, Volume 23, Issue 1, pp 91–99 | Cite as

Effect of copper on diesel degradation in Pseudomonas extremaustralis

  • María Antonela Colonnella
  • Leonardo Lizarraga
  • Leticia Rossi
  • Rocío Díaz Peña
  • Diego Egoburo
  • Nancy I. López
  • Laura J. Raiger IustmanEmail author
Original Paper


Environments co-contaminated with heavy metals and hydrocarbons have become an important problem worldwide, especially due to the effect of metals on hydrocarbon degrading microorganisms. Pseudomonas extremaustralis, a bacterium isolated from a pristine pond in Antarctica, showed high capabilities to cope with environmental stress and a very versatile metabolism that includes alkane degradation under microaerobic conditions. In this work, we analyzed P. extremaustralis’ capability to resist high copper concentrations and the effect of copper presence in diesel biodegradation. We observed that P. extremaustralis resisted up to 4 mM CuSO4 in a rich medium such as LB. This copper resistance is sustained by the presence of the cus and cop operons together with other efflux systems and porins located in a single region in P. extremaustralis genome. When copper was present, diesel degradation was negatively affected, even though copper enhanced bacterial attachment to hydrocarbons. However, when a small amount of glucose (0.05% w/v) was added, the presence of CuSO4 enhanced alkane degradation. In addition, atomic force microscopy analysis showed that the presence of glucose decreased the negative effects produced by copper and diesel on the cell envelopes.


P. extremaustralis Copper resistance Diesel degradation AFM 



This work was partially supported by grants from UBA, CONICET, and ANPCyT. NIL, LL, and LJRI are career investigators from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina). MAC has a postgraduate fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina).

Supplementary material

792_2018_1063_MOESM1_ESM.tif (6.7 mb)
Representative AFM images of live and hydrated P. extremaustralis cells grown on different conditions. PeakForce Error images of P. extremaustralis after 7 days of growth: with diesel as sole carbon source (A); diesel plus copper (B); glucose (C); glucose plus copper (D); glucose plus diesel (E); glucose with copper and diesel (F). (TIFF 6869 kb)


  1. Agnello AC, Bagard M, van Hullebusch ED, Esposito G, Huguenot D (2016) Comparative bioremediation of heavy metals and petroleum hydrocarbons co-contaminated soil by natural attenuation, phytoremediation, bioaugmentation and bioaugmentation-assisted phytoremediation. Sci Total Environ 563:693–703. CrossRefGoogle Scholar
  2. Arnoldi M, Fritz M, Bäuerlein E, Radmacher M, Sackmann E, Boulbitch A (2000) Bacterial turgor pressure can be measured by atomic force microscopy. Phys Rev E 62:1034–1044. CrossRefGoogle Scholar
  3. Ayub ND, Pettinari MJ, Ruiz JA, López NI (2004) A polyhydroxybutyrate-producing Pseudomonas sp. isolated from Antarctic environments with high stress resistance. Curr Microbiol 49:170–174. CrossRefGoogle Scholar
  4. Bai W, Zhao K, Asami K (2007) Effects of copper on dielectric properties of E. coli cells. Coll Surf B Biointerfaces 58:105–115. CrossRefGoogle Scholar
  5. Basu A, Apte SK, Phale PS (2006) Preferential utilization of aromatic compounds over glucose by Pseudomonas putida CSV86. Appl Environ Microbiol 72(3):2226–2230. CrossRefGoogle Scholar
  6. Basu A, Das D, Bapat P, Wangikar PP, Phale PS (2009) Sequential utilization of substrates by Pseudomonas putida CSV86: signatures of intermediate metabolites and online measurements. Microbiol Res 164:429–437. CrossRefGoogle Scholar
  7. Bender C, Cooksey D (1986) Indigenous plasmids in Pseudomonas syringae pv. tomato: conjugative transfer and role in copper resistance. J Bacteriol 165:534–541. CrossRefGoogle Scholar
  8. Benforte FC, Colonnella MA, Ricardi MM, Solar Venero EC, Lizarraga L, López NI, Tribelli PM (2018) Novel role of the LPS core glycosyltransferase WapH for cold adaptation in the Antarctic bacterium Pseudomonas extremaustralis. PLoS One 13(2):e0192559. CrossRefGoogle Scholar
  9. Bondarczuk K, Piotrowska-Seget Z (2013) Molecular basis of active copper resistance mechanisms in Gram-negative bacteria. Cell Biol Toxicol 29:397–405. CrossRefGoogle Scholar
  10. Chayabutra C, Ju LK (2000) Degradation of n-hexadecane and its metabolites by Pseudomonas aeruginosa under microaerobic and anaerobic denitrifying conditions. Appl Environ Microbiol 66:493–498. CrossRefGoogle Scholar
  11. Considine RF, Drummond CJ, Dixon DR (2001) Force of interaction between a biocolloid and an inorganic oxide: complexity of surface deformation, roughness and brushlike behavior. Langmuir 17:6325–6335. CrossRefGoogle Scholar
  12. Cooksey DA (1993) Copper uptake and resistance in bacteria. Mol Microbiol 7:1–5. CrossRefGoogle Scholar
  13. Dong ZY, Huang WH, Xing DF, Zhang HF (2013) Remediation of soil co-contaminated with petroleum and heavy metals by the integration of electrokinetics and biostimulation. J Hazard Mater 260:399–408. CrossRefGoogle Scholar
  14. Flemming CA, Trevors JT (1989) Copper toxicity and chemistry in the environment: a review. Water Air Soil Pollut 44:143–158CrossRefGoogle Scholar
  15. Francius G, Polyakov P, Merlin J, Abe Y, Ghigo JM, Merlin C, Beloin C, Duval JFL (2011) Bacterial surface appendages strongly impact nanomechanical and electrokinetic properties of Escherichia coli cells subjected to osmotic stress. PLoS One. Google Scholar
  16. Gaboriaud F, Gee ML, Strugnell R, Duval FL (2008) Coupled electrostatic, hydrodynamic, and mechanical properties of bacterial interfaces in aqueous media. Langmuir 24:10988–10995. CrossRefGoogle Scholar
  17. Kaczorek E, Sałek K, Guzik U, Jesionowski T, Cybulski Z (2013) Biodegradation of alkyl derivatives of aromatic hydrocarbons and cell surface properties of a strain of Pseudomonas stutzeri. Chemosphere 90:471–478. CrossRefGoogle Scholar
  18. Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G, Witholt B (1988) Formation of polyesters by Pseudomonas oleovorans: effect of substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. Appl Environ Microbiol 54:2924–2932Google Scholar
  19. Lee SM, Grass G, Rensing C, Barrett SR, Yates CJD, Stoyanov JV, Brown NL (2002) The Pco proteins are involved in periplasmic copper handling in Escherichia coli. Biochem Biophys Res Commun 295:616–620. CrossRefGoogle Scholar
  20. Li X, Wang X, Wan L, Zhang Y, Li N, Li D, Zhou Q (2016) Enhanced biodegradation of aged petroleum hydrocarbons in soils by glucose addition in microbial fuel cells. J Chem Technol Biotechnol 91:267–275. CrossRefGoogle Scholar
  21. Lim CK, Cooksey DA (1993) Characterization of chromosomal homologs of the plasmid-borne copper resistance operon of Pseudomonas syringae. J Bacteriol 175:4492–4498. CrossRefGoogle Scholar
  22. López NI, Pettinari MJ, Stackebrandt E, Tribelli PM, Põtter M, Steinbüchel A, Méndez BS (2009) Pseudomonas extremaustralis sp. nov., a poly(3-hydroxybutyrate) producer isolated from an antarctic environment. Curr Microbiol 59:514–519. CrossRefGoogle Scholar
  23. Luo H, Liu G, Zhang R, Jin S (2009) Phenol degradation in microbial fuel cells. Chem Eng J 147:259–264. CrossRefGoogle Scholar
  24. Montenegro IPFM, Mucha AP, Reis I, Rodrigues P, Almeida CMR (2017) Copper effect in petroleum hydrocarbons biodegradation by microorganisms associated to Juncus maritimus: role of autochthonous bioaugmentation. Int J Environ Sci Technol 14:943–955. CrossRefGoogle Scholar
  25. Mykytczuk NCS, Trevors JT, Ferroni GD, Leduc LG (2011) Cytoplasmic membrane response to copper and nickel in Acidithiobacillus ferrooxidans. Microbiol Res 166:186–206. CrossRefGoogle Scholar
  26. Nakajima M, Goto M, Hibi T (2002) Similarity between copper resistance genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato. J Gen Plant Pathol 68:68–74. CrossRefGoogle Scholar
  27. Nečas D, Klapetek P (2012) Gwyddion: an open-source software for SPM data analysis. Cent Eur J Phys 10:181–188. Google Scholar
  28. Norman RS, Frontera-suau R, Pamela J, Morris PJ (2002) Variability in Pseudomonas aeruginosa lipopolysaccharide expression during crude oil degradation. Appl Environ Microbiol 68:5096–5103. CrossRefGoogle Scholar
  29. Nunes I, Jacquiod S, Brejnrod A, Holm PE, Johansen A, Brandt KK, Priemé A, Sørensen SJ (2016) Coping with copper: legacy effect of copper on potential activity of soil bacteria following a century of exposure. FEMS Microbiol Ecol 92:fiw175. CrossRefGoogle Scholar
  30. Obuekwe CO, Al-Jadi ZK, Al-Saleh ES (2009) Hydrocarbon degradation in relation to cell-surface hydrophobicity among bacterial hydrocarbon degraders from petroleum-contaminated Kuwait desert environment. Int Biodeterior Biodegrad 63:273–279. CrossRefGoogle Scholar
  31. Ohki S, Arnold K (1990) Surface dielectric constant, surface hydrophobicity and membrane fusion. J Membr Biol 114:195–203. CrossRefGoogle Scholar
  32. Overbeek R, Begley T, Butler RM, Choudhuri JV, Chuang HY, Cohoon M, de Crécy-Lagard V, Diaz N, Disz T, Edwards R, Fonstein M, Frank ED, Gerdes S, Glass EM, Goesmann A, Hanson A, Iwata-Reuyl D, Jensen R, Jamshidi N, Krause L, Kubal M, Larsen N, Linke B, McHardy AC, Meyer F, Neuweger H, Olsen G, Olson R, Osterman A, Portnoy V, Pusch GD, Rodionov DA, Rülckert C, Steiner J, Stevens R, Thiele I, Vassieva O, Ye Y, Zagnitko O, Vonstein V (2005) The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res 33:5691–5702. CrossRefGoogle Scholar
  33. Radmacher M, Fritz M, Hansma PK (1995) Imaging soft samples with the atomic force microscope: gelatin in water and propanol. Biophys J 69:264–270. CrossRefGoogle Scholar
  34. Raiger Iustman LJ, Tribelli PM, Ibarra JG, Catone MV, Solar Venero EC, López NI (2015) Genome sequence analysis of Pseudomonas extremaustralis provides new insights into environmental adaptability and extreme conditions resistance. Extremophiles 19:207–220. CrossRefGoogle Scholar
  35. Ramadass K, Megharaj M, Venkateswarlu K, Naidu R (2016) Soil bacterial strains with heavy metal resistance and high potential in degrading diesel oil and n-alkanes. Int J Environ Sci Technol 13:2863–2874. CrossRefGoogle Scholar
  36. Rojo F (2010) Carbon catabolite repression in Pseudomonas: optimizing metabolic versatility and interactions with the environment. FEMS Microbiol Rev 34:658–684. CrossRefGoogle Scholar
  37. Rosenberg M, Gutnick D, Rosenberg E (1980) Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett 9:29–33. CrossRefGoogle Scholar
  38. Sandrin TR, Hoffman DR (2007) Bioremediation of organic and metal co-contaminated environments: effects of metal toxicity, speciation, and bioavailability on biodegradation. In: Singh SN, Tripathi RD (eds) Environmental bioremediation technologies. Springer, Heidelberg, pp 1–34. Google Scholar
  39. Sani RK, Peyton BM, Brown LT (2001) Copper-induced inhibition of growth of Desulfovibrio desulfuricans G20: assessment of its toxicity and correlation with those of zinc and lead. Appl Environ Microbiol 67:4765–4772. CrossRefGoogle Scholar
  40. Tribelli PM, Di Martino C, López NI, Raiger Iustman LJ (2012a) Biofilm lifestyle enhances diesel bioremediation and biosurfactant production in the Antarctic polyhydroxyalkanoate producer Pseudomonas extremaustralis. Biodegradation 23:645–651. CrossRefGoogle Scholar
  41. Tribelli PM, Raiger Iustman LJ, Catone MV, Di Martino C, Revale S, Méndez BS, López NI (2012b) Genome sequence of the polyhydroxybutyrate producer Pseudomonas extremaustralis, a highly stress-resistant antarctic bacterium. J Bacteriol 194:2381–2382. CrossRefGoogle Scholar
  42. Tribelli PM, Venero ECS, Ricardi MM, Gómez-Lozano M, Raiger Iustman LJ, Molin S, López NI (2015) Novel essential role of ethanol oxidation genes at low temperature revealed by transcriptome analysis in the antarctic bacterium Pseudomonas extremaustralis. PLoS One. Google Scholar
  43. Tribelli PM, Rossi L, Ricardi MM, Gomez-Lozano M, Molin S, Raiger Iustman LJ, López NI (2017) Microaerophilic alkane degradation in Pseudomonas extremaustralis: a transcriptomic and physiological approach. J Ind Microbiol Biotechnol. Google Scholar
  44. Van Liedekerke M, Prokop G, Rabl-berger S, Kibblewhite M (2014) Progress in the management of contaminated sites in Europe. JRC Ref Rep. Google Scholar
  45. Vullo DL, Ceretti HM, Daniel MA, Ramírez SAM, Zalts A (2008) Cadmium, zinc and copper biosorption mediated by Pseudomonas veronii 2E. Bioresour Technol 99:5574–5581. CrossRefGoogle Scholar
  46. Wang H, Wilksch JJ, Lithgow T, Strugnell RA, Gee ML (2013) Nanomechanics measurements of live bacteria reveal a mechanism for bacterial cell protection: the polysaccharide capsule in Klebsiella is a responsive polymer hydrogel that adapts to osmotic stress. Soft Matter 9:7560–7567. CrossRefGoogle Scholar
  47. Wang H, Wilksch JJ, Strugnell RA, Gee ML (2015) Role of capsular polysaccharides in biofilm formation: an AFM nanomechanics study. ACS Appl Mater Interfaces 7:13007–13013. CrossRefGoogle Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • María Antonela Colonnella
    • 3
  • Leonardo Lizarraga
    • 3
  • Leticia Rossi
    • 1
  • Rocío Díaz Peña
    • 2
  • Diego Egoburo
    • 2
  • Nancy I. López
    • 1
    • 2
  • Laura J. Raiger Iustman
    • 1
    • 2
    Email author
  1. 1.Departamento de Química Biológica, Facultad de Ciencias Exactas y NaturalesUniversidad de Buenos AiresBuenos AiresArgentina
  2. 2.IQUIBICEN, CONICETBuenos AiresArgentina
  3. 3.CIBION, CONICETBuenos AiresArgentina

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