The Succession of Dominant Culturable Hydrocarbon-Utilizing Bacteria During Bioremediation of Oil-Based Drilling Waste

Document Type : Research Article


Microbiology and Biotechnology Research Group, Research Institute of Petroleum Industry, Tehran, I.R. IRAN


Drilling operations of petroleum generate oily wastes. The disposal of a significant amount of oil-based drill muds has caused soil contamination and critical environmental impacts in the last decades.The current study aimed to investigate the potential of microbial remediation for an aged oil-based drilling waste and to monitor the fluctuation in microbial population throughout a 60-day microcosm experiment. A representative aged oil-based drilling waste sample was obtained randomly from a contaminated mud pit in the Khangiran district, Iran. Respiration measurement was performed according to the method described by standard ISO 17155. Total petroleum hydrocarbon (TPH) was measured by the gravimetric method. Microbial counts were measured at 10-day intervals during 60 days of incubation. Total heterotrophic bacteria were enumerated by standard plate count using R2A agar. Dominant heterotrophic and hydrocarbon-utilizing bacteria were selected for phylogenetic analysis. Statistical analyses of the experimental data, using one-way ANOVA were performed using Minitab 16. Following the biostimulation of the contaminated soil, both heterotrophic and hydrocarbon-utilizing bacterial counts increased to above three orders of magnitude in less than 20 days. The highest respiration level and hydrocarbon degradation efficiency were correlated and measured between the 10th and 20th days of the experiment to be 70.7 µg/g.soil.hand 23.13% respectively. Phylogenetic analyses indicated that the members of Actinobacteria (Georgenia, Brevibacterium, Micromonospora, and Streptomyces) were the major hydrocarbon-utilizing bacteria in the microcosm, among which the species of genus Georgenia were dominant throughout the experiments. Furthermore, the population of Alcanivorax species increased promptly and thrived in the microcosm during the active bioremediation phase which indicated their vital role for remediation of diesel range hydrocarbons in saline environments. In an overall view, elegant diversity of hydrocarbon utilizing bacteria along with the accomplished TPH removal efficiency of 45.4% (w/w) in the microcosms, confirmed the potential of indigenous microorganisms for bioremediation of the aged oil-based drilling waste.


Main Subjects

[1] Das N., Chandran P., Microbial Degradation of Petroleum Hydrocarbon Contaminants: an Overview, Biotechn. Res. Int., Article ID: 941810 (2011).
[2] Xie S., Jiang G., Chen M., Li Z., Huang X., Liang C., Jia X.P., Harmless Treatment Technology of Waste Oil-Based Drilling Fluids, Pet. Sci. Technol, 32(9): 1043-1049 (2014).
[4] Ji G., Yang Y., Zhou Q., Sun T., Ni J., Phytodegradation of Extra Heavy Oil-Based Drill Cuttings Using Mature Reed Wetland: an in Situ Pilot Study, Environ. Int.,30(4): 509-517 (2004).
[5] Yan P., Lu M., Guan Y., Zhang W., Zhang Z., Remediation of Oil-Based Drill Cuttings Through a Biosurfactant-Based Washing Followed by a Biodegradation Treatment, Bioresource. Technol., 102(22): 10252-10259 (2011).
[6] Balba M., Al-Awadhi N., Al-Daher R., Bioremediation of Oil-Contaminated Soil: Microbiological Methods for Feasibility Assessment and Field Evaluation, J. Microbiol. Methods., 32(2): 155-164 (1998).
[7] Rojas-Avelizapa N., Olvera-Barrera E., Fernández-Linares L., Feasibility Study of Bioremediation of a Drilling-Waste-Polluted Soil: Stimulation of Microbial Activities and Hydrocarbon Removal, J. Environ. Sci. Heal., Part A, 40(12): 2189-2201 (2005).
[8] Rojas-Avelizapa N., Roldan-Carrillo T., Arce-Ortega J., Ramirez-Islas M., Zegarra-Martinez H., Fernandez-Linares L., Enhancement of Hydrocarbon Removal in a Clay and Drilling-Waste Polluted Soil, Soil. Sediment. Contam., 15(4): 417-428 (2006).
[9] Rojas-Avelizapa N., Roldan-Carrillo T., Zegarra-Martinez H., Munoz-Colunga A., Fernandez-Linares L., A Field Trial for an Ex-Situ Bioremediation of a Drilling Mud-Polluted Site, Chemosphere, 66(9): 1595-1600 (2007).
[10] Ma J., Yang Y., Dai X., Chen Y., Deng H., Zhou H., Guo S., Yan G., Effects of Adding Bulking Agent, Inorganic Nutrient and Microbial Inocula on Biopile Treatment for Oil-Field Drilling Waste, Chemosphere, 150: 17-23 (2016).
[11] Ramírez M., Zapién B., Zegarra H., Rojas N., Fernandez L., Assessment of Hydrocarbon Biodegradability in Clayed and Weathered Polluted Soils, Int. Biodet. Biodegr., 63(3): 347-353 (2009).
[13] Steliga T., Jakubowicz P., Kapusta P., Changes in Toxicity During in Situ Bioremediation of Weathered Drill Wastes Contaminated with Petroleum Hydrocarbons, Bioresource. technol., 125: 1-10 (2012).
[14] Fan Y.Y., Wang G.C., Fu J.H., Zheng X.H., Bioremediation of Waste Drilling Fluid: Comparison of Biostimulation and Bioaugmentation, Desalin. Water. Treat.48(1-3): p. 329-334 2012.
[15] Okparanma R.N., A.J.M., Araka P.P., Bioremediation of Hydrocarbon Contaminated-Oil Field Drill-Cuttings ith Bacterial Isolates, Afr. J. Environ. Sci. Technol., 3(5): 131-140 (2009).
[17] Alavi N., Mesdaghinia A.R., Naddafi K., Mohebali G., Daraei H., Maleki A., Alaei L., Biodegradation of Petroleum Hydrocarbons in a Soil Polluted Sample by Oil-Based Drilling Cuttings. Soil. Sediment. Contam., 23(5): 586-597 (2014).
[18] Al-Razaq A.A.A., Al-Joubori M.H., AI-Hiti A.H., Biotreatment Technique to Treat Oil Wells Drilling Wastes. Iraqi. J. Chem. Petrol. Eng., 8(3): 37-41 (2007).
[19] Shaeyan M., Tirandaz H., Ghanbarpour S., Seyedipour N., Shavandi M., Dastgheib S.M.M., Bioremediation of a Drilling Waste-Contaminated Soil; Biotreatability Assessment and Microcosm Optimization for Developing a Field-Scale Remediation Process. Iran. J. Biotechnol., 16(3):193-199 (2018).
[20] Fan Y-Y., Wang G-C., Fu J-H., Zheng X-H., The Remediation of Waste Drilling Muds by a Combined Plant-microbe System. Petrol. Sci. Technol., 32(17):  2086-2092 (2014).
[21] Fiúza A.M., Vila M.C.C., An Insight into Soil Bioremediation Through Respirometry, Environ. Int., 31(2): 179-183 (2005).
[22] Plaza G., Ulfig K., Worsztynowicz A., Malina G., Krzeminska B., Brigmon R., Respirometry for Assessing the Biodegradation of Petroleum Hydrocarbons, Environ. Technol., 26(2): 161-170 (2005).
[23] Aspray T., Carvalho D., Philp J., Application of Soil Slurry Respirometry to Optimise and Subsequently Monitor ex Situ Bioremediation of Hydrocarbon-Contaminated Soils, Int. Biodeter. Biodegr., 60(4): 279-284 (2007).
[24] Nnubia C., Okpokwasili G., The Microbiology of Drill Mud Cuttings from a New Off-Shore Oilfield in Nigeria, Environ. Pollut., 82(2): 153-156 (1993).
[25] Benka-Coker M., Olumagin A., Waste Drilling-Fluid-Utilising Microorganisms in a Tropical Mangrove Swamp Oilfield Location, Bioresource. Technol., 53(3): 211-215 (1995).
[26] ChaIneau C.-H., Morel J.-L., Oudot J., Microbial Degradation in Soil Microcosms of Fuel Oil Hydrocarbons from Drilling Cuttings, Environ. Sci. Technol., 29(6): 1615-1621 (1995).
[27] Steliga T., Role of Fungi in Biodegradation of Petroleum Hydrocarbons in Drill Waste, Pol. J.  Environ. Stud., 21(2):471-479 (2012).
[28] Turner K.P., "Bioremediation of Drill Cuttings From Oil Based Muds", University of Nottingham (2002).
[29] Napp A.P., Pereira J.E.S., Oliveira J.S., Silva-Portela R.C.B., Agnez-Lima L.F., Peralba M.C.R., Bento F.M., Passaglia L.M.P., Thompson C.E., Vainstein M.H., "Comparative Metagenomics Reveals Different Hydrocarbon Degradative Abilities from Enriched Oil-Drilling Waste", Chemosphere, 209: 7-16 (2018).
[30] Kaplan C.W., Kitts C.L., Bacterial Succession in a Petroleum Land Treatment Unit, Appl. Environ. Microb., 70(3): 1777-1786 (2004).
[31] Militon C., Boucher D., Vachelard C., Perchet G., Barra V., Troquet J., Peyretaillade E., Peyret P., Bacterial Community Changes During Bioremediation of Aliphatic Hydrocarbon-Contaminated Soil, FEMS. Microbiol. Ecol., 74(3): 669-681 (2010).
[32] Vinas M., Sabaté J., Espuny M.J., Solanas A.M., Bacterial Community Dynamics and Polycyclic Aromatic Hydrocarbon Degradation During Bioremediation of Heavily Creosote-Contaminated Soil, Appl. Environ. Microb, 71(11): 7008-7018 (2005).
[33] Federation W.E., "Association A.P.H., Standard Methods for the Examination of Water and Wastewater", American Public Health Association (APHA): Washington, DC, USA, (2005).
[34] ASTM, "ASTM D2887, Standard Test Method for Boiling Range Distribution of Petroleum Fractions by Gas Chromatography”, ASTM International West Conshohocken, PA (2008).
[35] Margesin R., Schinner F., "Manual for Soil Analysis-Monitoring and Assessing Soil Bioremediation",  Vol. 5. Springer Science & Business Media (2005).
[36] Blodgett R., "Bacteriological Analytical Manual Appendix 2, Most Probable Number from Serial DilutionsUS Food and Drug Administration, Washington, DC, (2010).
[37] Wilson K., Preparation of Genomic DNA from Bacteria, Curr. Protoc. Mol. Biol., 56(1): 2.4.1-2.4.5 (2001).
[38] Kim O-S., Cho Y-J., Lee K., Yoon S-H., Kim M., Na H., Park S-C., Jeon Y-S., Lee J-H., Yi H., Won S., Chun J., Introducing EzTaxon-e: a Prokaryotic 16S rRNA Gene Sequence Database with Phylotypes that Represent Uncultured Species, Int. J. Syst. Evol. Micr., 62(3): 716-721 (2012).
[39] Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S., MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods, Mol. Biol. Evol., 28(10): 2731-2739 (2011).
[40] Timmis K.N., McGenity T., Van Der Meer J., De Lorenzo V., "Handbook of Hydrocarbon and Lipid Microbiology”, Springer Berlin (2010).
[41] Al-Awadhi H., Sulaiman R.H., Mahmoud H.M., Radwan S., Alkaliphilic and Halophilic Hydrocarbon-Utilizing Bacteria from Kuwaiti Coasts of the Arabian Gulf, Appl. Environ. Microb., 77(1): 183-186 (2007).
[42] Pizarro‐Tobías P., Niqui J.L., Roca A., Solano J., Fernández M., Bastida F., Garcia C., Ramos J.L., Field Trial on Removal of Petroleum‐Hydrocarbon Pollutants Using a Microbial Consortium for Bioremediation and Rhizoremediation, Environ. Microbiol. Rep., 7(1): 85-94 (2015).
[43] Jiménez N., Viñas M., Guiu-Aragonés C., Bayona J.M., Albaigés J., Solanas A.M., Polyphasic Approach for Assessing Changes in an Autochthonous Marine Bacterial Community in the Presence of Prestige Fuel oil and Its Biodegradation Potential, Appl.  Environ. Microb., 91(3): 823-834 (2011).
[44] Wang L., Wang W., Lai Q., Shao Z., Gene Diversity of CYP153A and AlkB Alkane Hydroxylases in Oil‐Degrading Bacteria Isolated from the Atlantic Ocean, Environ. Microbiol., 12(5): 1230-1242 (2010).
[45] Cappello S., Denaro R., Genovese M., Giuliano L., Yakimov MM., Predominant Growth of Alcanivorax During Experiments on “Oil Spill Bioremediation” in Mesocosms, Microbiol. Res., 162(2): 185-190 (2007).
[46] Kasai Y., Kishira H., Sasaki T., Syutsubo K., Watanabe K., Harayama S., Predominant Growth of Alcanivorax Strains in Oil‐Contaminated and Nutrient‐Supplemented Sea Water, Environ. Microbiol., 4(3): 141-147 (2002).
[47] Kleinsteuber S., Riis V., Fetzer I., Harms H., Müller S., Population Dynamics Within a Microbial Consortium During Growth on Diesel Fuel in Saline Environments, Appl. Environ. Microb., 72(5): 3531-3542 (2006).
[48] Kostka J.E., Prakash O., Overholt W.A., Green S.J., Freyer G., Canion A., Delgardio J., Norton N., Hazen T.C., Huettel M., Hydrocarbon-Degrading Bacteria and the Bacterial Community Response in Gulf of Mexico Beach Sands Impacted by the Deepwater Horizon Oil Spill, Appl. Environ. Microb., 77(22): 7962-7974 (2011).
[49] Shibata A.K., Robert F.M., Shifts in Alkane-Degrading Bacteria Genotypes During Bioremediation of a Vegetated Coastal Soil, World. J. Microbiol. Biotechnol., 25(9): 1667-1675 (2009).
[50] Dastgheib S.M.M., Amoozegar M.A., Khajeh K., Shavandi M., Ventosa A., Biodegradation of Polycyclic Aromatic Hydrocarbons by a Halophilic Microbial Consortium, Appl. Microbil. Biotechnol., 95(3): 789-798 (2012).
[51] Ali N., Dashti N., Al-Mailem D., Eliyas M., Radwan S., Indigenous Soil Bacteria with the Combined Potential for Hydrocarbon Consumption and Heavy Metal Resistance, Environ. Sci. Pollut. R., 19(3): 812-820 (2012).
[52] Gojgic-Cvijovic G.D., Milic J.S., Solevic T.M., Beskoski V.P., Ilic M.V., Djokic L.S., Narancic T.M., Vrvic M.M., Biodegradation of Petroleum Sludge and Petroleum Polluted Soil by a Bacterial Consortium: A Laboratory Study, Biodegradation, 23(1): 1-14 (2012).