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Electron Microscopy Protocols for the Study of Hydrocarbon-Producing and Hydrocarbon-Decomposing Microbes: Classical and Advanced Methods

  • Kamna Jhamb
  • Manfred AuerEmail author
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

One of the fascinating areas of hydrocarbon microbiology biology is the quest for an ultratstructural understanding of (macro)-molecular mechanisms underlying the degradation, synthesis, and intracellular storage of hydrocarbons, which due to their hydrophobic characteristics continuously threaten the integrity of biological membranes. Here we review classical and novel advanced electron microscopy approaches, including correlative light and electron microscopy that in combination with genetics and biochemical experimentation can be utilized to study such hydrocarbon–cell interactions.

Keywords:

Cellular inclusion Correlative microscopy Cryo-EM Electron microscopy Hydrocarbon Lipid 

References

  1. 1.
    Ladygina N, Deyukhina EG, Veinshtein MB (2006) A review on microbial synthesis of hydrocarbons. Process Biochem 41:1001–1014CrossRefGoogle Scholar
  2. 2.
    Head I, Aitken C, Gray N et al (2010) Hydrocarbon degradation in petroleum reservoirs. In: Timmis K (ed) Handbook of hydrocarbon and lipid microbiology. Springer, Berlin/HeidelbergGoogle Scholar
  3. 3.
    Sierra-Garcia I, de Oliveira V (2013) Microbial hydrocarbon degradation: efforts to understand biodegradation in petroleum reservoirs. In: Chamy R (ed) Biodegradation – engineering and technology. InTech, ISBN: 978-953-51-1153-5, doi: 10.5772/55920
  4. 4.
    Wenger L, Davis C, Isaksen G 2002 Multiple controls on petroleum biodegradation and impact on oil quality. In: Society for petroleum engineers (SPE) reservoir evaluation and engineering, pp 375–383Google Scholar
  5. 5.
    Roling W, Head I, Larter S (2003) The microbiology of hydrocarbon degradation in subsurface petroleum reservoirs: perspectives and prospects. Res Microbiol 154:321–328CrossRefPubMedGoogle Scholar
  6. 6.
    Atlas R, Bartha R (1993) Microbial ecology - fundamentals and applications. Benjamin-Cummings, Redwood CityGoogle Scholar
  7. 7.
    Atlas R (1981) Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiol Rev 45(1):180–209PubMedPubMedCentralGoogle Scholar
  8. 8.
    Van Hamme J, Singh A, Ward O (2003) Recent advances in petroleum microbiology. Microbiol Mol Biol Rev 67(4):503–549CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Muthuswamy S, Binupriya A, Baik S, Yun S (2008) Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium isolated from hydrocarbon contaminated areas. Clean 36(1):92–96Google Scholar
  10. 10.
    Martins L, Piexoto R (2012) Biodegradation of petroleum hydrocarbons in hypersaline environments. Braz J Microbiol 43(3):865–872CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Hazen T, Dubinsky E, De Santis T, Andersen G et al (2010) Deep-sea oil plume enriches indigenous oil-degrading bacteria. Science 330(6001):204–208CrossRefPubMedGoogle Scholar
  12. 12.
    Baelum J, Borglin S, Chakraborty R, Fortney J et al (2012) Deep-sea bacteria enriched by oil and dispersant from the deepwater horizon spill. Environ Microbiol 14(9):2405–2416CrossRefPubMedGoogle Scholar
  13. 13.
    Biological Agents. http://www2.epa.gov/emergency-response/biological-agents. Accessed 24 Nov 2014
  14. 14.
    Kostka J, Prakash O, Overholt W, Green S et al (2011) Hydrocarbon degrading bacteria and the bacterial community response in Gulf of Mexico beach sands impacted by the deepwater horizon oil spill. Appl Environ Microbiol 77(22):7962CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Scott C, Finnerty W (1976) A comparative analysis of the ultrastructure of hydrocarbon – oxidizing microorganisms. J Gen Microbiol 94:342–350CrossRefPubMedGoogle Scholar
  16. 16.
    Pinzon N, Aukema K, Gralnick J et al (2011) Nile red detection of bacterial hydrocarbons and ketones in a high throughput format. MBio 2(4):e00109-11. doi: 10.1128/mBio.00109-11 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Singer M, Tyler S, Finnerty W (1985) Growth of Acinetobacter sp. strain HO1-N on n-hexadecanol: physiological and ultrastructural characteristics. J Bacteriol 162(1):162PubMedPubMedCentralGoogle Scholar
  18. 18.
    Alvarez H, Steinbuchel A (2002) Triacylglycerols in prokaryotic microorganisms. Appl Microbiol Biotechnol 60:367–376CrossRefPubMedGoogle Scholar
  19. 19.
    Waltermann M, Steinbuchel A (2005) Neutral lipid bodies in prokaryotes: recent insights into structure, formation and relationship to eukaryotic lipid depots. J Bacteriol 187(11):3607CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Marin M, Pedregosa A, Laborda F (1996) Emulsifier production and microscopical study of emulsions and biofilms formed by the hydrocarbon-utilizing bacteria Acinetobacter calcoaceticus MM5. Appl Microbiol Biotechnol 44:660–667CrossRefGoogle Scholar
  21. 21.
    Waltermann M, Hinz A, Robenek H et al (2005) Mechanism of lipid body formation in prokaryotes: how bacteria fatten up. Mol Microbiol 55(3):750–763CrossRefPubMedGoogle Scholar
  22. 22.
    Meng X, Yang J, Xu X, Zhang L, Nie Q, Xian M (2009) Biodiesel production from oleaginous microorganisms. Renew Energy 34:1–5CrossRefGoogle Scholar
  23. 23.
    U.S. Bioenergy Statistics. http://www.ers.usda.gov/data-products/us-bioenergy-statistics. Accessed 10 Oct 2014
  24. 24.
    Shi S, Valle-Rodriguez J, Siewers V, Nielsen J (2011) Prospects for microbial biodiesel production. Biotechnol J 6:277–285CrossRefPubMedGoogle Scholar
  25. 25.
    Suzuki R, Ito N, Uno Y, Nishii I et al (2013) Transformation of lipid bodies related to hydrocarbon accumulation in a green alga, Botryococcus braunii (Race B). PLoS One 8(12), e81626. doi: 10.1371/journal.pone.0081626 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Davies S, Whittenbury R (1970) Fine structure of methane and other hydrocarbon-utilizing bacteria. J Gen Microbiol 61:227–232CrossRefPubMedGoogle Scholar
  27. 27.
    Kennedy R, Finnerty W, Sudarsanan K, Young R (1974) Microbial assimilation of hydrocarbons. I. The fine-structure of a hydrocarbon oxidizing Acinetobacter sp. Arch Microbiol 102:75–83CrossRefGoogle Scholar
  28. 28.
    Alvarez H, Mayer F, Fabritius D, Steinbüchel A (1996) Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630. Arch Microbiol 165(6):377–386CrossRefPubMedGoogle Scholar
  29. 29.
    Diestra E, Esteve I, Burnat M, Maldonado J, Sole A (2007) Isolation and characterization of a heterotrophic bacterium able to grow in different environmental stress conditions, including crude oil and heavy metals. In: Méndez-Vilas A (ed) Communicating current research and educational topics and trends in applied microbiology. Formex, BadajozGoogle Scholar
  30. 30.
    Osumi M (2012) Visualization of yeast cells by electron microscopy. J Electron Microsc 61(6):343–365CrossRefGoogle Scholar
  31. 31.
    Li Z (ed) (2002) Industrial application of electron microscopy. CRC Press, Boca Raton, p 362Google Scholar
  32. 32.
    Wigglesworth V (1975) Lipid staining for the electron microscope: a new method. J Cell Sci 19:425–437PubMedGoogle Scholar
  33. 33.
    Trent J (1984) Ruthenium tetraoxide staining of polymers: new preparative methods for electron microscopy. Macromolecules 17:2930–2931CrossRefGoogle Scholar
  34. 34.
    Khandpur A, Macosko C, Bates F (1995) Transmission electron microscopy of saturated hydrocarbon block copolymers. J Polym Sci B Polym Phys 33:247–252CrossRefGoogle Scholar
  35. 35.
    Richter H, Sleytr U (1971) Fettextraction bei −78°C: nachweis im Gefrieratzbild. Z Naturforsch 26b:470–473Google Scholar
  36. 36.
    Meyer H, Winkelmann H (1970) Die Darstellung von lipiden bei der gefrieratzpraparation und ihre beziehung zur strukturanalyse biologischer membranen. Exp Pathol 4:47–59Google Scholar
  37. 37.
    Moor H, Muhlethaler K (1963) Fine structure in frozen etched yeast cells. J Cell Biol 17:609–628CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Meyer H, Richter W (2001) Freeze-fracture studies on lipids and membranes. Micron 32:615–644CrossRefPubMedGoogle Scholar
  39. 39.
    Scott C, Finnerty W (1976) Characterization of intracytoplasmic hydrocarbon inclusions from the hydrocarbon-oxidizing Acinetobacter Species HO1-N. J Bacteriol 127(1):481–489PubMedPubMedCentralGoogle Scholar
  40. 40.
    Ishige T, Tani A, Takabe K, Kawasaki K et al (2002) Wax ester production from n-Alkanes by Acinetobacter sp. strain M-1: ultrastructure of cellular inclusions and role of acyl coenzyme A reductase. Appl Environ Microbiol 68(3):1192–1195CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Bleck C, Merz A, Gutierrez M, Alther P et al (2010) Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis. J Microsc 237:23–28CrossRefPubMedGoogle Scholar
  42. 42.
    Fujimoto K (1995) Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins - application to the immunogold labeling of intercellular junctional complexes. J Cell Sci 108:3443–3449PubMedGoogle Scholar
  43. 43.
    Severs N (1995) Freeze-fracture cytochemistry: an explanatory survey of methods. In: Severs N, Shotton D (eds) Rapid freezing, freeze fracture, and deep etching. Wiley-Liss, New York, pp 173–208Google Scholar
  44. 44.
    Robenek H, Severs N (2008) Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique. Biol Proced Online 10:9–19CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Scott C, Makula S, Finnerty W (1976) Isolation and characterization of membranes from a hydrocarbon-oxidizing Acinetobacter sp. J Bacteriol 127(1):469–480PubMedPubMedCentralGoogle Scholar
  46. 46.
    Kellenberger E, Johansen R, Maeder M, Bohrmann B et al (1992) Artefacts and morphological changes during chemical fixation. J Microsc 168:181–201CrossRefPubMedGoogle Scholar
  47. 47.
    Mc Donald K, Auer M (2006) High-pressure freezing, cellular tomography, and structural cell biology. Biotechniques 41(2):137, 139, 141Google Scholar
  48. 48.
    Djaczenko W, Muller M, Benedetto A (1990) Ultra-rapid high pressure freezing in high resolution EM of cell-cell and cell-substrate interactions. Cell Biol Int Rep 14Google Scholar
  49. 49.
    Dubochet J (1995) High-pressure freezing for cryoelectron microscopy. Trends Cell Biol 5(9):366–368CrossRefPubMedGoogle Scholar
  50. 50.
    Hurbain I, Sachse M (2011) The future is cold: cryo-preparation methods for transmission electron microscopy of cells. Biol Cell 103:405–420CrossRefPubMedGoogle Scholar
  51. 51.
    Paul T, Beveridge T (1994) Preservation of surface lipids and determination of ultrastructure of Mycobacterium kansasii by freeze substitution. Infect Immun 62(5):1542–1550PubMedPubMedCentralGoogle Scholar
  52. 52.
    Al-Amoudi A, Chang J, Leforestier A, McDowall A et al (2004) Cryo –electron microscopy of vitreous section. EMBO J 23(18):3583–3588CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Comolli L, Kundmann M, Downing K (2006) Characterization of intact subcellular bodies in whole bacteria by cryo-electron tomography and spectroscopic imaging. J Microsc 223:40–52CrossRefPubMedGoogle Scholar
  54. 54.
    Thomson N, Channon K, Mokhtar N, Staniewicz L et al (2011) Imaging internal features of whole, unfixed bacteria. Scanning 33(2):59–68CrossRefPubMedGoogle Scholar
  55. 55.
    (2010) Probes for lipids and membranes. In: The molecular probes® handbook: a guide to fluorescent probes and labeling technologies, 11th edn. http://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook/probes-for-lipids-and-membranes.html
  56. 56.
    Chen W, Zhang C, Song L, Sommerfeld M, Hu Q (2009) A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. J Microbiol Methods 77:41–47CrossRefPubMedGoogle Scholar
  57. 57.
    Elle I, Olsen L, Pultz D, Rødkær S, Færgeman N (2010) Something worth dyeing for: molecular tools for the dissection of lipid metabolism in Caenorhabditis elegans. FEBS Lett 584:2183–2193CrossRefPubMedGoogle Scholar
  58. 58.
    Govender T, Ramanna L, Bux R (2012) BODIPY staining, an alternative to the Nile Red fluorescence method for the evaluation of intracellular lipids in microalgae. Bioresour Technol 114:507–511CrossRefPubMedGoogle Scholar
  59. 59.
    Dantuma N, Pijnenburg M, Diederen J, Van der Horst D (1998) Electron microscopic visualization of receptor–mediated endocytosis of DiI–labeled lipoproteins by diaminobenzidine photoconversion. J Histochem Cytochem 46(9):1085–1089CrossRefPubMedGoogle Scholar
  60. 60.
    Cortese K, Diaspro A, Taccheti C (2009) Advanced correlative light/electron microscopy: current methods and new developments using Tokuyasu cryosections. J Histochem Cytochem 57(12):1103–1112CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Staubli W (1963) A new embedding technique for electron microscopy, combining a water soluble epoxy resin (Durcupan) with water insoluble Araldite. J Cell Biol 16:197–199CrossRefPubMedCentralGoogle Scholar
  62. 62.
    Mc Donald K, Webb R (2011) Freeze substitution in 3 hours or less. J Microsc 243(3):227–233CrossRefGoogle Scholar
  63. 63.
    Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain for electron microscopy. J Cell Biol 17:208CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Life Sciences DivisionLawrence Berkeley National LaboratoryBerkeleyUSA

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