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Eco-friendly Biodegradation of Hydrocarbons Compounds from Crude Oily Wastewater Using PVA/Alginate/Clay Composite Hydrogels

  • Genetic Engineering and Biotechnology Research Institute

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Immobilized microorganisms especially bacteria are most used rather than free cells to be protected from the environmental conditions when being used for the bioremediation of environmental pollutants. Herein, two marine’s bacterial isolates were tested for their ability to decompose crude oil. The optimum conditions for effective bacterial degradation e.g., pH, temperature, and inoculum size were investigated. PVA-alginate-clay composite hydrogel beads with different types of incorporated mineral clays were prepared and tested as bacterial carrier for potential bioremediation. Synthesized composite hydrogels were physico-chemically characterized by FTIR, SEM, and thermal analyses. Results showed that, embedded degrading bacteria in PVA-alginate beads recorded degradation rates as 74 and 66.6% for both tested bacterial isolates (S and R) compared to 61.2 and 53% degradation rates by free cells, respectively. Where, attapulgite clay-containing beads recorded maximum degradation% as 78.8 and 75% for both bacterial isolates, when added to immobilization matrices and these percentages could be enhanced under optimal conditions. The 16S rRNA gene of the two marine oils degrading bacterial isolates were amplified and sequenced, where both isolates were identified as Pseudomonas stutzeri and Rhodococcus qingshengii with submitted accession numbers of ON908963 and ON908962, respectively. These results are referring to the ability of using both tested isolates for crude oil bioremediation process and embedded them into PVA-alginate-clay beads as hydrogel carrier under the optimum conditions.
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Journal of Polymers and the Environment
Eco‑friendly Biodegradation ofHydrocarbons Compounds fromCrude
Oily Wastewater Using PVA/Alginate/Clay Composite Hydrogels
EmanFarid1· ElbadawyA.Kamoun2,3· TarekH.Taha4· AliEl‑Dissouky1· TarekE.Khalil1
Accepted: 5 July 2023
© The Author(s) 2023
Immobilized microorganisms especially bacteria are most used rather than free cells to be protected from the environmental
conditions when being used for the bioremediation of environmental pollutants. Herein, two marine’s bacterial isolates
were tested for their ability to decompose crude oil. The optimum conditions for effective bacterial degradation e.g., pH,
temperature, and inoculum size were investigated. PVA-alginate-clay composite hydrogel beads with different types of
incorporated mineral clays were prepared and tested as bacterial carrier for potential bioremediation. Synthesized compos-
ite hydrogels were physico-chemically characterized by FTIR, SEM, and thermal analyses. Results showed that, embedded
degrading bacteria in PVA-alginate beads recorded degradation rates as 74 and 66.6% for both tested bacterial isolates (S
and R) compared to 61.2 and 53% degradation rates by free cells, respectively. Where, attapulgite clay-containing beads
recorded maximum degradation% as 78.8 and 75% for both bacterial isolates, when added to immobilization matrices and
these percentages could be enhanced under optimal conditions. The 16S rRNA gene of the two marine oils degrading bacte-
rial isolates were amplified and sequenced, where both isolates were identified as Pseudomonas stutzeri and Rhodococcus
qingshengii with submitted accession numbers of ON908963 and ON908962, respectively. These results are referring to the
ability of using both tested isolates for crude oil bioremediation process and embedded them into PVA-alginate-clay beads
as hydrogel carrier under the optimum conditions.
Keywords Bioremediation· Immobilization· Attapulgite clay· Crude oil· Bacterial isolates· Optimization
PVA Polyvinyl alcohol
SA Sodium alginate
PAHs Poly aromatic hydrocarbons
SWR (%) Swelling ratio percentage
LB Luria Bertani broth
BH Bushnell-Hass broth
BEN Bentonite clay
MMT Montmorillonite clay
ATP Attapulgite clay
LRD Laponite RD clay
HAP Hydroxyapatite
Petrol related products, poly aromatic hydrocarbons
(PAHs)), and pesticide residues, are currently the most
hazardous contaminants represent a serious threat to the
environment and the aquatic life and considered as a major
challenge because of the advancement of industries and
modernization. Crude oil contaminated sites such as soil and
seas are a persistent problem with detrimental consequence
because of the stubborn, toxic, and carcinogenic constituents
* Elbadawy A. Kamoun
* Ali El-Dissouky
1 Chemistry Dep. Faculty ofScience, Alexandria University,
Alexandria, Egypt
2 Polymeric Materials Research Dep. Advanced Technology
andNew Materials Research Institute (ATNMRI), City
ofScientific Research andTechnological Applications
(SRTA-City), New Borg Al-Arab City, Alexandria21934,
3 Nanotechnology Research Center (NTRC), The British
University inEgypt, El-Shreouk City, Cairo, Egypt
4 Environmental Biotechnology Department, Genetic
Engineering andBiotechnology Research Institute (GEBRI),
City ofScientific Research andTechnological Applications
(SRTA-City), New Borg Al-Arab City, Alexandria21934,
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Journal of Polymers and the Environment
1 3
of crude oil [1]. Bioremediation or biodegradation process
is a waste treatment technology defined as the conversion
of chemical pollutants into biomass, H2O and CO2 can be
released by microorganisms [2], which is considered a low
cost and effective solution [3]. It was proven that immobiliz-
ing microorganisms into polymeric matrices is more efficient
than using free cells because the polymeric hydrogels have a
lot of advantages allowing long term and effective biodegra-
dation, in addition to protecting the microbes from the harsh
environmental conditions [4].
The immobilization carriers are vital for successful
bioremediation as they provide protection to the inoculated
microbes, and they should be environmentally friendly with
high affinity to the microorganisms and the PAHs [5]. Selec-
tion of bacterial supporting material is critical since the car-
rier can control the metabolic activity, maintain microbial
stability and safeguards from a hostile and dangerous exter-
nal environmental threats and hence improve the biodeg-
radation percentages [6]. Specific criteria must be fulfilled
in the chosen immobilization substrate such as having light
weight, low cost, chemically inert, physically and chemi-
cally stable, non-polluting, nontoxic, aqueous insoluble and
adapted to bacterial immobilization process [7].
PVA is a synthetic polyhydroxy polymer with high bio-
compatibility and mechanical qualities, PVA hydrogels are
safe and clean rubber like materials, so, they have been
widely used for drug delivery, tissue engineering, and other
biomedical applications. PVA hydrogels are also known
to be porous, so, they can be used as bacterial carriers [8]
but have high degree of swelling ability in water, so, they
must be chemically or physically cross linked to decrease
their hydrophilic nature to be more stable bacterial carri-
ers [9]. Alginate is a natural polysaccharide that extracted
from brown algae [10]. It is characterized by its high bind-
ing affinity to calcium ions, it also can form biodegradable
porous gel matrix, so, it is suitable as an inoculant carrier
Mixed matrices of PVA and alginate have desired prop-
erties such as high mechanical stability and reusability, so,
PVA- alginate beads showed better performance in PAHs,
bioremediation compared to alginate beads [12]. PVA is
incorporated in the PVA-alginate entrapping beads by inter-
molecular and intramolecular hydrogen bond between-OH
groups [13]. Recent studies showed that the immobilized
microbes have strong potentials to degrade environmen-
tal pollutants that their counterpart free cells [14], where
tested the immobilization of microbes into powders made
from nutshells, activated charcoal and organic materials in
order to remediate crude oil-polluted soils. Moreover, in
another study, the researchers reported the ability of Exig-
uobacteriumsp. AO-11 strain immobilized into bio-cord
carrier to effectively degrade crude oil of the contaminated
environment with the efficiency of the immobilized cells to
be reused for at least 5 cycles [15].
Different clay minerals were chosen as supportive materi-
als due to their nontoxicity to bacterial cells, low cost and
providing high surface area and mechanical stability, so,
they can be used to reinforce the PVA-alginate beads and
can be employed as a porous based material allowing the
oxygen diffusion into the embedded microbe in the pores
[16]. Also, the addition of a mineral clay acts as a physi-
cal crosslinker to improve the mechanical properties of the
composite beads [17].
Attapulgite (ATP) is a natural hydrophilic clay mineral
known for its reactive –OH groups on the surface and having
a layer chain like structure with exchangeable cations in its
framework channel. It was reported by Zhu etal. [18] that
modified hydrophobic ATP through cation exchange showed
high absorption capacity and selectivity to organic solvents
and oils owing to its mesoporous structure and hydrophobic
treatment which allow it to be effectively applied for crude
oil biodegradation issues.
This study aims to the isolation of effective biodegrad-
ing bacterial isolates that can be immobilized in polymeric
PVA-alginate beads for crude oil bioremediation purpose
and studying the optimum conditions such as the effect of
pH, temperature, inoculum size, incubation period, shak-
ing/static conditions, crude oil and clay concentration to
obtain the best degradation percentages of crude oil. Fur-
thermore, following up the degradation rates of crude oil
using gravimetric analysis and GC–MS investigation. Also,
the preparation of PVA- alginate composite hydrogel beads
as bacterial carrier and their instrumental characterization
using FTIR, SEM and TGA were also investigated. Moreo-
ver, further investigations were concerned by studying the
effect of using different types of clays such as Attapulgite,
Montmorillonite, Bentonite, Laponite and Apatite on the
enhancement of the crude oil bioremediation process and
the mechanical and thermal stability of the hydrogel beads.
Attapulgite had a great concern in the current study and has
been tested in different concentrations due to its low cost and
natural availability.
Materials andMethods
PVA (typically average Mw = 72,000g/mol; 98.9% hydro-
lyzed) was obtained from Biochemica, Germany. Sodium
alginate (SA) was purchased from DaeJung chemicals &
metals, Korea. Methylene chloride was purchased from
Fluka, Chemika. Calcium chloride (Fine GRD 90%) was
purchased from Fisher Scientific (Fairlawn, NJ, USA).
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Journal of Polymers and the Environment
1 3
Different types of clay were used, where Attapulgite was
obtained from Northwestern desert of Borg El-Arab, Egypt,
sodium Montmorillonite was obtained from Süd-Chemie
AG, Germany. Pure bentonite and Laponite-RD clays were
obtained from KREMER PIGMENTE GmbH, Aichstetten,
Germany. Hydroxyapatite was obtained from Nanoinglobal-
China, China. Na-MMT, ATP and Laponite RD were organi-
cally modified by a cation exchange with a quaternary alkyl
ammonium salts as intercalating agents, other chemicals were
used without further purification.
Culture Media
Luria Bertani (LB)
LB Broth was used for the cultivation of the bacterial strains
and was composed of (g/l): Yeast extract; 5, peptone; 10, and
NaCl; 10. Final pH (at 25°C) was adjusted as 7.0 ± 0.2.
Bushnell‑Hass (BH)
BH Broth was used as the basal medium for the crude oil deg-
radation experiments and was composed of (g/l): Magnesium
sulphate; 0.2, Calcium chloride; 0.02, Monopotassium phos-
phate; 1.0, Dipotassium phosphate; 1.0, Ammonium nitrate;
1.0and Ferric chloride; 0.05. Final pH (at 25°C) was adjusted
as 7.0 ± 0.2 using hydrochloric acid (0.01M) and Sodium
hydroxide (0.01M).
Sample Collection
Two different chemically polluted sites in Mediterranean Sea
named S (31.235772 N, 29.890912 E) and R (31.285874 N,
29.936795 E), Alexandria, Egypt were targeted for the col-
lection of samples. The collected samples from contaminated
sites were thought to have some bacterial strains with potent
capacity to decompose PAHs under controlled and specific
conditions. The sampling was performed according to [19]
with some modifications. Each sample was taken under aseptic
conditions using sterile 50ml falcon tubes which by directly
dipping the tubes into the water surfaces where both tubes
were opened and closed beneath the water surfaces by 30cm.
One milliliter of each sample was inoculated into 50ml LB
broth in sterile falcon tube. All the tubes were incubated at
30°C and 150rpm for 24h and were used for the bacterial
isolation process.
Isolation andPurification ofCrude Oil Degrading Bacterial
The capacity of microbes to grow on mineral salt medium
(BH Broth) supplemented with crude oil as a sole carbon
source was investigated according to Mishra etal. [20]
with minor modifications. Serial dilution for the previ-
ously collected and incubated samples was performed to
obtain pure bacterial colonies. From the lowest dilutions,
50µl were spread over BH agar plates supplemented with
100µl surface spread-crude oil followed by incubation
at 30°C for 7days.s until obvious growth of colonies
was observed. The growing colonies were considered as
presumptive crude oil degraders. Further purification of
these colonies on LB agar was performed and the purified
colonies were preserved in 4°C for subsequent use.
Screening ofCrude Oil Degrading Bacteria
The isolated bacteria that showed an ability to grow over
the crude oil containing BH plates were tested for their
ability to degrade the crude oil in liquid media according
to Mishra etal. [20] with minor modifications. Firstly,
20ml sterile BH broth was added to 50ml sterile fal-
con tubes followed by the addition of 1% crude oil. Each
tube was separately inoculated with 100µl of LB-growing
bacterial isolate (OD600 ~ 0.7). All tubes were incubated
at 30°C for 7days. After incubation, the residual oil of
each tube was gravimetrically investigated. The bacterial
isolates that showed the lowest oil residues were selected
for the rest of the work.
Gravimetric Determination ofOil Residues
The residues that were remaining after the biodegradation of
the crude oil by each bacterial isolate were estimated accord-
ing to the following method: 30ml of methylene chloride
were added to the whole content of each flask in a separat-
ing funnel. After well mixing for three minutes, two sepa-
rated immiscible layers were formed. The lower layer that
was composed of the solvent including the residual oil was
received into a clean and previously weighed glass beaker.
All beakers were heated at 60°C for two days till complete
evaporation of the solvent. After cooling, the beakers were
then weighted again and the difference between the two
weights of the empty and the oil-containing beakers was
The degradation percentage was calculated according to
the following equation:
Degradation%=(Wc−− WsWc)∗100
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Journal of Polymers and the Environment
1 3
Where, Wc is weight of control sample (g) and Ws is weight
of tested sample (g).
Optimization ofCrude Oil Degradation Conditions
bytheFree andImmobilized Bacteria
Different parameters were tested to optimize the best condi-
tions for oil degradation by the selected bacterial isolates.
Effect ofpH Value
Different pH values from 3 to 11 were tested for their effect
on the viability and crude oil biodegradation activity of the
tested bacterial cells. A volume of 100µl of each selected
bacterial isolate, previously cultured in LB broth, were
incubated with 10ml sterile BH broth containing 50µl of
crude oil in sterile falcon tubes. The pH of each tube was
formerly adjusted to the required pH value using 0.01M
NaOH and 0.01M HCl. All the tubes were incubated at
30°C and 150rpm for 3days. The remaining crude oil was
gravimetrically determined using methylene chloride as
mentioned earlier.
Effect ofDifferent Incubation Temperatures
Microbes ability to use hydrocarbons of the crude oil as a
carbon source is influenced by the surrounding temperature.
Different temperatures (25, 30 and 35°C) were tested to
determine the biochemical behavior of the bacterial isolates
towards crude oil biodegradation at the selected tempera-
tures. Each 100ml of sterile BH broth in 250ml conical
flasks were mixed with 1ml crude oil and 1ml of each
bacterial isolate previously grown in LB broth and immo-
bilized in the polymeric beads. In addition, un-inoculated
control flasks were also prepared. Each flask was incubated
separately at one of the previously mentioned temperatures
at 150rpm for 7days. At the end of the experiment, the
optimum temperature that resulted in the lowest crude oil
residues was investigated.
Effect ofCrude Oil Concentration
Different crude oil concentrations: 0.5, 1, 1.5, 2 and 2.5%
were amended to 100ml sterile BH broth followed by the
inoculation of 1ml of LB overnight culture of each bacte-
rial isolate, in addition to un-inoculated control flasks. All
flasks were incubated at 30°C and 150rpm for 28days in a
rotatory shaker, and then the residual oil was gravimetrically
measured to determine the optimum concentration of oil that
would be effectively degraded.
Effect ofShaking/Static Conditions
Depending on the examined bacteria, shaking of micro-
bial cultures would increases the amount and distribution
of the dissolved oxygen which might resulted in a negative
or beneficial impact on the overall process. In this experi-
ment, 100ml of sterile BH broth were amended with 1ml
crude oil and 1ml overnight culture of each bacterial isolate
already grown in LB broth. All the flasks were submitted
to static at 0 and/or shaking at 150rpm for 7days at 30°C.
After incubation, all flasks were removed from the incuba-
tors, and the crude oil residues of each flask were measured
and compared to control flasks (un-inoculated with bacteria)
to find out the best remediation condition. The residual oil
content was determined using gravimetric analysis as men-
tioned before.
Effect ofDifferent Incubation Periods
Time has a significant impact on the biodegradation pro-
cess as it is directly affecting the bacterial growth and their
subsequent breakdown of the inoculated carbon source. To
evaluate the effect of incubation time on the bioremediation
of crude oil, various incubation periods were tested from 0
to 28days. At the beginning, 1ml of each bacterial isolate
were cultured in LB broth and immobilized in the polymeric
beads. In 250ml flasks, 100ml of sterile BH broth were
mixed with 1ml of crude oil and the prepared beads fol-
lowed by incubation of the flasks at 30°C and 150rpm with
weekly sampling to determine the proper time for efficient
crude oil-biodegradation. The residual crude oil of each
treatment was gravimetrically determined and compared
with control flasks that included crude oil mixed media
without microbes.
Effect ofClay Incorporation intoBeads Hydrogels
Different types of clay such as Attapulgite, Montmorillon-
ite, Bentonite, Laponite and Apatite were tested as physical
cross-linkers or nanofillers to enhance the mechanical and
thermal stability of the PVA-alginate beads that have been
used as bacterial immobilization matrices. In this assay,
100ml of sterile BH broth were amended with 1ml of bac-
terial cultures previously entrapped in PVA/alginate/Clay
beads and 1ml of the tested crude oil. The flasks were then
incubated at 30°C and 150rpm for 28days to determine
the most mechanically and thermally stable beads that help
for the achieving of the higher biodegradation percentage
of the crude oil. The crude oil residues were gravimetri-
cally determined in both experimental flasks and bacterial-
free control flasks. Different concentrations of organically
modified Attapulgite clay (ATP: 0.25, 0.5, 1, and 2%) were
added into the polymeric beads to determine the optimum
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Journal of Polymers and the Environment
1 3
concentration that would result in the enhancement of the
crude oil bioremediation assay. Attapulgite clay has been
specifically selected for this experiment as it is one of the
most abundant and low-cost mineral clays in Egypt’s deserts.
It has also been tested to determine the ideal clay concentra-
tion that offer the highest bioremediation rate, mechanical,
and thermal stability with adequate hydrogel porosity for
sufficient interaction between imbedded bacteria and crude
oil in the surrounding media.
Effect ofInoculum Size
To evaluate the inoculum size effect on the degradation per-
centage of the crude oil; different inoculum sizes of 0.5, 1,
1.5, 2, and 2.5% of the selected isolates were amended into
100ml sterile BH broth followed by the addition of 1ml
crude oil. All flasks were incubated at 30°C and 150rpm
for 28days with interval sampling. After that, the residual
crude oil of each flask was gravimetrically meas28ured. The
optimum inoculum size that resulted in the effective biore-
mediation was determined.
Immobilization ofBacterial Isolates
intoPVA‑Alginate/Clay Composite Beads Hydrogel
PVA-Alginate beads were prepared via external ionic gela-
tion method [21, 22] by dissolving sodium alginate in dis-
tilled water (2% w/v) followed by co-dissolving of PVA (1%
w/v) in the same solution at 80°C. After complete solubility
and clarity of the solution, 1% (w/v) Clay was added as a
filler with continuous stirring to increase the homogeneity
and then the polymeric solution was let to cool to the room
temperature. After that, 1ml of each selected bacterial cul-
ture that was previously grown in LB broth was separately
added into10ml of the prepared alginate/PVA/Clay solu-
tion under aseptic conditions. The solution was mixed well
and was then dropped slowly through a syringe nozzle into
1% (w/v) CaCl2 solution as a cross linker. The solution was
stirred gently using magnetic stirrer for at least 10min as
a curing time to enhance the mechanical strength. Similar
procedures of using calcium chloride for crosslinking both
alginate and PVA were reported by Baigorria etal. [23] and
Narra etal. [24]. Different concentrations of PVA (0.5, 1,
and 3% w/v), Alginate (1, 2, and 3% w/v), CaCl2 (0.5, 1,
and 2%, w/v) and Attapulgite clay (0.25, 0.5, 1, and 2%,
w/v) were tested.
Biodegradation ofCrude Oil Using theImmobilized
The whole amount of the immobilized bacteria that were
entrapped inside the PVA/Alginate/Clay polymeric capsules
were added to a 250ml conical flask containing 100ml of
sterile BH broth mixed with 1ml crude oil as a sole carbon
and energy source. All the prepared flasks were then incu-
bated in a rotatory shaker at 150rpm and 30°C. The deg-
radation percentage was gravimetrically investigated every
7days for a maximum 28days successive.
Bioremediation ofCrude Oil byFree
andImmobilized Cells
It is thought that entrapped bacterial strains is more efficient
than free cells for potential bioremediation process, because
the encapsulation of bacteria in a polymeric matrix may offer
rapid and complete degradation of the tested waste, as it pro-
vides bacterial protection from the environment, extreme pH
and toxic materials that might existed in the contaminated
sites, and also can provide higher biological stability of the
tested strains [25]. To test that, 100ml of sterile BH broth
were incubated in 250ml conical flasks with 1ml of each
free bacterial strains and 1% crude oil. At the same time,
1ml of each tested bacterial isolate was entrapped in PVA/
alginate and PVA/alginate/clay beads and were added to
100ml sterile BH broth included 1% crude oil. After that,
all flasks were incubated at 30°C and 150rpm for 28days.
Oil residues were measured gravimetrically, and the effi-
ciency of the free and immobilized strains were compared
to determine the best bioremediation condition.
Molecular Identification ofBacterial Isolates
Genomic DNA extraction
Total genomic DNA from each bacterial culture was
extracted using Amshag kit according to the manual instruc-
tions of (SRTA-City, Egypt).
PCR amplification and sequencing of the 16S rRNA gene
Each extracted genomic DNA was used as a template for
the amplification of 16S rRNA, through PCR technique. A
multiplex PCR kit (Qiagen, USA) was used for the amplifica-
tion of the target gene using universal primers. The forward
(5`-GAA CGC GAA CCT TAC-3`) and reverse primers (5`-
to amplify 500bp of the 16S rRNA gene of each bacterial
isolate. The reaction program was started with a first dena-
turation step at 94°C for 4min followed by 35 cycles of 30s
at 94°C, 30s. at 55°C, and 30s at 72°C followed by a final
extension step at 72°C for 7min. The amplified genes were
submitted for electrophoresis in 1 × TBE buffer at 120V for
30min. The amplified genes were visualized and detected
using Gel documentation system, compared with 1kb DNA
ladder. The obtained genes were subsequently submitted for
purification and sequencing (SIGMA, Germany), and the
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Journal of Polymers and the Environment
1 3
obtained sequences were submitted in GenBank using new
accession numbers. The phylogenetic tree of the obtained
sequences and other related sequences in GenBank was per-
formed using MEGA 11.0 program.
Swelling Behavior ofPVA/SA Hydrogel Beads
The swelling ability of a hydrogel reflects its permeability
and represents an important parameter for hydrogel perfor-
mance and for immobilization process to ensure exchange
action between the immobilized bacteria and the surround-
ing media. In this experiment, 1g of dried PVA/SA beads
(Wd) was soaked in distilled water at room temperature with
interval sampling and weighting of the swelled beads (Ws)
till reaching equilibrium [26]. Water uptake percentage was
measured according to the following equation:
where Wd refers to the weight of the dry sample and Ws
refers to the weight of the swollen sample at specific time
Instrumental characterization
Vacuumed and dried samples of pure PVA, sodium alginate,
and PVA–Alginate hydrogel beads were examined by FTIR
on an EQUINOX 55 instrument (BRUKER, Germany). The
dry samples were ground together with infrared grade KBr
and then pressed to make Translucent KBr-disks. The FTIR
spectra were produced by recording 64 scans with a resolu-
tion of 2 cm−1 between 4000 and 400 cm−1. All samples
were freeze-dried using liquid nitrogen, crushed to a fine
powder (KBr: sample = 140mg: 2mg), and pressed into a
clear transparent disk with a diameter of 13mm by applying
a force of 105 N.
Scanning Electron Microscopy
Microstructure morphology and surface characterization
of immobilized S and R isolates in PVA-SA beads with/
without Clay were performed after beads lyophilization
using Analytical-SEM (type: JEOL, JSM-6360LA, Japan)
with 15kV voltage for secondary electron imaging. The
hydrogel beads were dehydrated by freeze-dryer and coated
with Au using an ion sputter coater (model: 11,430, USA,
combined with vacuum base unit or SPi module control,
model:11,425, USA).
Thermal Analysis
TGA thermograms were used to characterize the thermal
properties of a vacuumed dried PVA/SA hydrogel beads
using different clay concentrations (0, 0.25, 0.5, 1 and 2%).
The thermo-gravimetric analysis (TGA) was carried out
on a 204 Phoenix TGA instrument (NETZSCH, Germany)
at a heating rate of 10°C/min from room temperature to
600°C. The onset temperature Tonset was calculated using
TGA thermogram which are defined as the temperature at
the point of inflection or highest mass loss percentage at the
intersection of the baseline mass and the tangent drawn to
the mass curve.
GC–MS Analysis
Crude oil and its metabolites after 28days of biodegradation
were analyzed using GC–MS technique compared with con-
trol sample. The GC–MS test was carried out using a Trace
1300 GC Ultra/Mass Spectrophotometer ISQ QD (Thermo
Scientific) instrument, X-calibur 2.2 software (Thermo
X-calibur) equipped with a TG-5MS Zebron capillary col-
umn (length 30m × 0.25mm ID, 0.25µm film thickness;
Thermo). Helium (average velocity 39cm/s) was used as
the carrier gas. The oven temperature program was: Held at
80°C for 5min then increased from 80 to 200°C for 1min
(4°C/min), 200–300°C for 1min (7°C/min) and injector
temperature 300°C. Sample size was 1µl; injection split
ratio was 20:1 and run time was 51.29min. Data analysis
was carried out using NIST database.
Statistical Analysis
All the statistical analysis was performed using IBM SPSS®
software, 1997, according to Duncan’s multiple range test
It could be concluded that these methodologies target-
ing the proper immobilization of the tested bacterial cells
inside the polymeric materials supported by clay component
in order to enhance the crude oil bioremediation process
under tested optimized conditions.
Results andDiscussions
Isolation andPurification ofCrude Oil Degrading
Bacterial Strains
Contaminated samples that were collected from the Mediter-
ranean Sea, Alexandria, Egypt were used for the isolation
of microorganisms with crude oil degrading capacity. In
this case, two bacterial isolates identified as Pseudomonas
stutzeri and Rhodococcus qingshengii (S and R) were used
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Journal of Polymers and the Environment
1 3
as bacterial isolates from different locations that have been
tested for their ability to degrade crude oil as a sole carbon
and energy source. The obtained results revealed the ability
of both bacterial isolates to degrade crude oil effectively.
However, all other 8 bacterial isolates failed to show any
signs of crude oil biodegradation ability, and hence all of
them were ignored. On the other hand, both selected sam-
ples were then tested for their ability to degrade the tested
crude oil under different optimization conditions as will be
shown in the following experiments. Each experiment was
repeated 2 or 3 times.
One Variable atTime Optimization (OVAT)
Effect ofpH Value
Both crude oil degrading isolates were investigated for their
bioremediation capacity at wide pH ranges as described in
Fig.1a and b. It has been shown that, both S and R isolates
were able to grow in wide range of pH values including
neutral, slightly alkaline, and slightly acidic medium ranging
from 6 to 8 as the highest values resulted in higher biodeg-
radation percentages. In both isolates, 50% degradation was
recorded at slight acidity (6) and slight alkalinity (8). This
percentage was gradually decreased to reach 33.3% at pH 4,
5 and 9 for isolate S; and 5 and 9 for isolate R, with lower
degradation percentages at stronger acidic or alkaline con-
ditions. However, the highest degradation percentage was
recorded as 66.6% at neutral pH value (7) for both isolates.
Bacterial degradation in wide pH ranges is a good sign
for the bacterial stability even under varying pH conditions
or in different environments. Our results are almost matched
with Simarro etal. [28], who showed that the desired pH for
PAHs (Naphthalene, Phenanthrene and Anthracene) degra-
dation ranging from 5.5 to 7.8 using Enterobacter Pseu-
domonas and Stenotrophomonas bacterial consortium, On
contrary Das etal. [29] observed that the bacterial growth
increased in alkaline medium at pH 8.5 in the degradation
of P-Nitrophenol using potent Pseudomonas strain from the
textile dye industry effluent. The previous results indicate
that, pH regulates microbial enzyme activity, transport pro-
cess and nutrient solubility, and so governs the microbial
activity to great extent [30, 31].
Effect ofDifferent Incubation Temperatures
The effect of temperature on the growth of both tested bac-
terial isolates at the presence of crude oil as a sole carbon
and energy source was investigated. As shown in Fig.1c
and d, the optimum temperature was 30°C recording 80
and 76.5 degradation percentage for both S and R isolates,
respectively. This result consistent with the results obtained
by Paliulis etal. [32] who showed that 30°C is the optimum
Fig. 1 a Effect of pH on the degradation percentage of crude oil using S isolate and b using R isolate. c Effect ofdifferent incubation tempera-
tures on the degradation percentage of the tested crude oil using S isolate and d using R isolate
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Journal of Polymers and the Environment
1 3
temperature for the highest oil hydrocarbon degradation by
the use of Acinetobacter sp, and our results are also matched
with Huang etal. [33] in selenite bioremediation using the
highly selenite-tolerant strain (Providencia rettgeri HF16-
A). Also, 35°C showed high acceptable bioremediation
results while moderate growth was found at 25°C. These
results indicate the wide range application of the tested Sand
R isolates in different ranges of temperatures as crude oil
degraders. Extreme temperatures (either too low or too high)
have an impact on microbial growth and microbial enzyme-
catalyzed processes. When the temperature rises with a
reasonable range, the rate of bioremediation increases as
microbial metabolism rises, but to a certain limit that can
be bear with organisms. Moreover, the solubility of PAHs is
also increasing at higher temperature, as a result, bioavail-
ability increases [34].
Effect ofCrude Oil Concentration
The ability of S and R isolates to mineralize different crude
oil concentrations was tested as shown in Fig.2a and b.
Both showed a high degradation capacity at low crude oil
concentrations. However, their ability to degrade crude oil
was gradually reduced with increasing the oil concentra-
tions. Maximum degradation rates of 85 and 83.5% were
recorded at 0.5% crude oil concentration for S and R iso-
lates, respectively. At 1% crude oil concentration, S and
R isolates recorded 78.8 and 76.66% degradation rate,
respectively. At 1.5% crude oil concentrations, 74.1 and
71.6% degradation rate were recorded for S and R isolates,
respectively. Moderate degradation percentages of 71.6 and
69.6% were recorded at 2% crude oil, while 66.4 and 65%
were recorded when 2.5% crude oil was used for S and R
isolates, respectively. Our results are almost matched with
Taha etal. [35] who showed that the degradation percent-
age decreased with increasing phenanthrene concentration
using phenanthrene degrading bacteria (Enterobacter cloa-
cae, Bacillus sp. and Bacillus thuringiensis) isolated from
petroleum contaminated soil. Also, these results are consist-
ent with the results obtained by Abtahi etal. [36] where the
crude oil removal efficiency was decreased by increasing
petroleum initial concentration in their study of the effect
of competition between petroleum-degrading bacteria and
indigenous compost microorganisms on the efficiency of
petroleum sludge bioremediation using Acinetobacter radi-
oresistens strain KA5 and Enterobacter hormaechei strain
KA6 isolated from the petroleum waste sludge, as high levels
of petroleum compounds are poisonous to the bacteria so,
they preferred 1and 2% crude oil as optimum concentra-
tions. Also, El-Noubi etal. [37] reported that fast bacterial
growth occurred at low oil concentrations while the growth
rate decreased and suppressed at high oil concentration so,
they chose 0.6% crude oil concentration as optimum con-
centration. In the same context, Hazim etal. [38] reported
Fig. 2 a Effect of crude oil concentration on the degradation percentage using S isolate and b using R isolate. c Effect of shaking /static condi-
tions on the degradation percentage of crude oil, using S isolate and d using Risolate
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Journal of Polymers and the Environment
1 3
that decreasing the microbial degradation activity at high
hydrocarbon concentrations that can inhibit microorganisms
through toxic effects, resulting in a decrease in biodegra-
dation percentage in their study of the effect of petroleum
hydrocarbons contamination with Kerosene, Diesel and
lubricate oil on soil microorganisms and biodegradation.
Moreover, Abarian etal. [39] observed that, the number of
bacterial cells reduced by increasing phenol concentration
in their study of degradation of phenol at high concentra-
tions using immobilization of Pseudomonas putida P53 into
sawdust entrapped in sodium-alginate beads. So, low, and
moderate crude oil concentration was investigated as the
most preferred concentrations for effective bioremediation
using our tested isolates (S and R). Furthermore, Ibrahim
etal. [40] demonstrated that 1% crude oil concentration was
the optimum concentration as the sole source of carbon and
energy in their research of crude oil degradation, and heavy
metal tolerance. From these results, we can conclude that 0.5
and 1% crude oil can be considered as the optimum concen-
tration in our experiments.
Effect ofShaking/Static Conditions
Bacterial growth significantly affected by shaking conditions
which may subsequently affect the bioremediation process.
Figures2c and d show moderate degradation capability for
both S and R isolates under static condition. The maximum
crude oil degrading percentages were recorded when the
microbes were shaken at 150rpm. At this shaking rate, both
of S and R isolates recorded degradation percentages of 76.7
and 70.6%, respectively. While moderate degradation rate
of 61.2 and 58.8% were recorded using static conditions.
These results were attributed to the crude oil dissolution
and oxygen availability under shaking conditions which in
turn enhanced the tested crude oil degradation rate. These
results are consistent with A. Sed etal. [41] who explored
the biodegradation of phenanthrene by Klebsiella sp iso-
lated from organic contaminated sediment. Both S and R
isolates showed degradation activity using 150rpm shaking
condition when crude oil was amended as a sole carbon and
energy source. Accordingly, moderate shaking at 150rpm
was preferred as optimum condition than static condition for
effective bioremediation in the current experiment.
Effect ofDifferent Incubation Periods
By monitoring the crude oil degradation rates along 28days
as represented in Fig.3a and b; results showed that, by
increasing the incubation period from 7 to 28days; the
crude oil degradation percentages increased significantly.
After 7days of incubation, S and R isolates recorded 64.7
and 60% degradation rates, respectively. By increasing
the incubation period to 14days; the crude oil degrada-
tion rates increased to 68.2 and 64.7% for S and R isolates,
Fig. 3 a Effect of different incubation periods on the degradation percentage of crude oil, using S isolate and b using R isolate. c Effect of differ-
ent types of mineral clays on the degradation rates of crude oil using S isolate and d using R isolate
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Journal of Polymers and the Environment
1 3
respectively. Further increase in the degradation rates to 70.5
and 67% was recorded with increasing incubation period
to 21days for both S and R isolates, respectively. Maxi-
mum crude oil degradation rates of 76.66 and 70.5% were
recorded after 28days of incubation for S and R isolates,
respectively, which indicates that 28days of incubation are
the optimum incubation periods for the tested bacteria. In the
same context, Baoune etal. [42] reported that, after 28days
of incubation, significant decrease in crude oil concentra-
tion was observed compared to control flasks in petroleum
hydrocarbons degradation in their study of bioremediation
of crude oil-contaminated soils using Streptomyces sp. Hlh1.
Also, our results matched with El-Sheshtawy etal. [43] who
demonstrated that, the biodegradation percentage increased
with increasing the incubation time in the degradation of
petroleum hydrocarbons using Flavobacterium johnsoniae
and Shewanella baltica bacterial strains immobilized on
goethite-chitosan nanocomposite. Moreover, Usmani etal.
[44] reported that the degradation rate of lindane pesticide
was increased with the number of days of incubation in their
study of bioremediation of lindane contaminated soil and
exploring the potential of actinobacterial strains.
Effect ofClay Addition
Due to their high surface area, clays are commonly utilized
as adsorbents. In their natural form, clays have low sorp-
tion capacity to hydrophobic organic compounds in aque-
ous solutions because their exchangeable ions are highly
hydrated. Ionic exchange of their inorganic ions with cation
makes the clay surface hydrophobic and in turn increases
clays adsorption capacities towards hydrophobic compounds
[45]. In this case, clays can be used in cleaning up hydro-
carbon spills on the shoreline and land [46]. The effect of
different types of mineral clays such as: organically modi-
fied Attapulgite, Laponite RD, hydroxyapatite, pure Sodium
Montmorillonite, and pure Bentonite, on the degradation
percentages of crude oil can be shown in Fig.3c and d. It
was found that, the maximum degradation rates of 85.9 and
84.7% for S and R isolates, respectively were recorded when
apatite was added to the immobilization polymers. Second
higher degradation rates were recorded when Laponite was
used where it showed 82.4 and 78.33% for S and R isolates,
respectively. While at the presence of Attapulgite, the deg-
radation percentages were moderate and showed 78.8 and
75% for S and R isolates, respectively. Also, when Mont-
morillonite was incorporated, acceptable degradation rates
were recorded as 70.5 and 68% for S and R isolates, respec-
tively. On the other hand, the lowest degradation rates were
recorded through the presence of Bentonite. The degradation
percentages of 65.2 and 64.7% for S and R isolates; respec-
tively, were recorded at its presence. These results may be
attributed to the modification of the tested mineral clays by
ion exchange using intercalating agents which increased
their hydrophobic character and in turn enhance the inter-
action between the PVA- alginate beads and the hydropho-
bic crude oil in the surrounding media. These interactions
resulted in a significant increase in the degradation rates
that leading to more efficient degradation process. This find-
ing of the research is novel as it shed light on the effect of
different types of clays specially organically modified clay
such as hydroxyapatite, laponite RD and attapulgite due to
their higher sensitivity to hydrophobic crude oil compared to
hydrophilic natural clays such as bentonite and montmoril-
lonite making them a great addition into the hydrogel beads
composition and providing higher bioremediation rates of
the tested crude oil. A laboratory representative figure of
the crude oil before and after biodegradation by the bacterial
isolates at the presence of different clay types can be seen in
Fig. S1 (supplementary data).
Moreover, the suggested mechanism for the effectiveness
of bioremediation process through the addition of clay could
be summarized as follows: The clay minerals are known as
the most abundant minerals originated through the water–rock
interactions. They have a lot of advantageous such as hav-
ing surface charge, having large surface areas, and interlayer
spaces, which allows them to effectively adsorb different
types of polar and non-polar organic substances [47]; and
hence find their way in thousands of industrial applications,
environmental protection technologies, and remediation pur-
poses [48, 49]. The colloidal sized clays have been used for
the remediation of hydrocarbon contaminated solutions as
they able to aid dispersion, and their large surface areas can
positively accelerate the physical and chemical disaggregation
processes in addition to the size reduction of the oil globules
[50]. In addition, they potentially enhance the biodegradation
of hydrocarbon compounds and increasing the rate of bacte-
rial growth. This potentiality is linked to their ability adsorb
the protons released during the breakdown of the hydrocar-
bons which plays an important role in avoiding the change in
the optimal pH conditions, and hence sustaining the bacterial
growth. Moreover, some clays able to stimulate the bacte-
rial activity through the nutrient adsorption on the bacteria
cell walls by the formation of C–O–Na–Si complexes on the
outer surfaces of the bacterial cell walls which is associated
with the particle dissolution. This is highly matched with [47],
who reported that the addition of clay minerals with large
specific surface areas and high cation exchange capacities are
extremely improving the biodegradation of hydrocarbons by
bacterial cells over a significant time period.
Effect oftheAttapulgite Mineral Clay Concentration
ontheCrude Oil Degradation Rate
The effect of the concentration of the organically modified
Attapulgite clay mineral (ATP) (extracted and purified by
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Journal of Polymers and the Environment
1 3
Elbassyoni etal. [51] from (Northwestern desert of Borg El-
Arab, Egypt) on the bioremediation of crude oil was inves-
tigated as shown in Fig.4a and b. When the concentration
of ATP was 0.25%, the crude oil degradation rate reached
its maximum recording 74.1and 73% for S and R isolates,
respectively, and was gradually decreases by increasing
the clay concentration. This indicates that the higher clay
concentration in the prepared beads has a little and weak
effect on the bioremediation process. These higher concen-
trations might block the pores of the beads that immobiliz-
ing the bacteria, which in turn inhibit the metabolic activity
exchange between the bacteria and the outside medium and
negatively affecting the degradation process. As a result,
the addition of low clay concentrations to the beads could
enhance their mechanical stability with adequate perme-
ability and make the immobilized cells more efficient. Our
results are almost matched with Ruan etal. [52] in their deg-
radation of phenol using immobilized Sphingomonas sp. in
PVA-alginate- kaolin beads as they reported that high kaolin
content more than 1% in the hydrogel beads restricted the
cells' ability to develop, reducing the activity of the bacteria
and hence, decreasing the bioremediation rates. In the cur-
rent investigation, 0.25% ATP was the optimal concentration
in terms of degradation rate, mechanical strength, and cost
making attapulgite clay a novel candidate in our study pro-
viding higher bioremediation percentages even in very low
concentration. In the same context, Lin etal. [53] reported
similar results in the degradation of TNT using immobilized
Bacillus mycoides in PVA-alginate- kaolin beads as they
demonstrated that lower kaolin concentration was preferred
and the mechanical strength of the beads was much poorer
without kaolin. Also, it increased the adsorption capacity of
the beads and making the bacteria more active.
So, it could be concluded that it positively affected the
biodegradation process.
Effect ofInoculum Size
Initial inoculum size is an important factor in bioremedia-
tion processes. Results showed that the bioremediation of
crude oil was also inoculum size dependent. To evaluate
the effect of inoculum size as a critical parameter on crude
oil degradation rate, both S and R isolates were tested in
different concentrations as shown in Fig.4c and d. It was
observed that, the degradation percentage decreases sig-
nificantly by increasing inoculum size. It has been detected
that, at 0.5% inoculum size, maximum degradation rates of
87.1 and 88.33% for S and R bacteria, respectively were
recorded. At 1% inoculum size, the degradation rates
recorded 80 and 83.5% for S and R isolates, respectively.
Acceptable degradation rates of 74.1 and 64.7% for S and R
bacteria, respectively, were recorded when 1.5% inoculum
size was tested. Further decreases in the degradation rates
were measured at 2% inoculum size and resulted in 71.8
Fig. 4 a Effect of clay concentration on the degradation percentage of crude oil using S isolate and b using Risolate. c Effect of inoculum size on
the degradation rates of crude oil using S isolate and d using R isolate
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Journal of Polymers and the Environment
1 3
and 61.2% degradation percentages for S and R isolates,
respectively. The lowest degradation rates were recorded at
2.5% inoculum size and showed 69.4 and 57.65% for both
S and R bacteria, respectively. Accordingly, 0.5% inoculum
size was the optimum and preferred concentration in terms
of degradation rates and cost. In the same context, Costa
etal. [54] demonstrated that, higher inoculum concentration
did not improve the biomass production, so, lower inoculum
size was preferred (25µl) in their study to use the glycerol
from biodiesel production as a carbon source for biomass
production by actinobacteria withwell-known bioremedia-
tion abilities. In the current investigation, the results may be
attributed to the depletion of the carbon source (petroleum
oil components) along 28days in addition to the releasing of
poisonous metabolic products that might affect the activity
of the bacterial cells. However, the opposite has been proved
in other studies such as Philip etal. [55] who observed that,
the increase in bacterial cell concentrations would increase
the endosulfan degradation efficiency in their study using
Staphylococcus sp., Bacillus circulans enriched from con-
taminated soil collected from the vicinity of an endosulfan
processing industry.
Bioremediation ofCrude Oil byFree andImmobilized Cells
Comparing the crude oil degradation rates by free and
immobilized cells in PVA-alginate beads is represented in
Fig.5a and b. The maximum degradation percentages were
recorded by immobilized bacterial isolates in the presence of
ATP clay as nano-filler as 74 and 66.6% for S and R bacterial
isolates, respectively. While 68.24 and 55.5% were recorded
in the absence of clay by S and R isolates, respectively.
The lowest degradation percentages of 61.2 and 53% were
recorded by the free cells of S and R bacteria, respectively.
From the previous results we can conclude that, immobilized
bacterial cells acted more efficient than free ones. These
results may be attributed to the large surface area offered by
the beads which significantly increases the contact between
the microbes and the crude oil under protection conditions,
and hence resulted in high crude oil degradation percent-
ages even at much higher oil concentrations. In the same
context, Gouda etal. [56] observed that, the degradation rate
of kerosine increased and the time of degradation reduced
by immobilizing Gordonia sp. and Pseudomonas sp. in rice
straw compared to free cells in their case study of biore-
mediation of kerosene in liquid media. These results were
also matched with Talha etal. [57] in the bioremediation
of Congo red dye using a free cell and cell immobilized
on biochar of coconut shell. Moreover, Padmanaban etal.
[58] reported that, immobilized cell systems provide more
advantages over free cell systems in their study of degrada-
tion of reactive red 120 dye in bed reactor by bacillus cohnii
RAPT1 immobilized on poly urethane foam (PUF). Addi-
tionally, Bayat etal. [59] demonstrated that the trapping of
Pseudomonas aeruginosa UG14 in clay, alginate and skim
milk for phenanthrene degradation along 30days protected
bacterial cells survival compared to free cells that lasted for
only 18days.
Bacterial Identification Using 16S rRNA Gene
The molecular identification of the bacterial isolates was
depending on genomic DNA extraction, PCR amplification
and sequencing of the 16S rRNA gene. The obtained results
showed the successful amplification of 500bp of the tar-
get gene for both bacterial isolates. After purification and
sequencing of the amplified genes, the obtained sequences
were submitted in GenBank to have new accession numbers.
The isolate R was identified as Rhodococcus qingshengii
and was submitted in GenBank with the accession number
Fig. 5 a Bioremediation of crude oil by free and immobilized cells using S isolate and b using R isolate. A: Freecells, B: Immobilized cells, C:
Nano clay Containing-beads-immobilized cells
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Journal of Polymers and the Environment
1 3
ON908962. While the isolate S was identified as Pseu-
domonas stutzeri and was submitted in GenBank with the
accession number ON908963. The phylogenetic tree of the
two isolates compared with other similar sequences depos-
ited in GenBank as is illustrated in Graph 1.
Swelling Behavior ofHydrogel Beads
Swelling capacity is one of the undeniable assays required
for bioremediation process to ensure exchange between the
embedded bacteria inside the hydrogel beads and the sur-
rounding environment. So, adequate degree of water uptake
is required to facilitate the exchanging process. PVA-alginate
beads with different PVA concentrations were immersed in
distilled water to test their water uptake abilities as repre-
sented in Fig.6a. It was practically observed that, the weight
of the beads significantly increased with increasing the PVA
content in the tested hydrogel beads which may be attributed
to the higher hydrophilic nature of the PVA. In other words,
higher PVA ratio resulted in higher swelling capacity due
to increasing the number of –OH groups of the PVA com-
pound, and hence making the beads more hydrophilic [60].
So, we can conclude that, there is an inverse relation-
ship between swelling capacity and mechanical stability of
the hydrogel beads, 0.5% PVA has lower swelling capacity
with higher mechanical stability while 3% PVA has higher
water uptake capacity due to increasing hydrophilicity but
Graph 1 Phylogenetic tree of Rhodococous qingshengi and pseu-
domonas stutzeri strains within the relative strains. The tree was con-
stracted by Maximum likelihood tree method with bootstrap values
for 500 replicates using MEGA 11.0 software
Fig. 6 The effect of different a PVA, b alginate, c CaCl2, and d Attapulgite clay concentrations on the swellingbehavior of the hydrogel beads
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Journal of Polymers and the Environment
1 3
lower mechanical stability. So, 1% PVA was considered as
the optimal concentration in our study as it provides moder-
ate and adequate swelling capacity of the hydrogel beads and
hence acceptable mechanical stability.
By following up the swelling ratios of the PVA-alginate
composite beads with different alginate concentrations as
represented in Fig.6b; it was observed that increasing algi-
nate content significantly depressed the swelling capacities
of the tested beads. This may be attributed to the higher
network density of the alginate hydrogels which prevents
the swelling of the alginate chains in water as previously
reported by Omidian etal. [61] that higher crosslinking
density in alginate chains prevents them from expanding in
water in their study of elastic, super porous hydrogel hybrids
of polyacrylamide and sodium Alginate.
1 wt% alginate has higher swelling capacity and hence
lower mechanical stability while 3 wt% alginate has lower
water uptake and higher mechanical strength but condenser
hydrogel network and hence lower exchange capacity
between the embedded bacteria and the surrounding media.
So, 2 wt% alginate was considered as the optimal concen-
tration for acceptable exchange capacity and mechanical
The blending of Alginate with other polymers such as
polyvinyl alcohol (PVA) has been proposed to overcome
the quick breakdown of hydrated alginate beads This is also
consisting with Narra etal. [24] in their study ofRifampicin
Loading on PVA-alginate beads by ionotropic gelation
method. In addition, it is also proved that physical crosslink-
ing via intra molecular hydrogen bonding (entanglement)
occurs between the same PVA molecules and intermolecular
hydrogen bonding between PVA and alginate chains by Bai-
gorria etal. [23] in their study of arsenic removal.
CaCl2 concentration is another important factor that
influences the swelling rate of the alginate beads as Ca2+
ions act as an ionic cross linker which leading to the solidi-
fication process of the alginate beads. As represented in
Fig.6c, it was observed that increasing Ca2+ concentration
up to 2 wt% significantly decreased the swelling capacity
of the tested composite beads because of increasing the
crosslinking density and hence decreasing water uptake
ratios. 2 wt% CaCl2 has lower swelling capacity and higher
mechanical stability due to higher crosslinking degree
while 0.5 wt% CaCl2 has higher swelling ratio and in turn
lower mechanical stability because of lower crosslinking
density and then faster rapture of the hydrogel beads. So, 1
wt% CaCl2 was considered as optimal concentration to get
suitable exchange capacity required for the bioremediation
process, the same results were reported by Nunes etal.
[62] who reported that, cross-linked polymer networks can
be characterized by swelling measurements, which are use-
ful for understanding drug release and diffusion transport
processes via macromolecular materials. By monitoring
the effect of ATP clay on the swelling behavior of the clay
composite hydrogel beads; it was observed that, the swell-
ing ratios decreased by increasing the clay concentration
as represented in Fig.6d. These results can be explained as
the incorporation of ATP clay to the composite hydrogels
act as an additional physical cross linker leading to more
condenser network and hence decreasing the water uptake.
The same results were demonstrated by Golafshan etal.
[63] who found that laponite concentrations higher than
0.5% significantly reduced capacity of hydrogel beads to
swell during their study of nano hybrid hydrogels of PVA-
Alginate-Laponite as a potential wound healing material.
So, 0.25% clay was considered as optimum concentration
Fig. 7 FTIR spectra of pure
alginate, pure PVA, and cross-
linked PVA-alginate hydrogel
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Journal of Polymers and the Environment
1 3
Fig. 8 SEM images of a dry PVA-alginate beads, b lyophilized beads, c and d immobilized bacterial cells inthe hydrogel beads, e and f ATP clay distribution in the hydrogel composite beads
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Journal of Polymers and the Environment
1 3
in our study as it also provides higher degradation rate as
previously reported.
FTIR Analysis
The FTIR spectra of pure Alginate, PVA, and CaCl2
crosslinked PVA-alginate beads are represented in Fig.7.
The spectra associated with alginate showed that, Symmetric
and a symmetric stretching band of assigned to carboxylate
groups appeared at 1406 and 1612 cm−1, respectively. More-
over, the appeared peak at 1022 cm−1 is related to C–O–C
stretching vibration [64]. A characteristic broad strong band
assigned to hydroxyl group stretching vibration appeared at
3200–3600 cm−1 and the peak at 2987 cm−1 is correspond-
ing to C–H stretching [65].
The FTIR spectra associated with pure PVA showed
that, a broad band at 2887 cm−1 represents C–H stretch-
ing vibration of the alkyl groups of the PVA [59] while
carboxyl (C–O) stretching band appeared approximately
at 1107 cm−1. The peak at 1466 cm−1 represents the C-H
bending, while the band at 1718 cm−1 assigned from C=O
stretching vibration [66].
The spectrum of PVA-SA beads shows abroad absorption
band in the region of 3200 to 3600 cm−1 which is related to
stretching vibration of –OH groups of both PVA and alginate
[23]. Moreover, absorption bands at 1600 and 1410 cm−1
represents asymmetric and symmetric stretching vibrations
of –COO groups [21]. The band recorded at 1248 cm−1
may be related to H–bonding between –O–H groups of
alginates and PVA. A strong absorption peak that recorded
at 1066 cm−1 belongs to –C–O stretching [67]. Two peaks
were determined at 2980 and 2906 cm−1 which attributed to
–C–H stretching vibration. The incorporation of ATP clay
doesn’t chemically affect the polymer matrix as it acts as
physical filler.
SEM Investigation
The micrographs of PVA-alginate composite hydrogel beads
were investigated using SEM micrographs as are represented
in Fig.8. The surface morphology of the dry beads is rep-
resented in Fig.8a shows smooth surface that indicating
crosslinking reaction between polymers chains with tiny
pores in nano size. Figure8b represents porous network of
lyophilized wet PVA-alginate beads before bacterial immo-
bilization, which has a similar morphology as observed by
Shivakumara etal. [65]. However, the immobilized bacterial
cells showed irregular and rough surface indicating wide
distribution of bacterial cells inside the alginate composite
polymeric matrix and the proper immobilization process as
represented in Fig.8c and d. SEM images of the clay-con-
taining hydrogel beads as shown in Fig.8e and f, confirmed
the distribution of the ATP clay in the PVA-alginate matrix
Fig. 9 TGA results of PVA-
Alginate beads with different
ATP clay at (0, 0.25, 0.5, 1 and
2 wt. %)
Table 1 TGA thermal results of composite PVA-alginate beads with
different ATP clay concentrations at (0, 0.25, 0.5, 1and 2 wt%)
Clay con-
tent (wt%)
Tonset (oC) T50% (oC) Total weight
loss (%)
0 185 252 81.5 18.5
0.25 208 416 63.2 36.8
0.5 210 413 64.1 35.9
1 216 464 58.9 41.1
2 223 475 57 43
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Journal of Polymers and the Environment
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with improving the hydrogel physical properties (such as
the mechanical and thermal stability) because of sufficient
interaction between the ATP clay as a physical crosslinker
and the polymer matrix. Similar results were previously
determined by Golafshan etal. [63].
Thermal Gravimetric Analysis
TGA analysis was used to investigate the effect of ATP clay
on the thermal decomposition of PVA-alginate composite
hydrogel beads. TGA curves of dried PVA-alginate beads
with different ATP content (0, 0.25, 0.5, 1 and 2 wt%) are
represented in Fig.9 and summarized in Table1. The ther-
mal decomposition of PVA- alginate hydrogels occur in
three major steps. The first decomposition stage or dehydra-
tion stage between 100 and 200°C is due to the evaporation
of water and stored humidity. The second decomposition
stage or de-polymerization stage between 200 and 380°C is
related to the PVA elimination reactions and the cleavage of
the main chains of alginate [68].The third stage or pyrolysis
stage is between 380 and 600°C where NaHCO3 and car-
bonized residues formed [23].
For 0 wt% ATP clay there is a sharp steep step with an
onset temperature around 185°C which increases by increas-
ing ATP content recording 223°C for 2 wt% ATP. The final
residual yields are (18.5, 36.8, 35.9, 41.1 and 43 wt%) for
(0, 0.25, 0.5, 1and 2 wt%), respectively. Also, it is remarked
that, the total weight loss percentages after the third
decomposition stage decreased from 81.5 to 57 wt% indicat-
ing that the beads with higher clay content have higher onset
temperature, lower weight loss and hence higher residual
yield than clay free beads. So, we can conclude that incor-
poration of small amount of ATP clay into PVA-alginate
beads remarkably improve their thermal stability and pro-
longed their degradation time due to increasing the inorganic
matter content or the clay content, which is thermally most
stable and not decomposed. The same results were previ-
ously reported by Du etal. [69], who demonstrated that the
thermal stability of poly acrylic acid composite membranes
enhanced by increasing laponite concentration in the mem-
branes. Kamoun etal. [17] also reported that the interac-
tion between the polymers in the nanocomposite and clay as
nano-filler might result in enhancing thermal decomposition.
GC–MS Analysis
The most suitable approach to measure bioremediation effi-
ciency is to monitor the hydrocabons disappearance rates
which can be demonstrated using GC–MS analysis as rep-
resented in Figs.10 and 11. By comparing the crude oil
control sample (Figs.10a and 11a) with the bioremediation
metabolites ( Figs.10b and 11b) for Pseudomonas stutzeri
and Rhodococcus qingshengii, respectively; it can be seen
that after 28days of incubation under the optimium condi-
tions, the peaks associated with lighter hydrocarbons com-
ponents with low retention times have been disapeared. Also
Fig. 10 GC-MS chromatogrames for a crude oil control sample,and b S bacterial metabolites
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Journal of Polymers and the Environment
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Table 2 Examples of the major
hydrocarbons identified in the
crude oil sample that has been
completely degraded after the
bioremediation process
Retention time Compound name Molecular formula Molecu-
lar weight
14.21 Dodecane C12H26 170
14.21 Dodecane,2,6,11trimethyl C15H32 212
17.69 Tetradecane C14H30 198
21.00 1-Hexene C9H18 126
21.00 3,4,5-trimethy-n-Butyric acid C12H24O2200
22.95 2-pentyl undecyl ester C16H34O3S 306
24.12 Hexadecane C16H34 226
25.99 2-(Prop-2-enoyloxy)
27.06 3-Hexanone,
28.41 Heptadecane,
C21H44 296
29.96 Pyrrolidine,
37.37 n-Hexadecanoic acid C16H32O2256
34.97 Octadecane,1-iodo C18H37I 380
37.49 Sulfurous acid,octadecyl 2pentylester C23H48O3S 404
39.05 Tetrapentacontane,
40.97 Dodecane,1—cyclopentyl-4-(3-cyclopen-
C25H48 348
41.06 5-Heptadecene,1-bromo C17H33Br 316
41.91 Tetratetracontane C44H90 618
46.05 Octadecane,3-ethyl-5-(ethyl butyl) C26H54 366
50.54 Oleic acid,
3-(octadecyloxy)propyl ester
51.12 Ethyl isoallocholate C26H44O5436
Fig. 11 GC-MS chromatogrames for a crude oil control sample,and b R bacterial metabolites
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Journal of Polymers and the Environment
1 3
Pseudomonas stutzeri and Rhodococcus qingshengii, the
heavy compontents that represenet the long-chain hydro-
carbons were broken down into lower intermidates and less
complexity with lower concentrations that showed new
appearing peaks. The same results were reported by Lee
etal. [70] who reported that the long chain hydorcarbons
have been disappeared after their biodegradation, and new
peaks of short-chain hydrocarbons were appeared. Table2
show examples of the major hydrocarbons identified in the
crude oil sample that has been completely degraded after
the bioremediation process. However, some of the high
molecular weight hydrocarbons ranging from C15 to C44
were still present, but n-alkanes, iso-alkanes, alkenes and
some aromatic hydrocarbons were sussessfully disappered
in contrast to control sample. These results confirmed that,
both Pseudomonas stutzeri and Rhodococcus qingshengii
utilized different types of hydrocarbons as sole carbon and
energy sources but Pseudomonas stutzeri showed slightly
higher degradation potentail than Rhodococcus qingshengii
bacterial isolate. Our results are consistent with Popoola
etal. [71] and also Ilyas etal. [72] who demostrated the
same results in their study of crude oil bioremediation.
All these results conclude the effectivness of the immo-
bilization of the bacterial cells inside the PVA/Alginate/
Attapulgite clay mineral clay that has been fully character-
ized and showed higher crude oil biodegradation capability
compared with free bacterial cells.
In summary, crude oil degrading bacteria was success-
fully immobilized into PVA-alginate beads cross-linked
with CaCl2 and physically reinforced by different types
of clays especially ATP mineral clay. Also, the optimum
conditions required for the isolated bacterial isolates for
more efficient bioremediation process were studied and the
obtained results proved that moderate shaking at 150rpm
and incubation at 30°C at neutral pH are the most favora-
ble conditions for our tested bacterial isolates in terms
of the crude oil biodegradation. The prepared beads have
been characterized using FTIR, TGA, and SEM. The
GC–MS analyses of the tested crude oil before and after
bacterial biodegradation proved the ability of the tested
bacteria to degrade huge numbers of the existed hydro-
carbons as sole carbon and energy sources. The results
also proved that the incorporation of ATP clay into the
polymer matrix increased their mechanical, thermal sta-
bility, and decreased their swelling ability in addition to
improving the bioremediation efficiency. So, the above-
mentioned optimum conditions are recommended as an
ecofriendly approach to get rid of crude oil pollution in
case of sudden leakage. We recommend more attentions to
the use of different types of clays especially Hydroxyapa-
tite and Laponite RD in the bioremediation process for
future studies and improving the bioremediation efficiency
of the bacterial isolates using different conditions.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s10924- 023- 02991-y.
Author contributions EF conducted the experimental part and written
the original and final draft; EAK and AED: study design, characteri-
zation and supervision and reviewed the original manuscript; THT:
experimental design, microbiology experiments and supervision; and
TK: Supervision.
Funding Open access funding provided by The Science, Technology &
Innovation Funding Authority (STDF) in cooperation with The Egyp-
tian Knowledge Bank (EKB). The authors declare that no specific fund
was received for conducting this research.
Data Availability No data was used for the research described in the
Conflict of interest The authors declare that they have no conflict of
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1. Ejaz M, Zhao B, Wang X, Bashir S, Haider FU, Aslam Z, Khan
MI, Shabaan M, Naveed M, Mustafa A (2021) Isolation and
characterization of oil-degrading Enterobacter sp. from naturally
hydrocarbon-contaminated soils and their potential use against the
bioremediation of crude oil. Appl Sci 11:3504
2. Hassanshahian M, Emtiazi G, Kermanshahi R, Cappello S (2010)
Comparison of oil degrading microbial communities in sediments
from the Persian Gulf and Caspian. Sea Soil Sediment Contam
3. Abdeen Z, El-Sheshtawy HK, Moustafa Y (2014) Enhancement
of crude oil biodegradation by immobilizing of different bacterial
strains on porous PVA hydrogels or combining of them with their
produced biosurfactants. J Pet Environ Biotechnol 5:1
4. Mehrotra T, Zaman MN, Prasad BB, Shukla A, Aggarwal S,
Singh R (2020) Rapid immobilization of viable Bacillus pseudo-
mycoides in polyvinyl alcohol/glutaraldehyde hydrogel for bio-
logical treatment of municipal wastewater. Environ Sci Pollut Res
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Journal of Polymers and the Environment
1 3
5. Chen B, Yuan M, Qian L (2012) Enhanced bioremediation of
PAH-contaminated soil by immobilized bacteria with plant resi-
due and biochar as carriers. J Soils Sediments 12:1350–1359
6. Das M, Adholeya A (2015) Potential uses of immobilized bacteria,
fungi, algae, and their aggregates for treatment of organic and
inorganic pollutants in wastewater: water challenges and solutions
on a global scale. ACS Publications, Washington, pp 319–337
7. Mehrotra T, Dev S, Banerjee A, Chatterjee A, Singh R, Aggarwal
S (2021) Use of immobilized bacteria for environmental bioreme-
diation: a review. J Environ Chem Eng 9:105920
8. Liao H, Liu Y, Wang Q, Duan W (2018) Structure and prop-
erties of porous poly (vinyl alcohol) hydrogel beads prepared
through a physical–chemical crosslinking method. J Appl Polym
Sci 135:46402
9. Zain NAM, Suhaimi MS, Idris A (2011) Development and mod-
ification of PVA–alginate as a suitable immobilization matrix.
Process Biochem 46:2122–2129
10. Hu X, Long L, Gong T, Zhang J, Yan J, Xue Y (2020) Enhanced
alginate-based microsphere with the pore-forming agent for effi-
cient removal of Cu (II). Chemosphere 240:124860
11. Zommere Ž, Nikolajeva V (2017) Immobilization of bacterial
association in alginate beads for bioremediation of oil-contam-
inated lands. Environ Exp Bot 15:105–111
12. Partovinia A, Rasekh B (2018) Review of the immobilized
microbial cell systems for bioremediation of petroleum hydro-
carbons polluted environments. Crit Rev Environ Sci Technol
13. Sun Y, Lei C, Khan E, Chen S, Sang T, Lin D, Feng Y (2018)
Aging effects on chemical transformation and metal(loid) removal
by entrapped nanoscale zero-valent iron for hydraulic fracturing
wastewater treatment. Sci Total Environ 615:498–507
14. Zuo L (2020) Bioremediation of crude-oil polluted soil using
immobilized microbes. IOP Conf Ser 510(4):042047
15. Sakdapetsiri C, Kaokhum N, Pinyakong O (2021) Biodegradation
of crude oil by immobilized Exiguobacterium sp. AO-11 and shelf
life evaluation. Sci Rep 11(1):1–13
16. Cheng Y, Lin H, Chen Z, Megharaj M, Naidu R (2012) Biodeg-
radation of crystal violet using Burkholderia vietnamiensis C09V
immobilized on PVA–sodium alginate–kaolin gel beads. Ecotoxi-
col Environ Saf 83:108–114
17. Kamoun EA, Menzel H (2012) HES-HEMA nanocomposite poly-
mer hydrogels: swelling behavior and characterization. J Polym
Res 19:1–14
18. Zhu Z, Dai J, Liu Y, Sun H, Liang W, Li A (2015) Hydrophobic
spongy attapulgite for absorption of organics and oils from water.
19. Mulamattathil SG, Bezuidenhout C, Mbewe M, Ateba CN (2014)
Isolation of environmental bacteria from surface and drinking
water in Mafikeng, South Africa, and characterization using their
antibiotic resistance profiles. J Pathog. https:// doi. org/ 10. 1155/
2014/ 371208
20. Mishra A, Saxena A, Singh SP (2019) Isolation and characteri-
zation of microbial strains from refinery effluent to screen their
bioremediation potential. J Pure Appl Microbiol 13:2325–2332
21. Aljar MAA, Rashdan S, Abd El-Fattah A (2021) Environmentally
friendly polyvinyl alcohol−alginate/bentonite semi-interpenetrat-
ing polymer network nanocomposite hydrogel beads as an efficient
adsorbent for the removal of methylene blue from aqueous solu-
tion. Polymers 13:4000
22. Mollaei M, Abdollahpour S, Atashgahi S, Abbasi H, Masoomi F,
Rad I, Lotfi AS, Zahiri HS, Vali H, Noghabi KA (2010) Enhanced
phenol degradation by Pseudomonas sp. SA01: gaining insight
into the novel single and hybrid immobilizations. J Hazard mater
23. Baigorria E, Cano LA, Sanchez LM, Alvarez VA, Ollier RP
(2020) Bentonite-composite polyvinyl alcohol/alginate hydrogel
beads: preparation, characterization and their use as arsenic
removaldevices. Environ Nanotechnol Monit Manag 14:100364
24. Narra K, Dhanalekshmi U, Rangara G, Raja D, Kuma CS,
Reddy PN, Mandal AB (2012) Effect of formulation variables on
rifampicin loaded alginate beads. Iran J Pharm Res 11(3):715
25. Cunningham C, Ivshina I, Lozinsky V, Kuyukina M, Philp J
(2004) Bioremediation of diesel-contaminated soil by microor-
ganisms immobilised in polyvinyl alcohol. Int Biodeterior Bio-
degradation 54:167–174
26. Wang Y, Liu M, Ni B, Xie L (2012) κ-Carrageenan–sodium
alginate beads and superabsorbent coated nitrogen fertilizer with
slow-release, water-retention, and anticompaction properties. Ind
Eng Chem Res 51:1413–1422
27. Duncan DB (1955) Multiple range and multiple F tests. Biomet-
rics 11(1):1–42
28. Simarro R, González N, Bautista LF, Sanz R, Molina MC (2011)
Optimisation of key abiotic factors of PAH (naphthalene, phen-
anthrene and anthracene) biodegradation process by a bacterial
consortium. Water Air Soil Pollut 217:365–374
29. Das A, Dey A (2020) P-nitrophenol-bioremediation using potent
Pseudomonas strain from the textile dye industry effluent. J Envi-
ron Chem Eng 8:103830
30. Wong J, Lai K, Wan C, Ma K, Fang M (2002) Isolation and opti-
mization of PAH-degradative bacteria from contaminated soil for
PAHs bioremediation. Water Air Soil Pollut 139:1–13
31. Al-Hadithi H, Al-Razzaq E, Fadhil G (2017) Bioremediation of
polycyclic aromatic hydrocarbons by Acinetobacter species iso-
lated from ecological sources. J Environ Biol 38:785
32. Fatajeva E, Gailiūtė I, Paliulis D, Grigiškis S (2014) The use of
Acinetobacter sp. for oil hydrocarbon degradation in saline waters.
Biologija. https:// doi. org/ 10. 6001/ biolo gija. v60i3. 2971
33. Huang S, Wang Y, Tang C, Jia H, Wu L (2021) Speeding up
selenite bioremediation using the highly selenite-tolerant strain
Providencia rettgeri HF16-A novel mechanism of selenite reduc-
tion based on proteomic analysis. J Hazard Mater 406:124690
34. Tekere M, Jacob-Lopes E, Zepka LQ (2019) Microbial biore-
mediation and different bioreactors designs applied. Biotechnol
Bioeng. https:// doi. org/ 10. 5772/ intec hopen. 83661
35. Abdel-Razek A, El-Sheikh H, Suleiman W, Taha TH, Mohamed
M (2020) Bioelimination of phenanthrene using degrading bac-
teria isolated from petroleum soil: safe approach. Desalin Water
Treat 181:131–140
36. Abtahi H, Parhamfar M, Saeedi R, Villasenor J, Sartaj M, Kumar
V, Coulon F, Parhamfar M, Didehdar M, Koolivand A (2020)
Effect of competition between petroleum-degrading bacteria and
indigenous compost microorganisms on the efficiency of petro-
leum sludge bioremediation: field application of mineral-based
culture in the composting process. J Environ Manage 258:110013
37. El-Liethy MA, El-Noubi MM, A.L.K Abia,et al. (2022) Eco-
friendly bioremediation approach for crude oil-polluted soils
using a novel and biostimulated Enterobacter hormaechei ODB
H32 strain. Int J Environ Sci Technol 19(11):10577–10588
38. Hazim RN, Al-Ani MA (2019) Effect of petroleum hydrocarbons
contamination on soil microorganisms and biodegradation. Rafid-
ain J Sci 28:13–22
39. Abarian M, Hassanshahian M, Esbah A (2019) Degradation of
phenol at high concentrations using immobilization of Pseu-
domonas putida P53 into sawdust entrapped in sodium-alginate
beads. Water Sci Technol 79(7):1387–1396
40. Ibrahim IM, Konnova SA, Sigida EN, Lyubun EV, Muratova AY,
Fedonenko YP, Elbanna К (2020) Bioremediation potential of a
halophilic Halobacillus sp. strain, EG1HP4QL: exopolysaccha-
ride production, crude oil degradation, and heavy metal tolerance.
Extremophiles 24:157–166
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Journal of Polymers and the Environment
1 3
41. Sed A (2015) Biodegradation of Phenanthrene by Klebsiella sp.
isolated from organic contaminated sediment. J Adv Biol Biotech-
nol 4:1–12
42. Baoune H, Aparicio JD, Pucci G, Ould El Hadj-Khelil A, Polti
MA (2019) Bioremediation of petroleum-contaminated soils using
Streptomyces sp. Hlh1. J Soils Sediments 19:2222–2230
43. El-Sheshtawy H, Aman D, Nassar H (2022) A novel bioreme-
diation technique for petroleum hydrocarbons by bacterial con-
sortium immobilized on goethite-chitosan nanocomposite. Soil
Sediment Contam 31:176–199
44. Usmani Z, Kulp M, Lukk T (2021) Bioremediation of lindane con-
taminated soil: exploring the potential of actinobacterial strains.
Chemosphere 278:130468
45. Xu L, Zhu L (2009) Structures of OTMA-and DODMA-bentonite
and their sorption characteristics towards organic compounds. J
Colloid Interface Sci 331:8–14
46. Acikyildiz M, Gurses A, Yolcu H (2015) Synthesis of super
hydrophobic clay by solution intercalation method from aqueous
dispersions. Acta Phys Pol A 127:1156–1160
47. Warr LN, Perdrial JN, Lett MC, Heinrich-Salmeron A, Khodja
M (2009) Clay mineral-enhanced bioremediation of marine oil
pollution. Appl Clay Sci 46(4):337–345
48. Churchman GJ, Gates WP, Theng BKG, Yuan G (2006) Clays and
clay minerals for pollution control. Develop Clay Sci 1:625–675
49. Murray HH (2000) Traditional and new applications for kaolin,
smectite, and palygorskite: a general overview. Appl Clay Sci
50. Owens EH, Lee K (2003) Interaction of oil and mineral
fines on shorelines: review and assessment. Mar Pollut Bull
51. Elbassyoni S, Kamoun EA, Taha TH, Rashed MA, ElNozahi FA
(2020) Effect of Egyptian attapulgite clay on the properties of
PVA-HES–clay nanocomposite hydrogel membranes for wound
dressing applications. Arab J Sci Eng 45:4737–4749
52. Ruan B, Wu P, Chen M, Lai X, Chen L, Yu L, Gong B, Kang
C, Dang Z, Shi Z (2018) Immobilization of Sphingomonas sp.
GY2B in polyvinyl alcohol–alginate–kaolin beads for efficient
degradation of phenol against unfavorable environmental factors.
Ecotoxicol Environ Saf 162:103–111
53. Lin H, Chen Z, Megharaj M, Naidu R (2013) Biodegradation
of TNT using Bacillus mycoides immobilized in PVA–sodium
alginate–kaolin. Appl Clay Sci 83:336–342
54. Costa-Gutierrez SB, Aparicio JD, Delgado OD, Benimeli CS,
Polti MA (2021) Use of glycerol for the production of actinobac-
teria with well-known bioremediation abilities. 3 Biotech 11:1–10
55. Philip L (2006) Bioremediation of endosulfan contaminated soil
and water—optimization of operating conditions in laboratory
scale reactors. J Hazard Mater 136:354–364
56. Gouda MK, Omar SH, Chekroud ZA, Eldin HMN (2007) Biore-
mediation of kerosene I: a case study in liquid media. Chemos-
phere 69:1807–1814
57. Talha MA, Goswami M, Giri B, Sharma A, Rai B, Singh R (2018)
Bioremediation of Congo red dye in immobilized batch and con-
tinuous packed bed bioreactor by Brevibacillus parabrevis using
coconut shell bio-char. Biores Technol 252:37–43
58. Padmanaban V, Geed SR, Achary A, Singh R (2016) Kinetic stud-
ies on degradation of reactive red 120 dye in immobilized packed
bed reactor by Bacillus cohnii RAPT1. Biores Technol 213:39–43
59. Bayat Z, Hassanshahian M, Cappello S (2015) Immobilization of
microbes for bioremediation of crude oil polluted environments:
a mini review. Open Microbiol J 9:48–54
60. Knijnenburg JT, Kasemsiri P, Amornrantanaworn K, Suwanree
S, Iamamornphan W, Chindaprasirt P, Jetsrisuparb K (2021)
Entrapment of nano-ZnO into alginate/polyvinyl alcohol beads
with different crosslinking ions for fertilizer applications. Int J
Biol Macromol 181:349–356
61. Omidian H, Rocca JG, Park K (2006) Elastic, superporous hydro-
gel hybrids of polyacrylamide and sodium alginate. Macromol
Biosci 6:703–710
62. Nunes MA, Vila-Real H, Fernandes PC, Ribeiro MH (2010)
Immobilization of naringinase in PVA–alginate matrix using an
innovative technique. Appl Biochem Biotechnol 160:2129–2147
63. Golafshan N, Rezahasani R, Esfahani MT, Kharaziha M, Kho-
rasani S (2017) Nanohybrid hydrogels of laponite: PVA-Alg-
inate as a potential wound healing material. Carbohyd Polym
64. Mahdavinia GR, Mousanezhad S, Hosseinzadeh H, Darvishi F,
Sabzi M (2016) Magnetic hydrogel beads based on PVA/sodium
alginate/laponite RD and studying their BSA adsorption. Carbo-
hyd Polym 147:379–391
65. Shivakumara LR, Demappa T (2019) Synthesis and swelling
behavior of sodium alginate/poly (vinyl alcohol) hydrogels. Turk
J Pharm Sci 16:252
66. Hussein Y, El-Fakharany EM, Kamoun EA, Loutfy SA, Amin
R, Taha TH, Salim SA, Amer M (2020) Electrospun PVA/hyalu-
ronic acid/L-arginine nanofibers for wound healing applications:
nanofibers optimization and invitro bioevaluation. Int J Biol Mac-
romol 164:667–676
67. Rahman N, Wilfred CD (2018) Removal of Mn (VII) from indus-
trial wastewater by using alginate-poly (vinyl) alcohol as absor-
bent. J Phys. https:// doi. org/ 10. 1088/ 1742- 6596/ 1123/1/ 012067
68. Levic S, Djordjevic V, Rajic N, Milivojevic M, Bugarski B,
Nedovic V (2013) Entrapment of ethyl vanillin in calcium algi-
nate and calcium alginate/poly (vinyl alcohol) beads. Chem Pap
69. Du J, Zhu J, Wu R, Xu S, Tan Y, Wang J (2015) A facile approach
to prepare strong poly (acrylic acid)/LAPONITE® ionic nano-
composite hydrogels at high clay concentrations. RSC Adv
70. Lee DW, Lee H, Kwon B-O, Khim JS, Yim UH, Kim BS, Kim
J-J (2018) Biosurfactant-assisted bioremediation of crude oil by
indigenous bacteria isolated from Taean beach sediment. Environ
Pollut 241:254–264
71. Popoola LT, Yusuff AS (2021) Optimization and characterization
of crude oil contaminated soil bioremediation using bacteria iso-
lates: plant growth effect, South African. J Chem Eng 37:206–213
72. Saeed M, Ilyas N, Arshad M, Sheeraz M, Ahmed I, Bhattacharya
A (2021) Development of a plant microbiome bioremediation sys-
tem for crude oil contamination. J Environ Chem Eng 9:105401
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Petroleum oil contaminants have become severe ecological problems and negatively impact human health. It is, therefore, imperative to identify environmentally friendly approaches to remediate oil-polluted environments. Therefore, bacterial oil degradation stimulated with a nitrogen source under optimum conditions was assessed in this study. Based on the 16S rRNA analysis, strain ODB H32 recovered from oil-based mud of some petroleum drilling sites in the western desert, Egypt, was identified as Enterobacter hormaechei. The metabolic fingerprint of E. hormaechei, achieved using BIOLOG GEN III, revealed that the strain could utilize diverse carbon and chemical sources. Also, E. hormaechei could biodegrade 0.6% of oil under optimized pH (7.0) and temperature (30 °C) conditions. Analyzing different nitrogen stimulants revealed that peptone ˃ yeast extract ˃ ammonium nitrate ˃ urea enhanced the growth of E. hormaechei on mineral salts medium (MSM). Analysis by capillary gas chromatography revealed maximum (70.7%) degradation of peptone by E. hormaechei, indicating that peptone was a good biostimulant for oil degradation. These findings recommend using biostimulated E. hormaechei as an eco-friendly approach for remediating oil-polluted environments, under optimized conditions, especially in arid regions like the western desert of Egypt.
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Hazardous chemicals like toxic organic dyes are very harmful to the environment and their removal is quite challenging. Therefore there is a necessity to develop techniques, which are environment friendly, cost-effective and easily available in nature for water purification and remediation. The present research work is focused on the development` and characterization of the ecofriendly semi-interpenetrating polymer network (semi-IPN) nanocomposite hydrogels composed of polyvinyl alcohol (PVA) and alginate (Alg) hydrogel beads incorporating natural bentonite (Bent) clay as a beneficial adsorbent for the removal of toxic methylene blue (MB) from aqueous solution. PVA−Alg/Bent nanocomposite hydrogel beads with different Bent content (0, 10, 20, and 30 wt%) were synthesized via external ionic gelation method. The designed porous and steady structure beads were characterized by the use of Fourier transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDX), and scanning electron microscopy (SEM). The performance of the beads as MB adsorbents was investigated by treating aqueous solutions in batch mode. The experimental results indicated that the incorporation of Bent (30 wt%) in the nanocomposite formulation sustained the porous structure, preserved water uptake, and increased MB removal efficiency by 230% compared to empty beads. Designed beads possessed higher affinity to MB at high pH 8, 30 °C, and fitted well to pseudo-second-order kinetic model with a high correlation coefficient. Moreover, the designed beads had good stability and reusability as they exhibited excellent removal efficiency (90%) after six consecutive adsorption-desorption cycles. The adsorption process was found be combination of both monolayer adsorption on homogeneous surface and multilayer adsorption on heterogeneous surface. The maximum adsorption capacity of the designed beads system as calculated by Langmuir isotherm was found to be 51.34 mg/g, which is in good agreement with the reported clay-related adsorbents. The designed semi-IPN PVA−Alg/Bent nanocomposite hydrogel beads demonstrated good adsorbent properties and could be potentially used for MB removal from polluted water.
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Exiguobacterium sp. AO-11 was immobilized on bio-cord at 10 ⁹ CFU g ⁻¹ carrier for the removal of crude oil from marine environments. To prepare a ready-to-use bioremediation product, the shelf life of the immobilized cells was calculated. Approximately 90% of 0.25% (v/v) crude oil removal was achieved within 9 days when the starved state of immobilized cells was used. The oil removal activity of the immobilized cells was maintained in the presence of oil dispersant (89%) and at pH values of 7–9. Meanwhile, pH, oil concentration and salinity affected the oil removal efficacy. The immobilized cells could be reused for at least 5 cycles. The Arrhenius equation describing the relationship between the rate of reaction and temperature was validated as a useful model of the kinetics of retention of activity by an immobilized biocatalyst. It was estimated that the immobilized cells could be stored in a non-vacuum bag containing phosphate buffer (pH 7.0) at 30 °C for 39 days to retain the cells at 10 ⁷ CFU g ⁻¹ carrier and more than 50% degradation activity. These results indicated the potential of using bio-cord-immobilized crude oil-degrading Exiguobacterium sp. AO-11 as a bioremediation product in a marine environment.
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This study investigated the effect of soil pH, nitrogen-phosphorus ratio, crude oil concentration and bacteria isolates (from petroleum hydrocarbon-contaminated soil) concentration on the optimization of crude oil-contaminated soil bioremediation using central composite design. The responses were total petroleum hydrocarbons (TPH) and plant growth. A 60-day pot experiment was conducted. Natural soil, crude oil-contaminated soil and bioremediated soil were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), energy dispersive X-ray (EDX), carbon-hydrogen-nitrogen (CHN) analyser and gas chromatograph-mass spectroscopy (GC-MS). Optimum predicted values were 4.69, 7.68 g/g, 250.05 mL/L and 568.35 cell/g for soil pH, nitrogen-phosphorus ratio, crude oil concentration and bacteria isolates concentration respectively. Experimental run at optimum point affirmed the accuracy of the developed models. Characterization revealed contamination of natural soil by hydrocarbons and their biodegradation via the action of active functional groups present in bacteria isolates. Conclusively, the bacteria isolates could be applied as effective scavenger for hydrocarbon biodegradation in crude oil contaminated soil.
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The contamination of crude oil in soil matrices is a persistent problem with negative repercussions because of the recalcitrant, hazardous, and mutagenic properties of its constituents. To mitigate the effect of crude oil contamination in soil, the use of microorganisms is a cheap and feasible option. In the current study, bacterial species from numerous polluted oil field surfaces were isolated and examined for their ability to degrade crude oil. Random soil samples polluted with hydrocarbons were collected and various bacterial isolates were isolated. Results revealed that 40% of total isolates had potential use for hydrocarbon biodegradation, the synthesis of exopolysaccharides and the solubilization of phosphorous. Following isolation and characterization to degrade crude oil, a pot trial was conducted using maize inoculated with the four best strains—i.e., S1 (PMEL63), S2 (PMEL-67), S3 (PMEL-80), and S4 (PMEL-79)—in artificially hydrocarbon-polluted soil with concentrations of crude oil of 0, 1000, and 2000 ppm. Results revealed that S4 (PMEL-79) had significant potential to degrade hydrocarbon in polluted soils. The root length, shoot length, and fresh biomass of maize were increased by 65%, 45%, and 98%, respectively, in pots inoculated with S4 (PMEL-79) Enterobacter cloacae subsp., whereas the lowest root length was observed where no strain was added and the concentration of crude oil was at maximum. Moreover, S4 (PMEL-79) Enterobacter cloacae subsp. was found to be the most effective strain in degrading crude oil and increasing maize growth under polluted soil conditions. It was concluded that the isolation of microorganisms from oilcontaminated sites should be considered in order to identify the most effective microbial consortium for the biodegradation of naturally hydrocarbon-contaminated soils.
Environmental pollution by petroleum hydrocarbon is one of the significant concerns of the contemporary world. This paper deals with the relevant environmental issues concerning the oil pollution of the petroleum industry. There is a need for further development of sustainable remediation technologies. Nineteen petroleum-degrading bacteria were isolated from an oil-polluted soil in the Suez oil processing 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 biodegradation 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.
Bioremediation is traditionally carried out using ‘free’ bacterial cells; however, in recent years, utilization of ‘immobilized’ bacterial cells has gained attention as a promising technique due to multifarious benefits. This review collates a vast amount of existing literature on the myriad contaminants treated using immobilized bacteria. We also discuss various mechanistic aspects of using immobilized cells for environmental remediation applications, with special attention on cells encapsulated in hydrogels and their implementation in detoxifying harmful contaminants and environmental cleanup. We examine different methods/techniques for immobilizing viable bacterial cells in various supporting matrices, use of single- and multi-species bacterial communities, various growth substrates, and factors affecting the remediation process including mass transfer, kinetic processes and bioreactor configurations used in pilot and field-scale applications. The advantages and limitations associated with the use of immobilized bacteria in a bioreactor for contaminated water treatment are also discussed. From a sustainable futures perspective, resource recovery and retrieval of value-added products along with bioremediation could be an added benefit of the immobilized cell-based treatment system, making it a more cost-effective and viable treatment strategy as well as one that is amenable to the principles of circular economy.
Lindane, an organochlorine pesticide, causes detrimental impacts on the environment and human health owing to its high toxicity, low degradation, and bioaccumulation. Its toxic nature can be overcome by biological and eco-friendly approaches involving its degradation and detoxification. The biodegradation of lindane was assessed using actinobacterial species Thermobifida cellulosilytica TB100 (T. cellulosilytica), Thermobifida halotolerans DSM 44931 (T. halotolerans) and Streptomyces coelicolor A3 (S. coelicolor). The degradation conditions of Lindane such as pH, temperature, inoculum volume, glucose concentration and number of days were optimized under broth conditions. Lindane degradation at different concentrations was studied in soil using reverse phase-high performance liquid chromatography over a 30 day period. A bioassay test was performed on seeds of Lactuca sativa (Lettuce) to assess the success of bioremediated soil. Maximum lindane degradation in soil was observed using T. cellulosilytica sp. The degradation trend for different concentrations of lindane using T. halotolerans in sterilized soil was 55 mg kg⁻¹ (82 %) ˃ 155 mg kg⁻¹ (75 %) ˃ 255 mg kg⁻¹ (70 %) after an incubation period of 30 days. Lindane degradation in soil followed the first order reaction kinetics. Phytotoxicity test on seeds of Lactuca sativa showed considerably good vigor index values for the bioremediated sterilized and non-sterilized soil by T. cellulosilytica, T. halotolerans and S. coelicolor in comparison to the contaminated soil without bacteria. This confirms that these actinobacterial species can be implemented in bioaugmentation of contaminated sites to efficiently remediate high lindane concentrations.
Zinc oxide nanoparticles (nano-ZnO) are attractive as fertilizer materials but high concentrations may negatively affect the environment. To reduce their dispersion in the environment we entrapped nano-ZnO in biodegradable polymer beads consisting of alginate and polyvinyl alcohol (PVA). The alginate/PVA/ZnO beads were prepared via ionotropic gelation using two different crosslinking ions (Ca²⁺ and Zn²⁺), and the effect of alginate crosslinking ion and PVA content on bead structure, water absorption, water retention and zinc release was investigated. The pure CaAlg and ZnAlg beads demonstrated a poor water absorption and retention, which were strongly enhanced by the incorporation of PVA into the beads. The continuous Zn release was measured in a sand column, and it was found that the Zn-crosslinked beads rapidly released high concentrations of Zn followed by a more gradual Zn release, whereas Ca alginates showed only a gradual Zn release. The Zn dissolution kinetics could be tuned by the crosslinking ion composition. The prepared nano-ZnO-containing alginate/PVA beads may be attractive for Zn fertilizer applications under water-limited conditions.
Bacterial assisted phytoremediation is recently being considered to be an efficient technique for remediation of crude oil-contaminated soil. The present research was designed to establish a plant microbiome bioremediation system for treating crude oil contamination. 10 strains of plant growth-promoting rhizobacteria (PGPR) were isolated from oil-contaminated soil near Oil Refinery Rawalpindi, Pakistan. Based on plant growth-promoting characteristics and biosurfactant production, two strains (Pseudoarthrobacter phenanthrenivorans (MS2) and Azospirillum oryzae (MS6)) were selected. They showed a better emulsification index (54.2, 42.5%), oil displacement activity (3.4, 2.6 mm) and hydrophobicity content (78, 75%,). For the establishment of the plant microbiome system, both strains and their combination were inoculated in rhizospheric soil of maize in crude oil-contaminated soil. Better germination attributes of maize were observed by a combination of both strains with improved fresh (32 %) and dry biomass (26.5 %) as compared to control under oil stress (10 %). Plant microbiome bioremediation system improved the chlorophyll content (30.4 %), water potential (23.2 %), proline (32 %), amino acids (11.1 %), and antioxidant enzymes (catalase (21 %), peroxidase dismutase (30 %) and superoxide dismutase (22 %), as compared to control under oil stress (10 %). The hydrocarbons degradation efficiency of this system was 38.5%. Analysis of degradation products by GC-MS revealed the presence of low molecular weight hydrocarbons in the treated soil as compared to untreated soil. This study showed promising results by this plant microbiome system can be a way forward in bacterial assisted phytoremediation approaches at the field level in future.