Microbial Ecology

, Volume 57, Issue 2, pp 276–285 | Cite as

Identification and Genetic Characterization of Phenol-Degrading Bacteria from Leaf Microbial Communities

  • Amarjyoti Sandhu
  • Larry J. Halverson
  • Gwyn A. Beattie
Original Article


Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants. Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity provided evidence that the gram-negative strains had the C12O−, but not the C23O−, phenol catabolic pathway. Similarly, the Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways they employ for phenol degradation.


Rhodococcus Acinetobacter Phenol Degradation Phenol Hydroxylase Ortho Pathway 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We would like to thank Dr. Janice Thompson, Troy Bowman, and Iowa Select farms for their assistance in obtaining the green ash leaf samples. The work was supported by funds from the Iowa Space Grant Consortium, Department of Agronomy Endowment and the College of Agriculture and Life Sciences at Iowa State University.


  1. 1.
    Abd-El-Haleem D, Moawad H, Zaki EA, Zaki S (2002) Molecular characterization of phenol-degrading bacteria isolated from different Egyptian ecosystems. Microb Ecol 43:217–224PubMedCrossRefGoogle Scholar
  2. 2.
    Arutchelvan V, Kanakasabai V, Nagarajan S, Muralikrishnan V (2005) Isolation and identification of novel high strength phenol degrading bacterial strains from phenol-formaldehyde resin manufacturing industrial wastewater. J Hazard Mater 127:238–243PubMedCrossRefGoogle Scholar
  3. 3.
    Agency for Toxic Substances and Disease Registry (ATSDR) (1998) Toxicological profile for phenol. In: U. S. Department of Health and Human Services, Public Health Service, Atlanta, GAGoogle Scholar
  4. 4.
    Barac T, Taghavi S, Borremans B, Provoost A, Oeyen L, Colpaert JV, Vangronsveld J, van der Lelie D (2004) Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pollutants. Nat Biotechnol 22:583–588PubMedCrossRefGoogle Scholar
  5. 5.
    Barbe V, Vallenet D, Fonknechten N, Kreimeyer A, Oztas S, Labarre L, Cruveiller S, Robert C, Duprat S, Wincker P, Ornston LN, Weissenbach J, Marliere P, Cohen GN, Medigue C (2004) Unique features revealed by the genome sequence of Acinetobacter sp ADP1, a versatile and naturally transformation competent bacterium. Nucl Acids Res 32:5766–5779PubMedCrossRefGoogle Scholar
  6. 6.
    Basile LA, Erijman L (2008) Quantitative assessment of phenol hydroxylase diversity in bioreactors using a functional gene analysis. Appl Microbiol Biotechnol 78:863–872PubMedCrossRefGoogle Scholar
  7. 7.
    Chen CL, Wu JH, Liu WT (2008) Identification of important microbial populations in the mesophilic and thermophilic phenol-degrading methanogenic consortia. Water Res 42:1963–1976PubMedCrossRefGoogle Scholar
  8. 8.
    Cohen MF, Meziane T, Yamasaki H (2004) A photocarotogenic Rhodococcus sp. isolated from the symbiotic fern Azolla. Endocytobiosis Cell Res 15:350–355Google Scholar
  9. 9.
    De Kempeneer L, Sercu B, Vanbrabant W, Van Langenhove H, Verstraete W (2004) Bioaugmentation of the phyllosphere for the removal of toluene from indoor air. Appl Microbiol Biotechnol 64:284–288PubMedCrossRefGoogle Scholar
  10. 10.
    DeRito CM, Pumphrey GM, Madsen EL (2005) Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community. Appl Environ Microbiol 71:7858–7865PubMedCrossRefGoogle Scholar
  11. 11.
    El-Sayed WS, Ibrahim MK, Abu-Shady M, El-Beih F, Ohmura N, Saiki H, Ando A (2003) Isolation and characterization of phenol-catabolizing bacteria from a coking plant. Biosci Biotechnol Biochem 67:2026–2029PubMedCrossRefGoogle Scholar
  12. 12.
    Filonov AE, Duetz WA, Karpov AV, Gaiazov RR, Kosheleva IA, Breure AM, Filonova IF, van Andel JG, Boronin AM (1997) Competition of plasmid-bearing Pseudomonas putida strains catabolizing naphthalene via various pathways in chemostat culture. Appl Microbiol Biotechnol 48:493–498PubMedCrossRefGoogle Scholar
  13. 13.
    Futamata H, Harayama S, Watanabe K (2001) Diversity in kinetics of trichloroethylene-degrading activities exhibited by phenol-degrading bacteria. Appl Microbiol Biotechnol 55:248–253PubMedCrossRefGoogle Scholar
  14. 14.
    Futamata H, Harayama S, Watanabe K (2001) Group-specific monitoring of phenol hydroxylase genes for a functional assessment of phenol-stimulated trichloroethylene bioremediation. Appl Environ Microbiol 67:4671–4677PubMedCrossRefGoogle Scholar
  15. 15.
    Garcia MT, Ventosa A, Mellado E (2005) Catabolic versatility of aromatic compound-degrading halophilic bacteria. FEMS Microbiol Ecol 54:97–109PubMedCrossRefGoogle Scholar
  16. 16.
    Heinaru E, Truu J, Stottmeister U, Heinaru A (2000) Three types of phenol and p-cresol catabolism in phenol- and p-cresol-degrading bacteria isolated from river water continuously polluted with phenolic compounds. FEMS Microbiol Ecol 31:195–205PubMedCrossRefGoogle Scholar
  17. 17.
    Ilori MO, Amund OO, Ezeani CK, Omoijiahina S, Adebusoye SA (2006) Occurrence and growth potentials of hydrocarbon degrading bacteria on the phylloplane of some tropical plants. Afric J Biotechnol 5:542–545Google Scholar
  18. 18.
    Jiang HL, Tay STL, Maszenan AM, Tay JH (2006) Physiological traits of bacterial strains isolated from phenol-degrading aerobic granules. FEMS Microbiol Ecol 57:182–191PubMedCrossRefGoogle Scholar
  19. 19.
    Jiang Y, Wen JP, Li HM, Yang SL, Hu ZD (2005) The biodegradation of phenol at high initial concentration by the yeast Candida tropicalis. Biochem Eng J 24:243–247CrossRefGoogle Scholar
  20. 20.
    Jurkevitch EJ, Shapira G (2000) Structure and colonization dynamics of epiphytic bacterial communities and of selected component strains on tomato (Lycopersicon esculentum) leaves. Microb Ecol 40:300–308PubMedGoogle Scholar
  21. 21.
    Kadivar H, Stapleton AE (2003) Ultraviolet radiation alters maize phyllosphere bacterial diversity. Microb Ecol 45:353–361PubMedCrossRefGoogle Scholar
  22. 22.
    Larkin MJ, Kulakov LA, Allen CCR (2005) Biodegradation and Rhodococcus—masters of catabolic versatility. Curr Opin Biotechnol 16:282–290PubMedCrossRefGoogle Scholar
  23. 23.
    Lin CW, Lai CY, Chen LH, Chiang WF (2007) Microbial community structure during oxygen-stimulated bioremediation in phenol-contaminated groundwater. J Hazard Mater 140:221–229PubMedCrossRefGoogle Scholar
  24. 24.
    Maidak BL, Cole JR, Lilburn TG, Parker CT Jr, Saxman PR, Farris RJ, Garrity GM, Olsen GJ, Schmidt TM, Tiedje JM (2001) The RDP-II (Ribosomal Database Project). Nucl Acids Res 29:173–174PubMedCrossRefGoogle Scholar
  25. 25.
    Mandri T, Lin J (2007) Isolation and characterization of engine oil degrading indigenous microrganisms in Kwazulu-Natal, South Africa. Afric J Biotechnol 6:23–27Google Scholar
  26. 26.
    Muller RH, Babel W (1996) Growth rate-dependent expression of phenol-assimilation pathways in Alcaligenes eutrophus JMP134-the influence of formate as an auxiliary energy source on phenol conversion characteristics. Appl Microbiol Biotechnol 46:156–162CrossRefGoogle Scholar
  27. 27.
    O’ Brien RD, Lindow SE (1989) Effects of plant species and environmental conditions on epiphytic population sizes of Pseudomonas syringae and other bacteria. Phytopathology 79:619–627CrossRefGoogle Scholar
  28. 28.
    Paller G, Hommel RK, Kleber HP (1995) Phenol degradation by Acinetobacter calcoaceticus Ncib-8250. J Basic Microbiol 35:325–335PubMedCrossRefGoogle Scholar
  29. 29.
    Pankhurst ES (1965) A spot test for catechol 2:3-oxygenase in bacteria. J Appl Bacteriol 28:309–315PubMedGoogle Scholar
  30. 30.
    Parvanov D, Topalova Y (2008) Biodegradation potential of phenol-resistant bacteria localized in different stream habitats. Biotechnol Biotechnol Equip 22:709–715Google Scholar
  31. 31.
    Rivas R, Garcia-Fraile P, Mateos PF, Martinez-Molina E, Velazquez E (2007) Characterization of xylanolytic bacteria present in the bract phyllosphere of the date palm Phoenix dactylifera. Lett Appl Microbiol 44:181–187PubMedCrossRefGoogle Scholar
  32. 32.
    Saadoun I (2002) Isolation and characterization of bacteria from crude petroleum oil contaminated soil and their potential to degrade diesel fuel. J Basic Microbiol 42:420–428PubMedCrossRefGoogle Scholar
  33. 33.
    Sandhu A, Halverson LJ, Beattie GA (2007) Bacterial degradation of airborne phenol in the phyllosphere. Environ Microbiol 9:383–392PubMedCrossRefGoogle Scholar
  34. 34.
    Sei K, Inoue D, Wada K, Mori K, Ike M, Kohno T, Fujita M (2004) Monitoring behaviour of catabolic genes and change of microbial community structures in seawater microcosms during aromatic compound degradation. Water Res 38:4405–4414PubMedCrossRefGoogle Scholar
  35. 35.
    Shashirekha S, Uma L, Subramanian G (1997) Phenol degradation by the marine cyanobacterium Phormidium valderianum BDU 30501. J Indust Microbiol Biotechnol 19:130–133CrossRefGoogle Scholar
  36. 36.
    Song JM, Sung JH, Kim YM, Zylstra GJ, Kim E (2000) Roles of the meta- and the ortho-cleavage pathways for the efficient utilization of aromatic hydrocarbons by Sphingomonas yanoikuyae B1. J Microbiol 38:245–249Google Scholar
  37. 37.
    Tay ST, Moy BY, Maszenan AM, Tay JH (2005) Comparing activated sludge and aerobic granules as microbial inocula for phenol biodegradation. Appl Microbiol Biotechnol 67:708–713PubMedCrossRefGoogle Scholar
  38. 38.
    Thompson JD, Higgins DG, Gibson TJ (1994) Clustal-W: improving the sensitivity of progressive multiple Sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 22:4673–4680PubMedCrossRefGoogle Scholar
  39. 39.
    Van Aken B, Yoon JM, Schnoor JL (2004) Biodegradation of nitro-substituted explosives 2,4,6-trinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine, and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine by a phytosymbiotic Methylobacterium sp. associated with poplar tissues (Populus deltoides x nigra DN34). Appl Environ Microbiol 70:508–517PubMedCrossRefGoogle Scholar
  40. 40.
    van der Geize R, Dijkhuizen L (2004) Harnessing the catabolic diversity of rhodococci for environmental and biotechnological applications. Curr Opin Microbiol 7:255–261PubMedCrossRefGoogle Scholar
  41. 41.
    Wang YJ, Xiao M, Geng XL, Liu JY, Chen J (2007) Horizontal transfer of genetic determinants for degradation of phenol between the bacteria living in plant and its rhizosphere. Appl Microbiol Biotechnol 77:733–739PubMedCrossRefGoogle Scholar
  42. 42.
    Watanabe K, Teramoto M, Futamata H, Harayama S (1998) Molecular detection, isolation, and physiological characterization of functionally dominant phenol-degrading bacteria in activated sludge. Appl Environ Microbiol 64:4396–4402PubMedGoogle Scholar
  43. 43.
    Wei GH, Yu JF, Zhu YH, Chen WM, Wang L (2008) Characterization of phenol degradation by Rhizobium sp CCNWTB 701 isolated from Astragalus chrysopteru in mining tailing region. J Hazard Mater 151:111–117PubMedCrossRefGoogle Scholar
  44. 44.
    Whiteley AS, Wiles S, Lilley AK, Philp J, Bailey MJ (2001) Ecological and physiological analyses of Pseudomonad species within a phenol remediation system. J Microbiol Meth 44:79–88CrossRefGoogle Scholar
  45. 45.
    Xu Y, Zhang W, Chen M, Lin M, Li JM, Fang XJ (2000) Isolation and identification of a phenol-degrading bacterial strain. Acta Sci Circumstantiae 20:450–455Google Scholar
  46. 46.
    Yang CH, Crowley DE, Borneman J, Noel TK (2001) Microbial phyllosphere populations are more complex than previously realized. Proc Natl Acad Sci U S A 98:3889–3894PubMedCrossRefGoogle Scholar
  47. 47.
    Zahn JA, DiSpirito AA, Do YS, Brooks BE, Cooper EE, Hatfield JL (2001) Correlation of human olfactory responses to airborne concentrations of malodorous volatile organic compounds emitted from swine effluent. J Environ Qual 30:624–634PubMedGoogle Scholar
  48. 48.
    Zahn JA, Hatfield JL, Do YS, DiSpirito AA, Laird DA, Pfeiffer RL (1997) Characterization of volatile organic emissions and wastes from a swine production facility. J Environ Qual 26:1687–1696CrossRefGoogle Scholar
  49. 49.
    Zhang X, Gao P, Chao Q, Wang L, Senior E, Zhao L (2004) Microdiversity of phenol hydroxylase genes among phenol-degrading isolates of Alcaligenes sp. from an activated sludge system. FEMS Microbiol Lett 237:369–375PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Amarjyoti Sandhu
    • 1
  • Larry J. Halverson
    • 1
  • Gwyn A. Beattie
    • 1
    • 2
  1. 1.Department of Plant Pathology and Interdepartmental Microbiology ProgramIowa State UniversityAmesUSA
  2. 2.Department of Plant PathologyIowa State UniversityAmesUSA

Personalised recommendations