Journal of Plant Physiology 188 (2015) 1–8 Contents lists available at ScienceDirect Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph The coupling of the plant and microbial catabolisms of phenanthrene in the rhizosphere of Medicago sativa ∗ Anna Muratova , Ekaterina Dubrovskaya, Sergey Golubev, Vyacheslav Grinev, Marina Chernyshova, Olga Turkovskaya Institute of Biochemistry and Physiology of Plants and Microorganisms, RAS, 13 Prospekt Entuziastov, 410049 Saratov, Russia a r t i c l e i n f o a b s t r a c t Article history: We studied the catabolism of the polycyclic aromatic hydrocarbon phenanthrene by four rhizobacte- Received 14 May 2015 rial strains and the possibility of enzymatic oxidation of this compound and its microbial metabolites Received in revised form 13 July 2015 by the root exudates of alfalfa (Medicago sativa L.) in order to detect the possible coupling of the plant Accepted 14 July 2015 and microbial metabolisms under the rhizospheric degradation of the organic pollutant. A comparative Available online 10 September 2015 study of phenanthrene degradation pathways in the PAH-degrading rhizobacteria Ensifer meliloti, Pseu- domonas kunmingensis, Rhizobium petrolearium, and Stenotrophomonas sp. allowed us to identify the key Keywords: metabolites from the microbial transformation of phenanthrene, including 9,10-phenanthrenequinone, Medicago sativa 2-carboxybenzaldehyde, and 1-hydroxy-2-naphthoic, salicylic, and o-phthalic acids. Sterile alfalfa plants Root exudates −1 Rhizobacteria were grown in the presence and absence of phenanthrene (0.03 g kg ) in quartz sand under controlled Phenanthrene environmental conditions to obtain plant root exudates. The root exudates were collected, concentrated Biodegradation pathways by ultrafiltration, and the activity of oxidoreductases was detected spectrophotometrically by the oxida- tion rate for various substrates. The most marked activity was that of peroxidase, whereas the presence of oxidase and tyrosinase was detected on the verge of the assay sensitivity. Using alfalfa root exudates as a crude enzyme preparation, we found that in the presence of the synthetic mediator, the plant peroxidase could oxidize phenanthrene and its microbial metabolites. The results indicate the possibility of active participation of plants in the rhizospheric degradation of polycyclic aromatic hydrocarbons and their microbial metabolites, which makes it possible to speak about the coupling of the plant and microbial catabolisms of these contaminants in the rhizosphere. © 2015 Elsevier GmbH. All rights reserved. 1. Introduction contribute to the release of PAHs into the environment. Additional sources of these toxic, mutagenic, and carcinogenic substances are Plant–microbial associations and symbioses, with flexible wastes from oil-refining and byproduct-coking. metabolisms and unique enzyme systems, have great advantages Phytoremediation, an environmentally friendly biotechnology for survival in rugged environments, and their survival is due not based on the use of plants and associative microorganisms, has only to their increased tolerance to xenobiotics, but also to active demonstrated its success and strength because it is inexpensive, removal of toxic substances from the habitat. Despite the increased esthetically attractive, and efficient. The accumulated research and use of plants and their associated microorganisms in the form of field trial experience has led investigators to identify effective phytoremediation technology, biochemical interactions between remediating plants capable of intensive elimination of pollutants partners in the rhizosphere that are aimed at the degradation of from contaminated soil. One such plant is alfalfa (Medicago sativa organic pollutants are not fully understood. L.), which is effective for the cleanup of soils polluted with PAHs Polycyclic aromatic hydrocarbons (PAHs) are widespread and (Criquet et al., 2000; Schwab et al., 2006; Phillips et al., 2006; Martí hazardous environmental pollutants that are priority control sub- et al., 2009). stances. Both natural and anthropogenic sources, such as forest A principal factor of enhanced degradation of hydrocarbons in fires, vehicular emissions, and industrial combustion of fossil fuels the plant rhizosphere during phytoremediation is the rhizosphere effect, i.e., the increased numbers and activity of soil microorgan- isms in the plant-root zone. Alfalfa can selectively increase the ∗ number of PAH degraders in its rhizosphere, thereby intensifying Corresponding author. Fax:+7 8452 97 03 83. microbial degradation of pollutants in soil (Muratova et al., 2003; E-mail addresses: [email protected], [email protected] (A. Muratova). http://dx.doi.org/10.1016/j.jplph.2015.07.014 0176-1617/© 2015 Elsevier GmbH. All rights reserved. 2 A. Muratova et al. / Journal of Plant Physiology 188 (2015) 1–8 Kirk et al., 2005; Phillips et al., 2006). The diversity of microor- Na2SO4, 2.0; KNO3, 2.0; MgSO4, 0.5; traces of FeSO4; micronutri- −1 ganisms inhabiting the plant rhizosphere implies that there are ent solution, 1.0 mL . The micronutrient solution contained (g L ): different methods of microbial degradation of organic pollutants, H3BO3, 0.5; CuSO4·5H2O, 0.04; KI, 0.1; FeCl3, 0.2; MnSO4·H2O, 0.4; including PAHs, and that a variety of microbial metabolites of (NH4)6Mo7O24·4H2O, 0.2; ZnSO4·7H2O, 0.4. The growth medium these pollutants are present in the plant root zone. In this con- was inoculated with a fresh microbial culture grown in MSM with −1 text, the study of PAH degradation pathways by typical rhizosphere sodium succinate (1 g L ) to bring the initial absorbance of the microorganisms and the identification of their metabolites seems culture medium (A540) to about 0.5. Incubation was performed at ◦ important for our knowledge of plant-promoted microbial degra- 29 C with rotary shaking (130 rpm) for up to 14 days. The con- dation. trol flasks contained the same medium and equal concentrations In addition to microbial degradation, the involvement of plant of phenanthrene without the inocula. root-released enzymes in chemical reactions catalyzing the trans- Metabolites were analyzed at 0, 1, 3, 7, and 14 days. After cul- formation of organic xenobiotics in soil is an important mechanism tivation, phenanthrene and its metabolites were extracted with of PAH degradation in the rhizosphere (Adler et al., 1994; Schnoor ethyl acetate (50 mL of ethyl acetate per 200 mL of culture medium, et al., 1995; Siciliano et al., 1998). The enzymes released by the three times for 5 min each), first from the native culture medium plant roots include a large group of oxidoreductases (monophe- and then after acidifying the culture medium to pH 2 by adding nol monooxygenase, laccases, and peroxidases), certain esterases, 1 M hydrochloric acid. Neutral and acid extracts were dried over proteases, lipases, dehalogenase, nitroreductase, and nitrilase sodium sulfate and were concentrated by solvent evaporation at (Schnoor et al., 1995; Gramss and Rudeschko, 1998; Gramss et al., room temperature. 1999, 2000; Harvey et al., 2002) Gramss and Rudeschko, 1998; The key metabolites formed in the biotransformation of phenan- Gramss et al., 1999; Gramss et al., 2000 Harvey et al., 2002). Gramss threne were identified by different kinds of chromatography and et al. (1999) have shown that the roots of some plants release a suf- mass spectrometry. ficient amount of oxidoreductases to participate in the oxidative Thin-layer chromatography (TLC) was used for the prepar- degradation of soil organic matter. Hence, the involvement of plant ative isolation of metabolites. TLC was performed on Silufol extracellular enzymes in the rhizosphere degradation of organic UV-254 plates (Kavalier, Czech Republic), which were devel- pollutants can be significant. Being the primary oxidizing system oped in hexane:ethyl acetate:acetic acid (10:30:1) or in ben- in the root exudates of various plants, oxidoreductases can trans- zene:dioxane:acetic acid (90:10:2). The spots corresponding to form pollutants into compounds that are more available to plants metabolites were marked under ultraviolet light, scraped from the and/or rhizosphere microorganisms (Gramss and Rudeschko, 1998; plates, and extracted with methanol. Gramss, 2000; Gianfreda et al., 2006). Thus, the active involvement The residual concentration of phenanthrene in the medium was of plant oxidoreductases in the phytoremediation process has been analyzed by extraction with carbon tetrachloride and by gas chro- suggested. This assumption is also based on data showing that the matography (GC) on a Shimadzu 2010 chromatograph equipped peroxidase activity of some plants, including alfalfa, is high in PAH- with an Equity-1 (Supelco, USA) nonpolar capillary column, a flame contaminated soil (Criquet et al., 2000; Flocco et al., 2002; Muratova ionization detector, and He as a carrier and makeup gas. A solution et al., 2009). With this in mind, we hypothesized that the efficacy of of authentic phenanthrene was used as a standard. alfalfa in the phytoremediation of PAH-contaminated soil could be The concentration of phenanthrene metabolites were deter- connected not only with the selectively increased numbers of PAH mined by ethyl acetate extraction and by high-performance liquid degraders but also with the plant’s own enzymatic activity toward chromatography (HPLC) on a Thermo Scientific Dionex Ultimate aromatic contaminants. 3000 (USA) chromatograph with a diode array detector (DAD) The objective