Applied Microbiology and Biotechnology

, Volume 99, Issue 13, pp 5683–5696 | Cite as

Biostimulation of petroleum-hydrocarbon-contaminated marine sediment with co-substrate: involved metabolic process and microbial community

  • Zhen Zhang
  • Irene M. C. LoEmail author
Environmental biotechnology


This study investigated the effect of acetate and methanol as co-substrates on anaerobic biodegradation of total petroleum hydrocarbons (TPHs, C10–C40) in marine sediment. The findings evidenced that the degradation of TPH can be enhanced by adding acetate or methanol. The addition of acetate was generally more favorable than the addition of methanol for the TPH degradation. Both sulfate reduction and methanogenesis occurred in the acetate-treated sediment. However, the depletion of SO4 2− inhibited sulfate reduction over the incubation period. Only methanogenesis was prevalent in the methanol-treated sediment within the whole incubation period. The degradation of TPH fractions with higher carbon number ranges (C31–C40) was speculated to be more favored under sulfate-reducing condition, while TPH fractions with lower carbon number ranges (C10–C20) were preferentially degraded under methanogenic condition. The 16S rRNA clone library–based analysis revealed that the addition of different co-substrates led to distinct structures of the microbial community. Clones related to sulfate-reducing Desulfobacterales were the most abundant in the sediment dosed with acetate. Clones related to Clostridiales predominated in the sediment dosed with methanol. Acetoclastic methanogens were found to be the predominant archaeal species in the sediment dosed with acetate, while both acetoclastic methanogens and hydrogenotrophic methanogens accounted for large proportions in the sediment dosed with methanol. The results obtained in this study will contribute to more comprehensive knowledge on the role of acetate and methanol as co-substrates in biostimulation of petroleum-hydrocarbon-contaminated marine sediment.


Biostimulation Co-substrate Metabolic process Microbial community Total petroleum hydrocarbons (TPHs) 



The authors wish to thank the General Research Fund of Hong Kong Research Grants Council for providing financial support for this research study. We also thank Dr. Ming-fei Shao (Assistant Professor of Harbin Institute of Technology Shenzhen Graduate School) for his help in molecular identification and analysis of microbial community structure in sediment.

Supplementary material

253_2015_6420_MOESM1_ESM.pdf (307 kb)
ESM 1 (PDF 307 kb)


  1. Acosta-Gonzalez A, Rossello-Mora R, Marques S (2013) Characterization of the anaerobic microbial community in oil-polluted subtidal sediments: aromatic biodegradation potential after the Prestige oil spill. Environ Microbiol 15:77–92CrossRefPubMedGoogle Scholar
  2. Aitken CM, Jones DM, Maguire MJ, Gray ND, Sherry A, Bowler BFJ, Ditchfield AK, Larter SR, Head IM (2013) Evidence that crude oil alkane activation proceeds by different mechanisms under sulfate-reducing and methanogenic conditions. Geochimica Cosmochimica Acta 109:162–174CrossRefGoogle Scholar
  3. Ambrosoli R, Petruzzelli L, Minati JL, Marsan FA (2005) Anaerobic PAH degradation in soil by a mixed bacterial consortium under denitrifying conditions. Chemosphere 60:1231–1236CrossRefPubMedGoogle Scholar
  4. Babin J, Kau, P, Chan, L (2003) In situ sediment treatment to control odours and enhance biological breakdown of organic matter in Shing Mun River, the Hong Kong special administration region. 2nd International Symposium on Contaminated Sediments. Quebec City, CanadaGoogle Scholar
  5. Beck M, Brumsack HJ (2012) Biogeochemical cycles in sediment and water column of the Wadden Sea: the example Spiekeroog Island in a regional context. Ocean Coast Manag 68:102–113CrossRefGoogle Scholar
  6. Beolchini F, Rocchetti L, Regoli F, Dell'Anno A (2010) Bioremediation of marine sediments contaminated by hydrocarbons: experimental analysis and kinetic modeling. J Hazard Mater 182:403–407CrossRefPubMedGoogle Scholar
  7. Boopathy R (2003) Anaerobic degradation of No. 2 diesel fuel in the wetland sediments of Barataria-Terrebonne estuary under various electron acceptor conditions. Bioresource Technol 86:171–175CrossRefGoogle Scholar
  8. Caldwell ME, Garrett RM, Prince RC, Suflita JM (1998) Anaerobic biodegradation of long-chain n-alkanes under sulfate-reducing conditions. Environ Sci Technol 32:2191–2195CrossRefGoogle Scholar
  9. CEN (2004)Characterization of waste—determination of hydrocarbon content in the range of C10 to C40 by gas chromatography, European Committee for Standardization. EN 14039: 2004Google Scholar
  10. Chang BV, Chang SW, Yuan SY (2003) Anaerobic degradation of polycyclic aromatic hydrocarbons in sludge. Adv Environ Res 7:623–628CrossRefGoogle Scholar
  11. Chang BV, Chang IT, Yuan SY (2008) Anaerobic degradation of phenanthrene and pyrene in mangrove sediment. Bull Environ Contam Toxicol 80:145–149CrossRefPubMedGoogle Scholar
  12. Coates JD, Woodward J, Allen J, Philp P, Lovley DR (1997) Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments. Appl Environ Microbiol 63:3589–3593PubMedCentralPubMedGoogle Scholar
  13. Dell'Anno A, Beolchini F, Gabellini M, Rocchetti L, Pusceddu A, Danovaro R (2009) Bioremediation of petroleum hydrocarbons in anoxic marine sediments: consequences on the speciation of heavy metals. Mar Pollut Bull 58:1808–1814CrossRefPubMedGoogle Scholar
  14. Dolfing J, Larter SR, Head IM (2008) Thermodynamic constraints on methanogenic crude oil biodegradation. Isme J 2:442–452CrossRefPubMedGoogle Scholar
  15. Elferink SJWHO, Luppens SBI, Marcelis CLM, Stams AJM (1998) Kinetics of acetate oxidation by two sulfate reducers isolated from anaerobic granular sludge. Appl Environ Microbiol 64:2301–2303PubMedCentralGoogle Scholar
  16. Genthner BRS, Townsend GT, Lantz SE, Mueller JG (1997) Persistence of polycyclic aromatic hydrocarbon components of creosote under anaerobic enrichment conditions. Arch Environ Contam Toxicol 32:99–105CrossRefGoogle Scholar
  17. Gerlach R, Steiof M, Zhang CL, Hughes JB (1999) Low aqueous solubility electron donors for the reduction of nitroaromatics in anaerobic sediments. J Contam Hydrol 36:91–104CrossRefGoogle Scholar
  18. Handley KM, Wrighton KC, Piceno YM, Andersen GL, DeSantis TZ, Williams KH, Wilkins MJ, N'Guessan AL, Peacock A, Bargar J, Long PE, Banfield JF (2012) High-density PhyloChip profiling of stimulated aquifer microbial communities reveals a complex response to acetate amendment. FEMS Microbiol Ecol 81:188–204CrossRefPubMedGoogle Scholar
  19. Hasinger M, Scherr KE, Lundaa T, Brauer L, Zach C, Loibner AP (2012) Changes in iso- and n-alkane distribution during biodegradation of crude oil under nitrate and sulphate reducing conditions. J Biotechnol 157:490–498CrossRefPubMedGoogle Scholar
  20. Higashioka Y, Kojima H, Fukui M (2011) Temperature-dependent differences in community structure of bacteria involved in degradation of petroleum hydrocarbons under sulfate-reducing conditions. J Appl Microbiol 110:314–322CrossRefPubMedGoogle Scholar
  21. Holmer M, Kristensen E (1994) Coexistence of sulfate reduction and methane production in an organic-rich sediment. Mar Ecol Prog Ser 107:177–184CrossRefGoogle Scholar
  22. Johnson K, Ghosh S (1998) Feasibility of anaerobic biodegradation of PAHs in dredged river sediments. Water Sci Technol 38:41–48CrossRefGoogle Scholar
  23. Jonker MTO, Brils JM, Sinke AJC, Murk AJ, Koelmans AA (2006) Weathering and toxicity of marine sediments contaminated with oils and polycyclic aromatic hydrocarbons. Environ Toxicol Chem 25:1345–1353CrossRefPubMedGoogle Scholar
  24. Kendall MM, Liu Y, Boone DR (2006) Butyrate- and propionate-degrading syntrophs from permanently cold marine sediments in Skan Bay, Alaska, and description of Algorimarina butyrica gen. nov., sp nov. Fems Microbiol Lett 262:107–114CrossRefPubMedGoogle Scholar
  25. Kim TJ, Lee EY, Kim YJ, Cho KS, Ryu HW (2003) Degradation of polyaromatic hydrocarbons by Burkholderia cepacia 2A-12. World J Microb Biot 19:411–417CrossRefGoogle Scholar
  26. Kleikemper J, Pelz O, Schroth MH, Zeyer J (2002a) Sulfate-reducing bacterial community response to carbon source amendments in contaminated aquifer microcosms. Fems Microbiol Ecol 42:109–118CrossRefPubMedGoogle Scholar
  27. Kleikemper J, Schroth MH, Sigler WV, Schmucki M, Bernasconi SM, Zeyer J (2002b) Activity and diversity of sulfate-reducing bacteria in a petroleum hydrocarbon-contaminated aquifer. Appl Environ Microb 68:1516–1523CrossRefGoogle Scholar
  28. Lee N, Nielsen PH, Andreasen KH, Juretschko S, Nielsen JL, Schleifer KH, Wagner M (1999) Combination of fluorescent in situ hybridization and microautoradiography—a new tool for structure-function analyses in microbial ecology. Appl Environ Microb 65:1289–1297Google Scholar
  29. Leloup J, Fossing H, Kohls K, Holmkvist L, Borowski C, Jorgensen BB (2009) Sulfate-reducing bacteria in marine sediment (Aarhus Bay, Denmark): abundance and diversity related to geochemical zonation. Environ Microbiol 11:1278–1291CrossRefPubMedGoogle Scholar
  30. Liamleam W, Annachhatre AP (2007) Electron donors for biological sulfate reduction. Biotechnol Adv 25:452–463CrossRefPubMedGoogle Scholar
  31. Llobet-Brossa E, Rossello-Mora R, Amann R (1998) Microbial community composition of Wadden Sea sediments as revealed by fluorescence in situ hybridization. Appl Environ Microbiol 64:2691–2696PubMedCentralPubMedGoogle Scholar
  32. Lovley DR, Baedecker MJ, Lonergan DJ, Cozzarelli IM, Phillips EJP, Siegel DI (1989) Oxidation of aromatic contaminants coupled to microbial iron reduction. Nature 339:297–300CrossRefGoogle Scholar
  33. Lu X (2001) Biodegradation of polycyclic aromatic hydrocarbons in marine sediment under anoxic conditions. Hong Kong, University of Hong Kong. Doctor of PhilosophyGoogle Scholar
  34. Lv XM, Shao MF, Li CI, Li J, Xia X, Liu DY (2014) Bacterial diversity and community structure of denitrifying phosphorus removal sludge in strict anaerobic/anoxic systems operated with different carbon sources. J Chem Technol Biotechnol 89:1842–1849. doi: 10.1002/jctb.4265 CrossRefGoogle Scholar
  35. Manconi I, Carucci A, Lens P (2007) Combined removal of sulfur compounds and nitrate by autotrophic denitrification in bioaugmented activated sludge system. Biotechnol Bioeng 98:551–560CrossRefPubMedGoogle Scholar
  36. Mille G, Asia L, Guiliano M, Malleret L, Doumenq P (2007) Hydrocarbons in coastal sediments from the Mediterranean sea (Gulf of Fos area, France). Mar Pollut Bull 54:566–575CrossRefPubMedGoogle Scholar
  37. Miralles G, Grossi V, Acquaviva M, Duran R, Bertrand JC, Cuny P (2007) Alkane biodegradation and dynamics of phylogenetic subgroups of sulfate-reducing bacteria in an anoxic coastal marine sediment artificially contaminated with oil. Chemosphere 68:1327–1334CrossRefPubMedGoogle Scholar
  38. Okolo JC (2005) Effects of soil treatments containing poultry manure on crude oil degradation in a sandy loam soil. Appl Ecol Environ Res 3:47–53CrossRefGoogle Scholar
  39. Oremland RS, Polcin S (1982) Methanogenesis and sulfate reduction—competitive and noncompetitive substrates in estuarine sediments. Appl Environ Microbiol 44:1270–1276PubMedCentralPubMedGoogle Scholar
  40. Oremland RS, Taylor BF (1978) Sulfate reduction and methanogenesis in marine-sediments. Geochim Cosmochim Acta 42:209–214CrossRefGoogle Scholar
  41. Perelo LW (2010) Review: In situ and bioremediation of organic pollutants in aquatic sediments. J Hazard Mater 177:81–89CrossRefPubMedGoogle Scholar
  42. Phelps CD, Kerkhof LJ, Young LY (1998) Molecular characterization of a sulfate-reducing consortium which mineralizes benzene. Fems Microbiol Ecol 27:269–279CrossRefGoogle Scholar
  43. Phillips RL, Whalen SC, Schlesinger WH (2001) Influence of atmospheric CO(2) enrichment on methane consumption in a temperate forest soil. Glob Chang Biol 7:557–563CrossRefGoogle Scholar
  44. Qian J, Lu H, Cui Y, Wei L, Liu R, Chen G (2015) Investigation on thiosulfate-involved organics and nitrogen removal by a sulfur cycle-based biological wastewater treatment process. Water Res 69:295–306CrossRefPubMedGoogle Scholar
  45. Rueter P, Rabus R, Wilkes H, Aeckersberg F, Rainey FA, Jannasch HW, Widdel F (1994) Anaerobic oxidation of hydrocarbons in crude-oil by new types of sulfate-reducing bacteria. Nature 372:455–458CrossRefPubMedGoogle Scholar
  46. Sanchez-Andrea I, Rodriguez N, Amils R, Sanz JL (2011) Microbial diversity in anaerobic sediments at Rio Tinto, a naturally acidic environment with a high heavy metal content. Appl Environ Microbiol 77:6085–6093PubMedCentralCrossRefPubMedGoogle Scholar
  47. Siddique T, Fedorak PM, Foght JM (2006) Biodegradation of short-chain n-alkanes in oil sands tailings under methanogenic conditions. Environ Sci Technol 40:5459–5464CrossRefPubMedGoogle Scholar
  48. Siddique T, Penner T, Semple K, Foght JM (2011) Anaerobic biodegradation of longer-chain n-alkanes coupled to methane production in oil sands tailings. Environ Sci Technol 45:5892–5899CrossRefPubMedGoogle Scholar
  49. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599CrossRefPubMedGoogle Scholar
  50. Taylor LT, Jones DM (2001) Bioremediation of coal tar PAH in soils using biodiesel. Chemosphere 44:1131–1136CrossRefPubMedGoogle Scholar
  51. USEPA (1991) Draft analytical method for determination of acid volatile sulfide in sediment. EPA 821/R-91/100. Washington, DC, United States Environmental Protection AgencyGoogle Scholar
  52. USEPA (2005) Contaminated sediment remediation guidance for hazardous waste sites. EPA 540/R-05/012. Washington D.C., United States Environmental Protection AgencyGoogle Scholar
  53. Wang J, Shi MY, Lu H, Wu D, Shao MF, Zhang T, Ekama GA, van Loosdrecht MCM, Chen GH (2011a) Microbial community of sulfate-reducing up-flow sludge bed in the SANI (R) process for saline sewage treatment. Appl Microbiol Biol 90:2015–2025CrossRefGoogle Scholar
  54. Wang LY, Gao CX, Mbadinga SM, Zhou L, Liu JF, Gu JD, Mu BZ (2011b) Characterization of an alkane-degrading methanogenic enrichment culture from production water of an oil reservoir after 274 days of incubation. Int Biodeter Biodegr 65:444–450CrossRefGoogle Scholar
  55. Weijma J, Stams AJM (2001) Methanol conversion in high-rate anaerobic reactors. Water Sci Technol 44:7–14PubMedGoogle Scholar
  56. Yuan SY, Chang JS, Yen JH, Chang BV (2001) Biodegradation of phenanthrene in river sediment. Chemosphere 43:273–278CrossRefPubMedGoogle Scholar
  57. Zengler K, Richnow HH, Rossello-Mora R, Michaelis W, Widdel F (1999) Methane formation from long-chain alkanes by anaerobic microorganisms. Nature 401:266–269CrossRefPubMedGoogle Scholar
  58. Zhang M, Zhang T, Shao MF, Fang HHP (2009) Autotrophic denitrification in nitrate-induced marine sediment remediation and Sulfurimonas denitrificans-like bacteria. Chemosphere 76:677–682CrossRefPubMedGoogle Scholar
  59. Zhou JZ, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Department of Civil and Environmental EngineeringThe Hong Kong University of Science and TechnologyHong KongChina

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