Impacts of n-alkane concentration on soil bacterial community structure and alkane monooxygenase genes abundance during bioremediation processes

  • Yueqiao Liu
  • Aizhong DingEmail author
  • Yujiao Sun
  • Xuefeng Xia
  • Dayi ZhangEmail author
Research Article
Part of the following topical collections:
  1. Special Issue—Application of Environmental Genomics in Bioremediation of Persistent Pollutants


Petroleum hydrocarbons, mainly consisting of n-alkanes and polycyclic aromatic hydrocarbons (PAHs), are considered as priority pollutants and biohazards in the environment, eventually affecting the ecosystem and human health. Though many previous studies have investigated the change of bacterial community and alkane degraders during the degradation of petroleum hydrocarbons, there is still lack of understanding on the impacts of soil alkane contamination level. In the present study, microcosms with different n-alkane contamination (1%, 3% and 5%) were set up and our results indicated a complete alkane degradation after 30 and 50 days in 1%- and 3%-alkane treatments, respectively. In all the treatments, alkanes with medium-chain length (C11-C14) were preferentially degraded by soil microbes, followed by C27-alkane in 3% and 5% treatments. Alkane contamination level slightly altered soil bacterial community, and the main change was the presence and abundance of dominant alkane degraders. Thermogemmatisporaceae, Gemmataceae and Thermodesulfovibrionaceae were highly related to the degradation of C14- and C27-alkanes in 5% treatment, but linked to alkanes with medium-chain (C11-C18) in 1% treatment and C21-alkane in 3% treatment, respectively. Additionally, we compared the abundance of three alkane-monooxygenase genes, e.g., alk_A, alk_P and alk_R. The abundance of alk_R gene was highest in soils, and alk_P gene was more correlated with alkane degradation efficiency, especially in 5% treatment. Our results suggested that alkane contamination level showed non-negligible effects on soil bacterial communities to some extents, and particularly shaped alkane degraders and degrading genes significantly. This study provides a better understanding on the response of alkane degraders and bacterial communities to soil alkane concentrations, which affects their biodegradation process.


Petroleum hydrocarbon contaminated site n-alkane contamination level n-alkane biodegradation Soil bacterial community Alkane degraders Alkane-monooxygenase genes 



This work was supported by the National Scientific Foundation of China (No. 41672227).

Supplementary material

11783_2018_1064_MOESM1_ESM.pdf (16 kb)
Supplementary Material


  1. Acer O, Guven K, Bekler F M, Gul-Guven R (2016). Isolation and characterization of long-chain alkane-degrading Acinetobacter sp. BT1A from oil-contaminated soil in Diyarbakir, in the Southeast of Turkey. Bioremediation Journal, 20(1): 80–87Google Scholar
  2. Alonso-Gutiérrez J, Figueras A, Albaigés J, Jiménez N, Viñas M, Solanas A M, Novoa B (2009). Bacterial communities from shoreline environments (costa da morte, northwestern Spain) affected by the prestige oil spill. Applied and Environmental Microbiology, 75(11): 3407–3418Google Scholar
  3. Amouric A, Quéméneur M, Grossi V, Liebgott P P, Auria R, Casalot L (2010). Identification of different alkane hydroxylase systems in Rhodococcus ruber strain SP2B, an hexane-degrading actinomycete. Journal of Applied Microbiology, 108(6): 1903–1916Google Scholar
  4. Baek K H, Yoon B D, Cho D H, Kim B H, Oh H M, Kim H S (2009). Monitoring bacterial population dynamics using real-time PCR during the bioremediation of crude-oil-contaminated soil. Journal of Microbiology and Biotechnology, 19(4): 339–345Google Scholar
  5. Bamforth S M, Singleton I (2005). Bioremediation of polycyclic aromatic hydrocarbons: Current knowledge and future directions. Journal of Chemical Technology and Biotechnology (Oxford, Oxfordshire), 80(7): 723–736Google Scholar
  6. Binazadeh M, Karimi I A, Li Z (2009). Fast biodegradation of long chain n-alkanes and crude oil at high concentrations with Rhodococcus sp. Moj-3449. Enzyme and Microbial Technology, 45(3): 195–202Google Scholar
  7. Callaghan AV, Davidova I A, Savage-Ashlock K, Parisi VA, Gieg L M, Suflita J M, Kukor J J, Wawrik B (2010). Diversity of benzyl- and alkylsuccinate synthase genes in hydrocarbon-impacted environments and enrichment cultures. Environmental Science & Technology, 44(19): 7287–7294Google Scholar
  8. Campeão M E, Reis L, Leomil L, de Oliveira L, Otsuki K, Gardinali P, Pelz O, Valle R, Thompson F L, Thompson C C (2017). The deep-sea microbial community from the Amazonian Basin associated with oil degradation. Frontiers in Microbiology, 8: 1019Google Scholar
  9. Carls M G, Thedinga J F (2010). Exposure of pink salmon embryos to dissolved polynuclear aromatic hydrocarbons delays development, prolonging vulnerability to mechanical damage. Marine Environmental Research, 69(5): 318–325Google Scholar
  10. Chandra S, Sharma R, Singh K, Sharma A (2013). Application of bioremediation technology in the environment contaminated with petroleum hydrocarbon. Annals of Microbiology, 63(2): 417–431Google Scholar
  11. Cheng L, Shi S, Li Q, Chen J, Zhang H, Lu Y (2014). Progressive degradation of crude oil n-alkanes coupled to methane production under mesophilic and thermophilic conditions. PLoS One, 9(11): e113253Google Scholar
  12. Coleman N V, Yau S, Wilson N L, Nolan L M, Migocki M D, Ly M A, Crossett B, Holmes A J (2011). Untangling the multiple monooxygenases of Mycobacterium chubuense strain NBB4, a versatile hydrocarbon degrader. Environmental Microbiology Reports, 3(3): 297–307Google Scholar
  13. Das N, Chandran P (2011). Microbial degradation of petroleum hydrocarbon contaminants: An overview. Biotechnology Research International, 2011: 941810Google Scholar
  14. Deng S, Ke T, Li L, Cai S, Zhou Y, Liu Y, Guo L, Chen L, Zhang D (2018). Impacts of environmental factors on the whole microbial communities in the rhizosphere of a metal-tolerant plant: Elsholtzia haichowensis Sun. Environmental Pollution, 237: 1088–1097Google Scholar
  15. Ehrlich H L, Newman D K, Kappler A (2015) Ehrlich’s Geomicrobiology. Boca Raton: CRC PressGoogle Scholar
  16. Ekperusi O A, Aigbodion, F I (2015) Bioremediation of petroleum hydrocarbons from crude oil-contaminated soil with the earthworm: Hyperiodrilus africanus. 3 Biotech, 5(6), 957–965Google Scholar
  17. Elumalai P, Parthipan P, Karthikeyan O P, Rajasekar A (2017) Enzymemediated biodegradation of long-chain n-alkanes (C32 and C40) by thermophilic bacteria. 3 Biotech, 7, 116–126Google Scholar
  18. Feng K, Zhang Z, Cai W, Liu W, Xu M, Yin H, Wang A, He Z, Deng Y (2017). Biodiversity and species competition regulate the resilience of microbial biofilm community. Molecular Ecology, 26(21): 6170–6182Google Scholar
  19. Feng L, Wang W, Cheng J, Ren Y, Zhao G, Gao C, Tang Y, Liu X, Han W, Peng X, Liu R, Wang L (2007). Genome and proteome of longchain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir. Proceedings of the National Academy of Sciences of the United States of America, 104 (13): 5602–5607Google Scholar
  20. Genovese M, Crisafi F, Denaro R, Cappello S, Russo D, Calogero R, Santisi S, Catalfamo M, Modica A, Smedile F, Genovese L, Golyshin P N, Giuliano L, Yakimov M M (2014). Effective bioremediation strategy for rapid in situ cleanup of anoxic marine sediments in mesocosm oil spill simulation. Frontiers in Microbiology, 5: 162Google Scholar
  21. Haritash A K, Kaushik C P (2009). Biodegradation aspects of polycyclic aromatic hydrocarbons (PAHs): A review. Journal of Hazardous Materials, 169(1-3): 1–15Google Scholar
  22. Hasanuzzaman M, Ueno A, Ito H, Ito Y, Yamamoto Y, Yumoto I, Okuyama H (2007). Degradation of long-chain n-alkanes (C-36 and C-40) by Pseudomonas aeruginosa strain WatG. International Biodeterioration & Biodegradation, 59(1): 40–43Google Scholar
  23. Hasinger M, Scherr K E, Lundaa T, Bräuer L, Zach C, Loibner A P (2012). Changes in iso- and n-alkane distribution during biodegradation of crude oil under nitrate and sulphate reducing conditions. Journal of Biotechnology, 157(4): 490–498Google Scholar
  24. Hassanshahian M, Ahmadinejad M, Tebyanian H, Kariminik A (2013). Isolation and characterization of alkane degrading bacteria from petroleum reservoir waste water in Iran (Kerman and Tehran provenances). Marine Pollution Bulletin, 73(1): 300–305Google Scholar
  25. Hassanshahian M, Zeynalipour M S, Musa F H (2014). Isolation and characterization of crude oil degrading bacteria from the Persian Gulf (Khorramshahr provenance). Marine Pollution Bulletin, 82(1-2): 39–44Google Scholar
  26. Herlemann D P R, Labrenz M, Jürgens K, Bertilsson S, Waniek J J, Andersson A F (2011). Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea. ISME Journal, 5(10): 1571–1579Google Scholar
  27. Jia J L, Zong S, Hu L, Shi S H, Zhai X B,Wang B B, Li G H, Zhang D Y (2017). The dynamic change of microbial communities in crude oilcontaminated soils from oil fields in China. Soil & Sediment Contamination, 26(2): 171–183Google Scholar
  28. Jiang B, Li G, Xing Y, Zhang D, Jia J, Cui Z, Luan X, Tang H (2017). A whole-cell bioreporter assay for quantitative genotoxicity evaluation of environmental samples. Chemosphere, 184: 384–392Google Scholar
  29. Jiménez N, Viñas M, Bayona J M, Albaiges J, Solanas A M (2007). The Prestige oil spill: Bacterial community dynamics during a field biostimulation assay. Applied Microbiology and Biotechnology, 77 (4): 935–945Google Scholar
  30. Jiménez N, Viñas M, Guiu-Aragonés C, Bayona J M, Albaigés J, Solanas A M (2011). Polyphasic approach for assessing changes in an autochthonous marine bacterial community in the presence of Prestige fuel oil and its biodegradation potential. Applied Microbiology and Biotechnology, 91(3): 823–834Google Scholar
  31. Jurelevicius D, Alvarez V M, Peixoto R, Rosado A S, Seldin L (2013). The use of a combination of alkB primers to better characterize the distribution of alkane-degrading bacteria. PLoS One, 8(6): e66565Google Scholar
  32. Kilbane J J 2nd (2006). Microbial biocatalyst developments to upgrade fossil fuels. Current Opinion in Biotechnology, 17(3): 305–314Google Scholar
  33. King C E, King G M (2014). Description of Thermogemmatispora carboxidivorans sp. nov., a carbon-monoxide-oxidizing member of the class Ktedonobacteria isolated from a geothermally heated biofilm, and analysis of carbon monoxide oxidation by members of the class Ktedonobacteria. International Journal of Systematic and Evolutionary Microbiology, 64(Pt 4): 1244–1251Google Scholar
  34. Kulichevskaya I S, Ivanova A A, Baulina O I, RijpstraW I C, Sinninghe Damsté J S, Dedysh S N (2017). Fimbriiglobus ruber gen. nov., sp. nov., a Gemmata-like planctomycete from Sphagnum peat bog and the proposal of Gemmataceae fam. nov. International Journal of Systematic and Evolutionary Microbiology, 67(2): 218–224Google Scholar
  35. Li H, BoufadelMC (2010). Long-term persistence of oil from the Exxon Valdez spill in two-layer beaches. Nature Geoscience, 3(2): 96–99Google Scholar
  36. Liu C, Wang W, Wu Y, Zhou Z, Lai Q, Shao Z (2011). Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei B-5. Environmental Microbiology, 13(5): 1168–1178Google Scholar
  37. Liu Q, Tang J, Liu X, Song B, Zhen M, Ashbolt N J (2017). Response of microbial community and catabolic genes to simulated petroleum hydrocarbon spills in soils/sediments from different geographic locations. Journal of Applied Microbiology, 123(4): 875–885Google Scholar
  38. Liu X, Chen Y, Zhang X, Jiang X, Wu S, Shen J, Sun X, Li J, Lu L, Wang L (2015). Aerobic granulation strategy for bioaugmentation of a sequencing batch reactor (SBR) treating high strength pyridine wastewater. Journal of Hazardous Materials, 295: 153–160Google Scholar
  39. Lo Piccolo L, De Pasquale C, Fodale R, Puglia A M, Quatrini P (2011). Involvement of an alkane hydroxylase system of Gordonia sp. strain SoCg in degradation of solid n-alkanes. Applied and Environmental Microbiology, 77(4): 1204–1213Google Scholar
  40. Lu Z, Zeng F, Xue N, Li F (2012). Occurrence and distribution of polycyclic aromatic hydrocarbons in organo-mineral particles of alluvial sandy soil profiles at a petroleum-contaminated site. Science of the Total Environment, 433: 50–57Google Scholar
  41. Lueders T (2017). The ecology of anaerobic degraders of BTEX hydrocarbons in aquifers. FEMS Microbiology Ecology, 93(1): 1–13Google Scholar
  42. Magoc T, Salzberg S L (2011). FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics (Oxford, England), 27(21): 2957–2963Google Scholar
  43. Masy T, Demaneche S, Tromme O, Thonart P, Jacques P, Hiligsmann S, Vogel TM (2016). Hydrocarbon biostimulation and bioaugmentation in organic carbon and clay-rich soils. Soil Biology & Biochemistry, 99: 66–74Google Scholar
  44. Mehdi H, Giti E (2008). Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation. International Biodeterioration & Biodegradation, 62(2): 170–178Google Scholar
  45. Ni N, Song Y, Shi R, Liu Z, Bian Y, Wang F, Yang X, Gu C, Jiang X (2017). Biochar reduces the bioaccumulation of PAHs from soil to carrot (Daucus carota L.) in the rhizosphere: A mechanism study. Science of the Total Environment, 601-602: 1015–1023Google Scholar
  46. Pacwa-Plociniczak M, Plaza G A, Piotrowska-Seget Z (2016). Monitoring the changes in a bacterial community in petroleumpolluted soil bioaugmented with hydrocarbon-degrading strains. Applied Soil Ecology, 105: 76–85Google Scholar
  47. Pagé A P, Yergeau É, Greer C W (2015). Salix purpurea stimulates the expression of specific bacterial xenobiotic degradation genes in a soil contaminated with hydrocarbons. PLoS One, 10(7): e0132062Google Scholar
  48. Powell S M, Bowman J P, Ferguson S H, Snape I (2010). The importance of soil characteristics to the structure of alkane-degrading bacterial communities on sub-Antarctic Macquarie Island. Soil Biology & Biochemistry, 42(11): 2012–2021Google Scholar
  49. Powell S M, Ferguson S H, Bowman J P, Snape I (2006). Using real-time PCR to assess changes in the hydrocarbon-degrading microbial community in Antarctic soil during bioremediation. Microbial Ecology, 52(3): 523–532Google Scholar
  50. Ravin, N V, Rakitin A L, Ivanova A A, Beletsky AV, Kulichevskaya I S, Mardanov A V, Dedysh S N (2018) Genome analysis of Fimbriiglobus ruber SP5T, a planctomycete with confirmed chitinolytic capability. Applied and Environmental Microbiology, 84(7): e02645–17Google Scholar
  51. Ribeiro H, Mucha A P, Almeida C M R, Bordalo A A (2013). Bacterial community response to petroleum contamination and nutrient addition in sediments from a temperate salt marsh. Science of the Total Environment, 458-460: 568–576Google Scholar
  52. Sangwan P, Chen X, Hugenholtz P, Janssen P H (2004). Chthoniobacter flavus gen. nov., sp. nov., the first pure-culture representative of subdivision two, Spartobacteria classis nov., of the phylum Verrucomicrobia. Applied and Environmental Microbiology, 70 (10): 5875–5881Google Scholar
  53. Scherr K E, Lundaa T, Klose V, Bochmann G, Loibner A P (2012). Changes in bacterial communities from anaerobic digesters during petroleum hydrocarbon degradation. Journal of Biotechnology, 157 (4): 564–572Google Scholar
  54. Setti L, Lanzarini G, Pifferi P G, Spagna G (1993). Further research into the aerobic degradation of N-Alkanes in a heavy oil by a pure culture of a Pseudomonas sp. Chemosphere, 26(6): 1151–1157Google Scholar
  55. Silva E J, Rocha e Silva N M, Rufino R D, Luna J M, Silva R O, Sarubbo L A (2014). Characterization of a biosurfactant produced by Pseudomonas cepacia CCT6659 in the presence of industrial wastes and its application in the biodegradation of hydrophobic compounds in soil. Colloids and Surfaces. B, Biointerfaces, 117: 36–41Google Scholar
  56. Smits T H M, Witholt B, van Beilen J B (2003). Functional characterization of genes involved in alkane oxidation by Pseudomonas aeruginosa. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 84(3): 193–200Google Scholar
  57. Soman C, Li D,WanderMM, Kent A D (2017). Long-term fertilizer and crop-rotation treatments differentially affect soil bacterial community structure. Plant and Soil, 413(1–2): 145–159Google Scholar
  58. Sun Y, Lu S, Zhao X, Ding A, Wang L (2017). Long-term oil pollution and in situ microbial response of groundwater in Northwest China. Archives of Environmental Contamination and Toxicology, 72(4): 519–529Google Scholar
  59. Throne-Holst M, Markussen S, Winnberg A, Ellingsen T E, Kotlar H K, Zotchev S B (2006). Utilization of n-alkanes by a newly isolated strain of Acinetobacter venetianus: The role of two alkB-type alkane hydroxylases. Applied Microbiology and Biotechnology, 72(2): 353–360Google Scholar
  60. Tomasek A, Staley C, Wang P, Kaiser T, Lurndahl N, Kozarek J L, Hondzo M, Sadowsky M J (2017). Increased denitrification rates associated with shifts in prokaryotic community composition caused by varying hydrologic connectivity. Frontiers in Microbiology, 8: 2304Google Scholar
  61. Tourova T P, Sokolova D S, Semenova E M, Shumkova E S, Korshunova A V, Babich T L, Poltaraus A B, Nazina T N (2016). Detection of n-alkane biodegradation genes alkB and ladA in thermophilic hydrocarbon-oxidizing bacteria of the genera Aeribacillus and Geobacillus. Microbiology, 85(6): 693–707Google Scholar
  62. Trevathan-Tackett S M, Seymour J R, Nielsen D A, Macreadie P I, Jeffries T C, Sanderman J, Baldock J, Howes J M, Steven A D L, Ralph P J (2017). Sediment anoxia limits microbial-driven seagrass carbon remineralization under warming conditions. FEMS Microbiology Ecology, 93(6): fix033Google Scholar
  63. Vasileiadis S, Puglisi E, Arena M, Cappa F, Cocconcelli P S, Trevisan M (2012). Soil bacterial diversity screening using single 16S rRNA gene V regions coupled with multi-million read generating sequencing technologies. PLoS One, 7(8): e42671Google Scholar
  64. Wang X, Zhao X, Li H, Jia J, Liu Y, Ejenavi O, Ding A, Sun Y, Zhang D (2016). Separating and characterizing functional alkane degraders from crude-oil-contaminated sites via magnetic nanoparticlemediated isolation. Research in Microbiology, 167(9-10): 731–744Google Scholar
  65. Wang Y, Nie M, Wan Y, Tian X, Nie H, Zi J, Ma X (2017). Functional characterization of two alkane hydroxylases in a versatile Pseudomonas aeruginosa strain NY3. Annals of Microbiology, 67(7): 459–468Google Scholar
  66. Wentzel A, Ellingsen T E, Kotlar H K, Zotchev S B, Throne-Holst M (2007). Bacterial metabolism of long-chain n-alkanes. Applied Microbiology and Biotechnology, 76(6): 1209–1221Google Scholar
  67. Wu R R, Dang Z, Yi X Y, Yang C, Lu G N, Guo C L, Liu C Q (2011). The effects of nutrient amendment on biodegradation and cytochrome P450 activity of an n-alkane degrading strain of Burkholderia sp. GS3C. Journal of Hazardous Materials, 186(2-3): 978–983Google Scholar
  68. Zengler K, Heider J, Rossello-Mora R, Widdel F (1999). Phototrophic utilization of toluene under anoxic conditions by a new strain of Blastochloris sulfoviridis. Archives of Microbiology, 172(4): 204–212Google Scholar
  69. Zhang D, Berry J P, Zhu D, Wang Y, Chen Y, Jiang B, Huang S, Langford H, Li G, Davison P A, Xu J, Aries E, Huang W E (2015). Magnetic nanoparticle-mediated isolation of functional bacteria in a complex microbial community. ISME Journal, 9(3): 603–614Google Scholar
  70. Zhang D, Ding A, Cui S, Hu C, Thornton S F, Dou J, Sun Y, HuangWE (2013). Whole cell bioreporter application for rapid detection and evaluation of crude oil spill in seawater caused by Dalian oil tank explosion. Water Research, 47(3): 1191–1200Google Scholar
  71. Zhang S, Yao H, Lu Y, Yu X,Wang J, Sun S, Liu M, Li D, Li Y F, Zhang D (2017). Uptake and translocation of polycyclic aromatic hydrocarbons (PAHs) and heavy metals by maize from soil irrigated with wastewater. Scientific Reports, 7(1): 12165Google Scholar
  72. Zhao Q, Li R, Ji M, Ren Z J (2016). Organic content influences sediment microbial fuel cell performance and community structure. Bioresource Technology, 220: 549–556Google Scholar
  73. Zhu L, Wang Y, Jiang L, Lai L, Ding J, Liu N, Li J, Xiao N, Zheng Y, Rimmington G M (2015). Effects of residual hydrocarbons on the reed community after 10 years of oil extraction and the effectiveness of different biological indicators for the long-term risk assessments. Ecological Indicators, 48: 235–243Google Scholar

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© Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of Water SciencesBeijing Normal UniversityBeijingChina
  2. 2.School of EnvironmentTsinghua UniversityBeijingChina
  3. 3.Lancaster Environment CentreLancaster UniversityLancasterUK

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