Journal of Environmental Chemical Engineering 9 (2021) 105920

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Journal of Environmental Chemical Engineering

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

Use of immobilized bacteria for environmental bioremediation: A review

Tithi Mehrotra a, Subhabrata Dev b, c, Aditi Banerjee d, Abhijit Chatterjee e, Rachana Singh a, *,
Srijan Aggarwal b, f, **
a
Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, Uttar Pradesh 201313, India
b
Water and Environment Research Institute, University of Alaska Fairbanks, Fairbanks, AK 99775, United States
c
Mineral Industry Research Laboratory, University of Alaska Fairbanks, Fairbanks, AK 99775, United States
d
Center for Nanotechnology, Indian Institute of Technology, Guwahati, Assam, India
e
Department of Bio Engineering, National Institute of Technology, Agartala 799046, India
f
Department of Civil, Geological, and Environmental Engineering, University of Alaska Fairbanks, Fairbanks, AK 99775, United States

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

Editor: Dr Y Liu 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
Keywords: review collates a vast amount of existing literature on the myriad contaminants treated using immobilized
Bioreactor bacteria. We also discuss various mechanistic aspects of using immobilized cells for environmental remediation
Bioremediation
applications, with special attention on cells encapsulated in hydrogels and their implementation in detoxifying
Hydrogel
harmful contaminants and environmental cleanup. We examine different methods/techniques for immobilizing
Immobilized bacterial cells
Immobilizing carriers/matrices viable bacterial cells in various supporting matrices, use of single- and multi-species bacterial communities,
Kinetics various growth substrates, and factors affecting the remediation process including mass transfer, kinetic pro­
Mass transfer cesses and bioreactor configurations used in pilot and field-scale applications. The advantages and limitations
Wastewater treatment associated with the use of immobilized bacteria in a bioreactor for contaminated water treatment are also dis­
cussed. 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.

1. Introduction compounds like phenol, aniline, naphthalene, formaldehyde, dime­

Everyday substantial amount of wastewater gets discharged into the
environment from varied industrial and domestic sources. Even with a
most careful industry, with appropriate after-use treatment methods and
protocols implemented, waste production cannot be fully nullified or
avoided. With continuous growth in demand, water pollution is inevi­
table. Effective wastewater treatment and re-use strategies are essential
and critical for a sustainable environment. Biological treatment pro­
cesses have inherent advantages over harsh chemical treatments and
processes. With suitable techniques and design, biodegradation of pol­
lutants and biological treatment of wastewater have proven to be suc­
cessful, efficient, and cost-effective [1].
Different wastewaters comprise a diverse range of contaminants that
need to be remediated to create a prospect of water recycle and over­
come water scarcity. A principal class of contaminants include organic
thylformamide, triethylamine [2,3]. A related class of contaminants
include hydrocarbons like crude oil, diesel and petroleum products [4].
Dyes (majorly azo dyes), which are widely used in textile, printing,
leather, pharmaceutical, and cosmetic industries, form the next class of
contaminants [2,5]. Other categories of contaminants are toxic metals,
including arsenic (As), mercury (Hg), lead (Pb), copper (Cu), chromium
(Cr), and Selenium (Se), which are present in wastewater from mining
and metallurgical industries, and have significant adverse health re­
percussions [6,7]. Nitrogen and phosphorus compounds like NO3− ,
NH4+ and PO43− are nutrients present in any municipal wastewater and
domestic sewage, and form a separate class of pollutants that can lead to
eutrophication in natural waters upon discharge without proper treat­
ment [8]. There is also a class of micropollutants and emerging con­
taminants that include pharmaceutical compounds, endocrine
disruptors [202], per- and polyfluoroalkyl substrances (PFAS) [201] ,

* Correspondence to: Amity University Uttar Pradesh, Noida, Uttar Pradesh 201313, India.
** Corresponding author at: Water and Environment Research Institute, University of Alaska Fairbanks, Fairbanks, AK 99775, United States.
E-mail addresses: rsingh2@amity.edu (R. Singh), saggarwal@alaska.edu (S. Aggarwal).

https://doi.org/10.1016/j.jece.2021.105920
Received 19 March 2021; Received in revised form 3 June 2021; Accepted 21 June 2021
Available online 24 June 2021
2213-3437/© 2021 Elsevier Ltd. All rights reserved.
T. Mehrotra et al.
and more recently microplastics [203].
As compared to physical and chemical treatment processes, biore­
mediation utilizes microorganisms (including fungi, bacteria, and algae)
as free cells, biofilms, or aggregates (or activated sludge flocs), and plays
an important role in providing a greener, cost-effective, and sustainable
solution toward the treatment of contaminated water, air and soil [9].
Therefore, biological treatment methods have been extensively used to
treat organic and inorganic contaminants from wastewaters. Apart from
the conventional direct application of microorganisms wherein micro­
bial cells are freely exposed to the contaminated waste media in soil, air,
or water – an alternative approach is where microbial cells are cultured
and immobilized (or encapsulated) in a permeable polymeric gel matrix
[9], and the immobilized microbes within the gel matrix are exposed to
the contaminated media indirectly.
Historically, immobilized microbes were first developed for in-situ
production of enzymes. Back in 1969, immobilized enzymes (amino­
acylase) were first used industrially for continuous production of L-
amino acids from acyl-DL-amino acids [10]. Gradually the immobilized
enzymes were substituted with immobilized viable bacteria for in-situ
enzyme production and this was advantageous due to elimination of
expensive enzyme separation and purification steps, enhancement of
half-life and activity of intact bacterial enzymes, and increment in the
rate of regeneration of immobilized bacteria- leading to an additional
benefit of their possible reuse [11]. Within a couple of decades this
advancement attracted attention and several research groups adapted
the technology by immobilizing bacteria for the treatment of organic
contaminants [12] such as degradation of phenol [13,14], pentachlo­
rophenol [15], 4-chloro-2-nitrophenol [16], and 4-chlorophenol [17].
Recently, there have been other diverse applications of immobilized
bacterial cells such as in wastewater treatment [18], which has led to
enhanced investigation into more fundamental aspects of immobilized
cell systems such as the the effects of immobilization on bacterial con­
sortium [19] in case of multi-species systems.
Using free cells for remediation has some disadvantages, including
low biomass concentration in a particular place/time, easy washout of
microbes during application, and susceptibility to external stressors.
Employing immobilized or encapsulated bacterial cells has some specific
advantages over the free forms (single species or consortium) for
bioremediation e.g., efficient separation of biomass from treated water,
effective spatial-temporal control of biomass within the contaminated
media, longer survivability, stability, and reusability without loss of
activity [20]. Apart from bioremediation, immobilized cells have also
been widely used in other myriad applications including pharmaceutical
industry, chemical/process engineering, food industry, and biosensor
applications [20]. Advances in this area especially in the last decade
highlight the promise of this emerging technology for diverse applica­
tions. Another natural way that microbes are immobilized in nature is
via the formation of microbial biofilms [21–24] which immobilize mi­
crobes on surfaces within a self-secreted extracellular polymeric sub­
stance (EPS) matrix [25,26]. Role of biofilms in the water/remediation
sector hass also witnessed an exponential research growth in recent
times and biofilm impacts on water infrastructure [204] as well as
biofilm-based biofilters for drinking water and wastewater remediation
[27–30] are being more widely researched. To maintain the focus of this
article, however, we only consider bacteria immobilized within engi­
neered (or human-designed) matrices and not those within natural
biofilms; and the term ‘immobilized’ throughout the article refers to the
former.
In this review we summarize the current state of knowledge
regarding use of immobilized bacteria for environmental bioremedia­
tion applications and discuss different support matrices, immobilized
bacterial cultures and communities, immobilization techniques, nutri­
ents for immobilized bacteria, contaminants treated by immobilized
microbes, mass transfer considerations, factors that impact remediation
using immobilized cells (e.g., salinity, pH, toxicity of pollutants), ki­
netics and equilibrium analyses, and design of different biofilter
2
Journal of Environmental Chemical Engineering 9 (2021) 105920
configurations using immobilized cells. We conclude with a brief dis­
cussion on challenges, recent advances, and directions for future
developments.
2. Support matrices/carriers for bacterial cell immobilization
2.1. Desired properties of support material
Depending upon the type of wastewater, the type of contaminant,
and the expected outcome after the treatment process, selection of
carrier material is vital in the course of immobilization process, as it
controls the metabolic activity, provides operational stability, protects
from an aggressive and hazardous external environment – and thus helps
achieve more efficient biodegradation [9]. Biological treatment process
utilizing immobilized bacterial cells has the potential to degrade a large
fraction of biodegradable organic compounds in contaminated water.
However, during the immobilization process, some criteria need to be
fulfilled, i.e., the matrix should be light-weight, flexible, cost effective,
mechanically and chemically stable, inert, non-biodegradable,
non-polluting, non-toxic, insoluble in aqueous medium, amenable to a
simple method for bacterial immobilization, and possess high diffusivity
with high biomass retention. Thus, the selection of a good supporting
matrix/carrier for cell immobilization is an essential criterion.
2.2. Categories of carrier or support material
The carrier materials used for immobilization of varied types of cells
are classified into inorganic materials, natural organic polymers and
synthetic organic polymers [31]. Material of the support matrix must
provide enough support and mass transfer capability to achieve desired
bioremediation. Ceramics, lignocellulosic biomass, and polymers (nat­
ural or synthetic) are normally used as supporting matrices for immo­
bilization/entrapment of bacteria or biomacromolecules [9]. Physical
properties of the carrier/ supporting matrix are crucial and drive matrix
durability. It was also observed that varied cellular changes are stimu­
lated when the bacterial cells are immobilized on matrices in order to
enable attachment of cells (to the support material) and to facilitate the
cell growth within the immobilized matrix [37,38]. Additionally,
permeation provided by bacterial cell membrane assists in the uptake of
nutrients and remains unaffected even after immobilization [39]. For
whole cell immobilization, organic carriers are preferred over inorganic
carriers. Different natural as well as synthetic organic polymers have
been tested for their capability to be an effective carrier or matrix for
immobilization of viable bacteria for the purpose of remediation.
Natural organic carriers are easily available, inexpensive, biode­
gradable, non-toxic and usually have higher absorptivity as compared to
the inorganic carriers and the presence of wide-ranging functional
groups on the surface of organic matrices provides additional
enhancement in the absorption capacity. The matrices prepared from
natural organic polymers like carrageenan, pectate, agar, agarose, chi­
tosan, charcoal, cellulose, gelatin, collagen, bacterial cellulose, and
alginate (derived from algal polysaccharides) are prepared by gelation
of soluble polymers either by cooling process and/or in the presence of
various ions. Bacteria are able to sustain the process of immobilization
and grow well in the matrix prepared from natural polymers like
carrageenan and alginate [32]. Alginate immobilized cells do not un­
dergo significant physicochemical changes during the immobilization
process and the gel is permeable and transparent [35]. Chitosan is a
stable natural carrier as it has potentially reactive amino functional
groups that can improve the attachment of the bacteria with the sup­
porting matrix. Despite the widespread usage of natural carriers, there
are still some major disadvantages associated with them such as particle
disruption due to high bacterial concentration and excessive gas pro­
duction, low mechanical and chemical stability, low reproducibility, low
adaptability, high contamination rate, uninhibited rate of hydration and
loss of adhesiveness over storage for a long period of time
T. Mehrotra et al.
(approximately 20–25 days) [33,34]. Hence, to overcome the drawbacks
of the natural polymers, synthetic polymeric gels are used for cell
immobilization. The synthetic polymers are prepared by gelation of
monomeric units via diverse chemical and photochemical reactions.
These polymers are not easily biodegradable and have low diffusion
ability but their mechanical performance is superior in comparison to
the natural materials [9]. Commonly used synthetic gels for support are
polyvinyl alcohol (PVA), polyacrylamide (PAM), polycarbamoyl sulfo­
nate (PCS), polyethylene glycol (PEG). And prominent synthetic plastics
include polyethylene (PE), polyurethane (PU), polypropylene (PP),
polyacrylonitrile (PAN) and polyvinyl chloride (PVC) [9,40]. While the
synthetic organic polymers provide more stable matrix formation in
waste solutions of varying pH, the natural organic polymer matrix al­
lows higher diffusion [35]. To avail the best of both worlds, matrices
formed with natural and synthetic composite material show immense
potential. A composite with varying ratios of synthetic and natural
polymers was tested to achieve an optimal matrix with high mechanical
strength and diffusivity [36].
Hydrogels or aqueous polymeric gels are gaining immense attention
as support matrices for cellular immobilization. With the chief property
of swelling/water containment, hydrogels are also mechanically strong
to provide adequate structural support for encapsulation. Because of
their nature of formation and interlinking structure, hydrogels are not
homogeneous. The requirement of crosslinking agents depends upon
polymeric material and their characteristics. The water within the
hydrogel network generates osmotic pressure which encourages inter­
action between the pollutant, bacteria, and absorbent/carrier material.
Thus, hydrogels with immobilized bacterial cells is an enticing tech­
nology for bioremediation applications [41]. Overall, immobilized cells
in both natural or synthetic polymers show usefulness in the detoxifi­
cation of a broad range of contaminants like hydrocarbons, organic and
inorganic dyes, aromatic compounds like pyridine, toluene, heavy
metals and excess nutrients like nitrogen and phosphorus from waste­
water (an extensive list is provided in Table 1) [9,11,42–45].
3. Immobilized bacteria
Immobilization of a pure culture (single strain) or a laboratory-
formulated microbial consortium [43] is easier and more efficient for
a specific application as compared to immobilizing a naturally growing
microbial population obtained from a complex contaminated environ­
ment which may consist of numerous known and unknown microbial
species (pathogenic/non-pathogenic/tolerant/resistant to each other).
With high degree of heterogeneity, the synergistic/antagonistic action in
the naturally growing microbial community, their inter-dependent
growth patterns and extensive metabolic behavior likely makes it chal­
lenging to attain specific remediation goals [46]. For instance, a pure
atrazine degrading culture, Agrobacterium radiobacter J14a, and natural
mixed culture of microbes isolated from an atrazine-contaminated crop
field, were immobilized using phosphorylated-polyvinyl alcohol (PPVA)
hydrogel. At the proper cell-to-matrix ratio (3.5 mg/mL), the atrazine
biodegradation using immobilized J14a and the mixed culture was
approximately 50% and 40% within 120 h, respectively. Degradation
efficiency of mixed culture was reported to be lower in comparison to
the pure culture owing to lack of synergism in the natural consortium
[47]. In another research, a synthetic bacterial consortium (consisting of
17 strains) immobilized in chitosan beads, was studied for remediating
an oil-contaminated mangrove. The laboratory generated bacterial
consortium was successful in treating the contaminated site in 15 days
[48].
To avoid the extensive procedure of isolation and formation of bac­
terial consortium, the use of easily available activated or anaerobic
sludge, as free or immobilized in various macroporous polymeric gels/
cryogels for wastewater decontamination has also been reported. Acti­
vated sludge (a mixture of autotrophic and heterotrophic bacterial
cells), immobilized in polyurethane was used to efficiently treat
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Journal of Environmental Chemical Engineering 9 (2021) 105920
acrylonitrile wastewater [49]. Zhang et al. has also employed the
immobilized activated sludge in improvised polyvinyl alcohol hydrogel
for reduction of organics (measured as chemical oxygen demand, COD)
and nitrogenous compounds from synthetic wastewater [50]. In another
study by Sergio and Bustos, activated sludge encapsulated in
calcium-alginate beads was successful in biodegradation of nitrogen in
wastewater [51]. Some studies suggest that the sludge which includes
non-selective microbes that lack co-metabolism to carry out a targeted
application. For example, Senko et al. immobilized anaerobic sludge
microbial cells into macroporous cryogel of PVA polymer for converting
various wastes including xenobiotics into biogas, which resulted in
increased methanogenic activity to ~ 55% but was associated with
decreased acidogenic activity by ~ 16% [52]. Thus, these observations
suggest that immobilizing a mixed microbial culture or sludge is usually
less advantageous than a synthetic bacterial consortium developed in
laboratory due to lack of co-metabolic or synergistic degradation of the
pollutants in wastewater [52].
On the other hand, implementing genetically modified (GM) mi­
crobes has become a recent popular trend to enhance biosensing and
bioremediation by modifying the existing genes in the microbes towards
detection and degradation of specific target contaminants. Elcin and
Öktem in 2020 [53], immobilized a recombinant Escherichia coli
MG1655 (pBR-arsR773) strain in agar and alginate biopolymers for the
detection of arsenic in environmental water samples at safe drinking
water limits. The recombinant strain harbored a sensor plasmid used as
fluorescent bioreporter for targeted and sensitive detection of the heavy
metal. In another work, recombinant Escherichia coli with modified ge­
netic material was used for the specific detoxification of coumaphos,
chlorferon and diethyl-thiophosphate [54]. The uninhibited use and
release of such GM bacteria in the environment for field-scale biore­
mediation application, however, is a challenging task due to concerns
such as their stability, horizontal transfer of the engineered DNA, sur­
vival, cost in their development as well as ethical issues associated with
their application [55]. Examples of the immobilized bacteria in pure
culture form, as consortium, or as genetically modified form are
included in Table 1.
4. Nutrients for immobilized bacteria
Microbial cells (free or immobilized) require carbon, nitrogen, and
phosphorus for their growth, proliferation, and function. During biore­
mediation via immoblized bacterial cells, the cells obtain these nutrients
primarily from the wastewater, which is usually enriched with these
nutrients and also from the matrix they are immobilized in [9]. The main
necessity of carbon can be fulfilled by obtaining the same from organic
(natural or synthetic) polymeric matrices used as support material.
Natural polymeric gels like alginate, carrageenan, agar, gellan, xanthan
gum, guar gum, eladium, and bacterial cellulose that are used as support
carriers for bacterial cells are easy source of carbon and energy for the
cells during confinement. In a study by Saez et al., the performance of
Streptomyces strains was assessed for the degradation of a pesticide,
lindane, (in aqueous batch cultures) with free and immobilized cells in
four different matrices (agar cubes, PVA-alginate beads, agar in silicon
tube and cloth sachets). All the strains in free as well as immobilized
form could grow in media supplemented with lindane, which they used
as a carbon and energy source. Lindane removal was reported to be
higher for immobilized cells as compared to free cells. The best growth
of Streptomyces sp. A2, A5 and A11 was observed in silicon tube-based
support matrix that could possibly be due to adequate diffusion of
both oxygen and substrates provided by the medium [56].
During the treatment of wastewater, continuous availability of car­
bon and nitrogen are essential, hence the diffusion and availability of
these nutrients should be adequate and uninterrupted. For this, the pore
size of the immobilizing matrix plays an important role, as pore size is
vital for assessing the diffusion coefficient of the diffusing molecules
like, glucose, oxygen, etc. [20]. Further, substrate transport into the
T. Mehrotra et al. Journal of Environmental Chemical Engineering 9 (2021) 105920

Table 1
Microorganisms immobilized in polymeric matrices for bioremediation of contaminated water.
Bacteria Immobilizing material Immobilization Pollutants Degradation Ref.
technique efficiency

Alcanivorax sp. and Cycloclasticus sp. Laccase NM Long chain n-alkanes of C26-C35 and 79.2% and 78.7%, [178]
Klebsiella variicola strain SKV2 Polyurethane polymer Entrapment polycyclic aromatic hydrocarbons respectively [179]
Sphingomonas sp. scaffold and microcapsules of Entrapment Crude oil 44.31% and [180]
agar and paraffin Chlorpyrifos 71.87%,
Calcium-alginate respectively
75.4%
Bacillus pseudomycoides Polyvinyl alcohol- Entrapment BOD and COD 86% and 71%, [41]
Glutaraldehyde respectively
Pseudomonas putida YNS1 Alginate-silica Encapsulation Phenol 92%; [181]
Cadmium > 99%
copper > 97%
Pseudomonas sp. Silica NM Remazol dye 75% [94]
Methyl orange 79%
Benzyl orange 83%
Ochrobacterium sp. DGVK1 PVA-alginate Entrapment Dimethylformamide NM [182]
Klebsiella oxytoca Cellulose triacetate and NM Propionitrile 99% [97]
alginate
Arthobacter protophormiae Alginate NM Triethylamine 100% [183]
Arsenic oxidizing bacteria Polyvinyl alcohol (PVA NM Arsenic ~ 90% [184]
Pseudomonas putida montmorillonite clay, Adsorption Formaldehyde [185]
polyethyleneimine
Acinetobacter calcoaceticusSTB1 Electrospunpolysulfone (PSU) NM Ammonium 55% [186]
fibers methylene blue ~ 40%
Rhodococcus sp. Cellulose and ceramic NM Benzene 97% [4]
Pseudomonas putidaF1 Agave-fiber/polymer foamed- NM Benzene 100% [4]
composites (AFPFC) Toluene
RhodococcuserythropolisB4 Poly(vinylpyrrolidone) Adsorption Naphthalene 82% [4]
Anthracene 87%
Rhodococcus corynebacterioides QBT Chitin and chitosan flakes NM Crude oil 60% [4]
Prototheca zopfii Alginate, PUF Entrapment n-Alkane 100% [4]
Rhodococcus ruber PVA Entrapment Hexadecane 51% [4]
Bacillus subtilis Chitosan Entrapment Hexadecane 100% [4]
Pseudomonas fluorescens-CS2 Alginate, agar, Entrapment Ethyl benzene NM [4]
Polyacrylamide
Micrococcus sp. strain SMN-1 Agar Entrapment Nitrotoluene 74% [4]
Pseudomonas sp. YATO411 Alginate, PVA Entrapment Benzene 99% [4]
Comamonas sp. JB Gellan gum Entrapment Benzene, Toluene, Ethylbenzene and 100% [4]
Xylenes
Streptomyces sp. AB1 Alginate Entrapment Xylene 90% [4]
Burkholderia sp. Alginate Entrapment Quinoline 100% [4]
Pseudomonas sp.NGK1 Alginate Entrapment Naphthalene 100% [4]
RhodococcuserythropolisB4 Polyvinylpyrrolidone Entrapment Naphthalene 53% [4]
Pseudomonas stutzeri Alginate Entrapment Naphthalene 34% [4]
Sphingomonas sp. Alginate Entrapment Phenanthrene 99.8% [4]
Mycobacterium frederiksbergense Alginate Entrapment Pyrene NM [4]
Selenastrum capricornutum Alginate Entrapment Phenanthrene, fluorene, fluoranthene, 99% [4]
pyrene, benzo[a]pyrene, Copper,
cadmium, nickel, and zinc
Bacillus fusiformis Alginate, clay, PVA Entrapment Naphthalene 100% [4]
Rhodococcus sp. F92 PUF Entrapment Crude oil 90% [4]
Acinetobacter valentis Alginate Entrapment Crude oil 97% [4]
Bacillus lichenformis PVA Entrapment Crude oil 88% [4]
Bacillus pumilus Chitosan Entrapment Hexadecane 90.8% [4]
Pseudomonas aeruginosa NY3 PUF Entrapment Crude oil 90% [4]
Rhodococcus equi A4 Lentikats Gelation Nitrile compounds NM [187]
Agrobacterium radiobacter J14a phosphorylated-polyvinyl NM Atrazine 50% degradation in [95]
An oil degrading bacterial consortium alcohol (PPVA) Adsorption Petroleum 120 h [188]
puffed panicum miliaceum > 98%
(PPM),calcium alginate and
chitosan
Bacillus sp., Pseudomonas sp. and Serratia Alginate, Polyacrylamide NM Chromium, mercury, and nickel NM [34]
sp.
Anammox and ammonia-oxidizing PVA NM Nitrogen 100% [189]
bacteria
Aeromonas hydrophila MFB03 and Ca-alginate Encapsulation Tetradecyltrimethylammonium bromide 65% [190]
Pseudomonas putida A (ATCC 12633) (TTAB)
Exiguobacterium sp. ASW-1, Pseudomonas Ca-alginate Encapsulation Crude oil 75.1% [191]
aeruginosa strain ASW-2, Alcaligenes sp.
ASW-3, Alcaligenes sp. ASS-1, and
Bacillus sp. ASS-2
Rhodococcus pyridinivorans and Gordonia PVA Entrapment Diesel 92% [192]
alkanivorans
Ca-alginate Encapsulation Cationic surfactants NM [190]
(continued on next page)

4
T. Mehrotra et al. Journal of Environmental Chemical Engineering 9 (2021) 105920

Table 1 (continued )
Bacteria Immobilizing material Immobilization Pollutants Degradation Ref.
technique efficiency

Aeromonas hydrophila and Pseudomonas

putida
Activated sludge consortium PVA Entrapment Phenanthrene NM [193]
Activated sludge PVA Entrapment Furfural 100% [194]
Anammox sludge Alginate, PVA Entrapment Ammonium > 80% [195]
Acinetobacter sp., Bacillus circulans, Alginate Encapsulation Oil NM [96]
Bacillus licheniformis, Brevibacillus
brevis, Burkholderia cepacia, Leifsonia
aquatica and Sphingomonas paucimobilis
E. coli MG1655 (Pbr-arsR773) strain Agar and alginate Entrapment and Arsenic NM [53]
encapsulation
Recombinant Escherichia coli Ca-alginate Encapsulation Coumaphos, chlorferon and 10% more efficient [196]
diethylthiophosphate in degradation than
free cells
Recombinant E. coli BL 21 strain Ca-alginate Encapsulation Microcystins (MCs) NM [197]
Recombinant Escherichia coli (harboring Agarose Encapsulation Phenolic compounds NM [198]
the pLZCapR plasmid)
E. coli DH5α (pKAU17) Alginate atomization (MA), Urea Fastest urea [199]
inkjet printing (MI) diffusion and
and double- degradation were
encapsulation (DDMI) observed for MI
capsule
Pseudomonas putida MC4 (with cloned Ceramic rings Adsorption 1,2,3-Trichloropropane (TCP) > 95% [200]
dehalogenase gene (dhaA31))

Fig. 1. Various techniques and carriers used in the bacterial cell immobilization process.

5
T. Mehrotra et al.
support matrix is another essential factor for microbial cell growth
during immobilization. Effective diffusion coefficient for substrates like
oxygen and glucose in natural gels is 1.6–2 × 10− 9 m2 s− 1 and 0.6 ×
10− 9 m2 s− 1, respectively, whereas the same in synthetic gels is usually
lower due to higher polymer concentration [47] in synthetic gels than in
natural gels. Thus, the diffusion coefficients of glucose and oxygen are
usually lower in the synthetic gels [46].
On a physiological level, when a bacterial suspension is inoculated in
a medium containing organic polymer, some bacteria would be induced
to synthesize and secrete the extracellular enzyme(s) required to hy­
drolyze the polymer (in addition to some constitutively produced
extracellular enzymes(s) that may already be present in the inoculum).
As the enzyme acts on the polymer, it leads to polymer hydrolyzation
and further production of utilizable substrates. In case of natural poly­
mers, hydrolyzation leads to add-on in the substrate quantity over and
above the matrix itself. In the case of synthetic polymers, however,
hydrolyzation is the only way to produce utilizable carbon source for the
immobilized bacteria. With increase in the enzyme activity, there is an
increase in the concentration of usable substrate which in turn causes an
increase in the specific growth rate of the bacteria due to substrate
utilization [39].
5. Bacterial immobilization techniques
Through the years, there have been many methods/ techniques
developed for the immobilization of microbial cells in hydrogels (Fig. 1).
The most common and essential methods are adsorption and covalent
bonding on solid matrices. Others methods include cross-linking be­
tween biomolecules/biocatalysts/bacteria and the polymeric carrier,
encapsulation by membranes, and entrapment in a porous matrix [57].
The immobilization of the bacterial cells in hydrogel/support matrix
can be carried out in two ways: either by placing the prepared hydrogel
in a bacterial broth for adsorption of bacteria [58], or by simultaneous
gelation of hydrogel with pre-polymeric units and the bacterial cells
[41].
5.1. Adsorption
Adsorption/attachment of the cells on hydrogel for bioremediation
application is the simplest, inexpensive, and most often used immobi­
lization method. It is based on reversible physical interaction between
the bacterial and the carrier surface through weak binding forces [59].
The factors affecting adsorption include, first, the bacterial character­
istics like physiological conditions and age of the cells, bacterial surface
appendages, cell membrane charges, and hydrophobicity. Second, the
medium characteristics like, its composition and pH; and third, the
surface properties of support matrix, comprising of size and structure of
the adsorbent used and pores, if any, on the adsorbent [20]. Addition­
ally, nature of the adsorbent also plays an important role in assessing the
strength of the interaction in the adsorption process. Since weaker in­
teractions are involved in the adsorption processes, there is a high
possibility of leaking out of attached cells from the support into the
environment, thus, this technique of immobilization has limited appli­
cations [60].
5.2. Covalent bonding
The bacteria also get immobilized through covalent interactions
between reactive groups (–COOH or –NH2) at their surface and the
supporting matrix. On one hand, it leads to increase in the stability of the
covalently bonded bacteria, but, on the other hand, the bacterial
bioactivity decreases in the post-operational process. Covalent coupling,
however, is effective for enzyme entrapment but quite rarely used for
the immobilization of bacterial cells. The chemical agents used for co­
valent bonding are generally cytotoxic which diminish the bacterial
activity and viability, eventually leading to cell death [12,60].
6
Journal of Environmental Chemical Engineering 9 (2021) 105920
5.3. Cross-linking
Another method of cell immobilization is cross-linking that is ach­
ieved by covalent bond formation between the activated organic support
and bio-macromolecule(s)/bacterial cells through multifunctional
crosslinking agents like glutaraldehyde, hexamethylene-diisocyanate,
bis-diazo-benzidine and so on [12]. In a recent study by Mehrotra
et al., an unexplored bacterium, Bacillus pseudomycoides was immobi­
lized in a PVA hydrogel, and crosslinked with glutaraldehyde for the
biotreatment of municipal wastewater and demonstrated not just
magnificent bacterial viability for almost 2 months but successful
decontamination of wastewater without bacterial leaching [41]. Various
crosslinkers have been reported in the literature to crosslink polymers
like PVA; including sulfo-succinic acid [61], polystyrene sulfonic
acid-co-maleic acid (PSSA-MA) with glutaraldehyde [62], sulfo-phthalic
acid with sulfo-acetic acid [63,64], maleic acid [65], and glutaraldehyde
[66]. Bai et al. reported crosslinking of PVA hydrogels in the presence of
epichlorohydrin as cross-linking agent to immobilize nitrifying bacteria
in order to check the ability of the matrix to keep cells viable and with
the promising results of the study, the same could be used for varied
biotechnological applications [67]. Crosslinking method is easy but
challenging to carefully control. Similar to covalent bonding method,
crosslinking is also difficult for bacterial cell immobilization, because
the crosslinking agents are usually cytotoxic and the conditions are hard
to identify as to when the cells can be immobilized without any damage
[12].
The fabrication of cross-linked gels without using organic solvents as
cross linkers, is a promising means for encapsulation or entrapment of
living cells. Gamma irradiation-based crosslinking is reported as a
suitable technique for the polymerization of hydrogels during immobi­
lization. Such radiation-induced crosslinked hydrogels are formed at
room temperature without killing the bacterial cells. Some of the other
advantages of this method includes easy process control, polymerization
and sterilization, simultaneously, without the addition of any cross­
linking agent or initiator [4]. In a recent study by Singh et al., five
bacteria (BR-6, BR-14, BR-18, BR-21, and BR-26) were immobilized in
gamma-irradiated hydrogel and evaluated for biological removal of
strontium; 62–71% of remediation of 10 µg/mL strontium was observed
from the medium [68]. Rhodococcus erythropolis B4 was also successfully
immobilized in radiation based crosslinked poly(vinylpyrrolidone)
hydrogel for the degradation of polycyclic aromatic hydrocarbons from
the environment [69]. Crosslinking of some co-polymeric hydrogels is
also possible via radiation technique; however, they have been reported
to employ ultra-violet (UV) radiation induced photo-crosslinking of free
radicals. Hsueh et al. explained UV-initiated co-polymerization of
2-hydroxyethyl methacrylate/polyethylene glycol diacrylate (HEMA/­
PEGDA) hydrogel films to immobilize the bacterial cells which was used
for enhanced fermentation in biomedical, pharmaceutical, and food
industries [70].
5.4. Ionotropic gelation method
Entrapment of bacterial cells in alginate beads by ionotropic gela­
tion, utilizing various divalent and trivalent cations has found signifi­
cant use in the technology of immobilizing viable cells [71]. The main
drawback associated with calcium-alginate gels, however, is their
destabilization and subsequent solubilization by calcium-chelators
which results in low density and low mechanical strength. This has
been overcome with covalent crosslinking and alginate grafting with
synthetic polymers like PVA and further treatment with poly­
ethylenimine, followed by crosslinking [72]. A novel methodology has
also been developed at Bhabha Atomic Research Centre (BARC),
Mumbai (India), to stabilize the alginate beads with calcium-chelators
by gamma-irradiated polymerized polyacrylamide [73]. Besides cal­
cium, barium-alginate beads were also reported to be used for denitri­
fication under anoxic conditions due to their higher mechanical
T. Mehrotra et al.
compression and low oxygen diffusion potential [74].
5.5. Sol-gel method
For the whole bacterial cell encapsulation, sol-gel is one conven­
tional method adopted for over a decade. However, there are a few
limitations to this method, like, involvement of extreme pH conditions,
and formation of alcohol during the condensation process - which have
harmful effects on the immobilized bacteria leading to inhibition of
cellular division [75–78] and leaching of bacterial cells from the matrix
[79]. Therefore, certain modifications are carried out to overcome such
drawbacks, like executing the process under physiological conditions
using mainly neutral aqueous nanosols followed by mild solidification
and drying processes. The viability of the cells is affected greatly by this
as well as by the matrix rigidity. These factors can be further controlled
by the drying regime (air or freeze-drying), the residual water content,
the addition of fillers (e.g., ceramic fibers) [80], the admixture of soluble
and leachable pore-forming additives (e.g. sorbitol) [81], or the incor­
poration of water-retaining polymers such as polyethylene glycol [82].
Sol-gel technology has become increasingly significant for immobilizing
living cells such as bacteria, yeasts, and plant or animal cells [83]. Whole
cells of E. coli have been encapsulated within sol-gel silica matrices to
hydrolyze p-NPG (4-nitrophenyl-β-D-galactopyranoside) by their
β-galactosidase activity. The study showed that E. coli exhibited
noticeable enzymatic activity in wet gels even after aging for more than
a week at room temperature. Furthermore, sol-gel-derived silica has
been shown as a suitable matrix for cell growth [84] and protects the
viability of cells for over a year [85].
The technique that could be an extension and/or alternative to the
conventional alkoxide sol-gel technique is the freeze-cast/ freeze-gela­
tion technique for immobilization of bio-compounds/ cells. This method
was used to prepare ‘biocers’ (biological ceramic composites) which are
low-cost, porous, crack-free composites with almost zero shrinkage by
utilizing ceramic powder, colloidal SiO2, and the biocomponent in an
aqueous state. This customizable technique opens the possibility to
connect the freezing step which was necessary for sol-gel transition with
the preservation of immobilized bacteria with possible cell division
within the biocers. Freeze-gelation of biocers with immobilized Bacillus
sphaericus was utilized to analyze the viability, storage ability and
metabolic activity of cells [86]. In a similar study, freeze-gelation bio­
cers were shown to have the potential to be scaled up because of being
mechanically stable and maintained the viability of the immobilized
Rhodococcus ruber for several months [87].
5.6. LentiKats technology
Freeze–thaw method was employed for gelation of most widely used
polymers like PVA, that leads to the formation of H-bonds, resulting into
stronger and sturdier hydrogels [32]. However, polymerization below
0 ◦ C has negative impacts on the immobilized cells, resulting in loss of
their functionality and metabolic activity. Other varied methods for PVA
gelation have been developed, like, polymer formation using boric acid
[88], but the matrices were brittle with low stability [40]. Thus, the
most appropriate and effective process at room temperature, without the
use of any costly and harmful chemicals, is LentiKats® technology [57].
LentiKats® technology (lens shaped carriers), that makes use of the
gentle gelation technique is already scaled up to industrial use. Properly
developed LentiKats® can handle high temperatures of up to 50 ◦ C and a
broad range of pH [57], and combine the advantages of both large and
small beads. The lens shape of this carrier with 3–4 mm diameter and
200–400 µm thickness enables sufficient diffusion and easy separation
from the liquid media. Immobilization of enzymes or bacteria with
original shape and size is possible using PVA LentiKats® technique with
high mechanical and physical stability [57]. PVA carrier is
non-biodegradable and non-toxic to the immobilized bacteria, enzymes,
or the environment. Furthermore, the conditions for entrapment are
7
Journal of Environmental Chemical Engineering 9 (2021) 105920
very mild, which enables high rate of survival of the cells in the matrix
when immobilized [57]. The increased solidity and rigidity of the Len­
tiKats® allows nitrification and/or denitrification of varied wastewa­
ters. Nitrobacter europaea and Nitrobacter winogradskyi have been utilized
in LentiKats® technology for nitrification and Pseudomonas dinitrificans
and Pseudomonas fluorescens have been reported for denitrification in the
wastewater treatment process [57].
5.7. Electrospinning method
Bacterial encapsulation usually occurs in matrices which are of the
magnitude larger than thin films, making the nutrients and reactants
difficult to diffuse into the reach of immobilized bacteria, thereby
affecting the overall efficiency of the process. Thus, to overcome this
drawback as well as with some additional advantages of less space and
growth medium requirements, reusability potential and increased
resistance towards environmental stresses, the Brookhaven-Stony Brook
team in the United States used a technique known as ‘electrospinning’.
In this method, electrostatic force is applied to spin and produce poly­
mer filaments/fibers using the polymer solution containing the bacteria
of interest. This method is simple, inexpensive and versatile with high
potential for immobilizing viable bacterial strains [89]. Through a series
of experiments, fluorinated dimethylacrylate (FDMA) was chosen as the
encapsulating agent, which is an insoluble (by crosslinking with glyc­
erol), non-toxic and non-biodegradable fibrous polymer in which the
bacteria from genus Pseudomonas, Escherichia, and Zymomonas were
successfully encapsulated in a viable state. The viability results revealed
that the bacteria remained live for several months with their metabolic
activity intact in electro-spun fibers which could be used as reusable
biosensors, stable drug-delivery systems and as permeable reactive
barriers for de-contaminating wastewater [90]. The encapsulation of
E. coli and Staphylococcus albus in PVA nanofibers [91], and Lysinibacillus
sp. in water-soluble and biocompatible non-polymeric cyclodextrin (CD)
fibers using electrospinning have been recently reported [92]. However,
PVA and CD are water-soluble in nature, thus, could not be efficiently
applied for wastewater treatment. Thus, cellulose acetate/poly
(ethylene oxide) nanofibers with dimethyl sulfoxide (DMSO) as solvent
was used for immobilization of viable Bacillus paramycoides via elec­
trospinning for the removal of methylene blue dye [89]. Furthermore,
Hardick et al. through their work explained that electrospinning of
matrix like cellulose acetate (which is biodegradable, biocompatible,
water-insoluble, and highly porous) for immobilization of bacterial cells
leads to formation of nanofibers which display high surface area
(enhancing the overall adhesion ability) and are thereby useful in
immobilizing large number of bacterial cells for efficient bioremedia­
tion. This matrix can have possible industrial usage on account of its
several advantages like easy handling, affordability, and ability to be
reused multiple times [93].
5.8. Comparative analysis of different bacterial immobilization methods
On comparative analysis of the different bacterial immobilization
techniques discussed, adsorption, although being the most common,
simple, and inexpensive has several shortcomings like weak binding
force between the bacteria and support carrier, eventually leading to
loss of bacteria during the treatment and thus lower overall stability of
the process. Hence, covalent bonding was further adopted as immobi­
lization technique to overcome the gaps of adsorption. Such bonding
though provides increased bacterial stability but decreases bacterial
bioactivity during the operation. In addition, covalent bonding involves
chemical agents which are cytotoxic in nature, which would lead to
death of the immobilized bacteria or decrease its metabolic activity. One
of the most appropriate and effective process at room temperature,
without the use of any costly and harmful chemicals, is LentiKats®
technology, which can immobilize viable bacterial cells in their original
shape and size. Also, the potential to bear temperature up to 50 ◦ C and a
T. Mehrotra et al. Journal of Environmental Chemical Engineering 9 (2021) 105920

wide pH range, makes this technique by far a highly stable technique Table 2
both mechanically as well as physically. Finally, bio-integrated support Mathematical models for some common isotherms that describe the adsorption
matrices have been recently used for the encapsulation of viable bacteria process.
in nanofibers using electrospinning technique, which, owing to its Equation Model name Equation
simplicity, versatility, and low cost is a promising technique for immo­ number
bilizing viable bacteria with intact bioactivity for long durations. (2) Brunauer–Emmett–Teller qm . b . H
q =
isotherm (1 − H) . (1 + b . H − H)
6. Contaminants treated using bacteria immobilized hydrogels where q is pollutant adsorption at time
t, H is relative pressure, qm and b
represent the equation constants
Bacteria immobilized in 3D hydrogel scaffolds offer sustained and
(3) Freundlich isotherm 1
targeted biological responses, in addition to the unique elementary
q = kf . Cen
properties of porosity, swelling, tunable triggering, and elasticity, where q is pollutant adsorption at time
making them pertinent to a broad spectrum of potential applications. BI t, kf and n are equation constants, Ce is
hydrogels have qualified to be a smart material for efficient bioreme­ the equilibrium adsorbate
diation due to high degradation rate and adversity-resisting property. concentration in the solution

They have been successfully explored for biodegradation of pollutants (4) Frenkel–Halsey–Hill 1
q = qm . [− ln⁡(H)] r where q is
−
isotherm
like anions, heavy metals [46], dyes [89,94], pesticides [95], hydro­
pollutant adsorption at time t, H is
carbons [4,96], and organic compounds [97,98] from domes­ relative pressure, qm and r represent
tic/municipal as well as industrial water sources. The successful the equation constants
degradation of organic contaminants from wastewater by immobilized (5) Langmuir isotherm qm . L . Ce
q =
bacteria consequently also leads to decrease in biological oxygen de­ 1 + L . Ce
where q is pollutant adsorption at time
mand (BOD) and chemical oxygen demand (COD). Most studies are
t, qm and L are equation constants, Ce is
focused on the removal of a particular contaminant in each major class, the equilibrium adsorbate
mainly due to its widespread occurrence in the environment posing as a concentration in the solution
toxicant. Table 1 gives a detailed summary of the studies describing the (6) Temkin isotherm q = BT . ln⁡(KT Ce )
varied classes of contaminants remediated by different bacterial strains where q is pollutant adsorption at time
immobilized in polymeric support with their degradation efficiency to­ t, KT and BT are equation constants, Ce
wards each contaminant. is the equilibrium adsorbate
concentration in the solution
In addition to the contaminants mentioned in Table 1, some redun­ (
(7) Dubinin–Radushkevich
dant wastes like cellulose, produced from industrial food processing and isotherm q = qm .exp⁡ −
agricultural activities underwent biodegradation via immobilized bac­ [ ( )] )
1 2
terial cells and bio-composting to produce natural organic fertilizers and B . RT . ln⁡ 1 +
Ce
other useful products like single-cell protein (SCP) and protein-rich feed where q is pollutant adsorption at time
(PRF), as reported in a study in 1999 by Petre et al. [99]. t, qm and B are equation constants, Ce is
the equilibrium adsorbate
concentration in the solution, R is
7. Factors that influence bioremediation using immobilized universal gas constant and T is the
bacterial cells temperature

7.1. Mass transfer limitation

Bacteria immobilized in hydrogel enjoy higher tolerance towards
toxic contaminants due to protective encapsulation but suffer from slow
transfer of growth promoting materials (oxygen, and nutrients) due to
added mass transfer barrier. For example, studies on biodegradation of
diesel using immobilized cells showed that transport of substances
(nutrient, diesel) was inhibited in calcium alginate (CA) beads due to
their small pore size and dense network. This was rectified by combining
straw with sodium alginate, i.e., by forming straw-alginate beads [100,
101]. Degradation rate using free cells, however, was only half of that
found in immobilized cells. For bioremediation of toxic contaminant
using immobilized bacteria, several mechanisms may run concurrently
and may be affected differently by the toxic target contaminant. A part
of the contaminant may be removed by passive adsorption onto the
matrix material or the cell surface. An increase in the adsorbate con­
centration, regardless of its toxicity, may lead to higher adsorptive
removal following the pertinent adsorption isotherm relation (Table 2).
This may also stimulate accumulation inside the viable cells. However,
above a threshold concentration, both passive and active uptake of
pollutant will be ceased due to exhaustion of binding sites and lower
viability of cells, respectively. The dual role of matrix material (carrier
and adsorbent) was evaluated in uptake of Cd(II) ions by immobilized
Bacillus cells [102]. While immobilizing viable Bacillus cells onto CA gel
bead, different varieties of beads were prepared by incorporating
different types of biochar within the bead. High uptake capacity
(~159 mg/g) of the system was a result of bioaccumulation of Cd(II)
ions within the cells with partial contribution from its biosorption by
biochar and cells. The biochar material acted as a "protective buffer" by
triggering rapid passive uptake of Cd(II) ions, leading to less damage to
the viable cells inside. Under this situation, initial concentration of Cd
(II) may be increased to 180 mg/L with consequent increase in the total
uptake. For free suspension, the corresponding maximum tolerance level
was 150 mg/L only.
7.2. Toxicity of pollutants
Three different systems of PVA immobilized activated sludge (cry­
ogel beads with biomass only, cryogel beads with PAC on external sur­
face and biomass, cryogel beads with homogenized PAC and biomass)
were studied for biodegradation of chlorophenol [103]. It was found
that the highest removal of chlorophenol was obtained for cryogel beads
with PAC on external surface as toxic effect of the pollutant was mostly
reduced by its passive adsorption onto the outer layer of activated car­
bon followed by slow diffusion inside the bead for biodegradation. A
recent study on impact and biodegradation of sulfamethoxazole (SMX)
during anaerobically digested concentrate (ADC) treatment in a photo­
bioreactor was evaluated by comparing three different types of
microalgal-bacterial consortium: free suspension, CA immobilized
beads, CA immobilized beads with powdered activated carbon (CA-PAC)
[104]. It was found that SMX strongly inhibited the growth of free cells,
ADC treatment and SMX removal whereas satisfactory results were ob­
tained for both immobilized systems. As toxic effect of SMX was
extensively mitigated by its adsorption onto PAC, highest population of
living cells (86.2%), highest ADC treatment performance (COD, total

8
T. Mehrotra et al.
nitrogen, and total phosphorus removal of 72.12 ± 1.34%,
98.47 ± 0.69% and 98.49 ± 0.73%, respectively) and maximum SMX
removal (99.0 ± 0.2%) were achieved in the CA-PAC system.
7.3. Salinity
Biodegradation of hydrocarbon in high salinity wastewater depends
on salt tolerance ability of halophilic bacteria and the performance is
usually decreased beyond a certain threshold level of total dissolved
solids (TDS). A batch biodegradation study using free cells showed that
influent TDS higher than 145,000 mg/L reduced the bacterial growth
rate significantly [105]. Another study utilized walnut shell (WS) to
immobilize halophilic bacteria in the biological treatment of hypersaline
produced water in MBBR [106]. It was found that WS increased the
resistance of the system towards salinity shock and higher organic
loading rate at influent TDS higher than 90,000 mg/L. Halotolerant
bacterial cells immobilized onto polypropylene fiber achieved 4–7 times
higher biodegradation of crude oil compared to the free cells at the
highest salinity evaluated (180 g/L) using a batch reactor [107]. At this
salt concentration, free cells lost their biodegradation capacity.
Biodegradation of PAH (benzo[a]pyrene) contaminated soil samples
was analyzed using both free and vermiculite immobilized systems of
bacterial-fungal mixed consortium [108]. The pollutant removal per­
centage was found to be significantly high (95.3%) for immobilized
systems compared to that of free cells (79.6%). It was found that when
initial concentration of (benzo[a]pyrene) was above 400 mg/kg,
biodegradation capacity of free cells was almost inhibited whereas an
extended lag time was required for immobilized system.
7.4. Co-contaminants
Immobilization has also been found to protect the living cells from
the toxic effect of substances other than the target contaminant. For
example, the inhibitory effect of heavy metals towards denitrifying
bacteria is well documented. In a recent study, effect of various matrixes
(PVA-SA, PVA-SA-kaolin, SA-kaolin, ceramsite and activated carbon)
was compared for immobilization of a Pseudomonas strain to protect
denitrifiers from toxic effect of Cr(VI) [109]. It was found that free cells
only removed 4% of NO3− -N while the removal rate was increased to
86%, 88% and 100%, respectively, for cells entrapped in PVA-SA,
PVA-SA-kaolin and SA-kaolin. However, immobilization using adsorp­
tion carriers (activated carbon, ceramsite) was found to be inefficient.
Studies on biodegradation of phenol using CA immobilized bacterial
cells isolated from wastewater showed that the initial concentration of
the pollutant can be increased up to 800 mg/L whereas the corre­
sponding lethal dose for free cells was 400 mg/L only [110]. Still, there
are examples where contaminant concentration was not a prime factor.
Ji and Wang studied removal of Se (VI) in a packed bed reactor using a
Shigella fergusonii strain immobilized onto CA gel beads. The reactor bed
height and HRT were found to control the removal efficiency which was
not affected by the influent selenium concentration [111].
7.5. pH
For biosorption and bioaccumulation, pH of the medium controls
electrostatic interaction between the bacteria and pollutant by influ­
encing the speciation of the pollutant and the physicochemical proper­
ties (e.g., zeta potential) of bacterial cell surface which in turn affects the
availability of the binding sites on the surface of the bacterial cells. For
example, bioremediation of Cr(VI) using CA immobilized baker’s yeast
was studied by varying the pH of the solution from 1.5 to 7.5 and the
highest uptake capacity was found to be at pH 3.5 [112]. Similar
dependence was reported in other studies [113,114]. For biodegrada­
tion, the medium pH affects the enzyme activity and the bacterial
growth rate [115]. For a given bioreactor, the optimum pH for biore­
mediation can be determined by factorial experimental design.
9
Journal of Environmental Chemical Engineering 9 (2021) 105920
Typically, the immobilized cells showed a broad optimum pH profile by
shielding the biomass against unfavorable culture environment [116].
For example, phenol degradation ability of a Sphingomonas strain was
completely inhibited at pH 3; whereas the polyvinyl alcohol­
–alginate–kaolin immobilized cells were found to maintain 95%
removal efficiency [117]. In highly alkaline environments, such as at pH
10 and 12, free cells were only able to remove 70% and 50% phenol,
respectively, whereas immobilized cells still achieved 95% removal ef­
ficiency albeit at a slightly slower rate. Another study evaluated the
effect of pH and temperature on bioremediation of di-n-octyl phthalate
(DOP) contaminated soil using free and corncob-sodium alginate
immobilized bacteria [116]. Results showed that optimum pH and
temperature for both free cells and immobilized cells were 7 and 30 ◦ C,
respectively. But variation of temperature within 20–35◦ C showed that
degradation of DOP was affected severely (varied between 20% and
80%) for free cells, whereas the effect was less pronounced (50–90%) for
immobilized cells. Similarly, when pH is changed from 7 to 4, DOP
removal was reduced to only 18.1% for free cells, whereas immobilized
cells were still able to achieve 40.3% removal. The study also revealed
that the inoculated free cells may negatively impact the biodiversity of
the indigenous bacterial population. An analysis of bacterial community
structure showed that enhanced biodegradation of DOP by using
immobilized cells was due to better maintenance of biodiversity of
indigenous soil bacteria rather than the protection of the inoculated cells
through carrier material.
8. Kinetics and equilibrium analyses for hydrogels mass transfer
The process of hydrogel preparation and fabrication directly affects
their properties and nature in an application. While using as remediation
matrices, with or without bacterial entrapment, the efficiency of
contaminant removal depends on fabrication nature, behavior, and
pattern along with bond formation. To improve the application effi­
ciency of hydrogels, it is also important to understand their mass transfer
properties and adsorption behavior. Besides polymer intrinsic pattern,
mass transfer of any said particles changes substantially depending upon
the physical shape and size of the hydrogel. Post-application hydrogel
degradation is also an important factor to be considered.
The basic measure of mass transfer within a hydrogel can be repre­
sented by the equilibrium adsorption capacity q (mg/g). If c1 and c2
denote the pollutant concentration (mg/L) in water at initial time (t = 0)
and at equilibrium stage respectively, W is the weight of the hydrogel
(gm), and V is the volume of the solution (L), then
(c − c )
(1)
1 2
q=
W
∗V
Each polymer and structured hydrogel follow and fit a different
pattern. Some of the different isotherm mathematical models describing
the adsorption process are presented in Table 2.
The more pollutant gets adsorbed on the polymer site, the binding
energy starts to reduce, and the homogeneous adsorption tends to
become heterogeneous and non-linear. Temkin isotherm, however, as­
sumes linear mass transfer along with surface. Dubinin-Radushkevich
isotherm is the one that takes intrinsic characteristics of the material
into account and evaluates the adsorption rate by considering when
pollutant molecules fill up the pore space.
Mass transfer plays an important role for successful biodegradation
or adsorption/ biosorption of a pollutant using immobilized bacteria, as
supporting matrix used for immobilization adds another layer of mass
transfer resistance for the pollutant and other substances (such as nu­
trients and oxygen required for viable bacteria) to reach the active site.
For example, it has been found that addition of sodium alginate (SA) and
inorganic nanoparticles can reduce the strong mass transfer resistance
exhibited by polyvinyl alcohol (PVA) gel beads and prohibit leaking of
bacteria from the beads [118,119]. Studies on biological denitrification
using immobilized activated sludge within PVA-SA gel beads modified
T. Mehrotra et al. Journal of Environmental Chemical Engineering 9 (2021) 105920

by alumina nanoparticles (ALNP) showed that structural characteristics Table 3

of beads hindered the transport of oxygen within the bead [118]. This Mathematical models describing the rate of the contaminant transport from bulk
led to the formation of a dissolved oxygen (DO) concentration gradient liquid to immobilized bacterial cells.
from external surface of the bead to the bead interior. Biological nitro­ Eq. Model Differential Integrated Symbol legend
gen removal, which is a combination of both aerobic (nitrification) and No. form form
anaerobic processes (denitrification), was accomplished successfully by (8) Pseudo first dq q k1= PFO rate
= k1 (q∞ − ln(1 − )
utilizing the stratification of oxygen within the beads. In a subsequent order (PFO) dt q∞ constant, q=
q) = − k1 t
study on biodegradation of polycyclic aromatic hydrocarbons (phen­ uptake at time t,
q∞= uptake at
anthrene, fluoranthene, and pyrene) in soil washing effluent using
equilibrium
Klebsiella sp. immobilized within the same gel bead, the gel bead (9) Pseudo second dq k2=PSO rate
composition was optimized with respect to the mass transfer efficiency = q =
order (PSO) dt constant, q=
[119]. The mass transfer efficiency was evaluated by conducting a series k2 (q∞ − q)2 k2 q∞ 2 t uptake at time t,
of batch kinetic experiments to construct the concentration-time profile 1 + k2 q∞ t q∞= uptake at
equilibrium
of methylene blue adsorption within the bead with varying compositions (10) LDF (fluid ∂q kf= external or film
of gel bead components (SA, PVA, ALNP). The optimized gel bead ρ = kf S0 (C −
phase) ∂t mass transfer
composition was found to contain 10% PVA, 0.8% SA, and 0.7% ALNP Ci ) coefficient, ρ
[119]. =density of
adsorbent particle,
Diffusion is the primary mode for mass transfer. Researchers have
S0 =adsorbent
investigated the question regarding mode of mass transfer by various particle surface
experimental techniques such as protein adsorption, drug loading/ re­ area per unit
leases, dyes etc. While using in water treatment application, the mi­ volume, C = bulk
cropores of the hydrogel adsorb the pollutant while the interconnected aqueous
concentration of
capillary network helps the solute to reach the core and more accessible
adsorbate,
to bacteria, if involved. To understand the matrix efficiency, measuring Ci=aqueous
the mass transfer efficiency and kinetics is an important step. concentration of
In general, there are four steps in the mass transfer within a highly adsorbate at the
adsorbate-
porous polymer hydrogel network: a) transfer of the reacting substance
adsorbent
through bulk aqueous phase which is usually a fast process, b) move­ interface
ment of the substance through a fixed layer of liquid "film" surrounding (11) Weber–Morris
√̅̅
q = kid t + q= uptake at time
the gel bead (external mass transfer or film diffusion), c) diffusion of the Const t, kid=intra-
pollutant inside the gel bead (intraparticle diffusion), d) adsorption and/ particle rate
constant
or biodegradation reaction at the active site where the bacteria is located ∂q
(12) Shrinking-core rc=radius of the
(Fig. 2). For example, an investigation on degradation of petroleum =
∂t ⎛ shrinking core, R=
⎞
hydrocarbon by bacteria immobilized on various hydrogel structures radius of adsorbent
4π ⎜
⎜ Ds Ci ⎟,
⎟
concluded that the diesel was first adsorbed on the surface of hydrogel particle, M=mass
M ⎝ 1 1⎠ of adsorbent in the
followed by diffusion of adsorbed species along the pore surface to the −
rc R reactor, Ds=
active site causing subsequent degradation by bacteria [120]. R diffusion
All these elementary processes except the transfer through bulk of
=
rc coefficient of
( )1/3
the aqueous phase, i.e., step b to d, may influence the final rate of q∞ adsorbate within
removal but it is usually hypothesized that one of these steps is much q∞ − q the adsorbent, q=
uptake at time t,
slower than others and controls the overall rate. A theoretical mass
q∞= uptake at
transfer model is then derived accordingly which is tested by fitting equilibrium
against the experimental data (Table 3). A simplistic and frequently used
procedure assumes the reaction at the active site as the rate limiting step

Fig. 2. The mechanisms involving the transport of pollutants from bulk liquid bacterial cells immobilized inside a porous beads.

10
T. Mehrotra et al.
of the process ignoring the mass transfer resistance. For example, a
number of articles studying removal of contaminants through biomass
immobilized into various hydrogels found that batch kinetic data fol­
lowed pseudo first-order (PFO) or pseudo second-order (PSO) rate
equations (Eqs. (8) and (9)) [121,122]. The PFO or PSO model do not
provide any information regarding diffusional mass transfer within the
supporting matrix. To get an insight of diffusion limitation of mass
transfer, rate equations describing external (step b) and intraparticle
diffusion (step c) have to be considered. To determine the rate constant
for film diffusion, the rate of mass transfer is often assumed to follow
linear driving force (LDF), i.e., proportional to the difference in the
concentration between the bulk aqueous concentration of the solute and
the concentration at the interface (interphase boundary) leading to Eq.
(10). To determine the role of intraparticle diffusion, Weber–Morris
equation (Eq. (11)) is widely utilized.
A comparison between free and CA-immobilized yeast cells for
removal of phosphorous showed that PFO rate constant of former was 5
times lower than that for the latter [121]. For both cases, PFO rate
equation was found to fit better than PSO. Mass transfer within the bead
was modeled using Eqs. (10) and (11) to describe the external diffusion
and intraparticle diffusion, respectively. It showed multilinear plots in
both cases implying a multi-step mechanism of mass transfer. On the
other hand, each of four fungal strains immobilized into CA for removal
of arsenic were found to follow PSO rate equation better than PFO [122].
The remediation was found to be rapid as almost 90% of the uptake
capacity was exhausted within 100 min [122]. Another investigation for
removal of thulium, a rare earth metal, using brown alga Turbinaria
conoides revealed that free cells followed PFO rate law while the poly­
sulfone immobilized cells followed PSO rate law. For intraparticle
diffusion, a multi-linear plot was obtained consisting of 2 and 3 stages
for free cells and immobilized cells, respectively. The additional segment
for immobilized cells was attributed to the slow intraparticle diffusion
within the bead. Similar multistep mechanism was observed for bio­
sorption of Cu(II) from aqueous solution by Aspergillus sydowii immo­
bilized onto magnetic chitosan microspheres. The intraparticle mass
transfer within a gel bead is also described by shrinking-core model
(SCM), which is developed based on the observation that a partially
reacted sorbent particle contains a ‘reacted’ outer shell surrounding an
‘untreated’ core, whereby the reaction takes place only at the interface
between the shell and the core. This is because the pollutant needs to
diffuse through the saturated (no free binding sites are present) shell of
the particle into the unreacted core, to reach the binding site. As a result,
as remediation continues, the core keeps on ‘shrinking’ and rate of the
intra-particle diffusion is related to the rate of ‘shrinking’ of the core. For
such a system, a rate equation can be written as in Eq. (12). The bio­
sorption of nonylphenol on dead biomass of Rhizopus arrhizus encapsu­
lated in chitosan beads was modeled using SCM and was found to be
controlled by intraparticle mass transfer [123].
Another batch study compared the effect of incorporation of acti­
vated carbon fiber into CA beads to immobilize a Pseudomonas species
for enhanced adsorption of tetrahydrofuran followed by its biodegra­
dation [124]. The mass transfer was modeled by applying Fick’s second
law of diffusion assuming a steady state, i.e., rate of diffusion being
equal to the rate of biodegradation. It was found that Thiele modulus
values of the hybrid beads, i.e., beads containing carbon fibers, were
smaller than the original CA beads implying reduced diffusion limita­
tion. Also, the effective diffusion coefficients were larger by one order of
magnitude than those of CA beads only.
A batch reactor is easier to set up in laboratory to conduct the
adsorption/ biodegradation experiment whereas a fixed-bed reactor is
more suitable for commercial implementation. The shape of the break­
through curve, i.e., the concentration-time profile of the effluent after
adsorption, depicts the mass transfer zone within the column and is also
instrumental for scale-up of the system. However, the mathematical
modeling of the breakthrough curve is complex due to the unsteady state
operation and the heterogeneities in packing of gel beads. It typically
11
Journal of Environmental Chemical Engineering 9 (2021) 105920
employs numerical algorithm for simultaneous solution of mass balance,
isotherm and external and/ or intraparticle rate equation as analytical
solution is only available for a few simplified cases only. Most of the
published articles rely on these simplified models (Thomas model (Th),
Bohart–Adams model (BA), Yoon–Nelson model (YN) among others),
often ignoring the underlying assumptions [125]. A recent investigation
on removal of nickel ions from monometallic and simulated wastewater
using viable Bacillus sp. cells immobilized within CA gel analyzed the
breakthrough curves using the above three models and found fitting of
Th and YN models to be satisfactory [126]. The study also concluded
that the mass transfer in the initial phase (~ 7% of effluent/influent
concentration ratio) was controlled by intraparticle diffusion only while
the subsequent process was controlled by both film and pore diffusion.
Another investigation on removal of heavy metals using free and CA
immobilized Sargassum sp. showed that breakthrough curves for both
nickel and copper were well represented by Th model and corresponding
rate constants were evaluated through non-linear curve fitting method
[127]. Breakthrough curves for both free and immobilized cells were
found to be very similar in shape, implying no significant mass transfer
limitation due to immobilization. The affinity of copper ions was higher
than nickel ions and the dynamic uptake capacity was found to be higher
than batch uptake capacity for both metals. The Th model was also
found to fit better than YN model for the breakthrough curves in removal
of aluminum ions using agar immobilized Pseudomonas putida [128]. A
comparison with control (column packed with hydrogel without
biomass) showed a 10-fold increase in the removal percentage by
incorporation of biomass in the beads. It was also noted that the fixed
bed was suitable for 12 successive adsorption/desorption cycles using
dilute HCl as the eluent.
Another elegant analytical expression is available for modeling of
mass transfer in a fixed bed reactor where the pollutant is removed
through biodegradation under steady state, i.e., rate of biochemical re­
actions leading to the biodegradation (step-d) was assumed to be equal
to the LDF based rate of film diffusion (step-b) ignoring the intraparticle
diffusion [129]. The expression was linearly regressed to find mass
transfer correlations connecting Colburn J-factor and Reynolds number
for biodegradation of phenol by CA immobilized Bacillus cereus [130],
reduction of Cr (VI) by CA immobilized Bacillus sp. [131], and biodeg­
radation of malathion dye by CA immobilized Bacillus sp. [132].
It should also be considered that nature of organic pollutant
adsorption differs from inorganic pollutants. Tanaka and Fillmore have
studied the mass transfer and diffusivity by tracking the spherical par­
ticle radii and their swelling by water adsorption [133].
With a higher portion of water content, hydrolysis is the most
prevalent and dominant course of hydrogel degradation. Because of its
structural complexity, mass loss is the most reported degradation model
discussed [134,135]. Keeping polymer network formation, dissociation,
crosslinking capacity, and ester cleavage – and considering many envi­
ronmental factors, hydrolytic degradation can best be described with
hydrolysis rate constant (k), which can be represented via PFO kinetics.
Studies have shown that with minor changes in polymer concentration,
hydrolytic degradation pattern changes significantly. The hydrolysis
rate of hydrogel can be measured by measuring the ester bond cleavage
[136]. With analytical and experimental measurements (i.e. nuclear
magnetic resonance, NMR) of bond cleavage [137], Lau et al. showed
that hydrogel is formed by ester linkages and also validated the hydrogel
hydrolysis rate [136]. The hydrolysis kinetics can be written as a first
order equation: d[Ester]/dt = − k[Ester]. Here the rate of ester bond
dissociation can be calculated from the ester bonds present at any time t,
i.e, − ln(Et/E0) vs time. The key findings were: hydrolysis rate decreases
along with hydrogel degradation; and that the ionic charge of the
polymer in the sol-gel solution has a prominent effect on degradation. It
was also observed that negatively charged ions inhibit ester bond hy­
drolysis and act as a preventive layer. Finally, similar to previous reports
of polymer degradation patterns, solvent density and viscosity have a
substantial influence on matrix hydrolysis or degradation.
T. Mehrotra et al. Journal of Environmental Chemical Engineering 9 (2021) 105920

9. Design of biofiltration systems using bacteria immobilized
hydrogels
The use of immobilized bacteria in biofiltration systems has gained
significant attention for the treatment of wastewater [138–141] from
various industrial and domestic sources, including mining [142,143],
petroleum [144], pharmaceutical [145], textile [146], pulp and paper
[147], animal husbandry [148], and municipal sewage [149]. Different
bioreactor types including packed-bed bioreactor (PBR) [150], fluidized
bed bioreactor (FBR) [147], hybrid bioreactor (HBR) [151], anaerobic
membrane bioreactor (MBR) [152,153], and continuously stirred tank
bioreactor (CSTR) [154,155] have explored the feasibility of using
immobilized bacteria in beads/gels for the treatment of wastewater from
various sources (Table 4).
9.1. Packed-bed bioreactor (PBR)
The PBR is typically comprised of a column which is packed with
hydrogels immobilized with bacteria, and can be operated in either an
up-flow or a down-flow configuration (Fig. 3A). The PBR containing
Bacillus cereus immobilized in CA beads was used for the treatment of
wastewater generated from the petroleum industry [144]. The authors
reported more than 90% removal of COD, 96% removal of TOC, com­
plete removal of phenolics, 49% removal of ammonium nitrogen, and
42.6% removal of phosphate from the wastewater. Similar complete
removal of phenol from aqueous solution using Bacillus cereus immobi­
lized in CA beads has been reported in another study [130]. SRB sludge
immobilized in PVA and Na-alginate was used in PBR operating at 24 h
HRT to treat synthetic acid mine drainage wastewater (AMD) [156]. The
authors reported the supplementation of zero-valent iron (ZVI) in the
immobilized beads to create reducing condition which favored the
growth of sulfate reducing bacteria (SRB). Removal of Fe, Cu, Zn and Cd
by 99.9%, Mn by 42.1–99.3%, and increase in pH from 2.8 in influent to
7.8–8.3 in the treated effluent indicated successful treatment of AMD
[156]. Similar treatment of synthetic metal-contaminated wastewater
has been performed in another study using a mixed culture of SRB
immobilized in Ca-alginate beads [157]. The study showed that the
operation of the PBR at 48 h HRT resulted in the removal of 50–70% Ni,
90% Pb, 80–90% Zn, 95–99% Cu, 90–92% Cd, and 60–80% Fe. Textile
wastewater has also been treated using bioreactor packed with stainless
steel sponge-agar composites that contained immobilized mixed bacte­
rial consortia consisting of Brevibacillus laterosporus and Galactomyces
geotrichum [146]. The authors reported 80% removal of COD and 93%
removal of color during the treatment of the wastewater at 18.2 h HRT.
PBR has also been reported to remove 86–95% COD from cheese in­
dustry wastewater using pure culture of Lactobacillus bulgaricus immo­
bilized in Ca-alginate beads [158]. Although PBR has demonstrated
successful performance for the treatment of contaminated water from
different sources as discussed above, this bioreactor configuration faces
several operation challenges, including clogging [159],
cross-channeling [160], and mass transfer limitation [131,161].
9.2. Fluidized bed (bio)reactor (FBR)
The FBR comprises of immobilized bacteria in hydrogels which are
suspended in a column due to the up-flow of contaminated water at a

Table 4
Immobilized bacteria in different bioreactor types for the treatment of contaminated water.
Bioreactor Immobilizing Bacteria Wastewater HRT Performance Ref.
materials (h)

PBR Ca-alginate Bacillus cereus Petroleum refinery NM 99% COD removal, [144]
99.8% phenolics removal.
Phenol in Aqueous solution NM Complete removal [130]
PVA and Na-alginate Mixed culture consisting of Desulfovibrio and Synthetic AMD 24 99% removal of Fe, Cu, Zn, and [156]
Clostridium Mg,
42.1–99.3% sulfate removal.
Stainless steel Mixed consortium of Brevibacillus laterosporus Textile wastewater 18.2 93% decolorization, [146]
sponge-Agar and Galactomyces geotrichum 80% COD removal
Ca-alginate Mixed microbial culture containing Synthetic metallic wastewater 48 50–70% Ni removal, [157]
Desulfovibrio 90% Pb removal,
80–90% Zn removal,
95–99% Cu removal,
90–92% Cd removal,
60–80% Fe removal
Ca-alginate Lactobacillus bulgaricus Cheese whey NM 86–95% COD removal. [158]
FBR Ca-alginate Bacillus cereus Petroleum refinery NM > 95% removal of COD and [162]
phenolic compounds
PVA-cryogel Rhodococcus ruber, Rhodococcus opacus Petroleum contaminated water NM 70–100% removal of n-alkanes. [163]
(synthetic) 66–70% removal of PAH.
HBR PVA Sludge from wastewater treatment plant. Sewage wastewater (synthetic) NM 67–80% inorganic nitrogen [151]
removal.
73–92% COD removal.
PVA Anaerobic culture from upflow anaerobic Sewage wastewater 72 89% COD removal [165]
sludge blanket bioreactor
MBR PVA Mixed microbial community from anaerobic Domestic wastewater 24 84.7% COD removal. [153]
sludge containing. (synthetic)
Cellulose triacetate Microbial community from marine sediment Pharmaceutical 40–60 78–81% COD removal. [167]
14–22% TN removal.
PVA/SA Pseudomonas putida Hospital 12 h 48% TN removal. [152]
96% COD removal.
90% antibiotic (ciprofloxacin)
removal.
CSTR PEG Afipia sp. Wastewater containing dioxane 16 82–99% dioxane removal. [155]
(synthetic)
PVA Annamox sludge Reject water from wastewater 2 85–91% nitrogen removal. [169]
treatment plant
PEG Sewage sludge Sewage water (synthetic) 1.4 h 92% nitrogen removal. [154]

NM = not mentioned.

12
T. Mehrotra et al.
Fig. 3. Different bioreactors used for the treatment of contaminated water from various sources; (A) packed bed reactor (PBR), (B) fluidized bed reactor (FBR), (C)
hybrid bio-reactor (HBR), (D) anerobic membrane bioreactor (AnMBR), (E) continuous stirred tank reactor (CSTR).
certain velocity to promote efficient mixing (Fig. 3B). CA immobilized
pure culture of Bacillus cereus was used to treat wastewater from a pe­
troleum refinery [162]. The authors reported more than 95% removal of
COD and phenolics from the petroleum refinery wastewater. In a similar
study, Rhodococcus ruber and Rhodococcus opacus were immobilized in
PVA for the removal of 70–100% n-alkanes and 66–70% PAHs from
synthetically prepared petroleum-contaminated water [163]. The FBR
configuration provides a high rate of mass transfer to the immobilized
bacterial gel/beads leading to high biomass growth and an increase in
the rate of contaminants removal during wastewater treatment [164].
9.3. Hybrid bioreactor (HBR)
The hybrid bioreactor comprises of suspended and attached bed
bacterial growth to promote better bioreactor performance (Fig. 3C).
The anaerobic bacterial sludge immobilized in PVA has been used in
HBR for the treatment of synthetic sewage wastewater [151,165]. The
HBR system consisting of a mixture of anaerobic sludge immobilized in
PVA beads, suspended biomass, and biofilm carrier materials was re­
ported to remove 67–80% inorganic nitrogen and 73–92% COD [151].
In another study, HBR consisting of two-staged acidogenic and meth­
anogenic bioreactors packed with PVA removed 89% COD from a
sewage wastewater at 72 h HRT [165]. HBR configuration could protect
the immobilized bacteria from direct exposure to specific contaminants,
and improve treatment of wastewater [166].
9.4. Anaerobic membrane bioreactor (AnMBR)
The AnMRB comprises of membrane and bacteria immobilized on
hydrogels for the treatment of wastewater under anaerobic conditions
either in un-flow or down-flow configuration (Fig. 3D). Anaerobic
sludge immobilized in PVA has been reported to remove 84.7% COD
13
Journal of Environmental Chemical Engineering 9 (2021) 105920
from synthetically prepared domestic wastewater during the operation
of AnMBR at 24 h HRT [153]. In another study, the bacterial community
developed from the marine sediment was immobilized in cellulose
triacetate for the treatment of pharmaceutical wastewater [167]. The
authors reported the removal of COD and total nitrogen (TN) by 78–81%
and 14–22%, respectively, during the operation of AnMBR at 40–60 h
HRT. Treatment of hospital wastewater at 12 h HRT using a pure culture
of Pseudomonas putida immobilized in PVA/SA reported the removal of
TN, COD, and antibiotics (ciprofloxacin) by 48%, 96% and 90%,
respectively [152]. The application of immobilized beads in membrane
bioreactors retains the slow-growing bacteria, and prevents the attach­
ment of the suspended bacterial cells to the membrane surfaces [153].
The friction between immobilized beads and membrane surface reduces
cake formation on the membrane surface, and improves the MBR per­
formance for wastewater treatment [153,168].
9.5. Continuously stirred tank (bio)reactor (CSTR)
The CSTR contains bacteria immobilized hydrogels which is effi­
ciently mixed with wastewater under continuous stirring and aeration to
eliminate mass transfer limitation (Fig. 3E). A pure culture of Afipia sp.
immobilized on PEG was used in CSTR for the removal of 85–91%
dioxane from aqueous solution at 16 h HRT [155]. Anammox sludge
immobilized in PVA has been used in CSTR for the treatment of reject
water from wastewater treatment plants [169]. The authors reported
that the operation of the CSTR at 2 h HRT resulted in 88–92% nitrogen
removal. Another study using sewage sludge immobilized in PEG re­
ported removal of 92% total nitrogen during the CSTR operation at 1.4 h
HRT [154]. The configuration of CSTR provides high mass transfer of
substrates to the bacterial cells immobilized in the beads/gels, resulting
in a high rate of the contaminant removal from the wastewater [170].
T. Mehrotra et al.
10. Challenges, recent advances, and future scope
This review discusses the advances in immobilizing bacterial cells
using various polymers, unexplored bacterial strains, and new cell
immobilization techniques toward bioremediation of environmental
contaminants. As discussed in this article, bioremediation of industrial
wastes using immobilized bacterial beads has shown promising perfor­
mance in the removal of contaminants, but most extant work has been
largely limited to laboratory-scale studies. While immobilization of
microbes is an attractive strategy there are several associated challenges
including the complexity of operations, matrix stability, wastewater
characteristics, presence of multiple contaminants, process design for
industrial and large-scale operations, mass transfer limitation of sub­
strates into immobilized cells, build–up of toxic metabolic products
around the cells because of low diffusion rate (which can inhibit mi­
crobial growth), and formation of thick biofilm which can block the
pores of the beads and interrupt substrate transport from the bulk liquid
to immobilized cells. Despite these challenges, research continues to
progress in this field to further elucidate and advance these processes.
Some of the most recent developments in the field of immobilized
bacteria for bioremediation include investigation of various composite
materials such as biodegradable sponge-polycaprolactone, water soluble
sponge-NaCl-PEG [171], hydrogel-biochar-zerovalent iron [172], and
graphene oxide-polysulfone [173] for the treatment of wastewater
containing oily pollutants, Pb-contaminated water, and textile waste­
water, respectively. Moreover, immobilizing bacteria on matrices hel­
ped in not just simply remediating a contaminated site but also
accelerated pollutant biodegradation capacity, increased bacterial
enzyme activity, enhanced the durability of the immobilized strains, and
improved their tolerance to high pollutant loads. The study conducted
by Sun et al. [174] discussed enhanced tebuconazole degradation along
with enhanced bacterial bioactivity upon immobilizing bacteria onto
biochar. Similar immobilization of Bacillus sp. TZ5 on biochar enhanced
bacterial count and enzymatic activity, and increased Cd bioremediation
[175]. Another study conducted by Ma et al. [176] showed that
immobilizing bacteria with biochar accelerated polyacrylamide degra­
dation due to synergistic effect of bio-augmentation of the strain and
bio-stimulation of other indigenous polyacrylamide degraders. Also,
support matrix like natural coconut fibers have also been used for
immobilizing Pseudomonas aeruginosa for enhanced biodegradation of
n-hexadecane by reducing the half-life of the contaminant [177].
Among other aspects, future research should continue to explore
development of appropriate substrates to immobilize bacteria that are
cost-effectiveness, have stable physical and chemical properties, high
porosity and surface area, and non-toxic properties. Another direction of
the future research could focus on investigating appropriate ratios of
microbial biomass and immobilizing substrate dosing to prevent cell
overcrowding which is responsible for high biofilm formation and pore
clogging. Optimizing the flow rate through the bioreactor could be
another strategy to slough off the over-grown and mature biofilms to
prevent pore clogging. Recovering value-added products with simulta­
neous bioremediation processes such as recovering the metals during the
biological treatment of metal-rich wastewater could make the immobi­
lized cell-based treatment process more sustainable. Finally, standardi­
zation of bead-formation techniques and development of fundamental,
theory-based engineering design principles along with governing
equations and recommendations would go a long way to make immo­
bilized microbes a mainstream bioremediation tool. Overall, immobi­
lizing microbes is an intriguing concept and continues to attract research
leveraging next-generation smart materials and other reusable matrix
materials to remediate a wide variety of contaminants. We believe
research in this area will continue to grow swiftly in the coming decades
and there will be increasing examples of commercial and field adapta­
tion of this technology.
14
Journal of Environmental Chemical Engineering 9 (2021) 105920
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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