter calcoactius
Pseudomanas auroginosa
Figure 6:EffectofpHongrowthofAcinetobacterbaumani,Acinetobacterpitti,Acinetobacter calcoactius,andPseudomanasauroginosa.
e ISSN:2582 5208
InternationalResearchJournal of Modernization in Engineering TechnologyandScience ( Peer Reviewed, Open Access, Fully Refereed International Journal )
Volume:04/Issue:06/June-2022 Impact Factor- 6.752 www.irjmets.com
Effect of substrate concentration on bacterial growth: The effect of different concentrations (2%, 4% and 6%) of substrate is given in figure 7. It was noted that growth of all the isolated bacterial strains were higher in media containing 2% of diesel oil. Increasing the concentrationofdieseloil(4%and6%)ingrowthmedianegativelyaffectedthegrowthofisolatedstrains.
2% 4% 6%
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
1 Acinetobacter baumani Acinebacter pitti Acinetobacter cacoactius Psedudomonas auroginosa
Absorbance Bacterial Strains
0
Figure 7: Effectofdifferentconcentrationsofsubstrate(dieseloil)ongrowthofAcinetobacterbaumani, Acinetobacterpitti,Acinetobactercalcoactius,andPseudomanasauroginosa IV.
CONCLUSION
From this study, it has been concluded that oil polluted sites contain HCs degrading bacteria. Environmental condition has greater effect on the growth of HCs degrading bacteria. It is recommended that further study should be conducted in order to come across better hydrocarbon degrading bacteria. Thus,morediversified areasshouldbeselectedforisolationofsuitabledegradingmicroorganism.Alongwiththis,optimizationfactors likepH,temperatureandotherenvironmentalparameterssuchasoxygenavailability,nutrients(nitrogenand phosphorous), water contents may be studied for high rate of degradation. Extensive studies should be undertakentoprotectourenvironmentfromthreatenedofpollutionduetooilspillage.
V. REFERENCES
[1] Varjani, S. J. & Upasani, V. N. (2017). A new look on factors affecting microbial degradation of petroleumhydrocarbonpollutants.InternationalBiodeterioration&Biodegradation.,120:71 83.
[2] Egbeja, T. I., Oguche, J. U. & Bashir, A. A (2019). Biodegradation of gasoline polluted soil using goat dung.Journalof Applied Sciences and EnvironmentalManagement.,23(8): 1589 1594.
[3] Zabbey,N.andG.Olsson.2017.Conflicts oilexplorationandwater.GlobalChallenges.,1(5):1600015.
[4] Ivshina,I.B., Kuyukina,M.S.,Krivoruchko,A.V.,Elkin,A.A.,Makarov,S.O.,Cunningham,C.J., &Philp, J.C. (2015). Oil spill problems and sustainable response strategies through new technologies. Environmental Science: Processes &Impacts.,17(7):1201 1219.
[5] Wang,S.,Xu,Y.,Lin,Z.,Zhang,J.,Norbu,N.&Liu,W.(2017).Theharmofpetroleum pollutedsoilandits remediationresearch.InAIPConferenceproceedings.,1864(1):020222 1 020222 8.
[6] Nagkirti, P., Shaikh, A., Vasudevan, G., Paliwal, V. & Dhakephalkar, P. (2017). Bioremediation of TerrestrialOilSpills:FeasibilityAssessment.InOptimizationandApplicabilityofBioprocesses.,7:141 173.
@InternationalResearchJournalofModernizationinEngineering,TechnologyandScience [5689]
e ISSN:2582 5208
InternationalResearchJournal of Modernization in Engineering TechnologyandScience ( Peer Reviewed, Open Access, Fully Refereed International Journal )
Volume:04/Issue:06/June-2022 Impact Factor- 6.752 www.irjmets.com
[7] Li,J.,Deng,M,Wang,Y.&Chen,W.(2016).Productionandcharacteristicsofbiosurfactantproducedby Bacillus pseudomycoides BS6 utilizing soybean oil waste. International Biodeterioration & Biodegradation.,112:72 79.
[8] Narwal,S.K.&Gupta,R.(2017).Biodegradationofxenobioticcompounds:AnOverview.In Handbook of research on inventive bioremediation techniques., 8(1):186 212.
[9] Stroud, JL, Paton, G I & Semple, KT (2007). Microbe aliphatic hydrocarbon interactions in soil: implicationsforbiodegradationandbioremediation.JApplMicrobiol.,102:1239 1253.
[10] Chandra,S.,Sharma,R.,Singh,K.&Sharma,A.(2013).Applicationofbioremediationtechnologyinthe environmentcontaminatedwithpetroleumhydrocarbon.Annalsofmicrobiology.,63(2):417 431.
[11] Agarry, S. E. & Ogunleye, O. O. (2012). Box Behnken design application to study enhanced bioremediation of soil artificially contaminated with spent engine oil using biostimulation strategy. InternationalJournalofEnergyandEnvironmentalEngineering.,3(1):31.
[12] Guo, J, Feng, R, Ding, Y, et al. (2014). Applying carbon dioxide, plant growth promoting rhizobacterium and EDTA can enhance the phytoremediation efficiency of ryegrass in a soil polluted withzinc,arsenic,cadmiumandlead.JEnvironManage.,141:1 8.
[13] Das,S.& Dash, H.R.(2014).Microbialbioremediation:Apotentialtoolforrestoration of contaminated areas.InMicrobialbiodegradationandbioremediation.,(1):1 21.
[14] Malik, Z. & Ahmed, S. (2012). Degradation of petroleum hydrocarbons by oil field isolated bacterial consortium.AfricanJournalofBiotechnology.,11(3):650 658.
[15] Borai, E A.M.K., Eltayeb, M., Mostafa, A. R. & El Assar, S. A (2016). Optimization and Statistical Evaluation of Medium Components Affecting Crude Oil Biodegradation by Some Locally Isolated Bacteria.EgyptianJournalofBotany.,56(3):753 767.
[16] Sarkar,D.,Ferguson,M.,Datta,R.&Birnbaum,S.(2005) Bioremediationofpetroleumhydrocarbons in contaminated soils: comparison of biosolids addition, carbon supplementation, and monitored naturalattenuation.Environmentalpollution.,136(1):187 195. 28
[17] Leahy, J. G. & Colwell, R. R. (1990). Microbial degradation of hydrocarbons in the environment. MicrobiologyandMolecularBiologyReviews.,54(3):305 315.
[18] Zaidi, B. R. & Imam, S. H. (1999). Factors affecting microbial degradation of polycyclic aromatic hydrocarbonphenanthreneintheCaribbeancoastalwater.MarinePollutionBulletin.,38(8):737 742.
[19] Shah, M. P. 2014. Environmental bioremediation: A low cost nature's natural biotechnology for environmentalclean up.JournalofPetroleum&EnvironmentalBiotechnology.,5(4):1.
[20] Ivshina, I. B., Kuyukina, M. S. & Krivoruchko, A. V. (2017). Hydrocarbon oxidizing bacteria and their potentialineco biotechnologyandbioremediation.InMicrobialResources121 148.
[21] Sivagamasundari T. & Jayakumar, N. (2017). Optimization of Diesel oil degrading Bacterial strains at various culture parameters. International Journal of Research and Development in Pharmacy & Life Science,6(6):2840 2844.
[22] Parthipan,P.,Preetham,E.,Machuca,L.L.,Rahman,P.K.,Murugan,K.,&Rajasekar,2017.Biosurfactant and degradative enzymes mediated crude oil degradation by bacterium Bacillus subtilis A1. Frontiers inmicrobiology,8:193.
[23] Awasthi,M.K.,Selvam,A.,Chan,M.T.,&Wong,J.W.2018.Bio degradationofoilyfoodwasteemploying thermophilicbacterialstrains.Bioresourcetechnology,248:141 147.
[24] Luo Q, Zhang J G, Shen X R, Sui X, Fan Z Q. 2013. Characterization of a novel diesel oil degrading pseudomonassp.strainF4.FreseniusEnvironBullet,22:689 697.
[25] Ramasamy, S., P. Mathiyalagan, & P. Chandran, 2014. Characterization and optimization of EPS producing and diesel oil degrading Ochrobactrum anthropi MP3 isolated from refinery wastewater. PetroleumScience,11(3),439 445
[26] Mahalingam,P.U.&Nithya,S.2014.Optimizationofgrowthconditionfordieseloildegradingbacterial strains.Adv.App.Sci.Res.,5(6):91 96.
@InternationalResearchJournalofModernizationinEngineering,TechnologyandScience [5690]