ISSN 0003-6838, Applied Biochemistry and Microbiology, 2009, Vol. 45, No. 2, pp. 169–175. © Pleiades Publishing, Inc., 2009. Original Russian Text © N.A. Leneva, M.P. Kolomytseva, B.P. Baskunov, L.A. Golovleva, 2009, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2009, Vol. 45, No. 2, pp. 188–194.
Phenanthrene and Anthracene Degradation by Microorganisms of the Genus Rhodococcus N. A. Lenevaa, M. P. Kolomytsevab, B. P. Baskunovb, and L. A. Golovlevaa a
b
Pushchino State University, pr. Nauki 3, Pushchino, Moscow oblast, 142290 Russia Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5 Pushchino, Moscow oblast, 142290 Russia e-mail:
[email protected] Received January 30, 2008
Abstract—The cells of Rhodococcus opacus 412 and R. rhodnii 135 were adapted to phenanthrene and anthracene on a solid mineral medium. Preliminary adaptation of the strains accelerated the metabolism of polyaromatic hydrocarbons and provided for the ability of microorganisms to grow on pheanthrene as a sole carbon and energy source in a liquid mineral medium. It was shown that phenanthrene was mineralized by the strains through 7,8-benzocoumarin, 1-hydroxy-2-naphthoaldehyde, 1-hydroxy-2-naphthoic acid, salicylaldehyde, salicylate and catechol to the intermediates of tricarbonic acid cycle and partially transformed with the accumulation of the products of subsequent monooxygenation (3-hydroxyphenanthrene and phenanthrene dihydroxylated not in ortho-position). As a result of the adaptation of the strains to anthracene on a solid mineral medium, the obtained variant of strain R. opacus 412 was able to transform anthracene in a liquid mineral medium to anthraquinone and 6,7-benzocoumarin. DOI: 10.1134/S0003683809020094
Polycyclic aromatic hydrocarbons (PAH) are toxic pollutants [1]. The low water solubility of compounds of this class makes them highly resistant to degradation and accounts for their accumulation in various ecosystems. The most promising method of PAH detoxification is microbial degradation. As is known from literature, some microorganisms can transform and utilize phenanthrene as a sole carbon and energy source: Comamonas testosteroni GZ38A, GZ39, GZ42 [2], Burkholderia sp. [3], Alcaligenes faecalis AFK2 [4], Sphingomonas sp. P2 [5], Mycobacterium sp. PYR-1 [6], Pseudomonas putida NCBI98 [7], and Nocardioides sp. KP7 [8]. The reports on anthracene-metabolizing microorganisms are still fewer: Burkholderia sp. RP007 [3], Rhodococcus sp. [9], and bacteria of the genus Mycobacterium [6, 10]. Bioremediation of PAH-polluted soils may become a more efficient, economical, and universal alternative to physicochemical treatment due to the complete destruction of the pollutant, reduction of the cost of treatment, higher safety, and minimal impact on the environment. Therefore, the selection and study of new promising PAH-degrading strains and the optimization of PAH bioconversion is a relevant problem. Another quite important aspect is the application of microorganisms as biocatalysts for obtaining compounds, which are of great interest for biomedicine. These are first of all isomeric dihydrodihydroxy- and dihydroxyphenanthrenes, which are hardly liable to chemical synthesis but are necessary for subsequent
synthesis of preparations with antiallergenic and anticarcinogenic properties [11]. The goal of the work was to study the pathways of phenanthrene and anthracene transformation by the bacteria R. opacus 412 and R. rhodnii 135 and to optimize the bioconversion of the compounds under study for obtaining hydroxylated phenanthrene derivatives of biomedical interest. METHODS Strains. The strains used were Rhodococcus opacus 412 and Rhodoccocus rhodnii 135 from the rhodococci collection of E.L. Golovlev (Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences). Cultivation conditions. The strains were cultivated in a Gorlatov (G) mineral medium [12] and a Luria– Bertani (LB) nutrient medium (g/l): peptone–10; yeast extract–5; and NaCl–8. The microorganisms were adapted by repeated inoculations in the course of growth during 4 months on an agarized (1.5% agar) G mineral medium with 50 mg/l phenanthrene or anthracene as the sole carbon source. To obtain the inoculum with greater cell mass, the adapted variants of the strains were reinoculated on an agarized medium with 10% LB. After three days of cultivation, cells were washed off with 2 ml of G medium and used as inoculum in subsequent experiments.
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4
0.30 3
0.25 0.20
1
0.15
2 0.10
0
20
40
60
structure of the isolated intermediates was specified by comparing the obtained mass spectra with the literature data and standard compounds. The metabolites occurring in minor quantities in the culture liquid of unadapted and adapted variants of the strains were identified by HPLC analysis in a Waters 996 chromatograph (USA) with a Spherisorb Octyl column (100 × 4.6 mm) (LKB, Sweden) and by a UV detector at 254 nm. The analysis was performed in the methanol–water system with the addition of 1% acetic acid in the methanol gradient of 50 to 100%. For identification, the retention times of the studied and standard substances on the column were compared.
80 100 120 140 160 180 h
RESULTS AND DISCUSSION Fig. 1. Growth curves for the variants of rhodococcus strains unadapted and adapted to phenanthrene at intermittent introduction of phenanthrene, 12 mg/l (the arrows indicate substrate introduction). 1, unadapted variant of R. rhodnii 135; 2, unadapted variant of R. opacus 412; 3, adapted variant of R. opacus 412; 4, adapted variant of R. rhodnii 135.
The dynamics of formation of the key intermediates of phenanthrene and anthracene bioconversion were studied at cultivation in 100 ml of the liquid mineral G medium in 750 ml medical flasks at 29°ë and 160 rpm. The initial solution of 25 mg/ml phenanthrene and 10 mg/ml anthracene (Fluka, USA) in dimethyl formamide was used as a substrate. Cell growth was determined by the optical density at 545 nm in a Schimadzu UV-160 spectrophotometer (Japan). Isolation and identification of the key intermediates. For preparative isolation and identification of phenanthrene and anthracene bioconversion products, the culture liquid was initially extracted without acidification by ethylacetate (neutral extract) and then the residual liquid was acidified by hydrochloric acid to pH 2 and again extracted by ethylacetate to obtain an acid extract. The extracts were dehydrated with Na2SO4, evaporated in a rotor evaporator, and dissolved in methanol. The qualitative analysis of the extracts and isolation of the intermediates were performed by thin-layer chromatography (TLC) of the extracts on 60 F254 silica gel plates (Merck, Germany). The chromatography was performed in the following system of solvents: benzene–dioxane–acetic acid (90:10:1). The extracts were dispersed over the TLC plate at a distance of 5.5 cm. The UV light, benzidine reagent [13], and the Rf values of compounds were used for identification. For mass spectrometric analysis, the substances were isolated in preparative quantities. Purified preparations were analyzed in a Finnigan MAT 8430 (Germany) at an ionization energy of 70eV, with direct introduction of a sample into the ionization region. The
Growth characteristics of R. opacus 412 and R. rhodnii 135 on phenanthrene. Cultivation of microbial cells in a liquid medium with a xenobiotic usually entails the continuous contact of the whole cell mass with the toxic substrate, which results in undesirable consequences. Therefore, the resistance of microorganisms to stress impacts induced by xenobiotics is often improved by means of preliminary adaptation to the toxicant, which is accompanied by not only morphological changes of cells and cell colonies but also the changes in biochemical cell processes, including acceleration of xenobiotic metabolism due to enhanced expression of the enzymes involved in detoxification [14–18]. In the course of adaptation in our experiments, the first reinoculation of the culture on the agarized medium with phenanthrene was accompanied by weak growth on days 8–10 of cultivation. After four months of cultivation on the agarized medium with phenanthrene, the growth was observed already on day 2–3. The obtained variants of R. opacus 412 and R. rhodnii 135 adapted to phenanthrene could grow in the liquid mineral medium with phenanthrene as a sole carbon and energy source (Fig. 1), in contrast to the unadapted cultures that could not grow on this substrate and realized only partial transformation. Isolation and characterization of the intermediates of phenanthrene and anthracene transformation. The highest amount of phenanthrene transformation intermediates was observed in the first 24 h in the culture liquid of the unadapted variant of R. opacus 412. The neutral extract of the culture liquid contained a compound with Rf 0.64, which was stained reddishbrown by the benzidine reagent, suggesting the presence of a hydroxyl group in the aromatic ring. This compound was identified by mass spectrometric analysis as 1-hydroxy-2-naphthoaldehyde with å+172 (table). The intermediate with Rf 0.54 found in a minor quantity in the neutral extract of the unadapted variant of R. opacus 412 was stained dark-violet by benzidine and
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Products of phenanthrene and anthracene bioconversion by the strains R. opacus 412 and R. rhodnii 135 Intermediate
Rf, TAC
Gross formula
Mass spectrum peaks, m/z (%)
Phenanthrene transformation intermediates 3-hydroxyphenanthrene OH 0.54
C14H10O
M+ 194(100), 165(38), 139(4)
0.27
C14H10O2
M+ 210(100), 181(29), 149(40), 102(55)
0.7
C13H8O2
M+ 196(84), 168(100), 139(50), 113(5)
1-hydroxy-2-naphthoaldehyde OH CHO
0.64
C11H8O2
M+ 172(100), 143(13), 115(37)
1-hydroxy-2-naphthoic acid OH COOH
0.31
C11H8O3
M+ 188(35), 189(3), 171(13), 170(100), 142(15), 116(), 115(61), 114(11)
Dihydroxyphenanthrene (not in ortho-position) OH OH
7,8-benzocoumarin O O
Anthracene transformation intermediates Anthraquinone O 0.77
C14H8O2
M+ 208(96), 180(100), 152(83), 151 (43)
0.67
C13H8O2
M+ 196(100), 168(35), 139(38)
O 6,7-benzocoumarin O
O
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identified by mass spectrometric analysis as 3-hydroxyphenanthrene with å+194 (table). In addition, two compounds with R Rf 0.70 and 0.31 were isolated from the acid extract of culture liquid and characterized. The first ingredient was not stained by the benzidine reagent, fluoresced light-lilac in UV, and was identified by mass spectrometry as 7,8-benzocoumarin (å+196) (table). The intermediate with R Rf 0.31 was stained dark-brown by the benzidine reagent and consequently contained a hydroxyl group in the aromatic ring. This compound also had a carboxylic group as it was extracted at pH 2. Using mass spectrometry and TLC, this intermediate was identified as 1-hydroxy-2naphthoic acid (å+188) (table). Most of the above mentioned compounds were not revealed in the culture liquid of the adapted variant of R. opacus 412. However, significant accumulation of the intermediate with Rf 0.54 identified as 3-hydroxyphenanthrene (å+194) and a new compound with Rf 0.27 was observed on day 3. The latter compound was stained dark-cherry by the benzidine reagent like the former one, suggesting the presence of phenol hydroxyl. Mass spectrometric analysis showed that this metabolite had a molecular weight of 210 (table) and differed from the initial substrate by 32 atomic mass units (fragment mass), which usually corresponds to the inclusion of two oxygen atoms. The presence of an intensive molecular ion and a daughter fragment is evidence of maintenance of the cyclicity and probable symmetry of the molecule. The absence of the elimination of a water molecule from the molecular ion (i.e., the absence of the so-called “ortho-effect”) indicates possible hydroxylation to various positions of one ring or different rings, e.g., position 3,6. In previous work, it has been shown [19] that the strain R. rhodnii 135 during phenanthrene transformation in the Voroshilova–Dianova liquid mineral medium accumulates only one intermediate: 3-hydroxyphenanthrene. In the present work, we have shown that the neutral extracts of both the unadapted variants of the strain on the G medium and the phenanthreneadapted variants also accumulate this intermediate (Rf 0.31); however, the culture liquid of the adapted variant contains a new intermediate with Rf 0.27, also stained dark-cherry by the benzidine dye. The mass spectrometric analysis of the isolated intermediate shows the complete coincidence of its mass spectrum with dihydroxyphenanthrene (å+210) found in the culture liquid of the adapted variant of R. opacus 412. The formation and accumulation of phenanthrene dihydroxylated not in the ortho-position as an intermediate of phenanthrene transformation was shown for the first time for R. opacus 412 and R. rhodnii 135 and for rhodococci as a whole. Possibly, this compound is a product of subsequent monooxygenation, in contrast to dioxygenation performed by dioxygenases with the formation of 3,4-dihydroxyphenanthrene. Some of the currently known microorganisms demonstrate monooxygen-
ation in the course of transformation of different PAH, most likely associated with the presence of another type of enzymes—monooxygenases [20–23]. Monohydroxy- and dihydroxy-derivatives of phenanthrene, accumulated by the strains under study, may further be used for production of compounds with anticarcinogenic and antiallergenic properties [11]. In addition to the above-listed intermediates, trace quantities of salicylic aldehyde (Rt 10.9), salicylate (Rt 10.5), catechol (Rt 6.2), and hydroxymuconic semialdehyde (Rt 6.5) were revealed in the culture liquid of adapted and unadapted variants of both strains by HPLC analysis, evidencing more profound phenanthrene transformation. No growth was observed at cultivation of the studied microorganisms in the liquid mineral medium with anthracene as a sole carbon and energy source; however, the bioconversion of anthracene by the adapted variant of R. opacus 412 was indicated by the accumulation of several compounds in the culture liquid (table). Two intermediates of anthracene transformation, anthraquinone (Rf 0.77 and å+208) and 6,7-benzocoumarin (Rf 0.67 and å+196), were isolated from the neutral and acid culture liquid extracts of the adapted variant of R. opacus 412 and characterized (table). Supposed pathways of phenanthrene and anthracene degradation and transformation. The pathways of phenanthrene conversion by the studied rhodococci have been proposed on the basis of isolated and characterized intermediates of phenanthrene transformation by the strains R. opacus 412 and R. rhodnii 135 as well as the dynamics of their accumulation (Fig. 2). It is supposed that R. opacus 412 can utilize phenanthrene by the pathway (Fig. 2, a) that has been previously described in literature and often occurs in microorganisms [24]: through initial dihydroxylation of phenanthrene with the formation of 3,4-dihydrodiol and then 3,4-dihydroxyphenanthrene, followed by formation of 7,8-benzocoumarin, 1-hydroxy-2-naphthoaldehyde, and 1-hydroxy-2-naphthoic acid through salicylate and catchol to the tricarbonic acid cycle. The growth of the adapted variant of R. opacus 412 on phenanthrene as a sole carbon and energy source, the absence in the culture liquid of the primary metabolites of phenanthrene degradation (7,8-benzocoumarin, 1hydroxy-2-naphthoaldehyde, and 1-hydroxy-2-naphthoic acid), and trace amounts of the metabolites of its more profound transformation by pathway a (salicylic aldehyde, salicylate, catechol, and hydroxymuconic semialdehyde) are evidence of acceleration of the catalytic reactions of this pathway in the adapted variant of R. opacus 412. The absence of growth of the unadapted variant of the same strain on phenanthrene is most likely associated with accumulation of the primary intermediates of initial substrate transformation (some of them are probably toxic for cells).
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a
OH
Phenanthrene HO
173
H
HO H
3-hydroxyphenanthrene OH
OH HO
OH
Dihydroxyphenanthrene
O O
7,8-benzocoumarin OH CHO
1-hydroxy-2-naphthaldehyde OH COOH
1-hydroxy-2-naphthoic acid OH COOH
Salicylic acid OH OH
Catechol TAC
Fig. 2. Phenanthrene transformation by R. opacus 412 and R. rhodnii 135. Supposed intermediates known from the literature are given in brackets. TAC, tricarbonic acid cycle. APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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Anthracene O
OH H
OH H
ˆËÒ-1,2-dihydroxy1,2-dihydroanthracene
O
Anthraquinone
OH OH
ACKNOWLEDGMENTS The work was supported by Grant 1,2,06 on instructions of the Russian Ministry of Education, Russian Science Support Foundation, RFFI-Naukograd 04-0497266 and RFFI 05-04-49659.
1,2-dihydroxyanthracene
O
liquid of microorganisms. 6,7-benzocoumarin is a product of spontaneous closing of the ring of extradiol cleavage product, cis-1,2-dihydroxyanthracene, thereby suggesting the obligatory existence of anthracene cis-1,2-dihydrodiol—the first intermediate in the anthracene degradation pathway revealed in other microorganisms [24]. On the basis of intermediates of anthracene transformation by R. opacus 412, which have been isolated and identified in this work, and in consideration of the literature data, we have proposed two pathways of partial anthracene conversion by the strain (Fig. 3) including anthracene transformation into anthraquinone and the parallel transfer of successive anthracene conversion through cis-1,2-hydroxy-1,2dihydroanthracene and 1,2-dihydroxyanthracene into 6,7-benzocoumarin.
O
REFERENCES
6,7-benzocoumarin Fig. 3. Anthracene bioconversion by R. opacus 412. Supposed intermediates known from the literature are given in brackets.
The excess content of 3-hydroxyphenanthrene and the appearance and accumulation of phenanthrene hydroxylated not in ortho-position in the adapted variant of R. opacus 412 (Fig. 2, b) may be evidence of enhanced expression of monooxygenation enzymes after preliminary adaptation. The fact that such compounds accumulate with the lapse of time demonstrates the inability of the strain for their further utilization. Thus, one may distinguish a new, though dead-end pathway of phenanthrene transformation by the strain (Fig. 2, b). In consideration of growth of the adapted variant of R. rhodnii 135 and the presence of trace quantities of salicylic aldehyde, salicylate, catechol, and hydroxymuconic semialdehyde in the culture liquid, it is possible to suggest the existence of pathway a—the main pathway of phenanthrene mineralization by adapted cells. However, phenanthrene transformation by this pathway most likely occurs at higher rates as compared with the strain R. opacus 412. Preliminary adaptation of R. rhodnii 135 to phenanthrene also results in enhanced accumulation of the metabolites of pathway b, which may also evidence activation of the enzymes catalyzing subsequent monooxygenation of phenanthrene. As is known from literature data [19, 24, 25], anthraquinone is a dead-end compound of anthracene biotransformation and often accumulates in the culture
1. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Polynuclear Aromatic Compounds, part l, 1983, vol. 32, pp. 355–364. 2. Goyal, A.K. and Zylstra, G.J.., Environ. Microbiol., 1996, vol. 62, no. 1, pp. 230–236. 3. Laurie, A.D. and Lloyd-Jones, G., J. Bacteriol., vol. 181, no. 2, pp. 531–540. 4. Kiyohara, K., Nagao, K., Kouno, K., and Yano, K., Environ. Microbiol., 1982, vol. 43, no. 2, pp. 458–61. 5. Pinyakong, O., Habe, H., Supaka, N., Pinpanichkarn, P., Juntongjin, K., Yoshida, T., Furihata, K., Nojiri, K., Yamane, H., and Omori, T., FEMS Microbiol. Lett., 2000, vol. 191, no. 1, pp. 115–121. 6. Moody, J.D., Freeman, J., Doerge, D.R., and Cerniglia, C.E., Appl. Environ. Microbiol., 2001, vol. 67, no. 4, pp. 1476–1483. 7. Yang, Y., Chen, R.F., and Shiaris, M.P., J. Bacteriol., 1994, vol. 176, no. 8, pp. 258–2164. 8. Iwabuchi, T., Inomata-Yamoguchi, Y., Katsuta, A., and Harayama, S., J. Mar. Biotechnol., 1998, vol. 6, pp. 86–90. 9. Dean-Ross, D., Moody, J.D., and Cerniglia, C.E., FEMS Microbiol. Ecol., 2002, vol. 204, no. 1, pp. 1–7. 10. van Herwijnen, R., Sprigalen, D., Slot, P., Govers, K., and Parsons, J., Appl. Environ. Microbiol., 2003, vol. 69, no. 1, pp. 186–190. 11. Sheldrake, N., Chilarity in Industry. The Commercial Manufacture and Applications of Optically Active Compounds, Collins, A.N., Sheldrake, G.N., and Crosby, J., Eds., New York: Wiley-Intersci., 1992. 12. Gorlatov, S.N., Mal’tseva, O.V., Shevchenko, V.I., and Golovleva, L.A., Mikrobiologiya, 1989, vol. 58, no. 5, pp. 802–806. 13. Yamada, K., Horiguchi, S., Takahashi, J., Agric. Biol. Chem., 1965, vol. 29, no. 10, pp. 943–948.
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PHENANTHRENE AND ANTHRACENE DEGRADATION BY MICROORGANISMS 14. Mil’ko, E.S. and Egorov, N.S., Geterogennost’ populyatsii bakterii i protsess dissotsiatsii (Heterogeneity of Bacterial Populations and Dissociation Process), Moscow: Mosk. Gos. Univ., 1991. 15. Tsitko, I.V., Zaitsev, G.M., Lobanok, A.G., and Salkinoja-Salonen, M.S., Appl. Environ. Microbiol., 1999, vol. 65, no. 2, pp. 853–855. 16. Iwabuchi, N., Sunairi, M., Anzai, H., Nakajima, M., and Harayama, S.H., Appl. Environ. Microbiol., 2000, vol. 66, no. 11, pp. 5073–5077. 17. Iwabuchi, N., Sunairi, M., Urai, M., Itoh, C., Anzai, H., Nakajima, M., and Harayama, S., Appl. Environ. Microbiol., 2002, vol. 68, no. 5, pp. 2337–2343. 18. Kolomytseva, M.P., Solyanikova, I.P., Golovlev, E.L., and Golovleva, L.A., Prikl. Biokhim. Mikrobiol., 2005, vol. 41, no. 5, pp. 474–479. 19. Baboshin, M.A., Baskunov, B.P., Finkelmshtein, Z.I., and Golovleva, L.A., Mikrobiologiya, 2005, vol. 74, no. 3, pp. 357–364.
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175
20. Nojiri, H., Nam, J.-W., Kosaka, M., Morii, K.-L., Takemura, T., Furihata, K., Yamane, H., and Omori, T., J. Bacteriol,, 1999, vol. 181, no. 10, pp. 3105–3113. 21. Shindo, K., Ohnishi, Y., Chun, H.K., Takahashi, H., Hayashi, M., Saito, A., Iguchi, K., Furukava, K., Harayama, S., Horinouchi, S., and Misawa, N., Biosci. Biotechnol. Biochem., 2001, vol. 65, no. 11, pp. 2472– 2481. 22. Li, Q.S., Ogawa, J., Schmid, R.D., and Shimizu, S., Environ. Microbiol., 2001, vol. 67, no. 12, pp. 5735– 5739. 23. van Herwijnen, R., Wattiau, P., Bastiaens, L., Daal, L., Jonken, L., Springael, D., Govers, H.A.J., and Parsons, J.R., Res. Microbiol., 2003, vol. 154, pp. 199–206. 24. Habe, K. and Omori, T., Biosci. Biotechnol. Biochem., 2003, vol. 67, no. 2, pp. 225–243. 25. Dean-Ross, D., Moody, J.D., Freeman, J.P., Doerge, D.R., and Cerniglia, C.E., FEMS Microbiol. Letts., 2001, vol. 204, no. 1, pp. 205–211.
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