Biodegradation of Petroleum Sludge by Methylobacterium sp. Strain ZASH

Main Article Content

Zakuan Azizi Shamsul Harumain
Mohd Azrul Naim Mohamad
Noor Faizul Hadry Nordin
Mohd Yunus Abd Shukor


A bacterium was isolated from sludge-contaminated soil in a petroleum refinery and tested for its ability to degrade aliphatic hydrocarbon compounds present in petroleum sludge. The isolate was grown on minimal salt media agar supplemented with 1% (w/v) petroleum sludge. The isolate was tentatively identified as Methylobacterium s p. s train ZASH based on the partial 16s rDNA molecular phylogeny. The bacterium grew optimally between the temperatures of 30°C and 35°C, pH 7 and 7.5, 0.5% and 1.5% (v/v) Tween 80 as the surfactant, and between 1% and 2% (w/v) peptone as the nitrogen source. The constants derived from the Haldane equation were ?max = 0.039 hr–1, Ks = 0.385% (w/v) total petroleum hydrocarbons (TPH) or 3,850 mg/L TPH, and Ki =1.12% (w/v) TPH or 11,200 mg/L. The maximum biodegradation rate exhibited by this strain was 19 mg/L/hr at an initial TPH concentration of 10,000 mg/L. Gas chromatography analysis revealed that after 15 days the strain was able to degrade all aliphatic n-alkanes investigated with different efficiencies. Shorter n-alkanes were generally degraded more rapidly than longer n-alkanes with 90% removal for C-12 compared to only 30% removal for C-36. The addition of sawdust did not improve bacterial degradation of petroleum hydrocarbons, but it assisted in the removal of remaining undegraded hydrocarbons through adsorption.

Article Details

How to Cite
Biodegradation of Petroleum Sludge by Methylobacterium sp. Strain ZASH. (2023). Tropical Life Sciences Research, 34(2), 197–222.
Original Article


Abarian M, Hassanshahian M and Esbah A. (2019). Degradation of phenol at high concentrations using immobilization of Pseudomonas putida P53 into sawdust entrapped in sodium-alginate beads. Water Science and Technology 79(7): 1387–1396.

AL-Doury M M I. (2019). Treatment of oily sludge produced from Baiji oil refineries using surfactants. Petroleum Science and Technology 37(6): 718–726. https://

Alhefeiti M A, Athamneh K, Vijayan R and Ashraf S S. (2021). Bioremediation of various aromatic and emerging pollutants by Bacillus cereus sp. isolated from petroleum sludge. Water Science and Technology 83(7): 1535–1547. https://

Ali N, Eliyas M, Al-Sarawi H and Radwan S S. (2011). Hydrocarbon-utilizing microorganisms naturally associated with sawdust. Chemosphere 83(9): 1268–1272.

Arif N M, Ahmad S A, Syed M A and Shukor M Y. (2013). Isolation and characterization of a phenol?degrading Rhodococcus sp. strain AQ5NOL 2 KCTC 11961BP. Journal of Basic Microbiology 53(1): 9–19. jobm.201100120

Behera I D, Basak G, Kumar R R, Sen R and Meikap B C. (2020). Treatment of petroleum refinery sludge by petroleum degrading bacterium Stenotrophomonas pavanii IRB19 as an efficient novel technology. Journal of Environmental Science and Health, Part A 56(2): 226–239. 924

Cecotti M, Coppotelli B M, Mora V C, Viera M and Morelli I S. (2018). Efficiency of surfactant-enhanced bioremediation of aged polycyclic aromatic hydrocarbon-contaminated soil: Link with bioavailability and the dynamics of the bacterial community. Science of the Total Environment 634: 224–234. https://doi. org/10.1016/j.scitotenv.2018.03.303

Chen L, Lei Z, Luo X, Wang D, Li L and Li A. (2019). Biological degradation and transformation characteristics of total petroleum hydrocarbons by oil degradation bacteria adsorbed on modified straw. ACS Omega 4(6): 10921– 10928.

Cheng T, Liang J, He J, Hu X, Ge Z and Liu J. (2017). A novel rhamnolipid-producing Pseudomonas aeruginosa ZS1 isolate derived from petroleum sludge suitable for bioremediation. AMB Express 7(1): 1–14. 017-0418-x

Devi M P, Reddy M V, Juwarkar A, Sarma P N and Mohan S R V. (2011). Effect of co?culture and nutrients supplementation on bioremediation of crude petroleum sludge. Clean–Soil, Air, Water 39(10): 900–907. clen.201000588

Effendi A J, Kamath R, McMillen S, Sihota N, Zuo E, Sra K, Kong D, Wisono T and Syakir J. (2017). Strategies for enhancing bioremediation for hydrocarbon-impacted soils. Paper presented at the SPE Asia Pacific Health, Safety, Security, Environment and Social Responsibility Conference, Kuala Lumpur, Malaysia, April 2017.

Felsenstein J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39(4): 783–791. tb00420.x

Habib S, Ahmad S A, Johari W L W, Abd Shukor M Y, Alias S A, Khalil K A and Yasid N A. (2018). Evaluation of conventional and response surface level optimisation of n-dodecane (n-C12) mineralisation by psychrotolerant strains isolated from pristine soil at Southern Victoria Island, Antarctica. Microbial Cell Factories 17(1): 1–21.

Hamidi Y, Ataei S A and Sarrafi A. (2021). A highly efficient method with low energy and water consumption in biodegradation of total petroleum hydrocarbons of oily sludge. Journal of Environmental Management 293: 112911. https://doi. org/10.1016/j.jenvman.2021.112911

Hamzah A, Phan C-W, Abu Bakar N F and Wong K-K. (2013). Biodegradation of crude oil by constructed bacterial consortia and the constituent single bacteria isolated from Malaysia. Bioremediation Journal 17(1): 1–10. .1080/10889868.2012.731447

Hamzah A, Zarin M A, Hamid A A, Omar O and Senafi S. (2012). Optimal physical and nutrient parameters for growth of Trichoderma virens UKMP-1M for heavy crude oil degradation. Sains Malaysiana 41(1): 71–79.

Helmy Q, Kardena E and Wisjnuprapto. (2009). Performance of petrofilic consortia and effect of surfactant tween 80 addition in the oil sludge removal process. Journal of Applied Sciences in Environmental Sanitation 4(3): 207–218.

Hong S-H, Kim J-Y and Cho K-S. (2010). Isolation and characterization of a diesel-degrading bacterium, Gordonia sp. SD8. Microbiology and Biotechnology Letters 38(3): 335–339.

Huang Y, Pan H, Wang Q, Ge Y, Liu W and Christie P. (2019). Enrichment of the soil microbial community in the bioremediation of a petroleum-contaminated soil amended with rice straw or sawdust. Chemosphere 224: 265–271. https://doi. org/10.1016/j.chemosphere.2019.02.148

Islam B. (2015). Petroleum sludge, its treatment and disposal: A review. International Journal of Chemical Sciences 13(4): 1584–1602.

Ismail A S, El-Sheshtawy H S and Khalil N M. (2019). Bioremediation process of oil spill using fatty-lignocellulose sawdust and its enhancement effect. Egyptian Journal of Petroleum 28(2): 205–211. ejpe.2019.03.002

Ji L, Fu X, Wang M, Xu C, Chen G, Song F, Guo S and Zhang Q. (2019). Enzyme cocktail containing NADH regeneration system for efficient bioremediation of oil sludge contamination. Chemosphere 233: 132–139. https://doi. org/10.1016/j.chemosphere.2019.05.253

Johnson O A and Affam A C. (2019). Petroleum sludge treatment and disposal: A review. Environmental Engineering Research 24(2): 191–201. https://doi. org/10.4491/eer.2018.134

Jukes T H and Cantor C R. (1969). Evolution of protein molecules. Mammalian Protein Metabolism 3: 21–132.

Kaczorek E, Pacholak A, Zdarta A and Smu?ek W. (2018). The impact of biosurfactants on microbial cell properties leading to hydrocarbon bioavailability increase. Colloids and Interfaces 2(3): 35.

Lee G L Y, Ahmad S A, Yasid N A, Zulkharnain A, Convey P, Johari W L W, Alias S A, Gonzalez-Rocha G and Shukor M Y. (2018). Biodegradation of phenol by cold-adapted bacteria from Antarctic soils. Polar Biology 41(3): 553–562.

Lin C, Cheruiyot N K, Hoang H-G, Le T-H, Tran H-T and Bui X-T. (2021). Benzophenone biodegradation and characterization of malodorous gas emissions during co-composting of food waste with sawdust and mature compost. Environmental Technology & Innovation 21: 101351.

Liu Y, Wan Y Y, Wang C, Ma Z, Liu X and Li S. (2020). Biodegradation of n-alkanes in crude oil by three identified bacterial strains. Fuel 275: 117897. https://doi. org/10.1016/j.fuel.2020.117897

Margush T and McMorris F R. (1981). Consensus n-trees. Bulletin of Mathematical Biology 43(2): 239–244.

Mishra S, Jyot J, Kuhad R C and Lal B. (2001). In situ bioremediation potential of an oily sludge-degrading bacterial consortium. Current Microbiology 43(5): 328–335.

Muliadi F N A, Halmi M I E, Wahid S B A, Gani S S A, Zaidan U H, Mahmud K and Abd Shukor M Y. (2021). Biostimulation of microbial communities from Malaysian agricultural soil for detoxification of metanil yellow dye: A response surface methodological approach. Sustainability 13(1): 138. su13010138

Nikitina E V, Yakusheva O I, Zaripov S A, Galiev R A, Garusov A V and Naumova R P. (2003). Distribution and physiological state of microorganisms in petrochemical oily sludge. Microbiology 72(5): 621–627. https://doi. org/10.1023/A:1026063805092

Obayori O S, Ilori M O, Adebusoye S A, Oyetibo G O, Omotayo A E and Amund O O. (2009). Degradation of hydrocarbons and biosurfactant production by Pseudomonas sp. strain LP1. World Journal of Microbiology and Biotechnology 25(9): 1615–1623.

Ossai I C, Ahmed A, Hassan A and Hamid F S. (2020). Remediation of soil and water contaminated with petroleum hydrocarbon: A review. Environmental Technology & Innovation 17: 100526.

Page R D M. (1996). Tree View: An application to display phylogenetic trees on personal computers. Bioinformatics 12(4): 357–358. bioinformatics/12.4.357

Pugazhendi A, Abbad Wazin H, Qari H, Basahi J M A-B, Godon J J and Dhavamani J. (2017). Biodegradation of low and high molecular weight hydrocarbons in petroleum refinery wastewater by a thermophilic bacterial consortium. Environmental Technology 38(19): 2381–2391. 3330.2016.1262460

Rahman K S M, Rahman T J, Banat I M, Lord R and Street G. (2007). Bioremediation of petroleum sludge using bacterial consortium with biosurfactant. In: S N Singh and R D Tripathi (eds.). Environmental bioremediation technologies. New York: Springer, 391–408.

Roleda M Y and Hurd C L. (2019). Seaweed nutrient physiology: Application of concepts to aquaculture and bioremediation. Phycologia 58(5): 552–562. https://doi. org/10.1080/00318884.2019.1622920

Sabullah M K, Rahman M F, Ahmad S A, Sulaiman M R, Shukor M S, Shamaan N A and Shukor M Y. (2017). Assessing resistance and bioremediation ability of Enterobacter sp. strain saw-1 on molybdenum in various heavy metals and pesticides. Journal of Mathematical and Fundamental Sciences 49(2): 193– 210.

Saeedi M, Li L Y and Grace J R. (2019). Simultaneous removal of polycyclic aromatic hydrocarbons and heavy metals from natural soil by combined non-ionic surfactants and EDTA as extracting reagents: Laboratory column tests. Journal of Environmental Management 248: 109258. jenvman.2019.07.029

Saitou N and Nei M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4(4): 406–425.

Sarkar J, Kazy S K, Gupta A, Dutta A, Mohapatra B, Roy A, Bera P, Mitra A and Sar P. (2016). Biostimulation of indigenous microbial community for bioremediation of petroleum refinery sludge. Frontiers in Microbiology 7: 1407. https://doi. org/10.3389/fmicb.2016.01407

Shaban S M and Kim D-H. (2020). The influence of the Gemini surfactants hydrocarbon tail on in-situ synthesis of silver nanoparticles: Characterization, surface studies and biological performance. Korean Journal of Chemical Engineering 37: 1008–1019.

Shivanand P, Matussi N B A B and Lim L H. (2021). Microbial surfactants: Current perspectives and role in bioremediation. In: V Kumar (ed.), Rhizomicrobiome Dynamics in Bioremediation. Boca Raton, FL: CRC Press, 420–434. https://

Shukor M Y, Rahman M F, Shamaan N A and Syed M A. (2009). Reduction of molybdate to molybdenum blue by Enterobacter sp. strain Dr. Y13. Journal of Basic Microbiology 49(S1): S43–S54.

Shuler M L and Kargi F. (1992). Bioprocess engineering (1st Ed.). Upper Saddle River, NJ: Prentice-Hall, 412–420.

Somtrakoon K and Chouychai W. (2019). Effect of Triton X-100 and Tween 80 on removal of polycyclic aromatic hydrocarbons and possibility of cadmium accumulation by Siam weed (Chromolaena odorata). Songklanakarin Journal of Science & Technology 41(6): 1413–1420.

Srivastva N, Vishwakarma P, Bhardwaj Y, Singh A, Manjunath K and Dubey S K. (2017). Kinetic and molecular analyses reveal isoprene degradation potential of Methylobacterium sp. Bioresource Technology 242: 87–91. https://doi. org/10.1016/j.biortech.2017.02.002

Suganthi S H, Murshid S, Sriram S and Ramani K. (2018). Enhanced biodegradation of hydrocarbons in petroleum tank bottom oil sludge and characterization of biocatalysts and biosurfactants. Journal of Environmental Management 220: 87–95.

Tanee F B G and Jude K. (2017). Effect of detergent and sawdust addition on hydrocarbon reduction and growth of Abelmoschus esculentus L. (Okra) in a petroleum-contaminated soil. Nigerian Journal of Biotechnology 33: 24–33.

Thompson J D, Higgins D G and Gibson T J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22(22): 4673–4680.

Wang D, Lin J, Lin J, Wang W and Li S. (2019). Biodegradation of petroleum hydrocarbons by Bacillus subtilis BL-27, a strain with weak hydrophobicity. Molecules 24(17): 3021.

Wang G, Yin Q, Shen J, Bai Y, Ma X, Du Z and Wang W. (2017). Surface activities and aggregation behaviors of cationic-anionic fluorocarbon-hydrocarbon surfactants in dilute solutions. Journal of Molecular Liquids 234: 142–148.

Wang Y, Zhang X, Pan Y and Chen Y. (2017). Analysis of oil content in drying petroleum sludge of tank bottom. International Journal of Hydrogen Energy 42(29): 18681–18684.

Wongsa P, Tanaka M, Ueno A, Hasanuzzaman M, Yumoto I and Okuyama H. (2004). Isolation and characterization of novel strains of Pseudomonas aeruginosa and Serratia marcescens possessing high efficiency to degrade gasoline, kerosene, diesel oil, and lubricating oil. Current Microbiology 49(6): 415–422.

Xia M, Fu D, Chakraborty R, Singh R P and Terry N. (2019). Enhanced crude oil depletion by constructed bacterial consortium comprising bioemulsifier producer and petroleum hydrocarbon degraders. Bioresource Technology 282: 456–463.

Xu X, Liu W, Wang W, Tian S, Jiang P, Qi Q, Li F, Li H, Wang Q and Li H. (2019). Potential biodegradation of phenanthrene by isolated halotolerant bacterial strains from petroleum oil polluted soil in Yellow River Delta. Science of The Total Environment 664: 1030–1038.

Yan Z, Song N, Cai H, Tay J-H and Jiang H. (2012). Enhanced degradation of phenanthrene and pyrene in freshwater sediments by combined employment of sediment microbial fuel cell and amorphous ferric hydroxide. Journal of Hazardous Materials 199: 217–225. jhazmat.2011.10.087

Yang J, Zhang C-T, Yuan X-J, Zhang M, Mo X-H, Tan L-L, Zhu L-P, Chen W-J, Yao M-D and Hu B. (2018). Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor. Microbial Cell Factories 17(1): 1–15.

Yi T, Lee E-H, Park H and Cho K-S. (2011). Biodegradation of petroleum hydrocarbons by Neosartorya sp. BL4. Journal of Environmental Science and Health, Part A 46(14): 1763–1768.

Zhao G, Sheng Y, Wang C, Yang J, Wang Q and Chen L. (2018). In situ microbial remediation of crude oil-soaked marine sediments using zeolite carrier with a polymer coating. Marine Pollution Bulletin 129(1): 172–178. https://doi. org/10.1016/j.marpolbul.2018.02.030