Journal of Environmental Chemical Engineering 7 (2019) 103261

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Journal of Environmental Chemical Engineering

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

Potential and challenges of enzyme incorporated nanotechnology in dye T

wastewater treatment: A review
Johnny Kee Hong Wonga, Hong Koon Tana, Sie Yon Laua, , Pow-Seng Yapa,
⁎

Michael Kobina Danquahb

a
Faculty of Engineering and Science, Curtin University Malaysia, CDT 250, 98009, Miri, Sarawak, Malaysia
b
Department of Civil & Chemical Engineering, University of Tennessee, 615 McCallie Ave, Chattanooga, TN, 37403, United States

ARTICLE INFO ABSTRACT

Keywords: Enzymes are known to catalyze reactions at high efficiency, operate at milder conditions and are biodegradable.
Enzymes Due to enzyme limitations such as sensitivity to environmental conditions, enzyme immobilization is often used.
Immobilization The commonly employed immobilization methods include adsorption, entrapment, covalent attachment and
Nanotechnology cross-linking. Many research works have now focused on the immobilization of enzymes on nanoscale support
Wastewater treatment
due to the higher surface area to volume ratio, effective enzyme loading, significantly enhanced mass transfer
Nano-biocatalyst
efficiency and minimization of diffusional problems. The application of enzyme incorporated nanotechnology in
the treatment of dye wastewater is thus, of high interest. Therefore, this paper has critically reviewed (1) the
current technologies available for dye wastewater treatment; (2) different methods utilized for enzyme im-
mobilization; and (3) the application and performance of enzyme incorporated nanotechnology for dye waste-
water treatment. We identified that there is high potential for enzyme incorporated nanotechnology to be im-
plemented in dye wastewater treatment due to the high decolorization performance (e.g. laccase immobilized on
Fe3O4/SiO2 nanoparticles achieved 99% decolorization of Procion Red MX-5B in 20 min). We have also iden-
tified the key challenges faced by enzyme incorporated nanotechnology in dye wastewater treatment that in-
cludes: (i) realization of lab scale experiments to industrial applications; (ii) lack of understanding of enzymes
incorporated nanotechnology; (iii) recovery of immobilized enzyme; (iv) synthesis of hybrid nanoflowers; and
(v) sustainability of the nanomaterials used.

1. Introduction
In recent years, the textile and apparel industry in Malaysia has
been growing at a rapid pace in targeting the higher end of global value
chain with diversified production. The rapid growth of this sector poses
a great threat to the environment with the World Bank estimating that
up to 20% of industrial water pollution originates from this industry
alone [1]. The main constituent of the effluent from the textile industry
is dye, in which 20% of the dye used is lost or wasted due to incomplete
fixation on the fabric [2]. Dyes are also waste discharge from other
industries such as food and beverage, printing, and leather industries
[3]. Most dyes are persistent in the environment due to their complex
aromaticity, high stability and are highly resistant to light operation,
oxidants and moisture [4]. As they are designed for durability to meet
the demand of the consumer market, synthetic dyes can resist biode-
gradation naturally [5,6]. Dyes discharged to the marine systems can
cause devastating effects by reducing the penetration of light into water
which disrupts the photosynthetic activity of aquatic plants. With less
dissolved oxygen available, species heavily dependent on a healthy
supply of oxygen are threatened, and this may ultimately lead to a
process known as eutrophication [7,8]. Most dyes contain harmful
chemical compounds such as nitrates, sulfur, acetic acid, chromium
based compounds and heavy metals in trace quantities such as arsenic,
lead, mercury, cadmium, nickel, cobalt and zinc in their makeup [1,9].
These effluents collectively, cause dye effluent to be extremely toxic to
not only the environment, but also towards human health. Previous
studies have shown that some dyes can cause carcinogenic, mutagenic
and teratogenic effects [10,11]. Azo dyes that enter the human body
through the gastrointestinal tract, lungs or skin were reported to affect
the formation of hemoglobin adducts and causes disturbances to blood
formation [12]. A study carried out by Sudova and co-workers de-
monstrated that malachite green dye has carcinogenic effects by ad-
versely impacting the human immune and reproduction system [13].
The basic dye, methylene blue, is not a strongly poisonous dye but

⁎
Corresponding author.
E-mail address: johnlsy@curtin.edu.my (S.Y. Lau).

https://doi.org/10.1016/j.jece.2019.103261
Received 22 May 2019; Received in revised form 26 June 2019; Accepted 30 June 2019
Available online 02 July 2019
2213-3437/ © 2019 Elsevier Ltd. All rights reserved.
J.K.H. Wong, et al. Journal of Environmental Chemical Engineering 7 (2019) 103261

Table 1
Studies on physical, chemical and biological technique for dye wastewater treatment.
Typea Method Dye Efficiency Reference

P Adsorption of dyes onto apricot stones activated carbon Methylene blue (MB) qmax, MB : 36.28 mg/g [40]
Methyl orange (MO) qmax, MO : 32.25 mg/g
Removal : 88-98%
P Adsorption of cationic dyes onto olive stones activated carbon Methylene blue (MB) qmax, MB : 16.12 mg/g [41]
Removal : 97%
P Decolorization of textile effluent by nanofiltration processes Blue Bezaktiv S-GLD 150 Decolorization : 99% [42]
Black Novacron R
P-C Removal of reactive dyes by magnesium hydroxide coagulation process Reactive red (X-3B) Removal : < 97% [43]
Reactive yellow (X-R)
P-C Removal of textile dye using photovoltaic electrocoagulation Acid Red 336 Removal : < 87% [44]
P-C Decolorization of textile effluent by coagulation/flocculation Blue Bezaktiv S-GLD 150 Decolorization : 93-94% [42]
Black Novacron R
C Dye removal by electro-Fenton process using iron-doped mesoporous silica Rhodamine B (RhB) Removal : 97% [45]
C Azo dye degradation by Fenton-like reaction with Fe-impregnated sugarcane biochar Orange G (OG) Degradation : 89.3% [24]
catalyst
C Electrochemical dye degradation by electrospun activated carbon fibers modified with Methyl orange (MO) Degradation : < 90% [46]
carbon nanotubes
B Decolorization of textile dyes by ultrasonic/microbial treatment Reactive Red 2 (RR2) Decolorization (RR2) : 93% [47]
Reactive Blue 4 (RB4) Decolorization (RB4) : 88%
Basic Yellow 2 (BY2) Decolorization (BY2) : 40%
B Decolorization of monoazo dye using peroxidase from soybean seed hulls and luffa fruit Methyl orange (MO) Decolorization (SP) : 81.4% [48]
peels
Decolorization (LP) : 75.3%
B Decolorization of textile effluent using commercial laccase Blue Bezaktiv S-GLD 150 Decolorization : < 99% [42]
Black Novacron R
B Azo dye degradation using horseradish peroxidase immobilized on cross-linked Methyl orange (MO) Degradation : < 90% [49]
polyacrylamide gel
B Biodegradation of synthetic azo dye by horseradish peroxidase cross-linked on nano- Acid Blue 113 Degradation : < 95.4% [50]
composite support
Acid black 10 BX

a
P = Physical, P-C = Physico-chemical, C = Chemical, B = Biological.

ingestion of the dye can cause sicknesses including nausea, diarrhea,
methemoglobinemia and gastritis [10,14]. With growing health and
environmental concerns, it is crucial to search for effective treatment
methods for dye wastewater from industrial discharge.
Various dye treatment techniques have been developed in recent
years as a response to dye as an emerging contaminant. These techni-
ques can be categorized into either physical, chemical or biological
process, notwithstanding that some may utilize a combination of two or
more processes. Selected studies on the performance of the different dye
treatment techniques are presented in Table 1, with all treatment
techniques exhibiting comparable dye treating performance ranging
from 75% to 99%. The unique characteristics of each treatment
methods may be advantageous in one aspect but limited in another.
Factors that are frequently considered include dye treatment efficiency,
operation and investment costs, large-scale operation feasibilities and
human and environmental friendliness. Treatment techniques that in-
volves high installation and operation costs, extensive processing time,
low efficiency, and production of toxic by-products are often omitted
from consideration in industrial applications [15].
Physical treatment is one of the most commonly used techniques
due to its simplicity, flexibility and high efficiency [16]. This category
includes dye treatment methods like adsorption, ion exchange, coagu-
lation-flocculation and membrane filtration. Of all the physical tech-
niques available, adsorption is most widely used in treating dye was-
tewater as it can extract various types of water pollutants effectively.
Adsorption describes the process of solutes collecting and adhering onto
a sorbent whereby the dye can be easily removed along with the sorbent
from the reaction medium. Examples of high adsorption capacity ad-
sorbents extensively used include activated carbon, chitosan, resin and
carbon nanotubes [17]. Besides, there are no generation of harmful by-
products or involvement of any living organisms, thus allowing better
control on the processes to produce the desired results [18]. Ion ex-
change is commonly used to extract unwanted cations and anions from
effluent water using synthetic resins, which forms into larger flocs
complexes for easy separation. Because of this, it produces large
amounts of sludge and incurs higher recharge costs [19]. Coagulation-
flocculation has been widely used to treat dye wastewater, in which
polymer coagulants are added to reduce the zeta potential on the sur-
faces of dye particles to allow the formation of flocculated agglomer-
ates. Although efficient, extensive usage of chemicals for pH mod-
ifications produce high toxicity colored sludge that requires legislative
disposal [20]. Membrane technology such as ultrafiltration, nanofil-
tration and reverse osmosis utilizes a porous medium to separate sus-
pended substances in the solution by passing through it without the
need of dye degradation [21]. This signifies that the dyes, chemicals
and processed water are recycled in the system, thus minimizing was-
tages. There is also a type of membrane technology (i.e. membrane
bioreactor) that couples membrane process together with activated
sludge process. Yet, all types of membrane filtration technology are
susceptible to membrane fouling that prevents them from being used
for prolonged periods before backwash or cleaning operation are re-
quired. While this may be true, recent research conducted by Sepehri
and co-workers has established a new method in prolonging the op-
erational period of membrane bioreactor through enrichment of ni-
trifying bacteria in activated sludge [22]. The bacteria in the activated
sludge were enriched by limiting the C/N ratio to zero during their
growth. Not only could the enriched bacteria enhance sludge filter-
ability, but also significantly reduce the amount of soluble microbial
products and extracellular polymeric substance, which are primarily
responsible in membrane fouling. Their work has led to an increase in
permeation by 2.5 times and an increase in operational period by two-
fold compared to usage of conventional activated sludge in membrane
bioreactor.
Chemical dye treatment involves the utilization of chemicals and
application of processes for the reaction to occur. The techniques in-
clude but are not limited to chemical oxidation, electrochemical and
Fenton processes. During an oxidation process, the chemical composi-
tion of a dye molecule is altered under the presence of an oxidizing

2
J.K.H. Wong, et al.
agent such as ozone, hydrogen peroxide and permanganate. Catalysts
like iron, manganese and titanium dioxide may be added to increase the
oxidation rate [23]. The Fenton approach is a rapid and inexpensive
process to oxidize organic contaminants in effluent water with the
presence of hydrogen peroxide and ferrous iron as catalysts. However,
the Fenton reaction generates a significant amount of iron oxide sludge
at the end as the iron salts are unrecyclable [24]. As the Fenton ap-
proach dictates an acidic condition for the reaction to occur, more
acidic chemicals are expended. Chemicals of poorer stability also pro-
duce unwanted products and cause secondary pollution which harms
the environment [25]. The cost of operation for chemical dye treatment
methods is relatively higher compared to other approaches with an
exception of electrochemical dye degradation method [16]. In recent
years, dye degradation electrochemically has also proven to be a more
effective and rapid approach than the other conventional processes as
toxic dyes have been degraded into smaller colorless molecules without
any coagulant added [26]. The potential application of chemical
treatment is not only limited to chemical dyes but at the same time on
other types of pollutants in wastewater including phenols and micro-
organisms. Recent works performed by Vaiano and co-workers [27] as
well as by Rani and Shanker [28] have demonstrated photocatalytic
degradation of phenols in wastewater using silver-modified zinc oxide
and bimetallic metal oxide nanostructures respectively. The photo-
catalyst were able to remove nearly all phenol content in water treat-
ment through UV irradiation process. Whereas the same process has
also proven its efficiency with the degradation of orange G dye under
UV irradiation for at least three cycles [29]. The flexibility of chemical
treatment also allows its application onto coking and swine wastewater
as well as effluents from the dairy industry. Ozonation of phenols using
catalysts such as biochar and zinc ferrite for phenol removal in coking
wastewater has been achieved within a short period of time [30,31].
Through catalytic ozonation, any trace of reactive orange 4 dye in the
textile industry effluent has also been removed completely, governed by
direct ozone molecular mechanism [32]. In the ozonation process, clean
air is forced through an electric discharge of high voltage and subse-
quently bubbled up in wastewater to combine with unwanted pollu-
tants. The resulting water is free from taste and odor problems without
application of any chemicals.
Biological dye treatment is considered as one of the simplest and
inexpensive ways to treat dye wastewater with its removal efficiency
highly dependent on the growth of microorganisms. The term bior-
emediation is often used to represent a range of techniques that utilizes
either living or dead organisms to detoxify contaminated water [33].
These techniques include but are not limited to natural attenuation,
bioaugmentation, biostimulation, bioleaching and rhizoremediation
[34]. Commonly known bioremediation technology like biosorption
utilizes biological materials or biomass to form heavy metal ion com-
plexes through ligands and functional groups. Due to the higher affinity
of the sorbent for the ionic species, the dissolved species are attracted
and bound to the solid biomass [35]. Such non-metabolically mediated
metal binding process by biomass can effectively improve the purity of
dye-loaded wastewater to drinking water quality. The biosorption
performance is highly governed by both the nature of the biomass as
well as the application. The biomass responsible for this phenomena
usually involves changes in the cell wall of fungi, bacteria, or algae in
the means of structure, size or chemical composition, whereas the type
of application is determined by surrounding growth factors such as pH,
temperature, presence of cations and anions, metal speciation, and
pollutant solubility and form [36]. Biosorption by microbial biomass
are also used to treat dye wastewater as microorganisms like bacteria,
algae and yeast can remove various classes of dyes effectively. For in-
stance, Ali and co-workers used fungi to effectively decolorize acid and
reactive dyes [37]. As a matter of fact, using microbial biomass is ad-
vantageous as the technique is inexpensive, readily available and has a
high potential for bioremediation even under extreme conditions of pH
and temperature [38]. Moreover, cell walls usually contain cellulose,
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Journal of Environmental Chemical Engineering 7 (2019) 103261
hemicellulose, pectins and lignin which are the most prominent sorp-
tion sites [33]. Despite the large amount of fundamental research being
conducted, the commercialization of biosorption for dye wastewater
treatment has been progressively slow. The biological pathway of dye
treatment also employs aerobic and anaerobic process of microorgan-
isms. Aerobic treatment usually occurs in stabilization ponds, packed
bed reactors and aerated lagoon systems under the presence of free
oxygen and microorganisms, whereas anaerobic treatment converts
organic dyes into methane and carbon dioxide in the absence of free
oxygen [19]. Hence, the combination of aerobic and anaerobic pro-
cesses offers another feasible solution in dye wastewater treatment,
achieving high color removal and oxidation of aromatic amines [39].
Despite the ease of the processes, it can be difficult to introduce such
systems into small-scale business due to the requirement of special
bioreactors or stabilization ponds that usually incurs high investment
costs at the initial stage. As dye wastewaters often consist of different
dyes, the complexity may render biological method ineffective for dye
treatment. Above all, there is no single technology that can treat all
classes of dyes effectively without any limitations due to the complexity
of the dye effluents from industrial discharge. Therefore, it is para-
mount that each dye treatment technique is analyzed based on their
merits and demerits (as shown in Table 2). A specific scenario may
demand a specific dye treatment technique. Thus, it is entirely based on
the process and engineering requirements that a specific or combina-
tion of dye treatment technologies is selected for application.
2. Enzymes for dye treatment
2.1. Enzymes
Enzymes are biological catalysts that are extremely efficient in
catalyzing specific reactions that are commonly explained through the
lock and key model or the induced fit model. In theory, enzymes can
increase the rate of reaction by lowering the activation energy of the
reaction through stabilization of the transition state. The much-desired
properties of enzymes include their high efficiency, high selectivity and
operation in mild conditions compared to other chemical catalysts
[79,80]. Working under mild conditions allows significant cost reduc-
tion to be achieved as there is no need for expensive equipment to reach
extreme operating conditions such as high pressure or high temperature
often required by chemical catalysts. Due to their natural origins, the
use of enzymes minimally affects the environment as they are biode-
gradable. The application of enzymes in dye wastewater treatment has
been garnering increasing attention. Enzymes including lignin perox-
idase [4], soybean peroxidase [48,81], horseradish peroxidase [20] and
laccase [82] have been previously studied for their dye treatment po-
tential. For an oxidoreductase enzyme, an oxidizing agent (e.g. hy-
drogen peroxide, chlorine and potassium permanganate) plays a sig-
nificant role in catalyzing the reaction. As an example, peroxidase are
enzymes under the class of oxidoreductase that can catalyze the oxi-
dation of different compounds in the presence of the oxidizing agent,
hydrogen peroxide. Peroxidase can act on aromatic compounds espe-
cially synthetic dyes by degrading them into their respective con-
stituents and catalyze oxidative polymerization of phenolic compounds
to form insoluble polymers [80,83]. The presence of hydrogen peroxide
is crucial for the reaction as the first step of the catalytic cycle involves
the interaction between the resting state of Fe(III) of the peroxidase and
hydrogen peroxide. The reaction releases one water molecule along
with the generation of a high oxidation state intermediate comprising of
Fe(IV) oxoferryl center together with a porphyrin-based cation radical
[84]. The second step involves two similar one-electron reduction steps.
The first electron reduction step involves the reaction of porphyrin
cation in the presence of the reducing substrate. This leads to the for-
mation of compound II, an Fe(IV) oxoferryl species. The second electron
reduction step occurs with another reducing substrate. This reverts
compound II back to the resting state of the peroxidase. The free radical
J.K.H. Wong, et al. Journal of Environmental Chemical Engineering 7 (2019) 103261

Table 2
Advantages and disadvantages of various dye treatment techniques.
Method Advantages Disadvantages References
Physical Treatment

Adsorption - Suitable for a large variety of dye types - Regeneration of adsorbents like activated carbon is [18,51]
expensive
- Regenerable adsorbents - Incomplete dye removal
- Simplicity
- Readily available
Ion exchange - Regenerable - High cost of resins [19,52,53]
- No adsorbent loss - Effective only to limited dye types of opposite charges
- High cation exchange capacity (clay) - Most resins have poor hydrodynamic properties and hard
to tolerate with high wastewater pressures
- Produces water of high quality - Generates large volumes of sludge
Irradiation - Suitable for dyes that resist chemical oxidation/ - Requires huge amount of dissolved oxygen [18,19,54]
reduction
- Effective at low dye concentrations only - Expensive
Membrane Technology - Suitable for any dye types - High investment cost [19,22,55]
(reverse osmosis, nanofiltration, ultrafiltration, - High separation efficiency - Constant clogging of membrane pores
microfiltration, bioreactor)
- Low energy consumption - Membranes must have excellent chemical and thermal
stability to withstand high pressures and fluxes
- Facile operating conditions - Short life span due to membrane fouling
- Enrichment of nitrifying bacteria can reduce
fouling in membrane bioreactors
Physico-chemical Treatment
Coagulation-flocculation sedimentation - Cheap and robust process - Generates a lot of concentrated sludge which requires [43,56,57]
legislative disposal
- Suitable for disperse, sulphur and vat dye - High toxicity and TDS content in sludge
effluents
- Not suitable for acid, azo, basic and reactive dyes
- May be expensive due to usage of different chemicals
- Recycling of chemicals is not feasible
- pH dependent system
Chemical Treatment
Direct chemical oxidation - Degrade dyes completely - High cost [16,19]
- Short reaction time - Requires activation of hydrogen peroxide
- Straightforward and simple - pH dependent
- Reactive dyes require longer decolourization time
- Requires catalyst for efficient removal
- Produces toxic chlorinated organics
Ozonation - Fast and effective dye removal method - Extremely short half-life (20 minutes) [58,59,60]
- Can be used in gaseous state - High cost
- Does not increase wastewater volume - Produces unstable toxic by-products and carcinogenic
aromatic amines
- No chemical sludge
- Residual ozone can decompose to oxygen easily
Advanced oxidation process - Easy and rapid degradation of pollutants - Produces undesirable by-products [61,62]
- Eliminates toxic materials - pH dependent
- Removes dye under unusual conditions
Electrochemical destruction - Suitable for soluble and insoluble dyes - Additional production of hazardous materials [63,64]
- No consumption of chemicals - High cost of electricity
- No sludge build-up - High flow rates causing direct decrease in dye removal
Photochemical - Remove dye effectively - Expensive [65,66,67]
- No foul odours production - Formation of by-products
- No sludge production
Fenton reaction - Suitable for soluble and insoluble dyes - Cannot remove disperse and vat dyes [24,45,58]
- Suitable for dyes wastewater with solid content - High iron sludge production
- Removes all toxins in water - Long reaction time
- Works only at low pH
Ultraviolet irradiation - Do not require hazardous chemicals - Energy depletion [16,68]
- No sludge production - High cost
- Weakens foul odours - Limited treatment times
Biological Treatment
Adsorption by microbial biomass - Exceptional affinity towards microbial biomass - Not feasible for all dye types [37,38]
- High biodegradability adsorbents - Poor dye binding capacity and recyclability
- Low cost - Hard to transport large volume biomasses
Algae degradation - Consumes dyes - Unstable system [69,70]
- Inexpensive and easily assessable
- No harmful by-products
Aerobic-anaerobic combination - Robust process and suitable for a variety of dye - Incomplete dye elimination [39,71,72]
types
- High colour, COD removal - Forms methane and hydrogen sulphide
- Inexpensive - Generates sludge and requires land
- Do not form foams - Long reaction time and inflexible
(continued on next page)

4
J.K.H. Wong, et al.
Table 2 (continued)
Method Advantages
Physical Treatment
Enzyme degradation - Non-toxic
- Fast approach
- High specificity enzymes
- Cost-effectiveness
Fungal cultures - Eliminates a variety of dye types at once
- High flexibility
Microbial cultures - Fast decolorization of dyes (< 30 hours)
Pure and mixed cultures - Remove azo dyes only
- Good reusability
products then undergo polymerization. Notwithstanding the im-
portance of the oxidizing agent, high concentration of hydrogen per-
oxide may cause an inhibitory effect, leading to lower enzymatic ac-
tivity [85].
Enzymes function only under extremely specific environmental
conditions as they are very sensitive to changes in the local environ-
ment such as pH and temperature, which often lead to a loss in func-
tionality if the environment becomes unsuitable or harsh [86]. Enzymes
are polyionic polymers, in which a change in pH would result in
changes in the ionization stage of the active site and the charge dis-
tribution at the protein structure [87]. This might cause the affinity of
an enzyme for the substrate to be affected, resulting in the enzyme and
substrate to potentially repel one another due to similar surface charges
[88] Small changes in pH directly translates to relatively large changes
in [H+], which can affect the stability of the electrovalent bonds that
maintain the tertiary structure of protein molecules. Additionally, car-
bonate alkalinity has a synergistic effect with pH on the metabolic
enzymatic activity. In one study, Peng and co-workers investigated the
effect of carbonate alkalinity and pH on enzymatic activity in juveniles
of the razor clam, Sinonovacula constricta [89]. The carbonate alkalinity
stress at 10.38 mmol L−1 induced through addition of Na2CO3 and
NaHCO3 at pH 9 caused a lethal concentration of 50% within 48 h. The
increase in HCO3- led to an inhibition of sodium potassium adenosine
triphosphate but with the increase in CO32- ions, the activity of the
enzyme was subsequently increased. Other enzymes in the razor clam
responded differently to the alkaline conditions with one example, ly-
sozyme being unaffected. Likewise, Wang and co-workers studied the
impact of carbonate alkalinity on superoxide dismutase, acid phosphate
and alkaline phosphate activities in juvenile Gymnocypris przewalskii.
The study found that the activity of the three enzymes increased after
exposure to the carbon alkalinity stress but returned to control level
within 4 days. The study attributed the enzyme response to the car-
bonate alkalinity stress as vital in protecting the fish from the carbonate
alkalinity stress [90]. Therefore, enzyme activity greatly varies with pH
and can be influenced by carbonate alkalinity, so their impacts in dif-
ferent applications need to be ascertained.
The structure of an enzyme typically consists of the primary, sec-
ondary and tertiary structure in which the primary structure is bonded
by covalent bonds while both the secondary and tertiary structures are
bonded by weak solvation, non-covalent interactions, disulphide or
hydrogen bonds [88]. Any disruption to the secondary or tertiary
structure would cause conformational changes to the enzyme, causing
denaturation. The effect of temperature on an enzymatic reaction may
have different implications. According to the collision theory, the
supply of kinetic energy to a reaction will cause the reacting molecules
to move much more rapidly, to collide much more rapidly and to form
much more enzyme substrate complex. However, increasing the tem-
perature to sufficiently high levels would result in poorer stability of the
enzymes. The vibration at such high temperatures would become too
5
Journal of Environmental Chemical Engineering 7 (2019) 103261
Disadvantages References
- Limited recovery and reusability [48,50,73]
- Lengthy and unstable growth phase [6,74]
- Requires nitrogen atmosphere and large reactors for
complete dye removal
- Effective only to limited dye types [75,76]
- Large scale application is preferred
- Produces colourless toxic by-products [77,78]
- Generates sludge
- Requires conventional method as post-treatment
violent, resulting in the breaking of chemical bonds that hold the three
dimensional structure of the enzyme and therefore, disrupting it [91].
In short, the temperature that maximizes enzymatic activity is usually
detrimental in preserving the enzyme, rendering enzymes impractical
in prolonged reactions. As such, a clear understanding of the effect of
temperature on enzymatic reaction is needed to achieve a viable com-
promise between enzymatic activity and stability.
Equal importance must be placed on the impact of electrical pulses
on enzymatic activity. Direct exposure of enzymes to electrical pulses is
known to influence enzymatic activity, with the effect dependent on the
characteristics of the enzyme as well as local conditions. A study by Ho
and co-workers found that denaturation of enzymes in high electric
pulse environment differed from one enzyme to another [92]. In their
study, the enzymatic activity of lipase, glucose oxidase and α-amylase
were severely hampered and reduced by 70 to 85% by high electric
field pulses while peroxidase and polyphenol oxidase exhibited mod-
erate reduction at 30 to 40% in enzymatic activity. Reduction in en-
zymatic activity was attributed to the structural or conformational
changes of the enzymes caused by the high electrical field pulses. Si-
milar views were expressed in a later study by Zhong and co-workers
who investigated the effect of pulsed electric field on inactivation and
conformational change of horseradish peroxidase [93]. The study re-
ported that the inactivation of horseradish peroxidase was related to the
conformational change of α-helix and intrinsic fluorescence intensity
induced by pulsed electric field. The study also highlighted that an
increase of applied electrical field strength would cause a decrease in
enzymatic activity. Using a different experimental setup, Wang and co-
workers investigated the effect of direct current electric field on the
enzymatic activity of laccase [94]. They discovered that the activity of
laccase was greatly enhanced in the anodic region and that the anodic
environment can decelerate the degradation of enzyme in the solution.
However, the activity of laccase at the cathodic region was inhibited.
Current literature suggests that the mechanism involving the activation
and inactivation of enzymes due to electrical field has yet to be estab-
lished and remains an elusive area [95]. Nevertheless, it is certain that
the effect of electrical charges to enzymatic activity cannot be under-
mined.
The industrial application of enzymes in dye wastewater treatment
is often restricted because it increases production cost, lacks long term
operational stability, lacks reusability after the first run and has a
limited shelf life [81,96–98]. Also, enzymes in their crude form are
often limited by their stability in catalyzing reactions due to their
susceptibility to inhibition in complex dye wastewater [99]. For ex-
ample, different enzymes have varying degrees of sensitivity towards
heavy metals. A study by Effron and co-workers found that the presence
of cadmium, copper and lead had no effect on β-glucosidase but caused
severe reduction in enzymatic activity of arylsulfatase, acid phospha-
tase, protease and urease [100]. Other types of heavy metal such as
mercury can readily bind to sulfhydryl, phosphoryl, carboxyl, amide
J.K.H. Wong, et al.
and amine groups [101,102]. Consequently, enzymes with such groups
are susceptible to reaction with mercury once exposed. Once bound to
mercury, most enzymes are immediately rendered inactive in cata-
lyzing further reactions. In short, it is predicted that as the complexity
of effluents increases, the catalytic activity of enzyme will subsequently
decrease [80].
2.2. Enzyme kinetics
Enzyme kinetics are vital and significant as they describe the rates
of enzyme catalyzed reactions. Much of our understanding of enzyme
kinetics comes from the classical study by Michaelis and Menten [103].
The Michaelis-Menten (MM) theory first characterized the binding of an
enzyme E to substrate S to form an enzyme-substrate complex ES and
subsequent catalysis into the free enzyme and product P as presented in
Eq. (1.1).
k1 k cat
E+S ES E+P
k -1 (1.1)
Under quasi-steady-state approximation, the Michaelis-Menten
equation is derived and is as presented in Eq. (1.2):
Vmax [S]
v=
(KM+[S]) (1.2)
Commonly reported kinetic constants from enzyme related studies
are the Michaelis constant (KM), the maximum reaction velocity (Vmax)
and the turnover number (Vmax divided by concentration of enzyme).
Michaelis constant, KM is defined as the substrate concentration at
which the speed of the product formation is at half of its maximum
value. It describes the likelihood of the substrate to dissociate from the
enzyme. The turnover number describes how many substrates an en-
zyme can turn into product at maximum velocity. Experimental de-
termination of the kinetic constants is most commonly achieved based
on assaying the initial reaction rate as a function of the substrate con-
centration while keeping the enzyme concentration fixed in the enzyme
catalyzed reaction. Subsequently, the plots are fitted with the MM
model using non-linear regression or linearized functions such as
Lineweaver-Burk plot [104], Eadie-Hofstee plot and the Hanes-Woolf
plot [105]. These methods are currently most widely used for kinetic
studies in dye wastewater treatment using enzyme incorporated nano-
technology [106–110]. Typically, a large amount of experimental data
is required to accurately determine the enzyme kinetics using the noted
conventional plots. Thus, it becomes challenging especially in acquiring
data which has substrate concentrations much higher than that of KM.
As computational power has become readily available and accessible in
recent times, enzyme kinetics can be easily determined through the use
of single kinetic progress curves [111]. This alternative approach ex-
amines the entire enzyme catalyzed reaction and the evolution of
product concentration over time rather than solely on the initial reac-
tion rate, which is extremely useful if the initial reaction is extremely
rapid. The MM coefficients determined from the entire transient may be
preferred as a comprehensive indicator of adherence to classical MM
kinetics as compared to the conventional plots. Available analytical
programs such as FITSIM and DYNAFIT are widely used for progress
curve analysis in enzyme kinetics, occasionally coupled with Monte
Carlo simulations to reduce risk of progress curve analysis misuse
[112]. Many generalizations of the enzyme kinetics that account for
situations with the involvement of cofactor, inhibitor or allosteric are
also available [113].
Despite the standard models described thus far being commonly
used, Johnson and co-workers [114] argued that the models cannot be
directly adapted and may not be necessarily suitable for nanoparticle
systems. The reason being that the standard models are designed for
homogenous enzyme-substrate systems, but the nanoscale systems are
frequently heterogenous such as having localized regions of high
6
Journal of Environmental Chemical Engineering 7 (2019) 103261
concentrations on the nanoparticle surface [113]. In addition, the as-
sumptions made during the derivation of the standard models may not
be consistent with the physicochemical characteristics of the nanoscale
system, causing an overall mismatch. In order to improve the MM
model in nanoparticle systems, Johnson and co-workers reviewed four
major approaches available in recent literature. These include: (i)
augmenting the MM theory with concepts from the collision theory; (ii)
macroscopic approach in generalizing the chemical kinetics of the MM
model that retains the assumption of “well-mixed” conditions; (iii)
dealing with nanoparticle-enzyme system heavily affected by diffusion
whereby the “well-mixed” conditions are no longer valid; and (iv) the
potential of microscopic simulations of enzymatic processes where the
underlying molecular discreteness becomes prevalent. A further review
by Vranish and co-workers reasoned that although the MM model may
not be suitable for nanoparticle systems and that the meaning of the
parameters can be questioned, this does not hinder the simplicity and
familiarity offered by MM model and thus would still have considerable
practical value [113]. In addition, the lack of computational power in
simulating large amounts of atoms at the nanoscale means that mac-
roscopic or continuum models presents a compromise whereby the
fundamental physical and chemical phenomena are captured without
excessive details that cannot be checked experimentally due to the
limitation of current technologies [113].
2.3. Enzyme immobilization
To overcome the limitations associated with enzymes as biocatalysts
for dye treatment, a technique often used is immobilization. Enzyme
immobilization can be defined as the attachment of soluble enzymes to
a supporting matrix that results in the reduction or total loss in mobility
of the attached enzyme [115]. By immobilizing the enzyme, it gains
enhanced properties but on a strict case by case basis. The enhanced
properties might include but are not limited to stability to various de-
naturing conditions, pH tolerance, functional stability, easier separation
of enzyme and subsequent recovery, reusability, and increased catalytic
performance [96,115,116]. Mechanical rigidification of enzymes
through immobilization also helps in stabilizing the enzymes and pre-
vents dissociation-related inactivation [96]. For example, Gholami-
Borujeni and co-workers immobilized horseradish peroxidase on cal-
cium alginate gel beads for the decolorization of acid orange 7 dye [80].
The immobilized horseradish peroxidase was much more stable in
terms of pH and thermostability compared to the free enzyme. Mean-
while, Ali and co-workers immobilized ginger peroxidase on poly-
pyrolle-cellulose-graphene oxide composite for the removal of reactive
blue 4 dye [98]. Similarly, the immobilized ginger peroxidase could
achieve higher stability across a wider range of pH, higher reusability
and higher thermostability. On the contrary, the immobilization of
enzymes might lead to decreased catalytic activity compared to free
enzyme [96]. Chagas and co-workers immobilized soybean peroxidase
on chitosan beads using glutaraldehyde as cross-linker for phenolic
degradation [115]. However, the enzyme activity became more ther-
mally dependent after immobilization which the work attributed to the
destruction of active sites of the enzyme. This was due to the large
expansion coefficient of the chitosan support. In sum, enzyme im-
mobilization can either lead to an increase or decrease of a parameter
performance of an enzyme and it is only through studies that changes in
each parameter can be ascertained. Enzyme immobilization can yield
the desired properties needed for enzyme application when applied
correctly.
Four of the most commonly used techniques for enzyme im-
mobilization, either physical or chemical are entrapment, adsorption,
covalent bonding and cross-linking, as shown in Fig. 1. Based on
Table 3, each of these methods has varying performances. Adsorption
and entrapment are physical immobilization methods which rely on
weak interactions such as van der Waals forces, ion binding and phy-
sical constraint of the enzyme within the support [117,118]. The
J.K.H. Wong, et al. Journal of Environmental Chemical Engineering 7 (2019) 103261

Fig. 1. Different types of immobilization methods [97].

physical immobilization method has an advantage in terms of main-
taining the enzyme’s catalytic activity as the native structure of the
enzyme is not altered [79]. Among all of the developed techniques used
to carry out enzyme immobilization, adsorption is the most scalable in
the current industry due to its simpler mechanism, high efficiency and
affordability [119,120]. The process involves placing the support into
an enzyme solution for a specified duration for the enzyme to be im-
mobilized on the support. However, the weak non-specific forces
binding the enzyme and support together cause it to be easily disrupted.
Entrapment is an immobilization technique that is used to combat the
tendency of aggregation of enzymes that lowers the catalytic activities.
Enzymes are immobilized through this method, through micro-
encapsulation, metal organic frameworks (MOF) or gel/fiber entrap-
ping [121,122]. When the enzymes are immobilized through this
method, the substrates pass through the networks for the reaction to
take place while the enzyme is retained on the support. The outcome is
that the mechanical stability of the enzyme is improved while the
amount of enzyme leaching is reduced. As there are no covalent bonds
being formed between the enzyme and support, the enzyme con-
formations do not change. This ensures high catalytic activities. How-
ever, the disadvantage of entrapment is the presence of the high dif-
fusion barrier which prevents macromolecular substrates from passing
through the network of enzyme. In contrast, covalent attachment and
cross-linking enzymes are chemical immobilization methods which rely
on formation of covalent bonds between enzymes and the support using
bio-functional reagents like glutaraldehyde. This causes slight altera-
tion of the enzyme which might impact its activity but this is com-
pensated with its stronger chemical bonds and rigidity [97]. Thus, the
continuous demand for development of a novel immobilization strategy
to overcome the limitations still exist.
Recently, Ge and co-workers discovered an elegant immobilization
strategy by creating hybrid organic-inorganic nanoflowers with pro-
teins as the organic component and copper (II) ions as the inorganic
component [131]. The immobilization method involved three major
steps (as shown in Fig. 2). In the first step, the primary crystals were
formed through nucleation. The second step involved the growth of the
agglomerates of the primary crystals along with the biomolecules. The
final step involved anisotropic growth that caused the formation of
branched flower like structure, hence completing the nanoflower
structure in which the enzyme is encapsulated within [132]. The key
driving force behind the formation of the nanoflowers lies on the co-
ordination between the Cu2+ and the protein. Unlike conventional
immobilization methods, tests conducted by Ge and co-workers on
laccase incorporated nanoflower indicated that the immobilized

Table 3
Advantage and disadvantage of different immobilization methods.
Method Technique Advantages Disadvantages Reference

Physical Adsorption - Low-cost - Weak, reversible enzyme-substrate binding forces [97,99,123]

- Simple and most common - Poor stability against pH, temperature and ionic strength
changes
- Retains catalytic activity
- Maintains enzyme structure
- Requires no additional coupling agents or
modifications steps
- Regenerable supports
Entrapment - Low-cost - Prevents passing of macromolecular substrates [97,124,125,126]
- Improves mechanical and operational stabilities
- Maintains conformation
- Prevents enzyme aggregation
- Reduces enzyme leaching
Chemical Covalent attachment - Forms stable covalent bonds - Support surfaces must be activated prior to usage [15,97,127,128]
- Improves enzyme stability
- Reduces enzyme leaching
Cross-linking - Simple and robust - May lose catalytic activity after immobilization [50,129,130]
- Improves enzyme stability and promotes reusability
- Retains initial enzyme catalytic activity

7
J.K.H. Wong, et al.
Fig. 2. Mechanism of nanoflower synthesis with three steps: (1) nucleation and formation stage of primary crystals; (2) growth of crystals; and (3) formation of
nanoflowers [131].
enzyme exhibited 2.0–6.5 times more activity in phenol conversion and
syringaldazine oxidation, when compared to free enzyme [131]. At the
same time the laccase nanoflower showed high reusability and en-
hanced stability over the free enzyme. The enhanced activity of the
enzyme nanoflowers were attributed to the (1) high surface area of the
nanoflower that does not cause significant mass-transfer limitations; (2)
the cooperative effect of the nano-encapsulated enzyme; and (3) the
beneficial interaction between Cu2+ and laccase. Since then, other
types of supports have been used in the synthesis of hybrid nanoflowers
for various purposes which include calcium (II) ions, manganese (II)
ions, zinc (II) ions, cobalt (II) ions and iron (II) irons [133]. Discovery of
this new immobilization strategy involving hybrid nanoflowers is
therefore very interesting for wastewater treatment due to its superior
properties.
Other than the immobilization method, the selection of a support for
enzyme immobilization is crucial since it can affect the performance of
the immobilized enzyme and dictate its properties [134]. An ideal
support should include physical resistance to compression, resistance to
microbial attack, hydrophilicity, inertness towards enzymes, bio-
compatibility with the associated enzyme and accessibility at a low cost
[96,134]. In recent times, the increasing trend of smaller and compact
technology has brought development towards nanoscale technology. As
such, the use of smaller supports such as nanoparticles for enzyme
immobilization have become very interesting, especially in wastewater
treatment. Nanoscale support exhibits characteristics that are ideal such
as high surface area to volume ratio, effective enzyme loading, sig-
nificantly enhanced mass transfer efficiency and minimization of dif-
fusional problems [135]. It has also been shown that the interactional
patterns between the biological properties of the enzyme and nano-
support are largely influenced by the unique physicochemical proper-
ties of the nanosupport [136]. Optimizing both the immobilization
process and operational stage of the enzyme on nanosupport is key to
its performance. Optimization of such systems often edges out free
enzymes in four different aspects: (i) wider temperature range; (ii)
broader working pH; (iii) greater thermostability; and (iv) increased
reusability [135,136]. The modulation of enzyme activity via nano-
particles has been reviewed extensively by Arsalan and Younus, which
enables enzymatic process to occur much more efficiently and less
costly [137].
2.4. Enzyme incorporated nanotechnology
Enzymes immobilized on nanoscale support have been studied and
used for multiple applications such as enzyme pro drug therapy [138],
antimicrobial agent [139], glucose biosensor [140,141], and lactose
hydrolysis [142]. Although multiple reviews have reported on the
performance of enzymes immobilized on nanosupport for these dif-
ferent fields, no detailed review was found on the performance of en-
zymes immobilized on nanosupport for dye decolorization. The present
article will examine in detail and provide a review on the performance
of enzyme incorporated nanotechnology for dye decolorization appli-
cation. We present the optimized performance along with the kinetic
8
Journal of Environmental Chemical Engineering 7 (2019) 103261
parameters of enzymes incorporated nanotechnology on dye decolor-
ization from available literature, which is presented in Table 4.
Enzyme immobilized on nanosupport using conventional im-
mobilization methods evidently exhibited high activity at a broader
range of pH, greater thermal stability and higher reusability than their
free counterparts. For example, Ali and co-workers immobilized ginger
peroxidase on amino-functionalized silica-coated titanium dioxide na-
nocomposite whereby the immobilized ginger peroxidase was able to
achieve a much higher decolorization activity in both acidic and alkali
regions [143]. At the same time, the immobilized ginger peroxidase
could retain more than 80% of its activity after 100 min at 60 °C while
the free peroxidase retained less than 10% of its activity. The study
attributed the high retention of activity to the protection given by the
nanosupport against inhibition from the by-products of the reaction. In
another study, Sadighi and Faramarzi immobilized laccase through
chitosan nanoparticles on glass beads for congo red decolorization
[116]. The immobilized laccase was able to maintain nearly all of its
decolorization activity at 90 °C while the free laccase lost all of its de-
colorization activity due to enzyme denaturation at such high tem-
perature. Wang and co-workers immobilized laccase on Fe3O4/SiO2
nanoparticles for Procion Red MX-5B decolorization [144]. The im-
mobilized enzyme retained 79% of its initial activity at the 10th cycle,
highlighting excellent reusability. Notwithstanding that, Liu and co-
workers immobilized laccase on Poly(p-Phenylenediamine)/Fe3O4 na-
nocomposite for reactive blue 19 dye removal with only 43% of its
initial activity left at the 10th cycle [145]. The loss in activity was at-
tributed to accumulation of dye degradation products, mass transfer
limitations and loss of magnetic carriers during magnetic separation
from the reaction medium. Similarly, Othman and co-workers im-
mobilized laccase on functionalized multiwalled carbon nanotube
membranes for Reactive Black 5 decolorization [146]. The immobilized
laccase had poor reusability at only 6.89% of the initial activity after
the 4th cycle with the possible causes being inactivation by the selected
mediator and mass transfer limitations.
Some studies have also made progress using the recently discovered
hybrid nanoflowers immobilization strategy for dye decolorization with
positive results. For instance, Li and co-workers immobilized laccase in
a self-assembly hybrid nanocomposite consisting of copper phosphate,
graphene oxide and carbon nanotubes for the removal of crystal violet
and neutral red [147]. The hybrid nanocomposite exhibited excellent
dye removal at 100% removal while the free laccase managed less than
a 20% dye removal. The higher activity of the immobilized laccase was
attributed to the composite structure of the graphene oxide and carbon
nanotubes that ensured good affinity between laccase and substrates as
well as enhanced electron transfer efficiency. When stored at room
temperature for 6 days, the immobilized laccase was able to preserve
over 80% of its initial activity compared to the free enzyme at around
30%. In a different study, Altinkaynak and co-workers created perox-
idase-Cu2+ hybrid nanoflowers for the decolorization of victoria blue
dye in which the dye decolorization efficiency of the hybrid nano-
flowers increased by 35% [148]. The hybrid nanoflower also achieved
higher reusability and stability compared to the free enzyme.
 Table 4
Studies on dye decolorization by enzyme immobilized on nanoscale support.
a b
Enzyme Immobilization matrix Immobilization technique Dye Kinetic Parameters Conditions Decolorization (%) Time References
duration
J.K.H. Wong, et al.

LiP from Pleurotus ostreatus Carbon nanotubes Covalent attachment Remazol Brilliant Km = 12.3 μM pH = 5.0 > 65 12 h [149]
Blue R
Vmax = 451.9 U/mL T = 15°C
Dye conc. = 10 mg/L
LiP from Ganoderma lucidum Carbon nanotubes Covalent attachment Remazol Brilliant Km = 53.21 μM pH = 9.0 > 70
Blue R
Vmax = 756.86 U/mL T = 15°C
Dye conc. = 10 mg/L
Laccase from Pleurotus Fe3O4 / SiO2 nanoparticles Covalent attachment Procion Red MX- Km = 0.074 mmol pH = 5.0 99 20 min [109]
ostreatus 5B
Vmax = 0.051 mM/min T = 25°C
Kcat = 0.00196/min Dye conc. = 30 mg/L
Laccase from Trametes ZnO nanoparticles Adsorption Alizarin Red S Km = 2.75 mM pH = 7 83 60 min [150]
versicolor
Vmax = 146 U/mg Enzyme conc. = 50 mg
MnO2 nanoparticles Adsorption Alizarin Red S Km = 1.23 mM Dye conc. = 20 mg/L 71
Vmax = 65 U/mg
Laccase from Trametes Poly(MA-alt-MVE)-g-PLA/ODA-MMT Adsorption Reactive Red 3 – pH = 5.0 65 1.5 h [151]
versicolor nanocomposite
T = 20°C
Enzyme conc. = 0.05 mg/mL
Ginger peroxidase Amino-functionalized silica-coated titanium Covalent binding Acid Yellow 42 Km = 61.33 μM pH = 5.0 90 1.5 h [143]
dioxide nanocomposite
Vmax = 35.01 μM/min T = 40°C

9
Dye conc. = 25 mg/L
H2O2 conc. = 0.75 mM
1-hydroxybenzotriazole (HBT) = 1
mM
Laccase Magnetic graphene oxide Covalent binding Crystal Violet – pH = 3 94.7 3h [152]
T = 35°C
Malachite Green – Enzyme conc. = 5.0dmg/mL 95.6
Brialliant Green – Dye conc. = 50.0 mg/L 91.4
Laccase Magnetic poly(p-phenylenediamine) Covalent binding Reactive Blue 19 – pH = 3.5 80 1h [145]
nanocomposite
T = 65°C
Dye conc. = 12 mg/L
Chloroperoxidase Fe3O4 magnetic nanoparticles Entrapment Aniline Blue Km = 3.021 mM pH = 2.0 > 90 10 min [153]
From Vmax = 0.04363/min T = 30°C
Caldariomyces Kcat = 8.73 x 103/min Dye conc. = 0.1 mM

(continued on next page)

Journal of Environmental Chemical Engineering 7 (2019) 103261
J.K.H. Wong, et al. Journal of Environmental Chemical Engineering 7 (2019) 103261

References Collectively, these studies have shown promising results that most en-
zyme incorporated nanotechnology can decolorize dye effectively in a

[107]

[154]
short time. As a result, the potential for these biocatalysts to be applied
in industrial dye wastewater treatment is very promising.

3. Challenges and strategies

duration

8d
Decolorization (%) Time

3h
3.1. Implementation at industrial scale

As the global population continues to spiral upwards, the demand

> 60

> 20
> 95

for clean water is at an all-time high with millions of people still de-
100

100

prived of this daily necessity. Yet, it remains difficult to provide clean

water to areas where dye discharge from the industries are rampant.
Established and conventional technologies used in dye wastewater
treatment are often costly and ineffective, leading to irresponsible in-
dustries practicing illegal dumping of untreated dye wastewaters,
contaminating water catchment areas and groundwater resources.
Therefore, the application of enzyme for dye wastewater treatment is
NR conc. = 7.5 mg/L
CV conc. = 2.5 mg/L

Dye conc = 10 mg/L

Temperature = 25°C
H2O2 conc. = 4 mM

considered a leading candidate to eliminate most of the dye from

wastewater in a cheap and efficient manner. As presented previously,
much work has been done in verifying the ability of enzyme im-
Conditions

pH = 5.4

T = 25°C

mobilized on nanosupport for singular dye wastewater treatment and

pH = 6

that positive results have been discovered. However, the feasibility of

implementing such systems remains unclear as all studies are conducted
b

at lab scale. Limited number of works have made progress in treating

Kcat = 98.0 μmol/s g

Kcat = 44.4 μmol/s g

Kinetic Parameters

simulated industrial dye effluent and binary dye systems using enzyme
Km = 15.3 μM

incorporated nanotechnology. For example, graphene oxide (GO) based

Km = 9.9 μM

nanomaterials are excellent enzyme incorporated adsorbents which are

readily available to be commercialized, contributed by their impressive
regeneration and reusability properties [155]. Recent studies carried
–
a

out using magnetic GO-immobilized laccase [152], GO-immobilized

peroxidase [98] and GO-laccase nanoflowers [107] have proven to
degrade dyes from water efficiently in lab scale. Chen and co-workers
Crystal Violet

Crystal Violet
Neutral Red

Neutral Red
Congo Red

discovered that 59.8% enzymatic activity was retained even after 10

dye decolourization cycles in aqueous solution containing mainly dye
Dye

and phosphate buffer [152]. However, it is difficult to determine the

optimum conditions in the environmental bioremediation process as
Immobilization technique

their effectiveness in treating actual dye-loaded wastewater from in-

dustries has yet to be evaluated. The actual performances of the na-
Entrapment (Hybrid

Entrapment (Hybrid

Entrapment (Hybrid

notechnology in treating dye wastewater could differ from the results

produced in the laboratory. This means that the efforts made thus far
nanoflowers)

nanoflowers)

nanoflowers)

are deemed insufficient to ascertain the effectiveness of using enzyme

incorporated nanotechnology in actual dye wastewater treatment
where the composition of industrial dye discharge may be complex
[156]. Notwithstanding the potential of enzymes, the major challenge
therefore, would be in the realization of lab scale experiments into
actual industrial application. Hence, enzyme incorporated nano-
Copper phosphate / Carbon nanotubes /

technology should be implemented in actual industrial wastewater

Copper phosphate / Graphene oxide

conditions to evaluate its effectiveness in treating dye effluents. More

Graphene oxide nanocomposite

work should be done with the focus on treating actual industrial dye.
Immobilization matrix

3.2. Understanding enzymes incorporated nanotechnology

Copper foil surface

The dye decolorization studies using enzyme incorporated nano-

nanocomposite

technology described so far have made commendable progress in de-

signing solutions to enhance the enzyme catalytic activity. Much work
has been previously done to investigate and understand the individual
properties of nanomaterials [157,158] as well as of enzymes
[84,159–161]. However once incorporated, working with the combined
system at such minute scale becomes challenging to quantitatively
Table 4 (continued)

analyze and understand thoroughly [114]. It remains difficult to ex-

perimentally isolate each variable to determine the exact cause of the
change in the enzymatic activity of a specific system. For example,
enzymes after immobilization can lose diffusional mobility while at the
Enzyme

Laccase

Laccase
fumago

same time undergo conformational changes, making it difficult to es-

tablish the true cause of the change. Currently, there is no obvious

10
J.K.H. Wong, et al.
solution as to designing an experiment to separate these two effects of
diffusional mobility and conformation changes [114]. Also, it would be
flawed to generalize the root cause of an enhancement or limitation of
one system to another. Even the slightest change in combination of the
enzyme and support material used can cause significant differences. For
instance, Zhang, Luo, and Chen adsorbed catalase onto carbon nano-
tubes to determine the effect of the carbon nanotube surface properties
on enzymatic activity [162]. The study attributed the decreased activity
of the catalase structure after immobilization to conformational
changes. In another study, Pang, Li, and Zhang immobilized laccase on
carbon nanomaterials for the degradation of phenolic compounds
[163]. They conducted circular dichroism spectroscopy to investigate
the secondary structure of laccase after immobilization. Despite the
nanomaterial used being quite similar to the previous study by Zhang,
Luo, and Chen, they found out that there was no significant con-
formational change in the immobilized laccase and therefore concluded
that the loss in enzymatic activity was not due to conformational
change but rather, substrate accessibility. Ali and co-workers im-
mobilized ginger peroxidase on amino-functionalized silica-coated ti-
tanium dioxide nanocomposite for acid yellow 42 removal [143]. Si-
milarly, they conducted a circular dichroism spectroscopy to
demonstrate the conformational changes in the ginger peroxidase
structure, indicating it as a possible cause as to the enhanced perfor-
mance. In short, these studies have shown that immobilization of dif-
ferent enzymes on different nanomaterials will yield a system that may
be distinct from one another. The knowledge of how enzyme in-
corporated nanotechnology behaves is still very limited among scien-
tists. Consequently, more work should be done on understanding en-
zymes incorporated nanotechnology by focusing on how to investigate
the performance of individual variables. This however, does not dis-
credit the possibility that through deep understanding of the underlying
mechanisms involved in the improvement of enzymatic activity, that
better technologies can be designed and developed for dye wastewater
treatment as well as other industrial applications.
3.3. Recovery of immobilized enzyme
The ease of separation and subsequent recovery of immobilized
enzymes from a reaction medium mainly indicates an enzyme’s reusa-
bility, which is extremely crucial when intended for repeated use in
industrial applications. Enzymes immobilized at the microscale such as
on chitosan beads [115] and calcium alginate beads [80] can be easily
removed from a reaction medium. However, immobilizing enzymes on
nanoscale presents a unique challenge since conventional recovery
methods do not effectively recover the immobilized enzymes. Most
separation processes involving nanoscale support have resorted to
centrifugation, making the process rather tedious, inconvenient and
time-consuming [164].
An attractive method to overcome this shortfall is by immobilizing
enzyme on magnetic nanoparticles such as magnetite, Fe3O4 and ma-
ghemite, γ-Fe2O3. Owing to their superparamagnetism property, they
can be easily removed from the reaction medium via external magnetic
forces. Magnetic nanoparticles possess superior properties such as
having large surface-to-volume ratio, no toxicity and good biocompat-
ibility, which are ideal for enzyme immobilization [165]. Additionally,
magnetic nanoparticles, which are non-porous nanoparticles, do not
have external diffusion problems. This makes them better suited for
industrial applications especially in solid-liquid systems [166]. Ir-
onically, the large surface-to-volume ratio of the magnetic nano-
particles results in particle aggregation, whereby the surface energy is
minimized because of the strong magnetic interactions between parti-
cles. This ultimately limits their dispersion in aqueous solutions and
matrices, hence lowering the enzymatic activity [166]. Some other
disadvantages of immobilizing enzymes on magnetic nanoparticles in-
clude enzyme conformational changes and lowered efficacy against
insoluble substrates [167]. Magnetic nanoparticles are known for the
11
Journal of Environmental Chemical Engineering 7 (2019) 103261
abundance of hydroxyl groups on their surface that allows easy mod-
ification while simultaneously forming strong covalent bonds with the
enzymes [130]. Therefore, modifications of the magnetic nanoparticles
can be performed to minimize the drawbacks posed by naked magnetic
metal nanoparticles and to make them suit to their desired use better.
Such modifications include silica-coated magnetic nanoparticles, or-
ganic polymer-modified magnetic nanoparticles, mesoporous material-
modified magnetic nanoparticles and metal organic framework-mod-
ified magnetic nanoparticles, which have been extensively reviewed by
Liu and co-workers [97], Netto and co-workers [165] and Hu and co-
workers [168]. For example, Donadelli and co-workers successfully
immobilized soybean peroxidase on iron oxide nanoparticles with silica
coating [169]. The study highlighted that the silica shell coating played
a major role in preserving the enzymatic activity in terms of thermo-
stability and reusability.
In dye decolorization related studies, Dai and co-workers im-
mobilized laccase on Fe3O4/SiO2 nanoparticles to achieve 99% deco-
lorization of Procion Red MX-5B dye in 20 min, highlighting the out-
standing performance as a result of the synergy between laccase and the
magnetic Fe3O4/SiO2 support [109]. Due to the advantageous magnetic
recovery of the biocatalyst, high retention of enzymatic activity was
observed at nearly 50% of its initial activity at the 50th cycle. Despite
the excellent performance, the biocatalyst was not able to remove all
toxicity but only towards a considerably low level. In another study, Cui
and co-workers entrapped chloroperoxidase in a well-structured mag-
netic nanoparticles for the decolorization of aniline blue dye [153]. The
immobilized enzyme was able to achieve over 90% in decolorization of
dye in 10 min in which the performance was credited to the well-de-
fined layer by layer assembly architecture that reduced the diffusional
limitation of reactions and products. A sharp drop of about 20% and
10% in catalytic performance was observed at the 2nd and 3rd cycle
respectively, with the biocatalyst retaining about 70% of its initial ac-
tivity at the 20th cycle. The sharp drop was attributed to the leakage of
weakly bounded enzyme from the magnetic nanoparticles. Similar ob-
servations were made by another study during the magnetic separation
of the immobilized enzyme from the solution [145]. Despite the loss in
activity, the ease of separation of the immobilized enzyme from the
reaction medium was highlighted. Magnetic nanoparticles therefore,
provide a potentially feasible solution to the reusability and recovery
challenge associated with the use of nanosupport for enzyme im-
mobilization although improvements are still required.
Similar to enzyme immobilized on nanoscale support using con-
ventional immobilization strategies, it is also difficult to recover hybrid
nanoflowers from the reaction medium. Therefore, some researchers
have made progress in developing interesting strategies. Combined
nanoflower is a strategy that combines the conventional enzyme im-
mobilization methods such as adsorption, entrapment and covalent
binding with the hybrid nanoflowers (as shown in Fig. 3). For example,
Zhu and co-workers incorporated hybrid nanoflowers onto a membrane
for repeated use in detection of phenol [170]. The attachment of the
nanoflowers to the membrane through simple adsorption allowed it to
be easily recovered from the reaction medium. After each use, the filter
was injected with pure water to remove unreacted agents and then
followed by drying of the membrane for the next cycle. In another
study, Rong and co-workers immobilized laccase nanoflowers on a
copper foil surface [154]. The attachment of the nanoflowers onto the
copper foil surface allows easier recovery with the study reporting that
the laccase nanoflower lost less than 10% of its initial activity after 5
cycles of use.
3.4. Hybrid nanoflowers synthesis
The dye decolorization studies conducted using hybrid nanoflowers
have produced excellent results. Although hybrid nanoflowers can
achieve higher enzymatic activity, stability and reusability, the method
to synthesize the hybrid nanoflowers presented by Ge and co-workers
J.K.H. Wong, et al.
Fig. 3. Combined nanoflower [133].
requires the reaction mixture to be incubated for 3 days at room tem-
perature [131]. The considerable amount of time needed has severely
hampered the widespread applications of these nanoflowers. Therefore,
a new approach is needed to speed up the formation of the nanoflowers
to make it more applicable in industrial applications. A work by Batule
and co-workers was the first to greatly reduce the synthesis time from 3
days to only 5 min at room temperature [171]. The method relied on a
sonochemical method in which sonication facilitated the synthesis of
the nanoflowers. In addition to the reduced synthesis time, the nano-
flowers still possessed the advantages posed by nanoflowers synthesized
through incubation. The sonochemical method of synthesizing hybrid
nanoflowers has been confirmed by Chung and co-workers through
synthesizing glucose oxidase, laccase, and catalase nanoflowers for the
application of mediatorless glucose biofuel cell [172]. The study noted
that the sonication method yielded nanoflowers with hierarchically
structured flower-like morphology along with higher activity and sta-
bility, indicating great potential in the application of biosensor and
biocatalysts. With the overall reduced time required for synthesis and
superior advantages over conventional immobilization methods, the
potential application of hybrid nanoflowers in dye wastewater treat-
ment is thus, likely to be extremely exciting in the near future.
3.5. Sustainability of nanomaterials
Since the introduction of nanomaterials in dye removal, the effi-
ciency and performance of each nano-adsorbent type have been criti-
cally studied and reviewed in many studies. However, the real chal-
lenge that remains uncertain is whether the application of
nanomaterials is considered sustainable, in the sense that it does not
raise any environmental concerns on the life cycle. Based on previous
studies, it is prominent that the demand for nanomaterials such as
nano-TiO2 and carbon nanotubes (CNTs) is high for various dye deco-
lorization processes, and would exceed 1000 tons per annum, due to
their excellent catalytic properties. However, these nano-TiO2 and
CNTs are considered as two of the most expensive and highly hazardous
nanomaterials available that can be used extensively as dye adsorbents
[173]. Both single-walled (SWCNTs) and multi-walled nanotubes
(MWCNTs) cost about 10-fold higher compared to carbon fiber [174].
In addition, they possess several harmful effects under regular ex-
posures over a long period of time, as shown in Table 5. In addition,
both nanomaterials require high temperatures and pressures during
their synthesis processes. Viana and co-workers reported that the pro-
cess of synthesizing TiO2 nanoparticles requires centrifugation followed
by extreme heating at temperatures from 200 to 1100℃ [175]. While
there are numerous ways to produce CNTs, chemical vapor deposition
method proceeds with 427 to 1200℃ reaction temperature; laser
12
Journal of Environmental Chemical Engineering 7 (2019) 103261
vaporization method only produces CNTs of best quality at 1200℃ re-
action temperature; electric-arc discharge method requires tempera-
tures above 3000℃ to vaporize carbon atoms [176]. Since high tem-
peratures are strictly required, high power supply is consequently
needed to ensure the quality and quantity of the synthesized product
[177]. To decolorize dye effectively, nano-TiO2 normally requires
photocatalysis, in which its activity has to be activated with visible light
source in the range of 400 to 800 nm [66,67].
The development of sustainable adsorbents using nanomaterials
presents several research gaps that needs to be addressed in order to
reduce its negative impacts on living organisms. Recently, researchers
have focused on cheaper and less hazardous adsorbents like magnesium
oxide (MgO). Ge and co-workers discovered a low-cost and environ-
mental friendly nanomaterial which is magnetic Fe@MgO composites
that are highly capable of removing lead ions and methyl orange dye
from water [181]. Tian and co-workers also demonstrated an excellent
Congo red adsorption from dye-contaminated effluent by using porous
hierarchical MgO [182]. Another excellent example of a sustainable
approach was the pilot scale solar treatment method using zinc oxide
nanoparticles (nano-ZnO) as reported by Dhatshanamurthi and co-
workers [183]. The harnessing of solar energy from the sun for en-
vironmental clean up makes it highly suitable for continuous applica-
tions in hot and humid countries like Thailand and Malaysia. The use of
free nanomaterials in wastewater treatment has the possibility of
creating secondary pollution to the already contaminated effluents and
therefore defeats the purpose of treating dye effluents. Therefore, im-
mobilization supports are necessary in order to prevent such difficulties
[184]. Hadjltaief and co-workers reported a simple and inexpensive sol-
gel method to decolorize cationic Malachite Green (MG) and anionic
Red Congo (RC) dyes using nano-ZnO immobilized on natural Tunisian
clay [185]. Under simulated solar irradiation source, both dyes
achieved excellent degradation of 100% within 60 and 70 min respec-
tively. On the other hand, Nadaroglu and co-workers investigated a
low-cost and efficient approach to remove Trypan blue dye from water
using zinc nanoparticles (nano-Zn) immobilized on Luffa Sponge [184].
Such methods can be highly considered in treating real textile effluent
as the optimum conditions are discovered to be at pH 7 and the equi-
librium, achieved within 30 min. These methods can be considered as
highly spontaneous and feasible processes in the industrial scale. Be-
sides, numerous supports such as silica nanofibers, zeolite and glass
slides are widely used to immobilize silver nanoparticles (AgNPs) in
order to improve its stability and durability. AgNPs immobilized on
quartz beads have been used in the complete photocatalytic degrada-
tion of organic dyes including methylene blue, methyl orange and
rhodamine chloride, as reported by Zhou and Srinivasan [186]. Dong
and co-workers developed a novel fibrous nano-silica based
J.K.H. Wong, et al.
Table 5
Toxic effects of nano-TiO2 and CNTs.
Nanomaterial Test Species
TiO2 Mice
Algae and plants
SWCNT CD rat male
CD1 mouse
Male rat
DWCNT C57BL/6 mice male
MWCNT F344 rat male
TiO2: titanium dioxide; SWCNT: single-walled carbon nanotube; DWCNT: double-walled carbon nanotube; MWCNT: multi-walled carbon nanotube.
nanocatalyst immobilized with AgNPs to catalyse the reduction of toxic
4-aminoohenol and 2-nitroaniline, which can be regenerated and re-
used up to 10 cycles [187]. Liu and co-workers also presented AgNPs
immobilized onto tannic acid-modified eggshell membrane, which ex-
hibits high catalytic activity in the degradation of Congo red and me-
thyl orange dyes [188]. Apart from containing many functional groups
and still remaining mechanically stable, the use of eggshell membrane
is also able to reduce the amount of waste in the food industry.
In sum, nanomaterials such as nano-Zn, nano-ZnO and AgNPs are
highly sustainable owing to their potential applications as effective
catalysts. Compared to TiO2 and CNTs, they are less harmful to the
environment and human beings due to their non-toxic and eco-friendly
characteristics. In recent years, considerable attention has been placed
on photocatalytic-based processes due to their high performances in
treating dye water. Although photosensitive semiconductors like TiO2
and ZnO are able to conserve resources such as water, chemicals and
cleaning materials [189], further studies on the sustainability of na-
nomaterials are still required as long photocatalytic process is not
economical with the use of more energy on UV light and its application
is largely constricted by its own ultraviolet range [173,188].
4. Conclusion
Most of the technologies used today for dye wastewater treatment
are found to be hampered by their respective limitations. Enzyme in-
corporated nanotechnology is at present undergoing an evolutionary
phase that is reflected by the continuous development of these tech-
nologies using different combination of enzymes, support materials and
immobilization methods for a wide variety of applications. As dis-
covered through our review, these nanotechnologies offer a feasible
solution to the dye wastewater problem that is faced by multiple in-
dustries today. Despite the potential, enzyme incorporated nano-
technologies face different challenges that need to be tackled and there
is still much room for improvement. We recommend that more studies
should be done with focus on the feasibility of these systems in real
industrial applications, understanding how these systems behave, on
the systems’ recovery from the reaction medium and their environ-
mental sustainability. Once the challenges hindering the application of
this nanotechnology are resolved, the future remains optimistic for the
application of these biocatalysts in the treatment of dye wastewater.
Only then may we realize the true potential of these biocatalysts.
Declarations of interest
None.
Acknowledgement
Authors would like to thank Curtin University Malaysia for pro-
viding research fund to support the enzyme-immobilized wastewater
treatment research project.
13
Journal of Environmental Chemical Engineering 7 (2019) 103261
Toxic Effects Reference
Significant increase in body weight; [178]
Testis and testicular mass significantly reduced
Growth inhibition; [178]
Decreased photosynthetic efficiency
Transient inflammation [179]
Bone resorption; [179]
Fetuses with skeletal anomalies
Acute pulmonary and cardiovascular responses [180]
Persistent alveolitis and interstitial fibrosis [179]
Persistent inflammation and fibrosis [179]
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