1.

Introduction
Polycyclic aromatic hydrocarbons (PAHs) are a large
class of persistent, hydrophobic environmental pollutants
emitted into the environment by incomplete combustion
or pyrolysis of fossil fuels or organic materials [1]. PAHs
have high octanol-water partition coeffcients and low
vapor pressure, and therefore these environmental
pollutants are rapidly absorbed by living organisms
and particulate matter. PAHs may be present in the
vapor and/or dissolved phase, micelle form, sorbed to
colloidal organic matter or to particles, and incorporated
into biota in water [2-4]. These compounds as well as
the products of their transformation pose a hazard for
all living organisms with regard to their distribution in the
biosphere, risk properties and abilities of accumulation
in animal and plant tissue [5]. Among over a hundred
different PAH compounds, 16 have been classifed as
priority pollutants by the United States Environmental
Protection Agency [6].
PAHs consisted of two or more condensed benzene
(aromatic) rings which are linearly ordered. According
to their lipophilic nature they have a good absorption
by gastrointestinal tract of mammifers. Distribution to
body apparatus and tissues with accumulation tendency
in adipose tissues is relatively quick. During the photo-
Central European Journal of Chemistry
Biodegradation of Benzo[a]Pyrene
through the use of algae
* E-mail: miroslava.smolinska@yahoo.com
Received 21 November 2013; Accepted 5 February 2014
Abstract:
Versita Sp. z o.o.
Keywords: Algae Benzo[a]Pyrene Biodegradation Chlorella kessleri
1
VRUP, a.s., Vlie Hrdlo, 820 01 Bratislava, Slovakia
2
Department of Microbiology and Virology, Faculty of Natural Sciences,
Comenius University in Bratislava, 842 15 Bratislava, Slovakia
3
Department of Fibres and Textile Chemistry, Institute of Polymer Materials,
Faculty of Chemical and Food Technology, Slovak University of Technology in
Bratislava, 812 37 Bratislava, Slovakia
4
Department of Environmental Engineering, Institute of Chemical and
Environmental Engineering, Faculty of Chemical and Food Technology, Slovak
University of Technology in Bratislava, 812 37 Bratislava, Slovakia
5
Department of Microelectronics, Institute of Electronics and Photonics,
Faculty of Electrical Engineering and Information Technology, Slovak
University of Technology in Bratislava, 812 19 Bratislava, Slovakia
Albeta Takov
1
, Miroslava Smolinsk
2*
, Jozef Ryba
3
,
Tom Mackuak
4
, Jana Jokrllov
4
,
Pavol Hronec
5
, Gabriel k
4

Research Article
In this work for disposal of the biologically hard decomposed pollutant Benzo[a]Pyrene (BaP) photooxidation Chlorella kessleri was
used. The simulation model system under the different experimental conditions (varying biomass and light intensity) was evaluated. For
quantitative analysis of the decrease in BaP, GC/MS technique was used. The highest degradation effciency was achieved in the case
of biomass from the culture of live algae (29%) and light intensity at level of 13.5 W m
-2
. When the dry biomass was used, degradation
under the same conditions was lower because of lack of enzymatic activity in the system.
Cent. Eur. J. Chem. 12(11) 2014 1133-1143
DOI: 10.2478/s11532-014-0567-6
1133
Biodegradation of Benzo[a]Pyrene
through the use of algae
oxidation of PAHs in the presence of oxygen and a
sensitizing substance, photosensitizers absorb light
energy and reach an excited state. In the return to the
ground state, excited photosensitizers transfer their
energy to the ground-state oxygen, producing reactive
oxygen species (ROS) [7,8].
The excess of intracellular ROS may oxidize
macromolecules, leading to lipid peroxidation, protein
oxidation and DNA base oxidation [9]. ROS are reactive
species which generate oxidative stress and may
cause damage to the membranes, mitochondria and
chloroplasts and, therefore, to inhibit photosynthesis,
physiological activities and cell growth. The cells have
developed antioxidant defense systems, enzymatic
systems, to scavenge ROS, in response to the ROS
attack. This enzymatic defense system (catalase,
peroxidase and superoxide dismutase) may be
damaged from over-production of ROS, which is leading
to the cell death [10].
PAHs found in the alga-medium system have some
fate such as evaporation, adsorption and absorption
biomass uptake, cell biodegradation process and photo-
degradation. However, photo-degradation process may
be important in regard to the presence of detectable
photo-induced PAH concentrations in the liquid
phase extracts of heat-killed cell samples, where the
biodegradation was unlikely to occur [11]. The biomass
uptake of PAHs involved rapid adsorption to the cell
walls and dividing into lipophilic constituents such as
lipids [12].
Benzo[a]Pyrene (BaP) is an indirect carcinogen
which can initiate transformation of healthy cells into
cancerous pending its metabolic activation [13]. BaP
was frst identifed in 1932 by Cook as a carcinogenic
substance in laboratory animals [14]. The skin of those
animals was repeatedly coated by tar. Currently, BaP
is used as a marker for PAHs. It is believed that the
concentration of such a marker is at a level of approximate
standard of carcinogenic activity of samples. PAHs
are susceptible to oxidation in the presence of ozone,
UV radiation and as well as to the biological oxidation
caused by microorganisms.
Metabolic paths of microbiological oxidation of PAHs
consisting of two and three aromatic rings (naphthalene,
phenanthrene, anthracene and fuoranthene) were
deeply investigated and described. Their enzymatic and
genetic regulation is known [15]. The initiation step of
transformation of PAHs in the prokaryotic organisms
(bacteria) is caused by reaction of two atoms of oxygen
with two atoms of carbon from benzene ring, through
dioxogenases enzymatic system. As a consequence of
this reaction, the creation of cis-dihydrodiole in presence
of dehydrogenase takes place, which is subsequently
transformed to dihydrogenate intermediate
pyrocatechol [16,17].
The toxicology of BaP has been mainly studied with
regard to the carcinogenicity of its metabolites, but its
phototoxicity is not well understood. Although some
studies have indicated the lethal phototoxicity of BaP,
there have been no reports regarding the pattern of cell
death induced by this agent [18-20]. Aquatic ecosystems
are affected by a number of noxious organic compounds,
mostly of antropogenic origin, e.g. polychlorinated
biphenyls (PCBs), dibenzo-para-dioxines (PCDDs) and
polycyclic aromatic hydrocarbons (PAHs) [21].
The degradation of pollutants under these conditions
usually involves the combined actions of two or more
microorganisms. In the case of algae, it is clear that
the complete degradation of aromatic pollutants is rare
[22]. The application with microalgae for aquatic toxicity
assays, as a useful indicator, has been conducted
for the unique eco-niche in the aquatic food web and
high sensitivity to a wide spectrum of environmental
pollutants [23,24].
In this work the results of photooxidation of the model
sample BaP with algae Chlorella kessleri in anaerobic
conditions and different intensities of radiation are
discussed. From an ecological and economical point of
view this method of biological degradation of this type
of the environmental pollution can be subsequently
compared with an alternative common technology
(physical-chemical degradation).
2. Experimental procedure
2.1. Biomass modifcation
Biomass from the culture of live algae Chlorella kessleri
(LARG/1) was supplied on slant agar without bacterial
contamination from Botanic institute of Slovak Academy
of Science in Bratislava. The algae was batch cultured
in mineral medium (MM) containing the following stock
solutions (ST): (ST)
1
:NH
4
Cl 15 mg L
-1
, MgCl
2
6H
2
O
12 mg L
-1
, CaCl
2
2H
2
O 18 mg L
-1
, MgSO
4
7H
2
O
15 mg L
-1
, KH
2
PO
4
1.6 mg L
-1
; (ST)
2
: FeCl
3
6H
2
O
64 mg L
-1
, Na
2
EDTA2H
2
O 100 mg L
-1
; (ST)
3
: H
3
BO
3

185 g L
-1
, MnCl
2
4H
2
O 415 g L
-1
, ZnCl
2
3 g L
-1
,
CoCl
2
6H
2
O 1.5 g L
-1
, CuCl
2
2H
2
O 0.01 g L
-1
,
Na
2
MoO
4
2H
2
O 7 g L
-1
; (ST)
4
: NaHCO
3
50 mg L
-1
. Stock
solutions were mixed in the ratio (ST)
1
:(ST)
2
:(ST)
3
:(ST)
4

= 10:1:1:1, pH was modifed to 7.4. Experiments were
carried out in a sterile laminar box JOUAN MSC 12
(USA). The cell density of inoculum was 510
4
cells
per ml. Cultivation was carried out in a chamber with a
stabile temperature in the range of 22 3C with four
linear fuorescent bulbs (7400 lux; Testo 545, Germany).
1134
A. Takov et al.
The effect of BaP mixture on the growth of C. kessleri
was evaluated microscopically according the OECD test
201 (1984) by direct cells counting using the Brker grid.
Erlenmeyer fasks (250 mL) with MM were inoculated
with C. kessleri (510
4
cells per mL) and the mixture
of BaP (100 g mL
-1
) was added. Erlenmeyer fasks
were sealed with cotton wool and were cultivated under
shaking by an orbital platform shaker GFL 3020 (Czech
Republic) with the frequency of 120 rpm, 72 h in order
to assure aerobic conditions. The growth of C. kessleri
was quantifed until confuent growth microscopically (0,
24, 48, 72 h). All experiments were carried out in three
parallels.
Dry biomass was rinse with deionized water and
dried at 105C for 24 h. After drying, dust in grinding
mortar was prepared. Dry biomass with concentration
of 0.1 g L
-1
was added to solution of BaP (100 g L
-1
).
Volume for sorption was 100 mL, pH of solution (pH
= 7.5) was treated with solution NaOH (30 wt.%) and
solution of H
2
SO
4
(20 wt.%).
2.2. Preparation of BaP
For laboratory tests, the model sample of BaP was
prepared as follows: pure reference material (Fluka,
99 wt.% purity) was dissolved in acetone (Merck) and
deionized water (1:99) to the concentration of BaP
100 g L
-1
.
2.3. Preparation of model sample
The tested pollutant BaP was added to the growth culture
Chlorella kessleri as well as to the reference sample
contained biomass from dry algae. Inoculated fasks with
this culture were kept in the air-conditioned chamber
under the stable conditions: temperature 223C, light
source with changing light intensity (fuorescence lamp
6.2 and 13.5 W m
-2
), constant mixing on rotary shaker
at 120 rpm. Samples for the analysis of biodegradation
were taken in the specifc time period of 24, 48, 72 and
144 h. Algae biomass was separated from the solution
via membrane fltration (Millipore, size of pores =
0.45 m).
2.4. Chemical analysis
After the specifc time period, samples of algae and
pollutant were processed by liquid to liquid extraction. For
extraction hexane (Merck, purity p.a.) was used. Extracts
after condense (Kuder Danish) to volume of 1 liter
were consequently identifed via chromatography. For
identifcation of BaP method of inner standard Bifenyl D
10

(Fluka) and gas-chromatography with mass spectroscopy
GC/MS (Variant-Saturn 2100T, USA) were used.
The identifcation of BaP was carried out by gas
chromatography combined with mass spectroscopy GC/
MS (Varian - Saturn 2100 T, USA), using the method of
internal standard (Biphenyl D10, Fluka).
Samples were batched into an automatic
autosampler (Varian CP-8410, USA), with Varian type
1177 injector, split/splitless with a constant temperature
of 290C gas chromatograph worked in on-line
connection with the detector (Mass Spectrometric
Detector - MSD), which was the mass spectrometer -
ion trap. MSD worked with electron ionization in Full
Scan (FS). In the mass range 45-650 m/z. Data were
processed using MS Workstation. Separation was
carried out in a chromatographic column EZ-guard
VF-5 (Varian, USA) with dimensions L (30 m)ID
(0.25 mm) OD(0.39 mm)+10 m EZ - guard column.
Guard column is connected directly to the main column
without using the clutch. Helium was used as the carrier
gas (He 6.0, Messer, Slovakia) with a constant fow rate of
1 mL min
-1
. The volume of 1 L syringe (Hamilton 10 L) was
charged into the injector. Chromatographic separation
was performed with a temperature program: 50C (hold
time 1 min), next by the rate of 10C min
-1
to 290C (hold
time of 10 min). The total length of the analysis was
45 min.
2.5. Respirometric measurements
The rate of biological oxidation of organic carbon in
samples was monitored on-line with Micro-Oxymax
(M-O, Columbus Instruments, USA). Concentration of
CO
2
was measured by using of IR detector. Concentration
of O
2
was measured by paramagnetic detector. Obtained
data were evaluated by M-O software. Obtained results
represent the cumulative production and consumption
of CO
2
and O
2
.
2.6. Microscopic measurements
Images surface characterization of the samples were
evaluated by scanning electron microscopy (SEM)
JEOL JXA 840A device.
Microscopic measurements of biomass were
investigated with device Axio-IMAGER A (CARL-ZEISS,
Germany) which allowed us to compare the biomass
samples without contamination and samples of biomass
after biodegradation of BaP.
For microscopic observation (light: X-CITE, SERIE
120) the following flters were used:
flter (F1) - Filter set 02 - excitation G 365 nm,
Beam splitter FT 396, emission LP 420 nm
flter (F2) - Filter set 05 - excitation BP 395-440 nm,
Beam splitter FT 460 and emission LP 470 nm
1135
Biodegradation of Benzo[a]Pyrene
through the use of algae
3. Results and discussion
The aim of the present study was to gain more insight
into the process of degradation BaP under simulation
visible light (Fig. 1) and bioxidation mediated microalgae
Ch. kessleri.
Physico-chemical properties of PAHs can be changed
by modifying their structure by abiotic (photochemical)
and biotic processes (mono-oxygenated cytochrome
P450) [25]. One of the most important biotic factors is
sunlight.
Degradation of PAH in the aquatic ecosystem may
be affected by conditions of chemical oxidation and
biodegradation photooxidation aquatic organisms.
Microbial degradation is a relatively important route and
PAHs are partially or completely degraded by some
species of aquatic bacteria, algae and fungi [26,27].
Rate of biological oxidation of organic carbon was
monitored on-line with M-O (Fig. 2). Monitoring the
respiration of gasses after biodegradation of BaP by
using algae Ch. kessleri in dry and live form showed that
production of O
2
is highest after 48 h of biodegradation
in the case of biomass from the culture of the live algae.
This corresponds with cumulative production of CO
2
.
Oxidation of carbohydrates including PAHs to CO
2
and
H
2
O can be expressed with a general stoichiometric
formula:
C
n
H
m
+ (n + 0.25m) O
2
nCO
2
+ (0.5m) H
2
O (1)
Peng et al. [28] came to some interesting
conclusions. Their experiment gained
data and characteristics of a microorganisms catalysis
(Arthrobacter oxydans (B4) in the degradation of BaP
and designed a BaP biodegradation path (Fig. 3)
[28]. Peng et al. proposed that the products of PAH
degradation independent of their origin, potentially
caused a build up of toxic products in nature. In terms
of potential toxicity this is considered to be the most
important group of oxidized products of PAH [29].
Benzo[a]pyrene is one of the polycyclic aromatic
hydrocarbons (PAHs), which are widely present in
the environment. Traditionally, toxicological concern
regarding BaP has focused on its metabolites such
as ()-trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-
tetrahydrobenzo[a]pyrene, because BaP becomes
carcinogenic after metabolic activation [18-20].
In the Table 1 the effciency of biodegradation of
BaP is shown. Based on the results, the highest value
was reached by combination of biodegradation of BaP
with biomass from the culture of live algae (i.e., the
higher production of O
2
in comparison to the dry algae
production) and photodegradation of BaP. Several
studies strongly support the view point that PAHs were
unstable under sunlight [30]. Photo-oxidation of PAHs is
a potentially important pathway for PAHs modifcation
in the environment [31]. Upon absorbing sunlight, PAHs
can be rapidly transformed to a variety of compounds,
most of which are oxidation products. As photo-products
are likely to accumulate in the environment allowing
for more realistic analysis of PAHs loads [32]. In the
study [33], the phototoxicity of BaP was inhibited by
NaN
3
(quencher of singlet oxygen) but not by mannitol
(quencher of hydroxy radicals [34]), showing that BaP-
Figure 1. Absorption spectrum of BaP (solid line) and fuorescent lamp emission spectrum (dashed line); red circle overlay of the BaP absorption
spectrum and light source emission spectrum [61].

1136
A. Takov et al.
Figure 2. Cumulative production of CO
2
(L; a) and O
2
(L; b); degradation of BaP.
Figure 3. Degradation path of BaP by microorganism (Arthrobacter oxydans (B4) [28].


1137
Biodegradation of Benzo[a]Pyrene
through the use of algae
induced phototoxicity was due to the production of
singlet oxygen but not hydroxyl radicals.
PAHs generate singlet oxygen through type-II
photodynamic mechanism, in which the photoexcited
molecules (triplet state) transfers its energy to O2
resulting in energy rich singlet oxygen. Also, BaP can be
effciently excited by the light source and the resulting
photophysical processes of energy transfer to molecular
oxygen that leads to a variety of photoactive species
such as singlet oxygen (1O2) [35].
The effciency of biodegradation (%) was calculated
based on an equation:
= 1 (CC
0
-1
), (2)
where C
0
is the concentration of BaP at the beginning of
the biodegradation process and C is the concentration
of BaP at the end of the biodegradation process.
Fig. 4 shows amounts of cumulative decomposed
CO
2
during the biodegradation process of BaP via algae
biomass utilization and minimal production of CO
2
during
photodegradation of BaP. Initial biodegradation (frst
24 h) showed relatively lower effciency. After 48 and
144 h of monitoring of biodegradation process, values
of the day average biodegradation effciency reached
relatively constant tendency. Despite of constant
conditions, the effciency of biodegradation after 144 h
was lower when compared to the biodegradation without
use of photooxidation.
BaP is known to be transformed to diols and quinones
by marine algae in a period of 56 days. Warshawsky
et al. [36] found that Selenastrum capricornutum,
a freshwater green alga metabolizes BaP to cis-
dihydrodiols using a dioxygenase enzyme system.
Research in the feld of biodegradation and biosorption
processes of environmental organic pollutants has
mainly concentrated on microorganisms, such as
bacteria and fungi whith much less emphasis on algae
[37-41]. The limited number of experimental studies has
demonstrated the algae capability to accumulate and
degrade wastewater-borne organic pollutants, such as
dyes, phenol and BaP [22,37,42-45].
The rate of decrease of biodegradation can be
caused as a consequence of the continual decrease
of moisture, selective decomposition of better
decomposable substances, decrease of nutrient
content, accumulation of toxic intermediates as well as
by other factors [46]. As a consequence of splitting of
aromatic circle, the creation of organic acids occurred.
The biggest beneft of such systems can be described
as the consumption of created organic acids by
microbial metabolism followed by subsequent synthesis
of cell components and energy. Also water and CO
2
is
released [45].
Chemical analysis of model sample of water taken
during the biodegradation (after 24, 48, 72 and 144 h)
has shown not only a decrease in the concentration of
BaP (Fig. 4) but the creation of degrading products as
well. Before the analysis of samples via GC/MS method,
standard BaP was tested. Determination limit of BaP
was set to LOQ = 0.1 g L
-1
and retention time of BaP
was 32.35 min.
The values of concentration of BaP at the different
time intervals are given in Table 1. From these results
one can conclude that the best biodegradation effciency
of BaP (29%) was achieved with using combined method
of biodegradation with biomass from the culture of live
algae of Ch. kessleri and with radiation photooxidation
(13.5 W m
-2
).
In the present study, adsorption and absorption were
not distinguished. It is interesting to address that even
for the apenuated cells, the time course effect on the
Table 1. Concentration of BaP after different biodegradation conditions.
t (h) Biosorbent c
BaP
(g L
-1
) (%)
Initial concentration
(g L
-1
)
Light intensity
6.2 W m
-2
Light intensity
13.5 W m
-2
Light intensity
6.2 W m
-2
Light intensity
13.5 W m
-2
24
Dry algae 100 98 96 2 4
Live algae 100 89 86 11 14
48
Dry algae 100 93 90 7 10
Live algae 100 77 74 23 26
72
Dry algae 100 91 88 9 12
Live algae 100 75 71 25 29
144
Dry algae 100 89 86 11 14
Live algae 100 78 75 22 25
1138
A. Takov et al.
unaccounted-for BaP was present, which unlikely was
due to biodegradation. The results suggest that the
marine algae P. tricornutum is sensitive to the photo-
enhanced toxicity of PAHs, although it is capable of
utilizing solar light through photosynthesis [24].
The results of experimental study indicated that 89
90 g L
-1
of all the BaP was found in the biomass brown
algae solution (Fucus), the remainder (up to 4%) was not
recovered (considered to have been metabolized) and
the insignifcant part was in the solution. The proportion
Figure 4. Production of CO
2
and biodegradation effciency of BaP without algae (a), live (b) or dry (c) biomass (light intensity 13.5 W m
-2
).

1139
Biodegradation of Benzo[a]Pyrene
through the use of algae
of transformed PAHs was more essential (4249%) for
the green algae. The process of BaP transformation
is species specifc and depends on the presence of
enzymes localized in the plant cells in freshwater
and marine algae. Peroxidase, diphenol oxidase and
cytochrome P450 are the most important enzyme
systems for detoxifcation of BaP. The data obtained
indicate the important role of freshwater and marine
algae in the fate of carcinogenic PAHs in the environment
[49]. Exposure to BaP, both lysosomes and mitochondria
are involved in creating intracellular environment
(acidifcation and oxidative stress) necessary for the
occurrence of caspase dependent cell death [49].
Apart from BaP, gas-chromatography also identifed
other aromatic carbohydrates, namely diols, aldehydes
and aromatic acids. However, the higher intensity
of radiation of BaP is necessary. The effciency of
this radiation is lower when compared with the UVA
and UVB radiation. From the point of UV radiation,
photodegradation has always been better [50]. When
the BaP was exposed to the aggressive UV radiation the
dominant oxidation fragments were 3-hydroxy Benzo[a]
Pyrene, Benzo[a]Pyrene-9,10-dihydrodiol and Benzo[a]
Pyrene-6,12-dione.
Bacteria and algae which decompose PAHs, use
these pollutants as a source of carbon and energy.
On the other hand, fungi use PAHs for creation
water-soluble compounds. The main reason for
this phenomenon is the fact that bacteria and fungi
use different methods of metabolism [51]. Bacteria
degradation of PAHs generally starts by the attack of the
aromatic rings with dioxygenase and cis-dihydrodiole is
created. Consequently this product is dehydrogenated
for pyrocatechol. Pyrocatechol is the main intermediate
product of this splitting. The aromatic ring is split between
hydroxyl groups (orto-splitting) or appended to the one
of hydroxyls (meta-splitting). Another degradation of
the ring causes degradation of structure and molecules
and enables it to enter into the middle metabolic path
of bacteria. Photochemical degradation of PAHs can
cause creation of the same oxidation products as in
the case of elementary chemical oxidation of PAHs
(i.e., oxygen, alcoxyl radicals (RO

) and

OH radicals.
At the end of degradation a complex blend of ketones,
quinines, aldehydes, phenols, and carboxylic acids is
obtained [29].
The results obtained from GC-MS analysis of the
different cultures of the dry biomass and biomass
from the culture of live algae are shown. The activity
of pigment dyes, mainly chlorophyll(a), which acts as
the photosensitiser in photodynamic reactions was
used to analyse the reactions. In the biomass from the
culture of live algae activity is higher (enzyme activity)
and when compared to the dry (relatively metabolically
inactive), the difference is not great. This is because the
apenuated algae been partly removed moisture, but did
not pass as sterilization (120C, pressure of 120 kPa),
not be fully dampen enzyme activity.
In the Fig. 5 of the scanning electron microscope, we
see intact cell wall Ch. kessleri after apenuation at 105C.
Overview of the most number of publications shows that
the presence of PAHs may affect the absorption of light
[31,52], photosynthetic electron transport [53,54], gas
exchange [55,56], photosynthetic pigments [54,57].
For evaluation of photosynthesis the fuorescence
of living systems was used. This method used
analysis of potential damage of photosystem. In this
method, chlorophyll represents the internal indicator
of photosynthetic capacity of organisms. Under
optimal conditions the majority part of light energy
absorbed with chlorophyll is scattered via chemical
processes. A small part of this light is emitted in the
form of heat and fuorescence [58]. Capacity of photo-
synthesis (transformation of energy of protons to
chemical energy) can be controlled with adjusting the
conditions, i.e. under the stress condition that leads to
higher heat emission and fuorescence the capacity of
photosynthesis decreases [59]. This is refected in the
case of optimal invasion conditions for algae (stress -
temperature changes, decrease of nutrient content,
presence of toxic substance and changes in light
intensity). As a fuorescence response (Fig. 6). There are
two maximums are determining for chlorophyll: one at
690 nm and second one at 735 nm [60]. Concentration
of chlorophyll was evaluated via UV-VIS spectroscopy.
Biomass from the culture Ch. kessleri contained:
chlorophyll(a) = 1.1925 mg g
-1
and chlorophyll(b)
= 0.435 mg g
-1
, concentrations were determined in
accordance by norm STN ISO 10260 [61]. Outputs from
Figure 5. SEM image of apenueted culture of Chlorella kessleri at 105C.

1140
A. Takov et al.
fuorescence microscope are shown in Fig. 5. Obtained
results from fuorescence microscope shows (case of
live algae) intensive penetration of BaP into to cell of
algae (Fig. 6d). While in the case of lyophilized algae
occurs mainly sorption of BaP and partial penetration
into cells (Fig. 6c). On the other hand in dry algae
(Fig. 6a) is shown increasing of chlorophyll fuorescence
as compared to the biomass from the culture of live
algae (Fig. 6b). Fluorescence of chlorophyll is higher in
dry algae than in biomass from the culture of live algae
due to the ongoing photosynthesis.

4. Conclusion
Although resistant to biochemical degradation, PAHs
readily absorb UV light energy and are subject to
photolytic breakdown. A complete conversion of PAHs to
CO
2
and H
2
O by UV light alone is prohibitively costly, but
it seems reasonable that a relatively brief photolytic pre-
treatment would render the molecule more susceptible to
subsequent microbial attack. In this study, we examined
the biodegradation of BaP upon visible exposure.
We have shown a comparative analysis between
live a dry (apenuated) culture of Chlorella kessleri in
biodegradation process. The results indicated that of
all the BaP consumed, 8986 g L
-1
was found in the
solution with the biomass of dry algae. For solution with
the biomass from the culture of live algae the proportion
of transformed BaP was more essential, 7875 g L
-1
.
As follows from the results of degradation of BaP by
microalgae it is not clear what process prevails (photo-
oxidation, biodegradation). However, these processes
can operate in parallel. Due to its lipophilic character
and chemical stability of BaP need their cooperation.
Transmitation of photooxidation products of oxidation
in cells is successful. BaP molecules penetrate into
cells microalgae and images from the fuorescence
Figure 6. Fluorescence microscope; (a) StartDry algae Chlorella kessleri in medium; Red Fluorescence; Resolution 4010 (flter F1); (b) Start
Live algae Chlorella kessleri in medium; Red Fluorescence; Resolution 4010 (flter F1); (c) Dry algae Chlorella kessleri + BaP after 144
h; Blue Fluorescence; Resolution 4010 (flter F1); (d) Live algae Chlorella kessleri + BaP after 144 h; Blue Fluorescence; Resolution
4010 (flter F1); (e) Pure BaP; Blue Fluorescence; Resolution 4010 (flter F1).

1141
Biodegradation of Benzo[a]Pyrene
through the use of algae
microscope show their degradation after 144 h.
Fluorescence of chlorophyll is higher in dry algae than
in biomass from the culture of live algae due to ongoing
photosynthesis.
The results show that the degradation of BaP
was the most effective by light intensity at level of
13.5 W m
-2
and the degradation effciency was 29%
by using biomass from the culture of live algae.
Bioremediation by microbial processes in combination
with photo-oxidation can be considered as a promising
alternative to conventional technologies to remove BaP
from contaminated water.
Acknowledgement
This work was supported by the Slovak Research and
Development Agency under the contract No. APVV-
0665-10.
D. Desalme, P. Binet, N. Bernard, D. Gilbert,
M.L. Toussaint, Environ. Exp. Bot. 71, 146 (2011)
R. Krasnoschekova, U. Kirso, F. Perin,
P. Jacquignon, Polyc. Arom. Comp. 3, 41 (1992)
D. Broman, C. Nuf, I. Lundbergh, Y. Zebhr,
Environ. Toxicol. Chem. 9, 429 (1990)
J.F. McCarthy, L.E. Robertson, L. Burrus,
Chemosphere 19, 1911 (1989)
L.E. Sverdrup, P.H. Krogh, T. Nielsen, C. Kjr,
J. Stenersen, Chemosphere 53, 993 (2003)
D. Filgueira, D.P. Salomo de Freitas, A.P. de Souza
Votto, G. Fillmann, J.M Monserrat, L.A. Geracitano,
G. Santos Trindade, Photochem. Photobiol. 83,
1358 (2007)
M.F. Blum, Photodynamic Action and Diseases
Caused by Light (Reinhold, New York, 1941)
J.L. Newsted, J.P. Giesy, Environ. Toxicol. Chem.
6, 445 (1987)
C. Meewes, P. Brenneisen, J. Wenk, L. Kuhr,
W. Ma, J. Alikoski, A. Poswig, T. Krieg,
K. Scharffetter-Kochanek, Free Radic. Biol. Med.
30, 238 (2001)
L. Ke, L. Luo, P. Wang, T. Luan, N. Fung-Yee Tamb,
Biores. Technol. 101, 6950 (2010)
J. Kou, H. Zhang, Y. Yuan, Y. Li Wang, T. Yu, Z. Zou,
J. Physic. Chem. C 112, 4291 (2008)
B.F. Smets, B.E. Rittmann, Water Res. 24, 355
(1989)
Y. Zhang, Y.X. Zhu, K.K. Kwon, J.H. Park, S.J. Kim,
Chemosphere 55, 389 (2004)
J.W. Cook, J. Chem. Soc., 1472 (1932)
A.L. Juhasz, R. Naidu, Int. Biodeter. Biodegr. 45,
57 (2000)
C.E. Cerniglia, Adv. Appl. Microbiol. 30, 31 (1984)
D.T. Gibson, V. Subramanian, Appl. Environ.
Microbiol. 61, 3711 (1995)
I.B. Weinstein, A.M. Jeffrey, K.W. Jennette,
S.H. Blobstein, Science 193, 592 (1976)
P. Sims, P.L. Grover, A. Swaisland, K. Pal, A. Hewer,
Nature 252, 326 (1974)
R.E. Albert, M.L. Miller, T. Cody, A. Andringa,
R. Shukla, C.S. Baxter, Carcinogenesis 12, 1273
(1991)
C.-L. Lee, H.-T. Huang, L.-J. Kuo, Environ. Sci.
Health. A 35, 515 (2000)
K.T. Semple, R.B. Cain, S. Schmidt, FEMS
Microbiol. Lett. 170, 291 (1990)
Y. Nassiri, T. Ginsburger-Vogel, J.L. Mansot, J. Wry,
Biol. Cell. 86, 151 (1996)
L. Wang, B. Zheng, W. Meng, Ecotoxicol. Environ.
Safety 71, 465 (2008)
X.D. Huang, D.G. Dixon, B.M. Greenberg, Ecotox.
Environ. Safe. 32(2), 194 (1995)
G. Barankov, D. Fazekaov, P. Manko,
S. Torma, Chmia ivotnho prostredia (Preovsk
univerzita v Preove, Univerzitn kninica, Preov,
Slovakia, 2009) (in Slovak) ISBN 978-80-555-0082-
9
I. Holoubek, Crit. Rev. Anal. Chem. 29(3), 179
(1999)
H. Peng, H. Yin, J. Deng, J.-S. Ye, S.-N. Chen,
B.-Y. He, N. Zhang, Pedosphere 22(4), 554 (2012)
S. Lundstedt, Analysis of PAHs and their
transformation products in contaminated soil and
remedial processes, Ume: Solfjdern Offset AB,
56 (2003)
S. Mujtaba, A. Dwivedi, M.K.R. Mudiam, D. Ali,
N. Yadav, R.S. Ray, Photochem. Photobiol. 87,
1067 (2011)
A. Mallakin, D.G. Dixon, B.M. Greenberg,
Chemosphere 40, 1435 (2000)
J.J. Yin, Q. Xia, S.H. Cherng, W.I. Tang, P.P. Gu,
G. Lin, H. Yu, D.H. Saen, Int. J. Environ. Res.
Public. Health 5 1, 26 (2008)
Y. Ibuki, R. Goto, Environ. Toxicol. Pharm. 11, 101
(2002)
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
1142
A. Takov et al.
S. Goldstein, G. Czapski, Int. J. Radiat. Biol. 46,
725 (1984)
J. Regensburger, K. Lehner, T. Maisch, R. Vasold,
F. Santarelli, E. Engel, A. Gollmer, B. Konini,
M. Landthaler, W. Baumler, Exp. Der-matol. 19(8),
275 (2010)
D. Warshawsky, M. Radike, K. Jayasimhulu,
T. Cody, Biochem. Biophys. Res. Commun. 152,
540 (1988)
A. Aksu, Process Biochem. 40, 997 (2005)
B. Bhushan, S.K. Samanta, A. Chauhan,
A.K. Chakraborti, R.K. Jain, Biochem. Biophys.
Res. Commun. 275, 129 (2000)
A. Chauhan, A.K. Chakraborti, R.K. Jain, Biochem.
Biophys. Res. Commun. 270, 733 (2000)
D. Dean-Ross, J. Moody, C.E. Cerniglia, FEMS
Microbiol. Ecol. 41, 1 (2002)
S.K. Samanta, A.K. Chakraborti, R.K. Jain, Appl.
Microbiol. Biotechnol. 53, 98 (1999)
Z. Aksu, D. Akpinar, Biochem. Eng. J. 7, 183 (2001)
S.M.N. Chan, T. Luan, M.H. Wong, N.F.Y. Tam,
Environ. Toxicol. Chem. 25, 1772 (2006)
A.P. Lei, Y.S. Wong, N.F.Y. Tam, Water Sci. Technol.
46, 195 (2002)
A.P. Lei, Z.L. Hu, Y.S. Wong, N.F.Y. Tam, Biores.
Technol. 98, 273 (2007)
D. Kupka, P. Sekula, O. Tischler, J. Brianin, Acta
Montan. Slovaca 11, 314 (2006)
S.C. Wilson, K.C. Jones, Environ. Poll. 88, 229
(1993)
U. Kirso, N. Irha, Ecotox. Environ. Safe. 41, 83
(1998)
M. Gorria, X. Tekpli, M. Rissel, O. Sergent, L. Huc,
N. Landvik, O. Fardel, M.T.D. Boitrel, J.A. Holme,
D.L. Gossmann, Toxicol. Appl. Pharmacol. 228, 212
(2008)
A. Bednrikov, B. Sklrov, E. Kolek, P. imko,
Chem. Listy 104, 537 (2010)
C.E. Cerniglia, Biodegradation 3, 351 (1992)
X.D. Huang, B.J. McConkey, T.S. Babu,
B.M. Greenberg, Environ. Toxicol. Chem. 16, 1707
(1997)
K. Wakabayashi, P. Boger, Weed. Biol. Manage. 4,
8 (2004)
M. Kummerova, J. Krulova, . Zezulka, J. Tiska,
Chemosphere 65, 489 (2006)
M. Kummerova, L. Vaova, H. Fierova, M. Klem,
. Zezulka, J. Krulova, Plant. Growth Regul. 61,
161 (2010)
A. Aksmann, T. Shutova, G. Samuelsson, Z. Tukaj,
Aquat. Toxicol. 104, 205 (2011)
I. Oguntimehin, A.F. Eissa, H. Sakugawa,
Chemosphere 78, 877 (2010)
H.K. Lichtenthaler, J. Plant. Physiol. 148, 4 (1996)
K. Rohek, M. Bartk, Photosynthetica 37, 339
(1999)
C.S. French, In: W. Ruhland (Ed.), The chlorophylls
in vivo and in vitro (Berlin, Springer, 1960)
STN ISO 10260: Kvalita vody. Meranie
biochemickch parametrov. Spektrofotometrick
stanovenie koncentrcie chlorofylu a. Technical
standard (Slovakia, 1999) (in Slovak)
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
1143