Environ Chem Lett (2012) 10:287294 DOI 10.

1007/s10311-012-0354-6

ORIGINAL PAPER

Degradation of methyl-phenanthrene isomers during bioremediation of soil contaminated by residual fuel oil
Milan Novakovic Muftah Mohamed Ali Ramadan Tatjana Solevic Knudsen Malisa Antic Vladimir Beskoski Gordana Gojgic-Cvijovic Miroslav M. Vrvic Branimir Jovancicevic

Received: 17 May 2011 / Accepted: 13 January 2012 / Published online: 22 January 2012 Springer-Verlag 2012

Abstract Phenanthrene and methyl-phenanthrenes are major aromatic pollutants originating in particular from fuel oil. Phenanthrene is usually degraded faster than methylphenanthrenes under geological and environmental conditions. Here, we report a preferential and accelerated biodegradation of methyl-phenanthrenes versus phenanthrene in soil contaminated by fuel oil. The polluted soil was mixed with sawdust and sand to form a homogenized biopile. The biopile was continuously sprayed with microbial consortia isolated from crude oilcontaminated soil and treated by biosurfactants and nutritive substances for biostimulation. During a 6-month bioremediation experiment, a steady increase in the relative abundance of phenanthrene compared to methyl-phenathrenes was observed by gas chromatographymass spectrometry. The increase was the highest for trimethyl-phenanthrenes, with a phenanthrene/ trimethyl-phenanthrenes ratio increasing from 0.42 to 2.45. By contrast, the control, non-stimulated samples showed a

ratio decrease from 0.85 to 0.11. Moreover, the results showed that the level of degradability depends on the number of methyl groups. Keywords Bioremediation Soil Residual fuel oil Phenanthrene Methyl-phenanthrene isomers Degradation

Introduction Bioremediation is nowadays undoubtedly considered one of the effective approaches for removing organic pollutants from different parts of the environment, rst of all recent sediments, soils and surface waters. For example, the efciency of bioremediation processes is proved on the example of chlorinated organic solvents (Ferguson and Pietari 2000), polycyclic aromatic hydrocarbons (Bamfort and Singleton 2005), and pesticides (Gavrilescu 2005). However, in case of oil type pollutants (crude oil and renery products of petroleum rening), their biodegradation and removal from the environment are difcult to be classied in one category. Oil is a very complex mixture of hydrocarbons, but also nitrogen, sulfur, and oxygen compounds (NSO). Each class of compounds, and often individual compounds as well, require special study aimed to dene the type of microorganisms and optimal conditions for microbial degradation (Fritsche and Hofrichter 2008; Van Hamme et al. 2003). For degradation of some compounds, microorganisms and conditions for degradation are not known, and these compounds are considered non-biodegradable. This is especially true for compounds in the fraction of NSO compounds (Peters et al. 2005). According to previous organic geochemical studies (Volkman et al. 1983; Peters et al. 2005) and research

Electronic supplementary material The online version of this article (doi:10.1007/s10311-012-0354-6) contains supplementary material, which is available to authorized users.
M. Novakovic M. M. A. Ramadan M. M. Vrvic B. Jovancicevic (&) Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, P.O. Box 158, 11001 Belgrade, Serbia e-mail: bjovanci@chem.bg.ac.rs T. S. Knudsen V. Beskoski G. Gojgic-Cvijovic M. M. Vrvic B. Jovancicevic Center of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Njegoseva 12, P.O. Box 473, 11001 Belgrade, Serbia M. Antic Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11081 Belgrade, Serbia

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related to the fate of petroleum pollutants in the environ ment as well (for example: Jovancicevic et al. 2003; Antic et al. 2006; Ilic et al. 2011; Solevic et al. 2011), it was shown that in the fraction of saturated hydrocarbons, the most susceptible to microbial degradation are n-alkanes and isoprenoid aliphatic hydrocarbons. Polycyclic hydrocarbons are generally resistant to biodegradation. The aromatic fraction was examined together with saturated hydrocarbons in many organic geochemical studies (e.g., Lichtfouse et al. 1994). For this fraction can be said that it is generally more resistant to biodegradation than the fraction of saturated hydrocarbons (Volkman et al. 1983; Peters et al. 2005). However, microbiological degradation of individual polycyclic aromatic hydrocarbons has been proven. Additionally, mechanisms and pathways of degradation of aromatic hydrocarbons have been to the large extent explained. It has been known since the work of Davies and Evans (1964) that in aerobic conditions, mediated by bacteria, naphthalene can be decomposed to carbon dioxide via catechol according to the ring ssion mechanism. Also, fungi are capable of oxidation of anthracene and phenanthrene to trans-dihydrodiol (Cerniglia and Yang 1984). It was proven later that the same mechanism applies to other polycyclic aromatic hydrocarbons (naphthalene, uoranthene, pyrene, and benzo[a]pyrene). Moreover, it was proven that their degradation via cis-dihydrodiol, mediated by bacteria, can proceed to carbon dioxide (Cerniglia 1992). Recently, more detailed research has been conducted on phenanthrene isomers. It was shown that out of 29 types of bacteria in the soil, 11 types use for their development only methyl-phenanthrenes. For example, mycobacteriums use exclusively 2-methyl-phenanthrenes as their food, while sphingomonas use exclusively 1-methyl-phenanthrenes (Lamberts et al. 2008). In the present study, the changes in the distribution of phenanthrene and its methyl isomers (mono, di, and tri) during bioremediation of soils contaminated with heavy residual fuel oil (mazut) were investigated. The results of bioremediation experiment of soil that was treated with biomass (re-inoculation) and nutrients (biostimulation) were compared with the results of biodegradation of soil that was not subjected to these processes of stimulation.

Experimental During the period from September 2009 to March 2010, the biodegradation of soil contaminated with heavy residual fuel oil (mazut) was conducted. The crude oilpolluted soil was excavated contaminated soil from an energy power plant. Due to a break-down of the energy power plant, the soil had been polluted with heavy fuel oil and sediment from a heavy oil reservoir for a year. The level of contamination of soil

investigated in this paper, expressed through a set of parameters, including the content of the extract, is presented in the previous paper (Beskoski et al. 2011). The crude oilpolluted soil (approximately 150 t; 210 m3) was uniformly distributed over 300 m3 of not rinsed sand from the Sava River (settlement Ostruznica, Serbia). The sawdust from poplar, beech, and oak (approx. 60 m3) was added in order to increase the retention water capacity, but as alternative additional carbon (C) substrate as well. The entire material (volume of approx. 600 m3), dened as a bioremediation substrate, was homogenized and then formed into a biopile shape with dimensions of 75 9 20 9 0.4 m (length, width, height), with bulldozers. After formation of the biopile, it was continuously sprayed with biomass, from the tank of 5 m3. The biomass of microbial consortia, isolated from the crude oilcontaminated soil (re-inoculation) and nutritive substances (biostimulation), was applied on the biopile. Analytical prole index (API-Biomerieux) tests conducted with isolated cultures of microorganisms identied Pseudomonas aeruginos, Rhodococcus sp., Pseudomonas sp., Pseudomonas uorescens, Sphingomonas paucimobilis, Pseudomonas luteola, Achromobacter denitricans, Stenotrophomonas maltophilia and Aeromonas hydrophila. Biomass concentration was 1.44 9 107 cells/mL. An optimal ratio of C/N/P/K (approx. 100:10:1:0.1) was achieved by spraying a solution of dissolved ammonium nitrate (N), diammonium hydrogen phosphate (P and N) and potassium chloride (K) with agricultural spraying. Aeration and mixing were performed each 2 weeks with powerful construction machinery. Biomass and nutritive substances were added once a month by turning and mixing the biopile. Biosurfactant of Biosolve type was applied on the biopile at a concentration of 70 mL of the original solution per cubic meter of soil. After preparation, the biopile was covered with plastic foil to prevent direct inuence of precipitation and low temperatures on the bioremediation material. The average daily temperature during the sixmonth experiment was 7.6 6.3C (in the range from -2.3 to 23.5C). A detailed description of this procedure was discussed in the previous paper (Beskoski et al. 2011). Simultaneously with the sampling from biopile, at the beginning of the experiment, immediately after mixing, but before the addition of sawdust, biomass, nutrient substances, and biosurfactant, samples were taken from the control pile. The complete analytical procedure that was applied to the samples was also applied to the control samples. During the 6-month interval, the samples were taken ve times (07/09/2009, 06/10/2009, 09/11/2009, 12/01/ 2010, and 18/03/2010). Samples taken from soils that were treated with sawdust, biomass, nutrient, and biosurfactants were marked M1M5. Control test samples, taken at the same time, were marked M1kM5k.

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Organic substance from in total 10 soil samples was extracted with chloroform (HPLC, J.T., USA) using a Soxhlet apparatus. From these extracts, the hydrocarbons (saturated and aromatic) were isolated by column chromatography: the extracts were saponied with a 5% solution of KOH in methanol and neutralized (after standing overnight) with 10% hydrochloric acid. The products were dissolved in a mixture of dichloromethane (containing 1% methanol) and hexane (1:40) and separated by column chromatography on alumina and silica gel. The hydrocarbon fractions were eluted with hexane (saturated hydrocarbons) followed by dichloromethane (aromatic hydrocarbons). A detailed description of the analytical procedure was discussed in previous papers (Jovancicevic et al. 2003; 2005). Hydrocarbons were analyzed by the gas chromatographymass spectrometry (GCMS) techniques. An Agilent 7890N gas chromatograph tted with a HP5-MS capillary column (30 9 0.25 mm, 0.25 lm lm; temperature range: 80C for 0 min; then 2C min-1 to 300C and held for 20 min) with helium as the carrier gas (ow rate 1 cm3 min-1) was used. The GC was coupled to a HewlettPackard 5972 MSD operated at 70 eV in the 45550 scan range. Preliminary analyses of the investigated samples were conducted in the full-scan mode. Detailed analyses of the target compounds were conducted in the single-ion monitoring mode (SIM), comprising the following ion chromatograms: 178 (phenanthrene), 192 (methylphenanthrenes), 206 (dimethyl-phenanthrenes), and 220 (trimethyl-phenanthrenes). Peaks of the phenanthrene, methyl-phenanthrenes, and dimethyl-phenanthrenes were identied according to organic geochemical literature data (e.g., Peters et al. 2005), or based on the total mass spectra, using mass spectra databases (NIST/EPA/NIH mass spectral library NIST2000, Wiley/NBS registry of mass spectral data, 7th ed., electronic versions). The peaks of trimethyl-phenanthrenes were labeled using the hypothesized elution order according to Stojanovic et al. (2007). Phenanthrene and alkyl phenanthrene parameters were calculated from GCMS chromatogram peak areas (software GCMS Data Analysis).

Results and discussion Mass fragmentograms of phenanthrene, methyl-phenanthrenes, dimethyl-phenanthrenes, and trimethyl-phenanthrenes obtained by GCMS analysis of aromatic fractions isolated from extracts of samples M1M5 are shown in Fig. 1. These samples were subjected to re-inoculation and biostimulation with the addition of sawdust and biosurfactant. Fragmentograms of control tests (samples M1k M5k, without the addition of sawdust, biomass, nutrient substances and biosurfactant) are shown in Fig. 2.

In this study, the changes in the distribution of phenanthrene and its methyl isomers (mono-, di- and tri-) were investigated. These aromatic hydrocarbons are not at the so high level of toxicity (Simmon et al. 1979; Nousiainen et al. 1984; Henner et al. 1999). However, they are in most of the oils, and therefore in most of the oil-type pollutants, dominant aromatic hydrocarbons. Moreover, in general, as well as polycyclic aromatic hydrocarbons, they represent a signicant pollutant of all segments of the environment, including soils (Lichtfouse et al. 2005; Bryselbout et al. 2000; Henner et al. 1997). To this end, ratios of the relative concentrations of phenanthrene and the most abundant methyl, dimethyl and trimethyl isomers were calculated. Additionally, ratios of the relative concentrations of methyl and trimethyl isomers were calculated. The values of these parameters for samples M1M5 are shown in Fig. 3. The values of the parameters calculated for the control samples (M1kM5k) are shown in Fig. 4. Based on fragmentograms in Fig. 1, as well as on the values of numerous parameters shown in Fig. 3, it can easily be observed that during the process of bioremediation of soil contaminated by heavy residual fuel oil (mazut), there was a uniform increase in the relative abundance of phenanthrene compared to its methyl isomers. This increase was the most pronounced in the case of trimethyl-phenanthrenes (P/TMP = 0.422.45, Fig. 3) and the least in the case of methyl-phenanthrenes (P/ MP = 0.501.02, Fig. 3). The ratio of MP/TMP was also uniformly increased from 0.84 to 2.40 (Fig. 3). Based on these results, it can be drawn a general conclusion that the bioremediation process under the conditions described generally results in increase in the concentrations of phenanthrene, but also its lower methyl homologue compared to the higher homologues. From a total of 9 types of microorganisms identied in the zymogen consortium, 6 of them belong to the group of efcient petroleum hydrocarbons degraders. These are P. aeruginosa, Rhodococcus sp., Pseudomonas sp., P. uorescens, P. luteola and A. denitricans, S. maltophilia, and A. hydrophila (Bossert and Bartha 1984, Singh and Ward 2004). As already noted, the process of re-inoculation, that is, addition of biomass of microbial consortia isolated from crude oil-contaminated soil, was performed once a month after the biopile was mixed and turned around. At the same time, the nutrients (N, P, and K) necessary for cellular metabolism and successful development of microorganisms were added (Alexander 1994; Atagana et al. 2003). There is a possibility that the observed changes in the distribution of phenanthrene and its methyl isomers occurred as a result of demethylation (Huang et al. 2004) through the process of oxidative decarboxylation. In this way, an increase in absolute concentration of phenanthrene could have happened at the expense of degradation of methyl-, dimethyl-, and

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290 Fig. 1 Mass fragmentograms of phenanthrene (P, m/z 178), methyl-phenanthrenes (MP, m/z 192), dimethyl-phenanthrenes (DMP, m/z 206), and trimethylphenanthrenes (TMP, m/z 220), obtained by the gas chromatographymass spectrometry (GCMS) analysis (using the Single Ion Monitoring, SIM method) of aromatic fractions isolated from M1 to M5 extracts taken from soils that were treated with sawdust, biomass, nutrient, and biosurfactants during the 6-month bioremediation. Note the uniform decrease in the relative abundance of methyl isomers compared to phenanthrene. This trend is opposite to the typical biodegradation sequence of phenanthrene and its methyl isomers. Peaks used for parameter calculations (Fig. 3) are marked by dark points

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trimethyl-phenanthrenes. However, demethylation is a thermodynamically less favorable process. Therefore, it is more likely that the biostimulation process favored those bacteria strains in consortium that decompose methyl isomers (rst of all trimethyl-phenanthrenes). This biodegradation pattern can be a consequence of better interaction of reactive methyl groups with the active centers on the surface of bacterial cells

and, in this way, promoted decomposition of methyl-phenanthrene derivatives (Lamberts et al. 2008). The presence of biosurfactant that increases solubility and thus the availability of products with a higher degree of alkylation could also contribute this process. In this way, it could have happened a regular increase in ratios of P/MP, P/DMP, and P/TMP during the process of bioremediation of contaminated soils. On the

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Environ Chem Lett (2012) 10:287294 Fig. 2 Mass fragmentograms of phenanthrene (P, m/z 178), methyl-phenanthrenes (MP, m/z 192), dimethyl-phenanthrenes (DMP, m/z 206), and trimethylphenanthrenes (TMP, m/z 220), obtained by the gas chromatographymass spectrometry (GCMS) analysis (using the Single Ion Monitoring, SIM method) of aromatic fractions isolated from control sample extracts M1k M5k taken from soils that were not treated with sawdust, biomass, nutrient, and biosurfactants during the 6-month bioremediation. Note the uniform decrease in the relative abundance of phenanthrene compared to its methyl isomers. Peaks used for parameter calculations (Fig. 4) are marked by dark points

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other hand, during the bioremediation, a decrease in the relative concentration of trimethyl-phenanthrenes relative to the methyl-phenanthrenes was observed. This is reected through an increase in the ratio MP/TMP. This result could indicate that the interaction of phenanthrene isomers with bacterial cells is increased if phenanthrene contains a larger number of methyl substituents.

Monitoring changes in the distribution of phenanthrene and its methyl isomers in 5 samples belonging to the control trials (M1kM5k) actually gives an estimate of the transformations that occur during the natural microbial degradation of oil pollutant. Contrary to the bioremediation process (samples M1M5), sawdust, biomass, nutrient substances, and biosurfactant were not added to the control

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292 Fig. 3 Ratios of phenanthrene (P) and most intense methylphenanthrene (MP), dimethylphenanthrene (DMP), and trimethyl-phenanthrene (TMP), as well as MP and TMP isomers for M1M5 samples taken from soils that were treated with sawdust, biomass, nutrient, and biosurfactants during the 6-month bioremediation. The uniform increase in all ratios shows decrease in the relative abundance of methyl isomers compared to phenanthrene and also its higher methyl homologues compared to the lower homologues (See Fig. 1)

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Fig. 4 Ratios of phenanthrene (P) and most intense methylphenanthrene (MP), dimethylphenanthrene (DMP), and trimethyl-phenanthrene (TMP), as well as MP and TMP isomers for M1kM5k samples taken from soils that were not treated with sawdust, biomass, nutrient, and biosurfactants during the 6-month bioremediation. The decrease in all ratios shows decrease in the relative abundance of phenanthrene compared to methyl isomers and also its lower methyl homologues compared to the higher homologues (See Fig. 2)

samples. Under such conditions, comparing with the process of intensied biodegradation, an opposite trend can be noticed (Figs. 2, 4): the relative concentration of phenanthrene is reduced relative to methyl-phenanthrenes, dimethyl-phenanthrenes, and trimethyl-phenanthrenes. This decrease is the most pronounced in comparison with the trimethyl isomers (P/TMP = 0.850.11, Fig. 4) and the least pronounced in comparison with the methyl isomers (P/MP = 0.910.61, Fig. 4). MP/TMP was also uniformly decreased from 0.93 to 0.17 and 1.15 (Fig. 4). It can be concluded that, generally, in this process, a decrease in the relative concentration of phenanthrene and lower methyl

homologue comparing to the higher homologues occurred. These changes in the distribution of phenanthrene and its methyl isomers during biodegradation can be characterized as typical, and they are described in previous papers (for example, Solevic et al. 2011).

Conclusion During the 6 months of bioremediation of soils (re-inoculation, biostimulation, the addition of sawdust and biosurfactants) contaminated with heavy residual fuel oil (mazut),

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there was a uniform increase in the relative abundance of phenanthrene compared to its methyl isomers. Trend was observed in 5 samples that were taken at relatively regular intervals during the period from September 2009 to March 2010 (samples M1M5). This increase was most pronounced in comparison with the trimethyl-phenanthrenes and the least in comparison with the methyl-phenanthrenes (see Fig. 1 and ratios of P/TMP and P/MP in Fig. 3). The relative concentration of methyl-phenanthrenes increased relative to trimethyl-phenanthrenes as well (see ratio MP/TMP in Fig. 3). According to these results, a general conclusion can be drawn that this process of applied bioremediation generally resulted in an increase in the concentrations of phenanthrene, but also its lower methyl homologue compared to the higher homologues. During the process of natural microbial degradation of oil pollutants (control samples M1kM5k), a different trend was observed: the relative concentration of phenanthrene is reduced relative to methyl-phenanthrenes and dimethyl-phenanthrenes and especially relative to trimethyl-phenanthrenes. Similarly, the concentration of methyl-phenanthrenes is reduced relative to the trimethyl derivatives (see Figs. 2, 4). Demethylation is a process that could have led to the degradation of methyl derivatives of phenanthrene and an increase in the absolute concentration of phenanthrene. However, this process is thermodynamically less favorable. Accordingly, it was assumed that re-inoculation, biostimulation, and the addition of sawdust and biosurfactant promoted the process of decomposition of methyl-phenanthrene derivatives, by favoring the bacterial strains in the consortium (P. aeruginosa, Rhodococcus sp., Pseudomonas sp., P. uorescens, S. paucimobilis, P. luteola, A. denitricans, S. maltophilia, and A. hydrophila) that break down methyl isomers (rst of all trimethyl-phenanthrenes). This biodegradation pattern is explained as a result of better interaction between more reactive methyl groups with active centers on the surface of bacterial cells. Direct laboratory experiments would be useful for the conrmation of this assumption. On the other hand, changes in the distribution of phenanthrene and its methyl isomers during the unstimulated biodegradation can be characterized as typical, and they are described in earlier papers. On the basis of this research, a general conclusion can be drawn that an increase in the availability of phenanthrene and its methyl derivatives to microorganisms can increase degradability of methyl-phenanthrenes compared to phenanthrene. In this study, an increased availability of phenanthrene and its methyl derivatives to microorganisms was accomplished by re-inoculation, biostimulation, as well as by the addition of sawdust and biosurfactants. Additionally, it can be concluded that the level of

degradability in these conditions depends on the number of methyl groups, that is, on the level of alkylation.
Acknowledgments We thank the Ministry of Education and Science of the Republic of Serbia (Projects 176006 & III 43004) for supporting this research.

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