Inorganic Chemistry Communications 131 (2021) 108766

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Inorganic Chemistry Communications

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

Bacterial immobilization on cellulose acetate based nanofibers for

methylene blue removal from wastewater: Mini-review
Doaa Zamel a, *, Atta Ullah Khan b
a
Department of Chemistry, Faculty of Science, Helwan University, Egypt
b
Department of Biotechnology, University of Malakand, Pakistan

A R T I C L E I N F O A B S T R A C T

Keywords: Cellulose acetate (CA) has been reported as versatile substance that has wide applications ranging from diapers to
Cellulose Acetate Nanofibers membrane filters. In addition to that, cellulose acetate has many characteristics which make it unique and
Bacterial Immobilization excellent polymer for electrospinning and nanofibers preparations. The most important of them is that cellulose
Biofilm
acetate is biodegradable polymer which will not cause waste accumulation problem in the environment after
Methylene Blue Removal
usage and disposal, it also cost-effective polymer which is suitable for industrial applications, as well as it has
high affinity and adhesion properties in nanofibers morphology which strongly fit in the immobilization of
biomaterials by adhesion forces. In this review, the bacterial immobilization methods and possible applications
in the removal of methylene blue dye from wastewaters were summarized. Besides, cellulose acetate nanofibers’
capability in the immobilization of bacterial cells and their applications in decolorization and removal of
methylene blue dye from industrial wastewater will be explained here. As known, bacteria can be used in in­
dustry for pollutants removal from industrial wastewaters, and this process is called bioremediation. However,
free bacterial usage still not effectively applied in the industry because bacteria can make reproduction and
multiply by larger numbers and this may cause another source of pollution in water. Herein, immobilization of
bacteria is good manner to facilitate the handling of bacteria and harvesting during and after application in
wastewater. Furthermore, it increases their efficiency by increasing the overall exposure surface area. Immo­
bilization of bacteria will enrich their usage and applications in the removal of pollutants from industrial
wastewaters in the future.

1. Introduction
Recent advanced devices and technological apparatus have enabled
materials to be fabricated at the nanoscale [1]. Electrospinning has
extensive attention and motivation during the recent two decades for the
synthesis of nano and microscale fibers [2]. Nanomaterials have a lot of
merits when compared with bulk materials, such as; unique mechanical
properties, big surface area to volume ratio, adjustable surface
morphology, porosity, flexibility, and cost-effectiveness [3]. In partic­
ular, nanofibers with biodegradable and natural properties can be
applied in wide applications, such as; filtration [4], drug delivery [5],
adsorbents [6], tissue engineering [7], and wound healing [8]. Gener­
ally, nanofibers can be synthesized from high molecular weight mate­
rials like polymers, metal oxides, semiconductors, and carbon in several
forms, nanofibers, nanowires, and nanotubes [9]. The use of bacteria in
the industry is concerned with biotechnology while nanofibers synthesis
from polymeric materials is considered by nanotechnology. The inte­
gration of both branches of science which is called nano-biotechnology
that may lead to breakthroughs in many research areas such as envi­
ronment, medicine, engineering. Cellulose acetate is a biocompatible
and biodegradable material that is insoluble in water and only soluble in
organic solvents [2]. It is prepared by reaction of cellulose and acetic
anhydride together while the catalyst is sulfuric acid, whereas the sub­
stitution of the hydroxyl groups with acetate groups occurs on cellulose
[10]. That number of acetate groups gives the produced polymer its
definition as acetate, diacetate, and triacetate. Cellulose acetate can be
spun by electrospinning into nano-sized fibers however, natural cellu­
lose cannot. The reason is that cellulose acetate can dissolve in some

Abbreviations: CA, Cellulose acetate; DMF, N,N-dimethylformamide; DCM, Dichloromethane; DMSO, Dimethyl Sulfoxide; MB, Methylene blue; MeOH, Methanol;
DMAc, Acetone-dimethylacetamide; PVA, Poly(vinyl) alcohol; CD, Cyclodextrin; TFA, Trifluoroacetic acid; PEO, Poly(ethylene oxide.
* Corresponding author.
E-mail address: doaazamel@science.helwan.edu.eg (D. Zamel).

https://doi.org/10.1016/j.inoche.2021.108766
Received 21 May 2021; Received in revised form 24 June 2021; Accepted 26 June 2021
Available online 30 June 2021
1387-7003/© 2021 Elsevier B.V. All rights reserved.
D. Zamel and A.U. Khan
organic solvents in comparison with cellulose which is poorly soluble.
Cellulose acetate nanofibers have unique properties in bacterial immo­
bilization due to their high adhesion characteristics that enrich the
adhesion of bacterial cells on their surfaces [61]. Furthermore, cellulose
acetate nanofibrous webs can be commonly applied as membranes for
separation, filtration, food technology, and medical applications [11].
Scientists used bacteria in methylene blue dye removal from wastewater
for many years [27–33], but this biological treatment was not applied in
industrial fields as it lacks the bacterial cells handling in addition to how
to harvest and get rid of the bacterial cells after the treatment process till
the year of 2014 when San et al., [24] immobilized the bacteria on Poly
(vinyl) alcohol (PVA) nanofibrous membrane by attachment method
(adhesion) to it be handled, harvested and also reused for more than one
time. Electrospun cellulose acetate nanofibers are good supporting
material for the immobilization and encapsulation of bacterial cells
[13]. For instance, Tekinay et al. studied the immobilization of Aero­
monas eucrenophila, Pseudomonas aeruginosa and Clavibacter michi­
ganensis bacterial strains in cellulose acetate nanofibrous membrane for
decolorization ad removal of methylene blue dye from wastewaters
[14]. In another recent study, Zamel et al. [12] immobilized Bacillus
paramycoides bacterial strain in cellulose acetate nanofibers for decol­
orization of methylene blue dye from industrial wastewater, whereas the
results were promising and enriched the value of immobilization [13].
Immobilization of microorganisms, and regarding bacteria, have a lot of
merits; for example, it enhances the distribution of bacteria on the
support materials, also increases the working surface area, enhances the
handling of bacteria, and their washing and reusability for further times.
Furthermore, immobilization brings cost-effective merit for the bio­
materials as increases their chance for reusability as well as may
enhance efficiency in some cases. From here, scientists intended to
depend on the immobilization of microorganisms as solid and unique
solution for their applications and incorporation in many industrial
processes. In this review, cellulose acetate nanofibers and their appli­
cation in bacterial immobilization will be explained, and also their
possibility to be applied in the removal of methylene blue from indus­
trial wastewaters. Furthermore, the capability of bacterial immobiliza­
tion and encapsulation on polymeric nanofibers will be discussed
carefully.
2. Electrospinning of cellulose acetate
Cellulose acetate is the most common and wide ester derivative of
cellulose [1–5]. It has been a material of choice for electrospinning and
nanofibers production imputed to its biodegradability, biocompatibility,
good thermal stability, non-toxic, chemical persistence, and wide ap­
plications range starting from the good diapers to the advanced mem­
brane filters [7–9]. The electrospinning of cellulose acetate has opened
novel avenues for exploring its unique different properties (Fig. 1). It has
considered new applications to polymeric materials such as cellulose
acetate in the concept of multiple applications materials among many
Fig. 1. Electrospinning Diagram of Cellulose Acetate (CA).
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Inorganic Chemistry Communications 131 (2021) 108766
other chemical and natural substances [1,4,13]. These advantages make
the polymeric nanofibers excellent convenient for wide and different
ranges of applications such as; filters [9], drug carriers [5], composite
reinforcements [10], and tissue-engineered scaffolds [11]. Meanwhile,
cellulose acetate is commonly utilized in food and agricultural industries
because it is the most abundant and low-cost by-product [14].
2.1. Dissolving of cellulose acetate
The selection of a good solvent for the electrospinning of cellulose
acetate has been handled in research for many years. The selection of
suitable solvents has been subjected to several trials [15]. Previous
studies on CA electrospinning demonstrated using of different solvents
for cellulose acetate electrospinning. Rodríguez et al., [15] studied using
both single or mixed solvent systems and their effect on the fiber shape
and diameter. Previous studies on CA electrospinning showed individual
solvent systems such as acetone, chloroform, formic acid, chloroform-
MeOH, pyridine, N, N-dimethylformamide (DMF), dichloromethane
(DCM), and methanol (MeOH). Moreover, acetone-dimethylacetamide
(DMAc), Acetone: DMF (2:1) 12, and DCM-MeOH are considered to be
from the mixed solvent systems [16–20].
2.2. Preparation of cellulose acetate nanofibers
Cellulose acetate nanofibers could be synthesized from cellulose
acetate polymer dissolved in an organic solvent by electrospinning as
the most common technique for nanofibers preparation [5]. Some fac­
tors are controlling the electrospinning process including; polymer so­
lution concentration, applied voltage, feed rate, and the distance
between needle tip to the collector. Cellulose acetate nanofibers can be
synthesized from polymer concentration ranging from 10 to 20 wt%
depending on the polymer molecular weight and solvent viscosity.
Voltage can differ in a range between 20 and 35 kV, feed rate from 1 to 2
ml/h, and distance from 15 to 30 cm [16,17].
3. Immobilization of bacteria on nanofibers
Bacterial immobilization can be described by two methods; the
attachment method in which the bacterial cells adhere to the surface of
the nanofibers by covalent weak bonds. This method has a lot of dis­
advantages whereas the bacteria are weakly attached and they shall fall
of from nanofibers during the application. The encapsulation method is
another method where the bacterial cells can be trapped inside the
nanofibers matrix. Herein, the bacterial cells are wrapped by the
nanofibers and this enhances the pro-longed use and sustainability of the
bacteria/ nanofibers bio-composites. The difference between both
methods and bacterial position in nanofibers is shown in Fig. 2.
Fig. 2. Illustration of Bacterial Immobilization Methods.
D. Zamel and A.U. Khan
3.1. Attachment methods
In early reports, scientists investigated the immobilization of bacte­
ria by trapping them in a poly(tetrafluoroethylene) (PTFE) matrix.
Therefore, bacterial cells were blended with an aqueous solution of
PTFE while adding Triton X-100 as a surfactant to form a thick paste
[21]. Polymeric matrices (hydrogels) prepared of 2-hydroxyethyl
methacrylate and 2-hydroxyethyl acrylate while the solvent is water.
Glycerol and poly (vinyl alcohol) membranes were investigated as car­
riers for immobilization of bacterial cells in experiments of metal
decontamination [22]. Live bacteria have been immobilized on silicon
and gold substrates with high efficiency and specificity. The immobi­
lized bacterial cells are capable of dividing to form a bacterial mono­
layer on the patterned areas [23]. The disadvantages of those materials
which are explained as supporting materials are non-highly porous,
have low surface area/ volume ratio, and non-cost-effective due to the
input of high amounts of material. Therefore, scientists thought about
more effective nanomaterials which have several advantages to over­
come the previous drawbacks. Here come the nanofibers which have
been used in the immobilization of different substances such as metal
oxide nanoparticles, enzymes, and recently microorganisms. San et al.,
[24] reported the efficiency of the immobilized bacteria on poly(vinyl)
alcohol nanofibers by attachment method. They prepared the nanofibers
matrix and imbedded it inside bacterial media broth whereby the bac­
teria can adhere to the surface of PVA nanofibers. They also studied the
reusability of their first prepared membrane. Scanning electron micro­
scope images showed the PVA nanofibers before and after immobiliza­
tion of bacterial cells. Their study was considered the first study in
immobilization of bacteria on PVA nanofibers by attachment method as
they reported in their article. Gordegir et al, 2019 prepared nanofibers
from polyethyleneimine (PEI) and PCL polymer which helps in adding
functional amino groups on the surface. After that, Gluconobacter oxy­
dans was immobilized on the surface of the produced nanofibers and this
can be explained regarding the glutaraldehyde chemistry. The scanning
electron microscope, differential pulse voltammetry (DPV), and cyclic
voltammetry (CV) measurements were performed to evaluate the suc­
cess of the immobilization and the results showed that G. oxydans
immobilized nanofibers biosensor can be applied for sensing the glucose
molecule as metabolic analyte [18]. A recent study was performed on
the bacterial cellulose nanofibers and studying how to develop and
assess its capability to be considered as a carrier material for the adhe­
sion of the probiotic bacteria Lactobacillus acidophilus 016. Electro­
spinning of Bacterial cellulose (5%) and polyvinyl alcohol (PVA)
solutions was done by equal volume to produce nanofibers whereby
trifluoroacetic acid (TFA) was the solvent Bacterial cellulose (5%). L.
acidophilus 016 has successfully adhered to the prepared nanofibers
Fig. 3. Schematic Diagram for Immobilization of Bacteria by Attachment Method.
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Inorganic Chemistry Communications 131 (2021) 108766
through the adsorption-incubation technique. Scanning electron mi­
croscope images proved no damage to the immobilized bacteria on the
nanofibers. Furthermore, the results showed that the produced nano­
fibers could be applied in food technology for the delivery of probiotics.
See Fig. 3, showing the schematic diagram of bacterial immobilization
on nanofibers by attachment method.
3.2. Encapsulation methods
Sarioglu et al., 2017 prepared a bio-composite material by encap­
sulation of commercial strain of Pseudomonas aeruginosa within poly­
ethylene oxide (PEO) and polyvinyl alcohol (PVA) electrospun
nanofibrous membrane which has been applied in methylene blue (MB)
dye removal. Here, scanning electron microscopy (SEM) and fluores­
cence microscopy were used for monitoring of bacteria-encapsulated
nanofibers and the viability of encapsulated bacteria had been
confirmed using live/dead staining and cell counting assay [14]. Further
study showed that the efficiency of electrospinning technique in P.
agglomerans ISIB55 and B. caribensis ISIB40 immobilization and seed
coating with nanofiber-immobilized rhizobacteria while the viability or
unique properties of either rhizobacterium were not affected [20]. San
et al, 2017 [21] demonstrated the capability of immobilization of
Lysinibacillus sp. Bacterial strain into cyclodextrin nanofibers by
encapsulation method for heavy metals remediation (Ni (II), Cr (VI), and
RB5) from wastewater treatment. The bacterial cell viability in the
immobilized nanofibers extended for more than 7 days at 4 ◦ C. Recently,
Zamel et al, 2019 [12] studied the immobilization of Bacillus Para­
mycodis bacteria on cellulose acetate nanofibers using electrospinning
technique and the resulted nanofibrous membrane was applied in the
removal of methylene blue from wastewaters. In their study, the effi­
ciency of bacterial immobilization on nanofibers using the electro­
spinning technique was proved and the viability of the bacterial cells
after exposure to high voltage during the immobilization process using
the electrospinning. In Fig. 4, schematic diagram showing the bacterial
immobilization on nanofibers by encapsulation method.
4. Application of bacteria-immobilized cellulose acetate
nanofibers
4.1. Dye removal from wastewater
Dyes have been utilized in the industry for coloring several materials
such as leather, textiles, paper, plastics, and so on [34]. Every year, huge
amounts of commercial dyes worldwide are being manufactured
including different categories [35]. Among all of these dyes, azo dyes
come to be the most common dye category applied in the textile industry
D. Zamel and A.U. Khan
Fig. 4. Schematic Diagram for Immobilization of Bacteria by Encapsulation Method.
as they are stable and have low prices [29]. Most dyes are not efficient in
the process of dying as they remain some pigments that do not bind to
the targeted materials and this causes a percentage of 10–50% of dye as
a waste which after that enter the wastewater pathway [26]. Dyes
excreted in the textile industry may cause major problems not only to
wastewater resources but also on the environmental and human health
scales [36]. Around 5000 tons of dyes are discharged annually into the
wastewaters and the environment [37]. Their discharge to the envi­
ronment is a serious problem that has to be taken into consideration as
they may cause dangerous and toxic effects on both the environment and
human health [34]. These poisonous materials can decrease the oxygen
concentration from the water [24] and this is considered a major
problem as it threatens the environment and also human health. Major
problems accompanied with colored effluents are disruption of light
penetration which affects photosynthesis and distorts the beautiful and
pure scene of water resources [40]. As known, organic dyes have been
reported to be dangerous and threatens whole human beings, so we are
in a big need to remove the color from wastewater [25]. Methylene Blue
(MB) dye is the most popular dye utilized for dyeing silk, cotton, and
wood. Moreover, it may cause eye burns which may lead to permanent
injuries to the eyes of humans and animals [14,41]. In the case of
inhalation, it can give rise to short periods of rapid heartbeats or difficult
breathing. While ingestion through the mouth produces a burning
sensation and may cause nausea, vomiting, profuse sweating, mental
confusion, and methemoglobinemia [42]. Therefore, the treatment of an
effluent containing such dye (MB) is of great importance due to its
harmful impacts on receiving water [44,45]. Recently, many treatment
processes have been reported for dye-containing wastewaters coming
from the industry; such as chemical oxidation [26], biodegradation
[27], foam flotation [28], electro-coagulation [46], adsorption [47], and
photocatalysis by titanium dioxide [48].
The methods of methylene blue removal are classified into physical,
chemical, and biological methods.
4.1.1. Physical methods
Physical treatment methods include; membrane filtration, electrol­
ysis, reverse osmosis, and all adsorption processes. The known disad­
vantage in physical methods, especially in membrane filtration is the
limited life period of the membranes as well as the high cost of periodic
replacement which have to be calculated for the economic budget
[38,39,43,49,50]. In comparison with other physical methods, adsorp­
tion has been revealed to be the most effective method for the purifi­
cation of water. The adsorption method is an efficient and promising
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Inorganic Chemistry Communications 131 (2021) 108766
way to remove pollutants from wastewaters due to its low cost when
compared with other physical traditional methods [38,40,49,50,53].
Recently, several studies depended on the activated carbon like a good
absorbent and can be used in a wide range of various applications; for
example, the removal of different pollutants from industrial wastewaters
as dyes. However, reports on activated carbon still showing the high cost
of its manufacturing and preparation [38,40,50,51,53]. To get rid of the
toxic compounds present in the wastewater at low cost, great effort has
been performed to the natural adsorbent materials for instance; chito­
san, coal, zeolites, fly shy and sludge [54]. Scientists suggested that we
should restrict our usage of activated carbon due to its high cost. Also,
attempts should be performed to find less costive and effective materials
that can be used for the adsorption of contaminants from wastewaters
such as; clay materials, zeolites, agricultural wastes, siliceous materials
[54].
4.1.2. Chemical methods
The major materials of chemical methods of removing dyes from
wastewater are coagulants [55]. It includes the involvement of aiding
materials for instance; aluminum, ferric, or calcium ions onto the
effluent to stimulate the flocculation process. Furthermore, Mishra and
Bajpai, [56] have studied the utilization of other materials for chemical
processes for example; ferric sulfate, and other organic polymers. While
Shi et al., [55] suggested that the integration of two methods may
develop the removal process. Generally, chemical treatments have effi­
ciency and feasibility, but the major disadvantages of chemical methods
are that the cost of chemicals is very expensive also the price up to and
down in the market because of urgent demand on chemicals and the
number of chemicals produced [52,57,59]. However, the chemical
process is efficient, there is still a disadvantage which is the sludge
production which has some disposal problems [12].
4.1.3. Biological methods
Biological treatment is shown to be the most convenient and cost-
effective process while comparing with other physical and chemical
processes. Biological processes as the adsorption on the surface of mi­
croorganisms even living or dead, use of fungi for decolorization, and
bioremediation are widely utilized in industrial wastewater treatment
[29]. Microorganisms known as bacteria, fungi, yeasts, and algae can
reproduce, form biomass and degrade several pollutants. Biological
treatment may be aerobic and anaerobic [29]. Dye decolorization by
fungi and algae is achieved by adsorption instead of biodegradation
however it has slow growth and low decolorization efficiency which
D. Zamel and A.U. Khan Inorganic Chemistry Communications 131 (2021) 108766

minimizes their applications in the treatment of textile effluent [6,58].

In contrast, bacteria can grow faster and perform biodegradation pro­
cesses effectively [27]. Biological processes including different micro­
organisms whether bacteria, algae, or fungi have obtained great
attention as they are cost-effective, eco-friendly, and produce less sludge
when compared by chemical methods [60].
Azo dyes biodegradation may happen in aerobic and anaerobic ways
or both. The first step in the biodegradation reaction is an enzymatic
step which includes the reductive cleavage of azo bond under anaerobic
conditions which results in colorless aromatic amines as shown in Fig. 5.
The produced less-toxic materials for instance; aromatic amines undergo
other degradation reactions by multiple enzymes which are further
secreted by the microorganism [27,35,40]. From previous reports, the
N–CH3 bond is being broken first into HCHO or HCOOH. During the
degradation reactions, phenyl thiophene and other molecular structures
have been found in the produced materials. At the end of the degrada­
tion reactions, generation of some radical species has been found in the
methylene blue solution. These radical species are responsible for
oxidizing the residual organic molecular structures to transform into
inorganic ions, for example, H2O, CO2, NO–3 and SO−4 [62]. The major
advantage of biological treatment is that it is the most cost-effective
method in comparison with other removal methods; because we Fig. 5. Illustration of Biodegradation of Azo Dyes.
isolate organisms from the environment and use them in wastewater
treatment. It was proved that an increase in the production and synthesis
(2) They also should focus on blending other materials with cellulose
of dyes in the industry has occurred and this resulted in a big elevation of
acetate which has higher mechanical properties or preparing a
pollution in the environment. However, there are various methods for
double-layered membrane for supporting nanofibers.
dyes remediation, a practically efficient way to remove whole dyes has
not been proved. Moreover, although the physical and chemical
Declaration of Competing Interest
methods have high utilization and efficiency, and economic viewpoint
has limited their application in minor industries. In recent years, the
The authors declare that they have no known competing financial
treatment of wastewater getting out from industries has been consid­
interests or personal relationships that could have appeared to influence
ered, regulations have become definite. Furthermore, researchers have
the work reported in this paper.
shown the health, safety, and environmental problems that will be
increased if industrial wastewater was not properly treated before final
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D. Zamel and A.U. Khan
Doaa Zamel is young researcher from Egypt. She works at
Faculty of Science, Helwan University in Egypt and studying
her PhD at University of Chinese Academy of Sciences, China.
Doaa has a good experience in nanofibers preparation, char­
acterization and applications in wastewater treatment. She has
an excellent article in this specialty at Scientific Reports, Nature
in 2019 and others under review.
Inorganic Chemistry Communications 131 (2021) 108766
Atta Ullah Khan is teaching assistant at Malakand University,
Pakistan and studying his PhD at University of Chinese Acad­
emy of Sciences, China. His specialty is Nanobiotechnology and
he has more than eight published peer-reviewed articles in this
specialty.