A Study of the Abilities of Pseudomona Aeruginosa and Pseudomonas Fluorescens to Degrade Diesel Oil

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1 Journal of Emerging Trend in Engineering and Applied Science (JETEAS) 3 (3): 93 Scholarlink Reearch Intitute Journal, 1 (ISSN: 1171) jetea.cholarlinkreearch.org Journal of Emerging Trend in Engineering and Applied Science (JETEAS) 3(3):93 (ISSN: 1171) A Study of the Abilitie of Peudomona Aeruginoa and Peudomona Fluorecen to Degrade Dieel Oil 1 O.O. Olanipekun; 1 A.O. Ogunbayo; and S.K. Layokun 1 Department of Chemical Engineering, Univerity of Lago, Nigeria Department of Chemical Engineering, Obafemi Awolowo Univerity, IleIfe, Nigeria. Correponding Author: O.O. Olanipekun Abtract Peudomona aeruginoa and Peudomona fluorecen are among the common bacteria found to mediate in the biodegradation of hydrocarbon, a well a in cleaning up environment contaminated with uch. But little i known about their contribution, in term of their abilitie in degrading dieel oil. Therefore, thi tudy i with a view to etablih and compare their bioremediating potential on dieel oil. The pure and mixed culture of Peudomona aeruginoa and Peudomona fluorecen were incubated in mineral alt media with dieel oil a the ource of carbon and energy, at o C for 1 day in a Gyratory haker at rpm. Sample were withdrawn at hour interval, and centrifuged at 5 rpm for 3 minute. Bioma growth wa meaured on dry weight bai while dieel oil conumption wa etimated from the upernatant after treatment with petroleum ether by pectrophotometric method. The growth and ubtrate utilization parameter baed on integrated balance analyi were obtained and the reult obtained howed that P. aeruginoa and P. fluorecen, in both pure and mixed culture propagated well on dieel oil. It wa oberved that within 5 day of incubation, 5%, 51% and 75% of the dieel wa conumed by P. fluorecen, P. aeruginoa and the mixed culture, repectively. From the reparameterized Monod Kinetic model, the Monod contant value obtained were.5,. and. gl 1 for P. aeruginoa, P. fluorecen and the mixed culture, repectively. The maximum pecific ubtrate conumption rate of 1., 1. and. gg 1.day 1 were obtained for P. aeruginoa, P. fluorecen and their mixed culture, repectively. Thee reult howed that with relatively higher percentage of degradation and lower maximum pecific ubtrate conumption rate the P. aeruginoa and the mixed culture gave better degradation than the P. fluorecen. Keyword: biodegradingabilitie, peudomona aeruginoa, peudomona fluorecen, mixed culture, diauxic growth, dieel oil INTRODUCTION Environmental pollution with petroleum and petrochemical product (complex mixture of hydrocarbon) are widepread worldwide (Plohl et al., ; Margein et al., 3). The preence of hydrocarbon pollution in the environment, regardle of their amount, i potentially dangerou to the microfauna and microflora, and ha detrimental effect on the ecoytem. The impact, uch a evere ecoytem imbalance caued by thee pollutant i of great public concern (Whyte et al., 1997; Bicca et al., 1999; Margein et al., 3), and therefore there are worldwide concern about remediating and retoring the environment of hydrocarbon pollution (Bicca et al., 1999). Microbial biodegradation of pollutant play a pivotal role in the retoration and remediation of hydrocarboncontaminated environment. The biodegradingability of individual hydrocarbon will be influenced by everal factor among which are the hydrocarbonburden of the area and the ability 9 of the indigenou microorganim to utilize hydrocarbon. Thi ability of microbe to degrade organic contaminant into harmle contituent ha been explored a a mean to biologically treat contaminated environment ha been bai for the emergent field of bioremediation (Atla and Philp, 5). Thi proce ha become an accepted technology for retoration of hydrocarbon contaminated ite becaue of it cot effectivene and ecofriendly nature (Bicca et al., 1999; Halden et al., 1999; Plohl et al., ; Margein et al., 3; Olanipekun et al., 1). The capability of microorganim to degrade hydrocarbon can be meaured by uing material and energy balance in the analye of experimental data decribing cell growth, ubtrate utilization and product formation (Heijnen and Roel, 191; Solomon et al., 195; Olanipekun et al., 1). For any particular cae of biodegradation reaction in bioremediation, kinetic tudie give a meaure of how well the microbial ytem i carrying out it function

2 Journal of Emerging Trend in Engineering and Applied Science (JETEAS) 3(3):93 (ISSN: 1171) (Limbert and Bett, 1995). The kinetic of the growth of microorganim trongly influence how fat ubtrate (pollutant) will be utilized, removed or degraded. The microbial growth kinetic i the relationhip between the pecific growth rate and ubtrate concentration. Monod (199) propoed a k S S relatively imple model max for the concept of microbial growth kinetic which decribe the growth rate a a function of only the limiting ubtrate and aumed no dependence on the concentration of other ubtrate (nutrient) or other environmental factor. However, the model require a well defined ubtrate uptake rate. In many cae, growth of microorganim i a conequence of ubtrate uptake and not vice vera (Solomon et al., 199). Due to lape in the Monod model above, Pirt max q (195) ha identified a model q which k relate growth yield and the pecific ubtrate conumption rate and thi model for the growth proce which i baed on the Monod kinetic equation i dependent on the ubtrate uptake make more biological and mathematical ene (Solomon et al., 199). Thi i becaue it fully accounted for ituation at which ubtrate utilization approache zero. It ha been reported that the hydrocarbon preent in complex compound uch a in dieel oil, are not imultaneouly but erially metabolized (Atla, 191; Van Hamme et al.1). Thi i becaue the microorganim can only developed an enzymic ytem to degrade a ingle hydrocarbon at a time. Biodegradation of mixture of hydrocarbon proceed by the aid of two procee namely paring effect and cooxidation. Thee have marked influence on the peritence of particular hydrocarbon within a petroleum mixture and thu on the evolution of the weathered petroleum hydrocarbon mixture (Atla, 191). Sparing effect which involve the organim preferentially conuming ubtrate with fatet growing rate to le eaily metabolized carbon ource reult in hift in metabolim pattern in the midt of growth. The organim divert it energie from growth to retool for the new ubtance. Thi reult in catabolite repreion, which i a erial utilization phenomenon (Bailey and Olli, 19; Van Hamme, 1). Therefore, a mixture of hydrocarbon alo, require enzymic ytem which may be upplied by mixed culture (culture of heterogeneou population of microorganim) for better tranformation (Reifeld et al., 197). The conortium of organim complement the catalytic capabilitie of one another to degrade or tranform, even mixture of hydrocarbon (Forter and Wae, 199) and thi i referred to a ynergim. In thee microbial communitie many procee may occur which may enhance microbial metabolim, and thee include the production of growth factor, removal of toxic ubtance and cometabolim (Atla and Bartha, 197). An indicative increae in the poibilitie of hydrocarbon pollution due to pillage of dieel oil i enviaged with the recent everal report on oil pill in the environment, and dieel oil i conidered to be recalcitrant to microbial degradation due to it polynuclear tructure. It tend to perit in ecoytem becaue of it hydrophobic nature and low volatility and thu poe a ignificant threat to the environment (Abed et al., ). The contituent of dieel oil, are carcinogenic, mutagenic and are potent immunotoxicant, thu poing a eriou threat to human and animal health (Samanta et al., ). Therefore, there i a need to etablih the biodegrading ability of peudomona pecie, which ha been the ubject of many reearch invetigation due to their amazing metabolic diverity and wide ditribution in nature. The aim of thi work i to tudy the extent to which the Peudomona pecie uch a P. aeruginoa and P. fluorecen in pure and mixed culture can degrade dieel oil uing material and energy balance (integrated balance analyi) a well a kinetic model. MATERIAL AND METHOD MATERIAL Thi ection outline the material and Equipment ued in carrying out the experiment. Stock culture of P. aeruginoa and P. fluorecen, which are coded National Collection of Indutrial Bacteria, Scotland, NCIB95 and 375 repectively were obtained from Department of Microbiology, Obafemi Awolowo Univerity, IleIfe, Nigeria. Dieel oil wa obtained from African Petroleum Filling Station in Ile Ife Nigeria. New Brunwick Environment Controlled Incubator haker. Atell Heavon Model 19/ 9 SBV autoclave. Gallenkamp Centrifuge of 5 x 3 rpm capacity. MOl Viible Spectrophotometer Campec Cambridge. The Mineral alt medium compoed of (gl 1 ) Na HPO 1H O (3.), KH PO (3.), MgSO (.), FeSO 7H O (.1), CaCl H O (.1), NaCl (1.), KCl (1.), NaNO 3 (.) and. ml of trace element olution of whoe compoition (gl 1 ) wa H 3 BO 3 (.15), ZnSO 7H O (.75), NaMoO.H O (.5), MnSO H O (.75), CuSO.5H O (.75), FeCl 3 H O (.) and CoCl.H O (.) (Zhang et al. 5).. METHOD The method adopted in thi work i imilar to that that wa ued in a previou by Olanipekun et al., 3

3 Journal of Emerging Trend in Engineering and Applied Science (JETEAS) 3(3):93 (ISSN: 1171) (1). From the tock culture obtained, a loop of cell wa tranferred to ml conical flak containing 5 ml mineral alt medium and 1%(v/v) of dieel oil a ole carbon ource. Thi wa done eparately for each organim and incubated in rotary haker ( rpm) for 3 hour at 3 C to obtain primary culture. The econdary culture were prepared by tranferring 5 ml of the culture to 5 ml freh medium and incubated in ml conical flak in the rotary haker ( rpm) for 1 hour at 3 C. Inoculum for the experimental run were taken from the econdary culture, with the view that exponential growth phae i fully etablihed. The biodegrading abilitie of thee peudomona pecie on dieel oil, in pure and mixed culture were teted in 5 ml mineral alt medium, containing 35% (v/v) dieel oil, inoculated with 5 ml of econdary culture inoculum. The et up, which were in duplicate, were incubated at C on a New Brunwick Gyratory Shaker at rpm for 1 day. Sample were withdrawn at hour interval for analyi to obtain the dry bioma concentration and concentration of the dieel oil. The average value of the data were tended kinetic, model to obtain the parameter uch a required for thi tudy. into bioma were 5%, 3% and 19% for P. aeruginoa, P. fluorecen and their mixed culture repectively. While the average percentage bioma energetic yield of 1%, 3% and 1% were obtained for P. aeruginoa, P. fluorecen and their mixed culture repectively. Thi connote that the more the fraction of carbon converted into bioma the more the energy in the conumed ubtrate ued. Therefore, in the firt five day more fraction of the carbon in the ubtrate i converted into bioma in P. fluorecen than in the other culture. But le of it wa converted into bioma in their mixed culture. After the fifth day there were decreae in the bioma concentration. Similar decreae in the bioma concentration wa reported for the growth of peudomona train on glucoe after 7day by Stelmack et al. (1999). The decreae in the bioma concentration could be explained by three poible mechanim. Firtly, production of metabolite in the coure of degradation which may inhibit growth of the organim, econdly, the poibility of nutrient depletion due to utilization and thirdly cell adheion to the glaware. Approximately 39% of cell adheion to the glaware in the abence of urfactant wa reported in the work of Stelmack et al. (1999). EVALUATION OF PARAMETERS The carbon and energetic yield were etimated from the meaured data uing the following equation 1 and which are baed on the integrated balanced of batch proce analyi which i generally more conitent than intantaneou balance (Solomon et al., 199). From carbon balance the fraction of carbon (ubtrate) converted to bioma, y c, i etimated uing equation 1 while the fraction of ubtrate energy incorporated into bioma (that i the bioma energetic) i calculated from equation. y c b x f o b x x o f o x o b f f RESULT AND DISCUSSION Bioma Growth and Yield of the Organim with Dieel Oil a Subtrate The reult obtained for the bioma concentration, pecific growth rate, and yield aociated with growth uch a fraction of carbon converted into bioma and bioma energetic yield are preented in column,, and 7 repectively of the Table 1, and 3 for P. aeruginoa, P. fluorecen and their mixed culture repectively. The bioma concentration increaed with time for the firt 5 day in both pure and mixed culture of the P. aeruginoa and P. fluorecen. Within thi period the average percentage fraction of carbon converted 1 31 From the th day, increae in the bioma concentration were oberved for both P. aeruginoa and the mixed culture (Figure 1). Thi increae could be explained by what can be termed a retooling for the preence of either higher molecular weight hydrocarbon or xenobiotic (uch a polyaromatic hydrocarbon or metabolite). A the hydrocarbon preence in the dieel oil are not imultaneouly metabolized (Van Hamme et al., 1), the characteritic growth curve (Figure 1) reflect thi ituation a there are diauxic growth. Table 1: Variation of bioma growth and ubtrate conumption parameter aociated with the Growth of P. aeruginoa on Dieel oil. Degradation time (Day) 1 X (gl 1 ) (day 1 ) q (gg 1 day 1 ) y c Table : Variation of bioma growth and ubtrate conumption parameter aociated with the Growth of P. fluorecen on dieel oil. Degradation time (Day) 1 X (gl 1 ) (day 1 ) q (gg 1 day 1 ) y c

4 Journal of Emerging Trend in Engineering and Applied Science (JETEAS) 3(3):93 (ISSN: 1171) Table 3: Variation of bioma growth and ubtrate conumption parameter aociated with the Growth of mixed culture of P. aeruginoa and P. fluorecen on dieel oil. Degradation time (Day) 1 Bioma Concentration (gl 1 ) X (gl 1 ) (day 1 ) q (gg 1 day 1 ) y c The retooling or readaptation period wa more protracted in the pure culture of P. aeruginoa than the mixed culture. Thi could be a a reult of ynergic degradation. Thi i a complementary catalytic capability of the two organim (Amund et al., 197; Foter and Wae 199). After the ixth day, not much ignificant growth wa oberved in P. fluorecen. The earlier performance in term of bioma growth and ubtrate utilization oberved with P. fluorecen could be explained with the fact that P. fluorecen degrade nalkane more effectively than P. aeruginoa (Plohl et al., ). The hift in the growth (diauxic growth) oberved with the P. aeruginoa may be due to the two tage in the production of biourfactant oberved by Zhang et al. (5) in their work On biodegradation of crude oil. They oberved a rapid production tage within the firt day followed by a econd low production tage from day. P. aeruginoa P. fluorecen Mixed culture 1 Figure 1: Plot of Variation of Bioma Concentration of P. aeruginoa, P. fluorecen and their mixed culture on Dieel oil. Microbial Growth and Biodegradation of Dieel Oil Figure to howed that a the bioma increaed, the concentration of the dieel oil decreaed, thu, uggeting that the organim were able to grow and degrade the dieel oil. The pure and mixed culture grew quite quickly within the firt five day and thi period correpond to high rate of conumption of the ubtrate (Table 13): 5% by P. aeruginoa, 5% by P. fluorecen, and 71% by the mixed culture of the P. aeruginoa and P. fluorecen. Thi reult i i agreement with previou reearcher (Plohl et al. ; Zhang et al. 5) who found that the firt five day of incubation were the mot important for biodegradation. They aerted that during thi period majority of the compound (epecially nalkane) were degraded, and only a light increae followed in the remaining day of incubation. Bioma Concentration (gl 1 ) Bioma Concentration (gl1) Bioma Concentration Subtrate Concentration 1 Figure : Plot of Bioma Concentration and Subtrate Utilization of Growth of P. aeruginoa on Dieel oil Bioma Concentration Subtrate Concentration 1 Figure 3: Plot of Bioma Concentration and Subtrate Utilization of Growth Of P. fluorecen on Dieel oil It ha alo been reported previouly that after th day biodegradation of up to 5% wa oberved for the growth of P. fluorecen on dieel oil (Plohl et al. ) while in thi preent tudy about %, 7% and 9% of the dieel oil wa conumed by P. fluorecen, P. aeruginoa and their mixed culture repectively. Therefore, within the day of the experiment, % conumption by P. fluorecen Subtrate Concentration (gl 1 ) Subtrate Concentration (gl 1 ) 3

5 Journal of Emerging Trend in Engineering and Applied Science (JETEAS) 3(3):93 (ISSN: 1171) compare variably with 5% of P. fluorecen reported by Plohl et al. (). Bioma Concentration (gl 1 ) Bioma Concentration Subtrate Concentration 1 Figure : Plot of Bioma Concentration and Subtrate Utilization of Growth of Mixed culture of P. aeruginoa and P. fluorecen on Dieel oil The Growth Kinetic of the Organim on Dieel Oil From the reult obtained in etimating the kinetic parameter of growth in the Table, it i oberved that the mixed culture of P. aeruginoa and P. fluorecen increaed the affinity of the organim for the dieel oil. Therefore, the cavenging ability of the ynergetic metabolim i higher than pure culture of the organim. P. aeruginoa exhibited a lightly greater affinity (lower K value) for the dieel oil than P. fluorecen. That i P. aeruginoa i capable of high growth rate at low level of ubtrate (dieel oil) and, therefore, efficiently utilize, remove or degrade the dieel oil (Halden et al., 1999). Table : Kinetic Parameter for the Growth of the P. aeruginoa, P. fluorecen and mixed culture on dieel oil Kinetic Parameter P. P. Mixed Saturation Affinity Contant K (gl 1 ) Maximum Specific Subtrate conumption Rate (gg 1 day 1 ) Subtrate Concentrate (gl 1 ) aeruginoa fluorecen culture max Moreover, the value of q obtained for the culture are higher than the minimum ubtrate conumption rate for the organim during the coure of thi tudy. It i indication that the organim; P. aeruginoa, P. fluorecen and the mixed culture grew on the dieel oil a ubtrate. CONCLUSION Thi work wa a creening tet that did not conider the phyical and chemical condition for biodegradation. However, baed on the reult, the following inight ha been provided. Firtly, P. aeruginoa and P. fluorecen both in pure and mixed culture howed potency and poibility of being ued a agent for bioremediation. Secondly, P. aeruginoa had higher average pecific value than P. fluorecen an indication that P. aeruginoa i better than P. fluorecen in degrading dieel oil; defined not by microbial number but by performance which i hown in the degrading ability. Finally, the mixed culture of the P. aeruginoa and P. fluorecen performed better than the pure culture in degradation of dieel oil. NOTATION S Subtrate concentration gl 1 X Bioma concentration gl 1 K Monod contant gl 1 q Specific rate of ubtrate conumption (g ubtrate g bioma 1 hr 1 ) µ Specific growth rate (hr 1 ) µ max Maximum pecific growth rate (hr 1 ) y c fraction of carbon converted to bioma reductance degree ma fraction of carbon SUBSCRIPTS c carbon Subtrate b Bioma o Initial f Final REFERENCE Abed, R.M.M., Schonhuber, W., Amann, R. and GarciaPichel, F. (): Picobenthic cyanobacterial population revealed by 15 rrna targeted initu hybridization. Environ. Microbiol., Amund, O. O., Adebowale, and Ugoji, E. O. (197): Occurrence and Characteritic of Hydrocarbon Utilizing Bacteria in Nigeria Soil Contaminated with Spent Motor Oil. India Journal of Microbiology, 7, 37 Atla, R. M. (191): Microbial Degradation of Petroleum Hydrocarbon; an Environmental Perpective: Microbial Revteur, 5, 9 Atla, R. M. and Bartha, R. (197): Biodegradation of Petroleum in Seawater at Low Temperature: Canadian Journal of Microbiology 1: Atla, R.M. and Philip, J. (5): Bioremediation: Applied microbial olution for realworld environment cleanup. ASM Pre. Wahinghton, DC. Bailey, J.E. and Olli, D.F. (19): Biochemical Engineering Fundamental. nd Edition, McGraw Hill, Kogakuha, Ltd., pp. 1. Bicca, F.C., Fleck, L.C. and Ayub, M.A.Z. (1999): Production of biourfactant by hydrocarbon degrading Rhodococcu rubber and Rhodococcu erythropoli. Revita de Microbiologia 3,

6 Journal of Emerging Trend in Engineering and Applied Science (JETEAS) 3(3):93 (ISSN: 1171) Forter, C. and Wae, J. (199): Environmental biotechnology, ElliHorwood Publiher, England, pp. 951 Halden, R.U., Tepp, S.M., Halden, B.G. and Dwyer, D. (1999): Degradation of 3phenoxybenzoic Acid in Soil by Peudomona peudoalcaligen POB 3 (PBOB) and Two modified Peudomona train. Applied and Environmental Microbiology. 5, Heijnen, J.J. and Roel, J.A. (191): A microcopic model decribing yield and maintenance relationhip in aerobic fermentation proce. Biotechnology and Bioengineering, 3, Limbert, E.S. and Bett, W.B. (1995): Kinetic of biooxidation of a medium compriing phenol and a mixture of organic contaminant. Applied Microbiology and Biotechnology, 3, Margein, R., Labbe, D., Schinner, F., Greer, C.W. and Whyte L.G. (3): Characterization of HydrocarbonDegrading Microbial population in contaminated and Pritine Alpine Soil. Applied and Environmental Microbiology, 9, Monod, J. (199): The growth of bacterial culture. Annual Review Microbiology, 3, Solomon, B.O., Poten, C.; Harder, M.P.F.; Hecht, V. and Deckwer, W.D. (199): Energetic of P. Cepacia G Growth in a chemotat with phenol limitation. Journal of Chemical Technology and Biotechnology,, 75. Stelmack, P.L., Gray, M.R. and Pickard, M.A. (1999): Bacterial adheion to oil contaminant in the preence of urfactant. Applied and Environmental Microbiology, 5, 131. Van Hamme, J.D. and Ward, O.P. (1): Phyical and metabolic interaction of Peudomona pecie train JA5B5 and Rhodococu pecie train F9 D79 during growth on crude oil and effect of a chemical urfactant on them. Applied and Environmental Microbiology, 7(), 779. Whyte, L.G., Bourbonniere, L., Belleroe, C., Greer, C.W. and Nahir, M. (1997): Biodegradation of petroleum hydrocarbon by pychrotrophic Peudomonatrain poeing both alkane (UK) and naphthalene (nap) catabolic pathway. Applied Environmental Microbiology, 3, Zhang, G., Qian XinPnig, W. and Meng, Q. (5): Biodegradation of crude oil by Peudomona aeruginoa in the preence of rhamnolipid. Journal of Zhejiang Univerity of Science, B(): Olanipekun, O.O., Ogunbayo, A. O. and Layokun, S. K.( 1) Etimation of bioma Energetic Yield and Maintenance Energy of growth of P.aeruginoa and P. fluorencen on dieel oil. Int. J. Re. Chem. Environ. (1), 9. Pirt, S.J. (195): The maintenance energy of bacteria in growing culture. Proce Review Society, London. Serie B, 13, 31. Plohl, K., Lekovek, H. and Briceji, M. (): Biological Degradation of Motor Oil in Water. Act Chim Slovenia, 9, 799. Reifeld, A., Roenberg, E. and Gutnick, D. (197): Microbial degradation of crude oil: factor affecting the diperion in ea water by mixed and pure culture. Applied Microbiology,, 333. Samanta, S.K., Singh, O.V. and Jain, R.K. (): Polycyclic aromatic hydrocarbon: Environmental pollution and bioremediation. Trend Biotechnol,, Solomon, B.O., Layokun, S.K., Fatile, I.A. and Agho, G.N. (195): Analyi of the growth of Trichoporon cutaneum on glucoe. Yield and maintenance requirement. Journal of Chemical Technology and Biotechnology, 35B: 7. 3