Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Metabolic pathway of xenoestrogenic short ethoxy chain-nonylphenol to nonylphenol by aerobic bacteria, Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90


Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90 degrading short ethoxy (EO) chain-nonylphenol (NP) [NPEOav2.0 containing NP mono- ∼ tetraethoxylates (NP1EO ∼ NP4EO); average 2.0 EO units] were isolated by enrichment cultures. Both strains grew on NP but not on octyl- and nonylphenol polyethoxylates (NPEOs) (average 10 EO units). Growth and degradation of NPEOav2.0 was increased with increased concentrations of yeast extract (0.02–0.5%) in a culture medium. Culture supernatants of both strains grown on NPEOav2.0 were analyzed by high-performance liquid chromatography, showing degradation of NP4EO–NP1EO. The metabolites from nonylphenol diethoxylate (NP2EO) by resting cells of both strains were identified by gas chromatography–mass spectrometry as nonylphenoxyethoxyacetic acid, NP1EO, nonylphenoxyacetic acid (NP1EC), and NP, while those from NP1EO were identified as NP1EC and NP. Cell-free extracts from strain AS08 grown on NPEOav2.0 dehydrogenated NPEOs, NPEOav2.0, NP2EO, NP1EO, and PEG 400, but the extracts were inactive toward di- ∼ tetraethylene glycol. Aldehydes were formed in the reaction mixture of each substrate with cell-free extracts. From these results, the aerobic metabolic pathway for short EO chain-NP is proposed: A terminal alcohol group of the EO chain is oxidized to a carboxylic acid via an aldehyde, and then one EO unit is removed. This process is repeated until NP is produced.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Ahel M, Conrad T, Giger W (1987) Persistent organic chemicals in sewage effluents. 3. Determinations of nonylphenoxy carboxylic acids by high-resolution gas chromatography/mass spectrometry and high-performance liquid chromatography. Environ Sci Technol 21:697–703

  2. Ejlertsson J, Nilsson ML, Kylin H, Bergman A, Karlson I, Oquist M, Svensson BH (1999) Anaerobic degradation of nonylphenol mono- and diethoxylates in digestor sludge, landfill municipal solid waste and landfill sludge. Environ Sci Technol 33:301–306

  3. Frings J, Schramm Z, Schink B (1992) Enzymes involved in anaerobic polyethylene glycol degradation by Pelobacter venetianus and Bacteroides strain PG1. Appl Environ Microbiol 58:2164–2167

  4. Fujii K, Urano N, Ushio H, Satomi M, Iida H, Ushio-Sata N, Kimura S (2000) Profile of a nonylphenol-degrading microflora and its potential for bioremedial applications. J Biochem 128:909–916

  5. Giger W, Brunner PH, Schaffner C (1984) 4-Nonylphenol in sewage sludge: accumulation of toxic metabolites from nonionic surfactants. Science 225:623–625

  6. Hayashi S, Saito S, Kim J-H, Nishimura O, Sudo R (2005) Aerobic biodegradation behavior of nonylphenol polyethoxylates and their metabolites in the presence of organic matter. Environ Sci Technol 39:5626–5633

  7. Jobling R, Sumpter JP (1993) Detergent components in sewage effluent are weakly estrogenic to fish: an in vitro study using rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquat Toxicol 27:361–372

  8. John DM, White GF (1998) Mechanism for biotransformation of nonylphenol polyethoxylates to xenoestrogens in Pseudomonas putida. J Bacteriol 180:4332–4338

  9. Jonkers N, Knepper TP, de Voogt P (2001) Aerobic biodegradation studies of nonylphenol ethoxylates in river water using liquid chromatography–electrospray tandem mass spectrometry. Environ Sci Technol 35:335–340

  10. Jonkers N, Laane RW, de Voogt P (2003) Fate of nonylphenol ethoxylates and their metabolites in two Dutch estuaries: evidence of biodegradation in the field. Environ Sci Technol 37:321–327

  11. Kane MD, Poulsen LK, Stahl DA (1993) Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences. Appl Environ Microbiol 59:682–686

  12. Kawai F, Fukaya M, Tani Y, Ogata K (1977) Identification of polyethylene glycols (PEGs)-assimilable bacteria and culture characteristics of PEG 6000 degradation by a mixed culture. J Ferment Technol 55:429–435

  13. Kawai F, Kimura T, Tani Y, Yamada H, Ueno T, Fukami H (1983) Identification of reaction products of polyethylene glycol dehydrogenase. Agric Biol Chem 47:1669–1671

  14. Kvestak R, Ahel M (1994) Occurrence of toxic metabolites from nonionic surfactants in the Krka River estuary. Ecotoxicol Environ Saf 28:25–34

  15. Liu X, Tani A, Kawai F (2005) Determination of nonylphenol and short chain nonylphenol polyethoxylates by normal phase high performance liquid chromatography. Chin J Anal Chem 33:1189–1191

  16. Maeda S, Mikami I (1988) Degradation of the non-ionic surfactant (polyoxyethylene-p-nonylphenyl ether) by Pseudomonas species. J Water Wastes 30:1056–1063

  17. Maki H, Masuda N, Fujiwara Y, Ike M, Fujita M (1994) Degradation of alkylphenol ethoxylates by Pseudomonas sp. strain TR01. Appl Environ Microbiol 60:2265–2271

  18. Montgomery-Brown J, Drewes JE, Fox P, Reinhard M (2003) Behavior of alkylphenol polyethoxylate metabolites during soil aquifer treatment. Water Res 37:3672–3681

  19. Murray ERB, Doetsch RN, Robinow CF (1994) Determinative and cytological light microscopy. In: Gerhardt P, Murray ERB, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology. ASM, Washington, DC, pp 31–32

  20. Patoczka J, Pulliam GW (1990) Biodegradation and secondary effluent toxicity of ethoxylated surfactants. Water Res 24:965–972

  21. Planas C, Guadayol JM, Droguest M, Escals A, Rivera J, Caixach J (2002) Degradation of polyethoxylated nonylphenols in a sewage treatment plant. Quantitative analysis by isotopic dilution-HRGC/MS. Water Res 36:982–988

  22. Sato H, Shibata A, Wang Y, Yoshikawa H, Tamura H (2003) Characterization of biodegradation intermediates of nonionic surfactants by MALDI-MS. 2. Oxidative biodegradation profiles of uniform octylphenol polyethoxylate in 18O-labeled water. Biomacromolecules 4:46–51

  23. Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P, Murray, ERB, Wood WA, Krieg NR (eds) Methods for general and molecular bacteriology. ASM, Washington, DC, pp 615–640

  24. Soto AM, Justicia H, Wray JW, Sonnenschein C (1991) p-Nonylphenol: an estrogenic xenobiotic released from modified polystyrene. Environ Health Persp 92:167–173

  25. Sugimoto M, Tanabe M, Hataya M, Enokibara S, Duine JA, Kawai F (2001) The first step in polyethylene glycol degradation by sphingomonads proceeds via a flavoprotein alcohol dehydrogenase containing flavin adenine dinucleotide. J Bacteriol 183:6694–6698

  26. Thompson JD, Higgins DG, Gibson TJ (1994) Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

  27. Ushida Y, Takahara Y, Ohta H (2003) Sphingobium amiense sp. nov., a novel nonylphenol-degrading bacterium isolated from a river sediment. Inter J Syst Evol Microbiol 53:2045–2048

  28. Wintgens T, Gallenkemper M, Melin T (2003) Occurrence and removal of endocrine disrupters in landfill leachate treatment plants. Water Sci Technol 48:127–134

  29. Yuan SY, Chang JS, Yen J, Chang BV (2000) Biodegradation of phenanthrene in river sediment. Chemosphere 43:273–278

  30. Yuan SY, Yu CH, Chang BV (2004) Biodegradation of nonylphenol in river sediment. Environ Pollut 127:425–430

Download references


We are grateful to Dr. M. Hatta, Okayama University of Science, and to Mr. Y. Haba for their help with the GC–MS measurement. This work was partly supported by the Research Grant for Encouragement of Studies to F. K. and X. L. from the Graduate School of Natural Science and Technology, Okayama University. We appreciate the review of the manuscript prior to submission by KN International.

Author information

Correspondence to Fusako Kawai.

Additional information

English edition: The paper was edited by a native speaker through KN international (http://www.kninter.com)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Liu, X., Tani, A., Kimbara, K. et al. Metabolic pathway of xenoestrogenic short ethoxy chain-nonylphenol to nonylphenol by aerobic bacteria, Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90. Appl Microbiol Biotechnol 72, 552–559 (2006). https://doi.org/10.1007/s00253-005-0288-z

Download citation


  • Sewage Sludge
  • Nonylphenol
  • Nicotinamide Adenine Dinucleotide
  • Aerobic Degradation
  • Tetraethylene Glycol