Folia Microbiologica

, Volume 63, Issue 3, pp 273–282 | Cite as

Potential of the strain Raoultella sp. KDF8 for removal of analgesics

  • Andrea Palyzová
  • Jiří Zahradník
  • Helena Marešová
  • Lucie Sokolová
  • Eva Kyslíková
  • Michal Grulich
  • Václav Štěpánek
  • Tomáš Řezanka
  • Pavel Kyslík
Original Article


The bacterial strain KDF8 capable of growth in the presence of diclofenac and codeine analgesics was obtained after chemical mutagenesis of nature isolates from polluted soils. The strain KDF8 was identified as Raoultella sp. based on its morphology, biochemical properties, and 16S rRNA gene sequence. It was deposited in the Czech Collection of Microorganisms under the number CCM 8678. A growing culture efficiently removed diclofenac (92% removal) and partially also codeine (about 30% degradation) from culture supernatants within 72 h at 28 °C. The degradation of six analgesics by the whole cell catalyst was investigated in detail. The maximum degradation of diclofenac (91%) by the catalyst was achieved at pHINI of 7 (1 g/L diclofenac). The specific removal rate at high concentrations of diclofenac and codeine increased up to 16.5 mg/gCDW per h and 5.1 mg/gCDW per h, respectively. HPLC analysis identified 4′-hydroxydiclofenac as a major metabolite of diclofenac transformation and 14-hydroxycodeinone as codeine transformation product. The analgesics ibuprofen and ketoprofen were also removed, albeit to a lower extent of 3.2 and 2.0 mg/gCDW per h, respectively. Naproxen and mefenamic acid were not degraded.


Microbial degradation Raoultella sp. Analgesics Co-metabolism Whole cell catalyst 



The study was supported by the long-term research development project RVO 61388971 to the Institute of Microbiology of the Czech Academy of Sciences, the Epsilon programme TH02030337 of the Technology Agency of the Czech Republic and grant No. 720414 of the Grant Agency of Charles University, Prague.


  1. Behera SK, Kim HW, Oh JE, Park HS (2011) Occurrence and removal of antibiotics, hormones and several other pharmaceuticals in wastewater treatment plants of the largest industrial city of Korea. Sci Total Environ 409:4351–4360. CrossRefPubMedGoogle Scholar
  2. Benotti MJ, Trendholm RA, Vanderford BJ, Holady JC, Standford BD, Snyder SA (2009) Pharmaceuticals and endocrine disrupting compounds in U.S. drinking water. Environ Sci Technol 43:597–603CrossRefPubMedGoogle Scholar
  3. Bort R, Mace K, Boobis A, Gomez-Lechon MJ, Pfeifer A, Castell J (1999) Hepatic metabolism of diclofenac: role of human CYP in the minor oxidative pathways. Biochem Pharmacol 58(5):787–796. CrossRefPubMedGoogle Scholar
  4. Bouju H, Nastold P, Beck B, Hollender J, Corvini PFX, Wintgens T (2016) Elucidation of biotransformation of diclofenac and 4´hydroxydiclofenac during biological wastewater treatment. J Hazard Mater 301:443–452. CrossRefPubMedGoogle Scholar
  5. Bruce NC, Wilmot CJ, Jordan KN, Trebilcock AE, Gray Stevens LD, Lowe CR (1990) Microbial degradation of the morphine alkaloids: identification of morphinone as an intermediate in the metabolism of morphine by Pseudomonas putida M10. Arch Microbiol 154:465–470CrossRefPubMedGoogle Scholar
  6. Bueno MJM, Gomez MJ, Herrera S, Hernando MD, Agüera A, Fernández-Alba AR (2012) Occurrence and persistence of organic emerging contaminants and priority pollutants in five sewage treatment plants of Spain: two years pilot survey monitoring. Environ Pollut 164:267–273. CrossRefPubMedGoogle Scholar
  7. Carballeira JD, Quezada MA, Hoyos P, Simeó Y, Hernaiz MJ, Alcantara AR, Sinisterra JV (2009) Microbial cells as catalyst for stereoselective re-ox reaction. Biotechnol Adv 27:686–714. CrossRefPubMedGoogle Scholar
  8. Claus H, Bausinger T, Lehmler I, Perre N, Fels G, Dehner U, Preuß J, König H (2007) Transformation of 2,4,6-trinitrotoluene (TNT) by Raoultella terrigena. Biodegradation 18:27–35. CrossRefPubMedGoogle Scholar
  9. Commission implementing decision (EU) 2015/495 of 20 March 2015 establishing a watch list of substances for Union-wide monitoring in the field of water policy pursuant to Directive 2008/105/EC of the European Parliament and of the Council. Official Journal C311/8, 25/10/2013Google Scholar
  10. Domaradzka D, Guzik U, Hupert-Kocurek K, Wojcieszynska D (2016) Toxicity of diclofenac and its biotransformation by Raoultella sp DD4. Pol J Environ Stud 25(5):2211–2216.  10.15244/pjoes/62681 CrossRefGoogle Scholar
  11. Drancourt M, Bollet C, Carta A, Rousselier P (2001) Phylogenetic analyses of Klebsiella species delineate Klebsiella and Raoultella gen. nov., with description of Raoultella ornithinolytica comb. nov., Raoultella terrigena comb. nov and Raoultella planticola comb. nov. Int J Syst Bacteriol 51:925–932. CrossRefGoogle Scholar
  12. Ferrari B, Paxeus N, Lo Giudice R, Pollio A, Garric J (2003) Ecotoxicological impact of pharmaceuticals found in treated wastewaters: study of carbamazepine, clofibric acid, and diclofenac. Ecotoxicol Environ Saf 56(3):450–450. CrossRefGoogle Scholar
  13. Foster PL (1991) In vivo mutagenesis. Methods Enzymol 204:114–125CrossRefPubMedPubMedCentralGoogle Scholar
  14. Gavrilescu M, Demnerová K, Aamand J, Agathos S, Fava F (2015) Emerging pollutants in the environment: present and future challenges in biomonitoring, ecological risks and bioremediation. New Biotechnol 32:147–156. CrossRefGoogle Scholar
  15. Gomez MJ, Bueno MJM, Lacorte S, Fernandez-Alba AR, Agüera A (2007) Pilot survey monitoring pharmaceuticals and related compounds in a sewage treatment plant located on the Mediterranean coast. Chemosphere 66:993–1002. CrossRefPubMedGoogle Scholar
  16. Gröning J, Held C, Garten C, Claussnitzer U, Kaschabek SR, Schlomann M (2007) Transformation of diclofenac by the indigenous microflora of river sediments and identification of a major intermediate. Chemosphere 69(4):509–516. CrossRefPubMedGoogle Scholar
  17. Guzzella L, Feretti D, Monarca S (2002) Advanced oxidation and adsorption technologies for organic micropollutant removal from lake water used as drinking-water supply. Water Res 36:4307–4318CrossRefPubMedGoogle Scholar
  18. Hallare AV, Köhler HR, Triebskorn R (2004) Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO. Chemosphere 56:659–666. CrossRefPubMedGoogle Scholar
  19. Harder PA, Kunz DA (1989) Bacterial hydroxylation of codeine. United States Patent No 4,798,792Google Scholar
  20. Ikehata K, Naghashkar N, Ei-Din M (2006) Degradation of aqueous pharmaceuticals by ozonation and advanced oxidation processes: a review. Ozone Sci Eng 28:353–414CrossRefGoogle Scholar
  21. Jairaj M, Watson DG, Grant MH, Skellern GG (2003) The toxicity of opiates and their metabolites in HepG2 cells. Chem Biol Interact 146:121–129. CrossRefPubMedGoogle Scholar
  22. Ji S, Liu Z, Liu Z, Ren H (2007) Isolation and characterization of a bacterial strain that efficiently degrades sex steroid hormones. Front Environ Sci Engin China 1:325–328CrossRefGoogle Scholar
  23. Jones OA, Lester JN, Voulvoulis N (2005) Pharmaceuticals: a threat to drinking water. Trends Biotechnol 23(4):163–167. CrossRefPubMedGoogle Scholar
  24. Jux U, Baginski RM, Arnold HG, Kronke M, Seng PN (2002) Detection of pharmaceutical contaminations of river, pond, and tap water from Cologne (Germany) and surroundings. Int J Hyg Environ Health 205(5):393–398. CrossRefPubMedGoogle Scholar
  25. Kallio JM, Lahti M, Oikari A, Kronberg L (2010) Metabolites of the aquatic pollutant diclofenac in fish bile. Environ Sci Technol 44(19):7213–7219. CrossRefPubMedGoogle Scholar
  26. Kumar S, Samuel K, Subramanian R, Braun MP, Stearns RA, Chiu SL, Evans DC, Baillie TA (2002) Extrapolation of diclofenac clearance from in vitro microsomal metabolism data: role of acyl glucuronidation and sequential oxidative metabolism of the acyl glucuronide. J Pharmacol Exp Ther 303(3):969–978. CrossRefPubMedGoogle Scholar
  27. Kumar S, Stecher G, Tamura K (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–18744.
  28. Kyslíková E, Babiak P, Štepánek V, Zahradník J, Palyzová A, Marešová H, Valešová R, Hájíček J, Kyslík P (2013) Biotransformation of codeine to 14-OH-codeine derivatives by Rhizobium radiobacter R89-1. J Mol Catal B Enzym 87:1–5. CrossRefGoogle Scholar
  29. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, New York, pp 115–175Google Scholar
  30. Langenhoff A, Inderfurth N, Veuskens T, Schraa G, Blokland M, Kujawa-Roeleveld K, Rijnaarts H (2013) Microbial removal of the pharmaceutical compounds ibuprofen and diclofenac from wastewater. Bio Med Res Int.
  31. Liang Y, Zeng F, Qiu G, Lu X, Liu X, Gao H (2009) Co-metabolic degradation of dimethoate by Raoultella sp. X1. Biodegradation 20:363–373. CrossRefPubMedGoogle Scholar
  32. Liras P, Umbreit WW (1975) Transformation of morphine by resting cells and cell-free systems of Arthrobacter sp. Appl Microbiol 30:262–266PubMedPubMedCentralGoogle Scholar
  33. Liras P, Kasparian SS, Umbreit WW (1975) Enzymatic transformation of morhine by hydroxysteroid dehydrogenase from Pseudomonas testosterone. Appl Microbiol 30:650–656PubMedPubMedCentralGoogle Scholar
  34. Liu CY, Speitel GE, Georgiou G (2001) Kinetics of methyl t-butyl ether co-metabolism at low concentration by pure cultures of butane-degrading bacteria. Appl Environ Microbiol 67:2197–2201. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Madyastha KM, Reddy GVB (1994) Mucor piriformis, an efficient N -dealkylating reagent for thebaine and its N -variants. J Chem Soc Perkin Trans 1:911–912. doi:
  36. Meerburg F, Hennebel T, Vanhaecke L, Verstraete W, Boon N (2012) Diclofenac and 2-anilinophenylacetate degradation by combined activity of biogenic manganese oxides and silver. Microb Biotechnol 5:388–395. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Murdoch RW, Hay AG (2005) Formation of catechols via removal of acid side chains from ibuprofen and related aromatic acids. Appl Environ Microbiol 71:6121–6125. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Murdoch RW, Hay AG (2013) Genetics and chemical characterization of ibuprofen degradation by Sphingomonas Ibu-2. Microbiology 159:621–632. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Murdoch RW, Hay AG (2015) The biotransformation of ibuprofen to trihydroxyibuprofen in activated sludge and by Variovorax Ibu-1. Biodegradation 26:105–113. CrossRefPubMedGoogle Scholar
  40. Radjenovic J, Petrovic M, Barcelo D (2009) Fate and distribution of pharmaceuticals in wastewater and sewage sludge of the conventional activated sludge (CAS) and advanced membrane bioreactor (MBR) treatment. Water Res 43(3):831–841. CrossRefPubMedGoogle Scholar
  41. Richardson SD, Plewa MJ, Wagner ED, Schoeny R, deMarini DM (2007) Occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection by-products in drinking water: a review and roadmap for research. Mutat Res-Rev Mutat 636:178–242. CrossRefGoogle Scholar
  42. Swissa N, Nitzan Y, Langzam Y, Cahan R (2014) Atrazine biodegradation by a monoculture of Raoultella planticola isolated from a herbicides wastewater treatment facility. Int Biodeter Biodegr 92:6–11. doi:
  43. Vieno N, Sillanpää M (2014) Fate of diclofenac in municipal wastewater treatment plant—a review. Environ Int 69:28–39. CrossRefPubMedGoogle Scholar
  44. Zhang Q, Rich JO, Cotterill IC, Pantaleone DP, Michels PC (2005) 14-Hydroxylation of opiates: catalytic direct autoxidation of codeinone to 14-hydroxycodeinone. J Am Chem Soc 127:7286–7287. CrossRefPubMedGoogle Scholar
  45. Zhang YJ, Geissen SU, Gal C (2008) Carbamazepine and diclofenac: removal in wastewater treatment plants and occurrence in water bodies. Chemosphere 73(8):1151–1161. CrossRefPubMedGoogle Scholar
  46. Zharikova NV, Markusheva TV, Galkin EG, Korobov VV, Zhurenko EY, Sitdikova LR, Kolganova TV, Kuznetsov BB, Turova TP (2006) Raoultella planticola, a new strain degrading 2,4,5-trichlorophenoxyacetic acid. Appl Biochem Micro 42:258–262CrossRefGoogle Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2017

Authors and Affiliations

  • Andrea Palyzová
    • 1
  • Jiří Zahradník
    • 2
    • 3
  • Helena Marešová
    • 1
  • Lucie Sokolová
    • 1
  • Eva Kyslíková
    • 1
  • Michal Grulich
    • 1
  • Václav Štěpánek
    • 1
  • Tomáš Řezanka
    • 1
  • Pavel Kyslík
    • 1
  1. 1.Institute of Microbiology of the Czech Academy of SciencesPrague 4Czech Republic
  2. 2.Department of Genetics and Microbiology, Faculty of ScienceCharles University in PraguePrague 2Czech Republic
  3. 3.Laboratory of Biomolecular RecognitionInstitute of Biotechnology, v.v.i., BIOCEVVestecCzech Republic

Personalised recommendations