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Using Real-time PCR to assess changes in the


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contaminated seawater mesocosms
Article in International Biodeterioration & Biodegradation September 2014
Impact Factor: 2.13 DOI: 10.1016/j.ibiod.2014.06.006

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International Biodeterioration & Biodegradation 93 (2014) 241e248

Contents lists available at ScienceDirect

International Biodeterioration & Biodegradation


journal homepage: www.elsevier.com/locate/ibiod

Using Real-time PCR to assess changes in the crude oil degrading


microbial community in contaminated seawater mesocosms
Mehdi Hassanshahian a, *, Michail M. Yakimov b, Renata Denaro b, Maria Genovese b,
Simone Cappello b
a
b

Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran
Istituto per lAmbiente Marino Costiero (IAMC), CNR of Messina, Messina, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 4 December 2013
Received in revised form
24 April 2014
Accepted 9 June 2014

The real-time polymerase chain reaction (RT-PCR) was used to follow changes in the proportion of
hydrocarbonoclastic bacteria in the marine microbial community in oil polluted mesocosms during
bioremediation eld trial. Assay for alk-B1 of Alcanivorax borkumensis and alk-BT of Thalassolituus oleivorans were validated and found to be both sensitive and reproducible. Quantication of alk-B1 from
A. borkumensis SK2 in mesocosms show that in single bioaugmentation mesocosm (M1) this gene has
high quantity in fth day of sampling but in biostimulating mesocosm (M2) and consortium bioaugmentation mesocosm (M3) the high quantity of this gene was in tenth day of sampling. The comparison between expression of alk-BT and alk-B1 in M3 mesocosm show that alk-B1 copy number was
more than alk-BT. The proportion of alk-B1 or alk-BT containing bacteria was positively correlated to the
concentration of crude oil in the mesocosms. After the concentration of crude oil in the mesocosms
decreased the gene copy number of alkane monooxygenase genes also decreased.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Biodegradation
Crude-oil
Expression
Mesocosm
Quantication

1. Introduction
Oil spills occur frequently and are a major cause of marine
pollution. Crude oil is composed mainly of hydrocarbons, such as
saturated hydrocarbons, aromatics, resins and asphaltenes (Harayama
et al. 1999; Hassanshahian et al., 2013). Saturated hydrocarbons and
aromatics are easily biodegradable; for efcient remediation after
oil spills; we need to understand the behavior of microbial populations responsible for degrading crude oil (Hassanshahian et al.,
2012a, b, 2014a).
In bioremediation studies, the quantitative analysis of functional
genes have provided a valuable tool for studying the relationship
between specic microbial populations and the performance of the
degradation processes (Ringelberg et al., 2001; Piskonen et al.,
2005). In the environment, however, wide ranges of bacteria
participate in the degradation of organic contaminants. Since
diverse bacteria are associated with different phases of pollutant
degradation (Watanabe et al., 2002; Katsivela et al., 2004) better
interpretation of the microbial community dynamics occurring
during the progression of decontamination is important in the

* Corresponding author. Tel.: 98 9132906971; fax: 98 3413202032.


E-mail addresses: mshahi@uk.ac.ir, hasanshahi@gmail.com (M. Hassanshahian).
http://dx.doi.org/10.1016/j.ibiod.2014.06.006
0964-8305/ 2014 Elsevier Ltd. All rights reserved.

development of more efcient remediation processes (Hassanshahian


et al., 2010 ; Tebyanianet al., 2013).
The detection of functional catabolic genes, for evaluating potential micro-organisms capable to biodegrade hydrocarbons, has
been attempted much less often (Chandler and Brockman, 1996;
Joshi and Walia, 1996).
The ability to accurately quantify functional genes in the environment is an important step in understanding many ecological
processes. Existing techniques include hybridization methods such
as dot blots, and end-point polymerase chain reaction (PCR) methods
such as competitive PCR and most probable number (MPN-PCR).
Although these methods are useful, hybridization methods are often
only semi-quantitative and end-point PCR methods require many
reactions and a post-PCR analysis step, making them both laborintensive and expensive. Real-time PCR is fast becoming the most
popular alternative originally developed for gene expression studies;
it has found many applications in microbial ecology (Heid et al., 1996;
MacKay, 2004; Hassanshahian et al., 2014b). These include the
measurement of 16S rRNA genes as a measure of total and specic
cell populations (Smits et al., 2004), the measurement of catabolic
genes such as the bssA gene, coding for benzylsuccinate synthase, the
key enzyme of anaerobic toluene degradation (Beller et al., 2002) and
atrazine-degrading genes (atz) (Devers et al., 2004). The advantages

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M. Hassanshahian et al. / International Biodeterioration & Biodegradation 93 (2014) 241e248

that Real-time PCR offers include speed, sensitivity, accuracy,


simplicity, and the possibility of robotic automation (Heid et al.,
1996).
The alkane monooxygenases are a group of enzymes that catalyze the rst step in the aerobic degradation of alkanes. The genetics
of these enzymes are well characterized, particularly in Pseudomonas (Van Beilen. et al., 2001; Hasanshahian et al., 2008) and
Rhodococcus (Van Beilen et al., 2006). A more quantitative MPNPCR method was used to follow populations of alkane-degrading
microbes in seawater microcosms (Sei et al., 2003), for example
Heiss-Blanquet et al. (2005) used a competitive PCR method to
correlate alkB levels to n-heptane degradation in freshwater and
soil samples. All these studies, however, utilized several primer sets
to target different types of alkane monooxygenases (HeissBlanquet et al., 2005).
The present study focused on n-alkanes, a major component of
crude oil, and the aims of this study is development and validation
of a Real-time PCR method for quantication of alkB genes from
two marine hydrocarbonoclastic bacteria (HCB) include Alcanivorax
borkumensis and Thalassolituus oleivorans in contaminated mesocosms with different experimental conditions.
2. Materials and methods
2.1. Site description and sampling
During mesocosms experiments natural seawater (SW) was
collected, in November 2011, from the station Marisicilia
(38 12.23N, 15 33.10E) located the harbor of Messina (Italy). The
seawater has taken through use of a direct pipeline to sea and that
has consented the direct lling of the tanks of experimentations.
2.2. Set-up of experimental mesocosms systems
Experiments in mesocosms have been performed in a Mesocosm Facility planned and constructed at the Institute for Coastal
Marine Environment (IAMC) e CNR of Messina (Italy). The experiments were carried out in a rectangular tank of 11,250 L of capacity
(5000 cm long, 150 cm deep, 150 cm wide) lled with 10,000 L of
natural seawater (SW). Before the introduction in the reaction
tanks (mesocosms), natural seawater was ltered through a 100 mm
nylon mesh in order to screen large metazoans and detritus. The
seawater in this study was aerated and stirred during the all
experimental period. The seawater was maintained at 18 2  C
(Figs. 1 and 2) for all experimental period; pH values were also
measured to detect possible variation.
2.3. Bacterial strains
Two hydrocarbonoclastic bacterial A. borkumensis SK2 (Genebank
accession number NR_029340) and T. oleivorans MIL-1 (Genebank
accession number AJ431699) were used in this study. All bacteria
have been isolated from natural seawater from crude oil enrichments
carried out in ONR7a medium (Dykesterhouse et al., 1995) with crude
oil as only energy and carbon source as previously described
(Yakimov et al., 1998, 2004) and belong to collection of hydrocarbondegrading bacteria hold at the IAMC-CNR of Messina (Italy).
2.4. Growth conditions and inoculate amendments
As previously indicated (Hassashanin et al., 2014), starting cultures of bacteria selected (A. borkumenis SK2 and T. oleivorans MIL1) were prepared (separately) by inoculating microbial cells into
ONR7a mineral medium (Dykesterhouse et al., 1995) containing
0.1% (w/v) of sterile tetradecane (C14H30, SigmaeAldrich, Milan,

Fig. 1. Schematic representation of engineered and hydraulic system of Mesocosm


Facility of IAMC-CNR of Messina used in this study. (A) Fill line with water treatment
system (lter); B and C, system of water recirculation; the experimental tank is made
of a steel tower moulded-case covered with berglass. This structure is also covered by
a gel-coat whose chemical structure avoids the adhesion of oil to the interior walls of
the tank and the release of substances which are potentially toxic to marine microbial
ora. (D), seawater and crude oil.

Italy). After growth in a rotary shaker (New Brunswick C24KC,


Edison NJ, USA; 150 rpm) at 25  C for two days, 500 ml of the seed
culture broth were transferred into 100 mL of ONR7a medium
supplemented with 1% (w/v) of sterile crude oil (Arabian light
crude oil; ENI Technology S.P.A.). After 5 days of growth (25 1  C,
150 rpm), at the beginning (T0) of experiments selected microorganisms (A. borkumensis SK2 and T. oleivorans MIL-1) were added at
a nal density of 108 cell ml1 in experimental systems (Cappello
et al., 2012; Hassanshanian et al., 2014).

2.5. Experimental planning for mesocosms experimentations


Three different series of experimentations have been carried
out. In all experimentation natural seawater was supplemented
with crude oil (Arabian light crude oil) and inorganic nutrients
(KH2PO4 0.077 g l1, NH4Cl 0.2 g l1 and NaNO3 0.1 g l1). of Arabian
light crude oil (1000 mg) were add in all system in study after
physical weathering (100  g, 25  C for 48 h); 0.1% (v/v) of squalene
(C30H50, SigmaeAldrich, Milan) was insert to crude oil as internal
standard of biodegradation. System of control (natural seawater
whit oil and inorganic nutrients) was indicated as mesocosm 1
(indicated as M1). System whit addition of A. borkumensis SK2T was
indicated as mesocosm 2 (M2) and system whit simultaneous

M. Hassanshahian et al. / International Biodeterioration & Biodegradation 93 (2014) 241e248

243

Fig. 2. Bacterial densities determined by DAPI (cell mL1) staining during mesocosm experiment. Concentration of the cells observed in M1 (Seawater with crude oil and
inorganic nutrients); M2 (seawater with crude oil, inorganic nutrients and a single Alkanivorax borkumensis SK2 inoculum) and M3 (seawater with crude oil, inorganic nutrients and the bacterial consortium Alkanivorax borkumensis SK2 e Thalassolituus oleivorans MIL-1); experiment are depicted as white (M1), gray (M2) and black (M3)
bars respectively.

addition of A. borkumensis SK2T and T. oleivorans MIL-1T was indicated as mesocosm 3 (M3).
2.6. Sampling strategy and parameters assayed
All experimentations have been conducted for 20 days. To
monitor the succession bacterial activity on xed days (0, 3, 5, 10, 15
and 20) from the time zero (T0; introduction of oil, inorganic nutrients and bacteria) sub-volumes of sea-water were collected. Total
microbial abundance (DAPI count), measures of microbial activity
(screening of functional genes and quantication of alkane monooxygenase gene alkB by Real-Time PCR) were carried out from each
collected samples. The composition of total extracted and resolved
hydrocarbons and their derivates (TERHC) were also analyzed.
2.7. Total bacterial count
After short-time (3000 ) ultrasonic treatment, the total bacterial
cell counts were performed by DAPI (40 ,6-diamidino-2phenylindole 2HCl, SigmaeAldrich, Milan, Italy) staining on
samples xed with formaldehyde (2% nal concentration), according to Porter and Feig (1980). Slides were examined by epiuorescence with an Axioplan 2 Imaging (Zeiss) microscope (Carl
Zeiss, Thornwood, NY, USA). Results were expressed as number
of cells ml1.
2.8. DNA/RNA extraction and crDNA synthesis
Extraction of DNA and RNA from samples in study was performed using a DNA/RNA extraction kit (QIAGEN, Valencia, CA,
USA) according to manufacture instruction. The rRNA-crDNA heteroduplex was synthesized by reverse transcription using the
random primer (hexamer) oligonucleotides and SuperScript II
RNase H-free reverse transcriptase (Life Technologies). RT amplication was realized by using the protocol recommended by the
manufacture with 20 ml reaction mixtures containing 200 mM of
deoxynucleoside triphosphate, 5 mM of hexamer, 50e100 ng of
denatured RNA with a GeneAmp PCR System 2700 (Applied

Biosystems Foster City, CA, USA). The thermal cycler parameters


were as follows: 5 min at 65  C and 2 min at 4  C, followed by the
addition of 1 rst-strand buffer and 75-U of RNase inhibitor and
heating at 37  C for 2 min. Reverse transcriptase was added prior
to a 50-min incubation at 42  C. This reaction was then stopped
at 80  C for 5 min. No contaminating DNA was detected in any of
these reactions.
2.9. Screening of functional genes in extracted DNA and cDNA of
mesocosms samples
Screening of functional genes involved in biodegradation of
crude oil in mesocosms performed with specic primers. The
crDNA product was used as the template for a standard PCR.
Primers used for standard PCR amplication were listed in Table 1.
Positive control during PCR amplication was performed (by)
using total DNA strains A. borkumensis SK2 and T. oleivorans MIL-1
extracted as previously indicated (MasterPure Complete DNA&RNA
Purication Kit; Epicenter, Biotechnologies, Madison, WI). The
Table 1
List, characteristic and sequences of the primers used in this study for screening of
functionally genes.
Primer

Length (bp)

Sequence

PCR product (bp)

Alk-b1F
Alk-b1R
Alk-b2F
Alk-b2R
P450F
P450R
Alk-bTF
Alk-bTR
PhnAF
PhnAR
TMOAF
TMOAR
C23OF
C23OR
BPHF
BPHR

24
21
20
20
20
24
18
18
22
21
21
21
21
25
22
23

AATTGGCCTATATCTCGTATGCCA
GCTTAGGAACAACGGATTAGG
CGCCGTGTGAATGACAAGGG
CGACGGTTGGCGTAAGCATG
GTGGGCGGCAACGACACGAC
GCAGCGGTGGATGCCGAAGCCTAA
GACGTCGCCACACCTGCC
GGGCCATACAGAGCAAGC
CGTTGTGCGCATAAAGGTGCGG
CTTGCCCTTTCATACCCCGCC
GCTATGTTACCGAAGAGCAGC
GGAATAGATCCCAGTACCAGG
CAAGGCCCACGACGTGGCNTT
CGGTTACCGGACGGGTCGAAGAAGT
TGCAGCTACCACGGCTGGGCCTA
GCNGCRAAYTTCCARTTRCANGG

150
150
400
200
150
300
270
150

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M. Hassanshahian et al. / International Biodeterioration & Biodegradation 93 (2014) 241e248

PCR was performed by using Qiagen Taq Polymerase (QIAGEN,


Valencia, CA) according to the manufacturer's protocol. PCR products were puried and the amplicons analyzed at 1.0% agarose gels
run in Tris-borate-EDTA buffer stained with ethidium bromide
and UV illuminated.

2.13. Statistical analysis

2.10. Quantication of alkane monooxygenase gene alkB by


Real-time PCR

3. Results

Real-time PCR analysis was performed using the SYBR Greenbased detection system.
Real-time PCR was run in a 7300 Real-time PCR System
(Applied Biosystems, Foster City, CA) in triplicate. The reaction
mixture was prepared using SYBR Green PCR Master Mix Applied
Biosystems in a total volume of 25 ml containing 12.5 ml of Master
Mix, 0.5 ml of forward and reverse primers (at optimized concentrations), 1 ml of DNA containing 20 ng from pure culture
samples and 50 ng from tissue samples. Sterile MilliQ water was
used to adjust the volume of each reaction to 25 ml. A no-template
control (NTC) and a positive control DNA from pure culture were
included on each plate.
The thermal cycling protocol included an initial denaturation
step at 95  C for 10 min, followed by 38 cycles of denaturation at
95  C for 1 s and annealing/elongation at 57  C for 5 s. A dissociation
step was added to check for primer-dimer formation.

3.1. Total bacterial abundance

2.11. Construction of standard curves and copy number


determination
A tenfold serial dilution series ranging from 10 to 108 number
of cells of A. borkumensis SK2 and T. oleivorans MIL-1 per reaction
was used to create the standard curve for quantication. Serial
dilutions were prepared once for both targets and used for realtime quantication. The concentration of DNA was measured
using a NanoDrop ND-1000 spectrophotometer (Celbio) and
converted to the copy concentration using the following equation
(Crisa et al., 2011).
DNA (copy) 6.02  1023 (copies mol1)  DNA amount (g)/
DNA length (A. borkumensis SK2, 3,1 Mpb; T. oleivorans MIL-1, 3,9
Mpb)  660 (g mol1 bp1) (Schneiker et al., 2006; Rodkhum et al.,
2006; Golyshin et al., 2013).
Standard dilutions were analyzed in triplicate. Ct values were
plotted against the logarithm of their initial template copy concentration. Standard curves were generated by linear regression of
the plotted points. From the slope of each curve, PCR amplication
efciency (E) was calculated according to the following equation
(Rasmussen, 2001):

.
E 101 slope1

All data obtained, in different experimentations, from TERHC


and real time PCR analyses were detected by Analysis of variance (ANOVA).

The total bacterial counts obtained by DAPI staining are shown


in Fig. 2. In the control mesocosm (M1), from T10 on wards the
microbial abundance increased by one order of magnitude
(106 mL1), with respect to the bacterial abundance detected at the
beginning of the experiment. In the M2 system microbial abundance increased by two orders of magnitude (maximum value of
107 mL1, T10). In M3 mesocosm showed throughout the experiment the highest total bacterial density, being amended with the
bacterial consortium. At T0, the abundance of the total bacterial
community was approximately 105 cell mL1, while it increased
signicantly after the rst 24 h up to 109 cell mL1. Bacterial counts
remained on high magnitude orders until the end of experiment
(20 days).
3.2. Screening of functional genes
Some functional genes involved in crude oil biodegradation
screened by PCR in extracted DNA and cDNA of microbial population during mesocosm experiments (Table 2). These genes include:
i) genes responsible for n-alkanes degradation (e.g. alk-B1, alk-B2,
CytP450 from Alcanivorax sp. and alk-BT from Thalassolituus sp.)
and ii) genes responsible from PAHs degradation (e.g. C230, tmoA,
bpH, phnA from Cycloclasticus sp.). As shown positive amplication
is detected for alk-B1 and alk-B2 genes of Alcanivorax sp. from DNA
and cDNA in three mesocosms; although CytP450 gene did not
present in cDNA of three mesocosms samples. This pattern indicated that this gene had not expression in these mesocosms. From
genes that responsible for biodegradation of aromatic fraction of
crude oil, only phnA from Cycloclasticus was present in DNA of
mesocosms and other genes not present positive amplication.
3.3. Validation of Real-time PCR method
Melt-curve analysis was used to check for the production of
secondary products such as primer dimmers in the assays. It was
observed that there was a single peak in the melt curves of both the
alk-B1 and alk-BT genes around 75  Ce80  C. Although there was
smaller peak at 70  Ce75  C that attributed to primer dimmers
(Figs. 4 and 5). Electrophoresis of PCR product conrmed the single
band with expected size on 2% agarose gel (Fig. 3).

2.12. Hydrocarbon analysis


The composition of total extracted and resolved hydrocarbons
and their derivates (TERHC) was extracted from 1 L aliquots, analysed by high-resolution GCeMS and quantied according to previously described protocols (Dutta and Harayama, 2001; Cappello
et al., 2006). The TERHC composition was analysed by highresolution GCeMS using a PerkineElmer Turbo MS Auto System
XL GC (PerkineElmer Biosystems, Foster City, CA, USA) equipped
with a DB-TPH fused silica capillary column [30 m by 0.32 mm
(inner diameter); J and W Scientic (Folsom CA, USA)]. The samples
were quantied according to previously described protocols (Dutta
and Harayama, 2001).

Table 2
Results of screening of functional genes in DNA and cDNA of mesocosms samples,
Positive amplication is indicated as ; negative amplication is indicated as .
Mesocosm

M1 DNA

M2 DNA

M3 DNA

M1 cDNA

M2 cDNA

M3 cDNA

Gene
alk-B1
alk-B2
cyt-P450
alk-BT
c23O
bpH
tmoA
phnA

e
e
e
e

e
e
e

e
e
e

e
e
e
e
e
e

e
e
e
e
e
e

e
e
e
e

M. Hassanshahian et al. / International Biodeterioration & Biodegradation 93 (2014) 241e248

245

3.4. Quantication of the gene copy numbers of alk-B1 in three


mesocosms

Fig. 3. Gel electrophoresis (1.5%, agarose) of real time PCR products of gene alk-B1
(200 bp) and gene alk-BT (150 bp). Lane (L), 100 bp DNA size marker Ladder; lane
(1), positive control (Alkanivorax borkumensis SK2); lanes (2e7), mesocosm samples,
lane (8) negative control (Distill water), lane (9) positive control for alk-BT (Thalassolituus oleivorans MIL-1), lanes (10e15), mesocosm samples, lane (16) negative control.

The standard curves for both assays were linear (r2 > 0.95) over
8 orders of magnitude for the alkB and alk-BT genes assay (Fig. 4).
This equated to a lower limit of 3 copies mL1 for the alkB and alk-BT
genes assay. The r2 values were consistently greater than 0.95 and
usually greater than 0.99 for both assays. Once the primer concentration was optimized, the efciency of the reactions was between 0.9 and 1.1 (or 90e110%). Runs that fell outside these
parameters (r2 < 0.95 and efciency < 0.9 or >1.1) were repeated.
There was usually no signal in the no-template control for the alkB
and alk-BT genes assay.

RT-PCR was performed with specic primers for alk-B1 gene of


Alcanivorax. After validation of the method and control of melting
curve gene copy numbers of alk-B1 gene were quantied in
different samples (T 0 to T20) of three mesocosms. The results were
presented in Fig. 6. As shown in this gure the alk-B1 gene had the
highest quantity in fth day of sampling (1  106) at mesocosm one
(M1) however this gene had the highest quantity in tenth day of
sampling (1  106 for M2 and 1  107 for M3) at mesocosm two and
three (M2 and M3). The quantity patterns of alk-B1 were decrement
in the end of sampling period (T15 and T20) in all mesocosm. The
comparisons of gene copy number of alk-B1 in three mesocosms
were illustrated in Fig. 7. As shown in this gure in T3 and T 5 the
highest quantity of alk-B1 related to M1 mesocosm. But in T10, T15
and T20 the highest quantity of this gene belong to M3 mesocosm.
By comparison the gene copy number of alk-B1 Between three
mesocosm it was concluded that M3 mesocosm had the highest
copy number of this gene in tenth day of sampling (1  107) in
all mesocosms.
3.5. Quantication of the gene copy numbers of alk-BT in three
mesocosms
The results of screening functional genes in all mesocosms show
that alk-BT from Thalassolituus oleivoras MIL-1 was present only in

Fig. 4. Real-time PCR alk-B1 Alcanivorax borkumensis SK2 (1) and alk-BT Thalassolituus oleivorans MIL-1 dissociation plot measured during mesocosm experimentations.

246

M. Hassanshahian et al. / International Biodeterioration & Biodegradation 93 (2014) 241e248

Fig. 5. Standard curves for quantication, plotted from duplicate samples using Ct
values of tenfold dilutions for alkane monooxygenase gene (alk-B); empty triangle and
squares indicated different replicate.

Fig. 7. Comparison of copy number of alk-B1 of A. borkumensis SK2 gene measured


in three different experimental mesocosms (M1, black bars; M2, white bars and M3,
gray bars).

cDNA of M3 mesocosm, so RT-PCR were performed with cDNA of


M3 as template by specic primers. Quantication of this gene was
carried out only in M3 mesocosm. The results of quantication
were shown in Fig. 8. The highest quantity of this gene were
observed in fteen day of sampling (1  107). In contrast to alk-B1 of
Alcanivorax the alk-BT of Thalassolituus had the increment pattern
until T15 of sampling but the gene copy number of this gene
decreased in T20 of sampling in M3 mesocosm.
3.6. Comparison the quantity of alk-B1 and alk-BT gene copy
number in M3 mesocosm
Mesocosm three (M3) inoculated with two HCB bacteria (Alcanivorax and Thalassolituus). The quantity of alkane monooxgenase
gene from these two bacteria simultaneously quantied by RT-PCR
in this mesocosm. The results were presented in Fig. 9. By comparison the quantity of alk-B1 gene versus alk-BT gene in M3
mesocosm it was concluded that in all sampling times the gene
copy number of alk-B1 were higher than alk-BT. This result
conrmed the predominant of Alcanivorax than Thalassolituus in
M3 mesocosm. Thus in competition between these two bacteria in
M3 mesocosm Alcanivorax was winsome.
3.7. Crude oil biodegradation in mesocosms

Fig. 8. Copy number of alk-BT gene from T. oleivorans in three experimental mesocosms (M1, M2 and M3).

obtained in triplicate sub-samples were averaged. The results,


expressed as percentage of oil degradation, were presented
in Fig. 10.
In all experimental mesocosms an increment of crude oil
biodegradation is observed from fth day until 20th day of experiment. At the end of experiment (T20), the mesocosm M2 show the

Degradation of the crude oil in mesocosms experiment was


examined, by Gas Chromatography (GCeMS), at 3, 5, 10, 15 and 20
days of experiments; the concentration of crude oil components
was normalized using the pristane/phytane ratio, and the values

Fig. 6. Copy number of alk-B1 gene from A. borkumensis SK2 identied in experimental
mesocosms carried out in this study.

Fig. 9. The comparison between expression of alk-B1 from A. borkumensis SK2 (white
bars) and alk-BT from T. oleivorans (black bars) during M3 experimentation.

M. Hassanshahian et al. / International Biodeterioration & Biodegradation 93 (2014) 241e248

247

Fig. 10. Percentage of degradation of crude oil detected in Mesocosm 1 (black squares), Mesocosm 2 (black triangle) and Mesocosm 3 (dark circles) experiments; all data were
expressed as the percentages compared to initial petroleum sample (time zero). Control means the effect of petroleum weathering during mesocosm experiment (empty circles)
monitored in a separated tank lled with sterile seawater and Arabian light oil.

highest degradation of crude oil (~95%); that consortium bioaugmentation mesocosm (M3) and biostimulating mesocosm (M1)
present rate of bioremediation of ~70 and ~80%, respectively.
Effect of petroleum weathering during microcosm experiment
was monitored in separated tank lled with sterile seawater and
Arabian light oil. The obtained results indicated that evaporation of
light petroleum hydrocarbons did occur and resulted in a loss of 5%
of TERHC fraction (data not show).
4. Discussion
Real time PCR (RT-PCR) was mainly used for the detection and
quantication of pathogens such as Salmonella, Vibrio cholerae and
Escherichia coli (Zhang et al., 2006). More recently applications of
RT-PCR were extended to environmental samples analysis (Zhang
et al., 2006). For example Hall et al. (2002) use RT-PCR to monitor
the nitrifying bacteria population dynamics in wastewater treatment plants. Powell et al. (2006) an RT-PCR method for analysis the
changes in hydrocarbon-degrading microbial community in Antarctic soil during bioremediation.
In this study an RT-PCR method were used for understanding
the microbial changes and dynamic during crude oil biodegradation in mesocosms. The results of this study conrmed that the
alkane monooxygenase gene (alk-B1 and alk-BT) expressed in the
mesocosms until the crude oil was present and after biodegradation of crude oil the level of expression of these genes decreased but
the patterns in different mesocosms were variable.
Various studies have shown that the addition of fertilizer (biostimulation) or external bacteria (bioaugmentation) to oil contaminated seawater increased oil biodegradation (Hassanshahian et al.,
2012b). However, few studies have followed the microbial population dynamics in any detail, often only showing an increase in the
number of total or hydrocarbon-degrading bacteria. Here we
showed that there is a link between the decrease in the amount of
alkanes present with the number of alk-B and changes in the microbial community structure as shown in Fig. 5. The highest copy
number of this genes is on fth day of sampling and in the end of
experiment (T15 and T20) the quantity of this genes decreased that
this correlated to increment in biodegradation of crude oil in the
mesocosms that shown in Fig. 10. Similar patterns were seen for
alk-BT of Thalassolituus (Fig. 8). Thus the low quantity of alk-B1 in
T15 and T20 may be attributed to biodegradation of major fraction of

crude oil in the mesocosms, then pressure effect for induction of


this gene were removed in the mesocosms and quantity were
decreased. Thus there was a direct relationship between gene copy
number of alk-B1 and remaining of petroleum hydrocarbons in the
mesocosms.
Kazunari et al. (2003) measured the degradation of n-alkanes in
seawater microcosms over 60 days and observed that an increase in
the level of alk genes preceded the degradation of n-alkanes. This
was followed by a decrease in the level of alk genes after the alkane
had been degraded in a manner similar to the observation made in
this study.
By comparison between biostimulation strategies versus bioaugmentation (single or consortium) strategies in this study
we conclude that single bioaugmentation has the best effect
on crude oil biodegradation. The results of RT-PCR show that
copy number of alk-B1 gene was more than alk-BT in consortium
bioaugmentation mesocosm. Also the results of GCeMS for
biodegradation of crude oil conrmed that the single bioaugmentation mesocosm has the highest degradation in compare
to other mesocosms.

5. Conclusion
Real-time PCR is an ideal technique for ecological studies as a
single DNA extract can be analyzed for many genes, allowing a
quantitative approach to analyzing both community structure
and function. In this study, we found that Real-time PCR
was more reproducible, less labor-intensive, and quicker techniques for the study of dynamic of the microbial community in
the contaminated seawater mesocosms and data obtained
revealed that bioremediation actions (biostimulation and bioaugmentation) stimulated crude oil degrading bacteria than
other genus in the marine microbial community. However, it is
important that Real-time PCR assays are tested thoroughly for
specicity and reproducibility and that the limitations of the
method are recognized.

Acknowledgment
This work was supported by grants of Shahid Bahonar University
of Kerman and by National Counsel of Research (CNR) of Italy.

248

M. Hassanshahian et al. / International Biodeterioration & Biodegradation 93 (2014) 241e248

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