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A Novel Bioremediation Technique for Petroleum

Hydrocarbons by Bacterial Consortium
Immobilized on Goethite-chitosan Nanocomposite

H.S. El-Sheshtawy, D. Aman & H. N. Nassar

To cite this article: H.S. El-Sheshtawy, D. Aman & H. N. Nassar (2021): A Novel Bioremediation
Technique for Petroleum Hydrocarbons by Bacterial Consortium Immobilized on Goethite-
chitosan Nanocomposite, Soil and Sediment Contamination: An International Journal, DOI:
10.1080/15320383.2021.1916737

To link to this article: https://doi.org/10.1080/15320383.2021.1916737

Published online: 22 Apr 2021.

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SOIL AND SEDIMENT CONTAMINATION: AN INTERNATIONAL JOURNAL
https://doi.org/10.1080/15320383.2021.1916737

A Novel Bioremediation Technique for Petroleum

Hydrocarbons by Bacterial Consortium Immobilized on
Goethite-chitosan Nanocomposite
H.S. El-Sheshtawya, D. Amanb, and H. N. Nassara
a
Biotechnology Lab, Egyptian Petroleum Research Institute, Nasr City, Egypt; bRefining Department, Egyptian
Petroleum Research Institute (EPRI), Nasr City, Egypt

ABSTRACT KEYWORDS
Environmental pollution by petroleum hydrocarbon is one of the Bacterial consortium;
significant concerns of the contemporary world. This paper deals bioremediation; chitosan;
with the relevant environmental issues concerning the oil pollution immobilization;
of the petroleum industry. There is a need for further development of nanocomposite
sustainable remediation technologies. Nineteen petroleum-degrading
bacteria were isolated from an oil-polluted soil in the Suez oil proces­
sing company in Egypt. However, two bacterial species showed the
highest growth rate of oil hydrocarbons. These isolates were identified
by 16S rDNA gene sequence analysis into Flavobacterium johnsoniae
BS1 (NCBI Gene Bank Accession no. MT740243) and Shewanella baltica
BS2 (NCBI Gene Bank Accession no. MT740157). Ionic liquids prepared
the goethite-chitosan nanocomposite assisted synthetic hydrothermal
method. The antibacterial activity of synthesized nanocomposite on
BS1and BS2 was determined. The two oil-degrading bacterial strains
were immobilized onto the surface of the prepared nanocomposite.
Pure and bacterial consortium studied the bioremediation process
without/with nanocomposite. The remaining oil after bioremediation
was extracted. Study results demonstrated that the affinity between
the surface of bacterial cells and the prepared nanocomposite was
investigated using scanning electron microscopy (SEM). From the
antibacterial activity test of nanocomposite, there is no toxic effect
on the two biodegrading microorganisms. The remaining oil after
biodegradation showed that immobilized bacterial consortium
achieved the maximum degradation efficiency 93.32% after 3 days of
incubation. Biodegradation of different polyaromatic hydrocarbons
was also studied, and immobilized bacterial consortium showed
good biodegradation capabilities compared to those of free and
pure cells. The nanocomposite catalyst increases the microbiological
reaction rates by stimulating the activity of microbes during the bio­
degradation process. With this excellent biodegradation efficiency,
these results suggested that the immobilized BS1 and BS2 consortium
entailed high potential treatment for industrial applications for the
biodegradation of oil-contaminated soil.

CONTACT H.S. El-Sheshtawy hudaelsheshtawy@yahoo.com Biotechnology Lab, Egyptian Petroleum Research

Institute, Nasr City, Egypt
© 2021 Taylor & Francis
2 H. S. EL-SHESHATWY ET AL.

Introduction
Oil pollution results from exploration, production, and transportation activities in oil
industries leading to environmental pollution with substantial risk to the living flora and
fauna (Shahian, Emtiazi, and Cappello 2012). Crude oil is a naturally occurring complex
mixture of hydrocarbons, including polycyclic aromatic hydrocarbons (PAHs) and non-
hydrocarbon compounds, which possess measurable toxicity toward living systems. The
toxicity effect of crude oil products differs greatly, according to their composition, concen­
tration, environmental factors, and the biological state of the organisms at the time of the
contamination (Obire and Anyanwu 2009). Conventional remediation techniques, includ­
ing physical, chemical, and thermal treatments in most cases, are not economically feasible
and often generate secondary contamination. The biological remediation method has been
proved to be economical, versatile, and efficient for the cleanup of petroleum pollutants
with many advantages. Microorganisms can synthesize enzymes that can catalyze reactions
in which contaminants degrade and turn into simpler compounds and less toxic (Rizi et al.
2017). Bioremediation requires a long treatment time. It may not be effective if high
contaminant concentrations that are toxic to microorganisms exist.
The incorporation of nanomaterials and bioremediation has a great chance to be
efficacious and sustainable. Therefore, nanotechnology for bioremediation is a recently
emerging field. In addition, it plays an essential role in addressing innovative and dynamic
solutions to a wide range of environmental problems. The employment of nanomaterials for
remediation is more cost-effective and rapid than current conventional methods due to
their enhanced surface area, transport properties, and sequestration characteristics
(Subramaniam et al. 2019). Generally, nanoparticles can absorb the maximum amount of
pollutants due to large surface area and high surface energy. Besides, it catalyzes the
catabolic activity of biodegrading microorganisms at a faster compared to free cells, thus
reducing energy consumption during degradation or preventing the release of contami­
nants (Shin and Cha 2008).
Chitin is a biopolymer that includes a linear sequence of monomeric sugars with
N-acetylglucosamine. Chitin has considered a similar cellulose structure; the structural
difference is acetylamino groups in the chitin molecule that replace the hydroxyl groups
of carbon number two. The deacetylation of chitin produces chitosan by strong alkaline
aqueous solutions. During the reaction, the N-acetyl groups linked to chitin are lost,
forming D-glucosamine units that have an amine group. Chitosan is composed of partially
deacetylated polymeric chains with a deacetylation degree above 50%. Chitosan has been
widely used in several studies as an immobilizing agent for biodegrading microorganisms to
improve and stimulate the removal of organic pollutants, such as oil and its derivatives
(Bashan and Bashan 2010). Chitosan has been reported as a natural, low-cost alternative in
contaminated waters containing phenolic compounds (Milhome et al. 2009).
Soil contamination by petroleum hydrocarbons in the Suez oil processing company,
Egypt (SOPC) has the potential to threaten the surrounding natural environment/or
adversely affect human health. Suez Oil Processing Company is located in Suez, Egypt, on
the Gulf of Suez, at the Suez Canal entrance, which connects the Red Sea with the
Mediterranean Sea (Figure 1). Study’s main objective is to remedy petroleum soil pollution
in SOPC through the application of a new and innovative method based on biological
treatment and eco-friendly. This study aimed to isolate and characterize oil-degrading
 SOIL AND SEDIMENT CONTAMINATION 3

Figure 1. Represent position of Suez oil processing company (SOPC).

bacteria from petroleum-contaminated soil in (SOPC) and determine the degradation

capacity of the studied oil sample by bacterial consortium, and axenic cultures are studied.
Additionally, the effect of green synthetic nanocomposite as a carrier on the biodegradation
process with the free and immobilized bacterial consortium is specified.

Materials and methods

Soil sample
A soil sample (200 g) from surface soil (10 cm depth) was collected from the shoreline in the
Suez oil processing company, Egypt (SOPC), which was contaminated with crude oil. This
sample was transferred into sterile bottles using a sterile spatula for microbiological quality
determination and stored in the icebox to avoid contamination.

Crude oil sample

The crude oil used in the present work was supplied from Esh El-Malaha, Alexandria,
Egypt.

Physicochemical properties of crude oil sample

The physicochemical parameters of the crude oil sample obtained from Esh El-Malaha,
Egypt were studied. Density and specific gravity were determined according to ASTMD-
1298, and its API gravity was determined. Pour point following ASTM D-97 was measured.
4 H. S. EL-SHESHATWY ET AL.

Flash point in conformity with ASTM D-93 and also viscosity ASTM D-445 were recorded.
The sulfur content was calculated according to ASTM D-4294.

Preparation of goethite-chitosan composite

In the typical synthesis procedure of goethite-chitosan composite prepared via two
sequential steps: first, 1.0 M of FeCl3 and 3.0 M of NaOH were added to 25 mL of
deionized water. Then, 0.8 gm of methyl octyl imidazolium chloride was added to the
previously mentioned homogeneous solution. Second, adding 1.5 wt% chitosan in 2.0 v/v
% acetic acid to the above solution. The total solution was stirred for 2 h, then transferred
into a 50 mL autoclave, sealed, and heated to 150°C. The autoclave was cooled to room
temperature naturally. The resultant product was washed with distilled water and acetone
several times until the solution was neutral. The final products were dried at 100°C for
3 hours.

Characterization of the prepared nanocomposite

The prepared nanocomposite phases were examined via an X-ray diffractometer (XRD,
Shimadzu XD-l) with a Cu Ka radiation at a 2θ range of 10–60⁰ with a scanning rate of 5⁰
min−1. Morphology and particle size were determined by a high-resolution transmission
electron microscope (TEM-JEOL JEM 2100); dispersive X-ray spectroscopy (EDX) asso­
ciated with TEM device was employed to analyze the morphology and elemental composi­
tion of the adsorbents.

Isolation and identification of petroleum hydrocarbon degrading bacteria

Isolation of oil-degrading bacteria (ODB)
Two grams of oil-contaminated soil sample was added in a flask containing 200 ml of
sterile water, which was then shaken at 150 rpm for 30 min. Then, 0.1 ml of the flask was
loaded onto Luria-Bertani (LB) medium plates and incubated for 1 day at 30°C. The
different microbial colonies were transferred onto the new LB medium again for separa­
tion and purification. The constituents of LB medium (g/l): tryptone, 10, yeast extract, 5
and NaCl, 10 (Zheng et al. 2009). Bushnell Hass Mineral Salts Medium (BHMS) contains
the following (g/l): KH2PO4, 1; K2HPO4, 0.2; MgSO47H2O, 0.2; CaCl2, 0.02; NH4NO3, 1;
NaCl, 2; and 2 droplets of 60% FeCl3. The mineral salt media (MSM) (g/L): 0.2 MgSO4,
0.02 CaCl2, 1.0 KH2PO4, 1.0 K2HPO4, 1.0 NH4NO3, and 0.05 FeCl3. The pH of both types
of medium was adjusted to 7. This synthetic media was used for the isolation of crude oil-
degrading bacterial isolates. The BHMS (Bushell-Hass mineral salt) and MSM media were
supplemented with 2% (v/v) crude oil (Esh El-Malaha crude oil) as the sole source of
carbon and energy. The soil sample (1 g) was added to 100 ml of the two different media
types separately. The flasks were incubated for 7 days at 30 C, 150 rpm. Then, 10 ml
aliquots were transferred to fresh media. After a series of four further subcultures,
inoculums from the flask were taken, and ostensibly different colonies were purified on
the two different agar media for 3 days of the incubation period. The bacterial isolates
exhibited pronounced growth on crude oil were selected for further characterization (El-
Sheshtawy et al. 2017).
 SOIL AND SEDIMENT CONTAMINATION 5

Selection of the most promising bacterial strain

The most promising oil-degrading bacterial isolates (BS1 and BS2) were selected for
upcoming studies due to their higher growth on MSM supplemented with crude oil as
a sole carbon source.

Morphological observation
The colonies’ different morphological characteristics were observed, such as colony color,
shape, size, and elevation, and Gram staining was also performed (Yan et al. 2013).

Molecular identification of the most promising bacterial strain

The selected degrading bacterial isolates (BS1 and BS2) were identified using 16s rDNA
in Sigma Scientific Services Co., Egypt. The bacterial cells were harvested through the
enrichment medium up to 2 × 109 cells. DNA was extracted using the protocol of Gene
Jet genomic DNA purification Kit (Thermo) (Sigma Scientific Services Co., Egypt). To
amplify the 16S rDNA genes, a polymerase chain reaction (PCR) was achieved using
(5ʹ-AGA GTT TGA TCC TGG CTCAG-3ʹ) (5ʹ-GGT TAC CTT ACG ACT T-3ʹ) as
forward and reverse primers, respectively. The GeneJET™ PCR Purification Kit was used
for the cleanup of PCR to the PCR product. A 45 µl of binding buffer was added to the
complete PCR mixture. This mixture was then thoroughly transferred from step 1 to
the GeneJET™ purification column. After that, the mixture was centrifuged for 30–60
s at >12000 xg, and then the flow was discarded. A 100 µl wash buffer was added to the
GeneJET™ purification column, centrifuged for 30–60 s, neglected the flow-through, and
place the purification column back into the collection tube. The mixture was centri­
fuged at an empty GeneJET™ purification column for an additional 1 min to completely
remove any residual wash buffer. The purification column was transferred to a clean
1.5 ml microcentrifuge tube. A 25 µl of elution buffer was then added to the column
membrane center, which then centrifuged for 1 min, discarded the column, and stored
the purified DNA at −20°C. After the PCR products’ purification, the positive clone’s
DNA sequence was subjected to a similarity search BLAST on the NCBI website (http://
www.ncbi.nlm.nih.gov) and deposited into GenBank. Many relevant 16S rDNA gene
sequences with validly published names were selected as references from the Gen-
Bank.

Antimicrobial activity of the synthesized nanocomposite on the selected bacterial

strains
The antibacterial activity of the synthesized nanocomposite on the most promising bacterial
strains was determined using the agar well diffusion method (Rayn et al. 1996). The
bacterial strains were grown on a nutrient broth medium at 30 C for 18 h. After that, the
bacteria were homogeneously diffused into individual plates; wells of 3 mm diameter were
made on the agar using a cork borer. Four different nanocomposite concentrations at (100,
250, 500, and 1000 μg/ml) were separately poured in each well. Meanwhile, the antibiotic
disks (gentamicin and ampicillin) were used as controls. The antimicrobial activities were
determined by measuring the zone of inhibition’s diameter after a 24 h incubation period
(Bauer et al., 1966). The data were taken out in triplicate, and the zone of inhibition was
recorded.
6 H. S. EL-SHESHATWY ET AL.

Preparation of bacterial consortium

The oil degradation experiment was done with the individual bacterial strains (BS1 and
BS2) and their combination as a bacterial consortium. The initial inoculation quantity was
10 mL bacterial suspension (3 g wet weight cells mass per unit volume (WCW/L)) of each
set according to the principle of equal quantity.

Preparation of nanocomposite immobilized cells

Nanocomposite immobilized cells were prepared according to Ansari et al. (2009); Li et al.
(2009). Biodegrading bacterial isolates were grown in LB media up to the late-exponential
phase and harvested by centrifugation at 10,000 rpm for 15 min. The cell pellets were washed
twice with saline (8 g/l) to remove the residuals from the cell wall surfaces. The cells solution
concentration was about 3 g wet weight cells mass per unit volume (WCW/L). The pure and
mixed bacterial cells were immobilized on nano-composite as follows: the MSM was divided
into two parts separately. These two parts were mixed to keep the total reaction mixture
volume constant at 50 ml. Part a: 100 mg nano-composite in 40 ml MSM was sonicated for
15 min to better disperse the nano-composite using a Cole Parmer Ultrasonic Homogenizer
(model 8890, Germany) then sterilized by autoclaving for 20 min at 121°C. Part b: 10 ml of
sterile MSM with 2% (v/v) crude oil was inoculated by the pure and mixed bacterial cells
(about 3 g WCW/L). After mixing these two parts, the mixture was stirred at 50 rpm for 2 h to
allow the adhesion of biodegrading bacterial isolates on the surface of the nanocomposite.

Scanning electron microscopic (SEM) examination of immobilized cells

The nanocomposite and immobilized cells were firstly fixed with 2.5% glutaraldehyde for 2
h, in order to carry out SEM observation. Then, they were dehydrated for 30 min by a series
of gradually increasing ethanol concentrations: 10%, 30%, 50%, 70%, 80%, 90%, and 100%,
respectively. The morphology of immobilized bacterial cells was observed using scanning
electron microscopy Quanta 250 FEG according to a standard procedure with an accelerat­
ing voltage of 30 kV (Han et al., 2018).

Crude oil biodegradation efficiency of free and immobilized of the selected bacterial
strains
The strains were cultured in LB broth medium for 30°C up to the late-exponential phase
and the initial optical density was adjusted to A600 ≈ 1.5. The free and immobilized bacterial
consortium (BS1 and BS2) and individual cells were inoculated into a 250 ml Erlenmeyer
conical flasks. Each flask contains 100 ml of sterile MSM with 2% v/v crude oil (Liu,
Jacobson, and Luthy 1995). The experiment was carried out in triplicate, and un-
inoculated flasks were used as controls. All the flasks were incubated at 30°C at different
time intervals (24, 48, and 72 h) at 150 rpm and pH 7.5. The residual crude oil samples were
extracted from different cultural microcosms for gravimetrically and chromatographic
analyses (El-Sheshtawy et al. 2017).
 SOIL AND SEDIMENT CONTAMINATION 7

Analysis of crude oil biodegradation

At the end of the incubation period, the residual oil was extracted from each flask using
the US-EPA Gravimetric Method (1999). Briefly, a sample containing oil was collected
in a clean 500-mL separator funnel. The pH was brought down to ≤2 by adding 6 N
HCl to hydrolyze oil and prevent sodium sulfate interference. Oil in the samples was
exhaustively extracted twice with carbon tetrachloride. Then, the carbon tetrachloride
layer was dehydrated by passage through a funnel containing anhydrous sodium sulfate
and collected in a dry, pre-weighed receiving flask. The remaining solvent in the
solution was then evaporated in a rotary evaporator and by nitrogen gas with heating
at 70 C. Finally, the flask containing the concentrated oil was cooled and dried in
a desiccator. The residual oil recovered was determined gravimetrically (hiCohen 1957;
El-Sheshtawy et al. 2017). Reduction in oil content (%) was calculated as: (A – B)/A
x 100, where A is the initial weight of crude oil and B is the residual crude oil weight.

Determination of n- and iso-paraffins

The degradation of residual hydrocarbon components was quantified chromatographically
via capillary gas chromatography (GC) using Agilent 6890 plus; the characterization Gas
chromatography was as described by in El-Sheshtawy et al. (2014). The peak area of each
resolved component (consisting of either n- and iso-paraffins) was detected individually.
While the unresolved complex mixtures (UCMs) consisted of non-n-paraffins fundamentally
cycloparaffins and aromatics with long side chains and were detected only as a total hump.

Determination of 16 polyaromatics hydrocarbons (PAHs)

The residual concentrations of 16 PAHs (listed by US-EPA) were estimated using HPLC.
Ten microliters of each sample were injected into HPLC Waters (600E, USA) analytic
System, using C18 column, and the flow rate was set at 1 ml min. The mobile phase was
composed of water: acetonitrile (1:1). All the PAH compounds selected were detected by
UV absorbance at 254 nm (Chen et al. 2006).

Results and discussion

Physicochemical properties of crude oil sample
Different physicochemical parameters of the crude oil sample obtained from Esh El-Malaha,
Alexandria, Egypt were studied as shown in Table 1. Density, specific gravity, and API
gravity were determined at 0.900 g/cm3, 0.901 and 25.5, respectively. Pour point was
calculated at 6°C. Flash point was measured at 58°C and viscosity was determined at
23.03 cSt. The sulfur content was recorded at 1.49%. The crude oil sample was fractionated
into saturates, aromatics, resins and asphaltenes. It is used as an indication for the
characterization and identification of the Esh El-Malaha sample through its hydrocarbon
type’s distribution. Table 1 shows that saturates content at 55.653%. Meanwhile, the
aromatic hydrocarbons content was detected at 19.093%. In addition, resin components
are considered as a polar fraction. The value of resins contents were calculated at 17.840%.
Asphaltene was observed at 7.414%, asphaltene is a significant fraction of the higher
molecular weight consisting of hydrocarbons, oxygen, sulfur, and nitrogen compounds
(Odebunmi, Ogunsakin, and Iiukhor 2002).
8 H. S. EL-SHESHATWY ET AL.

Table 1. Physicochemical characteristics of Esh El-Malaha crude oil

sample.
Properties ASTM method Results
Density at 15°C ASTM D-1298 0.900 g/cm3
Specific gravity at 15°C ASTM D-1298 0.901
API gravity ASTM D-1298 25.5
Pour point ASTM D-97 6°C
Flash point ASTM D-93 58°C
Viscosity at 40°C ASTM D-445 23.03 cst
Sulfur content ASTM D-4294 1.49%
Components analysis (wt %) ASTM D-2549
Total Saturates 55.653
Total Aromatics 19.093
Total Resins 17.840
Asphaltene fraction 7.414

Gas chromatography analysis of crude oil sample

The GC analysis of saturates crude oil shows many peaks over the hump; these peaks
represent the paraffinic hydrocarbon (iso- and n-alkane). The hump or UCM represents the
heavy non-eluted compounds. The crude oil studied sample has an iso-component besides
the normal one starting from C11 to max carbon number C37. The hydrocarbon distribution
of the crude oil’s saturated fraction as illustrated in (Figure 2) showed a gradual increase in
paraffinic hydrocarbons and a carbon number ranging from C11- C37.

Isolation of oil-degrading bacterial strains and identification of the selected bacteria

This study revealed that 19 bacterial strains were isolated from the shoreline in the Suez oil
processing company (SOPC), Egypt. The bacterial isolates were isolated by two different
media types (BHMS and MSM); the bacterial count was reached into 7.0 and 5.07 log count
on MSM and BHMS, respectively. Of the nineteen bacterial isolates, only two bacterial

Figure 2. Gas chromatography analysis for crude oil sample.

 SOIL AND SEDIMENT CONTAMINATION 9

strains (BS1 and BS2) were isolated from the MSM media. They were chosen as the most
promising bacterial strains according to the crude oil substrate’s growth rate (see Table 2).
Identification of the most promising biodegrading isolates (BS1 and BS2) was first done
based on their morphological and physiological properties, as illustrated in Table 3. The
bacterial strain (BS1) was found to be Gram-negative, rod-shaped with round, yellow color,
and opaque colony. In comparison, the bacterial strain (BS2) was found to be Gram-
negative, rod-shaped with round, creamy color, and opaque colony. BS1 and BS2 were
identified by 16S rDNA gene sequence analysis to be Flavobacterium johnsoniae BS1 (NCBI
Gene Bank Accession no. MT740243) and Shewanella baltica BS2 (NCBI Gene Bank
Accession no. MT740157) with a similarity of 98.64% and 98.55%, respectively. Figure 3
illustrates the phylogenetic tree reconstructed by the Neighbor-Joining method of the 16S
rDNA gene from isolates BS1, BS2, and closely related bacteria. Chaudhary et al. (2019)
reported the ability of Flavobacterium sp. to degrade petroleum oil. Joe et al. (2019) also
determined the efficiency of Shewanella sp. in the degradation of petroleum pollutants. This
is consistent with the results of this research.

Preparation mechanism of goethite-chitosan nanocomposite

Herein, goethite chitosan composite was prepared by the environmentally benign green
route via (Jaiswall et al. 2013) an ionic liquid (ILs)-assisted hydrothermal synthetic method.
This method has advantages because ionic liquid can form extended hydrogen systems in
the liquid structure with phenomena that make it like an entropic driver to prepare a big
series of nano and microstructure of inorganic materials. Also, ILs structure can self-
assemble and form well-ordered structure because it contains a hydrophilic ionic head
group and a hydrophobic organic chain that looks like the surfactant (Lian et al. 2009; Zhao
et al. 2017). For instance, micro/mesoporous silica materials were prepared using protic
ionic liquids as a model (Zhao et al. 2015). Many photocatalytic catalysts like pure rutile and
rutile-anatase composite nanoparticles are fabricated via an ionic liquid-assisted method
and a series of shape-controllable ZnO nanocrystals stabilized by different ionic liquids
(Wang et al. 2008; Zheng et al. 2009).
The principal ions species in the solution medium is Fe(OH)33−, and ionic liquid micelles
play an essential role in the well-uniform morphology of goethite nanomaterial. This role
can occur via various mechanisms, like self-assembly, hydrogen bonds, electrostatic attrac­
tion, etc. At this stage, the mechanism is the electrostatic attraction between the cation of
ionic liquid, especially at H-C(2) of the imidazole ring and Fe(OH)33−. In this regard, the
cations of the ionic liquid may be adsorbed on the surface of goethite nuclei during the
goethite nuclei formation step and consequently affect the structure of goethite (Zhao et al.
2017).
Afterward, amino groups of chitosan solution were cationized and electrostatically
attracted by negative charges on the surface of iron hydroxide and due to the reduction
properties of chitosan, which lead to the formation of goethite-chitosan nanocomposite
(Xie et al. 2013). A possible diagram illustration of the goethite-chitosan nanocomposite
formation process is shown in Scheme 1.
 10

Table 2. Morphological characteristics of different bacterial isolates.

Code of isolate Morphological characteristic
Color Edge Elevation Shape Transparency Abundance* growth rate on MSM
(CFU/100 ml) **
H. S. EL-SHESHATWY ET AL.

SB1 Yellow Entire Raised Circular, large Opaque Abundant 8*10−8

SB2 Creamy Entire Flat Circular, medium Opaque Moderate 6*10−8
SB3 Light yellow Entire Raised Circular, irregular Opaque Rare 8*10−6
SB4 Creamy Entire Flat Circular, medium Opaque Moderate 7*10−6
SB5 Buff Branched Raised Irregular Opaque Moderate 8*10−7
SB6 Buff Entire Flat Circular, medium Transparent Moderate 6*10−6
SB7 Creamy Entire Flat Irregular Opaque Moderate 5*10−7
SB8 Creamy Branched Flat Irregular Opaque Rare 7*10−6
SB9 Creamy Entire Raised Circular, medium Transparent Abundant 8*10−5
SB10 Mucous Entire Raised Circular, large Transparent Moderate 6*10−6
SB11 White Entire Raised Circular, medium Opaque Moderate 8*10−7
SB12 Creamy Entire Raised Circular, medium Opaque Abundant 9*10−4
SB13 Light yellow Entire Raised Pin-shape Transparent Moderate 8*10−7
SB14 Light creamy Branched Flat Irregular, big Opaque Moderate 2*10−5
SB15 Mucous creamy Entire Flat Irregular Transparent Abundant 8*10−7
SB16 Light yellow Entire Raised Circular, medium Transparent Moderate 9*10−7
SB17 Creamy Entire Flat Pin-shape Opaque Rare 4*10−7
SB18 Light orange Branched Flat Irregular Opaque Rare 6*10−5
SB19 Simon Entire Raised Circular, small Opaque Rare 7*10−7
Abundance*: Abundant, Moderate, and Rare colony count on the growth media
CFU/100 ml**: Colony Forming Unit/100 ml MSM
Table 3. Morphological characteristics and molecular identification of the isolated bacterial strains.
Code of isolate Microscopic characteristic Morphological characteristic Identification 16S rDNA
Gram stain Cell morphology Color Edge Elevation Shape Transparency Growth rate on MSM
CFU/100 ml*
BS1 G-ve Rod shaped Yellow Entire Raised Circular, large Opaque 8*10−8 Flavobacterium johnsoniae
BS2 G-ve Rod shaped Creamy Entire Flat Circular, medium Opaque 6*10−8 Shewanella baltica
BS1: Bacterial strain no. 1, BS2: Bacterial strain no. 2
CFU/100 ml*: Colony Forming Unit/100 ml MSM
SOIL AND SEDIMENT CONTAMINATION
11
12 H. S. EL-SHESHATWY ET AL.

Figure 3. Illustrates the phylogenetic tree reconstructed by the Neighbor-joining method of the 16s rDNA
gene from isolating Flavobacterium johnsoniae (BS1) and Shewanella baltica (BS2) and closely related
bacteria.

Characterization of prepared nanocomposite and antimicrobial activity

The x-ray diffraction pattern of the goethite chitosan sample (Figure 4) was in good
agreement with the standard pattern of orthorhombic goethite (JCPDS No. 81–0464).
The peaks at 2θ = 9.24° and 20.18° were representative peaks of semicrystalline chitosan.
The weaker diffraction lines of goethite/chitosan in the XRD pattern indicate that the
goethite nanoparticles were covered by chitosan polymer (Zhang et al. 2012).
 SOIL AND SEDIMENT CONTAMINATION 13

(110)
Chitosan

(130)

(111)

(221)
Intensity (a.u)

(020)

(140)

(151)
JCPDS No. 81-0464

10 20 30 40 50 60
2 theta

Figure 4. XRD pattern for (Goethite/Chitosan) nanocomposite.

Scheme 1. The diagram illustration of the nanocomposite (Goethite/Chitosan) formation.

14 H. S. EL-SHESHATWY ET AL.

Figure 5. TEM micrographs of Goethite chitosan composite.

All the nanocomposite particles were spherical and opaque, with no aggregation and the
particle size not exceeding 20 nm as illustrated in Figure 5. The TEM-EDS photographs of
goethite/chitosan nanocomposite for the elemental mapping at the microstructural level
obtained were used to view the formation and distribution of goethite in the composite
micro-particle specimens as demonstrated in Figure 6. The mapping image depicted in dots
represents the elemental maps of O, C, N, and Fe atoms, respectively. The results confirm
that goethite/chitosan nanocomposite exists and is homogeneously distributed inside the
synthesized composite particles (Yang et al. 2016).
Antimicrobial activity of the tested nanocomposite exhibited no significant activity
against BS1 and BS2 at all studied concentrations. This may be explained by blocking
antibacterial amino groups of chitosan composites during the synthesis of nanocomposite.
A similar observation was reported by Zheng and Zhu (2003). In this study, acetic acid with
adjusted pH 6.0 was used as a control and showed no inhibitory activity. Consequently, this
result confirms that the synthesized nanocomposite is suitable for the improvement of the
bioremediation process.

SEM of immobilized oil degrading bacterial strains on the synthesized

nanocomposite
Scanning Electron Microscopy (SEM) analysis was conducted to test the bacterial adhesion
on the surface of the nanocomposite, as shown in Figure 7. It could be seen after the
 SOIL AND SEDIMENT CONTAMINATION 15

Figure 6. TEM-EDS photographs of FeO(OH) chitosan composite.

immobilization process; the bacterial consortium has been adhered and attached to the
interior surface of the nanocomposite and is ready for biodegradation. Due to the larger
specific surface area and high adsorption capacity of chitosan polymer, bacterial coloniza­
tion and in-suit bioremediation were promoted. By supplying an available surface area to
support bacterial growth and adsorbing of sufficient substrates to have direct contact
between bacterial cells and petroleum hydrocarbon. This eventually resulted in making
a better performance in crude oil biodegradation.

Biodegradation of crude oil sample

Gravimetric analysis
In the present study, the efficiency of crude oil degradation by the individual bacterial
cultures and the bacterial consortium in the presence and absence of nanocomposite was
determined qualitatively by estimating the consumed hydrocarbons after the end of each
incubation period (24, 48 and 72 h) at 30 C (see Table 4). The results clearly showed that the
values of residual oil decrease gradually with increasing incubation period up to 72 h (as
a result of the biodegradation of crude oil). Their values after biodegradation by the
immobilized consortium were the highest.
16 H. S. EL-SHESHATWY ET AL.

Figure 7. SEM micrographs showing bacterial adhesion on the surface of prepared nanocomposite under
different magnification power: (A) BS1 (B) BS2 (C) nanocomposite.

Gas chromatographic analysis (GC)

The percentage biodegradation of oil sample by the two bacterial strains BS1 and BS2 and
their mixture in the presence or absence of nanocomposite was estimated by (GC) analysis
within 3 days listed in (Table 5). The chromatograms are detected as several peaks
representing the residual paraffinic hydrocarbons over a hump, representing the unresolved
complex mixture (UCM) of high molecular hydrocarbons. The results demonstrated that
the biodegradation percentage increases with increasing the incubation time. The max­
imum percentage biodegradation was reached after 3 days in microcosm containing
immobilized bacterial consortium on nanocompsite at 93.32% as represented in Table 5.
Several investigators, for instance, Chhatre et al. (1996); Vasudevan and Rajaram (2001)
reported bacterial consortia’s ability to degrade 51% of saturates and 18% of aromatics
present in crude oil or up to 60% of crude oil. While in a study by Thahira-Rahman,
 SOIL AND SEDIMENT CONTAMINATION 17

Table 4. Residual of crude oil after biodegradation by two bacterial strains as pure and their mixture
without/with nanocomposite.
Incubation period (h) Experiment condition Original weight crude oil (g/l) Residual crude oil
(g/100 ml)
BS1+ crude oil 1.6
BS1+ crude oil+ nano-composite 1.5
BS2+ crude oil 1.6
24 BS2+ crude oil+ nano-composite 2 1.5
BS1+ BS2+ crude oil 1.6
BS1+ BS2+ crude oil+ nano-composite 1.4
BS1+ crude oil 1.5
BS1+ crude oil+ nano-composite 1.4
BS2+ crude oil 1.6
48 BS2+ crude oil+ nano-composite 2 1.4
BS1+ BS2+ crude oil 1.4
BS1+ BS2+ crude oil+ nano-composite 1.3
BS1+ crude oil 1.4
BS1+ crude oil+ nano-composite 1.3
BS2+ crude oil 1.3
72 BS2+ crude oil+ nano-composite 2 1.4
BS1+ BS2+ crude oil 1.2
BS1+ BS2+ crude oil+ nano-composite 0.9

Lakshmanaperumalsamy, and Banat (2002), the mixed bacterial culture gave the maximum
degradation percentage because no pure bacterial strain can degrade all the components
found within the crude oil. The microbial community’s mixed culture is required to
complete biodegradation of oil pollutants because the hydrocarbon mixtures vary markedly
in volatility, solubility, and susceptibility to degradation, and the necessary enzymes needed
cannot be found in a single organism (Adebusoye et al. 2007). This concept agrees with
Bordenave et al. (2007); AL-Saleh, Drobiova, and Obuekwe (2009) were investigated in
which individual microorganisms metabolize only a limited range of hydrocarbon sub­
strates and crude oil is made of a mixture of compounds. Therefore, its biodegradation
requires mixtures of different bacterial groups to degrade a wider range of hydrocarbons.
Zhang et al. (2010) reported that the percentage biodegradation of total polyaromatic
hydrocarbons of crude oil about 87.5% by a consortium of seven strains after 7 days.
The obtained data in Table 5 also observed with higher biodegradation of iso-paraffins
than n-paraffins in all different microcosms. The best biodegradation of iso-paraffins was
observed in microcosm containing the immobilized bacterial consortium with different
incubation periods (24–72 h). These results indicate that nanocomposite helps the
bacterial isolates to consume the iso-paraffins more than n-paraffins increase with
increasing the incubation time and reached its maximum after the end of the incubation
period (72 h).
Overall, the results seem to be a new and valuable bioremediation trend. Petroleum
hydrocarbons differ in their susceptibility to microbial attack and in the past, they had
generally been ranked in the following order of decreasing susceptibility: n-alkanes >
branched alkanes > low molecular weight aromatics > cycloalkanes (Medić et al. 2020;
Mohamed et al. 2006; Paudyn et al. 2008). From the above results, it can be concluded that
the presence of nanocomposite is capable of assisting bacterial strains activities for improv­
ing the biodegradation of iso-paraffins rather than n-paraffins. The results agree with El-
Sheshtawy et al. (2014) who reported that the presence of different nanoparticles can
 18

Table 5. Percentage degradation of crude oil sample by best bacterial strains and their mixture without/with nanocomposite using GC chromatography.
Sample (%) (%) biodegradation of total paraffin (%) biodegradation of UCM* (%) degradation of total paraffin
Total biodegradation Iso N
24 h
Control 0 0 0 0 0
H. S. EL-SHESHATWY ET AL.

Crude oil + BS1 5.79 5.14 0.65 4.6 0.54

Crude oil + BS1+ nano-composite 57.42 34.62 22.8 20.85 13.77
Crude oil + BS2 6.9 5.97 0.93 5.17 0.80
Crude oil + BS2+ nano-composite 34.95 23.41 11.54 15.68 7.73
BC+ crude oil 15.52 11.27 4.25 8.19 3.08
BC+ crude oil+ nano-composite 65.25 51.4 13.85 40.49 10.91
48 h
Control 0 0 0 0 0
Crude oil + BS1 10.35 8.89 1.46 7.64 1.25
Crude oil + BS1+ nano-composite 76.75 31.16 45.59 12.65 18.51
Crude oil + BS2 14.69 14.54 0.15 14.39 0.15
Crude oil + BS2+ nano-composite 40.61 22.51 18.1 15.63 6.88
BC+ crude oil 30.15 18.91 11.24 10.15 8.76
BC+ crude oil+ nano-composite 84.24 62.95 21.29 36.92 26.03
72 h
Control 0 0 0 0 0
Crude oil + BS1 14.37 9.4 4.97 6.15 3.25
Crude oil + BS1+ nano-composite 77.79 16.09 61.70 3.33 12.76
Crude oil + BS2 24.31 16.99 7.32 11.87 5.12
Crude oil + BS2+ nano-composite 55.21 30.99 24.22 20.97 10.02
BC+ crude oil 60.82 57.72 3.1 54.78 2.94
BC+ crude oil+ nano-composite 93.32 64.72 28.60 37.80 26.92
UCM*: Unresolved Complex Mixture, BS1: Bacterial strain no. 1, BS2: Bacterial strain no. 2, BC: Bacterial consortium
 SOIL AND SEDIMENT CONTAMINATION 19

Figure 8. The comparison consumed of crude oil between the control flask and mixed bacterial culture
with nanocomposite after 72 h.

improve the bacterial strain’s ability to degrade or to degrade iso-paraffins more than
n-paraffins. Additionally, in Figure 8 represents that the bacterial consortium had greater
consumption of crude oil than the control sample in the presence of nanocompsite.

High performance liquid chromatography (HPLC)

Polycyclic aromatic hydrocarbons (PAHs) contain two or more fused aromatic rings made
up of carbon and hydrogen atoms. There are many natural and anthropogenic sources of
PAHs in the environment. These hydrocarbon compounds have a potential risk to animal,
plant, and human health, as many of them are carcinogens (Deziel et al. 1996). This result
was performed using two bacterial isolates BS1 and BS2, and their consortium in the
presence or absence of nanocomposite at different time intervals (24, 48, and 72 h) as
shown in Figures 9–11.
The consumption of different membered rings of PAHs was calculated and compared to
that remaining in the un-inoculated control. Different membered rings of polyaromatics
were fully utilized in some bacterial microcosms. It also showed an increased bioremedia­
tion rate of other membered rings of polyaromatics with other bacterial microcosms.
Meanwhile, the biodegradation of different polyaromatics was increased gradually with
increasing the incubation period from 24 to 72 h in microcosms containing bacterial
consortium of two bacterial strains and nanocomposite.
20 H. S. EL-SHESHATWY ET AL.

member ring 2 member ring 3 member ring 4 member ring 5 member ring 6
90
80
% Residual polyaromatics

70
60
50
40
30
20
10
0
BS1 BS1 BS2 BS2 BC BC
Control Free Immboilized Free Immboilized Free Immboilized

Figure 9. Percentage residual of polyaromatic hydrocarbons after treatment by two bacterial strains as
pure and consortium with/without nanocomposite after 24 h.

member ring 2 member ring 3 member ring 4 member ring 5 member ring 6
90
80
% Residual polyaromatics

70
60
50
40
30
20
10
0
BS1 BS1 BS2 BS2 BC BC
Control Free Immboilized Free Immboilized Free Immboilized

Figure 10. Percentage residual of polyaromatic hydrocarbons after treatment by two bacterial strains as
pure and consortium with/without nanocomposite after 48 h.

The highest percentage degradation of high member rings polyaromatics (6-membered

rings considered highly toxic and carcinogenic polyaromatics) was observed after 72 h in
microcosms containing the bacterial consortium and nanocomposite (Figure 11).
It must be to mention that the increased degradation percentage of any hydrocarbon
compound may be attributed to the relative accumulation of such hydrocarbons into the
lower molecular weight polyaromatics (El-Sheshtawy et al. 2014, 2017). Only limited data
are currently available on high PAHs’ bacterial biodegradation with five or more rings and
pure or mixed microbial cultures (Kanaly and Harayama 2000).
 SOIL AND SEDIMENT CONTAMINATION 21

member ring 2 member ring 3 member ring 4 member ring 5 member ring 6
90
80
% Residual polyaromatics
70
60
50
40
30
20
10
0
BS1 BS1 BS2 BS2 BC BC
Control Free Immboilized Free Immboilized Free Immboilized

Figure 11. Percentage residual of polyaromatic hydrocarbons after treatment by two bacterial strains as
pure and consortium with/without nanocomposite after 72 h.

Role of nanocomposite for enhancing the microbial reaction

Nanocomposite can enhance the bacterial strain’s activities. Only a limited number of
studies have been reported on the nanocomposite effect on the microbiological reaction
rates. The higher activity of nanoparticles is usually referred to by their unique properties
and highly available active specific surface areas. Generally, nanoparticle catalysts increase
the microbiological reaction rates by locating on the cells to stimulate microbes’ activity
(Shan et al. 2005). In the previous work, El-Sheshtawy et al. (2017) investigated how
nanoparticles can enhance the bacterial growth rate and the performance of biosurfactants.
Biosurfactants were able to accelerate the rate of the biodegradation of crude oil by
increasing their bioavailability.
This article can use specific type and concentration of nanoparticles or nanocomposites
in contaminated soil and water with crude oil. In upscale soil and water bioremediation, the
nanocomposites may add with the nutrients source (N, P) to stimulate growth, reaction to
indigenous microbial strains, and improve the bioremediation process. Finally, the micro­
bial strains can be consumed or degraded these nanoparticles as a nutrient source.

Conclusions
Petroleum contaminants consider one of the critical problems that face global nature,
which causes the decline of environmental health. The bioremediation technology was
simple and effective in this study to minimize the hazards of toxic materials.
Nanocomposite material, which seems to be new and valuable for the biodegradation
trend, studied the bio-stimulation process. In the present study, all different microcosms
affect the degradation of paraffinic hydrocarbons and UCM in the crude oil under study.
The individual bacterial strains have less biodegradation abilities than their combination
(consortium). The main reason for this is that petroleum hydrocarbons have the cap­
ability of volatility, solubility, and susceptibility to degradation. It is worth noting that the
necessary enzymes needed for biodegradation cannot be found in a single organism. The
microcosm containing mixed bacterial culture could carry out a maximum degradation at
22 H. S. EL-SHESHATWY ET AL.

93.3% for the studied crude oil after 3 days of incubation period in the presence of the
nanocomposite material. Furthermore, the nanocomposite can enhance the bacterial
consortium strains’ ability to improve the biodegradation of iso-paraffins compared to
n-paraffins. Likewise, this material improved the bacterial consortium for degradation of
highly membered rings polyaromatics (5- and 6-membered rings), which consider highly
toxic polyaromatic hydrocarbons after 72 h. Our research has underlined that the selected
bacterial strains could effectively clear or remove oil contaminate. A mixed bacterial
culture can efficiently degrade the crude oil contaminate of the soil sample in Suez Oil
Processing Company (SOPC).

Acknowledgments
The authors would like to thank Egyptian Petroleum Research Institute (EPRI) for supporting every
facility to achieve the work.

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