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Use of Taguchi design for optimization of diesel-oil biodegradation using consortium of Pseudomonas stutzeri, Cellulosimicrobium cellulans, Acinetobacter baumannii and Pseudomonas balearica isolated from tarball in Terengganu Beach, Malaysia


A consortium of bacteria capable of decomposing oily hydrocarbons was isolated from tarballs on the beaches of Terengganu, Malaysia, and classified as Pseudomonas stutzeri, Cellulosimicrobium cellulans, Acinetobacter baumannii and Pseudomonas balearica. The Taguchi design was used to optimize the biodegradation of diesel using these bacteria as a consortium. The highest biodegradation of diesel-oil in the experimental tests was 93.6%, and the individual n-alkanes decomposed 87.6—97.6% over 30 days. Optimal settings were inoculum size of 2.5 mL (1.248 OD600nm); 12% (v/v) the initial diesel-oil in a minimal salt medium of pH 7.0, 30.0 gL−1 NaCl and 2.0 gL−1 NH4NO3 concentration, incubated at 42 °C temperature and 150 rpm agitation speed. Parameters significantly improved diesel-oil removal by consortium as shown by the model determination coefficient (R2 = 90.89%; P < 0.001) with a synergistic effect of agitation speed significantly contributing 81.03%. Taguchi design determined the optimal settings for the parameters under study, which significantly improved diesel-oil removal by consortium. This can be used to design a novel bioremediation strategy that can achieve optimal decontamination of oil pollution in a shorter time.


  • Hydrocarbon-degraders in Tarball were isolated and identified by their 16S rRNA gene sequence as Pseudomonas stutzeri, Cellulosimicrobium cellulans, Acinetobacter baumannii and Pseudomonas balearica.

  • Taguchi method was applied to optimize effects of parameters such as initial diesel concentration, salinity (NaCl concentration), nitrate (NH4NO3) concentration, pH, temperature, agitation speed and inoculum size on diesel-oil removal in 30 days.

  • Maximum diesel-oil biodegradation by experimental runs was 93.6% with individual n-alkanes degraded between 87.6% – 97.6% in 30 days

  • Optimal settings were 2.5 mL (1.248 OD600nm) inoculum size; 12% (v/v) initial diesel-oil in MSM media with 7.0 pH, 30.0 gL−1 NaCl and 2.0 gL−1 NH4NO3 concentration, incubated at 42 °C temperature and 150 rpm agitation speed

  • Parameters significantly improved diesel-oil removal by this consortium as indicated by model determination coefficient (R2 = 90.89%; P < 0.001) with synergistic effect of agitation speed significantly contributing 81.03%

  • Taguchi design established optimal settings of investigated parameters that produced significant improvement on diesel-oil removal by consortium

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Data availability

We further confirm that datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.




16S rRNA:

16S ribosomal RNA

A. baumannii :

Acinetobacter baumannii

Adj. SS:

Adjusted sum of squares

Adj. MS:

Adjusted mean squares


Analysis of Variance


Basic Local Alignment System Tool


Degree Celsius

C. cellulans :

Cellulosimicrobium cellulans


Confidence interval






Degree of freedom


Deoxyribonucleic Acid


Deoxy nucleoside triphosphates




Gas Chromatography Mass Spectrometry

gL 1 :

Gram per litre




High performance liquid chromatography




Luria Bertani


Molecular Evolutionary Genetics Analysis






Minimal salt media


Sodium chloride


Nominal is best


National Center for Biotechnology Information


Nanogram per milligram

NH4NO3 :

Ammonium nitrate

OD600nm :

Optical density at 600 nm



P. balearica :

Pseudomonas balearica


Polymerase chain reaction


Parts per million

P. stutzeri :

Pseudomonas stutzeri


Revolutions per minute

SE Coefficients:

Standard error coefficients




Surrogate internal standard


Signal noise


Volume per volume


  1. Abatenh E, Gizaw B, Tsegaya Z, Wassie M. Application of microorganisms in bioremediation-review. J Environ Microbiol. 2017;1(1):2–9.

    Google Scholar 

  2. Abbasian F, Lockington R, Mallavarapu M, Naidu R. A comprehensive review of aliphatic hydrocarbon biodegradation by bacteria. Appl Biochem Biotechnol. 2015;176:670–99.

    CAS  Article  Google Scholar 

  3. Adzigbli L, Yuewen D. Assessing the impact of oil spills on marine organisms. J Oceanogr. 2018;3(6):179.

    Google Scholar 

  4. Agarry SE. Statistical optimization and kinetic studies of enhanced bioremediation of crude oil - contaminated marine water using combined adsorption-biostimulation strategy. J Appl Sci Environ Manag. 2017;21(1):59.

    CAS  Google Scholar 

  5. Aghamiri SF, Kabiri K, Emtiazi G. A novel approach for optimization of crude oil bioremediation in soil by the taguchi method. J Pet Environ Biotechnol. 2011;2(2):1–7.

    Article  CAS  Google Scholar 

  6. Al-Sayegh A, Al-Wahaibi Y, Joshi S, Al-Bahry S, Elshafie A, Al-Bemani A. Bioremediation of heavy crude oil contamination. Open Biotechnol J. 2016;10(1):301–11.

    CAS  Article  Google Scholar 

  7. Amer R, Abdel-Fattah YR. Hydrocarbonoclastic marine bacteria in Mediterranean Sea, El-Max, Egypt: isolation, identification and site characterization. Jokull J. 2014;64(4):42–58.

    Google Scholar 

  8. Armstrong JA, Schulz JR. Agarose Gel Electrophoresis. Current Protocols in Essential Laboratory Techniques 2015; 2015. 721-7222.

  9. Avanzi IR, Gracioso LH, Baltazar MDPG, Karolski B, Perpetuo EA, Nascimento CAO. Aerobic biodegradation of gasoline compounds by bacteria isolated from a hydrocarbon-contaminated soil. Environ Eng Sci. 2015;32(12):990–7.

    CAS  Article  Google Scholar 

  10. Ayed BH, Jemil N, Maalej H, Bayoudh A, Hmidet N, Nasri M. Enhancement of solubilization and biodegradation of diesel oil by biosurfactant from Bacillus amyloliquefaciens An6. Int Biodeterior Biodegradation. 2015;99:8–14.

    Article  CAS  Google Scholar 

  11. Azubuike CC, Chikere CB, Okpokwasili GC. Bioremediation techniques–classification based on site of application: principles advantages limitations and prospects. World J Microbiol Biotechnol. 2016;32(11):1–18.

    CAS  Article  Google Scholar 

  12. Bacosa HP, Thyng KM, Plunkett S, Erdner DL, Liu Z. The tarballs on Texas beaches following the 2014 Texas City “Y” Spill: Modeling, chemical, and microbiological studies. Mar Pollut Bull. 2016;109(1):236–44.

    CAS  Article  Google Scholar 

  13. Baharudzaman EH, Tuah PM. Distribution and abundance of stranded tarballs in Marintaman Beach, Sipitang, Sabah, Malaysia. Int J Adv Res Sci Eng Technol. 2018;5(3):5396–400.

    Google Scholar 

  14. Baird RB, Eaton AD, Rice EW. American Water Works Association and Water Environment Federation In Standard Methods for the Examination of Water and Wastewater. 23rd ed. Washington: American Public Health Association; 2017. 2017.

  15. Bellas J, Saco-Alvarez L, Nieto O, Bayona JM, Albaiges J, Beiras R. Evaluation of artificially-weathered standard fuel oil toxicity by marine invertebrate embryogenesis bioassays. Chemosphere. 2013;90(3):1103–8.

    CAS  Article  Google Scholar 

  16. Bennasar-Figueras A, Salvà-Serra F, Jaén-Luchoro D, Seguí C, Aliaga F, Busquets A, Lalucat J. Complete genome sequence of Pseudomonas balearica DSM 6083T. Genome Announc. 2016;4(2):1–2.

    Article  Google Scholar 

  17. Bhattacharya M, Biswas D, Guchhait S. Applied growth kinetic models for crude oil spill bioremediation in a batch scale bioreactor. J Environ Hazards. 2018;1(1):1–6.

    Google Scholar 

  18. Bio-Rad. PCR troubleshooting. Bio-Rad - Life Sci Res. 2016;2016:1–22.

    Google Scholar 

  19. Blackburn M, Mazzacano CAS, Fallon C, Black SH. Oil in our oceans: A review of the impacts of oil spills on marine invertebrates. Portland, Oregon. The Xerces Society for Invertebrate Conservation. 2014. 2014: 152.

  20. Borah D, Yadav RNS. Bioremediation of petroleum based contaminants with biosurfactant produced by a newly isolated petroleum oil degrading bacterial strain. Egypt J Pet. 2017;26(1):181–8.

    Article  Google Scholar 

  21. Borowik A, Wyszkowska J. Remediation of soil contaminated with diesel oil. J Elementol. 2018;23(2):767–88.

    Google Scholar 

  22. Brown LD, Cologgi DL, Gee KF, Ulrich AC Bioremediation of Oil Spills on Land. In Oil Spill Science and Technology. 2nd ed. 2017. 2017: 699–729.

  23. Brzeszcz J, Kaszycki P. Aerobic bacteria degrading both n-alkanes and aromatic hydrocarbons: an undervalued strategy for metabolic diversity and flexibility. Biodegradation. 2018;29:359–407.

    Article  Google Scholar 

  24. Cappuccino JG, Welsh C. Microbiology. A Laboratory Manual. Pearson Education Limited. 11th ed. 2017. 2017.

  25. Catania V, Cappello S, Di Giorgi V, Santisi S, Di Maria R, Mazzola A, Quatrini P. Microbial communities of polluted sub-surface marine sediments. Mar Pollut Bull. 2018;131:396–406.

    CAS  Article  Google Scholar 

  26. Challa S, Neelapu NRR. Phylogenetic Trees: Applications Construction and Assessment In K R Hakeem N A Shaik B Banaganapalli and R Elango (Eds). Essentials of Bioinformatics Volume III: In Silico Life Sciences: Agriculture. Cham: Springer International Publishing; 2019. 2019: 167–192.

  27. Chen Q, Li J, Liu M, Sun H, Bao M. Study on the biodegradation of crude oil by free and immobilized bacterial consortium in marine environment. PLoS One. 2017;12(3):1–23.

    Google Scholar 

  28. Coskun O. Separation techniques: chromatography. North Clin Istanb. 2016;3(2):156–60.

    Google Scholar 

  29. Costa JC, Oliveira JV, Alves MM. Response surface design to study the influence of inoculum particle size and inoculum to substrate ratio on the methane production from Ulex sp. Renew Energy. 2016;96(B):1071–7.

    CAS  Article  Google Scholar 

  30. Daghio M, Tatangelo V, Franzetti A, Gandolfi I, Papacchini M, Careghini A, Bestetti G. Hydrocarbon degrading microbial communities in bench scale aerobic biobarriers for gasoline contaminated groundwater treatment. Chemosphere. 2015;130:34–9.

    CAS  Article  Google Scholar 

  31. Dashtbozorg M, Riyahi Bakhtiari A, Shushizadeh MR, Taghavi L. Quantitative evaluation of n-alkanes PAHs and petroleum biomarker accumulation in beach-stranded tarballs and coastal surface sediments in the Bushehr Province Persian Gulf (Iran). Mar Pollut Bull. 2019;146:801–15.

    CAS  Article  Google Scholar 

  32. Dashti N, Ali N, Eliyas M, Khanafer M, Sorkhoh NA, Radwan SS. Most hydrocarbonoclastic bacteria in the total environment are diazotrophic which highlights their value in the bioremediation of hydrocarbon contaminants. Microbes Environ. 2015;30:70–5.

    Article  Google Scholar 

  33. Deng MCC, Li J, Liang FRR, Yi M, Xu XMM, Yuan JPP, Wang JHH. Isolation and characterization of a novel hydrocarbon-degrading bacterium Achromobacter sp HZ01 from the crude oil-contaminated seawater at the Daya Bay southern China. Mar Pollut Bull. 2014;83:79–86.

    CAS  Article  Google Scholar 

  34. Dong L, Lv LB, Lai R. Molecular Cloning A laboratory Manual. 4th ed. 2012. 33: 11–115.

  35. Dou TY, Luan HW, Ge GB, Dong MM, Zou HF, He YQ, Yang L. Functional and structural properties of a novel cellulosome-like multienzyme complex: efficient glycoside hydrolysis of water-insoluble 7-xylosyl-10-deacetylpaclitaxel. Sci Rep. 2015;5:13768.

    Article  Google Scholar 

  36. Drabik A, Bodzoń-Kułakowska A, Silberring J. Gel Electrophoresis. In Proteomic Profiling and Analytical Chemistry: The Crossroads. 2nd ed. 2016. 2016: 115–143.

  37. Environment Canada HC. Final Screening Assessment Report for Pseudomonas stutzeri. Pseudomonas Stutzeri ATCC 17587. 2015. 2015: 1–40.

  38. Eppendorf,. OD600 measurements using different photometers. White Paper. 2015;28:1–4.

    Google Scholar 

  39. Farag S, Soliman NA, Abdel-Fattah YR. Statistical optimization of crude oil bio-degradation by a local marine bacterium isolate Pseudomonas sp SP48. J Genet Eng Biotechnol. 2018;16(2):409–20.

    Article  Google Scholar 

  40. Ferreira TF, Coelho MAZ, da Rocha-Leão MHM. Factors influencing crude oil biodegradation by Yarrowia lipolytica. Braz Arch Biol Technol. 2012;55(5):785–91.

    Article  Google Scholar 

  41. Fingas MF. The basics of oil spill cleanup. 3rd ed. Boca Raton: CRC Press; 2013. 2013: 225.

  42. Fu Y, Cheng L, Meng Y, Li S, Zhao X, Du Y, Yin H. Cellulosimicrobium cellulans strain E4–5 enzymatic hydrolysis of curdlan for production of (1 → 3)-linked β-d-glucan oligosaccharides. Carbohyd Polym. 2015;134:740–4.

    CAS  Article  Google Scholar 

  43. Gao YC, Wang JN, Guo SH, Hu YL, Li TT, Mao R, Zeng DH. Effects of salinization and crude oil contamination on soil bacterial community structure in the Yellow River Delta region China. Appl Soil Ecol. 2015;86:165–73.

    Article  Google Scholar 

  44. Gopinath LR, Divya D, Geitha TR, Bhuvaneswari R, Archaya S, Merlin CP. Hydrocarbon degradation and biogas production efficiency of bacteria isolated from petrol polluted soil. Res J Recent Sci. 2015;4(9):60–7.

    CAS  Google Scholar 

  45. Hamzah A, Manikan V, Abd Aziz NAF. Biodegradation of tapis crude oil using consortium of bacteria and fungi: Optimization of crude oil concentration and duration of incubation by response surface methodology. Sains Malaysiana. 2017;46(1):43–50.

    CAS  Article  Google Scholar 

  46. Hassanshahian M, Abarian M, Cappello S. Biodegradation of aromatic compounds biodegradation and bioremediation of polluted systems. New Adv Technol. 2015;6:110–8.

    Google Scholar 

  47. Ibrahim M, Makky EA, Azmi NS, Ismail J. Optimization parameters for Mycobacteria confluentis biodegradation of PAHs. MATEC Web Conf. 2018;06035(150):1–5.

    Google Scholar 

  48. Ikner L, Schmitz B, Gerba C, Pepper I. Bacterial growth curve analysis and its environmental applications. JoVe. 2015;2015:1–11.

    Google Scholar 

  49. Imron MF, Titah HS. Optimization of diesel biodegradation by Vibrio alginolyticus using Box-Behnken design. Environ Eng Res. 2018;23(4):374–82.

    Article  Google Scholar 

  50. Islam B. Petroleum sludge its treatment and disposal: a review. Int J Chem Sci. 2015;13(4):1584–602.

    CAS  Google Scholar 

  51. Jiang Y, Qi H, Zhang X. Novel method for separation and screening of lubricant-degrading microorganisms and bacterial biodegradation. Chin J Chem Eng. 2016;24(3):353–9.

    CAS  Article  Google Scholar 

  52. Kaczorek E, Bielicka-Daszkiewicz K, Héberger K, Kemény S, Olszanowski A, Voelkel A. Best conditions for biodegradation of diesel oil by chemometric tools. Braz J Microbiol. 2014;45(1):117–26.

    Article  Google Scholar 

  53. Kamaruzzaman JA, Zain AM. Coral Bay shore zones tarball distribution. Procedia Eng. 2016;148:437–43.

    CAS  Article  Google Scholar 

  54. Kaur R, Kumari A, Kaur R. Microbial degradation of crude-petroleum-oil: factors and strategies affecting the bioremediation process. Pollut Res. 2018;37(4):1053–7.

    CAS  Google Scholar 

  55. Krasowska A, Sigler K. How microorganisms use hydrophobicity and what does this mean for human needs. Front Cell Infect Microbiol. 2014;4:112.

    Article  Google Scholar 

  56. Kumar V, Kumar M, Prasad R. Microbial degradation of hydrocarbons in the environment: an overview. Microbial Action Hydrocarbons. 2019;2019:353–86.

    Google Scholar 

  57. Lee PY, Costumbrado J, Hsu CY, Kim YH. Agarose gel electrophoresis for the separation of DNA fragments. J Vis Exp. 2012;62(e3923):1–5.

    Google Scholar 

  58. Lee CR, Lee JH, Park M, Park KS, Bae IK, Kim YB, Lee SH. Biology of Acinetobacter baumannii: Pathogenesis antibiotic resistance mechanisms and prospective treatment options. Front Cell Infect Microbiol. 2017;7(55):1–35.

    Google Scholar 

  59. Liu Z, Liu J. Evaluating bacterial community structures in oil collected from the sea surface and sediment in the northern Gulf of Mexico after the Deepwater Horizon oil spill. Microbiol Open. 2013;2(3):492–504.

    CAS  Article  Google Scholar 

  60. Liu L, Mishchenko MI. Modeling study of scattering and absorption properties of tar-ball aggregates. Appl Opt. 2019;58(31):8648–57.

    CAS  Article  Google Scholar 

  61. Liu S, Wang B, Wang Y. Identification of Diesel Residues by GC / MS / MS. International Conference on New Energy and Renewable Resources. 2017. 2: 341–345.

  62. Liu H, Xu J, Liang R, Liu J. Characterization of the medium- And long-chain n-alkanes degrading Pseudomonas aeruginosa strain SJTD-1 and its alkane hydroxylase genes. PLoS One. 2014;9(8):1–14.

    Google Scholar 

  63. Liu B, Ju M, Liu J, Wu W, Li X. Isolation identification and crude oil degradation characteristics of a high-temperature hydrocarbon-degrading strain. Mar Pollut Bull. 2016;106(1–2):301–7.

    CAS  Article  Google Scholar 

  64. McGenity TJ, Timmis KN, Nogales FB. Hydrocarbon and Lipid Microbiology Protocols. Activities and Phenotypes. Springer Protocols Handbooks; 2017;2017: 7-67.

  65. Messaoudene NA. Taguchi design of experiments. Analysis. 2010;2010:71–6.

    Google Scholar 

  66. Mishra PR, Gupta EN, Joshi D. Prediction of moulding sand properties using multiple regression methodology. J Adv Comput Commun Technol. 2016;4(1):1–4.

    Google Scholar 

  67. Mukherjee AK, Bhagowati P, Biswa BB, Chanda A, Kalita B. A comparative intracellular proteomic profiling of Pseudomonas aeruginosa strain ASP-53 grown on pyrene or glucose as sole source of carbon and identification of some key enzymes of pyrene biodegradation pathway. J Proteomics. 2017;167:25–35.

    CAS  Article  Google Scholar 

  68. Muthukamalam S, Sivagangavathi S, Dhrishya D, Sudha Rani S. Characterization of dioxygenases and biosurfactants produced by crude oil degrading soil bacteria. Braz J Microbiol. 2017;48(4):637–47.

    CAS  Article  Google Scholar 

  69. Mwaura AN. Screening and characterization of hydrocarbonoclastic bacteria isolated from oil-contaminated soils from auto garages. Int J Microbiol Biotechnol. 2018;3(1):1–11.

    Article  Google Scholar 

  70. Nazifa TH, Ahmad MAB, Hadibarata T, Salmiati S, Aris A. Bioremediation of diesel oil spill by filamentous fungus Trichoderma reesei H002 in aquatic environment. Int J Integrated Eng. 2018;10(9):14–9.

    Google Scholar 

  71. Nkem BM, Halimoon N, Yusoff FM, Johari WLW, Zakaria MP, Medipally SR, Kannan N. Isolation identification and diesel-oil biodegradation capacities of indigenous hydrocarbon-degrading strains of Cellulosimicrobium cellulans and Acinetobacter baumannii from tarball at Terengganu beach Malaysia. Mar Pollut Bull. 2016;107(1):261–8.

    CAS  Article  Google Scholar 

  72. Nkem BM, Halimoon N, Yusoff FM, Johari WLW. Isolation and optimization of diesel-oil biodegradation using Cellulosimicrobium cellulans from tarball. Pertanika J Sci Technol. 2019;27(3):1031–40.

    Google Scholar 

  73. Notowidjaja MSI, Ekawati Y, Noya S. Taguchi experimental design to optimize the sugar content of candied carrot. IOP Conf Ser Mater Sci Eng. 2019;528: 012068.

    CAS  Article  Google Scholar 

  74. Omotoyinbo O. Effect of Varying NaCl Concentrations on the Growth Curve of Escherichia coli and Staphylococcus aureus. Cell Biol. 2016;4(5):31–4.

    CAS  Google Scholar 

  75. Palanisamy N, Ramya J, Kumar S, Vasanthi NS, Chandran P, Khan S. Diesel biodegradation capacities of indigenous bacterial species isolated from diesel contaminated soil. J Environ Health Sci Eng. 2014;12(1):1–8.

    Article  CAS  Google Scholar 

  76. Pandey P, Pathak H, Dave S. Biodegradation of diesel oil by Pseudomonas balearica strain UKMS3P3 isolated from soil around mathura refinery. Indian J Environ Prot. 2018;38(6):467–76.

    Google Scholar 

  77. Paniagua-Michel J, Fathepure BZ. Microbial consortia and biodegradation of petroleum hydrocarbons in marine environments. Microbial Action Hydrocarbons. 2019;2019:1–20.

    Google Scholar 

  78. Parthipan P, Elumalai P, Sathishkumar K, Sabarinathan D, Murugan K, Benelli G, Rajasekar A. Biosurfactant and enzyme mediated crude oil degradation by Pseudomonas stutzeri NA3 and Acinetobacter baumannii MN3. 3 Biotech. 2017;7(5):1–1.

    Article  Google Scholar 

  79. Payne JR, Phillips CR. Tarball Formation and Distribution. Petroleum Spills in the Marine Environment Boca Raton CRC Press. 1st ed. 2018. 2018: 83–97.

  80. Pereira MR, Mercaldi GF, Maester TC, Balan A, De Macedo Lemos EG. Est16 a new esterase isolated from a metagenomic library of a microbial consortium specializing in diesel oil degradation. PLoS One. 2015;10(7): e0133723.

    Article  CAS  Google Scholar 

  81. Pereira TM, Merçon J, Passos LS, Coppo GC, Lopes TOM, Cabral DS, Chippari-Gomes AR. Effects of the water-soluble fraction of diesel oil (WSD) on the fertilization and development of a sea urchin (Echinometra lucunter). Ecotoxicol Environ Saf. 2018;162:59–62.

    CAS  Article  Google Scholar 

  82. Polmear R, Stark JS, Roberts D, McMinn A. The effects of oil pollution on Antarctic benthic diatom communities over 5 years. Mar Pollut Bull. 2015;90(1–2):33–40.

    CAS  Article  Google Scholar 

  83. Pundir R, Chary GHVC, Dastidar MG. Application of Taguchi method for optimizing the process parameters for the removal of copper and nickel by growing Aspergillus sp. Water Resour Ind. 2018;20:83–92.

    Article  Google Scholar 

  84. Qin W, Fan F, Zhu Y, Huang X, Ding A, Liu X, Dou J. Anaerobic biodegradation of benzo(a)pyrene by a novel Cellulosimicrobium cellulans CWS2 isolated from polycyclic aromatic hydrocarbon-contaminated soil. Braz J Microbiol. 2018;49(2):258–68.

    CAS  Article  Google Scholar 

  85. Rajabnasab M, Khavari-Nejad RA, Shokravi S, Nejadsattari T. Investigating the physiological responses of three endaphic strains of Cyanobacteria to crude oil concentrations in limited salinity and irradiation conditions. Appl Ecol Environ Res. 2018;16(4):4559–73.

    Article  Google Scholar 

  86. Ramasamy S, Arumugam A, Chandran P. Optimization of Enterobacter cloacae (KU923381) for diesel oil degradation using response surface methodology. J Microbiol. 2017;55(2):104–11.

    CAS  Article  Google Scholar 

  87. Rao S, Samant P, Kadampatta A, Shenoy R. An Overview of Taguchi Method: Evolution Concept and Interdisciplinary Applications. Int J Sci Eng Res. 2013;4(10):621–6.

    Google Scholar 

  88. Reddy CM, Arey JS, Seewald JS, Sean PS, Lemkau KL, Nelson RK, Carmichael CA, McIntyre CP, Fenwick J, Ventura GT, Van Mooy BAS, Camilli R. Composition and fate of gas and oil released to the water column during the Deepwater Horizon oil spill. Proc Natl Acad Sci USA. 2012;109:20229–34.

    CAS  Article  Google Scholar 

  89. Rengarajan T, Rajendran P, Nandakumar N, Lokeshkumar B, Rajendran P, Nishigaki I. Exposure to polycyclic aromatic hydrocarbons with special focus on cancer. Asian Pac J Trop Biomed. 2015;5(3):182–9.

    CAS  Article  Google Scholar 

  90. Roslee AFA, Zakaria NN, Convey P, Zulkharnain A, Lee GLY, Gomez-Fuentes C, Ahmad SA. Statistical optimisation of growth conditions and diesel degradation by the Antarctic bacterium Rhodococcus sp strain AQ5-07. Extremophiles. 2019;24(2):277–91.

    Article  CAS  Google Scholar 

  91. Roy A, Dutta A, Pal S, Gupta A, Sarkar J, Chatterjee A, Kazy SK. Biostimulation and bioaugmentation of native microbial community accelerated bioremediation of oil refinery sludge. Biores Technol. 2018;253:22–32.

    CAS  Article  Google Scholar 

  92. Sadeghi HZM, Ebrahimipour G, Shahriari MM, Fakhari J, Abdoli T. Bioremediation of crude oil using bacterium from the coastal sediments of Kish Island Iran. Iran J Public Health. 2016;45(5):670–9.

    Google Scholar 

  93. Sawadogo A, Otoidobiga HC, Nitiema LW, Traoré AS, Dianou D. Optimization of hydrocarbons biodegradation by bacterial strains isolated from wastewaters in ouagadougou burkina faso: case study of SAE 40/50 Used Oils and Diesel. J Agric Chem Environ. 2016;5(1):1–11.

    CAS  Google Scholar 

  94. Shaieb FM, Elghazawani AH, Issa A. Studies on crude oil degrading Bacteria isolated from Libyan desert. Int J Curr Microbiol App Sci. 2015;4(2):920–7.

    CAS  Google Scholar 

  95. Shine H, Samant LR, Tulaskar V, Vartak D. Isolation of potent hydrocarbon degrading microorganisms and its application in bioremediation. Int J Curr Pharm Res. 2017;9(3):65.

    CAS  Article  Google Scholar 

  96. Shirneshan G, Bakhtiari AR, Memariani M. Identification of sources of tarballs deposited along the Southwest Caspian Coast, Iran using fingerprinting techniques. Sci Total Environ. 2016;568:979–89.

    CAS  Article  Google Scholar 

  97. Singh P, Parmar D, Pandya A. Parametric optimization of media for the crude oil degrading bacteria isolated from crude oil contaminated site. Int J Curr Microbiol App Sci. 2015;4(2):322–8.

    CAS  Google Scholar 

  98. Sivagamasundari T, Jayakumar N. Optimization of diesel oil degrading bacterial strains at various culture parameters. Int J Res Dev Pharm Life Sci. 2017;6(6):2840–4.

    CAS  Google Scholar 

  99. Stagars MH, Emil Ruff S, Amann R, Knittel K. High diversity of anaerobic alkane-degrading microbial communities in marine seep sediments based on (1-methylalkyl) succinate synthase genes. Front Microbiol. 2016;6(1511):1–17.

    Google Scholar 

  100. Stauffer E. Gas Chromatography-Mass Spectrometry. Encyclopedia of Forensic Sciences Elsevier Inc. 2nd ed. 2013;2013: 596–602.

  101. Suneel V, Vethamony P, Naik BG, Krishna MS, Jadhav L. Identifying the source of tarballs deposited along the beaches of Goa in 2013 and comparing with historical data collected along the West Coast of India. Sci Total Environ. 2015;528:313–21.

    Article  CAS  Google Scholar 

  102. Tan YH, Chiang EW, Chan HYL, Ling S, Hii, Kit K, Woo. Behavioral properties of Acinetobacter species in degrading oil. Malays J Biochem Mol Biol. 2018;21(3):72–6.

    Google Scholar 

  103. Tanzadeh J, Ghasemi MF. the Use of Microorganisms in Bioremediation of Oil Spills in Sea Waters and Shoreline. Chem Biol Interface. 2016;5(6):282–9.

    Google Scholar 

  104. Tripathi NK, Shrivastava A. Scale up of biopharmaceuticals production. Nanoscale Fabrication Optimization Scale-up and Biological Aspects of Pharmaceutical Nanotechnology. 2017. 2017: 133–172.

  105. Uba BO, Chukwura EI, Okoye EL, Ubani O, Chude CO, Akabueze UC. In vitro degradation and reduction of aromatic hydrocarbons by marine bacteria isolated from contaminated marine environments of Niger delta. Adv Res. 2019;18(3):1–17.

    Article  Google Scholar 

  106. Umar ZD, Nor Azwady AA, Zulkifli SZ, Muskhazli M. Effective phenanthrene and pyrene biodegradation using Enterobacter sp MM087 (KT933254) isolated from used engine oil contaminated soil. Egypt J Pet. 2018;27(3):349–59.

    Article  Google Scholar 

  107. Urakawa H, Rajan S, Feeney ME, Sobecky PA, Mortazavi B. Ecological response of nitrification to oil spills and its impact on the nitrogen cycle. Environ Microbiol. 2019;21(1):18–33.

    CAS  Article  Google Scholar 

  108. Valdor PF, Gómez AG, Puente A. Environmental risk analysis of oil handling facilities in port areas application to Tarragona harbor (NE Spain). Mar Pollut Bull. 2015;90:78–87.

    CAS  Article  Google Scholar 

  109. Varjani SJ, Gnansounou E, Pandey A. Comprehensive review on toxicity of persistent organic pollutants from petroleum refinery waste and their degradation by microorganisms. Chemosphere. 2017;188:280–91.

    CAS  Article  Google Scholar 

  110. Vulin C, Leimer N, Huemer M, Ackermann M, Zinkernagel AS. Prolonged bacterial lag time results in small colony variants that represent a sub-population of persisters. Nat Commun. 2018;9:1–7.

    CAS  Article  Google Scholar 

  111. Wood JL, Osman A, Wade SA. An efficient cost-effective method for determining the growth rate of sulfate-reducing bacteria using spectrophotometry. Methods X. 2019;6:2248–57.

    CAS  Google Scholar 

  112. Woodman ME, Savage CR, Arnold WK, Stevenson B. Direct PCR of intact bacteria (colony PCR). Curr Protocols Microbiol. 2016;2016:A3D1-A3D7.

    Google Scholar 

  113. Wu Y, Zeng J, Zhu Q, Zhang Z, Lin X. PH is the primary determinant of the bacterial community structure in agricultural soils impacted by polycyclic aromatic hydrocarbon pollution. Sci Rep. 2017;7(40093):1–7.

    Google Scholar 

  114. Yetti E, A’La A, Mercuriani IS, Yopi Y. Potency of microbial consortium F14 from three selected bacterial strains (Labrenzia agregata LBF-1-0016 Pseudomonas balearica LBF 1-0062 and Lysobacter concretionis LBF-1-0080) for oil degradation. AIP Conference Proceedings. 2018; 2024(1): 020043.

  115. Zakaria MP, Bong CW, Vaezzadeh V. Fingerprinting of petroleum hydrocarbons in Malaysia using environmental forensic techniques: a 20-year field data review. Oil Spill Environ Forensics Case Stud. 2018;2018:345–72.

    Article  Google Scholar 

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This study was sponsored by Geran Putra IPB (UPM Reference Code: UPM/700-2/1/GP-IPB/2013/9412400) and Geran Putra IPB (UPM Reference Code: UPM/700-2/1/GP/2018/9592200) awarded by Universiti Putra Malaysia (UPM). We also acknowledge the contributions of International Institute of Aquaculture and Aquatic Sciences/Department of Aquaculture, Universiti Putra Malaysia for laboratory equipment provided for conducting experiments.


Research on this publication was funded by: Geran Putra IPB (UPM Reference Code: UPM/700–2/1/GP-IPB/2013/9412400).

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Correspondence to Normala Halimoon.

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Nkem, B.M., Halimoon, N., Yusoff, F.M. et al. Use of Taguchi design for optimization of diesel-oil biodegradation using consortium of Pseudomonas stutzeri, Cellulosimicrobium cellulans, Acinetobacter baumannii and Pseudomonas balearica isolated from tarball in Terengganu Beach, Malaysia. J Environ Health Sci Engineer (2022).

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  • Bacteria consortium
  • Biodegradation
  • Diesel-oil
  • Optimization
  • Taguchi design
  • Tarball