Environ Chem Lett (2012) 10:287–294
DOI 10.1007/s10311-012-0354-6
ORIGINAL PAPER
Degradation of methyl-phenanthrene isomers
during bioremediation of soil contaminated by residual fuel oil
Milan Novaković • Muftah Mohamed Ali Ramadan •
Tatjana Šolević Knudsen • Mališa Antić • Vladimir Beškoski •
Gordana Gojgić-Cvijović • Miroslav M. Vrvić • Branimir Jovančićević
Received: 17 May 2011 / Accepted: 13 January 2012 / Published online: 22 January 2012
Ó Springer-Verlag 2012
Abstract Phenanthrene and methyl-phenanthrenes are
major aromatic pollutants originating in particular from fuel
oil. Phenanthrene is usually degraded faster than methyl-
phenanthrenes under geological and environmental condi-
tions. Here, we report a preferential and accelerated bio-
degradation 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 oil–contaminated 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 com-
pared to methyl-phenathrenes was observed by gas chro-
matography–mass 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
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. Novaković
M. M. A. Ramadan
M. M. Vrvić

B. Jovančićević (&)
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. Š. Knudsen
V. Beškoski
G. Gojgić-Cvijović

M. M. Vrvić
B. Jovančićević
Center of Chemistry, Institute of Chemistry, Technology and
Metallurgy, University of Belgrade, Njegoševa 12,
P.O. Box 473, 11001 Belgrade, Serbia
M. Antić
Faculty of Agriculture, University of Belgrade, Nemanjina 6,
11081 Belgrade, Serbia
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, first of all recent
sediments, soils and surface waters. For example, the
efficiency 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
refinery products of petroleum refining), their biodegrada-
tion and removal from the environment are difficult to be
classified in one category. Oil is a very complex mixture of
hydrocarbons, but also nitrogen, sulfur, and oxygen com-
pounds (NSO). Each class of compounds, and often indi-
vidual compounds as well, require special study aimed to
define the type of microorganisms and optimal conditions
for microbial degradation (Fritsche and Hofrichter 2008;
Van Hamme et al. 2003). For degradation of some com-
pounds, microorganisms and conditions for degradation are
not known, and these compounds are considered non-bio-
degradable. 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
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related to the fate of petroleum pollutants in the environ-
ment as well (for example: Jovančićević et al. 2003; Antić
et al. 2006; Ilić et al. 2011; Šolević 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 hydro-
carbons are generally resistant to biodegradation.
The aromatic fraction was examined together with satu-
rated 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 pro-
ven. 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 bac-
teria, naphthalene can be decomposed to carbon dioxide via
catechol according to the ‘‘ring fission’’ mechanism. Also,
fungi are capable of oxidation of anthracene and phenan-
threne to trans-dihydrodiol (Cerniglia and Yang 1984). It
was proven later that the same mechanism applies to other
polycyclic aromatic hydrocarbons (naphthalene, fluoranth-
ene, 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 oil–polluted 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
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Environ Chem Lett (2012) 10:287–294
investigated in this paper, expressed through a set of
parameters, including the content of the extract, is presented
in the previous paper (Beškoski et al. 2011).
The crude oil–polluted 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),
defined 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 oil–contami-
nated soil (re-inoculation) and nutritive substances (bi-
ostimulation), was applied on the biopile.
Analytical profile index (API-Biomerieux) tests con-
ducted with isolated cultures of microorganisms identified
Pseudomonas aeruginos, Rhodococcus sp., Pseudomonas
sp., Pseudomonas fluorescens, Sphingomonas paucimobi-
lis, Pseudomonas luteola, Achromobacter denitrificans,
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 influence of
precipitation and low temperatures on the bioremediation
material. The average daily temperature during the six-
month experiment was 7.6 ± 6.3°C (in the range from -2.3
to 23.5°C). A detailed description of this procedure was
discussed in the previous paper (Beškoski 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
five 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 M1–M5. Control test samples, taken at the
same time, were marked M1k–M5k.
Environ Chem Lett (2012) 10:287–294
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 chro-
matography: the extracts were saponified with a 5% solu-
tion 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 hydrocar-
bon fractions were eluted with hexane (saturated hydro-
carbons) followed by dichloromethane (aromatic
hydrocarbons). A detailed description of the analytical
procedure was discussed in previous papers (Jovančićević
et al. 2003; 2005).
Hydrocarbons were analyzed by the gas chromatogra-
phy–mass spectrometry (GC–MS) techniques. An Agilent
7890N gas chromatograph fitted with a HP5-MS capillary
column (30 9 0.25 mm, 0.25 lm film; temperature range:
80°C for 0 min; then 2°C min-1 to 300°C and held for
20 min) with helium as the carrier gas (flow rate
1 cm3 min-1) was used. The GC was coupled to a Hewlett-
Packard 5972 MSD operated at 70 eV in the 45–550 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 moni-
toring mode (SIM), comprising the following ion chro-
matograms: 178 (phenanthrene), 192 (methyl-
phenanthrenes), 206 (dimethyl-phenanthrenes), and 220
(trimethyl-phenanthrenes). Peaks of the phenanthrene,
methyl-phenanthrenes, and dimethyl-phenanthrenes were
identified 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-phe-
nanthrenes were labeled using the hypothesized elution order
according to Stojanović et al. (2007). Phenanthrene and alkyl
phenanthrene parameters were calculated from GC–MS
chromatogram peak areas (software GC–MS Data Analysis).
Results and discussion
Mass fragmentograms of phenanthrene, methyl-phenan-
threnes, dimethyl-phenanthrenes, and trimethyl-phenan-
threnes obtained by GC–MS analysis of aromatic fractions
isolated from extracts of samples M1–M5 are shown in
Fig. 1. These samples were subjected to re-inoculation and
biostimulation with the addition of sawdust and biosur-
factant. Fragmentograms of control tests (samples M1k–
M5k, without the addition of sawdust, biomass, nutrient
substances and biosurfactant) are shown in Fig. 2.
289
In this study, the changes in the distribution of phen-
anthrene 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
significant 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 M1–M5 are shown in Fig. 3. The
values of the parameters calculated for the control samples
(M1k–M5k) 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 bioremedia-
tion of soil contaminated by heavy residual fuel oil
(mazut), there was a uniform increase in the relative
abundance of phenanthrene compared to its methyl iso-
mers. This increase was the most pronounced in the case of
trimethyl-phenanthrenes (P/TMP = 0.42–2.45, Fig. 3) and
the least in the case of methyl-phenanthrenes (P/
MP = 0.50–1.02, 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 identified in
the zymogen consortium, 6 of them belong to the group of
efficient petroleum hydrocarbons’ degraders. These are P.
aeruginosa, Rhodococcus sp., Pseudomonas sp., P. fluo-
rescens, P. luteola and A. denitrificans, 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 microorgan-
isms 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 hap-
pened at the expense of degradation of methyl-, dimethyl-, and
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Fig. 1 Mass fragmentograms

of phenanthrene (P, m/z 178),
methyl-phenanthrenes (MP, m/z
192), dimethyl-phenanthrenes
(DMP, m/z 206), and trimethyl-
phenanthrenes (TMP, m/z 220),
obtained by the gas
chromatography–mass
spectrometry (GC–MS) 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

trimethyl-phenanthrenes. However, demethylation is a ther-
modynamically less favorable process. Therefore, it is more
likely that the biostimulation process favored those bacteria
strains in consortium that decompose methyl isomers (first 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-phen-
anthrene 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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Fig. 2 Mass fragmentograms

of phenanthrene (P, m/z 178),
methyl-phenanthrenes (MP, m/z
192), dimethyl-phenanthrenes
(DMP, m/z 206), and trimethyl-
phenanthrenes (TMP, m/z 220),
obtained by the gas
chromatography–mass
spectrometry (GC–MS) 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

other hand, during the bioremediation, a decrease in the rel-
ative concentration of trimethyl-phenanthrenes relative to the
methyl-phenanthrenes was observed. This is reflected 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 (M1k–M5k) actually gives an estimate of the
transformations that occur during the natural microbial
degradation of oil pollutant. Contrary to the bioremediation
process (samples M1–M5), sawdust, biomass, nutrient
substances, and biosurfactant were not added to the control

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Fig. 3 Ratios of phenanthrene

(P) and most intense methyl-
phenanthrene (MP), dimethyl-
phenanthrene (DMP), and
trimethyl-phenanthrene (TMP),
as well as MP and TMP isomers
for M1–M5 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)

Fig. 4 Ratios of phenanthrene

(P) and most intense methyl-
phenanthrene (MP), dimethyl-
phenanthrene (DMP), and
trimethyl-phenanthrene (TMP),
as well as MP and TMP isomers
for M1k–M5k 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 pro-
cess of ‘‘intensified’’ 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 = 0.85–0.11, Fig. 4) and the
least pronounced in comparison with the methyl isomers
(P/MP = 0.91–0.61, 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 phenan-
threne and its methyl isomers during biodegradation can be
characterized as typical, and they are described in previous
papers (for example, Šolević et al. 2011).
Conclusion
During the 6 months of bioremediation of soils (re-inocu-
lation, biostimulation, the addition of sawdust and biosur-
factants) 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 M1–M5). 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 con-
centration 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 gener-
ally resulted in an increase in the concentrations of phen-
anthrene, but also its lower methyl ‘‘homologue’’ compared
to the higher homologues.
During the process of natural microbial degradation of
oil pollutants (control samples M1k–M5k), a different
trend was observed: the relative concentration of phenan-
threne is reduced relative to methyl-phenanthrenes and
dimethyl-phenanthrenes and especially relative to tri-
methyl-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, biosti-
mulation, and the addition of sawdust and biosurfactant
promoted the process of decomposition of methyl-phen-
anthrene derivatives, by favoring the bacterial strains in the
consortium (P. aeruginosa, Rhodococcus sp., Pseudomo-
nas sp., P. fluorescens, S. paucimobilis, P. luteola, A.
denitrificans, S. maltophilia, and A. hydrophila) that break
down methyl isomers (first 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 confirma-
tion of this assumption.
On the other hand, changes in the distribution of phen-
anthrene 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 phenan-
threne 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
293
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 Sci-
ence of the Republic of Serbia (Projects 176006 & III 43004) for
supporting this research.
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