Advertisement

Measuring the Impact of Hydrocarbons on Rates of Nitrogen Fixation

  • Florin MusatEmail author
  • Niculina Musat
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Crude oils consist of complex mixtures of hydrocarbons, chemicals which are composed exclusively of carbon and hydrogen atoms. With a very low nitrogen content, crude oil contamination through natural seepage or anthropogenic activities leads to an overload of the affected environment with organic carbon. Degradation of oil hydrocarbons by microorganisms is therefore limited by the availability of fixed nitrogen. Studies of bioremediation of crude oil spills have shown that addition of nitrogen fertilizers or nitrogen salts led to an enhancement of crude oil degradation. Fixed nitrogen in crude oil-contaminated environments could also be provided by N2 fixation, a process carried out by diverse groups of chemoautotrophic, heterotrophic, and phototrophic microorganisms. N2-fixing heterotrophic bacteria have been isolated from environments contaminated with crude oil. Some of these bacteria were able to fix N2 while growing with hydrocarbons as sole substrates. Microcosm studies showed that crude oil alters the N2-fixing microbial populations or offers a substratum for the growth of N2-fixing phototrophic microorganisms. Also, crude oil can indirectly stimulate the growth of N2-fixing microorganisms by reducing the grazing pressure. In this chapter we describe methods to measure N2 fixation rates using sediment or water microcosms. We present methods for preparation of microcosms with addition of crude oil. We describe two methods to measure the bulk N2 fixation by the entire microcosm: the acetylene reduction assay and the analysis of bulk N isotope ratios following incubations with 15N2. Also, we present a method to determine N2 fixation at cellular level, based on nanoSIMS analysis of individual cells from microcosms incubated with 15N2.

Keywords:

15N assimilation 15N isotope labeling Acetylene reduction assay Crude oil NanoSIMS Nitrogen fixation Single cell 

References

  1. 1.
    Tissot BP, Welte DH (1984) Petroleum formation and occurrence, 2nd edn. Springer, BerlinCrossRefGoogle Scholar
  2. 2.
    Leahy JG, Colwell RR (1990) Microbial degradation of hydrocarbons in the environment. Microbiol Rev 54(3):305–315PubMedPubMedCentralGoogle Scholar
  3. 3.
    Widdel F, Musat F (2010) Energetic and other quantitative aspects of microbial hydrocarbon utilization. In: Timmis KN (ed) Handbook of hydrocarbon and lipid microbiology. Springer, Heidelberg/Berlin pp 731–763Google Scholar
  4. 4.
    Atlas RM (1981) Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiol Rev 45(1):180–209PubMedPubMedCentralGoogle Scholar
  5. 5.
    Swannell RP, Lee K, Mcdonagh M (1996) Field evaluations of marine oil spill bioremediation. Microbiol Rev 60(2):342–365PubMedPubMedCentralGoogle Scholar
  6. 6.
    Bragg JR, Prince RC, Harner EJ, Atlas RM (1994) Effectiveness of bioremediation for the Exxon Valdez oil spill. Nature 368:413–418CrossRefGoogle Scholar
  7. 7.
    Röling WFM, Milner MG, Jones DM et al (2002) Robust hydrocarbon degradation and dynamics of bacterial communities during nutrient-enhanced oil spill bioremediation. Appl Environ Microbiol 68(11):5537–5548CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Medina-Bellver JI, Marin P, Delgado A et al (2005) Evidence for in situ crude oil biodegradation after the Prestige oil spill. Environ Microbiol 7(6):773–779CrossRefPubMedGoogle Scholar
  9. 9.
    Zehr JP, Waterbury JB, Turner PJ et al (2001) Unicellular cyanobacteria fix N2 in the subtropical North Pacific Ocean. Nature 412(6847):635–638CrossRefPubMedGoogle Scholar
  10. 10.
    Zehr JP, Jenkins BD, Short SM, Steward GF (2003) Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 5(7):539–554CrossRefPubMedGoogle Scholar
  11. 11.
    Fay P (1992) Oxygen relations of nitrogen fixation in cyanobacteria. Microbiol Rev 56(2):340–373PubMedPubMedCentralGoogle Scholar
  12. 12.
    Ploug H, Musat N, Adam B et al (2010) Carbon and nitrogen fluxes associated with the cyanobacterium Aphanizomenon sp. in the Baltic Sea. ISME J 4(9):1215–1223CrossRefPubMedGoogle Scholar
  13. 13.
    Musat F, Harder J, Widdel F (2006) Study of nitrogen fixation in microbial communities of oil-contaminated marine sediment microcosms. Environ Microbiol 8(10):1834–1843CrossRefPubMedGoogle Scholar
  14. 14.
    Musat F, Wieland A, Widdel F (2004) Marine sediment with surface contamination by oil in microcosms for microbiological studies. Ophelia 58(3):217–222CrossRefGoogle Scholar
  15. 15.
    Harper HJ (1939) The effect of natural gas on the growth of micro-organisms and the accumulation of nitrogen and organic matter in the soil. Soil Sci 48(6):461–466CrossRefGoogle Scholar
  16. 16.
    Schollenberger CJ (1930) Effect of leaking natural gas upon the soil. Soil Sci 29(4):261–266CrossRefGoogle Scholar
  17. 17.
    Toccalino PL, Johnson RL, Boone DR (1993) Nitrogen limitation and nitrogen fixation during alkane biodegradation in a sandy soil. Appl Environ Microbiol 59(9):2977–2983PubMedPubMedCentralGoogle Scholar
  18. 18.
    Knowles R, Wishart C (1977) Nitrogen fixation in arctic marine sediments: effect of oil and hydrocarbon fractions. Environ Pollut 13(2):133–149CrossRefGoogle Scholar
  19. 19.
    Thomson A, Webb K (1984) The effect of chronic oil pollution on salt-marsh nitrogen fixation (acetylene reduction). Estuaries 7(1):2–11CrossRefGoogle Scholar
  20. 20.
    Chronopoulou P-M, Fahy A, Coulon F et al (2013) Impact of a simulated oil spill on benthic phototrophs and nitrogen-fixing bacteria in mudflat mesocosms. Environ Microbiol 15(1):242–252CrossRefPubMedGoogle Scholar
  21. 21.
    Taketani RG, Dos Santos HF, Van Elsas JD, Rosado AS (2009) Characterisation of the effect of a simulated hydrocarbon spill on diazotrophs in mangrove sediment mesocosm. Antonie Van Leeuwenhoek 96(3):343–354CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Davis JB, Coty VF, Stanley JP (1964) Atmospheric nitrogen fixation by methane-oxidizing bacteria. J Bacteriol 88:468–472PubMedPubMedCentralGoogle Scholar
  23. 23.
    Coty VF (1967) Atmospheric nitrogen fixation by hydrocarbon-oxidizing bacteria. Biotechnol Bioeng 9(1):25–32CrossRefGoogle Scholar
  24. 24.
    Roy I, Shukla S, Mishra A (1988) n-Dodecane as a substrate for nitrogen fixation by an alkane-utilizing Azospirillum sp. Curr Microbiol 16(6):303–309CrossRefGoogle Scholar
  25. 25.
    Chen Y, Lopez-De-Victoria G, Lovell C (1993) Utilization of aromatic compounds as carbon and energy sources during growth and N2-fixation by free-living nitrogen fixing bacteria. Arch Microbiol 159(3):207–212CrossRefGoogle Scholar
  26. 26.
    Prantera M, Drozdowicz A, Gomes Leite S, Soares RA (2002) Degradation of gasoline aromatic hydrocarbons by two N2-fixing soil bacteria. Biotechnol Lett 24(1):85–89CrossRefGoogle Scholar
  27. 27.
    Laguerre G, Bossand B, Bardin R (1987) Free-living dinitrogen-fixing bacteria isolated from petroleum refinery oily sludge. Appl Environ Microbiol 53(7):1674–1678PubMedPubMedCentralGoogle Scholar
  28. 28.
    Eckford R, Cook FD, Saul D, Aislabie J, Foght J (2002) Free-living heterotrophic nitrogen-fixing bacteria isolated from fuel-contaminated antarctic soils. Appl Environ Microbiol 68(10):5181–5185CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Al-Awadhi H, Al-Mailem D, Dashti N, Khanafer M, Radwan S (2012) Indigenous hydrocarbon-utilizing bacterioflora in oil-polluted habitats in Kuwait, two decades after the greatest man-made oil spill. Arch Microbiol 194(8):689–705CrossRefPubMedGoogle Scholar
  30. 30.
    Audinot JN, Cabin-Flaman A, Philipp P, Legent G, Wirtz T, Migeon HN (2011) NanoSIMS50 imaging of thin samples coupled with neutral cesium deposition. Surf Interface Anal 43:302–305CrossRefGoogle Scholar
  31. 31.
    Rosner BM, Rainey FA, Kroppenstedt RM, Schink B (1997) Acetylene degradation by new isolates of aerobic bacteria and comparison of acetylene hydratase enzymes. FEMS Microbiol Lett 148(2):175–180CrossRefPubMedGoogle Scholar
  32. 32.
    Montoya JP, Voss M, Kahler P, Capone DG (1996) A simple, high-precision, high-sensitivity tracer assay for N2 fixation. Appl Environ Microbiol 62(3):986–993PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Department of Isotope BiogeochemistryHelmholtz Centre for Environmental Research – UFZLeipzigGermany

Personalised recommendations