APPLIED MICROBIAL AND CELL PHYSIOLOGY

Novel mechanisms of biotransformation of p -tert-amylphenolby bacteria and fungi with special degradation abilitiesand simultaneous detoxification of the disinfectant

Rabea Schlueter & Anja Röder & Nadine Czekalski &Daniel Gliesche & Annett Mikolasch & Frieder Schauer

Received: 28 June 2013 /Revised: 27 September 2013 /Accepted: 28 September 2013 /Published online: 26 October 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract The compound p -tert-amylphenol (p -(1,1-dimethylpropyl)phenol) is a widely used disinfectantbelonging to the group of short branched-chain alkylphenols.It is produced in or imported into the USAwith more than onemillion pounds per year and can be found in the environmentin surface water, sediments, and soil. We have investigated forthe first time the biotransformation of this disinfectant and theaccumulation of metabolites by five bacterial strains, threeyeast strains, and three filamentous fungi, selected becauseof their ability to transform either aromatic or branched-chaincompounds. Of the 11 microorganisms tested, one yeast strainand three bacteria could not transform the disinfectant despiteof a very low concentration applied (0.005 %). None of theother seven organisms was able to degrade the short branchedalkyl chain of p -tert-amylphenol. However, two yeast strains,two filamentous fungi, and two bacterial strains attacked thearomatic ring system of the disinfectant via the hydroxylatedintermediate 4-(1,1-dimethyl-propyl)-benzene-1,2-diolresulting in two hitherto unknown ring fission products withpyran and furan structures, 4-(1,1-dimethyl-propyl)-6-oxo-6-H-pyran-2-carboxylic acid and 2-[3-(1,1-dimethyl-propyl)-5-oxo-2H-furan-2-yl]acetic acid. While the disinfectant wastoxic to the organisms applied, one of the ring cleavageproducts was not. Thus, a detoxification of the disinfectantwas achieved by ring cleavage. Furthermore, one filamentousfungus formed sugar conjugates with p -tert-amylphenol asanother mechanism of detoxification of toxic environmentalpollutants. With this work, we can also contribute to the

allocation of unknown chemical compounds withinenvironmental samples to their parent compounds.

Keywords Disinfectant . p -tert-amylphenol .

Detoxification . Biodegradation . Alkylphenols

Introduction

The compound p -tert-amylphenol belongs to thealkylphenols, a group of chemical compounds consisting ofa phenol ring substituted with a linear or branched alkyl chainof various lengths. The most commercially used members ofthe alkylphenol family are the long chain isomers, especiallynonylphenol (NP). A technical mixture of NP with more than20 highly branched isomers of the alkyl chain is the basis forthe production of NP ethoxylates, widely used as industrialsurfactants (Nguyen Ngoc et al. 2011). p -tert-amylphenol andNP are both endocrine disrupting chemicals (Guenther et al.2006) and both are widespread in the environment but due tocompletely different applications. While NPs are used asintermediates or additives for many industrial products andprocesses (Saito et al. 2004), p -tert-amylphenol is used asdisinfectant. These days disinfectants are used not only formedical purposes but are ever more important in households,as components of cosmetics, and for industrial purposes(Kümmerer 2001). These broad applications result in theirincreasing input into the environment. In particular,disinfectants with aromatic structure accumulate in theenvironment because their toxicity can discouragebiodegradation. The compound p -tert-amylphenol is mainlyused in medical facilities as an ingredient of surfacedisinfectants but also in the household and especially in theUSA, there are more than 30 products on the market withactive ingredient concentrations of p -tert-amylphenol

R. Schlueter (*) :A. Röder :N. Czekalski :D. Gliesche :A. Mikolasch : F. SchauerInstitute of Microbiology, Ernst-Moritz-Arndt-University ofGreifswald, Friedrich-Ludwig-Jahn-Str. 15, 17487 Greifswald,Germanye-mail: rabea.schlueter@uni-greifswald.de

Appl Microbiol Biotechnol (2014) 98:373–384DOI 10.1007/s00253-013-5312-0

between 0.0028 % (Coldcide, 0.25 % disinfectin wipes;National Library of Medicine TDN 2010), 1.2 % (CleanCarpet Sanitizer; National Library of Medicine TDN 2010),and 7.6% (LpHMaster product; National Library ofMedicineTDN 2010). Given the broad usage, it is listed as a HighProduction Volume Chemical in the USA being produced inor imported into the USAwith more than one million poundsper year. However, it is also applied by mushroom growers todisinfect spent mushroom compost prior to disposal (Davorenand Fogarty 2005), in the manufacture of oil-soluble resinsand as an intermediate in the manufacture of chemicals used inthe rubber and petroleum industries (Gangolli 1999). In thisway, p -tert-amylphenol reaches the surface water in lakes andrivers as well as polluting sediments and soils. If thecompound is released to soil, it is expected to have slightmobility and after releasing into water it is expected to adsorbto suspended soils and sediment (National Library ofMedicine TDN 2010) due to a log KOW of 4.03 (Schultz1987). Ecotoxicity studies showed that this compound hassignificant toxicity to aquatic organisms (Davoren andFogarty 2005). The bioconcentration factor has beencalculated to be 210 (National Library of Medicine TDN2010) suggesting a high potential for bioconcentration inaquatic organisms (Franke et al. 1994). This raises questionsabout the microbial degradability of this disinfectant, thebiotransformation mechanisms, and the toxicity of itsdegradation products. In water and soil, microorganisms playan important role in the degradation of natural compounds,and they also may have a considerable potential to degradeenvironmental pollutants like disinfectants. Therefore, wehave investigated the transformation of p -tert-amylphenol bybacteria, yeasts, and filamentous fungi mainly isolated fromenvironmental samples and with a high potential for theoxidation of aromatic ring systems or branched alkyl chains,respectively. Yeasts of the genus Trichosporon have aconsiderable potential to degrade aromatic environmentalpollutants (Hammer et al. 1998; Schauer et al. 1995;Sietmann et al. 2002). Trichosporon species are widespreadin the environment with 50 different species, including 16species with clinical relevance, and can be isolated fromwater,soil, sludge, plants, animals, and food (Colombo et al. 2011;Motaung et al. 2013). Considering the special features andabilities of many Trichosporon species, these organismsbecame ever more the focus of investigations of appliedmicrobiology such as bioremediation of soils and watercontaminated with metals (Bajgai et al. 2012) or lipidproduction with microbial lipid as alternative feedstock forbiodiesel production (Hu et al. 2011). Given their ability for aneffective transformation of aromatic environmental pollutants(Awe et al. 2009; Sietmann et al. 2000, 2001), Trichosporonasahii and Trichosporon mucoides were included in our studyof the degradation of p -tert-amylphenol. Furthermore,Ralstonia sp. has been chosen as a prokaryotic organism able

to grow with biphenyl and to degrade dibenzofurancometabolically by biphenyl-grown cells (Becher et al.2000). However, the structure of p -tert-amylphenol exhibitstwo possible positions for microbial attack: the aromatic ringsystem as well as the branched alkyl side chain. Therefore wealso included organisms able to oxidize hydrocarbons andbranched chain hydrocarbons like Rhodococcus ruber ,Mycobacterium neoaurum (Nhi-Cong et al. 2009), andNocardia cyriacigeorgica (Nhi-Cong et al. 2010) todeterminewhether there are different transformation pathwaysavailable to organisms using different carbon and energysources for growth.

Materials and methods

Microorganisms, media, as well as growth and incubationconditions

Strains of three yeasts (Candidamaltosa SBUG 700; T. asahiiSBUG 833, T. mucoides SBUG 801), three filamentous fungi(Fusarium oxysporum SBUG 1142, Mucor hiemalis SBUG255, Trametes versicolor SBUG 1401) and five bacteria (M.neoaurum SBUG 109, N. cyriacigeorgica SBUG 1472,Ralstonia sp. SBUG 290, Rhodococcus erythropolis SBUG251, and R. ruber SBUG 82), deposited at the strain collectionof the Department of Biology of the University of Greifswaldwere tested.

Biotransformation experiments to investigate theconversion of p -tert-amylphenol by different microorganismswere carried out with resting cells and the disinfectant in aminimal medium in the absence of a growth substrate. Priorbiotransformation, cells have grown with different carbon andenergy sources for biomass production and induction ofenzymes potentially involved in the transformation of p -tert-amylphenol.

For biotransformation experiments 500-ml-flasks with100 ml mineral salts medium (MM for yeasts and filamentousfungi and mineral salts medium for bacteria (MMB); seebelow) and p -tert-amylphenol at a final concentration of0.005 % (equivalent to 0.3 mM) to 0.025 % (equivalent to1.5 mM; w /v ) were shaken for 12 h at 30 °C and 180 rpm tosaturate the liquid medium with the compound, after whichthe cells were applied.

The yeast cells were grown in 500-ml-flasks containing100 ml of MM, pH 5.4, according to Hornei et al. (1972) witheither glucose or phenol. For growth with glucose, cells werecultured for 48 h in MM supplemented with 2 % glucose and1 ml vitamin solution (Van der Walt and Van Kerken 1961) ona rotary shaker at 30 °C and 180 rpm. For growth on phenol,the yeast cells (T. mucoides and T. asahii only) were firstgrown in 100 ml of MM supplemented with 1 % glucose and0.4 % yeast extract. After 24 h at 30 °C and 130 rpm on a

374 Appl Microbiol Biotechnol (2014) 98:373–384

rotary shaker 5 (T. mucoides ) or 10 ml (T. asahii ) cellsuspensions were transferred into 500-ml-flasks containing100 ml of MM supplemented with 0.05 % phenol and 0.4 %yeast extract. The cells were induced again with 0.05 %phenol for an additional 3 h. Furthermore, C. maltosa wasgrown in 500-ml-Erlenmeyer flasks with 100 ml MMsupplemented with 1 % n -dodecane and 1 % vitamin solutionand inoculated with cell material from a 48-h old malt agarculture. The flask was incubated for 3 days at 30 °C and180 rpm. Afterwards cells grown with glucose, phenol, or n -dodecane were harvested by centrifugation at 4 °C and6,000×g (T. mucoides and C. maltosa ) or 16,000×g (T.asahii) for 5 min and washed twice with sterile MM. The cellpellet was resuspended in 5 to 10 ml MM and added to theflasks with the disinfectant until an optical density (A600) of6.0 was reached. Cultures were incubated with the substratesfor 120 h on a rotary shaker at 30 °C and 130 rpm.

For the cultivation of T. versicolor a nitrogen-rich medium(NRM) was used (NRM; 5.0 g l−1 glucose, 0.52 g l−1

asparagine, 0.5 g l−1 KH2PO4, 0.5 g l−1 yeast extract,0.5 g l−1 KCl, 0.5 g l−1 MgSO4×7H2O, 50ml mineral solution(0.16 g l−1 Mn(CH3COO)2×4H2O, 0.04 g l

−1 ZnSO4×7H2O,1.0 g l−1 Ca(NO3)2×4H2O, and 0.06 g l−1 CuSO4×5H2O),50 ml FeSO4-solution (0.2 g l−1 FeSO4×7H2O)). The pH ofthe NRM was adjusted to 4.5. One piece of overgrown maltagar from a 7-day-old culture was added to a 500-ml-Erlenmeyer flask containing 100 ml NRM, and the culturewas incubated for 7 days at 30 °C under static conditionsbefore the mycelium was homogenized (IKA Ultra-Turrax,Janke & Kunkel, Staufen, Germany). For the cultivation ofstrains of the genera Mucor and Fusarium Sabouraud-medium (SM; 20 g l−1 glucose and 10 g l−1 peptone) wasused. Three overgrown malt agar pieces of a 7-day-old culturewere transferred into a 100-ml-Erlenmeyer flask containing40 ml SM and the culture was incubated for 3 days at 30 °Cand 180 rpm after which the mycelium was homogenized;2.5 ml of the homogenized mycelium of T. versicolor, M.hiemalis , or F. oxysporum were transferred into 100 ml MMsupplemented with 1 % glucose and 1 % vitamin solution andgrown at 30 °C and 150 rpm. After reaching the latelogarithmic phase, cells were harvested by centrifugation at4 °C and 4,000×g for 5 min and washed twice with sterileMM. The cell pellet was resuspended in 5 to 10 ml MM andadded to the flasks with the disinfectant until an opticaldensity (A600) of 6.0 was reached. Cultures were incubatedwith the substrates for 264 h to 312 h on a rotary shaker at30 °C and 130 rpm.

The bacteria R. erythropolis and R. ruber were grown inMMB (5.0 g l−1 NH4H2PO4, 2.5 g l−1 K2HPO4, 0.5 g l−1

MgSO4×7H2O, 0.5 g l−1 NaCl, 0.46 g l−1 K2SO4, 0.07 g l−1

CaCl2, 2.0 mg l−1 FeCl3×6H2O, and 10 ml micro-elementsolution (Fritsche 1968), pH 6.8). For cultivation with glucosecells were grown for 48 h in MMB supplemented with 2 %

glucose and 1 ml vitamin solution (Van der Walt and VanKerken 1961) on a rotary shaker at 30 °C and 180 rpm. Cellswere then harvested by centrifugation at 4 °C and 4,000×g for5 min and washed twice with sterile MMB. The cell pellet wasresuspended in 5 to 10 ml MMB and added to the flasks withMMB and disinfectant to an optical density (A600) of 6.0.Cultures were incubated with the substrates for 120 h on arotary shaker at 30 °C and 130 rpm.

To cul t ivate R. ruber , M. neoaurum , and N.cyriacigeorgica with n -hexadecane or pristane, 48-h-oldculture from nutrient agar were plated on mineral salts agar(MMwith 18 g l−1 agar-agar, pH 6.8) in Petri dishes. Sterilizedfilters in the cover of the plates were saturated with 300 μlhexadecane or 100 μl pristane, respectively, and the cultureswere incubated for 3–4 (n -hexadecane) and 7 days (pristane)at 30 °C. The cells of 5 plates were resuspended in a smallvolume of MMB and transferred into flasks with MMB anddisinfectant. Cultures were incubated with the substrates for78 to 120 h on a rotary shaker at 30 °C and 130 rpm.

Ralstonia sp. was grown with biphenyl according toBecher (Becher et al. 2000). Briefly, cells were cultivated innutrient broth for 6 h on a rotary shaker at 30 °C and 180 rpmand then transferred into MMB with 0.1 % biphenyl. After60 h cultivation at 30 °C and 130 rpm cells were harvested bycentrifugation at 4 °C and 10,000×g for 8 min and washedtwice with sterile MMB. The cell pellet was resuspended in 5to 10 ml MMB and added to the flasks with MMB anddisinfectant to an optical density (A600) of 3.0. Cultures wereincubated with the substrates on a rotary shaker for 120 h at30 °C and 130 rpm.

Flasks with cells in MM or MMB without substrate, andsubstrate in MM orMMBwithout cells were used as controls.All experiments were carried out at least in duplicate.

Analysis of the formation and extraction of transformationproducts

Samples of 1 ml cell suspension were taken under sterileconditions and 100 μl of the centrifuged culture supernatantwas analyzed by high-performance liquid chromatography(HPLC). To identify transformation products the centrifugedculture supernatant of additional cultures containing 100 mlcell suspension were extracted three times with equal volumesof ethyl acetate at pH 7 and again three times afteracidification of the aqueous residue to pH 2. The organicphases were dried over anhydrous sodium sulfate and thesolvent was evaporated. The residues obtained were dissolvedin methanol and stored at −20 °C until further analysis. Oneproduct was purified by solid phase extraction with an RP-18(60 mL and 55 μm) solid-phase extraction column(Phenomenex, Torrance, USA) activated with 60 ml methanoland equilibrated with 60 ml double-distilled water. 60 ml ofthe cell-free supernatant was applied to the column which was

Appl Microbiol Biotechnol (2014) 98:373–384 375

washed with 60 ml of 5 % methanol in 0.1 % acetic acid,60 ml of 10 % methanol in 0.1 % acetic, 90 ml of 15 %methanol in 0.1 % acetic acid, and 60 ml of 20 % methanol in0.1 % acetic acid to remove undesired impurities. Thetransformation product was then eluted with 60 ml of 30 %methanol in 0.1 % acetic acid. The isolated product wasconcentrated by rotary evaporation. The residue was dissolvedin methanol and stored at −20 °C until further analysis.

Toxicity studies

The toxicity of p -tert-amylphenol and its transformationproducts was estimated by their inhibition of the growth ofT. mucoides with glucose in MM. The cell cultivation wascarried out in 500-ml-flasks containing 100 ml MMsupplemented with 2 % glucose and 1 % vitamin solution asdescribed above. Cultures were incubated for 24 h at 30 °Cwith rotary shaking (130 rpm).

Various concentrations of the disinfectant (0.005 to 0.25%)or of its transformation products (0.005%) were added to 500-ml-flasks containing 100 ml of MM. Flasks were shaken for24 h at 30 °C and 130 rpm to achieve saturation of thecompounds in the liquid medium. 1 % glucose and 1 %vitamin solution were then added to the flasks as along withthe culture described above to an optical density (A600nm) of0.2. Flasks were shaken for 48 h to 72 h on a rotary shaker at30 °C and 130 rpm. At different intervals, samples wereremoved for spectrometric analysis (OD600nm) to determinecell growth. Supplemented flasks without disinfectants ortransformation products were used as controls.

Chemical analyses and identification of metabolites

HPLC was carried out for detection and quantification oftransformation products on an Agilent (Santa Clara, USA)HPLC apparatus 1200 series equipped with a quaternarypump system G1311A, a diode array detector G1315D set at220, 254, and 280 nm, a degasser G1322A, an auto samplerG1329A and an Agilent ChemStation. Transformationproducts were separated on a LiChroCart 125-4 RP-18endcapped (5 μm) column (Merck, Darmstadt, Germany)using a solvent system of methanol and phosphoric acid(0.1 %, v /v ) with a linear gradient from 30 to 100 %methanolover a period of 14 min at a flow rate of 1 ml min−1.

Semipreparative HPLC for the purification oftransformation products was performed on a Merck-Hitachiapparatus (Darmstadt, Germany) equipped with a L-6200 Apump, a L-4250 ultraviolet–visible (UV–vis) detector set at280 nm and a D-2500 Chromato-Integrator. Transformationproducts were separated on a LiChroCart 125–4 RP-18endcapped (5 μm) column (Merck, Darmstadt, Germany)using a solvent system of methanol and acetic acid (0.1 %,v /v ) with a linear gradient from 30 to 100 % methanol over a

period of 14 min at a flow rate of 1 ml min−1. The isolatedproducts were concentrated by rotary evaporation and theresidues dissolved in methanol.

Gas chromatography–mass spectrometry (GC/MS) wascarried out on a gas chromatograph GC 8000 linked to aMD 8000 mass selective detector (Fisons Instruments, Mainz,Germany) operating at 70 eV, fitted with a 30-m Zebron ZB5-ms column (0.25 mm by 0.25 μm film; Phenomenex®,Aschaffenburg, Germany). Separation on the column wasachieved by using a temperature program from 80 to 280 °C(10 °C min−1). To analyze acid-extractable products, thecompounds were derivatized by methylation withdiazomethane as described by De Boer and Backer (De Boerand Backer 1956) in a micro-apparatus (Aldrich-Chemie,Steinheim, Germany).

The p roduc t s we r e cha r ac t e r i z ed by l i qu i dchromatography-mass spectrometry (LC/MS) using aBruker-Daltoniks micrOTOF instrument (ionization method,electrospray ionization; dry and nebulizer gas, nitrogen;software, HyStar (Bruker)) coupled with an Agilent 1100HPLC-system (solvent system consisting of formic acid,0.1 % (eluent A) and acetonitril (eluent B), starting from aninitial ratio of 90 % A and 10 % B and reaching 100 % Bwithin 14 min, flow rate 0.5 ml min−1) or by direct infusionand electro spray ionisation using a QSTAR Elite instrument(Applied Biosystems/MSD Analytical Technologies, USA).The nuclear magnetic resonance (NMR) spectra wererecorded on a Bruker AVANCE 600 MHz (Rheinstetten,Germany). The transformation products were dissolved inmethanol-d4 (MeOH-d4), dimethylsulfoxide-d6 (DMSO-d6),or acetonitril-d3.

Deconjugation experiments

Deconjugation was carried out using β-glucosidase accordingto Casillas et al. (1996), β-glucuronidase and arylsulfataseaccording to Cerniglia et al. (1982), and xylosidase accordingto Sutherland et al. (1992). The assay contained 50 mglyophilizate and assays without the addition of enzyme wereused as controls. HPLC analyses of the enzyme treatedsamples enabled the identification of the sugar moiety of theconjugate.

Chemicals

p -tert-amylphenol (purity>99 %) was obtained from Aldrich(Steinheim, Germany). All other chemicals were of highestpurity available.

All experiments were carried out at least in duplicate. Justthe determination of the toxicity of the biotransformationproducts was done once only due to the limited amount ofpurified products.

376 Appl Microbiol Biotechnol (2014) 98:373–384

Results

Biotransformation of p -tert-amylphenol by yeasts

Irrespective of the carbon source used for pre-cultivation(glucose, phenol, or n -dodecane), C. maltosa was not ableto transform p -tert-amylphenol. In contrast, during theincubation of T. mucoides and T. asahii with p -tert-amylphenol, three transformation products were detected byHPLC analysis. These we refer to as product I (R f (HPLC)10.3 min, UV–vis (MeOH) λmax 280 nm), product II (R f

(HPLC) 7.2 min, UV–vis (MeOH) λmax 300 nm), and productIII (R f (HPLC) 6.5 min, UV–vis (MeOH) without λmax)(Table 1). Glucose-grown cells and phenol-grown cells of T.mucoides oxidized 94 % of the substrate within 120 h andformed the same products, but differed in regard to thereaction time and the amount of products formed. Phenolgrown cells formed product I and II much faster and in higherconcentrations than did cells grown on glucose.

Product I was extracted at pH 7, while the other twoproducts were enriched only by the extraction at pH 2. Forstructure elucidation product I and III were separated by semipreparative HPLC and product II by solid phase extractionprior to identification by GC/MS and NMR analyses (Table 2).

Identification of product I

GC/MS analysis of the derivatized product I showed amolecular mass (m /z ) of 208. 1H-NMR (MeOH-d4, Table 2):

δ 0.67 (t, J =7.4 Hz, 3H, H-10), 1.21 (s, 6H, H-8), 1.58 (m, J =7.4 Hz, 2H, H-9), 6.63 (dd, J =8.3 Hz, J =1.9 Hz, 1H, H-5),6.68 (d, J =8.3 Hz, 1H, H-6), 6.77 (d(s), J =1.9 Hz,1H, H-3).13C-NMR (MeOH-d4, Table 2): 9.2 (C-10), 28.9 (C-8), 37.8(C9), 37.9 (C-7), 114.3 (C-3), 115.6 (C-6), 118.0 (C-5), 142.1(C-4), 143.3 (C-1), and 145.5 (C-2). HMBC correlations(MeOH-d4; Table 2): H-3 (C-1, C-2, C-5, and C-7), H-5 (C-1, C-2, C-3, and C-7), H-6 (C-1, C-2, and C-4), H-8 (C-3, C-4,C-5, C-7, C-8, C-9, and C-10), H-9 (C-4, C-7, C-8, and C-10),and H-10 (C-7 and C-9). The data obtained by GC/MS andNMR analysis led to the identification of product I as 4-(1,1-dimethyl-propyl)-benzene-1,2-diol (Table 2).

Identification of product II

GC/MS analysis of the derivatized product showed amolecular mass (m /z ) of 224. 1H-NMR (acetonitril-d3;Table 2): δ 0.72 (t, J =7.5 Hz, 3H, H-11), 1.19 (s, 6H, H-9),1.60 (m, J =7.5 Hz, 2H, H-10), 6.30 (d(s), J =1.5 Hz, 1H, H-5), and 7.14 (d(s), J =1.4 Hz, 1H, H-3). 13C-NMR (acetonitril-d3, Table 2): 8.9 (C-11), 26.1 (C-9), 34.4 (C-10), 40.0 (C-8),110.2 (C-3), 115.5 (C-5), 150.0 (C-2), 161.4 (C-7), 161.8 (C-6), and 166.3 (C-4). HMBC correlations (acetonitril-d3;Table 2): H-3 (C-2, C-5, C-7, and C-8), H-5 (C-3, C-6, andC-8), H-9 (C-3, C-4, C-5, C-8, C-9, C-10, and C-11), H-10 (C-4, C-8, C-9, and C-11), and H-11 (C-8 and C-10). 1H-NMR(DMSO-d6; Table 2): δ 0.67 (t, J =7.5 Hz, 3H, H-11), 1.16 (s,6H, H-9), 1.56 (m, J =7.5 Hz, 2H, H-10), 6.31 (d(s), J =1.7 Hz, 1H, H-5), and 7.12 (d(s), J =1.6 Hz, 1H, H-3). 13C-

Table 1 Products formed duringthe incubation of yeasts,filamentous fungi, and bacteriawith 0.005 % (equivalent to0.3 mM) p-tert-amylphenol aftercultivation with different carbonand energy sources and detectedby HPLC analysis

Abbreviations: “+” detected, “−”not detected, P product, P I4-(1,1-dimethyl-propyl)-benzene-1,2-diol, P II 4-(1,1-dimethyl-propyl)-6-oxo-6-H-pyran-2-carboxylic acid, P III 2-[3-(1,1-dimethyl-propyl)-5-oxo-2H-furan-2-yl]acetic acid, P IV sugarconjugatea Incubation period differedbetween the organisms (see“Materials and methods”)

Microorganisms Carbon sourcefor cultivation

Substrate degradationduring incubationperioda (%)

Transformation productsof p-tert-amylphenol

P I P II P III P IV

Candida maltosa Glucose 0 − − − −Phenol 0 − − − −n-dodecane 0 − − − −

Trichosporon asahii Glucose 71 + + + −Phenol 77 + + + −

Trichosporon mucoides Glucose 78 + + + −Phenol 88 + + + −

Fusarium oxysporum Complex medium 94 − + − −Mucor hiemalis Complex medium 92 + + − −Trametes versicolor Complex medium 100 − − − +

Mycobacterium neoaurum Pristane 0 − − − −Nocardia cyriacigeorgica Pristane 0 − − − −Ralstonia sp. Biphenyl 68 + + − −Rhodococcus erythropolis Glucose 0 − − − −Rhodococcus ruber Glucose 97 + + + −

n-hexadecane 100 + + + −Pristane 63 + + + −

Appl Microbiol Biotechnol (2014) 98:373–384 377

NMR (DMSO-d6, Table 2): 8.6 (C-11), 25.6 (C-9), 33.2 (C-10), 38.3 (C-8), 108.6 (C-3), 114.0 (C-5), 149.4 (C-2), 160.5(C-7), 160.6 (C-6), and 165.0 (C-4). HMBC correlations(DMSO-d6; Table 2): H-3 (C-2, C-5, C-7, and C-8), H-5 (C-3, C-6, and C-8), H-9 (C-4, C-5, C-8, C-9, C-10, and C-11), H-10 (C-4, C-8, C-9, and C-11), and H-11 (C-8 and C-10). Byusing GC/MS and NMR analysis, product II was identified as4-(1,1-dimethyl-propyl)-6-oxo-6-H-pyran-2-carboxylic acid(Table 2).

Identification of product III

The molecular mass of the non derivatized product has beendetermined by LC/MS analysis: (C11H16O4) calcd: [M+H]+

213.112684; found: pos. ion mode [M+H]+ m /z 213.113359(−7.5 ppm); [M+Na]+ 235.094627; found: pos. ion mode [M+Na]+ m /z 235.098267 (−17.8 ppm); calcd: [M−H]−

211.097034; found: neg. ion mode [M−H]− m /z 211.093921(17.4 ppm). The identification of product III could only be

Table 2 Oxidation products formed during the biotransformation of p-tert-amylphenol by different bacteria, yeasts and filamentous fungi asrevealed by coupling of gas chromatography andmass spectrometry (GC/

MS), coupling of liquid chromatography and mass spectrometry (LC/MS), and nuclear magnetic resonance spectroscopy (NMR) analysis

Compounds Molecular mass (m/z) Identification Structure

GC/MS LC/MS

I 208

(methylated)

Not

determined

4-(1,1-Dimethyl-propyl)-

benzene-1,2-diol

II 224

(methylated)

Not

determined

4-(1,1-Dimethyl-propyl)-

6-oxo-6-H-pyran-2-

carboxylic acid

III Not

determinable

212 2-[3-(1,1-Dimethyl-

propyl)-5-oxo-2H-furan-

2-yl]acetic acid

III

Methylated

Not

determinable

Not

determined

2-[3-(1,1-Dimethyl-

propyl)-5-oxo-2H-furan-

2-yl]acetic acid methyl

ester

56

OH

OH1

23

47

89

10

OOOH

O1

235

6

7

8910

11

4

O

O

O

OH

1

2

3

5

67

8910

11

4

O

O

O

O

1

2

3

5

67

8910

11

412

378 Appl Microbiol Biotechnol (2014) 98:373–384

achieved by NMR analyses of both the nonderivatized and themethylated product. 1H-NMR (DMSO-d6; Table 2): δ 0.77 (t,J =7.4 Hz, 3H, H-11), 1.13 (s, 3H, H-9), 1.15 (s, 3H, H-9),1.51 (m, J =7.0 Hz, 2H, H-10), 2.36 (dd, J =9.2 Hz, J =16.3 Hz, 1H, H-6), 3.15 (dd, J =3.1 Hz, J =16.3 Hz, 1H, H-6), 5.34 (m, J =1.5 Hz, J =3.0 Hz, J =9.2 Hz, 1H, H-2), 5.99(d(s), J =1.5 Hz, 1H, H-4). 13C-NMR (DMSO-d6; Table 2):8.6 (C-11), 26.0 (C-9), 26.4 (C-9), 33.3 (C-10), 36.7 (C-8),38.0 (C-6), 79.5 (C-2), 116.5 (C-4), 170.5 (C-7), 171.8 (C-5),and 178.8 (C-3). HMBC correlations (DMSO-d6; Table 2): H-2 (C-3, C-4, C-6, and C-7), H-4 (C-2, C-3, C-5, C-8, and C-9),H-6 (C-2, C-3, and C-7), H-9 (C-3, C-4, C-8, C-9, C-10, andC-11), H-10 (C-3, C-8, C-9, and C-11), H-11 (C-8 and C-10).1H-NMR (methylated product, DMSO-d6, Table 2): δ 0.76 (t,J =7.4 Hz, 3H, H-11), 1.12 (s, 3H, H-9), 1.14 (s, 3H, H-9),1.49 (m, J =7.2 Hz, 2H, H-10), 2.52 (dd, J =9.1 Hz, J =16.3 Hz, 1H, H-6), 3.25 (dd, J =3.1 Hz, J =16.3 Hz, 1H, H-6), 3.63 (s, 3H, H-12), 5.37 (m, J =1.5 Hz, J =3.1 Hz, J =9.1 Hz, 1H, H-2), and 6.00 (d(s), J =1.6 Hz, 1H, H-4). 13C-NMR (methylated product, DMSO-d6, Table 2): 8.5 (C-11),26.0 (C-9), 26.4 (C-9), 33.3 (C-10), 36.7 (C-8), 37.4 (C-6),51.6 (C-12), 79.3 (C-2), 116.8 (C-4), 169.8 (C-7), 171.8 (C-5),and 178.8 (C-3). HMBC correlations (methylated product,DMSO-d6, Table 2): H-2 (C-3, C-4, C-6, and C-7), H-4 (C-2, C-3, C-5, and C-8), H-6 (C-2, C-3, and C-7), H-9 (C-3, C-4,C-8, C-9, C-10, and C-11), H-10 (C-3, C-8, C-9, and C-11),H-11 (C-8 and C-10), H-12 (C-7). LC/MS: (C11H16O4) calcd:[M+H]+ 213.112684; found: pos. ion mode [M+H]+ m /z213.113359 (−7.5 ppm); [M+Na]+ 235.094627; found: pos.ion mode [M+Na]+ m /z 235.098267 (−17.8 ppm); calcd: [M−H]− 211.097034; found: neg. ion mode [M−H]− m /z211.093921 (17.4 ppm).

From these data, we postulate product III to be 2-[3-(1,1-dimethyl-propyl)-5-oxo-2H-furan-2-yl]acetic acid (Table 2).

Biotransformation of p -tert-amylphenol by filamentous fungi

All three fungi tested were able to oxidize the disinfectant.M.hiemalis formed 4-(1,1-dimethyl-propyl)-benzene-1,2-diol(product I) and 4-(1,1-dimethyl-propyl)-6-oxo-6-H-pyran-2-carboxylic acid (product II), F. oxysporum accumulated 4-(1,1-dimethyl-propyl)-6-oxo-6-H-pyran-2-carboxylic acid(product II) only, and T. versicolor transformed p -tert-amylphenol to product IV (R f (HPLC) 10.4 min, UV–vis(MeOH) λmax 220, 270 nm).

Identification of product IV

Product IV eluted 1 min earlier from the RP-18-column incomparison to the disinfectant but the UV–vis-spectrum ofproduct IV was similar to that of the substrate (UV–vis(MeOH) λmax=225, 275 nm). In deconjugation experiments,the lyophilized culture supernatant containing product IVonly

was incubated with β-glucosidase, xylosidase, β-glucuronidase, or arylsulfatase. HPLC analyses showed onlyproduct IVafter treatment withβ-glucosidase, xylosidase, andarylsulfatase, while p -tert-amylphenol was detected afterincubation with β-glucuronidase. Based on these resultsproduct IV is identified as a sugar conjugate of p -tert-amylphenol, it is highly probable glucuronic acid. However,given the unspecific reaction of glucuronidase towards severalsugar conjugates, other possible sugar moieties besidesglucuronic acid are glucose, rhamnose, mannose, and xylose.

Biotransformation of p -tert-amylphenol by bacteria

HPLC analyses of the culture supernatant during theincubation of pristane-grown cells of M. neoaurum and N.cyriacigeorgica as well as glucose-grown cells of R.erythropolis showed neither a substrate decrease nor productformation. Ralstonia spec. is not able to grow with glucosebut after cultivation with biphenyl it transformed p -tert-amylphenol to (1,1-dimethyl-propyl)-benzene-1,2-diol(product I) and 4-(1,1-dimethyl-propyl)-6-oxo-6-H-pyran-2-carboxylic acid (product II), while R. ruber, independent ofthe growth substrate, additionally accumulated 2-[3-(1,1-dimethyl-propyl)-5-oxo-2H-furan-2-yl]acetic acid (productIII; Table 1).

Toxicity of p -tert-amylphenol and the products formedduring the microbial transformation of p -tert-amylphenol

p -tert-amylphenol, the hydroxylated intermediate 4-(1,1-dimethyl-propyl)-benzene-1,2-diol (product I) as well as thering fission product 4-(1,1-dimethyl-propyl)-6-oxo-6-H-pyran-2-carboxylic acid (product II) were tested for theirinfluence on the growth of T. mucoides with glucose.Considering the small amount of 2-[3-(1,1-dimethyl-propyl)-5-oxo-2H-furan-2-yl]acetic acid (product III) available itcould not be tested for toxicity. The Trichosporon speciesand R. ruber form the same products, but the toxicity hasbeen investigated only on the yeast strain since the bacteriumforms pellets and hence growth cannot be measured by opticaldensity. The disinfectant is very toxic to the growing cells ofthe yeast strain and inhibited the growth in the lowestconcentration applied (0.005 %) over the first 8 h. Toinvestigate whether the transformation products are less toxicthey were added at the same concentration. The hydroxylatedintermediate is also very toxic at this concentration. However,after 30 h of cultivation cells started growing, though thegrowth was considerably delayed compared with the controlcells grown with glucose. By contrast, no growth inhibitionwas detected in the presence of 0.005 % of the ring fissionproduct 4-(1,1-dimethyl-propyl)-6-oxo-6-H-pyran-2-carboxylic acid (Fig. 1).

Appl Microbiol Biotechnol (2014) 98:373–384 379

Discussion

There are different kinds of compounds comprising anaromatic ring substituted with an alkyl chain: (1)phenylalkanes with an aromatic ring substituted with a linearalkyl chain, (2) alkylphenols with a phenolring substitutedwith a linear alkyl chain, especially nonylphenol (nNP), (3)alkylphenols with long branched alkyl chains (bAP), and (4)short branched-chain alkylphenols (sbAP). The disinfectantp -tert-amylphenol belongs to the last group.

The microbial metabolism of phenylalkanes (Awe et al.2008; Bhatia and Singh 1996; Dutta and Harayama 2001;Fedorak and Westlake 1986; Smith and Ratledge 1989) andnNP (Corti et al. 1995; Iwaki et al. 2012; Jeong et al. 2003;Vallini et al. 2001) is well investigated. In this process, eitherthe aromatic ring is oxidized or the alkyl chain is attacked. Incontrast, bAP is a very resistible group to microbial

degradation. Most investigations focused on the technicalmixture of nonylphenol (bNP) with more than 20 highlybranched isomers of the C9 alkyl chain (Nguyen Ngoc et al.2011). The quaternary α-carbon atom on the branched alkylchain prevents these compounds from microbial ω- and β-oxidation and therefore bNP are highly persistent in theenvironment. Only a few organisms mainly belonging to thegenera Sphingomonas , Sphingobium and Pseudomonas areable to use bNP as sole source of carbon and energy (Corviniet al. 2004; deVries et al. 2001; Kolvenbach and Corvini 2012;Tanghe et al. 1999). All these organisms refer to gram-negative bacteria and degrade bNP by ipso -hydroxylationand 1,2-C,O shift. In the process, the alkyl substituent of anα-quaternary nonylphenol isomer is detached as a C9 alcoholand the resulting aromatic ring system serves as carbon andenergy source (Gabriel et al. 2005b). The detachedhydroxylated branched alkyl chain (C9 alcohol) is notdegraded and observed decreasing concentrations of thisstructure are due to volatilization (Kolvenbach and Corvini2012). Thus, organisms of the genera Sphingomonas ,Sphingobium , and Pseudomonas can grow with bNP, butnone of these bacteria seems to be able to attack the branchedside chain.

Only little is known about the metabolism of sbAP. Thecompound p -tert-amylphenol is a member of this group and awidely used disinfectant highly released into the environment.Thus, p -tert-amylphenol was used in this study to investigatethe microbial transformation. The structure exhibits twopossible positions for microbial attack: the aromatic ringsystem as well as the branched alkyl side chain. Therefore,we included organisms able to attack the aromatic ring likeRalstonia sp. (Becher et al. 2000) and yeasts of the genusTrichosporon (Hammer et al. 1998; Schauer et al. 1995;Sietmann et al. 2001, 2002) as well as organisms able tooxidize hydrocarbons and branched chain hydrocarbons likeR. ruber , M. neoaurum (Nhi-Cong et al. 2009), and N.cyriacigeorgica (Nhi-Cong et al. 2010) in our investigationsto determine the possible transformation pathways of p -tert-amylphenol by microorganisms. The yeasts T. asahii and T.mucoides, known for their high potential in the transformationof aromatic environmental pollutants (Awe et al. 2009;Hammer et al. 1998; Schauer et al. 1995; Sietmann et al.2002) hydroxylated p -tert-amylphenol next to the phenolichydroxyl group and the dihydroxylated intermediateunderwent ring fission resulting in two products with lactonestructures – one contained a pyrone ring and the other a furanring (Fig. 2). Analogous to the oxidation of otherenvironmental pollutants like biphenyl and diphenyl ether byyeasts of the genus Trichosporon (Schauer et al. 1995;Sietmann et al. 2001) the formation of the pyrone is assumedto take place via the introduction of a third hydroxyl groupinto the molecule ortho to those already present. This isfollowed by an ortho -ring cleavage and a subsequent

0 10 20 30 40 50

0 10 20 30 40 50

0 10 20 30 40 50

OD

[600

nm]

OD

[600

nm]

OD

[600

nm]

Cultivation time [h]

Cultivation time [h]

Cultivation time [h]

a

b

c

0.1

0.01

1

10

0.1

0.01

1

10

0.1

0.01

1

10

Fig. 1 Influence of 0.005 % a p-tert-amylphenol (filled squares), b0.005 % of the hydroxylated intermediate 4-(1,1-dimethyl-propyl)-benzene-1,2-diol (filled triangles), and c 0.005 % of the ring fissionproduct 4-(1,1-dimethyl-propyl)-6-oxo-6-H-pyran-2-carboxylic acid(filled circles ) on the growth of T. mucoides with 1 % glucose incomparison to the control without disinfectant or transformation products(empty circles)

380 Appl Microbiol Biotechnol (2014) 98:373–384

lactonization with the loss of water (Fig. 2). This assumptionis supported by the fact that application of 3,4,5-trihydroxybiphenyl to T. mucoides results in a pyrone ofsimilar structure (Sietmann et al. 2001) as oxidation productas well as by the cleavage of pyrogallol by non-heme iron-containing dioxygenases resulting in 2-pyrone-6-carboxylicacid (Saeki et al. 1980). By contrast, the formation of thefuran is thought not to require a third hydroxylation prior toring fission, because this product is formed in an analoguesfashion to the generation of [5-oxo-2,5-dihydrofuran-2-yl]acetic acid from catechol via cis ,cis -muconic acid(Vollmer et al. 1994). In case of biphenyl, the formation ofthe ring fission product with a furan ring has been shown to beformed by a single enzyme only (Schlueter et al. 2013). Thelactones are dead end products.

Yeasts of the genus Trichosporon are well known for theirability to grow with phenol and structurally relatedmonoaromatic compounds and the enzymes involved suchas phenol monooxygenase, catechol-1,2-dioxygenase andcis ,cis -muconate cycloisomerase are well investigated(Middelhoven 1993). To what extent these enzymes areinvolved in the degradation of similar compounds like p -tert-amylphenol is not yet validated but there are strong hints

that these enzymes are involved because the transformationoccurs much faster by phenol-grown cells compared withglucose-grown cells. In addition, the enzyme involved in theformation of lactones during the biotransformation ofhydroxylated biphenyls by T. mucoides—having the samestructure like the lactones formed during the transformationof p -tert-amylphenol by yeasts of the genus Trichosporon—has been purified and characterized (Schlueter et al. 2013).Thus, hydroxylating enzymes as well as two different ringcleavage enzymes are described for yeasts of the genusTrichosporon and affirm the transformation pathway of p -tert-amylphenol by yeasts of this genus. Even though theenzymes are well known, the genes have hardly been studied.

For gram-negative bacteria able to grow with long-chainalkylphenols the existence of genes encoding phenoldegrading enzymes has been shown, too (Merimaa et al.2006; Nguyen Ngoc et al. 2011), but the participation of theseenzymes in the degradation of alkylphenols was not proved.In addition, the bacteria R. ruber and Cupriavidus basilensisseem to express phenol degrading enzymes as they oxidizedp -tert-amylphenol in the same way as the yeasts did thatmeans by a cleavage of the dihydroxylated aromatic ringsystem resulting in different lactones as dead end products.

p-tert-Amylphenol

4-(1,1-Dimethyl-propyl)-benzene-1,2-diol

2-[3-(1,1-Dimethyl-propyl)-5-oxo-2H-furan-2-yl]

acetic acid

4-(1,1-Dimethyl-propyl)-6-oxo-6H-pyran-2-carboxylic acid

sugar

(a) (b)

(c)

(Product I)

(Product III) (Product II)

(Product IV)

CCH2

CH3CH3

CH3

OH COOHCOOH

O

CCH2

CH3CH3

CH3

OHOOC

C

OH

CH2

CH3CH3

CH3

OH OH

CCH2

CH3CH3

CH3

OO

HOOC

C

OH

CH2

CH3CH3

CH3

OH

C

OH

CH2

CH3CH3

CH3

C

O

CH2

CH3CH3

CH3

Fig. 2 Proposed pathway for the transformation of p-tert-amylphenol by the yeasts T. asahii and T. mucoides (a , b), the filamentous fungiM. hiemalis,F. oxysporum (b), and T. versicolor (c) as well as the bacteria R. ruber (a , b) and Ralstonia sp. (b)

Appl Microbiol Biotechnol (2014) 98:373–384 381

Whether this is realized by an ortho - or meta -cleavage couldnot be determined, but no colored intermediates were formed.By contrast, similar compounds like p -tert-butylphenol or 2-sec -butylphenol are oxidized via meta -cleavage only bySphingobium fuliginis (Toyama et al. 2010b) andPseudomonas sp. strain MS-1 (Toyama et al. 2010a),respectively, and support growth.

It is well known that the branched side chain ofalkylphenols is very resistant to microbial attack due to thequaternary α-carbon atom (Kolvenbach and Corvini 2012).Even though we used organisms able to attack branched alkylchains no metabolites pointed to a microbial side chain attack.M. neoaurum and N. cyriacigeorgica are able to oxidizebranched alkyl chains without quaternary carbon atoms(Nhi-Cong et al. 2010), but did not oxidize the disinfectantat all. Our results confirm the high persistence of quaternarycarbon atom containing substances in the environment. Theshort chain length is crucial, too. While Sphingomonasxenophaga can utilize bNP as sole carbon and energy source,it is not able to grow with tert-butylphenol (Gabriel et al.2005a). The same has been observed in the present study forthe anamorphic ascomycetous yeast C. maltosa . Even thoughC. maltosa is able to grow with aromatics like phenol(Hammer et al. 1996) as well as alkanes (Awe et al. 2008)this yeast is not able to oxidize p -tert-amylphenol, but usesphenylalkanes with side chains longer than C7 as growthsubstrates (Awe et al. 2008). Neither glucose nor n -dodecanecould induce enzymes in C. maltosa capable of oxidativeattack on p -tert-amylphenol.

All species tested have in common that they are not able touse p -tert-amylphenol as a carbon and energy source. Asshown with T. mucoides , the oxidation of this disinfectantserves as a mechanism to drastically reduce its toxicity. Whilethe hydroxylated intermediate still inhibits growth of the yeastinitially even more than the substrate (Fig. 1b), the ring fissionproduct 4-(1,1-dimethyl-propyl)-6-oxo-6-H-pyran-2-carboxylic acid does not (Fig. 1c). The disinfectant and thehydroxylated intermediate totally inhibit cell growth duringthe first 8 or 30 h of cultivation, so the hydroxylatedintermediate seems to be more toxic than p -tert-amylphenol.The onset of growing after a certain time (Fig. 1a, b) is basedon a diminished concentration of the compounds tested due totheir transformation into ring fission products which have notoxic effects on the cells. Thus, a detoxification of the toxic p -tert-amylphenol was achieved by the yeasts as well as by thebacteria via cleavage of the aromatic ring system. Bycomparison, T. versicolor did not form ring fission productsbut instead sugar conjugates with p -tert-amylphenol as analternative mechanism for detoxification. The formation ofsugar or sulfate conjugates is a common detoxificationstrategy used by many eukaryotes from fungi to mammalsfor dealing with other aromatic environmental pollutants suchas biphenyl (Dodge et al. 1979; Golbeck et al. 1983; Meyer

and Scheline 1976; Romero et al. 2005), chlorinated biphenyls(Block and Cornish 1959; Schnellmann et al. 1984), ordibenzofuran (Hammer et al. 2001).

Hospital disinfection is very important to prevent infectionsand thus the utilization of disinfectants is necessary. However,the effectiveness of a disinfectant dictates the usageindependent on the microbial degradability or environmentalimpact. Thus, the microbial degradation of disinfectants needsto be investigated. Bacteria, yeasts, and filamentous fungiused in our studies were able to transform p -tert-amylphenolto ring cleavage products with lactone structures or conjugatesbeing less toxic than the disinfectant or nontoxic. In addition,in natural environments, there are many other organismsunknown so far maybe able to use these transformationproducts as growth or transformation substrates. Furthermore,we could show that these uncommon products consisting of alactone ring substituted with a short branched alkyl chainhitherto not found in bacteria during degradation experimentswith branched chain alkylbenzenes or alkylphenols resultfrom the transformation of aromatic disinfectants.

Acknowledgments The authors thank Michael Lalk for providingNMR data and Robert Jack for reviewing the manuscript.

References

Awe S, Mikolasch A, Hammer E, Schauer F (2008) Degradation ofphenylalkanes and characterization of aromatic intermediates actingas growth inhibiting substances in hydrocarbon utilizing yeastCandida maltosa . Int Biodeterior Biodegrad 62(4):408–414. doi:10.1016/j.ibiod.2008.03.007

Awe S, Mikolasch A, Schauer F (2009) Formation of coumarines duringthe degradation of alkyl substituted aromatic oil components by theyeast Trichosporon asahii . Appl Microbiol Biotechnol 84(5):965–976. doi:10.1007/s00253-009-2044-2

Bajgai RC, Georgieva N, Lazarova N (2012) Bioremediation ofchromium ions with filamentous yeast Trichosporon cutaneumR57. J Biol Earth Sci 2:B70–B75

Becher D, Specht M, Hammer E, Francke W, Schauer F (2000)Cometabolic degradation of dibenzofuran by biphenyl-cultivatedRalstonia sp strain SBUG 290. Appl Environ Microbiol 66(10):4528–4531. doi:10.1128/AEM.66.10.4528-4531.2000

Bhatia M, Singh HD (1996) Biodegradation of commercial linear alkylbenzenes by Nocardia amarae . J Biosci 21(4):487–496. doi:10.1007/bf02703213

Block WD, Cornish HH (1959) Metabolism of biphenyl and 4-chlorobiphenyl in the rabbit. J Biol Chem 234(12):3301–3302

Casillas RP, Crow SA, Heinze TM, Deck J, Cerniglia CE (1996) Initialoxidative and subsequent conjugative metabolites produced duringthe metabolism of phenanthrene by fungi. J Ind Microbiol 16(4):205–215. doi:10.1007/BF01570023

Cerniglia CE, Freeman JP, Mitchum RK (1982) Glucuronide and sulfateconjugation in the fungal metabolism of aromatic hydrocarbons.Appl Environ Microbiol 43(5):1070–1075

Colombo AL, Padovan ACB, Chaves GM (2011) Current knowledge ofTrichosporon spp. and Trichosporonosis. ClinMicrobiol Rev 24(4):682–700. doi:10.1128/cmr.00003-11

382 Appl Microbiol Biotechnol (2014) 98:373–384

Corti A, Frassinetti S, Vallini G, Dantone S, Fichi C, Solaro R (1995)Biodegradation of nonionic surfactants. 1. Biotransformation of 4-(1-nonyl)phenol by a Candida maltosa isolate. Environ Pollut90(1):83–87. doi:10.1016/0269-7491(94)00080-w

Corvini PFX, Vinken R, Hommes G, Mundt M, Hollender J, Meesters R,Schroeder HF, Schmidt B (2004) Microbial degradation of a singlebranched isomer of nonylphenol by Sphingomonas TTNP3. WaterSci Technol 50(5):189–194

Davoren M, Fogarty AM (2005) Ecotoxicological evaluation of thebiocidal agents sodium o -phenylphenol, sodium o -benzyl-p -chlorophenol, and sodium p -tertiary amylphenol. EcotoxicolEnviron Saf 60(2):203–212. doi:10.1016/j.ecoenv.2004.04.004

De Boer TD, Backer HJ (1956) Diazomethane. In: Leonard NJ (ed)Organic synthesis, vol 36. Wiley, New York, NY, pp 14–16

deVries YP, Takahara Y, Ikunaga Y, Ushiba Y, HasegawaM, Kasahara Y,Shimomura H, Hayashi S, Hirai Y, Ohta H (2001) Organic nutrient-dependend degradat ion of branched nonylphenol bySphinogomonas sp. YT isolated from a river sediment sample.Microbiol Environ 16:240–249. doi:10.1264/jsme2.2001.240

Dodge RH, Cerniglia CE, Gibson DT (1979) Fungal metabolism ofbiphenyl. Biochem J 178(1):223–230

Dutta TK, Harayama S (2001) Biodegradation of n-alkylcycloalkanesand n-alkylbenzenes via new pathways in Alcanivorax sp strainMBIC 4326. Appl Environ Microbiol 67(4):1970–1974. doi:10.1128/aem.67.4.1970-1974.2001

Fedorak PM, Westlake DWS (1986) Fungal metabolism of normal-alkylbenzens. Appl Environ Microbiol 51(2):435–437

Franke C, Studinger G, Berger G, Böhling S, Bruckmann U, Cohors-Fresenborg D, Jöhncke U (1994) The assessment ofbioaccumulation. Chemosphere 29(7):1501–1514. doi:10.1016/0045-6535(94)90281-x

Fritsche W (1968) Effect of carbon sources on growth rate, proteincontent and enzyme pattern in Candida guilliermondii . Z AllgMikrobiol 8(2):91–99

Gabriel FLP, Giger W, Guenther K, Kohler HPE (2005a) Differentialdegradation of nonylphenol isomers by Sphingomonas xenophagaBayram. Appl Environ Microbiol 71(3):1123–1129. doi:10.1128/aem.71.3.1123-1129.2005

Gabriel FLP, Heidlberger A, Rentsch D, Giger W, Guenther K, KohlerHPE (2005b) A novel metabolic pathway for degradation of 4-nonylphenol environmental contaminants by Sphingomonasxenophaga Bayram—ipso-hydroxylation and intramolecularrearrangement. J Biol Chem 280(16):15526–15533. doi:10.1074/jbc.M413446200

Gangolli S (1999) The dictionary of substances and their effects. RoyalSociety of Chemistry, Cambridge, UK

Golbeck JH, Albaugh SA, Radmer R (1983) Metabolism of biphenyl byAspergillus toxicarius—induction of hydroxylating activity andaccumulation of water-soluble conjugates. J Bacteriol 156(1):49–57

Guenther K, Kleist E, Thiele B (2006) Estrogen-active nonylphenolsfrom an isomer-specific viewpoint: a systematic numbering systemand future trends. Anal Bioanal Chem 384(2):542–546. doi:10.1007/s00216-005-0181-8

Hammer E, Kneifel H, Hofmann K, Schauer F (1996) Enhancedexcretion of intermediates of aromatic amino acid catabolism duringchlorophenol degradation due to nutrient limitation in the yeastCandida maltosa . J Basic Microbiol 36(4):239–243. doi:10.1002/jobm.3620360406

Hammer E, Krowas D, Schäfer A, Specht M, Francke W, Schauer F(1998) Isolation and characterization of a dibenzofuran-degradingyeast: identification of oxidation and ring cleavage products. ApplEnviron Microbiol 64(6):2215–2219

Hammer E, Schöfer L, Schäfer A, Hundt K, Schauer F (2001) Formationof glucoside conjugates during biotransformation of dibenzofuranby Penicillium canescens SBUG-M 1139. Appl MicrobiolBiotechnol 57(3):390–394

Hornei S, Weide H, Köhler M (1972) Fatty-acids of Candida strain aftergrowth on alkanes. Z Allg Mikrobiol 12(1):19–27

Hu C, Wu S, Wang Q, Jin G, Shen H, Zhao Z (2011) Simultaneousutilization of glucose and xylose for lipid production byTrichosporon cutaneum . Biotechnol Biofuels 4(1):25. doi:10.1186/1754-6834-4-25

Iwaki H, Takada K, Hasegawa Y (2012) Maricurvus nonylphenolicusgen. nov., sp nov., a nonylphenol-degrading bacterium isolated fromseawater. FEMS Microbiol Lett 327(2):142–147. doi:10.1111/j.1574-6968.2011.02471.x

Jeong JJ, Kim JH, Kim CK, Hwang I, Lee K (2003) 3- and 4-alkylphenoldegradation pathway in Pseudomonas sp strain KL28: geneticorganization of the lap gene cluster and substrate specificities ofphenol hydroxylase and catechol 2,3-dioxygenase. Microbiol SGM149:3265–3277. doi:10.1099/mic.0.26628-0

Kolvenbach BA, Corvini PFX (2012) The degradation of alkylphenols bySphingomonas sp strain TTNP3-a review on seven years of research.New Biotechnol 30(1):88–95. doi:10.1016/j.nbt.2012.07.008

Kümmerer K (2001) Drugs in the environment: emission of drugs,diagnostic aids and disinfectants into wastewater by hospitals inrelation to other sources—a review. Chemosphere 45(6–7):957–969. doi:10.1016/S0045-6535(01)00144-8

Merimaa M, Heinaru E, Liivak M, Vedler E, Heinaru A (2006) Groupingof phenol hydroxylase and catechol 2,3-dioxygenase genes amongphenol- and p-cresol-degradingPseudomonas species and biotypes.Arch Microbiol 186(4):287–296. doi:10.1007/s00203-006-0143-3

Meyer T, Scheline RR (1976) Metabolism of biphenyl. II. Phenolicmetabolites in rat. Acta Pharmacol Toxicol 39(4):419–432

Middelhoven WJ (1993) Catabolism of benzene compounds byascomycetous and basidiomycetous yeasts and yeastlike fungi.Antonie Van Leeuwenhoek 63:125–144

Motaung T, Albertyn J, Kock JF, Lee C-F, Suh S-O, Blackwell M, Pohl C(2013) Trichosporon vanderwaltii sp. nov., an asexualbasidiomycetous yeast isolated from soil and beetles. Antonie VanLeeuwenhoek 103:313–319. doi:10.1007/s10482-012-9811-2

N a t i o n a l L i b r a r y o f Me d i c i n e TDN ( 2 0 1 0 ) 4 - ( 1 , 1 -Dimethylpropyl)phenol. Publisher. http://toxnet.nlm.nih.gov/cgi-bin/sis/search/a?dbs+hsdb:@term+@DOCNO+5236. Accessed 05November 2012

Nguyen Ngoc T, Hsieh H-C, Lin Y-W, Huang S-L (2011) Analysis ofbacterial degradation pathways for long-chain alkylphenolsinvolving phenol hydroxylase, alkylphenol monooxygenase andcatechol dioxygenase genes. Bioresour Technol 102(5):4232–4240. doi:10.1016/j.biortech.2010.12.067

Nhi-Cong LT, Mikolasch A, Klenk HP, Schauer F (2009) Degradation ofthe multiple branched alkane 2,6,10,14-tetramethyl-pentadecane(pristane) in Rhodococcus ruber and Mycobacterium neoaurum .Int Biodeterior Biodegrad 63(2):201–207. doi:10.1016/j.ibiod.2008.09.002

Nhi-Cong LT, Mikolasch A, Awe S, Sheikhany H, Klenk HP, Schauer F(2010) Oxidation of aliphatic, branched chain, and aromatichydrocarbons by Nocardia cyriacigeorgica isolated from oil-polluted sand samples collected in the Saudi Arabian Desert. JBasic Microbiol 50(3):241–253. doi:10.1002/jobm.200900358

Romero MC, Hammer E, Hanschke R, Arambarri AM, Schauer F (2005)Biotransformation of biphenyl by the filamentous fungusTalaromyces helicus . World J Microbiol Biotechnol 21(2):101–106. doi:10.1007/s11274-004-2779-y

Saeki Y, Nozaki M, Senoh S (1980) Cleavage of pyrogallol by non-hemeiron-containing dioxygenases. J Biol Chem 255(18):8465–8471

Saito T, Kato K, Yokogawa Y, Nishida M, Yamashita N (2004)Detoxification of bisphenol A and nonylphenol by purifiedextracellular laccase from a fungus isolated from soil. J BiosciBioeng 98(1):64–66. doi:10.1016/s1389-1723(04)70243-1

Schauer F, Henning K, Pscheidl H, Wittich RM, Fortnagel P, Wilkes H,Sinnwell V, Francke W (1995) Biotransformation of diphenyl ether

Appl Microbiol Biotechnol (2014) 98:373–384 383

by the yeast Trichosporon beigelii SBUG 752. Biodegradation 6(2):173–180. doi:10.1007/BF00695348

Schlueter R, Lippmann R, Hammer E, Gesell Salazar M, Schauer F(2013) Novel insights into the fungal oxidation of monoaromaticand biarylic environmental pollutants by characterization of twonew ring cleavage enzymes. Appl Microbiol Biotechnol 97(11):5043–5053. doi:10.1007/s00253-013-4742-z

Schnellmann RG, Volp RF, Putnam CW, Sipes IG (1984) Thehydroxylation, dechlorination, and glucuronidation of 4,4'-dichlorobiphenyl (4-DCB) by human hepatic microsomes.Biochem Pharmacol 33(21):3503–3509. doi:10.1016/0006-2952(84)90127-8

Schultz TW (1987) The use of the ionization constant (pKa) in selectingmodels of toxicity in phenols. Ecotoxicol Environ Saf 14(2):178–183

Sietmann R, Hammer E, Moody J, Cerniglia CE, Schauer F (2000)Hydroxylation of biphenyl by the yeast Trichosporon mucoides .Arch Microbiol 174(5):353–361. doi:10.1007/s002030000219

Sietmann R, Hammer E, Specht M, Cerniglia CE, Schauer F (2001)Novel ring cleavage products in the biotransformation of biphenylby the yeast Trichosporonmucoides . Appl EnvironMicrobiol 67(9):4158–4165. doi:10.1128/aem.67.9.4158-4165.2001

Sietmann R, Hammer E, Schauer F (2002) Biotransformation of biaryliccompounds by yeasts of the genus Trichosporon . Syst ApplMicrobiol 25(3):332–339. doi:10.1078/0723-2020-00131

Smith MR, Ratledge C (1989) Catabolims of alkylbenzenes byPseudomonas sp. NCIB-10643. Appl Microbiol Biotechnol 32(1):68–75

Sutherland JB, Selby AL, Freeman JP, Fu PP, Miller DW, Cerniglia CE(1992) Identification of xylose conjugates formed from anthraceneby Rhizoctonia solani. Mycol Res 96:509–517

Tanghe T, Dhooge V, Verstraete W (1999) Isolation of a bacterial strainable to degrade branched nonylphenol. Appl Environ Microbiol65(2):746–751

Toyama T, Maeda N, Murashita M, Chang Y-C, Kikuchi S (2010a)Isolation and characterization of a novel 2-sec-butylphenol-degrading bacte r ium Pseudomonas sp s t ra in MS-1.Biodegradation 21(2):157–165. doi:10.1007/s10532-009-9290-y

Toyama T, Momotani N, Ogata Y, Miyamori Y, Inoue D, Sei K, Mori K,Kikuchi S, Ike M (2010b) Isolation and characterization of 4-tert-butylphenol-utilizing Sphingobium fuliginis strains fromPhragmites australis rhizosphere sediment. Appl EnvironMicrobiol 76(20):6733–6740. doi:10.1128/aem.00258-10

Vallini G, Frassinetti S, D'Andrea F, Catelani G, Agnolucci M (2001)Biodegradation of 4-(1-nonyl)phenol by axenic cultures of the yeastCandida aquaetextoris : identification of microbial breakdownproducts and proposal of a possible metabolic pathway. IntBiodeterior Biodegrad 47(3):133–140. doi:10.1016/s0964-8305(01)00040-3

Van der Walt JP, Van Kerken AE (1961) The wine yeasts of the Cape. PartV. Studies on the occurrence of Brettanomyces intermedius andBrettanomyces schanderlii . Antonie Van Leeuwenhoek 27(1):81–90

VollmerMD, Fischer P, Knackmuss HJ, SchlömannM (1994) Inability ofmuconate cycloisomerases to cause dehalogenation duringconversion of 2-chloro-cis , cis -muconate. J Bacteriol 176(14):4366–4375

384 Appl Microbiol Biotechnol (2014) 98:373–384