Int. J. Environ. Sci. Technol. (2015) 12:1243–1252 DOI 10.1007/s13762-014-0498-y ORIGINAL PAPER Insights into polyaromatic hydrocarbon biodegradation by Pseudomonas stutzeri CECT 930: operation at bioreactor scale and metabolic pathways F. Moscoso • F. J. Deive • M. A. Longo • M. A. Sanroma´n Received: 11 February 2013 / Revised: 22 November 2013 / Accepted: 3 January 2014 / Published online: 30 January 2014 Ó Islamic Azad University (IAU) 2014 Abstract Contamination of the environment with poly- Introduction cyclic aromatic hydrocarbons is one of the major problems facing the industrialized nations today. In this work, Polycyclic aromatic hydrocarbons (PAHs) are common Pseudomonas stutzeri CET 930 was studied for the first environmental pollutants that contain at least two or more time as bioremediation agent for the degradation of efflu- fused aromatic rings in linear, angular or cluster arrange- ents containing phenanthrene, pyrene and benzanthracene, ments. They represent a large and heterogeneous group of both individually and mixed. The promising results of hydrophobic organic pollutants, and their presence in the degradation obtained at flask scale (92, 63 and 94 % in environment is caused by natural and anthropogenic 7 days, respectively) marks the onset of the operation at events, such as forest and rangeland fires or the incomplete bench scale bioreactor. The overall biotransformation of combustion of fossil fuels and petroleum (Haritash and phenanthrene, pyrene and benzanthracene in batch opera- Kaushik 2009). tion mode was 95, 78 and 82 % when present individually, Among the major pollutants, PAHs are widely distrib- and 100, 98 and 100 % when carrying out the biological uted environmental contaminants that are known to exert process in cometabolic conditions, respectively. We have acutely toxic effects and/or possess mutagenic, teratogenic demonstrated the great versatility of this strain for the or carcinogenic properties (Simarro et al. 2011). Due to all degradation of structurally different contaminants, such as these reasons, the US Environmental Protection Agency metal working fluids, polycyclic aromatic hydrocarbons or (USEPA) and the Agency for Toxic Substances and Dis- insecticides such as chlorpyrifos, which makes it a suitable ease Registry have proposed some of them as apriority candidate to be applied at industrial scale. In all cases, the pollutants (Moscoso et al. 2012d). experimental data were successfully fitted to models, which Although PAHs may be removed by physical (volatili- turned out to be valuable tools to classify the metabolites zation, photolysis and adsorption), chemical (chemical involved in the biodegradation process as biomass-related. oxidation, photocatalysis) or biological (biosorption or Since a complete mineralization was pursued, the meta- biodegradation) techniques, the latter are considered a bolic pathways of the studied contaminants have been major route for their complete degradation. Microbial proposed based on gas chromatography–mass spectrometry activities allow the mineralization of this kind of com- data. pounds into carbon dioxide and water. The potentiality of microbes as degradation agents of several PAHs thus points Keywords Bioremediation Á Pseudomonas stutzeri Á to biological treatment as the major promising alternative Polyaromatic hydrocarbons Á Bioreactor Á Metabolic to attenuate the environmental impact caused by PAHs pathway (Janbandhu and Fulekar 2011). Usually, there are many factors that influence the outcome of a biodegradation process. However, the microbial agent, the chemical & F. Moscoso Á F. J. Deive ( ) Á M. A. Longo Á M. A. Sanroma´n structure of the pollutant and the environmental conditions Department of Chemical Engineering, University of Vigo, Isaac Newton Building, 36310 Vigo, Spain are the most outstanding ones (Li et al. 2008). Biodegra- e-mail: [email protected] dation pathways involve breakdown of organic 123 1244 Int. J. Environ. Sci. Technol. (2015) 12:1243–1252 compounds, being ring fission by intracellular oxidation separately, the process was approached with a mixture of and hydroxylation the typical initial steps. the three compounds. The bioremediation strategy was The efficiency of PAH biodegradation under natural scaled-up from shake flasks to stirred tank bioreactor. Up to conditions is limited by their poor bioavailability, as a our knowledge, this is the first time that the bacterial strain consequence of their low aqueous solubility and high P. stutzeri was used for proposing a viable degradation hydrophobicity, which together with their high adsorption treatment of BaA, individually and mixed with other coefficient and high thermodynamic stability make up PAHs. This research work was conducted in the University inherent features of this kind of pollutants (Cao et al. of Vigo and was completed in October 2012. 2009). Bearing in mind these properties, considerable efforts have been focused on the enhanced solubility of these compounds with the addition of surfactants, amphi- Materials and methods philic molecules that increase the solubility of hydrophobic compounds by decreasing the interfacial surface tension at Microorganism the aqueous/organic interphase. In this way, Sponza and Gok (2010) reported that surfactants have been shown to The bacterial strain P. stutzeri CECT 930, used in this enhance both biodegradation and reaction rate, and Bau- study was obtained from the Spanish Type Culture Col- tista et al. (2009) have even confirmed that Tween 80, a lection (ATCC 17588). P. stutzeri was inoculated in plates non-ionic surfactant, was used as carbon source by a containing PS medium. The composition of this medium is Pseudomonas strain, thus being considered biodegradable (per litre)as following: yeast extract 2 g, meat extract 1 g, (A´ lvarez et al. A´ lvarez et al. 2012a, b; Ulloa et al. 2012a, casein peptone 5 g, NaCl 5 g and agar 15 g (pH 7.2). The b). plates were incubated at 26 °C for 5 days. P. stutzeri is a During the last years, a wide phylogenetic diversity of gram-negative, rod-shaped and single-polar flagellated bacteria belonging to genus Arthrobacter (Guo et al. 2008), bacterium (Lalucat et al. 2006). Burkholderia (Kim et al. 2003), Pseudomonas (Cha´vez et al. 2004), Rhodococcus (Dean-Ross et al. 2002) and Chemicals and culture media Sphingomonas (Madueno et al. 2011), among others, have been reported to be able to degrade PAHs. More specifi- Phenanthrene (PHE), pyrene (PYR) and benzo[a]anthra- cally, bacteria from the genus Pseudomonas, which is cene (BaA) (purity higher than 99 %) used in degradation present in a large number of different natural and con- experiments were purchased from Sigma-Aldrich (Ger- taminated environments, have been the subject of a great many). Relevant properties of these compounds are shown scientific interest due to both their high degree of physio- in Table 1. The non-ionic surfactant Tween 80 was sup- logical and genetic adaptability and their efficient capacity plied by Merck. All chemicals used were at least reagent to aerobically degrade a wide range of aromatic com- grade or better. PAHs stock solutions were 5 mM in pounds. From previous results of our group and informa- acetone. tion coming from the literature, Pseudomonas stutzeri Minimal medium (MM) was used for biodegradation strains have been demonstrated to be a promising microbial experiment. The medium composition was as follows (per agent able to metabolize compounds such as PHE and PYR litre): Na2HPO4Á2H2O 8.5 g, KH2PO4 3.0 g, NaCl 0.5 g, (Moscoso et al. 2012a; Seo et al. 2009; Kazunga and Ait- NH4Cl 1.0 g, MgSO4Á7H2O 0.5 g and CaCl2 14.7 mg. MM ken 2000). However, this is the first time that P. stutzeri is also contained trace elements (per litre) that are as follows: proposed for benzo[a]anthracene (BaA) degradation. CuSO4 0.4 mg, KI 1.0 mg, MnSO4ÁH2O 4.0 mg, Frequently, contaminated sites and effluents are char- ZnSO4Á7H2O 4.0 mg, H3BO3 5.0 mg, H2MoO4Á2H2O acterized by the occurrence of complex mixtures of several 1.6 mg and FeCl3Á6H2O 2.0 mg. A concentrated stock pollutants which is translated into an increased complexity solution containing these salts was prepared and added to of the biodegradation process. Since PAHs make up a the medium (2 mL/L). group of persistent compounds, different strategies have been tackled to succeed in their biodegradation, such as Biodegradation cultures cometabolism (Pathak et al. 2008; Klankeo et al. 2009). In this work, PHE (a three aromatic ring molecule), PYR Flask scale and BaA (both with four aromatic rings) have been used as models of low molecular weight (LWM) and high molec- Biodegradation experiments were carried out in 250-mL ular weight (HWM) hydrocarbons to propose a viable Erlenmeyer flasks with 50 mL of MM, containing 1 % w/v biodegradation strategy. After assessing the biodegrad- of surfactant Tween 80 and 2 % v/v of a stock solution, in ability of an effluent containing each contaminant order to achieve the solubilization of the PAHs (up to 123 Int. J. Environ. Sci. Technol. (2015) 12:1243–1252 1245 Table 1 Main physical properties of used PAHs Compound M.F. Structure M.W. B.Pt. (°C) M.Pt. (°C) V.P. (Pa at 25°C) Aqueous solubility (mg/L) -4 PHE C14H10 178.23 340 105.5 6.8 9 10 1.20 -6 PYR C16H10 202.26 393 156 2.5 9 10 0.077 -8 BaA C18H12 228.29 400 162 2.2 9 10 0.010 100 lM). The non-ionic surfactant Tween 80 was chosen of cell dry weight per litre using an experimental calibra- since non-ionic surfactants are less toxic to bacteria than tion curve. anionic and cationic surfactants (Sartoros et al. 2005). Each flask, capped with cellulose stoppers, was inoculated (3 %) PAHs analysis with actively growing cells in exponential phase (24 h). Cultures were incubated in the darkness for 7 days in an PHE, PYR and BaA concentrations in the culture media orbital shaker at 37 °C, initial pH 8.0 and 150 rpm. Sam- were analysed by reversed-phase high-performance liquid ples were withdrawn at different times to monitor PAH chromatography (HPLC) equipped with a reversed-phase biodegradation and cell density.