Isolation and Characterization of a Rhodococcus Strain Able to Degrade 2-Fluorophenol

Appl Microbiol Biotechnol (2012) 95:511–520 DOI 10.1007/s00253-011-3696-2 ENVIRONMENTAL BIOTECHNOLOGY Isolation and characterization of a Rhodococcus strain able to degrade 2-fluorophenol Anouk F. Duque & Syed A. Hasan & Vânia S. Bessa & Maria F. Carvalho & Ghufrana Samin & Dick B. Janssen & Paula M. L. Castro Received: 15 October 2011 /Accepted: 31 October 2011 /Published online: 19 November 2011 # Springer-Verlag 2011 Abstract A pure bacterial culture able to utilize 2- Introduction fluorophenol (2-FP) as sole carbon and energy source was isolated by selective enrichment from sediments collected The rapid growth of industrial organic synthesis has led to from a contaminated site in Northern Portugal. 16S rRNA the widespread production and release of xenobiotic gene analysis showed that the organism (strain FP1) compounds, whose fate in biological waste treatment belongs to the genus Rhodococcus. When grown aerobi- processes and in the environment is often problematic and cally on 2-FP, growth kinetics of strain FP1 followed the unknown. Halogenated compounds are frequently detected Luong model. An inhibitory effect of increasing 2-FP in waste streams, and their recalcitrant and toxic properties concentrations was observed with no growth occurring at 2- have stimulated studies on their microbial metabolism and FP levels higher than ca. 4 mM. Rhodococcus strain FP1 mineralization. The biodegradation of many chlorinated was able to degrade a range of other organofluorine organic compounds has been studied (Chaudhry and compounds, including 2-fluorobenzoate, 3-fluorobenzoate, Chapalamadugu 1991; Fetzner 1998; Haggblom 1992; 4-fluorobenzoate, 3-fluorophenol, 4-fluorophenol, 3- Janssen et al. 1994; van Pée and Unversucht 2003). The fluorocatechol, and 4-fluorocatechol, as well as chlorinated behavior of fluorinated compounds, such as fluorophenols compounds such as 2-chlorophenol and 4-chlorophenol. and fluorobenzenes, has been less investigated (Key et al. Experiments with cell-free extracts and partially purified 1997; Murphy et al. 2009; Murphy 2010), even though enzymes indicated that the first step of 2-fluorophenol their use in agricultural and industrial processes is large, metabolism was conversion to 3-fluorocatechol, suggesting and cases of environmental contamination are becoming an unusual pathway for fluoroaromatic metabolism. To our more frequent. knowledge, this is the first time that utilization of 2-FP as a Fluorinated aromatics, including compounds with fluo- growth substrate by a pure bacterial culture is reported. rophenol structural units, are used as agrochemicals (Theodorides 2006), pharmaceuticals (Dimagno and Sun Keywords 2-Fluorophenol (2-FP) . Rhodococcus sp. 2006; Sun and Adejare 2006), and in many other open Biodegradation . Luong model . Monooxygenase applications (Key et al. 1997). Such compounds were considered to be biologically more inert and therefore less A. F. Duque : V. S. Bessa : M. F. Carvalho : P. M. L. Castro (*) likely to have an impact on human health or the CBQF/Escola Superior de Biotecnologia, environment. On the other hand, inert molecules tend to Universidade Católica Portuguesa, persist and accumulate in trophic chains and are more Rua Dr. António Bernardino de Almeida, difficult to remove by bioremediation. Fluorinated com- 4200-072 Oporto, Portugal e-mail: [email protected] pounds have significant biological effects as enzyme – : : inhibitors and modifiers of cell cell communication, and S. A. Hasan G. Samin D. B. Janssen they may disrupt membrane transport and processes for Laboratory of Biochemistry, Groningen Biomolecular Sciences energy generation (Key et al. 1997). Regardless of the little and Biotechnology Institute, University of Groningen, Nijenborgh 4, information available about biological degradation of 9747 AG Groningen, The Netherlands fluoroaromatic compounds, aerobic biodegradation of fluo- 512 Appl Microbiol Biotechnol (2012) 95:511–520 robenzoic acids (Engesser and Schulte 1989; Harper and 0.44 mM of 2-FP as the sole carbon source. To confirm the Blakley 1971; Oltmanns et al. 1989; Schlömann et al. capacity to grow on 2-FP, growth and fluoride releases were 1990), fluorobenzene (Carvalho et al. 2002, 2005), and 4- again monitored. One strain, designated FP1, able to fluorophenol (Ferreira et al. 2008) by cultures using these degrade 2-FP as a sole carbon and energy source was compounds as the sole source of carbon and energy has isolated. Strain FP1 was deposited at BCCM/LMG Bacteria been investigated. Collection, Gent, Belgium (accession number LMG Only a few studies related to biodegradation of 2- 26251), and at DSMZ German Collection of Microorgan- fluorophenol (2-FP) can be found in the literature. Aerobic isms and Cell Cultures (Braunschweig, Germany) (acces- biotransformation of 2-FP by several Rhodococcus strains sion number DSM 45581). (Bondar et al. 1999; Finkelstein et al. 2000), acclimated activated sludge (Chaojie et al. 2007), Gloeophyllum Classification of the 2-fluorophenol-degrading strain striatum (Kramer et al. 2004)andPenicillium strains (Hofrichter et al. 1994; Marr et al. 1996), has been reported. Strain FP1 was classified by 16S rRNA gene sequencing as In all cases, 2-FP was degraded by cometabolism in the follows. Pure bacterial colonies were suspended in 300 μl presence of other carbon sources. Recently, a strain of of a 0.85% sodium chloride solution and incubated for Pseudonocardia benzenivorans, originally isolated from 15 min at 95 °C. The suspension was then kept on ice for an enrichment with tetrachlorobenzene (Kampfer and 7 min and vortexed. Subsequently, the sample was Kroppenstedt 2004),wasreportedtogrowon4-FPas centrifuged at 14,000 rpm for 5 min and the supernatant the sole carbon source and to convert 2-FP and 3-fluorophenol was stored at −20 °C. Then, 16S rRNA gene DNA was (3-FP) when added to a resting cells suspension (Kim et al. amplified with polymerase chain reaction (PCR) using the 2010). However, no growth of P. benzenivorans on 2-FP was universal primers set 27f-1492r (Lane 1991) in a reaction found. Studies on the anaerobic transformation of 2-FP and mixture of 25 μl containing 1x PCR buffer (Promega), 3-FP by methanogenic cultures and by a 2-chlorophenol- 2.5 mM MgCl2, 2.5% dimethylsulfoxide, 200 μM of each degrading anaerobic consortium have also appeared (Londry dNTP, 25 pmol of each primer, 1.25 U Taq polymerase and Fedorak 1993;SharakGenthneretal.1989; 1990). (Promega, US), and 50–100ngofDNA.ThePCR To our knowledge, growth of a pure bacterial culture on 2- conditions were as described by Rainey et al. (1996). The FP has not been yet reported. This work describes the reactions were carried out in a Bio-Rad iCycler thermal enrichment, isolation, and characterization of a strain able cycler (Bio-Rad Laboratories, Richmond, CA, USA). PCR to degrade 2-FP and use it as sole carbon and energy products were purified using GE purification kit, according source. to the instructions of the manufacturer. Cloning of the amplicons into pGEM T-Easy vector (Promega) and sequencing was done using an Applied Biosystems type Materials and methods 3730xI automatic sequencer under BigDyeTM terminator cycling conditions by Macrogen Inc. (Seoul, Republic of Enrichment and isolation of a 2-FP-degrading bacteria Korea) with universal bacterial 16S rRNA primers (f27, f518, r800, and r1492 (Lane 1991)). To determine the Soil and rhizosphere samples collected from a contaminated phylogenetic affiliation, similarity searches were done site in Northern Portugal, which has received the discharge using the BLAST program (Altschul et al. 1997). Phyloge- of chemical industry effluents for more than 50 years (fine netic analysis was performed to accurately determine the chemistry, agrochemicals), were combined as the initial taxonomic position of strain FP1. For that, 16S rRNA gene inocula for the 2-FP enrichments. This soil (approximately sequences were aligned using the Clustal-X program 5 g) was used to inoculate 250-ml flasks containing 50 ml (Thompson et al. 1997) and trees were constructed with of sterile mineral salts medium (MM) (Caldeira et al. 1999) PAUP* version 4.0b10 (Swofford 2003)usingthe and 2-FP supplied as the sole carbon and energy source at a neighbor-joining method (Kimura two-parameter distance- concentration of 0.44 mM. Cultures were incubated in an optimized criteria). The resulting sequence was submitted orbital shaker (100 rpm) at 25 °C. Half of the suspension to GenBank (accession number HM210775). was removed and replaced with fresh medium at 6- to 7-day intervals. Growth was monitored by measuring the optical Growth kinetics density (OD) at 450 nm and liberation of fluoride was monitored using an ion-selective electrode. Bacterial strains Strain FP1 was examined for its capacity to grow in batch in the degrading culture were purified by repetitive culture using 1-l flasks filled with 250 ml of MM and 2-FP streaking onto nutrient agar (NA) medium (Lab M). These concentrations varying between 0 and 4 mM. Each flask pure cultures were then re-inoculated into MM containing was inoculated with a preculture of strain FP1 to an initial Appl Microbiol Biotechnol (2012) 95:511–520 513 optical density at 600 nm (OD600) of about 0.05, measured equilibrated with TEMG buffer (50 mM Tris–SO4,5% on a Helios Gamma spectrophotometer (Unicam Instru- glycerol, 1 mM β-mercaptoethanol, and 0.5 mM EDTA, pH ments, UK). The cultures were incubated on an orbital 7.0). After washing the column with two column volumes shaker at 150 rpm and 25 °C. Samples were taken of buffer, elution was carried out with a linear gradient of periodically for fluoride, 2-FP, and optical density measure- 0% to 100% of 0.5 M (NH4)2SO4 in the same buffer. ments. The experiments were run in triplicate and for each Fractions were collected, concentrated using 10-kDa Milli- 2-FP concentration and average growth rates were deter- pore filters (Amicon, USA), and tested for oxygenase mined. Growth rate data were fitted with nonlinear activities. Fractions containing 2-fluorophenol monooxyge- regression using SPSS statistics 17.0 (SPSS Inc., Chicago, nase were desalted and loaded separately onto a gel IL, USA).

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