Marine Environmental Research xxx (2013) 1e11

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Marine Environmental Research

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Bioremediation (bioaugmentation/biostimulation) trials of oil polluted

seawater: A mesocosm simulation study
Mehdi Hassanshahian a, *, Giti Emtiazi b, Gabriella Caruso c, Simone Cappello c
a
Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran
b
Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran
c
Istituto per l’Ambiente 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: Bioaugmentation (amendment with selected bacterial strains) and/or biostimulation (nutrients addition
Received 27 October 2013 and/or air supply) are relatively new fields in environmental microbiology for preventing pollution and
Received in revised form cleanup contamination. In this study, the efficiency of application of bioaugmentation/biostimulation
13 December 2013
treatments, for recovery of crude oil-polluted seawater, was evaluated. Three different series of exper-
Accepted 16 December 2013
iments were performed in a “Mesocosm Facility” (10.000 L). Natural seawater was artificially polluted
with crude oil (1000 ppm) and was amended with inorganic nutrients (Mesocosm 1, M1), inorganic
Keywords:
nutrient and an inoculum of Alcanivorax borkumensis SK2T (Mesocosm 2, M2) and inorganic nutrient and
Oil spill
Bioremediation
an inoculum of A. borkumensis SK2T and Thalassolituus oleivorans MIL-1T (Mesocosm 3, M3), respectively.
Bioaugmentation/biostimulation During the experimental period (20 days) bacterial abundance (DAPI count), culturable heterotrophic
Mesocosm bacteria (CFU count), MPN, microbial metabolic activity [Biochemical Oxygen Demand and enzymatic
Alcanivorax activity (leucine aminopeptidase LAP, b-glucosidase BG, alkaline phosphatase AP)] and quali-, quanti-
Thalassolituus tative analysis of the composition of total extracted and resolved hydrocarbons and their derivates
(TERHCs) were carried out. The microbiological and physiological analysis of marine microbial com-
munity found during the three different biostimulation and bioaugmentation assays performed in
mesocosms show that the load of crude oil increases total microbial abundance, inhibits the activity of
some enzymes such as LAP while stimulates both AP and BG activities. The biodegradation results show
that bioaugmentation with A. borkumensis SK2T alone is able to produce the highest percentage of
degradation (95%) in comparison with the biostimulation treatment (80%) and bioaugmentation using an
Alcanivorax-Thalassolituus bacterial consortium (70%). This result highlights the reduced biodegradation
capability of the consortium used in this study, suggesting an unfavourable interaction between the two
bacterial genera.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction biostimulation and bioaugmentation, has proven to be an effective

Petroleum hydrocarbons are the most widespread contami-
nants in the environment (Santas et al., 1999; Yakimov et al., 2007)
entering into aquatic environments because of catastrophic acci-
dents (shipping disasters or pipeline failures), chronic pollution
(ships, ports, oil terminals, freshwater runoff, rivers and sewage
systems), natural oil seepages and natural sources (Floodgate, 1972;
Cappello et al., 2007a; Hassanshahian et al., 2012a).
Many physical, chemical and biological technologies have been
developed to remove hydrocarbon pollutants from soils and marine
environments. However, these techniques often are not able to fully
remove pollutants from environments. Bioremediation, including
* Corresponding author. Tel.: þ98 9132906971; fax: þ98 3413202032.
E-mail addresses: hasanshahi@gmail.com, mshahi@uk.ac.ir (M. Hassanshahian).
method for cleaning up residual oil in a variety of environments
(Van Hamme et al., 2003) and has been proposed as the only viable
management option that can be implemented on a large scale in
marine environments (Snape et al., 2001; Hassanshahian et al.,
2012b).
At sea, petroleum hydrocarbon degradation is mainly performed
by microorganisms, and the microbial communities of marine
ecosystems involved in this process have been extensively studied
(Harayama et al., 1999; Yakimov et al., 2004).
Different studies have shown that marine communities of
hydrocarbon-degrading bacteria are composed of indigenous
members (Cappello et al., 2007b; Gertler et al., 2012) able to degrade
mixtures of hydrocarbons, like crude oil. It has been well-
documented that the addition of nitrogen and phosphorus signifi-
cantly enhances the growth of hydrocarbon-degrading bacteria, with

0141-1136/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.marenvres.2013.12.010

Please cite this article in press as: Hassanshahian, M., et al., Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: A
mesocosm simulation study, Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.010
2 M. Hassanshahian et al. / Marine Environmental Research xxx (2013) 1e11
consequent stimulation of metabolic processes involved in oil
biodegradation (Gertler et al., 2012). Moreover, as a single species can
metabolize only a limited range of hydrocarbon substrates, a con-
sortium of many different bacterial species, with broad enzymatic
capacities, is usually involved in oil degradation (Rölling et al., 2002).
To understand how the composition of microbial community affects
the process of biodegradation, it is necessary to analyse the microbial
response to oil pollution both at genetic and metabolic levels. It is
also important to develop a multidisciplinary approach for the study
of microorganisms having high specificity for recalcitrant com-
pounds, dealing with their structure and function. Knowledge of
microbial diversity and metabolism in oil-polluted sites can be
helpful for bioremediation of oil spills, as human intervention by
using specific microbial consortia can be planned for cleaning up oil
pollution (Denaro et al., 2005; Hassanshahian and Emtiazi, 2008).
The application of bioremediation techniques (bioaugmentation
and/or biostimulation) for the recovery of polluted marine envi-
ronments relies on the knowledge of oil weathering processes,
biological dynamics (e.g. variation of the microbial community) and
rates of degradation of hydrocarbons (Genovese et al., 2013).
However, despite its proven utility, natural and non-destructive
character, bioremediation remains a controversial technology (Lee
and Merlin, 1999; Genovese et al., 2013; Hassanshahian et al.,
2013). The effectiveness of bioremediation practice depends on
several factors, such as the presence of suitable hydrocarbon-
degrading microbial consortia as well as of favourable environ-
mental conditions (Hassanshahian and Emtiazi, 2008). Moreover,
advancements in this topic are often difficult due to the insufficient
consistency between data obtained from laboratory experiments
and those obtained in “in situ” experiments (Reilly, 1999). The
“in vitro” experiments sometime produce few realistic data, which
cannot be exported to natural environments. On the other hand, “in
situ” experiments are difficult to be carried out and the obtained
data are not simple to be interpreted because of the complexity of
the analysed biological processes (Cappello and Yakimov, 2010). As
previously indicated (Cappello and Yakimov, 2010) the need to link
the “in vitro” experiments e that can be statistically replicated but
are not performed under real conditions e and the experiments
performed in large-scale e in which intrinsic difficulties are related
to the inability to control environmental factors-, has lead to the
increasing use of medium-scale systems, like microcosms and/or
mesocosms. Different studies have demonstrated the great versa-
tility and potentiality of mesocosm systems in reproducing the
same processes occurring in nature (Alldredge et al., 1995; Reilly,
1999; Takeuchi et al., 2000; Lebaron et al., 2001) through the
control over both dependent/independent variables and the in-
teractions among the chemical, physical and biological parameters
(Cappello and Yakimov, 2010).
However, no study has ever been carried out to test, in meso-
scale systems in the size (10,000 L) similar to those used in this
work, efficiency of bioremediation processes and biostimulation.
The aim of the present study was to monitor the efficiency of
application of bioaugmentation/biostimulation strategies, in the
mesoscale (mesocosm) systems, for the recovery of crude oil-
polluted marine area. Some physiological, biochemical and enzy-
matic assays occurring in the structure and composition of seawater
natural microbial communities were carried out to reach this goal.
2. Materials and methods
2.1. Site description and sampling
During mesocosm experiments, natural seawater (SW) was
collected, in November 2011, from the station “Marisicilia”
(38 12.230 N, 15 33.100 E) located in the harbour of Messina (Italy).
Please cite this article in press as: Hassanshahian, M., et al., Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: A
mesocosm simulation study, Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.010
2.2. Set-up of experimental mesocosms systems
Experiments in mesocosms were performed in a “Mesocosm
Facility” designed and built at the Institute for Coastal Marine
Environment (IAMC) e CNR of Messina, Italy (Fig. 1). The experi-
ments were carried out in a rectangular tank of 11250 L of capacity
(5000 cm long, 150 cm deep, 150 cm wide) filled with 10000 L of
natural seawater (Fig. 2). The mesocosm system was filled with
natural sea water (SW) collected directly from the Straits of Mes-
sina through a direct pipeline. Before the introduction in the
mesocosms, natural seawater was filtered through a 100 mm nylon
mesh to remove large metazoans and detritus. During the study the
seawater was aerated and kept under agitation during the all
experimental period. Mesocosm water was mixed by a pump
(35 l h 1), placed at the exit of each tank, that takes water from two
opposite bottom corners and drives it below the surface. Seawater
temperature (18  2  C) was checked for all experimental period;
pH values were also measured to detect possible variations.
2.3. Bacterial strains
Two hydrocarbonoclastic bacterial strains Alcanivorax borku-
mensis strain SK2T (Genebank accession number Y12579; ¼ DSM
11573T; Yakimov et al. (1998) and Thalassolituus oleivorans strain
MIL-1T (Genebank accession number AJ431699; ¼ DSM
14913T, ¼ LMG 21420T; Yakimov et al. (2004) were used in this
study (Fig. 3). Both strains belong to a collection of hydrocarbon-
degrading bacteria hold at IAMC-Messina.
2.4. Growth conditions of the bacterial inocula amendments
Starting cultures of bacteria selected for bioaugmentation
(A. borkumensis SK2T and T. oleivorans MIL-1T) were prepared
separately for each strain by inoculating one loop of bacterial cells
into 10 mL of ONR7a mineral medium (Dyksterhouse et al. (1995)
containing 0.1% (w/v) of tetradecane (C14H30, SigmaeAldrich,
Milan, Italy) sterilized by filtration through a 0.2-mm syringe filter
(Sartorius). 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 a 250 mL Erlenmeyer flask
containing 100 mL of ONR7a medium supplemented with 1% (w/v)
of sterile Arabian Light Crude Oil (ENI Technology S.P.A.). The cul-
ture was incubated in a rotary shaker (150 rpm) at 25  C for 5 days.
At values of microbial abundance (measured by DAPI count) of
108 cell mL 1 cultures were added in experimental mesocosms
(Beginning of experiment, t0).
2.5. Design of mesocosm experiments
Three different series of experiments were set up. In all the
experiments, natural seawater was supplemented with Arabian
Light Crude Oil and inorganic nutrients. 1 L of Arabian Light Crude
Oil (ENI Technology S.P.A.) were added to all the mesocosms; 0.1%
(v/v) of squalene (C30H50, SigmaeAldrich, Milan) was also added to
crude oil as an internal spike/control to monitor the biodegradation
process. Inorganic nutrients were added to reach concentrations
higher than those in natural water (final concentrations: KH2PO4
0.077 g L 1, NH4Cl 0.2 g L 1 and NaNO3 0.1 g L 1). The “control”
mesocosm, consisting of natural seawater with oil and inorganic
nutrients, was indicated as “mesocosm 1 (M1)”. The mesocosm
amended with the isolate A. borkumensis SK2T only was indicated as
“mesocosm 2” (M2), while the mesocosm where A. borkumensis
SK2T and T.oleivorans MIL-1T were simultaneously added was
indicated as “mesocosm 3 (M3)”.
 M. Hassanshahian et al. / Marine Environmental Research xxx (2013) 1e11 3

Fig. 1. Schematic representation of engineered and hydraulic system of “Mesocosm Facility” of IAMC-CNR of Messina used in this study. (1) Fill line with water treatment system
(filter); 2 and 3, system of water recirculation; the experimental tank (4) is made of a steel tower moulded-case covered with fibreglass. 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 flora. (5), seawater
and (6), crude oil.

2.6. Sampling strategy and parameters assayed
All experiments lasted 20 days. To monitor changes in the mi-
crobial community from the time zero (t0; introduction of oil,
inorganic nutrients and bacteria) to the end of the experiment in
terms of bacterial abundances (total bacteria, heterotrophic bac-
teria and hydrocarbon degrading bacteria) and microbial activities
(enzymatic activity and Biochemical Oxygen Demand), sub-
volumes (1 L) of sea-water were drawn (in triplicate) from the
surface layer (5  1 cm) of the mesocosms on fixed days (day 0, 5,
10, 15 and 20). At the same time of sampling, the composition of
total extracted and resolved hydrocarbons and their derivates
(TERHCs) was also analysed. Triplicate measurements of all pa-
rameters were carried out.
2.7. Total bacterial count (DAPI)
stored at 4  C until analysis. After short-time (30 s) ultrasonic
treatment (Ultrasonic Bath Branson 1200, Branson, USA), samples
were filtered on Nuclepore black polycarbonate filters (pore size
0.2 mm) and the filters stained with 4, 6-diamidino-2-phenylindole
(DAPI; SigmaeAldrich S.R.L., Milan, Italy, 5 mg L 1 final concen-
tration), according to standard analytical protocols (Zampino et al.,
2004; Porter and Feig, 1980). Slides were examined under a Zeiss
Axioplan 2 Imaging epifluorescence microscope (Zeiss; Carl Zeiss
Inc., Thornwood, N.Y.) and the labelled cells present in at least 30
microscopic fields counted. The bacterial counts were expressed as
the total number of cells mL 1.
2.8. Culturable heterotrophic bacteria (CFU)
The abundance of culturable heterotrophic bacteria was esti-
mated by spreading 100 mL of 10-fold dilutions of water samples on

For total bacterial counting, suitable volumes of seawater (1e

2 mL) were fixed in formaldehyde (2% final concentration) and

Fig. 2. In principal picture image of the mesocosm used during this study. In particular
in (A) is shown particular of superficial seawater at the begging of experimental period,
in (B) is shown particular of superficial seawater at the end experimental period after
introduction of (Alcanivorax borkumensis strain SK2T).
Fig. 3. Phylogenetic tree based on 16S rRNA gene sequences for bacterial strains
(Alcanivorax borkumensis strain SK2T (Genebank accession number Y12579; Thalasso-
lituus oleivorans strain MIL-1T (Genebank accession number AJ431699) used in this
study and recognized species of the genus Alcanivorax and principal hydro-
carbonoclastic bacteria. Percentages of 100 bootstrap resembling that supported the
branching orders in each analysis are shown above or near the relevant nodes. The tree
was rooted and out grouped (black arrow) by using the 16S rRNA sequences of
Methanococcus jannaschii (M59126). Evolutionary distance is indicated by vertical
lines; each scale bar length corresponds to 0.05 fixed point mutations per sequence
position.

Please cite this article in press as: Hassanshahian, M., et al., Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: A
mesocosm simulation study, Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.010
4 M. Hassanshahian et al. / Marine Environmental Research xxx (2013) 1e11
the surface of Marine agar (Difco 2216, Milan, Italy) plates, further
incubated at 20  C for 7 days. The colonies growing on plate were
counted and the results expressed as colony forming units
(CFU mL 1).
2.9. Hydrocarbon-degrading bacterial count (MPN)
Hydrocarbon-degrading bacteria present in the mesocosms
were enumerated by a miniaturized Most Probable Number (MPN)
method, according to Brown and Braddock (1990). As previously
reported (Cappello et al., 2007a), MPN test was performed using
sterile 24-wells (3 mL each) tissue culture test plates containing
2 mL per well of sterile Bushnell-Hass (B-H) medium (Difco) added
with 2% NaCl, pH 7. Tenfold serial dilution of samples (10 2e10 9)
was made in B-H medium. Wells were inoculated by pipetting
100 mL from the sample dilutions. Following sample inoculation,
10 mL of Arabian light weathered crude oil, sterilized by filtration
through a 0.2-mm syringe filter (Sartorius) was added, with care, in
the central area of each well. The remaining four wells of each plate
were used as a reference (B-H medium þ oil) as a negative control.
Plates were incubated at 20  C for 20 days. Wells were scored as
positive when oil emulsification was clearly indicated by the
disruption of the oil film and/or the oil ‘ring’ produced on the
surface of the medium in contact with the wall of the well,
accompanied with changes in the oil colour. MPN index was
calculated according to the American Public Health Association
(APHA, 1992).
2.10. Microbial enzymatic activities
The metabolic activity of the marine microbial community was
studied by the analysis of three enzymes: leucine aminopeptidase
(LAP), b-glucosidase (BG) and alkaline phosphatase (AP). LAP is
involved in protein degradation, BG in carbohydrate degradation
and AP in the release of phosphorus from organic compounds.
Microbial extracellular enzymes were estimated according to the
Hoppe’s method (Hoppe, 1993), using different fluorogenic sub-
strates, each of them specific for the enzyme to be assayed
(Cappello et al., 2007a). The substrates for the determination of LAP,
AP and BG were L-leucine-7-amido-4-methylcoumarine (Leu-
MCA), 4-methylumbelliferyl phosphate (4-MUF) and b-D-gluco-
side, respectively. Fluorescence measurements were carried out
with a Turner TD 700 fluorimeter immediately after substrate
addition and after 2 h of incubation at ‘in situ’ temperature, at 365/
445 nm (excitation/emission wavelengths), except for LAP (380/
440 nm). Enzymatic activity rates were expressed in terms of
maximum velocity of substrate hydrolysis (Vmax), in nanomoles of
product released from the substrate per litre and per hour
(nmol l 1 h 1).
2.11. Biochemical Oxygen Demand (BOD)
To assess the indirect capability to degrade pollutants, mea-
surements of Biochemical Oxygen Demand (BOD) were carried out
using a BOD sensor (VELP Scientific, Milan), in accordance with the
manufacturer’s instructions.
2.12. Hydrocarbon analysis
Total extracted and resolved hydrocarbons and their derivates
(TERHCs) were obtained by extraction from 1-L of superficial
seawater; TERCHs composition was analysed by high-resolution
GCeMS and quantified according to previously described pro-
tocols (Cappello et al., 2007a; Denaro et al., 2005; Dutta and
Harayama, 2001). After collection with using of clean beaker
Please cite this article in press as: Hassanshahian, M., et al., Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: A
mesocosm simulation study, Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.010
superficial seawater samples were acidized; TERHCs from samples
were extracted on a shaking table using dichloromethanee
seawater (10% v/v). This procedure was repeated three times, and
the CH2 Cl2 phase was treated with anhydrous Na2 SO4 to remove
residual water. Extracts were concentrated by rotary evaporation
(Rotavapor model R110; Büchi Labortechnik, Flawil, Switzerland) at
room temperature, followed by evaporation under a stream of ni-
trogen and taken up in a solution containing heptamethyl-nonane
as an internal standard (79 mg mL 1). A high-resolution 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
Scientific (Folsom CA, USA)] was used for the analysis. The samples
were quantified according to previously described protocols (Dutta
and Harayama, 2001) and as described by Denaro et al. (2005). Data
obtained were normalized by squalene (internal spike/control of
biodegradation) and indices Pristane/Phytane (Pr/Ph) were used in
order to evaluate the relative biodegradation of n-alkanes.
Moreover, the percentage of degradation of n-alkanes was
expressed as the percentage of TERCHs degraded in relation to the
amount of the remaining fractions in the appropriate control. The
biodegradation efficiency (BE), based on the decrease in the total
concentration of hydrocarbons, was calculated by using the
expression described by Michaud et al. (2004):
100 ðAs*100=AacÞ
Where As ¼ total area of peaks in each sample, Aac ¼ total area of
peaks in the appropriate control.
2.13. Statistical analysis
All the DAPI, MPN, CFU, BOD and enzymatic activity data ob-
tained during the experiments in the mesocosms M1, M2 and M3
were subjected to Analysis of variance (ANOVA).
3. Results
3.1. Total bacterial abundance
The total bacterial counts obtained by DAPI staining are shown
in Fig. 4. In the control mesocosm, indicated as “mesocosm 1” (M1),
from the10th day onwards the microbial abundance increased by
one order of magnitude (106 mL 1), with respect to the bacterial
abundance detected at the beginning of the experiment.
In the “mesocosm 2” (M2) total cell numbers increased by two
orders of magnitude, reaching on the 10th day a maximum value of
107 mL 1; at the end of experimental period, they decreased by one
order of magnitude.
The “mesocosm 3” (M3) 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 mL 1, while it increased significantly
after the first 24 h up to 109 cell mL 1. Bacterial counts remained on
high magnitude orders until the end of experiment (20 days).
A general decrease in total bacterial numbers was noticed from
the 15th day onwards in all the mesocosms.
3.2. Culturable heterotrophic bacterial count
Culturable heterotrophic bacterial counts obtained from the
mesocosms are reported in Fig. 5. The concentration of culturable
heterotrophic bacteria in the mesocosms M1 and M2 increased
from the starting day to the 3rd day (1  105 CFU mL 1); it remained
almost constant in the mesocosm M1, while in the mesocosm M2 it
 M. Hassanshahian et al. / Marine Environmental Research xxx (2013) 1e11 5

Fig. 4. Bacterial densities determined by DAPI (cell mL 1) staining during mesocosm experiment. Concentration of the cells observed in “Mesocosm 1” (Seawater with crude oil and
inorganic nutrients; empty squares), “Mesocosm 2” (seawater with crude oil, inorganic nutrients and a single Alkanivorax borkumensis inoculum; empty triangles) and “Mesocosm
3” (seawater with crude oil, inorganic nutrients and the bacterial consortium Alkanivorax borkumensis-Thalassolituus oleivorans; empty circles).

decreased progressively until the 20th day. The highest concen-
tration of culturable heterotrophic bacteria was detected in the
mesocosm M3, like what observed for total bacterial counts. On Day
10, culturable heterotrophic bacteria reached a peak value of
1  106 CFU mL 1 which corresponded to a three orders of
magnitude increase compared to the counts obtained at t0. From
the 15th day onwards, in all the mesocosms, the concentration of
culturable heterotrophic bacteria depicted a decreasing trend
similar to that described by total bacterial numbers.
In the mesocosm M2, hydrocarbon-degrading bacteria
increased their numbers from t0 to the 5th day (5.2  102 and
1.1  105 MPN index mL 1, respectively). After the 5th day onwards,
the abundance of oil-degrading bacteria dramatically decreased
(103 and 102 MPN index mL 1 at t10 and t15, respectively).
In the mesocosm M3, like in M1, the number of hydrocarbon-
degrading bacteria showed a rapid increase during the first days
of the experiment and reached a maximum concentration on day
10 (106 MPN index mL 1).

3.3. Hydrocarbon-degrading bacteria count 3.4. Biochemical oxygen demand (BOD) measurements

Hydrocarbon-degrading bacterial counts obtained by MPN In all tested mesocosms the consumption of oxygen, as indicated
method are shown in Fig. 6. In the mesocosm M1, MPN index values by BOD data, was at its maximum level on the 5th day (Fig. 7). After
remained constant during all the experimental period, and only on this day until the end of the experiment, a decreasing trend in BOD
the 10th day an increase to 103 MPN index mL 1 took place. values was found in all the mesocosms. A peak value of BOD

Fig. 5. Distribution of culturable heterotrophic bacteria grown on Marine Agar plates (Colony Forming Units, CFU mL 1) obtained during mesocosm experiments. M1, M2 and M3
mesocosms are indicated with squares, triangles and empty circles, respectively. Refer to Fig. 4 legend for the description of mesocosms’ amendments.

Please cite this article in press as: Hassanshahian, M., et al., Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: A
mesocosm simulation study, Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.010
6 M. Hassanshahian et al. / Marine Environmental Research xxx (2013) 1e11

Fig. 6. Most Probable Number (MPN) index obtained during mesocosm experiments. M1, M2 and M3 mesocosms are indicated with squares, triangles and empty circles,
respectively. Refer to Fig. 4 legend for the description of mesocosms’ amendments.

(670 ppmO2 mL 1) was reached in the mesocosm M1 on the 5th
day; on the other hand, among all the mesocosms, M1 exhibited the
highest BOD values, while the lowest ones were recorded in the
mesocosm M2, where values never exceeded 90 ppmO2 mL 1 and
showed mostly an unchanged trend over the whole experimental
period.
3.5. Relationship between the total bacterial community and BOD
BOD measures the amount of oxygen consumed by bacteria to
degrade the organic matter present in waters, thus when there is a
high concentration of biologically degradable organic matter also
BOD increases. Fig. 8 shows the relationship between the total
bacterial community and BOD. Similar patterns, with a temporal
shift between the peaks in BOD and DAPI counts, were observed in
all three mesocosms. Particularly, in mesocosm M1, BOD peaked
was on the 3rd day, while the peak in bacterial abundance was
observed on the 15th day. For mesocosm M2 and M3 similar
pattern were found that BOD peak was on the 5th day, while the
peak for bacterial abundance was on the 10th day. Thus the bac-
terial community increased in abundance after that the biochem-
ical demand for oxygen required by organic matter decomposition
decreased.
3.6. Microbial enzymatic activities
The results of enzymatic activity measurements are shown in
Fig. 9. For the three enzymes, the highest enzymatic activities were
measured in the mesocosm M3. LAP decreased its metabolic ac-
tivity over mL in all the mesocosms, with the exception of the
mesocosm M3; this decreasing trend suggested that oil amend-
ment caused enzymatic inhibition, so impairing the proteolytic

Fig. 7. Dynamics of Biochemical Oxygen Demand (B.O.D.) recorded in “Mesocosm 1” (white grey), “Mesocosm 2” (grey) and “Mesocosm 3” (dark grey) experiments.

Please cite this article in press as: Hassanshahian, M., et al., Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: A
mesocosm simulation study, Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.010
 M. Hassanshahian et al. / Marine Environmental Research xxx (2013) 1e11
Fig. 8. The relation between total bacterial population and biochemical oxygen de-
mand (BOD) as determined in three mesocosms.
activity. This negative effect was partially overcome by the
amendment with the bacterial consortium (mesocosm M3) at least
until day 5, while afterwards the bacterial inocula were no more
able to stimulate the protein decomposition. Conversely, both AP
and BG activities increased in the three mesocosms, particularly in
the mesocosm M3. Particularly, AP increased from a value of
38 nmol L 1 h 1 recorded at t0 to a peak of
350 nmol L 1 h 1measured on the 10th day after bioaugmentation
with the bacterial consortium. BG increased from 125 nmol L 1 h 1
to a maximum of activity (1440 nmol L 1 h 1) measured on the
15th day of experiment.
3.7. Crude oil biodegradation in mesocosms
The concentration of crude oil components (n-alkanes) was
normalized using the squalene and pristane/phytane ratio, and the
values obtained in triplicate sub-samples were averaged. The re-
sults, expressed as percentage of oil degradation, are presented in
Fig. 10 n-alkanes biodegradation described increasing patterns in
all the mesocosms from the 5th to the 20th day of the experiment.
The mesocosm M2 showed the highest degradation of n-alkanes
(95% on the 20th day) among all the mesocosms. It is interesting to
Please cite this article in press as: Hassanshahian, M., et al., Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: A
mesocosm simulation study, Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.010
7
note that consortium bioaugmentation mesocosm (M3, 70%)
showed lower degradation of n-alkanes compared to single bio-
augmentation mesocosm (M2) and biostimulating mesocosm (M1,
80%).
Effect of petroleum “weathering” during the mesocosm exper-
iment was monitored in a separated tank filled with sterile
seawater and Arabian light oil. The obtained results indicated that
evaporation of light petroleum hydrocarbons did occur and resul-
ted in a loss of 5% of TERHCs fraction (data not shown).
4. Discussion
Biodegradation of hydrocarbons by natural microbial pop-
ulations is the primary mechanism by which oil contaminants are
removed from the marine environment (Yakimov et al., 2007).
However, one of the main reasons for the prolonged persistence of
hydrophobic hydrocarbons in polluted environments is their low
water solubility, which limits their availability to biodegrading
microorganisms (Cappello et al., 2012).
Research concerning the degradation of petroleum in natural
environments has been intensified and different alternative tech-
nologies have been proposed to treat oil polluted systems; how-
ever, bioremediation methods (which use specific bacteria to break
down hydrocarbons) are more attractive because of this low cost
compared to other technologies (Cappello et al., 2012). Bioreme-
diation is based on the enhancement of the activity of indigenous
organisms (biostimulation) and/or on the addition of microbial in-
oculates (bioaugmentation) to enhance the clean-up pills
(Syutsubo et al., 2001; Hassanshahian et al., 2010).
Different studies have indicated that many chemical and phys-
ical parameters (e.g. pH, T, oxygen concentration) of seawater may
affect bioremediation of crude oil in marine environments. Many
researchers use scale systems for mimicry of natural processes
occurring in marine environments, such as effect of radiation or
pollution on ecosystem etc (Pukall et al., 1999; Troussellier et al.,
2005). The use of experimental mesocosms gives important infor-
mation about the temporal effects of addition of various pollutants
on the structure and functioning of natural aquatic ecosystems
(Cappello and Yakimov, 2010). The growth of bacteria in a natural
environment is regulated by insufficient levels of energy and nu-
trients and physiological and phylogenetic changes, in a bacterial
community, are the result of specific survival strategies (Cappello
et al., 2008). Experimental or natural (during accidental oil spills)
addition of crude oil to natural microbial populations results in a
significant decline of the metabolic activity and physiological index.
These changes lead to a strong selection of petroleum-degrading
bacteria that are usually found in low numbers in pristine marine
environments (Cappello et al., 2007a; Harayama et al., 1999; Rölling
et al., 2002; Cappello et al., 2012; Syutsubo et al., 2001; Kasai et al.,
2001).
In natural marine environments nutrient availability is insuffi-
cient to support a significant increase of microbial growth when
considerable inputs of organic matter such as crude oil are intro-
duced/discharged. Thus the process of petroleum degradation un-
der nutrient depletion is very slow and hardly operable (Rölling
et al., 2002).
In the present study, the effectiveness of three designed meso-
cosms on marine microbial community and biodegradation of
crude oil has been investigated, through the set up of three
different biostimulation/bioaugmentation methods. In all experi-
mentations some abundance parameters (total bacterial counts,
culturable heterotrophic and hydrocarbon-degrading bacterial
counts) as well as metabolic (enzymatic activity, BOD) and chemical
parameters (TERHCs) have been assayed.
8 M. Hassanshahian et al. / Marine Environmental Research xxx (2013) 1e11

Fig. 9. Enzymatic activity patterns measured in M1, M2 and M3 mesocosm experiments (indicated with empty triangles, circles and squares, respectively): (a) leucine amino-
peptidase; (b) b-glucosidase; (c) alkaline phosphatase.

Please cite this article in press as: Hassanshahian, M., et al., Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: A
mesocosm simulation study, Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.010
 M. Hassanshahian et al. / Marine Environmental Research xxx (2013) 1e11 9

Fig. 10. Relative degradation values of major TERHC fractions of crude oil detected in “Mesocosm 1” (white grey), “Mesocosm 2” (grey) and “Mesocosm 3” (dark grey) 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
monitored in a separated tank filled with sterile seawater and Arabian light oil.

Bioaugmentation is based on the increase of metabolic activity
of indigenous microbial community. Bioaugmentation performed
on a mesocosm scale is a combination of microbial ecology and
biological engineering and has been the topic of recent research in
the last decades. Ruberto et al., 2009 designed some microcosms
systems to study the effect of biostimulation (with N and P) and
bioaugmentation for recovery of Antarctic soil oil polluted.
Vanbroekhoven et al., 2004, used the microcosms system for study
the effect of bioaugmentation in polluted soil and evidence as the
addition of Acinetobacter strain B22 resulted in hydrocarbon reme-
diation (estimated around 75%), with contemporaneous increase of
the fraction of heterotrophic bacteria in the soil and progressive
decrease of microbial diversity. Biostimulated mesocosms have also
been used for bioremediation of oil-polluted seawater. Schafer
et al., 2001 designed biostimulating mesocosms for bioremedia-
tion of oil contaminated Mediterranean Sea water and concluded
that biostimulation induce major changes in marine microbial
community and select for specific bacterial groups (Schafer et al.,
2001).
The results of the present study confirm that the abundances of
total bacteria, hydrocarbon-degrading and culturable heterotrophic
bacteria increase in the bioaugmented mesocosms from t0 (oil
addition) until the 10th day of experiment. The measured abun-
dances are correlated with the amounts of microbial inoculate
amended to each mesocosm. The decrease recorded in their counts
after the 10th day until the end of experiment may be explained by
the fact that, as soon as the degradation of oil proceeds in the
mesocosms, the substrate for bacterial growth disappears. Also
BOD measurements, giving the amount of oxygen consumed for
organic decomposition, prove that the bacterial community is
metabolically active; BOD values are inversely related with total
bacterial counts, suggesting that the total bacterial density in-
creases as soon as the demand for oxygen e needed to sustain the
heterotrophic metabolism e is decreasing.
Particularly, in the mesocosm M1, BOD peaked on the 5th day,
while a peak in bacterial abundance was found later, on the 15th
day. Thus the bacterial community increased in abundance after
that the biochemical demand for oxygen required by organic
matter decomposition decreased. The reason for this inverse trend
was that the consumption of oxygen by the bacterial community
increased after t0, following the introduction of oil into the meso-
cosm, but the increase of total bacteria was inhibited by oil toxic
effects. Only a few days later, on the 10 and 15th days, the bacterial
community becomes adapted to oil and its abundance increased.
In order to measure the physiological properties of the meta-
bolically active members of the microbial community, the activity
of some microbial enzymes involved in organic matter decompo-
sition, although not specifically related to hydrocarbons degrada-
tion, has been quantified (Hoppe et al., 2002). In fact, the enzymatic
measurements performed in the current study have aimed only at
assessing the effects induced by oil and bacterial amendment on
the decomposition of polymers such as proteins, polysaccharides
and organic phosphates, which are among the most frequent
organic substrates in aquatic environments; furthermore, it must
be noted that in the natural seawater not all the micro-organisms
may express metabolic pathways involved in hydrocarbon
decomposition with potential bioremediation. Although not
directly associated with primary oil degradation, microbial extra-
cellular enzymes, like peptidase and b-glucosidase, have been
assayed as indicators of heterotrophic microbial activities in oil-
contaminated surface seawater from the Deepwater Horizon oil
spill and enhanced activity rates have been recorded (Ziervogel
et al., 2012).
The different behaviours among the three enzymes measured in
this study suggest a diversified metabolic response of the microbial
community to oil amendment. Both BG and AP activities are
stimulated significantly by oil and bacterial inocula amendments
(ANOVA F-values 8.33, P < 0.01, and 3.51, P < 0.05, respectively),
compared to the control mesocosm. The highest activity rates of
these enzymes are recorded in the mesocosm M3 on the 10e15th
day after the beginning of the experiment. Conversely, LAP activity
is inhibited by oil contamination, but interestingly, the amendment
with the bacterial consortium allowed a recovery of this enzymatic
activity at least within a few days from the amendment with the
inocula. A significant enhancement of AP activity was recorded in
soil microcosms after treatment with chemical pollutants
(Dougherty and Lanza, 1989); some enzymes involved in break-
down of organic matter are considered useful probes to assess the
degree of ecological damage occurring in polluted areas (Lanza and
Dougherty, 1991). Increased leucine aminopeptidase and beta-

Please cite this article in press as: Hassanshahian, M., et al., Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: A
mesocosm simulation study, Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.010
10 M. Hassanshahian et al. / Marine Environmental Research xxx (2013) 1e11
glucosidase activity rates together with bacterial densities were
measured in a coupled bioaugmentation/biostimulation experi-
ment (Gallizia et al., 2005). The decrease in enzymatic levels
recorded after the 10e15th day of sampling suggested that since
this mL some functional changes occurred in the microbial com-
munity composition or in the metabolism of some microflora
members. Similar time variability was observed from the eighth
day after oil pollution in a microcosm experiment (Cappello et al.,
2007a), when the composition of the microbial flora confirmed
the selection of HCB, having no metabolic potentialities towards
organic polymers.
Concerning crude oil biodegradation, the results obtained in this
mesocosm study show that the best remediation treatment for
crude oil is associated with the single-bioaugmented mesocosm
(M2), where the observed rates of biodegradation are comprised
between those measured in the biostimulated mesocosm (M1) and
in the bacterial consortium- bioaugmented mesocosm (M3).
5. Conclusions
The microbiological and physiological analysis of marine mi-
crobial community obtained during the three different bio-
stimulation and bioaugmentation treatments performed in
mesocosms show that the load of petroleum increase total micro-
bial community but suppress some certain enzymes. In the same
time, the data obtained in this paper suggest that using a single
bacterium (Alcanivorax sp.) for recovery of oil contaminated marine
environment have advantage than using of consortium of bacteria;
it is presumably due at the interaction between different genera of
bacteria may be suppress oil bioremediation in the marine envi-
ronment, that confirmed in this study.
Acknowledgement
This work was financially supported by Shahid Bahonar Uni-
versity of Kerman and grants from EC Project “Unraveling and
exploiting Mediterranean Sea microbial diversity and ecology for
XEnobiotics’ and pollutants’ clean up” (ULIXES-FP7-KBBE-2010-
3.5-03).
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mesocosm simulation study, Marine Environmental Research (2013), http://dx.doi.org/10.1016/j.marenvres.2013.12.010