Biodegradation of aromatic hydrocarbons and phenols by bacteria isolated from caspian waters and soils

Article abstract of DOI:10.1134/S0965544113050101

Experimental studies have been carried out on the degradability of monoaromatic hydrocarbons (benzene, toluene, and ethylbenzene) and phenols (phenol, pyrocatechol, hydroquinone, tetrachloropyro- catechol) by bacteria isolated from coastal waters and soil

Full text of DOI:10.1134/S0965544113050101

ISSN 0965ꢀ5441, Petroleum Chemistry, 2013, Vol. 53, No. 6, pp. 426–430. © Pleiades Publishing, Ltd., 2013.
Original Russian Text © M.G. Veliev, M.A. Salmanov, A.A. Babashly, S.R. Alieva, N.R. Bektashi, 2013, published in Neftekhimiya, 2013, Vol. 53, No. 6, pp. 476–480.
Biodegradation of Aromatic Hydrocarbons and Phenols by Bacteria
Isolated from Caspian Waters and Soils
M. G. Velieva, M. A. Salmanovb, A. A. Babashlyb, S. R. Alievab, and N. R. Bektashia
a Institute of Polymer Materials, National Academy of Sciences of Azerbaijan, Sumgait, Azerbaijan
b Institute of Microbiology, National Academy of Sciences of Azerbaijan, Baku, Azerbaijan
eꢀmail: mveliev@mail.ru
Received February 25, 2013
Abstract—Experimental studies have been carried out on the degradability of monoaromatic hydrocarbons
(benzene, toluene, and ethylbenzene) and phenols (phenol, pyrocatechol, hydroquinone, tetrachloropyroꢀ
catechol) by bacteria isolated from coastal waters and soils of the Absheron peninsula at the Caspian Sea. It
has been shown that the degradation of pyrocatechol, in particular, by Pseudomonas sp. bacteria occurs with
the cleavage of the aromatic ring in two directions (in the metaꢀ and orthoꢀpositions), and that of tetrachloꢀ
ropyrocatechol involves the cleavage of the aromatic ring in the orthoꢀposition and the formation of tetrachloꢀ
romuconic acid. By reversedꢀphase highꢀperformance liquid chromatography, more than ten individual
compounds were detected and identified in the biodegradation products under the test conditions. It has been
also shown that mainly linear functional compounds formed by aromatic ring opening in the metaꢀ or ortho
position are found in the case of pyrocatechol and hydroquinone.
ꢀ
Keywords: biodegradation, phenols, aromatic hydrocarbons, microorganisms, microbial synthesis, liquid
chromatography
DOI: 10.1134/S0965544113050101
It is known that aromatic compounds are widely and hydroquinone) by bacteria isolated from water
and soil of the Absheron peninsula at the Caspian Sea.
distributed in the nature [1–4] and they are major
environmental pollutants [5–7]. Of all approaches to
the problem of removal of aromatic pollutants from
environment, their microbial degradation is the most
promising. Destructor microorganisms catabolize
aromatic substances, transforming them into hydroxꢀ
ylated derivatives with the subsequent opening of the
benzene ring and the formation of numerous easily
utilized substrates [1, 8–12]. The biodegradation
products formed are of interest from the standpoint of
research in the basic principles of utilization (biodegꢀ
radation) of aromatic compounds and prospects for
their use in biotechnologies of environmental remediꢀ
ation. The study of microbial degradation of petroꢀ
leum hydrocarbons, including phenolic compounds,
is important for the development of methods for bioreꢀ
mediation of the environment from crude oil and pheꢀ
nolic contaminants [13–15], so we have continued
research in this area [13, 16, 17].
EXPERIMENTAL
The Stolbunov’s medium was used to isolate the
bacteria [18]. The salinity of the medium was adjusted
to 13%. Wellꢀknown microbiological methods were
used for isolation of the bacteria on plain agar (PA)
agarized media [19]. The isolated bacteria on the basis
of their morphological, physiological and biochemical
characteristics were attributed to the Micrococcus,
Bacillus, Pseudomonas, Arthrobacter, Mycobacterium,
Acinetobacter, Aeromonas, Vibrio, Sarcina, and Alcaliꢀ
genes genera according to the Bergey’s manual [20].
The intensity of the biodegradation process was
indirectly assessed by the accumulation of the biomass
of bacteria growing on individual aromatic and pheꢀ
nolic compounds during a month. In this experiment,
1–2 day cultures of microorganisms grown on the PA
were used as the inoculum. The cells were washed off
with 10 mL of distilled water; then, 5 mL of the resultꢀ
ing suspension was placed in flasks with 0.1 L of a
nutrient medium containing benzene, toluene, pheꢀ
The aim of the present work is to study the products
of biodegradation of some monoaromatic hydrocarꢀ
bons (benzene, toluene, and ethylbenzene) and pheꢀ
nols (phenol, pyrocatechol, tetrahloropyrocatechol, nol, pyrocatechol, or tetrahloropyrocatechol as the
426

BIODEGRADATION OF AROMATIC HYDROCARBONS AND PHENOLS BY BACTERIA
427
sole carbon source. In all the experiments the cultures
were incubated at a temperature of 28 . Further, the
Yield of dry biomass, g/L
°С
g
5.0
4.5
4.0
3.5
3.0
dynamics of the bacterial population and processes of
biodegradation of aromatic compounds added to the
medium in concentrations of 100, 300, and 500 mg/L
were studied. After incubation, the biodegradation
products were extracted with an equal volume of chloꢀ
roform from the culture medium freed from cells by
filtration.
Products of the degradation of aromatic and pheꢀ
nolic compounds were investigated by reversedꢀphase
highꢀperformance liquid chromatography (HPLC).
Samples of initial and residual (after biodegradation)
aromatic compounds were analyzed by the NMR and
IR spectroscopic techniques as well [21–23].
f
g
f
c
c
b
g
f
b
e
e
a
2.5
d
g
c
e
a
d
a
2.0
1.5
1.0
0.5
0
f
d
b
c
b
de
a
1
2
3
4
Weeks
a
b
c
d
e
f
g
Chromatographic investigations were carried out
on a Kovo highꢀperformance liquid chromatograph
(Czech Republic) with an UV spectrophotometric
The yield of Pseudomonas sp
.
biomass upon growth on aroꢀ
matic ((a) toluene, (b) benzene, (c) ethylbenzene) and
phenolic ((d) hydroquinone, (e) tetrachloropyrocatechol,
(f) pyrocatechol, (g) phenol) compounds.
detector operating at a wavelength of
3.3 150 mm columns packed with the Separon SGXꢀ
C18 reverse stationary phase (particle size 7 m; temꢀ
perature, 20–25 ) were used. Eluent, methanol :
λ
= 254 nm. Two
×
µ
The results show that the degradation of the test
compounds by the action of the above mentioned
microorganisms is accompanied by the transformaꢀ
tion of the compounds to corresponding phenols, carꢀ
boxylic and phenolcarboxylic acids, polyhydric pheꢀ
nols, benzoquinones, as well as to small quantities of
polymers. Note that the identification of the biotransꢀ
formation products by HPLC analysis was carried out
at the final step of the microbiological synthesis with
the formation of individual organic compounds.
°С
water (75 : 25 vol. %). Mobile phase flow rate,
0.3 mL/min. The components were identified by
comparing the retention parameters for a standard
mixture and biotransformation products. Standard
solutions with a concentration of 1–1.5 mg/mL were
prepared in the methanol : water (75 : 25% vol.%)
eluting system by the conventional procedure [23, 24].
The structural composition of the products of biodegꢀ
radation of individual hydrocarbons and phenolic
compounds was determined by the methods of infraꢀ
red spectroscopy (URꢀ20) (thin layer) in the spectral
As follows from the chromatographic data, starting
from the oxidation of the aromatic ring the degradaꢀ
tion of benzene after 6 days results in the formation of
pyrocatechol and phenol in amounts of 30% and 60%,
respectively.
1
range 4000–700 cm–1 and H NMR spectroscopy
(Tesla BSꢀ487B spectrometer with an operating freꢀ
quency of 80 MHz; solvent, ССl4; internal standard,
hexamethyldisiloxane (HMDS)). Control tests (withꢀ
out introducing biodestructor bacteria) were perꢀ
formed for all the experiments.
Along with the chromatographic determination
1
data, the IR and H NMR data were examined; they
confirmed the structure of the products. The IR specꢀ
tra of the benzene biodegradation products exhibited
absorption bands in the region of 3590–3650 cm–1
typical of the hydroxyl group along with the bands
characterizing the phenyl ring. The chemical shifts of
aromatic ring and hydroxyl protons in their 1HꢀNMR
RESULTS AND DISCUSSION
The experiments showed that all investigated aroꢀ
matic hydrocarbons and phenolic compounds served
as the only carbon source for the growth of each of the
isolated group of bacteria. Of the isolated bacteria
strains, 47% grew on benzene; 54%, on ethylbenzene;
43%, on toluene; 100%, on phenol and pyrocatechol;
70%, on hydroquinone; and 13%, on tetrachloropyroꢀ
catechol. The accumulation of biomass in the medium
with admixed aromatics (benzene, toluene, ethylbenꢀ
zene) and phenolic compounds (phenol, pyrocateꢀ
chol, hydroquinone, tetrachloropyrocatechol) at a
concentration of 500 mg/L is shown in the figure. As
can be seen in the figure, the accumulation of biomass
for the Pseudomonas sp. strain occurred more
intensely upon utilization of phenolic compounds
than with aromatic ones, with the biomass varying
from 1.6 g/L to 4.5 g/L.
spectra were detected at
δ
= 6.25–7.2 ppm and
δ
=
7.65–9.00 ppm, respectively, [21, 22].
In the case of toluene degradation, basically three
individual compounds were detected; ꢀcresol (50%),
o
benzoic acid (30%), and 2,3ꢀdihydroxytoluene (10%)
prevailed in the products. The conversion occurred
through the formation of intermediates; and, thus,
based on the known theoretical concepts, we can proꢀ
pose the following scheme of toluene biodegradation
by the action of Pseudomonas sp.:
PETROLEUM CHEMISTRY Vol. 53
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2013

428
VELIEV et al.
CH2OH
COOH
CH3
CHO
]
[
[
CH3
CH3
CH3
H
OH
O
Pseudomonas sp.
O
H
H
]
H
CH3
CH3
CH3
H
H
OH
OH
OH
O
OH
H
O
H
]
[
The IR spectra of the toluene biodegradation products
displayed absorption bands characteristic of hydroxyl
(3590–3650 cm–1) and carboxyl (1680–1700 cm–1)
groups along with the Ar–CH3 bands. The H NMR
spectrum of the degradation products displayed signals
Thus, the transformations of toluene and ethylbenꢀ
zene under the conditions developed in this work
occur in two directions simultaneously, involving the
oxidation of the side chain and the aromatic ring.
Despite the fact that the degradation of toluene via the
mixed mechanism is known from the literature [8], the
composition of the oxidation products in this case difꢀ
fers from the one found previously. Thus, in [12, 13] it
was shown that toluene degradation occurs in different
directions, depending on the microorganism species.
Interestingly, the compounds formed in the prior
experiments [13, 16, 17] were not detected in this
study. This suggests that the same bacteria cultured
under different conditions and isolated from different
sources may differ in the mechanism of action.
1
at
δ
2.1–2.8 ppm,
δ
7.70–9.00 ppm, and
δ
9.5–
10.5 ppm characteristic of the protons of the Ar–CH3
,
Ar, Ar–OH, and Ar–COOH groups.
In [13], we showed that ethylbenzene is converted
into acetophenone and phenylacetic acid by the
action of micromycetes under coꢀoxidative condiꢀ
tions. In contrast, experiments in this study have
shown that the degradation of ethylbenzene by the
action of Pseudomonas sp. bacteria yields benzoylforꢀ
mic acid (30%) in addition to acetophenone (28%)
and phenylacetic acid (45%), according to the followꢀ
ing scheme:
The chromatographic analysis of phenol biodegraꢀ
dation by the action of phenolꢀdigesting bacteria
showed that phenol under the test conditions is transꢀ
formed into polyhydric phenols: pyrocatechol (40%),
pyrogallol (20%), and hydroquinone (10%). Among
the phenol biotransformation products (ca. 15%), a
polymer fraction with a relatively diffuse band was
detected; the fraction corresponds to oligophenylene
with a molecular weight of ~3500.
COOH
CH2
CH3
CH2
COOH
C O
CH3
C O
Pseudomonas sp.
+
+
OH
OH
OH
OH
OH
OH
OH
OH
Pseudomonas sp.
+
+
It was found that unlike aromatic hydrocarbons mainly via the route involving aromatic ring opening
and phenol investigated in the study, the degradation in the orthoꢀ and metaꢀpositions. By analysis of the
in the case of pyrocatechol and hydroquinone biotransꢀ chloroform extracts of pyrocatechol and hydroꢀ
formation by Pseudomonas sp. bacteria proceeded quinone, linear compounds relating to muconic acid
PETROLEUM CHEMISTRY Vol. 53
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BIODEGRADATION OF AROMATIC HYDROCARBONS AND PHENOLS BY BACTERIA
429
and its various derivatives were successively revealed. hydroxyꢀcis,cisꢀmuconic acid (20%), cis,cisꢀmuconic
Thus, 2ꢀketoꢀcis,cisꢀmuconic acid (10%), 2ꢀhydroxyꢀ acid (40%) and pyrogallol (5%) are formed in the case
cis,cisꢀmuconic acid (20%), semialdehyde of 2ꢀ of pyrocatechol, as shown in the following scheme:
O
OH
OH
OH
OH
OH
OH
OH
COOH
COOH
COOH
COOH
Arthrobacter sp.
COOH
COOH
CHO
COOH
+
+
+
+
But in the case of hydroquinone, 3ꢀketoꢀcis,cis
ꢀ
isolated from coastal waters and soils of the Absheron
muconic acid (25%), 4ꢀhydroxyꢀcis,cisꢀmuconic acid peninsula in the Caspian Sea has been studied. More
(35%), semialdehyde of 4ꢀhydroxyꢀcis,cisꢀmuconic than ten individual compounds, biodegradation prodꢀ
acid (30%), and hydroxyhydroquinone (4%) are ucts, were discovered and identified by reversedꢀphase
formed.
The IR spectra of the pyrocatechol and hydroꢀ
liquid chromatography in the biodegradation products
of the test compounds. Of these products, only the
compounds detected in the case of pyrocatechols,
which degrades via aromatic ring opening in the metaꢀ
and orthoꢀpositions, were found to be linear. It is
shown that unlike the case of pyrocatechol, the
decomposition of tetrachloropyrocatechol occurs
only in the orthoꢀposition with the formation of tetraꢀ
chloromuconic acid. The results can be used in fine
organic synthesis for the production of practically useꢀ
ful functional compounds (acids, ketones, aldehydes,
etc.) [25].
quinone biodegradation products showed the absorpꢀ
tion bands characteristic of the aromatic ring, the douꢀ
ble bond (1600–1660 cm–1), and the carboxyl group
(1700–1725 cm–1 and 3560–3650 cm–1). In this case,
the absorption bands of the aldehyde group are found
at 1685 cm–1. In their 1H NMR spectra, doubleꢀbond
protons appear at
tons of the carboxyl group appear as a singlet at
10.5 ppm; protons of the aromatic ring are identified
as a multiplet at 6.25–7.25 ppm, and those of the
aldehyde group are manifested as a singlet at
δ
6.44–6.8 ppm (
J
= 9.3 Hz). Proꢀ
δ
9.5–
δ
δ
9.25 ppm.
ACKNOWLEDGMENTS
The further study showed that unlike pyrocatechol,
The work was supported by the Science Developꢀ
ment Foundation under the President of Azerbaijan
Republic, project no. EIF—2011ꢀ1(3)ꢀ82/66/4.
tetrachloropyrocatechol undergoes biodegradation
only in one direction to form tetrachloromuconic acid
(yield up to 13%), the structure of which was also conꢀ
firmed by IR and 1H NMR data. The IR spectrum of
the acid exhibits absorption bands at 1720 cm–1 due to
the carboxyl group in addition to the absorption bands
of the C–Cl (650 cm–1) and C=C– (1625 cm–1)
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Translated by V. Makhaev
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