ISSN 00036838, Applied Biochemistry and Microbiology, 2014, Vol. 50, No. 7, pp. 722–729. © Pleiades Publishing, Inc., 2014. Original Russian Text © D.O. Egorova, T.I. Gorbunova, M.G. Pervova, V.A. Demakov, 2013, published in Biotekhnologiya, 2013, No. 4, pp. 56–64.

ECOLOGY

Bacterial Degradation of a Mixture Obtained through the Chemical Modification of Polychlorinated Biphenyls by Polyethylene Glycols D. O. Egorovaa, T. I. Gorbunovab, M. G. Pervovab, and V. A. Demakova a

Institute of Ecology and Genetics of Microorganisms, Russian Academy of Sciences, the Ural Branch, Perm, 614081 Russia b Postovskii Institute of Organic Synthesis, Russian Academy of Sciences, the Ural Branch, Yekaterinburg, 620990 Russia email: [email protected] Received May 28, 2013

Abstract—Polychlorinated biphenyls (PCBs), also known by the trade name Sovol, are toxic industrial wastes. They have been subjected to chemical treatment by polyethylene glycols (PEGs) and potassium hydroxide. As a result of the interaction of the Sovol with various molecular mass PEGs (MMPEG4 ~ 200, MMPEG22 ~ 1000), watersoluble mixtures M1 and M2 containing mono(polyethylene glycol)oxyderiva tives (PCBPEG4 and PCBPEG22), polychlorobiphenylols, and unreacted PCB congeners (PCB 44, PCB 47, PCB 49, PCB 52, and PCB 66) were obtained. It was shown for the first time that mixtures M1 and M2 are susceptible to bacterial degradation without their fractionation. According to the gasliquid chroma tography with flameionization and massspectrometric detection, the Rhodococcus wratislaviensis KT1127 strain degraded all of the chemical compounds occurring in the mixtures. In a 5day experiment, it was found that the KT1127 strain decomposes mono(polyethylene glycol)oxyderivatives completely (by 100%) and polychlorobiphenylols and PCB congeners by 90–95% in the M1 and M2 mixtures. The culture medium did not contain transformation products, whereas free chlorine ions were accumulated (72–94% of the maximum possible amount). Thus, the use of the chemical modification and consecutive bacterial degradation provided an effective destruction of technical PCB mixtures with a high content of highly chlorinated congeners. Keywords: biological degradation, chemical treatment, congeners, polychlorinated biphenyls, polyethylene glycols, Rhodococcus, utilization DOI: 10.1134/S0003683814070023

INTRODUCTION Human industrial activity on the planet is con nected with the accumulation of large volumes of environmentally hazardous synthetic substances. In 2001 the international community adopted the Stock holm convention, according to which a number of persistent organic pollutants are subject to the elimi nation of their production and use and must be destroyed [1]. Polychlorinated biphenyls (PCBs) are included on the list of compounds regulated by the Stockholm convention. According to various estimates, disposal sites on the territory of Russia contain from 180 to 500 kt of PCBs [2]. The utilization of PCBs is difficult because of their unique persistence and the composi tional inhomogeneity of industrial mixtures, which combine several dozen PCB congeners with various Abbreviations: amu—atomic mass unit; CBA—chlorobenzoic acid; CM—culture medium; GCFID—gas chromatograph with flame ionization detection; GCMS—gas chromatograph/mass spectrometer; IUPAC—International Union for Pure and Applied Chemistry; MM—molecular mass; OD—optical den sity; PCBOH—pentachlorobiphenylol; PCBPEG4—mono substituted derivatives of PCB and PEG4; PCB—polychlori nated biphenyls; PEG—polyethylene glycol.

chlorination degrees. Due to these facts, it is impossi ble to perform recycling of PCB wastes using a single method, including energyconsuming burning (the approximate cost of burning of 1 t of PCBs is about 2000 US dollars) [2]. Analysis of the literature data shows that polyethyl ene glycol (PEG) treatment in the presence of a base is an efficient method for cleaning traces of PCBs from equipment [3–5]. The process results in the formation of both mono [4, 5] and dipolyethylene glycol deriv atives of PCBs [6], which are watersoluble in contrast to PCBs [4–6]. However it was revealed that tri, tetra, and some pentachlorinated biphenyls have low reactivity and practically do not interact with PEG. As a result, the final products of the transformation of PCB technical mixtures by PEG always contain a range of the initial PCB congeners and polychlorobi phenylols, which are formed upon hydrolysis of the main PCBs. Consequently, products of PCB chemical modification by PEG cannot be considered safe for the environment or humans [2, 7, 8]. On the other hand, a number of aerobic bacteria are capable of degrading PCB congeners and their hydroxylderivatives [7, 9–16]. The literature contains a description of the bacterial transformation of PCBs

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through the formation of hydroxyoxophenylhexa dienic acids and then chlorobenzoic acids (CBAs) [7, 9]. CBAs are toxic, but they are less dangerous than PCBs (second and fourth toxicity classes, respec tively). At the same time, there are bacterial strains with the unique ability to decompose PCBs into non toxic compounds [10–15]. We previously suggested a method of PCB utiliza tion that combines chemical (hydroxydechlorination) and microbiological (bacterial transformation) pro cesses [17]. The utilization of Sovol industrial PCBs resulted in the formation of a mixture of hydroxy derivatives of PCBs containing residual quantities of (trihexa)chlorinated PCBs. It was separated into two fractions: hydroxyderivatives of PCBs and the initial congeners. The hydroxyderivatives of PCBs then underwent reductive dechlorination; the reaction resulted in the formation of a mixture of biphenyls, which are more watersoluble than the chlorinated derivatives. The fraction of (trihexa)chlorinated biphenyls was then bacterially degraded. Microbes degraded all the PCB congeners that did not enter the reaction. Moreover, environmentally hazardous com pounds were not accumulated. The purpose of the work is to study the possibility of using the bacterial degradation of the mixture formed during the chemical modification of technical PCBs under the action of PEG in the presence of potassium hydroxide without fractionation of the reaction products. METHODS Bacterial degrader. The strain used in the work, Rhodococcus wratislaviensis KT1127 (Russian Col lection of Microorganisms, RCM Ac2623D), was previously isolated by us from soils subjected to tech nogenic burden. The strain has active systems of deg radation of biphenyl and benzoic acid and can degrade these substrates in unfavorable conditions (saliniza tion, low temperatures) [18]. The synthesis of PCB and PEG derivatives was per formed using a Sovol PCB technical mixture with PEG4 (MMPEG4 ~200) or PEG22 (MMPEG22 ~1000), according to the method, [6] at 120–130°C for 6–8 h. As a result, we obtained: a) a mixture of compounds after the reaction with PEG4, designated M1; b) a mixture of compounds after the reaction with PEG22, designated M2. The quantities of the main elements (carbon, oxy gen, hydrogen, and chlorine) in mixtures M1 and M2 were determined by elemental analysis using a PE 2400 Series II CNH automatic analyzer (Perkin Elmer, United States). Analysis of the interaction products was performed using a Shimadzu GC 2010 gas chromatograph with a flame ionization detector (GCFID) and quartz capillary column ZB5 (length of 30 m, diameter of 0.25 mm); the stationary phase film was 0.25 mm thick (polymethyl APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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siloxane, 5% of bonded phenyl groups) (Shimadzu, Japan). The initial column temperature was 40°C (3 min); then it was heated at a speed of 10°/min up to the final temperature of 290°C. The temperatures of the vaporizer and the detector were 280 and 320°C, respectively. Identification of the reaction products and registra tion of mass spectra was performed using an Agilent GC 7890A MSD 5975C inert XL EI/CI gas chro matographmass spectrometer (GCMS) (Agilent Technologies, United States) with a quartz capillary column HP5MS with a length of 30 m and a diameter of 0.25 mm; the stationary phase film was 0.25 mm thick (polydimethylsiloxane, 5% of bonded phenyl groups) and from a quadrupole massspectrometric detector. Total ion current scanning was performed in the mass range of 20–1000 amu in the regime of elec tron ionization (70 eV). Helium served as a carrier gas; the split was 1 (through the column) : 50 (for detec tion). The initial column temperature was 40°C (iso therm of 3 min); heating was performed at a speed of 10°/min up to 290°C (isotherm of 40 min). The tem peratures of the vaporizer, transfer chamber, mass spectrometric source, and quadrupole were 250, 280, 230, and 250°C, respectively. Calculation of the content of the types of compounds in M1 and M2 was made by internal normalization of the shares of these substances according to the peak squares on chromatograms of each component in the resulting mixture (GCFID). Bacterial degradation of the mixture formed from the reaction of PCB with PEG was performed in the experiment with “washed” cells. The bacterial culture was preliminary grown in 250mL flasks filled with 50 mL of Raymond liquid mineral medium [19] with the addition of biphenyl as a carbon source (1 g/L) on a shaker (120 rpm) at 28°C up to the OD600 = 1.0. Cells washed twice with the Raymond medium (1 mL, OD600 = 2.0) were transferred into vials with Teflon lids. M1 and M2 were added as water solutions into the vials with the bacterial culture up to the final concen trations of 0.3 mg/mL (M1) and 0.6 mg/mL (M2) and incubated on a thermostated shaker (120 rpm) at 28°C for 5 days. Samples for an analysis of the bacterial destruction were taken immediately after the addition of M1 and M2 and on days 1, 3, and 5. Qualitative and quantitative analysis of the samples was performed using GCMS and GCFID under the conditions described above. The presence of intermediate products of PCB bac terial destruction and free chlorine ions in the culture medium without bacterial cells was determined using a UVvisible BioSpecmini Shimadzu spectropho tometer as described in [20]. Statistical analysis of the results was performed using the standard Microsoft Excel and Statistica 6.0 software. All of the experiments on bacterial degrada tion were repeated three times.

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Time (min) Fig. 1. Chromatogram (GCFID) of the Sovol PCB technical mixture. Numbers of the PCB congeners according to the IUPAC classification [22] are shown in bold type.

Cly

Cly−1

y = 4–6 I

Cly−1

OH

HO(CH2CH2O)4H KOH

O(CH2CH2O)4H

+

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IV

Fig. 2. Reaction of PCBs (I) with PEG4. The reaction products: II—tetrachlorobiphenylols; III—pentachlorobiphenylols; IV—monosubstituted derivatives of PEG4 and penta and hexachlorobiphenyls.

RESULTS AND DISCUSSION A Sovol PCB technical mixture consisting of sev eral dozen congeners containing from four to six chlo rine atoms in a molecule was used as a substrate for degradation (Fig. 1) [21, 22]. It was revealed that the change ratio of the initial mixture in the reaction of the Sovol with PEG4 reached 70% (Figs. 2, 3a). Analysis of the toluol and chloroform extracts of the mixture obtained during the chemical reaction showed that the extracts contained the same range of products (Fig. 3a). The scanning of every peak in the chromatogram in Fig. 3a under con ditions of electron ionization (70 eV) showed that the hexachlorobiphenyls present in the Sovol entirely

underwent chemical transformation into pentachloro biphenyls; however, no derivatives that react with PEG4 were found for them. Eighty percent of pen tachlorobiphenyls react with PEG4 and form tetra chlorobiphenylols and monosubstituted derivatives. Ten percent of tetrachlorobiphenyls are transformed by PEG4 into monosubstituted derivatives. Conge ners such as PCB 52, PCB 49, PCB 47, and PCB 44 remain unchanged under the conditions of the process (Fig. 3a), while PCB 66 partly reacts. Consequently, the interaction of the Sovol and PEG4 results in the formation of M1 containing tetra and pentachlorobi plenylols (PCBOH), monosubstituted PCB deriva tives (PCBPEG4), and unreacted PCB congeners

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Time (min) Fig. 3. Chromatogram (GCFID) of M1 (a) before and (b) 5 days after the beginning of the bacterial degradation: II—tetrachlo robiphenylols; III—pentachlorobiphenylols; IV—monosubstituted derivatives of PEG4 and penta and hexachlorobiphenyls. Numbers of the unreacted PCB congeners are shown in bold type.

(table). All of the results are in accordance with the early data on the higher reactivity of highly chlorinated PCB congeners upon nucleophilic substitution in comparison with those that are lowchlorinated [2, 23]. APPLIED BIOCHEMISTRY AND MICROBIOLOGY

Analysis of the literature shows that microorgan isms, particularly bacteria of the Rhodococcus genus, are able to transform lowchlorinated biphenyls [7, 9, 10]. We proposed to degrade M1 using the Rhodococ

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Compositions of mixtures M1 and M2 obtained from the chemical reaction of Sovol technical PCBs with PEG4 and PEG22, respectively, in the presence of potassium hydroxide before and after the bacterial degradation (5 days) Concentration of components in the initial mixture (mg/g)

Concentration of components in the samples undergone bacterial degradation (mg/mL)

Mixture

M1 M2

PCB

PCBOH

PCB PEG

PCB

PCBOH

PCBPEG

300 ± 1 100 ± 2

280 ± 1 160 ± 2

420 ± 2 740 ± 1

0.090 ± 0.001 0.060 ± 0.002

0.084 ± 0.002 0.097 ± 0.002

0.126 ± 0.001 0.443 ± 0.003

cus wratislaviensis KT1127 strain, which has a high degrading activity for biphenyls and the products of its transformation [18]. We analyzed the CM after the microbial degrada tion of M1 by GCMS and revealed that the KT1127 strain disrupted all of the PCBPEG4, residual con geners, and tetra and pentachlorobiphenylols during a 5day incubation (Fig. 3b). In 24 h, 100% degrada tion of monopolyethyleneglycoloxy derivatives (PCB PEG4) and pentachlorobiphenylols was noted; trace amounts of PCB 49, PCB 52, and tetrachlorobipheny lols were registered. By the end of the fifth day of incu bation, polychlorobiphenylols were not registered in M1, and the degradation of PCB congeners was 90% (the PCB residual amount in the culture medium was 0.0088 ± 0.004 mg/mL) (Fig. 3b). It should be noted that the PCB 49 and PCB 52 are the most resistant to microbial degradation, because they contain substituting groups in the ortho and meta positions. However, their decrease is indicative of the ability of the KT1127 strain to transform these conge ners. According to the degradation levels of PCB 49 and PCB 52, the R. wratislaviensis KT1127 strain is as good as other Rhodococcus strains studied earlier and is better than the degrading strains described in other works [7, 11, 24–26]. The quantities of PCBs and polychlorobiphenylols in M1 (see table) allowed us to suppose that bacterial destruction is accompanied by the accumulation of intermediate products that are characteristic of the classic pathway of the aerobic bacterial transformation of these compounds in the medium [7, 11, 16, 20]. It is known that bacterial enzymes oxidize biphenyl or its chlorinated derivatives into biphenyldihydrodiol, dihydroxybiphenyl, and hydroxyoxophenylhexadienic

Cly

0.300 ± 0.002 0.600 ± 0.003

acid during aerobic degradation; eventually hexa dienic and benzoic acids are formed [7, 11, 16, 20]. However, spectrophotometric analysis of the CM did not reveal the presence of chlorohydroxyoxophenyl hexadienic and chlorobenzoic acids. An accumulation of chlorine ions was registered (0.39 ± 0.02 mM on the third day of the experiment, 0.83 ± 0.03 mM on the fifth day). According to the elemental analysis (data not shown), M1 contains 13.58% of chlorine ions. Considering the quantity of M1 used for degradation, the maximum quantity of chlorine ions in the medium in the case of total degradation and dechlorination of the mixture can reach 1.15 mM. In spite of the fact that the amount of chlorine ions in the medium (0.83 ± 0.03 mM) was lower than the possible maximum by the end of the experiment, we can suppose that degra dation of M1 is not accompanied by the accumulation of chlorinated products. The difference between the disengaged and contained amounts of chlorine in M1 results from the fact that chlorine ions are partly bound in the PCB 49 and PCB 52 molecules. Consequently, the use of the R. wratislaviensis KT1127 strain resulted in almost complete utilization of the mixture obtained during the interaction of the Sovol technical PCB mixture with PEG4 in the pres ence of potassium hydroxide. The conversion ratio in the reaction of the Sovol technical PCB mixture with PEG22 was 90% (Figs. 4, 5a). Analysis of the chloroform extract of the reaction products revealed tetra and pentachlorobi phenylols analogous to those formed in the course of the Sovol reaction with PEG4 and the nonreactive PCB 52, PCB 49, PCB 47, PCB 44, and PCB 66 on the chromatogram. PCBPEG22 monosubstituted derivatives did not elute from the chromatographic

Cly−1 HO(CH2CH2O)22H KOH

y = 4–6 I

Concentration of the mixture in the samples under gone bacterial degra dation (mg/mL)

Cly−1

OH

O(CH2CH2O)22H

+

II, III

V

Fig. 4. Reaction of PCBs (I) with PEG22. The reaction products: II—tetrachlorobiphenylols; III—pentachlorobiphenylols; V—monosubstituted derivatives of PEG22 and penta and hexachlorobiphenyls. APPLIED BIOCHEMISTRY AND MICROBIOLOGY

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Time (min) Fig. 5. Chromatogram of M2 (a) before and (b) 5 days after the beginning of the bacterial degradation: II—tetrachlorobipheny lols; III—pentachlorobiphenylols. Numbers of the unreacted PCB congeners are shown in bold type.

column. Consequently, the chemically obtained mix ture M2 consists of monosubstituted PCBPEG22, polychlorobiphenylols, and untransformed PCB con geners (see table). M2 also underwent bacterial degradation. The composition of M2 changes after the first day of the APPLIED BIOCHEMISTRY AND MICROBIOLOGY

incubation with the KT1127 strain; only traces of polychlorobiphenylols and PCB congeners nonreac tive at the chemical stage are registered 5 days later (Fig. 5b). It should be noted that intermediates of the bacterial degradation of monosubstituted PCBPEG 22 derivatives, PCB congeners, and polychlorobiphe

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nylols were not registered in the medium, as in the case of M1 degradation. However, chlorine ions accumu lated in the system (1.38 ± 0.02 mM on the third day of culturing and 1.65 ± 0.02 mM on the fifth day). Consequently, the amount of free chlorine ions is close to the maximum possible in the case of total M2 dechlorination (the portion of chlorine ions in the mixture is 10.41%, and in the used amount of M2 this value is 1.76 mM). The difference between the maxi mum concentration of chlorine in the medium was calculated for the case in which all of the M2 com pounds are dechlorinated, and the experimental value was 0.11 mM. Moreover, it is known that the mixture still contains some PCB 49 and PCB 52 5 days after destruction. These congeners contain chlorine as a substitute. The revealed difference between the actual and possible maximum chlorine concentrations is caused by the presence of chlorine bound in the PCB 49 and PCB 52 molecules (corresponding math ematical calculations not shown). So we can conclude that the formed metabolites cannot contain chlorine. It was revealed that the level of bacterial degrada tion of residual PCBs in M2 is higher in comparison with the destruction of the analogous PCB congeners in M1 (see table). The PCB congeners containing sub stitutes in ortho and parapositions in one of the rings of the molecule (PCB 47, PCB 66) are the most actively degraded; their 100% destruction occurs in 5 days. The conversion of PCB 49 and PCB 52, which are the most resistant to bacterial degradation, was 95% by the end of the experiment. This result is possi bly caused by the fact that the PCB concentration in M2 is lower (2/3 of the PCB amount in M1, see Table), and, consequently, the mixture is less toxic for the bacterial culture. Analysis of the literature data showed that the KT1127 strain degrades all tetrachlo rinated biphenyls present in M1 and M2 more effi ciently than other PCB degrading Rhodococcus strains [7, 11, 24–26]. It is known that polychlorobiphenylols as well as PCBs are toxic compounds; their accumulation in the organisms of humans and animals causes severe dis eases [8]. Polychlorobiphenylols are present in the environment in regions polluted by PCBs, where they form as a result of the enzymatic action of some microorganisms, fungi, plants, and animals [27–29]. However the literature contains sparse data on the degradation of mono and dichlorobiphenylols by bacterial strains [16]. One work [16] reports the ability of the biphenyl dioxygenase (the enzyme performing the first oxidative reaction of substituted biphenyls) isolated from Burkholderia sp. LB400 and Comamonas testosterone B356 strains to oxidize the unsubstituted rings of 3chlorobiphenyl2ol, 5chlorobiphenyl2 ol, and 3,5dichlorobiphenyl2ol. Due to this fact, our data on the activity of the R. wratislaviensis KT1127 strain towards medium and highly substi tuted chlorobiphenylols (see Figs. 3, 5) are very inter esting and allow us to conclude that, if the KT1127

strain is able to degrade more toxic, highly chlorinated biphenylols, it must provide degradation of lowchlo rinated biphenylols presenting in nature. Consequently, we have shown the possible degra dation of toxic wastes—PCBs—using a combination of chemical and biological processes. Chemical mod ification of the Sovol PCB technical mixture under the action of PEG in the presence of potassium hydroxide allows us to obtain watersoluble mixtures containing mono(polyethylene glycol)oxy derivatives, polychlo robiphenylols, and residual PCB congeners. The use of the R. wratislaviensis KT1127 strain allows for the nearly total degradation of the reaction products. The developed method is an alternative way of cleaning industrial PCBs from various objects. ACKNOWLEDGMENTS This work was supported by the Ural Branch of the Russian Academy of Sciences (interdisciplinary project no. 12M342036) and the Russian Founda tion for Basic Research (grant no. 110496028 r_ural_a). REFERENCES 1. Final Act of the Conference of Plenipotentiaries on the Stockholm Convention on Persistent Organic Pollutants, Stockholm, May 22–23, 2001, UNEP/POPS/CONF/4, United Nations Environment Programme, Geneva, 2001. http://chm.pops.int 2. Gorbunova, T.I., Pervova, M.G., Zabelina, O.N., Saloutin, V.I., and Chupakhin, O.N., Polikhlorbifenily: Problemy ekologii. analiza i khimicheskoi utilizatsii (Polychlorinated Biphenyls: Environmental Problems, Chemical Analysis, and Utilization), Charushin, V.N., Ed., Moscow: KRASAND; Yekaterinburg: UrO RAN, 2011. 3. McGraw, M.G., The PCB problem: separating fact from fiction, Electrical World, 1983, vol. 197, no. 2, pp. 49–72. 4. De Filippis, P., Chianese, A., and Pochetti, F., Removal of PCBs for mineral oils, Chemosphere, 1997, vol. 35, no. 8, pp. 1659–1667. 5. De Filippis, P., Scarsella, M., and Pochetti, F., Dechlo rination of polychlorinated biphenyls: a kinetic study of removal of PCBs from mineral oils, Ind. Eng. Chem. Res., 1999, vol. 38, no. 2, pp. 380–384. 6. Gorbunova, T.I., Zapevalov, A.Ya., Kirichenko, V.E., Saloutin, V.I., and Chupakhin, O.N., Polychlorinated biphenyls in reactions with alcohols, Zh. R. Kh., 2000, vol. 73, pp. 610–614. 7. Pieper, D.H., Aerobic degradation of polychlorinated biphenyls, Appl. Microbiol. Biotehcnol., 2005, vol. 67, no. 2, pp. 170–191. 8. Masuda, Y., Health effect of polychlorinated biphenyls and related compounds, Int. J. Health Sci., 2003, vol. 49, pp. 333–336. 9. Furukawa, K. and Fujihara, H., Microbial degradation of polychlorinated biphenyls: biochemical and molecu

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Vol. 50

Translated by O. Maloletkina

No. 7

2014