International Biodeterioration & Biodegradation 110 (2016) 235e244

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International Biodeterioration & Biodegradation

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

Oil-degrading bacteria from a membrane bioreactor (BF-MBR) system

for treatment of saline oily waste: Isolation, identification and
characterization of the biotechnological potential
Simone Cappello a, *, Anna Volta a, b, Santina Santisi a, c, Claudia Morici d,
Giuseppe Mancini b, Paola Quatrini e, Maria Genovese a, Michail M. Yakimov a,
Michele Torregrossa d
a
Institute for Coastal Marine Environment (IAMC)-CNR of Messina, Messina, Italy
b
Dep. of Industrial Engineering, Faculty of Engineering, University of Catania, Catania, Italy
c
Faculty of Science MM. FF. NN. Ph.D School in “Biology and Cellular Biotechnology”, University of Messina, Italy
d
Dep, of Civil, Environmental, Aerospatial, Material Engineering, Faculty of Engineering, University of Palermo, Palermo, Italy
e
Dept. of “Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche” (STEBICEF), University of Palermo, Italy

a r t i c l e i n f o a b s t r a c t

Article history: A collection of forty-two (42) strains was obtained during microbiological screening of a Membrane
Received 6 May 2015 Bioreactor (MBR) system developed for the treatment of saline oily waste originated from marine
Received in revised form transportation. The diversity of the bacterial collection was analyzed by amplification and sequencing of
29 December 2015
the 16S rRNA gene. Taxonomic analysis showed high level of identity with recognized sequences of seven
Accepted 31 December 2015
Available online 23 April 2016
(7) distinct bacterial genera (Alcanivorax, Erythrobacter, Marinobacter, Microbacterium, Muricauda, Rho-
dococcus and Rheinheimera). The biotechnological potential of the isolates was screened considering an
important factor such as the biosurfactant production. In particular fourteen (14) biosurfactant producing
Keywords:
Alcanivorax
bacteria were selected and further tested, for growth on crude oil and hydrocarbon degradation. Data
Oil-degrading bacteria obtained from this study confirmed the high activity of bacteria related to genera Alcanivorax (isolates
Membrane bioreactor (MBR) system MBR-B11 and MBR-G10), Rheinheimera (isolates MBR-H02 and MBR-H05), Rhodococcus (isolates MBR-F04
Saline oily waste and MBR-G05) and Muricauda (isolate MBR-G04) and underline the possible application of these bacteria
Oil pollution in remediation of saline oily waste water.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Marine environment are constantly assaulted by a very wide
range of hydrocarbons, which can make their appearance from the
exploration, production, refining, transport and storage of petro-
leum and its derivates (Mnif et al., 2009). The oil pollution, espe-
cially deriving from anthropogenic activities, seriously endangers
marine biodiversity (Gertler et al., 2009a; Nogales et al., 2011). It is
well known that several marine bacterial strains have acquired the
ability to biodegrade hydrocarbons, using them as a source of car-
bon and energy source (Yakimov et al., 2007; Paisse et al., 2008;
Gertler et al., 2009b), although this process is limited by oligo-
trophy, generally typical of seawater. To optimize these completely
* Corresponding author.
E-mail address: simone.cappello@iamc.cnr.it (S. Cappello).
natural processes, researchers are always looking for more effective
ways to use microorganisms, in order to promote bioremediation,
mitigating marine pollution.
The bioremediation of oil in marine environments is a kind of
biotechnology that promotes the catabolism of hydrocarbons by
specific strains of indigenous bacteria. This requires a good
knowledge of these organisms and their nutritional needs
(Mahmoud et al., 2009). Bioremediation can be considered as green
technologies with low effective cost. The high salinity of water
bodies can often affect growth and metabolism of bacteria; how-
ever, in high concentration of salt, halophilic microorganisms can
survive (Woolard and Irvine, 1995). Some researchers use halo-
philic bacteria in biological systems to treat saline produced water.
Several biological methods are employed in the treatment of pe-
troleum impacted environmental media including bioreactor-
based treatments (Young and Cerniglia 1995; Siciliano et al.,
2003; Montiel et al., 2009). The treatments based on bioreactor

http://dx.doi.org/10.1016/j.ibiod.2015.12.028
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236 S. Cappello et al. / International Biodeterioration & Biodegradation 110 (2016) 235e244
technology present an advantage over other methods, because it
reduces or eliminates the problems arising from limiting factors or
variables such as oxygen supply, temperature variations, pH and
specific nutritional formulations (Van Hamme et al., 2003). The use
of membrane filtration processes is considered a promising method
for the separation of emulsions (Leiknes et al., 2006; Guo et al.,
2007; Bouju et al., 2008) but the attention is mainly paid to the
possibility of removal of dissolved organic compounds and
biomass, which must be removed from the crude liquid waste to
limit membrane's fouling and clogging. One of the advantages of
MBR systems resides in their ability to operate with the values of
cell retention time (SRT, sludge retention time) and concentrations
of total suspended solids (TSS) much higher than those of the
conventional activated sludge process, and accordingly with
significantly lower values of the sludge load (F/M) (Torregrossa
et al., 2010). Another advanced treatment technology is the Mov-
ing Bed Biofilm Reactor (MBBR), in such systems the biomass can
grow both as suspended flocs and as biofilm, allowing a higher total
biomass concentration in the reactor. Recently, MBR and MBBR
have been employed jointly (known as BF-MBR), replacing the
secondary settler by means of MBR (Di Trapani et al., 2014). These
conditions are likely to result in removal rates of hydrocarbons
refractory considerably higher. The bioreactors employed in
bioremediation strategies are nothing more than tanks in which
living organisms carry out their biological reactions. A reactor
should be easy to maintain and operate (Evangelho et al., 2001),
and should be able to function both in aerobic and anaerobic con-
ditions. Their efficiency is ensured by the ability of bacteria to join
on inert material, such as granular activated carbon, to generate
high biomass at interfaces (Bouwer and McCarty 1982; Teitzel and
Parsek, 2003). Through the breaking of solid aggregates and the
dispersion of insoluble supports, desorption of hydrocarbons and
contact with the aqueous phase is fostered, with consequent in-
crease of biodegradation. Various types of bioreactors are widely
used in a large variety of aerobic bioprocesses such as aerobic
fermentation, biological waste water and hydrocarbon impacted
soil/sediments treatments (Van Hamme et al., 2003). The filtration
of oil contaminated water with a porous membrane bioreactor
(MBR) is a recent advancement in bioremediation. A membrane
bioreactor combines the activated sludge process with a membrane
separation process. The operation of the reactor is similar to a
conventional activated sludge process but without the need for
secondary clarification and tertiary steps like sand filtration. Spe-
cifically, in MBR treatment, generally performed by indigenous,
water-borne micro-organisms in a managed habitat, the selection
and identification of microbial consortia with high capability to
degrade hydrocarbons is a primary step for the optimization of the
process.
During this study a laboratory biofilm-membrane bioreactors
was used to investigate the catabolic potential of natural marine
bacteria; in particular, the aim of this work was the isolation,
identification and characterization of the biotechnological potential
of adapted/selected microorganisms in a BF-MBR.
2. Materials and methods
2.1. BF-MBR system set-up
A biofilm BF-MBR pilot plant was used to carry out the experi-
mental investigations. A BF-MBR process is a combination of
moving bed biofilm reactor and membrane filtration unit (MBR) in
the same tank; in details, the biofilm grows upon carriers that move
in the reactor pushed by aeration and in this way degrades the
organic matter fed in the tank.
The pilot plant, realized in the Laboratory of Sanitary and
Environmental Engineering of Palermo University, was composed
of a 20 L bioreactor. The reactor was equipped with an ultrafiltra-
tion (UF) hollow fiber membrane module (Zee-Weed™01, with
specific area equal to 0.1 m2 and nominal porosity of 0.04 mm) and
Mutag BioChipTM carriers (active surface 3 m2 L
1) were used, with
a 17.5% filling fraction. The bioreactor was continuously aerated and
provided with level sensors that activated the feeding pump,
allowing a semi-continuous supply.
Through a suction pump, an average flux of permeate equal to
7 L m
2 h
1 was extracted from the membrane and transferred in a
apposite permeate tank, then every 5 min a fraction of permeate
was pumped back to backwash the membrane for a period of 1 min.
The suction-backwashing cycles were managed by the means of a
Programmable Logical Controller (PLC). A schematic diagram of the
pilot plant setup used, is reported in Fig. 1, while the overall oper-
ational condition are summarized in Table 1.
In this paper, the BF-MBR process was applied to treat synthetic
ship slops. The slop was obtained as a mixing of diesel oil (as only
carbon source), sodium dodecyl benzenesulfonic (SDBS, Sigma-
eAldrich, Milan; as surfactant) and (non sterile) natural seawater
based on the composition of real slop samples recovered from the
Augusta (SR) harbor. Diesel oil was the only carbon source, natural
seawater was added to ensure the presence of the inorganic nu-
trients (NH4Cl, KH2PO4) and the metal of concern in slops (PbCl2,
CuCl2, MnCl2, MnCl2 and MgSO4). In Table 2, the composition of the
synthetic slop and the main pollution parameter are reported.
2.2. Bacterial community characteristics
To monitor the total abundance of microbial population present
in the BF-MBR system in study, 500 mL of sample was taken
aseptically, using sterile bottles, (under sterile conditions) at the
end of experimental period (seven day, T7) and analyzed for mea-
sures of direct bacterial counts (DAPI count), dilution plate counts
(CFU) and Most Probable Number (MPN). During the sampling the
synthetic slop was in vigorously mixed by aeration apparatus pre-
sent in principal tank.
2.2.1. Total bacterial abundance (DAPI count)
After a short-time (3000 ) ultrasonic treatment, the total bacterial
cell counts were performed by DAPI (40 ,6-diamidino-2- phenyl-
indole 2HCl, SigmaeAldrich, Milan, Italy) staining on samples fixed
with formaldehyde (2% final concentration), according to Porter
and Feig (1980). Slides were examined by epifluorescence with
Axioplan 2 Imaging (Zeiss; Carl Zeiss Inc., Thornwood, N.Y.) mi-
croscope. Results were expressed as number of cells ml
1.
2.2.2. Plate counts
For enumeration of total heterotrophic bacteria and
hydrocarbon-degrading bacteria, cells present in MBR water sam-
ples were serially diluted in sterile physiological solution (0.2 mM
Millipore filter; Millipore, Milan, Italy) and plated (100 ml) on Ma-
rine agar 2216 medium (Difco S.p.a, Milan, Italy), ONR7a
(Dyksterhouse et al., 1995) and ONR7a added with sterilized diesel
oil (100 ml) as unique energy and/or carbon source. Culture media
and diesel oil were autoclaved separately for 20 min at 120  C. All
agar plates were incubated at 25 ± 1  C for 7 days. Results were
expressed as colony forming units (CFU) in ml (CFU ml
1).
2.2.3. Most probable number (MPN)
Hydrocarbon-degrading bacteria were enumerated by a minia-
turized Most Probable Number (MPN) method (Brown and
Braddock, 1990), slightly modified (Cappello et al., 2007). The
MPN of hydrocarbon-degrading microorganisms was determined
from the appropriate MPN tables according to American Public
 S. Cappello et al. / International Biodeterioration & Biodegradation 110 (2016) 235e244 237

Fig. 1. Configuration of theBF-MBR pilot plant used throughout this study.

Table 1
Operational condition of BF-MBR system.

Surface reactor (m2 L
1) Carriers filling ration (%) Average flow (L h
1) HRT (h) Average OD (mg L
1) Average pH

10.5 ± 0.5 17.5 ± 0.5 0,4 48 8 8.2 ± 0.3

Table 2 characterization of the isolated strains. Total DNA was extracted

(A), Principal slop features of BF-MBR used in this study. (B), values of cultivable from the bacterial strains using the CTAB method
bacteria fraction on different culture media. TOC, Total Organic Carbon; MA, Marine
Agar medium.
(Winnepenninckx et al., 1993). The bacterial 16S rRNA loci were
amplified (Lane, 1991) using the domain-specific forward primer
Synthentic slop Concentration Bac27_F (50-AGAGTTTGATCCTGGCTCAG-30) and the universal
(A) Diesel Oil (mg L
1) 170 reverse primer Uni_1492R (50-TACGYTACCTTGTTACGACTT-30). The
Surfactant SDBS (mg L
1) 17 amplification reaction was performed in a total volume of 50 ml
Mineral Salt medium (mg L
1) 112
containing 1 solution Q (Qiagen, Hilden, Germany), 1 Qiagen reac-
TOC (mg L
1) 21
TC (mg L
1) 50 tion buffer, 1 mM of each forward and reverse primer, 10 mM dNTPs
IC (mg L
1) 29 (Gibco, Invitrogen Co., Carlsbad, CA), and 2 U of Qiagen Taq poly-
TN (mg L
1) 31.4 merase (Qiagen). Amplification for 35 cycles was performed in a
GeneAmp 5700 thermocycler (PE Applied Biosystems, Foster City,
(B) pH 7.9 ± 0.5
CA, USA). The temperature profile for PCR was 95 C for 5 min (1
Temp ( C) 20 ± 1
CFU (MA, cell mL
1) 3  107 cycle); 94  C for 1 min and 72  C for 2 min (35 cycles); and 72  C for
CFU (ONRA7a, cell mL
1) 5  105 10 min after the final cycle (Troussellier et al., 2005). The amplified
CFU (ONRA7a þ Diesel Oil, cell mL
1) 4  104 16S rRNA fragment was sequenced using Macrogen Service (Korea).
The similarity rank from the Ribosomal Database Project (RDP)
(Maidak et al., 1997) and FASTA Nucleotide Database Queries were
Health Association (A.P.H.A. 1992). used to estimate the degree of similarity to other 16S rRNA gene
sequences. Phylogenetic analysis of the sequences was performed
2.3. Isolation and characterization of bacteria in study as previously described by Yakimov et al. (2006).

After isolation, bacterial strains were characterized by 16S rRNA
sequencing. All strains in study were screened for biosurfactant
production, emulsification activity and cellular adhesion.
2.3.1. Isolation of bacteria
Individual colonies obtained from enumeration of cultivable
bacteria, in ONR7a medium with diesel oil, were selected and pu-
rified by repeatedly streaking under identical growth conditions.
The ONR7a mineral medium was used for the isolation of oil-
degrading bacteria. ONR7a media were supplemented with sterile
1% (v/v) diesel oil as the sole carbon source. Phenotypically
different colonies obtained from the plates were purified and
transferred to fresh medium. The procedure was repeated, and only
isolates exhibiting growth on diesel oil were stored in stock me-
dium with glycerol at 20 ± 1  C for further characterization
(Hassanshahian et al., 2012).
2.3.2. Molecular identification
Analysis of the 16S rRNA gene was performed for the taxonomic
2.4. Screening of biosurfactant production and emulsification
activity of bacteria
Biosurfactant production was screened by two distinct
methods: (i) the Drop collapse test, and ii) in the oil spreading
method. In the Drop collapse test which 7 ml of mineral oil was
added to each well of a 96-well microtiter plate. Covered plates
were equilibrated for 24 h at room temperature before adding
culture supernatant. To induce biosurfactant production, bacterial
isolates were grown at 25 ± 1  C for 1 week on rotator shaker
(180 g) on liquid TSB medium (SigmaeAldrich, Milan) supple-
mented with 2% glucose. To test the presence of biosurfactant, 20 ml
of the culture supernatant was added on the oil surface in the
microtiter well. Sodium dodecyl sulfate (SDS, SigmaeAldrich,
Milan) with the sterile distilled water was used as control sus-
pension (Mahjoubi et al., 2013). In the oil spreading method 20 ml
of distilled water was added to an empty Petri dish followed by
addition of 10 ml of crude oil to the surface of the water. Ten mi-
croliters of bacterial culture on TSB medium were then added to the
238 S. Cappello et al. / International Biodeterioration & Biodegradation 110 (2016) 235e244
 S. Cappello et al. / International Biodeterioration & Biodegradation 110 (2016) 235e244
oil surface. The diameter of the clear zone around the bacterial
suspension corresponds to the biosurfactant concentration
(Youssef et al., 2004).
The emulsification index (E24), of different bacterial culture, was
determined by adding 2 ml of hydrocarbon (benzene, C6H6 and
diesel) to same volume of culture. After mixing with a vortex for
2 min and leaving to stand for 24 h, the E24 index is given as per-
centage of height of emulsified layer (in millimeters) divided by
total height of the liquid column (in millimeters) (Iqbal et al., 1995;
Cappello et al., 2012).
2.5. Adhesion to polystyrene
Cellular adhesion was measured by Bacterial Adherence To
Hydrocarbons (BATH) test. The BATH assay was performed as
described in Rosenberg (1984) and Cappello & Guglielmino (2007)
4 ml of the bacterial suspension (5  107 cells ml
1) was vigorously
mixed with 1 ml of n-hexadecane for 2 min. The water and organic
phase were allowed to separate for 15 min and 0.1 ml of the
aqueous phase was sampled and the amount of cells determined
spectrophotometrically (OD600nm, Shimadzu UV-160, Japan). The
hydrophobicity was expressed as:
(a-b)/a  100
where “a” is the initial cell concentration in the aqueous phase and
“b” is the concentration in the aqueous phase after partitioning.
Samples were run in duplicate and the results are presented as
mean values.
2.6. Capability of growth and hydrocarbons degradation
According to data obtained from biosurfactant production,
emulsification activity of bacteria and cellular adhesion, four-teen
(14) bacteria were selected and further screened, for growth on
crude oil and hydrocarbon degradation.
2.6.1. Growth
Bacterial isolates were grown at 25 ± 1  C for 1 week on rotator
shaker (180 g) on ONR7a medium whit sodium acetate and ONR7a
whit diesel oil, respectively. The growth of the isolates was
routinely assessed indirectly by measuring the turbidity (OD600nm)
using a UVevisible spectrophotometer (Shimadzu UV-160, Japan)
as indicated in Hassanshahian et al. (2012).
2.6.2. Hydrocarbon analysis (HC degradation test)
The residual Total Extracted and Resolved Hydrocarbons
(TERHC) and their derivates in different bacterial cultures were
analyzed after 10 days of incubation. The total volume of the bac-
terial culture was used in the hydrocarbon extraction. Hydrocar-
bons were analyzed using high-resolution GCeMS and quantified
according to previously described protocols (Denaro et al., 2005).
After acidification, TERHC were extracted from bacterial cultures at
ambient temperature on shaking table using dichl oromethane-
seawater (10% v/v). This procedure was repeated three times, and
the CH2Cl2 phases were combined and treated with anhydrous
Na2SO4 to remove the residual water. Extracts were concentrated
by rotary evaporation (Rotavapor model R110; Buchi Labortechnik
AG, Switzerland) at ambient temperature, followed by evaporation
under a stream of nitrogen. The residue was then dissolved in a
Fig. 2. Phylogenetic tree based on 16S rRNA gene sequences for bacterial strains isolated from the MBR treatment systems used in this study bilge water used in this study.
Percentages of 100 bootstrap re-sampling that supported the branching orders in each analysis are shown above or near the relevant nodes (, values > 85%; , values < 85%).
Evolutionary distance is indicated by vertical lines; each scale bar length corresponds to 0.05 fixed point mutations per sequence position.
239
solution containing heptamethyl-nonano as an internal standard
(79 ng ml
1). The TERHC composition was analyzed by high-
resolution GCeMS using a PerkineElmer TurboMS autosystem 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)]. The samples were quantified according to previously
described protocols (Hassanshahian et al., 2012).
3. Results and discussion
3.1. Bacterial community characteristics
The analysis related to microbial abundance showed that total
bacterial abundance (DAPI count) is significantly higher
(4.1  104 cells ml
1) than that related to the cultivable bacteria
fraction on different culture media (Marine Agar, ONR7a and
ONR7a with addition of diesel as only carbon and energy source).
Measure of MPN count showed values of 2.7  102 MPN ml
1
(Table 1).
3.2. Bacterial isolation and taxonomic identification
Culture-dependent approach based on the isolation and iden-
tification of hydrocarbon degrading bacteria allowed us to isolate
42 strains from contaminated samples of BF-MBR system. The
molecular identification of the isolates was performed by
sequencing the 16S rRNA gene and comparing the sequences to the
16S rRNA database; the results are shown in Fig. 2.
Three phylogenetic groups could be recognized: Bacteroidetes-
Chlorobi (4.6%), Actinobacteria (32.5%) and Proteobacteria. This
latter was the most dominant one, with 69.7% of the isolates, and
was represented by two subclasses, Alphaproteobacteria and the
most abundant Gammaproteobacteria (37.2%). Our results are in
line with those of Mahjoubi et al. (2013) who have indicated that
these microorganisms are naturally detected in marine environ-
ments polluted with hydrocarbons and could play an important
ecological role in bioremediation.
Alphaproteobacteria group is known to be very heterogeneous
and characterized by the ability to degrade the polyaromatic hy-
drocarbons (Mahjoubi et al., 2013). In our study, isolates related to
Alphaproteobacteria were identified exclusively as Erythrobacter
(Erythrobacteraceae). Two different strains, Erythrobacter citreus
and Erythrobacter vulgaris were detected, showing about the same
percentage abundance (14%). Erythrobacter is an anoxygenic pho-
totrophic bacterium (Kolber et al., 2001), that may contribute to
aromatic hydrocarbon degradation in oil-contaminated beaches
(MacNaughton et al., 1999; Chung and King, 2001; Ro € ling et al.,
2004; Beazley et al., 2012).
Gammaproteobacteria are represented by the three families of
Chromatiaceae, Alteromonadaceae and Alcanivoracaceae. Chro-
matiaceae cluster (28%), in this experiment, encompassed 12 iso-
lates high related with Rheinheimera aquimaris, with mostly 100% of
sequence identity (Fig. 2 and Table 2). Genus Rheineimera is
generically known as hydrocarbons (BTEX) degraders and non-
bioemulsifier producer that doesn't grow under anaerobic condi-
tions. Alteromonadaceae Family was represented by the unique
strain related to common oil-degrading Marinobacter genus (Head
et al., 2006), specifically, Marinobacter guinaeae. Alcanivorax die-
selolei, the only detected member of Alcanivoracaceae family, rep-
resents 7% of the entire amount. This group of marine bacteria is
240 S. Cappello et al. / International Biodeterioration & Biodegradation 110 (2016) 235e244
well known to use petroleum oil hydrocarbons as sources of carbon
and energy, and has been employed for bioremediative in-
terventions in polluted marine and coastal systems (Schneiker
et al., 2006; Cappello and Yakimov, 2010). It was reported that A.
dieselolei can produce biosurfactants (Dastgheib et al., 2011). In
addition, Alcanivorax species have clear potential in producing
biocatalysts in non-polluting industrial processes and to act as a
biosensor for in situ monitoring of aromatic or aliphatic compounds
(Golyshin et al., 2003).
Bacteroidetes-Chlorobi group was represented just with two
isolates belonging to Muricauda beolgyonensis (Flavobacteriaceae,
98% of sequence identity). Lastly, the Actinobacteria group included
families of Microbacteriaceae and Nocardiaceae, in this study rep-
resented respectively by Microbacterium chocolatum (4.6%) and
Rhodococcus erythropolis (27.9%). Even if the role of Gram-positive
bacteria in hydrocarbon biodegradation is less known, both of
these organisms have been identified as promising candidates for
biotechnological applications (Aniszewski et al., 2010; Pacheco
et al., 2010). It has long been known that some HCB species, such
as Alcanivorax borkumensis and Pseudomonas aeruginosa, are able to
degrade hydrocarbons, alone, with an optimal yield.
But it is also true that, in most cases, the environmental con-
ditions and the extreme variety of pollutants involve the formation
of bacterial consortia. These associations of microorganisms are
more efficient, because individual species do not possess all the
enzymatic systems to degrade pollutants until their complete
mineralization. So, although the ability to degrade hydrocarbons
was not found for all bacterial strains isolated in this study, it is
conceivable that all together contribute to the degradation of the
pollutants present in their own medium (McGenity et al., 2012).
This would explain why, in the present study, some bacterial strains
isolated from polluted water in a BF-MBR, don't grow selectively on
minimal medium (ONR) supplemented with diesel oil. In fact, of the
42 bacterial isolates in study, only 18 have demonstrated ability to
grow on mineral medium with the addition of hydrocarbons. These
isolated are largely attributable to the group of Actinobacteria (R.
erythropolis), while among the Gamma Proteobacteria more
diversified growth were found among species A. dieselolei and a
strain (MBR-H02) related to R. aquimaris. Lastly, E. vulgaris was the
only member of the Alpha Proteobacteria to grow on ONR7a me-
dium plus oil. This data could be interpreted as a provision of the
strains, to tolerate the presence of hydrocarbon compounds in the
medium, which does not involve their active degradation capacity,
which could be specifically expressed in their collective synergy.
3.3. Biosurfactant production, emulsification activity and cellular
adhesion
Many bacteria are able to produce a different kind of surface
active agents, known as bio-surfactants, which increase the cell
adhesion to the substrate (Banat, 1995). The action mechanism of
bio-surfactants lies in their accumulation at the interface of
immiscible compounds, reducing the surface tension and thereby
increasing their surface area; this leads to a higher bioavailability
that ease the degradation of the pollutants (Batista et al., 2006).
Bio-emulsifiers belong to various classes including glycolipids,
lipopeptides, fatty acids, phospholipids, neutral lipids and lipo-
polysaccharides (Thavasi et al., 2011). The various applications of
this surfactant agents have lead to the development of rapid and
reliable methods to screen for efficient producing bacteria. In this
study, it has been verified the biosurfactants production with
different methods: the drop collapse test, the oil spreading test and
the emulsification activity (E24). The drop collapse test showed
positive results for all samples, after an hour of settling of inocula in
the wells supplemented with crude oil. This data are not aligned
with the official protocol of the test (Jain et al., 1991), which pro-
vides for the observation of emulsifiers production 1 min after the
addition of the bacterial culture. It was observed that, after a
minute, only samples MBR-B11, MBR-G10, G02-MBR, MBR-G05
showed positive results with 4 mm of drop diameter (Table 2).
These are related to A. dieselolei, E. vulgaris and R. erythropolis, well
known strains in producing biosurfactants (Qiao and Shao, 2009,
Pacheco et al., 2010; Liu and Liu, 2013), It is conceivable that the
amount of biosurfactants, in the remaining strains, was not such as
to trigger an immediate reaction. The fact remains that it is possible
to find a reference in literature that provides for the observation of
the drop formation after 1 h (Phan et al., 2013). The Oil Spreading
method was used to detect the low biosurfactant production. This
test consists in detecting the diameter of the clear zone which is
proportional to the concentration of the biosurfactant (Youssef
et al., 2004). The results revealed that the 83% of the isolates
were positive. This is in agreement with those obtained by Mahn-
jubi et al. (2013). Biosurfactant production is sometimes detected
by measuring emulsification (Van Dyke et al., 1993; Makkar and
Cameotra, 1998). The screening of emulsifier potential has indi-
cated the presence of such activity in all the isolates, revealing a
correlation between biosurfactants production and emulsifying
activity. In the subclass of the Alpha Proteobacteria, E. citreus has
proved to be the most active strain, with 48.5% of the emulsification
(Table 3). Among the fraction Gamma Proteobacteria, the E24 test
showed contrasted results. Is well known that hydrocarbonoclastic
strains as A. dieselolei actively produce biosurfactants (Qiao and
Shao, 2010). Yet the results obtained by the two related strains
were not among the highest. Standing out instead the values ob-
tained from R. aquimaris, whose production of biosurfactants has
never been investigated extensively; a reference mentions it as
non-emulsifiers producer. For Bacteroidetes-Chlorobi group, the
two strains related to M. beolgyonensis, for which no data relate to
bioeemulsifier activity have been found in the literature, exhibited
values of approximately 67%. This suggests that this species may
find use in biotechnology field. The emulsifying activity was also
found in significant percentages for Gram-positive bacteria detec-
ted in the source array (Membrane bioreactor). M. chocolatum
showed average values of 49%, in agreement with the study of
Aniszewski et al. (2010) that, for the first time, have demonstrated
the capacity of organisms belonging to the genus Microbacterium to
produce bio-emulsifiers, especially for the removal of heavy metals.
The same authors suggested that the presence of large amounts of
organic matter can enhance the biosurfactant production due to the
metabolic stimulation. For this, the strains well grow in a medium
(TSB) added with glucose as a carbon source for screening of bio-
emulsifier, because this substratum supports and stimulates in situ
production of bio-surfactants for remediation (Bodour et al., 2003).
Perhaps for this was not detected significative activity production
of bio-emulsifiers on minimal ONR plus-oil medium. As for R.
erythropolis, the results obtained (about 57%) are in full agreement
with other literature data (Perfumo et al., 2010; Pacheco et al., 2010;
Mahjoubi et al., 2013) that ascribe Rhodococcus as a significant
producer of surface active agents. By R. erythropolis excellent results
from all three screening tests were obtained, in both kind of media
(TSB and ONR þ oil) and the conjugation of each of these activities
gives these strains good prerequisites for the hydrocarbons
degradation (Hassanshahian et al., 2012), which makes them po-
tential candidates, either in cultures or in consortia, for bioreme-
diation experiments. A further analysis was carried out to check the
bacterial affinity for hydrophobic substrates (hexane, in this case,
also used in the test E24).
Cell-surface hydrophobicity is recognized as one of the deter-
mining factors in bacterial adhesion to the surface of hydrophobic
substances and, among the many methods to estimate it (Hamada
 S. Cappello et al. / International Biodeterioration & Biodegradation 110 (2016) 235e244 241

Table 3
Identification, biosurfactant production (Drop collapse test, Oil spreading), emulsification activity (E24), cellular adhesion (percentage of hydrophobicity, % HP) of bacteria
isolated from the membrane bioreactor (MBR) system in study. þ1 positive/- negative.

Isolate code Accession number Closest relative Sequence similarity (%) Drop collapse test (mm) Oil spreading E24 (%) HP (%)

MBR-A11 KC534412 Erythrobacter citreus 99 4 þ 48.5 56.8

MBR-F10 KC534412 Erythrobacter citreus 100 4 þ 46 56
MBR-D04 KC534412 Erythrobacter citreus 100 3 þ 47.3 54
MBR-D05 KC534412 Erythrobacter citreus 99 3 þ 48.8 50.9
MBR-E05 KJ702642 Erythrobacter citreus 100 3 þ 45 49.5
MBR-B05 KJ702642 Erythrobacter citreus 100 4 þ 47 49.5
MBR-F01 KM387388 Erythrobacter vulgaris 100 4 þ 46.2 45
MBR-F03 KM387388 Erythrobacter vulgaris 100 4 þ 43 48
MBR-D02 KM387388 Erythrobacter vulgaris 100 3 þ 42 48.2
MBR-G01 KM387388 Erythrobacter vulgaris 100 4 þ 46 58
MBR-G02 KM387388 Erythrobacter vulgaris 99 3 þ 20 71.4
MBR-B11 KJ849833 Alcanivorax dieselolei 100 4 þ 46 88.7
MBR-G10 KJ849833 Alcanivorax dieselolei 100 4 þ 48 90.3
MBR-A02 KC420682 Marinobacter guineae 100 4 þ 20 56.5
MBR-H03 HM059656 Rheinheimera aquimaris 99 3 e 55.5 44
MBR-H05 HM059656 Rheinheimera aquimaris 100 3 þ 40 45.1
MBR-B03 HM059656 Rheinheimera aquimaris 100 3 e 38 43
MBR-D03 HM059656 Rheinheimera aquimaris 100 3 e 38 43
MBR-G03 HM059656 Rheinheimera aquimaris 100 3 þ 38,2 44.5
MBR-A05 HM059656 Rheinheimera aquimaris 100 3 e 32 44.7
MBR-H01 FJ429321 Rheinheimera aquimaris 100 3 e 40 51
MBR-H02 FJ429321 Rheinheimera aquimaris 99 3 þ 42 71.7
MBR-E02 FJ429321 Rheinheimera aquimaris 100 2 þ 42.3 69
MBR-A09 EF076758 Rheinheimera aquimaris 100 4 þ 38 65
MBR-F09 EF076758 Rheinheimera aquimaris 99 4 þ 40 66
MBR-E04 EF076758 Rheinheimera aquimaris 99 4 þ 42 67
MBR-G04 NR_117844 Muricauda beolgyonensis 100 4 þ 59.2 67.2
MBR-C02 NR_117845 Muricauda beolgyonensis 100 4 þ 59 66.9
MBR-H10 JX007959 Microb. chocolatum 100 3 þ 48 39
MBR-E10 JX007959 Microb. chocolatum 100 3 e 50 43.7
MBR-G05 KM670434 Rhodococcus erythropolis 98 4 þ 60 85.7
MBR-F13 KM670434 Rhodococcus erythropolis 96 4 þ 58 78
MBR-D10 KM670434 Rhodococcus erythropolis 99 4 þ 58.5 78.3
MBR-B02 KM670434 Rhodococcus erythropolis 100 4 þ 57 76.6
MBR-B09 KM670434 Rhodococcus erythropolis 99 3 þ 56 74
MBR-F05 KM670434 Rhodococcus erythropolis 99 3 þ 54 70
MBR-F04 KM670434 Rhodococcus erythropolis 100 4 þ 56 65
MBR-A10 KM670434 Rhodococcus erythropolis 99 4 þ 56 65
MBR-B10 KM670434 Rhodococcus erythropolis 100 4 þ 55.5 67
MBR-C10 KM670434 Rhodococcus erythropolis 99 4 þ 56 65
MBR-G09 KM670434 Rhodococcus erythropolis 99 4 þ 56.4 67
MBR-H09 KM670434 Rhodococcus erythropolis 99 3 þ 55.5 67.3

et al., 2008) in this study was proposed the measure of bacterial
adhesion to hydrocarbon (BATH). As regards the Alpha Proteobac-
teria E. citreus and E. vulgaris showed values of cell surface hydro-
phobicity respectively equal to 52.8% and 54.1%. The results of BATH
test for A. dieselolei showed that the cells had very hydrophobic
surface, in accordance with Dastgheib et al. (2011). These values are
higher than those obtained by the other belonging to the group of
Gamma Proteobacteria, M.guineae (20%) and R. aquimaris (average
value of 50,2%) as expected. M. chocolatum, related to Actino-
bacteria groups, showed 41.3% of hydrocarbon adhesion. The BATH
test showed, for R. erythropolis, maximum values of 85.7%. That is in
agreement with the data provided by Yamashita et al. (2007),
although the organic solvent used was different (n-Tetradecane).
Emerges, in this way, that R. erythropolis is a species highly tolerant

Table 4
Taxonomic identification of the bacteria used in this study.

Isolate code Closest relative Genus Family Accession number Sequence similarity (%)

MBR-A11 Erythrobacter citreus Erythrobacter Erythrobacteraceae KP863902 99

MBR-D05 Erythrobacter citreus Erythrobacter Erythrobacteraceae KP863904 99
MBR-G01 Erythrobacter vulgaris Erythrobacter Erythrobacteraceae KP863907 100
MBR-G02 Erythrobacter vulgaris Erythrobacter Erythrobacteraceae KP863908 99
MBR-B11 Alcanivorax dieselolei Alcanivorax Alcanivoracaceae KP863903 100
MBR-G10 Alcanivorax dieselolei Alcanivorax Alcanivoracaceae KP863911 100
MBR-A02 Marinobacter guineae Marinobacter Alteromonadaceae KP863915 100
MBR-H05 Rheinheimera aquimaris Rheinheimera Chromatiaceae KP863913 100
MBR-H02 Rheinheimera aquimaris Rheinheimera Chromatiaceae KP863912 99
MBR-G04 Muricauda beolgyonensis Muricauda Flavobacteriaceae KP863909 100
MBR-H10 Microbacterium chocolatum Microbacterium Microbacteriaceae KP863914 100
MBR-E10 Microbacterium chocolatum Microbacterium Microbacteriaceae KP863905 100
MBR-G05 Rhodococcus erythropolis Rhodococcus Nocardiaceae KP863910 98
MBR-F04 Rhodococcus erythropolis Rhodococcus Nocardiaceae KP863906 100
242 S. Cappello et al. / International Biodeterioration & Biodegradation 110 (2016) 235e244

Fig. 3. Percentage (%) of oil present after cultivation of strains in study in ONR7a whit 0.1% of crude oil (30 ± 1  C for 1 week on rotator shaker, 180  g). Time zero, beginning of
experiment; AC, abiotic control.

Table 5 present in the crude oil. In contrast, strains CS-2, PG-26 and PG-24
Measures of growth (optical density, OD600nm) in ONR7a mineral medium whit have low n-alkane degradation abilities. For all strains, n-alkanes
sodium acetate (A) and in ONR7a mineral medium with 0.1% of diesel oil (B) of with a medium length (C12eC18) were degraded to a greater extent
strains isolated in study.
(rate of degradation > of 50%) than short (C9eC11) and long chains
Isolate code Closest relative A (OD600nm) B (OD600nm) (C19eC25) because short-chain n-alkanes are toxic to bacteria and
MBR-A11 Erythrobacter citreus 0.465 0.09 dissolve the cellular membrane and long-chain n-alkanes are solid
MBR-D05 Erythrobacter citreus 0.471 0.1 and their low solubility inhibits degradation by bacteria.
MBR-G01 Erythrobacter vulgaris 0.677 0.279
MBR-G02 Erythrobacter vulgaris 0.939 0.282
MBR-B11 Alcanivorax dieselolei 0.512 0.453 4. Conclusion
MBR-G10 Alcanivorax dieselolei 0.596 0.455
MBR-A02 Marinobacter guineae 0.998 0.267
MBR-H05 Rheinheimera aquimaris 0.653 0.535
This study represents the first holistic microbiological investi-
MBR-H02 Rheinheimera aquimaris 0.588 0.538 gation of microbial biodiversity occurring at BF-MBR system used
MBR-G04 Muricauda beolgyonensis 0.708 0.784 for recovery treatment of saline oily waste taking advantage of both
MBR-H10 Microbacterium chocolatum 0.946 0.1 molecular and cultivation techniques. A collection of forty-two (42)
MBR-E10 Microbacterium chocolatum 0.801 0.1
strains was obtained during microbiological screening of a Mem-
MBR-G05 Rhodococcus erythropolis 1.408 0.511
MBR-F04 Rhodococcus erythropolis 2.011 0.518 brane Bioreactor (BF-MBR) system was carried out. In particular, 14
strains, selected to different bacterial genera, showed one or more
metabolic traits of interest for bioremediation purposes (e.g., the
capability to grow on single hydrocarbon molecules, presence of
to different types of hydrocarbons (Bruheim, 1997; Bredholt et al.,
genes involved in detoxification systems, and traits related to the
2002).
production of biosurfactants).
Data obtained from this study confirmed the high activity of
3.4. Growth rate and crude-oil removal by the studied strains bacteria related to genera Alcanivorax, Rheinheimera Rhodococcus
and Muricauda and underline the possible application of these
All bacterial strains (Table 3) were grown in 1% diesel oil for 1 bacteria in remediation of saline oily waste water.
week with shaking. After 1 week, the levels of microbial growth Particularly interesting is the simultaneous presence of bacteria
and diesel oil biodegradation were analyzed using spectrometry- associated to the genus Alcanivorax (the most studied among the
based methods. As reported in Table 4, the strains BS, PG-12 and obligate hydrocarbonoclastic bacteria) with other bacteria able of
PG-11 exhibit highest levels of diesel-oil biodegradation, degrading degrading hydrocarbons (eg. Rhodococcus) and bacteria apparently
82%, 71% and 68% of the oil, respectively. The strains PG-26 and AS not involved in phenomena of bio-degradation. This condition
exhibit the lowest levels of oil removal (33% and 36%, respectively) leads us to assume that although the selected bacterial strains are in
among all isolates (Fig. 3) (Table 5). direct dependence on the type of pollutant and the operating
Rate of degradation of n-alkanes The percentage degradation of conditions (e.g those of the BF-MBR system) these are in close
n-alkanes (C10eC25) present in the crude oil was calculated by metabolic correlation between them and that therefore their
comparison of the gas chromatograms of the abiotic control and the development and their dominance in the presence of pollutant is a
degraded sample for each strain (Table 3). The data obtained show condition closely related to the development of the normal mi-
that strains BS, PG-3 and PG-12 degraded almost all n-alkanes crobial community. In any case bacteria isolated appear to be
 S. Cappello et al. / International Biodeterioration & Biodegradation 110 (2016) 235e244
extremely versatile and potentially applicable to all those engi-
neering systems (eg. BF-MBR) or natural (polluted environments)
in which these could be applied to recovery processes.
Conflicts of interest
Authors have declared that no competing interests exist.
Acknowledgments
This work was supported by grants of 1) University of Palermo
and 2) National Counseil of Research (CNR) of Italy and by: i) Italian
Project PRIN2010-2011 “La “System Biology” nello studio degli
effetti di xenobiotici in organismi marini per la valutazione dello
stato di salute dell'ambiente: applicazioni biotecnologiche per
potenziali strategie di ripristino”; ii) National Operative Project
PON R&C 2007-2013 “Sviluppo di Tecnologie Innovative per il
trattamento dei rifiuti liquidi della navigazione finalizzate alla
Tutela dell’Ambiente Marino” (STI-TAM); iii) National Operative
Project PON R&C 2007-2013 “Sviluppo di tecnologie innovative per
la Sostenibilit
a Energetica ed Ambientale di cantieri nautici ed aree
Portuali” (SEA-PORT).
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