Separation and Purification Technology 219 (2019) 186–207

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Separation and Purification Technology

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

A review on phenolic wastewater remediation using homogeneous and T

heterogeneous enzymatic processes: Current status and potential challenges
Muntathir Alshabiba, Sagheer A. Onaizia,b,
⁎

a
Department of Chemical Engineering, King Fahd University of Petroleum and Minerals, Dhahran 31216, Saudi Arabia
b
Center of Excellence in Nanotechnology, King Fahd University of Petroleum and Minerals, Dhahran 31261, Saudi Arabia

ARTICLE INFO ABSTRACT

Keywords: Water pollution is one of the serious environmental problems threatening the sustainable development of human
Enzymatic wastewater treatment process civilization. Many phenolic compounds are hazardous, toxic, endocrine disrupting, mutagenic, teratogenic, and/
Phenolic pollutants or carcinogenic. They also cause severe damages to marine ecosystem. Accordingly, their removal from polluted
Enzyme immobilization wastewaters prior to its discharge to the environment is a mandatory task. Several processes have been proposed
Homogeneous enzymatic reactions
for treating phenol-contaminated waters. Among the proposed treatment processes is the enzymatic method.
Heterogeneous enzymatic reactions
Thus, the key objective of this review article is to present the recent progress in utilizing peroxidases and laccases
for the remediation of phenolic wastewaters. Both homogenous and heterogeneous enzymatic processes to re-
move different phenolic pollutants from wastewaters will be reviewed. Recent studies on the effects of the key
operational factors (i.e., temperature and pH) will also be presented. Additionally, emerging trends in enzymatic
wastewater treatment will be addressed. The obstacles and challenges facing the large scale applications of
enzymatic remediation of phenolic wastewaters will be highlighted. More importantly, several ideas for ad-
dressing the limitations of the process and improving its efficiency and viability will be provided. These ideas
might form the basis for future studies on developing a more effective enzymatic process for treating waste-
waters contaminated with phenolic pollutants, which is a growing environmental problem worldwide.

1. Introduction
Chemical and allied industries generate large quantities of water
polluted with high concentrations of several phenolic components. The
concentration of phenolic compounds in the discharged wastewaters
can be as high as thousands part per million (ppm), depending on the
industrial source of discharge [1]. Besides the high levels of phenolic
compounds in the generated wastewaters, large quantities of phenolic
wastewaters are also generated annually. It has been estimated, for
instance, that more than 10 million tons of phenols are yearly dis-
charged to the environment [2]. The discharge of phenolic wastewaters
to the environment without a prior satisfactory treatment leads to many
health problems (see Table 1). Additionally, the discharge of phenolic
wastewaters to the environment leads to the contamination of soil,
surface water, or/and groundwater [3,4], threatening the harmony of
the ecosystem. Accordingly, industrial wastewater effluents should not
contain more than 5 ppm if these effluents are to be discharged into
public sewage systems; however, if phenolic wastewater effluents are to
be released into inland water bodies, the maximum permissible dis-
charge level of phenols in these effluents is only 1 ppm [5,6]. Fur-
thermore, the level of phenols in potable drinking water should not
exceed 1 part per billion according to the United States Environmental
Protection Agency [7]. Thus, proper and effective treatment of phenolic
wastewaters before reuse or discharge is an imperative task.

Abbreviations: BPA, bisphenol A; ppm, part per million; DNA, deoxyribonucleic acid; HRP, horseradish peroxidase; SBP, soybean peroxidase; 2,4-DCP, 2,4-di-
chlorophenol; MPx, mesquite peroxidase; CP, chlorophenol; PCP, pentachlorophenol; 2,4,6-TCP, 2,4,6-trichlorophenol; 2-CP, 2-chlorophenol; 3-CP, 3-chlorophenol;
4-CP, 4-chlorophenol; NLP, nonylphenol; TC, triclosan; MOF, metal-organic framework; Zr-MOF, Zr-metal organic framework; PAN, polyacrylonitrile; CMMC,
carbon-based mesoporous magnetic composite; PVDF, polyvinylidene fluoride; EE2, 17a-ethinylestradiol; E2, 17b-estradiol; 2,3,4,5-TTCP, 2,3,4,5-tetrachlorophenol;
BPB, bisphenol B; BPF, bisphenol F; BPC, bisphenol C; BPE, bisphenol E; BPO, bisphenol O; BPT, bisphenol T; BPZ, bisphenol Z; TCBPA, tetrachlorobisphenol A; SMZ,
sulfamethoxazole; CBZ, carbamazepine; DIC, diclofenac; 2,4-DNP, 2,4-dinitrophenol; 3-MEP, 3-methoxyphenol; 2-MEP, 2-methoxyphenol; 4-C-3,5-DMP, 4-Chloro-
3,5-dimethylphenol; 2-B-4-CP, 2-benzyl-4-chlorophenol; 4-AAP, 4-acetamidophenol; PA, phenylacetate; 2,6-DMEP, 2,6-dimethoxyphenol; PEG, polyethylene glycol;
CMC, critical micelle concentration; FMNP, ferromagnetic nanoparticles; GMP, guanosine monophosphate; SDS, sodium dodecyl sulfonate; CTAB, hexadecyl-
trimethyl ammonium bromide
⁎
Corresponding author at: Department of Chemical Engineering, King Fahd University of Petroleum and Minerals, Dhahran 31216, Saudi Arabia.
E-mail address: onaizi@kfupm.edu.sa (S.A. Onaizi).

https://doi.org/10.1016/j.seppur.2019.03.028
Received 29 October 2018; Received in revised form 28 February 2019; Accepted 8 March 2019
Available online 09 March 2019
1383-5866/ © 2019 Elsevier B.V. All rights reserved.
M. Alshabib and S.A. Onaizi Separation and Purification Technology 219 (2019) 186–207

Table 1
Noxious health effects of phenolic pollutants on human and aquatic creatures.
Phenolic pollutants Noxious effects Ref(s)

Phenols • Cause muscle fatigue, skin rashes, and diarrhea [8–12]

• Modify aquatic biota such as algae and other microorganisms
• Lead to bronchoconstriction and adverse effects in rat and human lungs
• Induce suicidal death in human red blood cells
Bisphenols • Lead to metabolic disorders and abnormalities in human babies [13–17]
• Cause cancer in breast and prostate glands
• Induce mutations and disruption in reproduction systems in animals
Chlorophenols • Disturb organ and endocrine system in aquatic organisms [18,19]
• Adversely affect cell growth or induce genetic mutations in fish
• Cause digestive tract infections, asthma, heart diseases, and sarcoma to humans
• Lead to oxidative stress, deoxyribonucleic acid (DNA) damage, and lung cancer in living organisms
Alkylphenols • Cause disturbance in testicular development, and damage of some sustentacular cells [20–22]
• Adversely impact the secretion of progesterone and androstenedione in males
Triclosans • Disturb immune system, reduce the production of reactive oxygen species, and lead to malfunctioning in cardiovascular system [23,24]
• Lead to uncoupling of mitochondria, and induce lethal effects on cells
Cresols • Lead to abnormalities in gap junction and adherens junction [25,26]
• Suppress the formation of blood clots
• Reduce the production of Reactive Oxygen Species in humans, leading to bleeding disorders
Nitrophenols • Suppress the pathway of androgen receptor (AR) signaling [27–30]
• Induce changes in testicular tissues
• Sharply decrease the plasma amounts in some hormones
• Hinder transcription process, thereby affecting the level of genes in thyroid system
Aminophenols • Decrease the level of haemoglobin and volume percentage of red blood cells in fish [31–33]
• Lead to malfunctioning in both reproductive and respiratory systems in humans
• Lead to premature death of cells in liver and also damage to kidney
There are a number of conventional methods for treating phenolic conditions and remain active even when conditions are quickly
wastewaters such as adsorption [34,35], distillation [36,37], chemical changed [62–65].
oxidation [38,39] and extraction [40,41] (see Table 2). Additionally, In some cases even simple pollutants become recalcitrant, making
some advanced techniques such as membrane separation [42,43] and the use of microbial treatment ineffective [61,66]. Such cases are not
photocatalytic oxidation [44,45] have been proposed as alternative usually encountered with using enzymes [46]. Furthermore, enzymes
methods for treating phenolic wastewaters. Besides these physico-che- are biodegradable proteins and do not generate toxic waste (i.e.,
mical treatment methods, biological treatment using microorganisms sludge) as is the case with microbial processes [67,68]. Some side-
has been also utilized. In such a treatment, microorganisms degrade products generated in microbial treatment of wastewater might be very
phenolic compounds by opening the aromatic rings while consuming harmful to human health or/and environment. Although microbe mu-
energy and carbon from the targeted pollutants [46]. The metabolism tation is usually beneficial, it sometimes leads to undesirable con-
of phenolic components by the microorganisms is catalyzed by certain sequences [69]; some microbes might become invasive and resistant to
intracellular enzymes (i.e., oxidoreductases). Accordingly, a relatively antibiotics, for instance [70]. Microbes are usually ineffective when the
new trend has emerged in the past years on utilizing extracellular en- pollutants are present at low concentrations. To enhance the bio-
zymes instead of the whole microbial cells for treating wastewaters availability of pollutants, organic co-solvents or surfactants might be
polluted with organic matters. added. However, the addition of co-solvents or surfactants might ne-
Despite that the biological treatment (using living microorganisms) gatively impact the biological wastewater treatment. Enzymes are
of wastewater carries some advantages over enzymatic treatment (using usually more tolerant towards such additives than microorganisms
extracellular enzymes), it has also some very significant drawbacks. The [71]. Not only that but some additives have also resulted in enzymatic
key advantages of microbial over enzymatic remediation of waste- remediation enhancement [4,72–75]. In addition to the above limita-
waters are: microbes can reproduce and increase their population, tions of microbial wastewater treatment, some phenolic pollutants
providing higher order of pollutant consumption and thus more effi- might be toxic to the utilized microbes even when present at low
cient treatment. Contrarily, enzymes are not living species and thus concentrations. For instance, it has been reported that phenols with a
they cannot reproduce themselves. Therefore, any increase in enzyme concentration slightly above 200 ppm can modify microbial structure
concentration must be added externally. Additionally, enzyme may lose and adversely affect microbial growth [56,57].
activity either partially or totally if enzyme inhibitors present in the Therefore, enzymatic remediation is increasingly becoming attrac-
system or when some enzymes undergo autolysis [47–49]. Further- tive environmentally-friendly and sustainable alternative wastewater
more, some microbes can mutate and, thus, adapt themselves to new treatment technology. Additionally, the potential to produce enzymes
(e.g., more harsh) conditions and new food (e.g., new pollutants) on a higher scale, with enhanced stability and/or activity, and at a
sources [50]. lower cost by using recombinant-DNA technology further encourages
Despite the abovementioned advantages of microbial over enzy- the utilization of enzymes in wastewater remediation [76,77]. The fact
matic treatment, the latter carries more valuable advantages in other that enzymes do not require nutrients, do not require biomass accli-
aspects than microbial treatment. For example, although microbes can mation, do not generate sludge, has greatly lower mass transfer lim-
reproduce and increase their population, it is always difficult to itation to contaminants compared to the living microorganisms, form
maintain/control the optimal level of growth conditions (i.e., appro- simpler systems which are easy to control, and are effective at very low
priate moisture and nutrients, adequate temperature, adequate pH, and concentrations make them even more attractive than the living micro-
sustainable availability of sufficient oxygen in case of aerobic micro- organisms.
organisms). Therefore, microbes might not survive under certain con- Despite the attractiveness of enzymatic wastewater treatment, it is
ditions, leading to inefficient wastewater treatment process [61]. Con- still immature and uncompetitive with biological one. However, as is
trarily, enzymes are effective on a wide range of environmental mostly the case with new technologies, progresses and breakthroughs in

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M. Alshabib and S.A. Onaizi Separation and Purification Technology 219 (2019) 186–207

Table 2
Common phenolic wastewater treatment techniques and their major drawbacks.
Treatment method Description Major drawbacks References

Distillation • Phenols are separated from water based on relative • High energy consumption [9,51]
volatility • Inefficient in treating low levels of phenols
Adsorption • Adsorptive materials like activated carbons, zeolites,
graphene, and clays are used to selectively remove
• Regeneration
costly
of adsorbents is complex and [9,51–53]

phenols from wastewater streams via physical or

chemical interactions
• Regeneration
phenolic wastes
of adsorbents creates new

• Chemicals used in modification steps of

adsorbents might be toxic.
• Adsorption capacity might be low, requiring
a large adsorbent quantity
liquid-liquid extraction • Phenols are recovered from aqueous solutions based on • Low selectivity to some phenolic compounds [9,51,52,54]
their relative solubilities with the aid of appropriate
extractive solvents
• Some used solvents are harmful to the
environment
• Solvent regeneration might be expensive and
challenging
Wet air oxidation • Phenols are oxidized when exposed to oxygen or air at
high pressure and high temperature
• Requires high temperature and high pressure
to attain the desired oxidation
[9,51]

• Reaction is slow (i.e., high residence time)

Catalytic Wet Air Oxidation • Phenols are oxidized by either oxygen or air at high
pressure and high temperature in the presence of
• Post-treatment
degrade
products are not feasible to [9,51,52]

catalysts • Requires reactors manufactured by expensive

Biodegradation (Aerobic or Anaerobic)
materials resisting acidic conditions at high
temperatures
• Phenols are degraded by various microorganisms like
bacteria, yeast, and fungi
• Difficult to control the optimal level of
growth media
[55–57]

• Results in toxic by-products after treatment

• Not suitable for high phenol concentrations
• Might require co-solvent when phenol
concentration is low
• Generation
sludge)
of secondary pollution (i.e.,

Chemical Oxidation • Destructive chemicals (i.e. ozone, hydrogen peroxide,

chlorines, permanganates, and ferrates) are used to
• Formation of recalcitrant chemicals when
using some metal oxides
[9,51]

oxidize phenols in wastewater • Ozone is expensive to generate, has low

Electrochemical oxidation (direct or indirect)
soluble in water, and ineffective to oxidize
phenolic compounds
• Safety concerns due to the handling of
harmful chemicals
• Direct oxidation: phenols are directly adsorbed on the
anode surface (i.e. physically by hydroxyl radicals or
• Costly in terms of equipment and energy
consumption
[9,51]

chemically by the active oxygen) • Low capacity process

• Indirect oxidation: chlorine or hypochlorite with
hydrogen peroxide and metal ions are used as
• Safety concerns due to the handling of
harmful chemicals
intermediates in the destruction process
Photo-oxidation (Non-catalytic or catalytic) • Phenols are degraded under UV irradiation with the • Difficult to attain effective charge separation [8,9,51,58,59]
presence of oxidizing agents • The use of UV light is not economical
• Photocatalysts like titanium dioxide are sometimes used
to enhance the oxidation efficiency
• Economic synthesis and regeneration of the
photocatalysts are nott always feasible
• Dopants are used to reduce the band gap of the
photocatalyst, making it possible to carry the photo-
• Disposal of spent photocatalysts is another
cost factor
oxidation reaction under visible light
Membrane-based separation techniques • Separation of phenols from wastewater streams is driven • Short lifetime of membranes due to fouling [8,9,51,60]
(nanofiltration/reverse osmosis, liquid
membranes, and pervaporation
by the change in concentration gradient or
transmembrane pressure gradient
• Separation effectiveness of some polymeric
membranes is lowered when subjected to
high temperatures or under severe conditions
• High energy requirement
research and development related to this area will speed up the de- phenolic pollutants using mobile enzymes (i.e., homogeneous enzy-
ployment of this technology on a commercial scale. Thus, we focus in matic treatment), recent studies on heterogeneous enzymatic treatment
this article on reviewing recent progress in utilizing enzymes for phe- (using immobilized enzymes) of phenolic wastewaters will also be re-
nolic wastewater remediation. Enzymatic treatment of phenolic com- viewed. Enzymatic reaction temperature and the pH of the medium
pounds involves the polymerization of phenolic substrates into water- where the enzymatic reaction takes place are also among the key factors
insoluble products [9], which are, then, easily removed from the affecting the enzyme stability. Thus, recent studies on the optimal va-
treated water by simple separation methods such as centrifugation or lues of these parameters will be addressed. Additionally, new trends
filtration [78,79]. These polymeric products can be valuable materials (e.g., enzyme immobilization on nano-sized supports, surface active
that might be separated and sold to increase the revenue of the process. additives, and mimicking approaches) in enzymatic wastewater will be
One of the key limitations of enzymatic treatment of phenolic presented. Finally, the unresolved challenges will be highlighted with
wastewater that severely affects the economy of the process is the proposals for tackling them in future research in order to improve the
lifetime (i.e., stability) of the utilized enzyme. Enzyme immobilization effectiveness and the feasibility of the process.
has been proposed as a useful strategy to address this problem. Thus,
besides reviewing recently published studies on the remediation of

188
M. Alshabib and S.A. Onaizi
2. Homogeneous enzymatic reactions for the remediation of
phenolic wastewaters
Homogeneous enzymatic reactions involve the utilization of dis-
solved enzymes in wastewater sample to catalyze the removal of phe-
nolic pollutants. The most prevalent enzymes, which have been suc-
cessfully used to eradicate phenolic pollutants, are peroxidases and
laccases. Peroxidases have either a heme cofactor in their active sites or
redox-active cysteine/selenocysteine residues [80]. Owing to the easy
access to their active sites, the removal of several pollutants from
wastewaters, including phenol and its derivatives, can be catalyzed by
peroxidases.
The most widely utilized peroxidase for the removal of phenolic
compounds from wastewaters is horseradish peroxidase (HRP) [81–84].
A recent study, for example, evaluated the use of HRP for the de-
gradation of phenols from a biorefinery wastewater sample. The en-
zymatic system removed over 99% of phenols within 35 min of treat-
ment [85]. Another study revealed that increasing HRP concentration
enhanced Bisphenol A (BPA) degradation to more than 98% within 3 h
of treatment [86]. This extent of BPA removal was achieved in the
presence of hydrogen peroxide (H2O2), which is an essential electron
acceptor additive for effective degradation of phenolic substrates using
HRP. However, at high H2O2 dose, HRP was subjected to deactivation,
resulting in the reduction of BPA removal [86].
The potential use of HRP on an industrial scale is severely limited by
the high enzyme production cost, its high vulnerability to deactivation
[2,46,79], and its limited availability as a result of the laborious cul-
tivation and extraction processes [87]. An alternative to HRP is soybean
peroxidase (SBP), which is more abundant [87,88], has the potential to
be produced more cheaply than HRP, and it exhibits a lower vulner-
ability to irreversible deactivation by H2O2. In terms of catalytic power,
it has been reported that SBP is more powerful than HRP in removing
Triclosan (TC) [89], which is a recalcitrant phenolic pollutant, from
wastewater. For example, SBP effectively removed 98% of this pollu-
tant within 30 min while only 36.5% removal was obtained using HRP
within the same time frame [89].
Although SBP might be produced from soybean hulls (a low-value
by-product of soybean processing), most of other peroxidases, are
mainly extracted from agricultural sources, which might divert crops
and agricultural lands [2]. Thus, researchers are seeking alternative
sources. Hairy roots extracts [90–92], white radish (Raphanus sativus)
[93] and waste Brassica oleracea [94] are good non-food sources for
peroxidases production, which also do not compete with crops for
agricultural land use. Additionally, wastes produced from food pro-
cessing plants can also be used for peroxidases production. Accordingly,
Kurnik et al. [79] utilized potato pulp, a waste by-product produced in
potato starch factories, to extract peroxidases and used the produced
peroxidases for the removal of phenol from synthetic and real waste-
water samples. The authors reported that more than 95% of phenol was
removed from synthetic wastewater samples containing optimized
phenol concentrations; the term “optimized phenol concentrations” was
used by the authors [79] to refer to the initial phenol concentrations in
synthetic wastewater samples, which correspond to more than 95%
phenol removal at the end of the treatment process. Furthermore, over
90% of phenol was removed from industrial wastewater samples con-
taining phenol concentrations in the range of 0.02–0.1 mM [79]. An-
other study tested the efficiency of peroxidases derived from potato
waste in removing 2,4-dichlorophenol (2,4-DCP) from a synthetic
wastewater sample [95]. At a pollutant concentration ranging from 1 to
3 mM, the removal extent reached 98%. The above studies demonstrate
the potential production of peroxidases from food wastes and their
utilization for the remediation of phenolic compounds from waste-
waters.
Recently, scientists extracted a peroxidase from a mesquite tree
(Prosopis juliflor) and compared this low purity peroxidase with HRP
[2]. Mesquite peroxidase (MPx) provided 90–92% phenol and
189
Separation and Purification Technology 219 (2019) 186–207
chlorophenol (CP) removal from wastewater while HRP could not re-
move more than 40–60% of these phenolic pollutants. In terms of ac-
tivity, MPx possesses 6-fold oxidation power higher than that of the
purified HRP. Moreover, the MPx exhibited a higher residual activity
and more resistance to inhibition by the reaction products. In addition
to its higher catalytic efficiency over HRP, MPx is also more sustainable
enzyme since it is produced from a source that is widely and sustainably
available in nature. Furthermore, the extraction of MPx is easier re-
lative to HRP [2].
One important drawback of using peroxidases is the requirement of
adding H2O2 to the reaction medium in order for these enzymes to
function properly. However, even low concentrations of H2O2 can
lower the enzymatic reaction rate [96] while high H2O2 concentrations
could render these enzymes totally inactive [96,97]. Furthermore, it
has been reported that the oxidation power of peroxidases highly de-
pends on the type of phenolic substrates and also on the source of the
utilized peroxidase [98]. Accordingly, alternatives have been sought by
several researchers. Laccases represent good alternatives to peroxidases
since these enzymes can function well in the absence of H2O2. Unlike
peroxidases which require H2O2 to function properly, the oxidant re-
quired in the case of laccases is oxygen, which is harmless, does not
inhibit enzyme activity and can be easily obtained from free and
abundant sources (e.g., air) [99].
Laccases, mainly produced by fungi, contain four copper atoms,
which are categorized into three groups (i.e., type 1, type 2 and type 3)
according to analysis using UV–visible and electron paramagnetic re-
sonance spectroscopy. The laccase mechanism begins when electrons
are extracted from a phenolic substrate by the type 1 (T1) copper site.
These electrons are then transferred internally into T2 and T3 copper
sites followed by the reduction of oxygen (i.e., externally transfer of
electrons to oxygen) to form water [100–102]. The oxidation of phe-
nolic compounds leads to the formation of phenoxy radicals, which in
turn undergoes non-enzymatic coupling with each other to form dimers.
Eventually, these dimers combine to produce polymers after some en-
zymatic reaction cycles [9].
Laccases have been extensively used to treat wastewaters con-
taminated with phenols. One of the key reasons for the popularity of
laccases for the remediation of wastewaters contaminated with phe-
nolic compounds is their non-specificity [103]. These multicopper
oxidases [104] are capable of catalyzing a wide variety of substrates
[9,80,105,106], including phenolic and nonphenolic compounds [109].
There are several studies on utilizing homogeneous enzymatic re-
actions based on laccases for the removal of phenolic compounds from
wastewaters. A study of such was conducted by Asadgol et al. [104]
where phenol and BPA were separately removed from wastewater
samples using laccase. The initial concentration of each pollutant was
4 mM and the treatment was carried out for 30 min. After 30 min in-
cubation of each of these two pollutants in 5 U/mL concentration of the
enzyme, the extents of phenol and BPA removal were 80 and 60%,
respectively. With optimizing the conditions of the degradation of these
two phenolic pollutants, the removals of phenol and BPA were in-
creased to about 96% and 88%, respectively, within the same treatment
duration [104]. The removal of BPA (44 μM) from wastewater using a
laccase cocktail obtained from Pycnoporus sanguineus CS43 at a con-
centration of 0.1 U/mL has been also studied by Garcia-Morales et al.
[17]. The researchers found that about 90% of BPA was removed by the
cocktail within about 5 h of treatment [17]. In the above two studies,
almost the same extent of BPA removal was achieved; however, the
laccase used by Asadgol et al. [104] was much faster in removing BPA.
Despite that the initial BPA concentration used by Asadgol et al. [104]
was more than 90 folds higher, those researchers also utilized a higher
enzyme concentration, which might explain their observation of faster
BPA degradation rate. Furthermore, the source and the purity of the
utilized laccase, and the treatment conditions (i.e., pH, temperature,
presence of organic/inorganic co-pollutants) might also play roles in
the observed big difference in the treatment duration of BPA reported
M. Alshabib and S.A. Onaizi
by these two studies.
Laccase has been also reported to be effective in removing phenol
from a refinery wastewater sample [107]. However, although laccase
was able to remove 90% of phenol from the above sample, the rate of
phenol remediation was less than that obtained using SBP [107]. The
90% removal of phenol from the refinery wastewater sample was
achieved at the optimal (0.12 U/mL) laccase concentration. Although
phenol is, supposedly, easier to degrade than BPA, it has been reported
that a laccase concentration of 0.12 U/mL was sufficient to almost
completely remove BPA from a synthetic wastewater sample within 3 h
[86], relative to 90% removal of phenol from a refinery wastewater
sample within the same treatment duration [107]. The presence of
other contaminants and the complexity of the industrial wastewater
sample (i.e., the refinery sample) is probably the reason for such higher
remediation of BPA than phenol. In line with the complete removal of
BPA from synthetic wastewater sample reported by Escalona et al. [86],
a complete removal of BPA within 4 h of treatment was also reported by
Daâssi et al. [108], who utilized a laccase produced by Coriolopsis gallica
fungi. Laccase produced by other fungi (Bjerkandera adusta and T. ver-
sicolor) was less effective [108], demonstrating the effect of laccase
source on the removal of phenolic pollutants from wastewaters.
In addition to the removal of phenol and BPA from synthetic and
industrial wastewaters, the removal of chlorophenols (CPs) has been
also reported in the literature. For example, Zhang et al. [31] have
investigated the separate removal of 4-chlorophenol (4-CP), 2-chlor-
ophenol (2-CP), and 2,4-DCP from synthetic wastewater samples using
laccase, which was obtained from Coriolus versicolor. The initial con-
centrations of each of the tested pollutants and laccase were 50 and
80 ppm, respectively. After 10 h of treatment, this enzyme was able to
remove 69% of 4-CP, 75% of 2-CP, and 94% of 2,4-DCP [31]. In another
study, a mixture of CPs (i.e. the initial concentration of each pollutant
was 15 ppm) was treated with laccase produced from Trametes pub-
escens [110]. After 4 h of contact between laccase and the CPs pollu-
tants, the enzyme (its activity was 10 U/L) completely removed 2-
chlorophenol (2-CP) and 2,4-DCP from the treated wastewater [110].
The better removal extents of 2-CP and 2,4-DCP achieved by Gaitan
et al. [110] could be attributed to the utilization of a crude enzymatic
extract containing two laccase isoenzymes along with the use of lower
concentrations of the targeted pollutants. As highlighted previously, the
source of enzyme might play a role in the extent and the rate of phe-
nolic pollutants removal. However, there are other variables between
these two studies (e.g., the level of the utilized enzymes, the presence of
co-pollutants, and the treatment conditions), which might have also
contributed to such a difference.
The remediation of wastewater contaminated with other phenolic
compounds has been also reported in the literature. It has been claimed
that laccase from Trametes trogii completely removed hydroxytyrosol,
tyrosol, guaiacol and p-coumaric acid within 1 h of treatment [111].
Despite its efficiency in degrading the above four phenolic compounds,
laccase obtained from the same microorganism (i.e., Trametes trogii)
was less effective in degrading catechol [111]. The complete degrada-
tion of catechol required 2 h of enzymatic treatment in contrast to the 1-
h treatment required to completely eradicate the above four phenolic
compounds from synthetic wastewater samples. Although the in-
vestigators have proposed that the slower removal of catechol com-
pared to the other four phenolic components is related to the variations
in the structures of these pollutants (see Fig. 1), no direct evidence was
provided. Other researchers have reported more than 40% degradation
of catechol by laccase (obtained from Trametes versicolor) within only
72 s [112]. This might intuitively suggest that prolonged treatment time
of 1 h (more than 50-fold longer) could provide a complete removal of
this pollutant. However, no data were provided for longer treatment
(residence) time by Tušek et al. [112]. Additionally, Chakroun et al.
[111] did not provide the extent of catechol degradation within a
minute or so. Thus, it is difficult to correlate the difference in the de-
gradation rate of catechol relative to the other four phenolic
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Separation and Purification Technology 219 (2019) 186–207
compounds reported by Chakroun et al. [111] to merely the variations
in the pollutant structure, even though the pollutant structure usually
plays an important role in the extent and the rate of its degradation.
This proposition is supported by the findings of Chang et al. [84] who
reported that HRP was able to completely remove 2,4-DCP within 2 h of
treatment relative to only 70% phenol removal within the same treat-
ment time despite that 2,4-DCP is more structurally complex than
phenol. Menale et al. [113] have also reported a similar observation,
where the more complex 2,4,6-TCP and PCP (see Fig. 1) were easily
removed by laccase (from Trametes versicolor) relative to 2-CP.
3. Heterogeneous enzymatic reactions for the remediation of
phenolic wastewaters
One of the key limitations of homogenous enzymatic reactions is the
recovery of the biocatalysts. Such limitation might be overcome via the
immobilization of the biocatalysts on/within a suitable insoluble sup-
port, facilitating the enzyme recovery and reusability [15,114,115].
Another advantage of using heterogeneous enzymatic reactions (i.e.,
immobilized enzyme systems) is the improved enzyme stability
[100,116–118]. It has been reported that the insoluble enzymes have
the ability to resist denaturing agents [79,119,120] and that they pos-
sess a better storage stability [101,120,121].
Several studies (see, for example, Table 3) have reported the im-
mobilization of enzymes on various solid supports and then used the
immobilized enzymes for the degradation of phenolic compounds in
wastewaters. The utilized solid supports include silica, organic gels,
chitosan, kaolinite, graphene oxide and recently metal–organic frame-
works (MOFs), among others [122,123]. Enzyme immobilization on/
within insoluble supports can be achieved via chemical or physical
means [16,100]. The common physical immobilization methods are
physisorption (i.e., the binding of enzymes to solid supports via elec-
trostatic or hydrophobic interactions), entrapment or encapsulation
while the chemical immobilization is achieved through the attachment
of the enzyme of interest to a solid support via the formation of covalent
bonds between the enzyme and the insoluble support [116,124]. Al-
though covalent bonding usually provides a robust enzyme im-
mobilization, it is often more complicated and more expensive than
physical methods. Accordingly, several insoluble supports and im-
mobilization approaches have been utilized for enzyme immobilization,
with mixed outcomes and occasionally contradicting conclusions on the
effect of enzyme immobilization on its activity despite that most of the
published studies in this regard reported an improvement in enzyme
stability upon immobilization.
Physisoprtion was utilized by Nguyen et al. [125] for the im-
mobilization of laccase on a granular activated carbon support. The
immobilized enzyme, which was obtained from the genetically mod-
ified Aspergillus oryzae, was used to remove Sulfamethoxazole (SMZ),
Carbamazepine (CBZ), Diclofenac (DIC) and BPA from wastewater
samples in a batch enzymatic reactor. After 24 h of treatment, 59%,
40%, 60% and 98% of SMZ, CBZ, DIC and BPA were removed, re-
spectively. It has been observed by Nguyen et al. [125] that the removal
of phenolic compounds by the insoluble enzyme was higher than that
achieved using the free enzyme. The higher phenolics removal using the
immobilized enzyme has been attributed to the combined effects of
enzymatic reaction and pollutants adsorption on the solid support. Al-
though it is expected that products would likely compete with pollu-
tants in adsorption on the solid support, the authors did not tackle such
competitive adsorption or quantify (via, for instance, washing the ad-
sorbent and analyzing the after-wash solution) the impact of products
adsorption on the enzymatic activity. Nonetheless, it has been reported
that even for enzyme-free adsorbent, the adsorption capacity (i.e., ex-
tent of phenolic pollutant removal) decreased with the repeated use of
the adsorbent, suggesting that irreversible products/pollutants adsorp-
tion might play a role in the overall process, particularly with the re-
peated use of the immobilized enzyme. The removal of any irreversibly
M. Alshabib and S.A. Onaizi
Fig. 1. Structure of some common phenolic pollutants in wastewaters.
adsorbed pollutants/products, which has been addressed by Nguyen
et al. [125], will likely improve the efficacy of the immobilized enzyme.
Nguyen et al. [125] also studied the removal of these pollutants by
laccase, which has been immobilized in a packed bed reactor, as a
function of the treated wastewater volume. A complete removal of BPA,
DIC, SMZ and CBZ was observed until the volume of the treated was-
tewater sample reached 6000, 8000, 4000 and 5000 times the bed
volume, respectively, beyond which a decrease in the removal of these
phenolic pollutants was observed, most likely due to the saturation of
the adsorption sites of the solid support [125].
Gassara et al. [126] immobilized manganese and lignin peroxidases
via encapsulation, which is more robust than physical adsorption
techniques, within three different polymeric (pectin, gelatin and car-
boxymethylcellulose) matrices and then used these immobilized en-
zymes to study the removal of BPA from wastewater samples. The au-
thors reported that enzyme immobilization within the pectin polymeric
matrix has provided a higher removal of BPA relative to enzyme im-
mobilization within the other two matrices. The higher removal effi-
ciency of BPA using the encapsulated enzyme within the pectin matrix
has been correlated to the higher level of protection provided by this
matrix against inhibitory components in the reaction medium. Alem-
zadeh and Nejati [127] also used the encapsulation method to im-
mobilize HRP within an alginate matrix and reported a positive effect
on the enzyme stability and phenol removal efficiency. Sol-gel
191
Separation and Purification Technology 219 (2019) 186–207
technique was utilized by Mohidem and Mat [136] to encapsulate
laccase, which was purified from Tremetes sp., within a tetraethyl or-
thosilicate (TEOS) matrix. The immobilization has improved the en-
zyme stability. The enzyme activity towards the conversion of 6-di-
methoxyphenol has been also improved, which was more significant at
high enzyme loadings [136]. Instead of polymeric matrices, Ai et al.
[97] immobilized HRP on a hydrous-titanium surface and reported that
the immobilized enzyme is more efficient than the mobile enzyme in
removing phenol from wastewater. The increased removal of phenol
using the immobilized enzyme has been attributed to the combined
effects of pollutant adsorption on the solid support used to immobilize
the enzyme and the enzymatic degradation of the pollutant.
In terms of enzyme stability, Ai et al. [97] reported that the im-
mobilized HRP enzyme retained the same activity even when the con-
centration of H2O2 has increased from 0.2 to 1 mM [97].
Despite the reported improvement of phenolic components removal
using physically immobilized enzymes, the leakage of these enzymes
from the surface of the solid support or from the polymeric matrix is
common. Any leaked enzyme molecules will be lost to the reaction
medium, rending the biocatalytic solid materials less effective with the
repeated use. A feasible alternative to physical immobilization methods
is cross-linking the molecules of two or more enzymes in order to form
insoluble enzyme aggregates. Taboada-Puig et al. [130] have utilized
this approach where they cross-linked a peroxidase produced from
 Table 3
Enzyme immobilization on various supports, the utilization of the immobilized enzymes for the remediation of phenolic wastewaters, and the reported enzyme activity and stability.
Enzyme Support/Carrier Immobilization Pollutant(s) Maximum Treatment Storage Stability Thermal Stability Ref
Method Removal (%) Time

HRP Calcium alginate Encapsulation Phenol ∼53% 100 min Not Reported (NR) NR [127]
HRP Hydrous-titanium coatings Encapsulation Phenol > 92% 15 min NR At temperatures ranging from 60 to 90 °C, [97]
M. Alshabib and S.A. Onaizi

the remaining activity for insoluble

enzyme was 50%
HRP Magnetic poly(glycidylm- Covalent Phenol 86% 2h At 4 °C, the insoluble enzyme retained ∼84% of At 65 °C, the retained activity for mobile [128]
ethacrylate-co- 4-CP 59% its original activity after 56 days and immobilized HRP were 11% and
methylmethacr-ylate) 42%, respectively
HRP Polyacrylonitri-le (PAN)- Covalent Phenol 99.9% 5h NR NR [82]
based beads
HRP and SBP Aldehyde-modified glass Covalent 4-CP 90% HRP 1h NR NR [129]
95% SBP 0.5 h
Versatile peroxidase & Enzyme aggregates Covalent BPA, ∼96% 10 min NR NR [130]
glucose oxidase NLP, ∼100 %
TC, ∼26%
EE2, ∼93%
E2 ∼90%
Laccase PAN-based beads Covalent 2-CP Individual 1.5 h NR NR [113]
PCP 85%,
2,3,4,5-TTCP 70%
91%
Mixed
65%,
91%
93%

192
Laccase PAN-based beads Covalent BPA 100% 1.5 h NR At 70 °C, the immobilized and soluble [78]
BPB 100 % enzymes kept 80% and 15% of their
BPF 100% original activities, respectively.
TCBPA 96%
Laccase Chemically modified Covalent Phenol 40% 0.5 h NR After 1.5 h and at 60 °C, the free and [131]
polypropylene membrane 2-CP 44% insoluble laccases kept 30% and 65% of
3-CP 42% their original activities, respectively.
4-CP 42%
2,4-DCP 60%
BPA 50%
NLP 42%
3-MEP 38%
4-C-3,5-DMP 24%
2-B-4-CP 42%
4-AAP 12%
Laccase Enzyme aggregates Covalent P353NP (a > 95% 400 min NR NR [132]
branched isomer > 95% 400 min
of NLP) 80% 425 min
TC
BPA
Laccase Glutaraldehyde-modified Covalent 2,4-DCP 88.8% NR At room temperature, the mobile laccase lost its NR [133]
chitosan entire activity after 17 days, while the insoluble
laccase maintained its whole activity during the
same period.
Laccase Silica beads Covalent BPA 90% 1h NR At 70 °C, the remaining activities of the [124]
NLP 30% free and immobilized enzymes after 1 h
TC 50% were 5% and 35%, respectively
Laccase Alginate beads Entrapment Phenol 95% 0.5 h NR NR [134]
Laccase Encapsulation 3h [135]
(continued on next page)
Separation and Purification Technology 219 (2019) 186–207
M. Alshabib and S.A. Onaizi Separation and Purification Technology 219 (2019) 186–207

Bjerkandera adusta with glucose oxidase and then utilized the enzyme

[125]
aggregates for BPA removal from wastewater samples. The authors
Ref

investigated the removal of this phenolic pollutant using batch and

retained 82% of its original activity after

continuous treatment processes. Both treatments resulted in almost

insoluble and free enzyme were > 85%

At 70 °C, the free laccase lost the whole

At 60 °C, the retained activities of the

complete removal of BPA from the polluted water despite the huge

and 43% of their original activities,

activity, while the insoluble laccase

difference in the treatment time.

Although enzyme cross-linking could potentially give reactive ag-
gregates, high amounts of enzymes would be required to produce big
aggregates that can be easily recovered from the reaction medium using
simple separation methods such as filtration or centrifugation.
Thermal Stability

Additionally, enzyme molecules buried within these aggregates might

respectively

not be accessible, rendering these aggregates less effective. These lim-

itations might be overcome by covalent immobilization of the enzyme
of interest to a flat support or within the pores, which must be easily
4h

accessible by the phenolic substrates, of the support. To enable such

immobilized and free laccases were ∼72% and

immobilization, both the enzyme and the insoluble support must pos-
∼16% of the original activity after 6 weeks,

sess reactive functional groups. Nonreactive supports might be acti-

vated using a suitable chemical coupling method [48,70,137]. Fig. 2
At 25 °C, the retained activities of the

illustrates the common covalent immobilization strategies depending

on the functional group of the enzyme and the support.
Xu et al. [138] covalently immobilized HRP on electrospun micro-
fibrous membranes and used it for BPA removal from synthetic was-
tewater samples. The authors reported that the immobilized enzyme
system was able to remove 93% of BPA within 3 h of treatment relative
Storage Stability

to 61% achieved by the free enzyme within the same treatment time.
respectively.

HRP stability has also significantly improved upon immobilization ac-

cording to Xu et al. [138]. Furthermore, Bódalo et al. [129] modified a
NR

glass support with aldehyde and then immobilized both SBP and HRP
on the modified glass support through chemical coupling of the primary
amine group on the enzymes to the aldehyde group of the modified
Treatment

glass. The immobilized enzymes were used to remove 4-CP from syn-
thetic wastewater samples. Interestingly, HRP immobilization has im-
Time

24 h

proved the extent of 4-CP removal relative to the free enzyme while the
immobilized SBP showed lower removal efficiency compare to the
Removal (%)

mobile SBP. The authors have attributed the reduction in the re-
Maximum

mediation efficiency of the immobilized SBP to a possible enzyme de-

99.6%
87.3%

activation during the immobilization process.

59%
40%
60%
98%

In addition to HRP and SPB immobilization, laccase was also im-

mobilized on chitosan by Zhang et al. [133]. Prior to the immobiliza-
tion of laccase, which was obtained from Coriolus versicolor, chitosan
Pollutant(s)

was activatated through the treatment with glutraldehyde. The im-

2,4,6-TCP
2,4-DCP

mobilized enzyme was then utilized for the remediation of 2,4-DCP

SMZ

BPA
CBZ
DIC

from a synthetic wastewater sample with about 89% removal of this

pollutant within 6 h of treatment. The researchers have stated that the
stability of the enzyme was improved upon immobilization. For ex-
ample, immobilized laccase retained more than 50% of its initial ac-
Immobilization

Physisorption

tivity while the mobile laccase became totally inactive after 17 days of
storage at room temperature. The researchers also observed that the
Method

stability of the immobilized enzyme can be tuned with the variations of

glutaraldehyde concentration and cross-linking time. However, despite
the reported improvement in the enzyme stability, the immobilized
Granular activated carbon

enzyme only retained less than 50% of its original activity after six
cycles of use. According to Zhang et al. [133], the decrease in the 2,4-
DCP removal efficiency with use might be a result of the adsorption of
Support/Carrier

the reaction products on the laccase surface. If this is the case, enzyme
activity might be restored with frequent washing [133]. Nonetheless, it
was not demonstrated that such washing would fully restore the ori-
Silica
glass

ginal enzyme activity.

Besides the aforementioned solid supports, PAN beads have been
also used for the covalent immobilization of enzymes. Nicolucci et al.
Table 3 (continued)

[78] utilized PAN beads for the immobilization of laccase, which was
obtained from Trametes versicolor. The immobilized enzyme was, then,
used for the separate removal of BPA, BPB and BPF from synthetic
wastewater samples. The researchers also immobilized another enzyme
Enzyme

Laccase

(tyrosinase) on the same support and used it for the same purpose. It
has been observed that the immobilized laccase almost completely

193
M. Alshabib and S.A. Onaizi Separation and Purification Technology 219 (2019) 186–207

removed, in separate experiments, the three bisphenol components
while tyrosinase only removed 90% of each of these pollutants within
90 min of treatment. Both immobilized enzymes exhibited a good op-
erational stability as they retained more than 80% of their original
activities after storage at 4 °C for 30 days [78]. The removal of 2-CP,
PCP, and 2,4,6-TCP using laccase-PAN beads system was also reported
in the literature [113]. Up to 65% of 2-CP, 91% of PCP, and 93% of
2,4,6-TCP were removed within 90 min of contact between the mixture
of these pollutants (1 mM each) and the immobilized laccase. However,
the removal efficiency of these chlorophenols decreased with increasing
their initial concentrations, which is an obvious limitation. None-
theless, when circulation of the reaction mixture to the reactor was

(a) Aldehyde surface

Enzyme Enzyme
O
N NH

Enzyme NH2 NaBH3CN

(b) Carboxylic acid surface

NH O
Enzyme
N
OH + O N
N O NH
O - O O O
Cl H O

+
N N N
-
Cl H O
N
O
Enzyme NH2
H

(c) Epoxide surface (X is NH for lysine or S for cysteine)

Enzyme
X

O
OH

Enzyme XH

(d) Maleimide surface

Enzyme
S
O
O
O N
O N

Enzyme SH

(e) Amine surface

Enzyme
NH O NH2 O
N
OH + O N
N O NH
O - O O
Cl H O

Enzyme N
+
N N
Enzyme Enzyme
- O O
Cl H N
H

(caption on next page)

194
M. Alshabib and S.A. Onaizi Separation and Purification Technology 219 (2019) 186–207

Fig. 2. Enzyme immobilization onto solid supports with different functional groups. (a) Covalent binding of enzyme molecules to surfaces having exposed aldehyde
functional groups via the formation of imine bond between the enzyme molecules and the support. The unstable imine bond is then converted to a stable amine bond
through the addition of a suitable reducing agent such as sodium cyanoborohydride (NaBH3CN) [202,203]. (b) The covalent immobilization of enzyme to a
carboxylic acid-terminated solid support through the activation of the carboxylic acid group with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride
(EDC), followed by the addition of N-hydroxysuccinimide (NHS) before the introduction of the enzyme. A peptide bond between the primary amine on the enzyme
and the carboxyl of the support is formed, resulting in the covalent attachment of the enzyme to the support. (c) The covalent attachment of enzyme with accessible
XH (where X stands for S of the cysteine or NH of the lysine amino acid residue) to a solid support terminated with an epoxide functional group [203] via nucleophilic
epoxide-ring opening by the primary amino or thiol group of the enzyme. (d) The reaction of the enzyme thiol group (on the enzyme cysteine residue) with the α,β-
unsaturated carboxyl of maleimide-terminated surfaces, leading to the covalent binding of the enzyme to the maleimide-functionalized supports. (e) Supports with a
terminal primary amine can be used to covalently bind enzymes via their carboxylic groups. The carboxylic group of the enzyme is first activated through the addition
of EDC and then NHS, followed by incubating the amine-functionalized supports in the solution of the activated enzyme. (f) Enzymes with accessible primary amine
groups can be covalently bound to supports bearing isothiocyanate functional groups. (g) Supports bearing accessible thiol groups can covalently bind enzymes with
exposed cysteine residue via the formation of disulfide bond between the cysteine and the support thiols [204]. (h) Supports bearing alcohol functional groups can be
utilized to covalently immobilize enzymes via two different routes. In the first route, the hydroxyl group of the support is derivatized with an amino alcohol (e.g.,
ethanolamine) to impart a primary amine on the support; this primary amine covalently binds the enzyme with an activated carboxylic group. In the second route,
the hydroxyl group of the support is derivatized with a component bearing aldehyde groups (e.g., glutaraldehyde) and then the steps in part (a) are followed.

utilized, the remediation level was improved. Another interesting ob-
servation reported in this study is the insensitivity of 2-CP degradation
to the presence of the co-substrate, PCP. Contrarily, the removals of
2,4,6-TCP and PCP were adversely affected by the presence of 2-CP.
Regarding the stability of the immobilized enzyme, it has been reported
that the insoluble laccase maintained 70% of its original activity after
20 days while the mobile laccase lost its entire activity after 12 days of
storage at 4 °C [113].
PAN beads were also utilized by Wang et al. [82] for the covalent
immobilization of HRP; the immobilized enzyme was then utilized for
the removal of phenol from synthetic wastewater samples. The re-
searchers stated that the immobilized HRP had a better activity at a
wider range of pH and showed an excellent stability. A complete re-
moval of phenol was achieved using the immobilized enzyme within 5 h
of treatment when the initial concentration of phenol in the wastewater
sample was 1 mM. The extent of phenol removal, however, dropped to
80% when the initial concentration of phenol was increased to 5 mM
while keeping all other variables unchanged [82]. Such significant drop
in the extent of phenol removal with increasing the pollutant con-
centration is one of the issues that have to be rectified in order to im-
prove the efficacy of the enzymatic wastewater treatment processes.
The treatment time of 5 h is also long and has to be reduced to improve
the feasibility of the enzyme-based wastewater treatment technologies.
Enzyme immobilization has been proved to be a good strategy to
improve enzyme stability. However, most of the above cited studies
reported the positive effect of enzyme immobilization on its storage
stability. Although storage stability is important, immobilized enzymes
should also, ideally, sustain uncompromised activity even when they
are subjected to harsh treatment conditions. Thus, the following two
sections are devoted to discuss the effect of temperature and pH on the
activity and stability (during the reaction time) of immobilized en-
zymes; benchmarking with those of mobile enzymes will be presented.
4. Effect of operating temperature
Operating temperature is one of the most important operational and
economic factors that must be considered in any enzymatic wastewater
treatment process. It is known that the rate constant of enzyme-cata-
lyzed reactions increases with increasing the reaction temperature, re-
sulting in a higher rate of pollutants removal from wastewaters.
However, this positive effect of temperature is counteracted by its ne-
gative effect on enzyme stability since enzymes might undergo some
structural changes, especially at high temperatures, leading to a re-
duction in, or even a complete loss of, their activity [139,140] and,
thus, their effectiveness in removing phenolic pollutants [16,117].
Accordingly, there is always an optimal temperature where the enzyme
activity is the highest, above which thermal deactivation becomes more
pronounced. However, this optimum temperature can be very narrow
or it might span over a relatively wider range depending on the
characteristics of the utilized enzyme, substrate and the conditions of
the reaction medium. Optimal temperature for different enzyme-phe-
nolic substrate systems is summarized in Table 4.
Several studies have reported the effect of reaction temperature on
the removal of phenolic pollutants from wastewaters. For example,
Dehghanifard et al. [117] investigated the effect of temperature on the
removal of 2,4-DNP from a wastewater sample using laccase and ob-
served that the optimal temperature is 40 °C, at which more than 90%
2,4-DNP removal was obtained within 3 h of treatment. However, when
the reaction temperature was increased to 60 °C, 2,4-DNP removal
dropped to 33% within the same treatment duration [117]. Phenol
removal also dropped significantly from more than 96% at the optimal
reaction temperature of 50 °C to about 47% when the temperature of
the laccase-containing reaction medium was raised to 60 °C [104]. Si-
milarly, the extent of BPA removal using laccase dropped from about
90% at 50 °C (the optimal temperature) to less than 50% with 10 °C
increment in the reaction temperature [104]. The same adverse effect
was also observed for phenol removal by peroxidase extract (obtained
from jicama) where the increase in the reaction temperature to above
50 °C (the optimal temperature is in the range of 30–40 °C) led to a
reduction in the extent of phenol removal to less than 50% [96].
Contrarily, a peroxidase derived from mesquite tree showed a good
resistance to thermal deactivation, where the enzyme was able to re-
move 75% of chlorophenols even at high temperature (i.e., 80 °C) [2],
despite that the highest chlorophenol removal (> 90%) was obtained at
25 °C. This finding demonstrates the possibility of producing biocata-
lysts that have reasonably good thermal stability.
A useful approach to improve enzyme thermal stability is through
enzyme immobilization [125]; immobilized enzymes have also shown,
in some cases, a wider optimal temperature range [121,131] as a result
of enhanced enzyme rigidity [131,135]. In this regard, Lloret et al.
[140] assessed the effect of temperature on the catalytic activity of free
and immobilized laccase and reported that the insoluble laccase pos-
sessed a broader temperature optima and a better activity compared to
the free enzyme [140]. Laccase immobilization within nano-porous
cage resulted in an increased thermal stability; such improved stability
has been attributed to the formation of intermolecular stabilizing forces
between the enzyme molecules and the support [140]. Additionally,
laccase immobilization (via physisorption) on a bimodal micro-meso-
porous Zr-metal organic framework has enhanced the enzyme activity
by more than 2-fold over the free laccase [122]. Such mesoporous
support has also promoted enzyme resistance against thermal deacti-
vation and resulted in a higher optimal temperature relative to the
mobile enzyme (40 versus 30 °C) [122]. Improved activity at high
temperatures has been also observed for laccase that has been im-
mobilized on a magnetic bimodal mesoporous carbon via physisorption,
despite that both the free and the immobilized enzymes displayed an
optimal temperature of 45 °C [150]. Covalent immobilization of laccase
on PAN beads has been also reported to enhance enzyme activity [78],

195
M. Alshabib and S.A. Onaizi Separation and Purification Technology 219 (2019) 186–207

(f) Isothiocyanate surface

Enzyme
Enzyme

S NH
S
N NH

Enzyme
Enzyme NH2

(g) Thiol Surface

Enzyme
Enzyme

S
SH S

Enzyme
Enzyme SH

(h) Alcohol Surface

Fig. 2. (continued)

where the immobilized enzyme preserved about 80% of its original
activity even when the reaction temperature was set to 70 °C. Unlike
this remarkable activity of the immobilized enzyme at this harsh tem-
perature, the activity of the free enzyme dropped by about 7-fold [78],
even though the mobile and the insoluble enzymes have the same op-
timal temperature (40 °C). In line with this observation, Songulashvili
et al. [124] reported that immobilized laccase was much more active
than the free enzyme at 80 °C; however, both the free and the anchored
enzymes (covalently bound onto silica beads) showed relatively high
temperature optima of 70 °C.
In the above-mentioned studies, laccases were utilized and the
general observation is that enzyme immobilization enhanced their

196
M. Alshabib and S.A. Onaizi Separation and Purification Technology 219 (2019) 186–207

Table 4
Temperature optima for different mobile and immobilized enzymes utilized for the removal of various phenolic pollutants.
Enzyme Source Mobile/Immobilized Substrate Optimal Temp (0C) Ref

Laccase Trametes versicolor Both 2,4-DNP 40 [117]

Laccase Trametes versicolor Mobile BPA, BPB, BPC, BPE, BPF, BPO, BPT, BPZ 40 [141]
Laccase Trametes versicolor Immobilized BPA, 30 [142]
BPF, 40
Bisphenol S (BPS) 30

Laccase Trametes versicolor Both BPA 40 [78]

Laccase Pycnoporus sanguineus CS43 Mobile BPA 25 [17]
Laccase Trametes versicolor Both Phenol 25 [123]
Laccase Trametes versicolor Both BPA 35 [16]
Laccase Trametes versicolor Both 2,4-DCP 35 (Mob) [143]
60 (Imm)
Laccase Paraconiothyrium variabile (PvL) Mobile Phenol, BPA 50 [104]
Laccase Trametes pubescens Mobile 2-CP, 2,4-DCP, 2,4,6-TCP, PCP 40 [110]
Laccase Coriolus versicolor Immobilized 2-CP, 4-CP 50 [113]
Laccase Coriolus versicolor Both 2,4-DCP 35–45 (Imm) [133]
35 (Mob)
Laccase Trametes versicolor Mobile BPA 40 [141]
Laccase Trametes versicolor Both Phenol, 3-MEP, NLP, BPA, 2-CP, 3-CP, 4-CP, 2,4-DCP, 4-AAP, 2-B-4-CP, 4-C- 40 (Mob) [131]
3,5-DMP 55 (Imm)
Laccase Ascomycete Trichoderma atroviride Mobile 2,6-DMEP 50 [144]
Laccase Trametes versicolor Mobile BPA 22–26 [145]
Laccase Trametes versicolor Immobilized 2,4-DCP 50 [146]
Peroxidase Horse Radish Both Phenol, 4-CP 25 (Mob) [128]
35 (Imm)
Peroxidase Horse Radish Immobilized Phenol 45 [81]
Peroxidase Horse Radish Mobile 2-MEP and other phenolic compounds 28 [85]
Peroxidase Horse Radish Immobilized 2,4-DCP 25 [147]
Peroxidase Horse Radish Both 2,4-DCP 25 [148]
Peroxidase Horse Radish Both 2-CP, 4-CP, 30 [83]
2,4-DCP
Peroxidase Horse Radish Mobile BPA 30 [139]
Peroxidase Horse Radish Both BPA 30 (Mob) [138]
40 (Imm)
Peroxidase Horse Radish Both Phenol 37 [97]
Peroxidase Horse Radish Mobile Phenol 30 [149]
Peroxidase Horse Radish Both 2,4-DCP 25 [147]
Peroxidase Jicama Skin Peels Mobile Phenol 30–40 [96]
Peroxidase Soybean hulls Mobile TC 25 [89]
Peroxidase White Radish (Raphanus sativus) Mobile α-naphthol and other phenolic compounds 40 [93]

thermal stability, and in some cases broadened and raised their optimal
temperature (see Table 4). Similar observation was also reported for
peroxidases. For example, Xu et al. [138] have reported that the op-
timal temperature of the immobilized HRP on electrospun microfibrous
membranes is 40 °C while that of the free HRP is 10 °C lower. Ad-
ditionally, the enzyme immobilization has resulted in a remarkable
improvement in the HRP thermal stability. Zhang et al. [151] have also
observed a significant enhancement in the HRP thermal stability upon
the immobilization onto a nanostructured graphene oxide support.
However, the positive effect of enzyme immobilization on its thermal
stability significantly dropped at relatively high temperatures. For in-
stance, Chang et al. [83] observed that the activity of the immobilized
HRP on a superparamagnetic Fe3O4/graphene oxide nanocomposite
dropped to 40% of the original activity at 70 °C, which is 40 °C above
the optimal value. Similarly, Ai et al. [97] reported that the im-
mobilized HRP on hydrous titanium coatings maintained only 50% of
its original activity when it is exposed to a temperature in the range of
60–90 °C (the highest activity was observed at 37 °C). Nonetheless,
looking into the process from economic and practical perspectives, the
enzymatic wastewater treatment has to be carried out at or close to
room temperature in order to save heating cost of wastewater. Thus, the
reduction in the immobilized enzyme activity at these high tempera-
tures is unlikely to be a serious concern. This proposition is supported
by the reported observations in the literature that operating in the
temperature range of 5–55 °C does not usually lead to a significant re-
duction in the activity of the immobilized oxidoreductive enzymes (e.g.,
peroxidases and laccases) [152].
5. Effect of operating pH
In addition to the effect of temperature, the pH value of the enzyme-
containing medium has also a great effect on the enzyme stability and
activity [145,153]. Accordingly, there is always a pH range (which
might be relatively narrow or wide depending on the characteristics of
the utilized enzyme and substrate) through which the enzyme activity is
at its maxima (i.e., optimal pH). Table 5 illustrates the pH optima for
different enzyme-phenolic substrate systems. The presence of pH op-
tima is related to the degree of the ionization of the amino acids of the
enzyme; such ionization is pH-dependant. Thus, changing the medium
pH might drive the formation/breakdown of some intramolecular ionic
bonds, leading to the alteration of the tertiary structure of the enzyme
molecules. Such alteration could potentially alter the enzyme activity.
For example, it has been reported that the ionic groups of laccase
formed a strong electrostatic repulsion when the pH value was varied,
leading to the destruction and the degeneration of the enzyme active
site and, thus, the enzyme was almost completely inactive in alkaline
media [122]. Furthermore, HRP has undergone a significant activity
loss at low pH values as a result of the chemical modification in the
heme group of the peroxidase [94], most likely due to the physical
changes in the enzyme tertiary structure [85].
Besides the possible alteration of the enzyme ionization state and,
thus, its tertiary structure, the change in pH could also alter the ioni-
zation state of the polar substrates, leading to a significant change in
the enzyme affinity for those substrates [136] (i.e., a change in the
enzyme-substrate interaction). Accordingly, the removal of phenol from

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Table 5
Optimal pH values for the degradation of various phenolic pollutants by different immobilized and free enzymes.
Enzyme Source Mobile/Immobilized Substrate Optimal pH Ref

Laccase Trametes versicolor Both 2,4-DNP 5 [117]

Laccase Trametes versicolor Mobile BPA, BPB, BPC, BPE, BPF, BPO, BPT, BPZ 7 [141]
Laccase Trametes versicolor Immobilized BPA, BPF, BPS 5,5,4 [142]
Laccase Trametes versicolor Both BPA 5 [78]
Laccase Pycnoporus sanguineus CS43 Mobile BPA 5 [17]
Laccase Trametes versicolor Both Phenol 6 (Imm) [123]
5 (Mob)
Laccase Trametes versicolor Both BPA 6 (Mob) [16]
5 (Imm)
Laccase Trametes versicolor Both 2,4-DCP 3 [143]
Laccase Paraconiothyrium variabile (PvL) Mobile Phenol, BPA 5 [104]
Laccase Trametes pubescens Mobile 2-CP, 2,4-DCP, 2,4,6-TCP, PCP 6 [110]
Laccase Coriolus versicolor Immobilized 2-CP, 4-CP 5 [113]
Laccase Coriolus versicolor Both 2,4-DCP 5.5 (Imm) [133]
6 (Mob)
Laccase Trametes versicolor Mobile BPA 7 [141]
Laccase Trametes versicolor Both Phenol, 3-MEP, NLP, BPA, 2-CP, 3-CP, 4-CP, 2,4-DCP, 4-AAP, 2-B-4-CP, 4-C- 4.6 (Mob) [131]
3,5-DMP 5.5 (Imm)
Laccase Ascomycete Trichoderma atroviride Mobile 2,6-DMEP 5 [144]
Laccase Trametes versicolor Both 2, 6-DMEP 6 (Mob) [136]
5 (Imm)
Laccase Trametes versicolor Mobile BPA 6 [145]
Laccase Trametes versicolor Immobilized 2,4-DCP 6 [146]
Peroxidase Soybean hulls Mobile Phenol 6–8 [107]
Peroxidase Horse Radish Both Phenol, 4-CP 7 [128]
Peroxidase Horse Radish Immobilized Phenol 4.2 [81]
Peroxidase Horse Radish Mobile 2-MEP and other phenolic compounds 6.3 [85]
Peroxidase Horse Radish Immobilized 2,4-DCP 8 [147]
Peroxidase Horse Radish Both 2,4-DCP 6 (Mob) [148]
7 (Imm)
Peroxidase Horse Radish Both 2-CP, 4-CP, 2,4-DCP 6.4 [83]
Peroxidase Horse Radish Mobile BPA 6 [139]
Peroxidase Horse Radish Both BPA 4 (Mob) [138]
5 (Imm)
Peroxidase Horse Radish Both Phenol 7 [97]
Peroxidase Horse Radish Mobile Phenol 7.4 [149]
Peroxidase Horse Radish Both 2,4-DCP 6.5 (Mob) 7 (Imm) [147]
Peroxidase Jicama skin peels Mobile Phenol 7 [96]
Peroxidase Soybean hulls Mobile TC 7 [89]
Peroxidase White Radish (Raphanus sativus) Mobile α-naphthol and other phenolic compounds 6.5 [93]

a wastewater sample by peroxidase extracted from jicama skin peels has
dropped from more than 80% at pH 7 (the optimal pH) to less than 20%
at pH 8. Such sharp decrease in the removal efficiency might be linked
to the variations in enzyme conformation induced by protonation and
hydroxylation effects [96]. Similarly, the removal efficiency of α-
naphthol by HRP significantly decreased at low and high pH values
(> 90% at pH 7 (the optimal value); ∼10% at pH 2 and ∼0% at pH
10). The substantial decrease in α-naphthol removal efficiency at pH 2
is correlated to the HRP deactivation due to the release of heme pros-
thetic group from the polypeptide chain of the enzyme. On the other
hand, the null removal of α-naphthol at pH 10 might be attributed to
the formation of α-naphthol conjugated base (the pKa of α-naphthol is
9.38), leading to a negligible binding of the substrate to the enzyme
active site [93].
The negative effect of the medium pH can be mitigated, to certain
extents, via enzyme immobilization. For example, it has been reported
that laccase immobilization onto a granular activated carbon support
enhanced its stability and widened the effective pH range; 3–9 for the
immobilized laccase versus 6–9 for the free enzyme [125]. Similarly,
laccase, which has been encapsulated within a methyltrimethoxysilane-
tetramethoxysilane matrix prepared by the sol-gel technique, showed
an improved stability over a wider pH range (2–8) relative to the free
enzyme, with more than 70% and almost 0% residual activity of the
immobilized and the free enzyme, respectively, upon incubation for 1 h
in an aqueous medium having pH 2 [140]. Moreover, the immobiliza-
tion of laccase onto a bimodal mesoporous Zr-MOF via physisorption
has shifted its optimal pH from 3 to 4, and resulted in a more enzyme
stability [122]. Contrarily, the immobilization of HRP onto Fe3O4/
graphene oxide nanocomposite did not alter its optimal pH (6.4 for both
the free and the immobilized enzymes). Nonetheless, the enzyme im-
mobilization has resulted in a slower rate of enzyme deactivation at
extreme pH values; e.g., the immobilized enzyme retained about 85% of
its original activity at pH 9.4 relative to less than 55% in the case of the
free HRP [83].
In line with the above observation, Xu et al. [138] reported that the
free and the immobilized HRP onto electrospun microfibrous mem-
branes have the same pH optima of 5; however the enzyme im-
mobilization has improved its resistance to pH deactivation (e.g., the
retained activities of the free and the immobilized HRP at pH 3 were 48
and 73%, respectively). Additionally, the immobilization of HRP onto
hydrous titanium coatings preserved about 50% of the original enzyme
activity relative to less than 25% in the case of the mobile enzyme when
these enzymes were stored at pH 3 and 25 °C for 4 h [97]. However,
when these enzymes were stored at pH 7 (the optimal value) and 25 °C
for 4 h, the immobilized enzyme preserved about 85% of its original
activity while the preserved activity of the mobile enzyme was less than
75% [97]. Improved enzyme activity upon immobilization has been
also reported by Zhai et al. [154], who observed that the activity of
HRP in both acidic (pH 4) and alkaline (pH 9) media has been sig-
nificantly boosted upon immobilizing HRP onto chitosan-halloysite
hybrid-nanotubes; the maximum activity was obtained at pH 8 (the
optimal value).

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M. Alshabib and S.A. Onaizi
6. Recent trends in enzymatic treatment of phenolic wastewaters
New research trends in enzymatic wastewater treatment have
evolved in the past few years. One of the interesting research activities
in this regard is the development of nano-sized supports for enzyme
immobilization [117,155–157]. Such supports offer two key advantages
over the traditional supports. The first one is the minimization, if not
the elimination, of diffusion limitation and the second advantage is the
maximization of surface area available for contact between the phenolic
substrates and the immobilized biocatalysts [84,147]. In addition to
enzyme immobilization on nanomaterials, the addition of surface active
agents (i.e., polymers and (bio)surfactants) into the enzymatic reaction
media has been also proposed recently with the aim to enhance the
enzyme activity and stability. Furthermore, mimicking approach to
design materials with enzyme-like catalytic activity but at a lower cost
has emerged as a new attractive trend in enzymatic wastewater treat-
ment. These three trends will be discussed further in the subsequent
sections.
6.1. Nanomaterials for enzyme immobilization
It has been argued that enzyme immobilization on nanomaterials
could make wastewater treatment more feasible and cost-effective
[152]. Accordingly, a number of researchers have immobilized enzymes
on nano-supports (see Table 6) and used them for the remediation of
phenolic pollutants from wastewater. For example, Liu et al. [150]
immobilized laccase, which was obtained from Trametes versicolor, on
carbon-based mesoporous magnetic composite (CMMC) using physi-
soprtion approach. It has been reported that the physically immobilized
enzyme retained 91% of its original activity. The immobilized enzyme
was used to remove phenol and 4-CP from synthetic wastewater sam-
ples. Within 12 h of treatment, 78% of phenol and 84% of 4-CP were
eliminated. It has been reported that the highest removal rate was
obtained in the first hour of treatment and then the removal rate sig-
nificantly decreased afterward. Such observation has been attributed to
the significant contribution of pollutants adsorption on the CMMC be-
sides the enzymatic degradation of the pollutants. After 1 h, the ad-
sorption would have approached equilibrium, making the enzymatic
reaction the main cause of pollutants disappearance. However, with the
progress of reaction time, the immobilized enzyme would experience
higher level of inactivation as a result of the accumulation of enzyme
inhibition products [150]. Such observation demonstrates that the im-
mobilized enzyme does not possesses high stability. One of the possible
reasons is the desorption of the immobilized enzyme to the reaction
medium since the physical bonds holding the enzyme molecules to the
surface of the support are usually week [70].
To overcome such immobilization limitation encountered with
physisorption, and also with other physical immobilization methods,
Dehghanifard et al. [117] used chemical coupling method to covalently
bind laccase, from Trametes versicolor, onto nano-porous silica beads
and, then, used the immobilized enzyme to remove 2,4-dinitrophenol
(2,4-DNP) from synthetic wastewater samples. It was observed that the
maximum removal of 2,4-DNP is 91% within 12 h treatment time at
40 °C. Although this treatment time is still practically long, the im-
mobilized enzyme retained over 85% of its original activity even after
30 cycles of use over a 30-day span with a use duration of 12 h per day.
In comparison, the free enzyme only retained about 15% of its original
activity [117]. Thus, it is obvious that immobilizing laccase on the
nano-support has enhanced both the stability and the reusability of the
enzyme, which are key factors for the applications of heterogeneous
enzymatic processes on a large scale.
Covalent method was also used by Hou et al. [158] in order to
immobilize laccase, from Trametes versicolour, onto titanium nano-
particles, which have been coated on polyvinylidene fluoride (PVDF)
membranes. The immobilized enzyme was, then, used to remove BPA
from synthetic wastewater samples [158]. After 5 h of treatment, 90%
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Separation and Purification Technology 219 (2019) 186–207
BPA removal was achieved when PVDF membrane pore size was 0.1 μm
with a laccase loading of 120 mg/cm2. The removal extent of BPA was
reported to be significantly affected by the membrane pore size and the
coating cycles of the titanium nanoparticles on the membrane; the latter
has direct impact on the level of enzyme loading. The authors stated
that their approach would lead to an optimized enzyme immobilization
on the PVDF membrane surfaces that have been already modified with
nanoparticles, resulting in the improved treatment of wastewaters
contaminated with hazardous pollutants [158]. In another study, Yao
et al. [143] used halloysite nanotubes along with chitosan to prepare
hybrid porous microspheres, which were utilized as support for laccase
immobilization by physical adsorption. This heterogeneous enzymatic
reactor was able to remove 76% of 2,4-DCP initially present in a syn-
thetic wastewater sample in the first 4 h of treatment. Extending the
treatment time to 10 h resulted in 95% removal of this phenolic pol-
lutant [143]. Other researchers, such as Zhai et al. [154], have also
used chitosan-halloysite nanotubes as porous nanocomposites for HRP
covalent immobilization. The results showed that the immobilized en-
zyme was able to remove more than 94% of phenol with an initial
pollutant concentration of 1 mM within 30 min; this value increased to
almost 99% when the pollutant concentration was dropped to 0.5 mM.
The prepared nano-structured enzyme system remained fully active
after 35 days of storage at 4 °C. However, despite this high storage
stability, the immobilized enzyme activity has significantly dropped to
about 60% after 4 cycles of use, demonstrating that the storage stability
and reusability are highly uncorrelated.
One of the interesting nanotechnological approaches for enzyme
immobilization is the use of magnetic supports, which facilitates the
easy recovery of the insoluble biocatalytic materials from the reaction
medium by simply applying a magnetic field [88]. Owing to such im-
portant advantage, Lin et al. [16] prepared metal-ion-chelated magnetic
microspheres and then laccase was immobilized onto these micro-
spheres via physical adsorption. This enzymatically active microspheres
were able to provide more than 85% removal of BPA within 12 h of
treatment. The immobilized enzyme has also shown a reasonable sta-
bility. It has been reported that the immobilized enzyme retained more
than 60% of its original activity after 14 days of storage in phosphate
buffer (pH 5) at 4 °C. Interestingly, the immobilized enzyme stability
has significantly improved when the storage medium contained 10 mg/
L copper ions (i.e., the immobilized enzyme retained 99% of its original
activity after 14 days of storage) [16]. The enhanced laccase stability in
the presence of copper ions has been attributed to the saturation of the
laccase histidine clusters via the formation of stable copper-laccase
complexes. Despite the improved storage stability of laccase in the
presence of copper ions, the authors did not report the remaining ac-
tivity of the immobilized enzyme with the repetitive use, which is also
an important operational factor.
Chang et al. [84] immobilized HRP on a graphene oxide/Fe3O4
support, which has a magnetic character and reported that the insoluble
enzyme preserved 60% of its original activity after four cycles of use.
The immobilized enzyme was used to remove both phenol and 2,4-DCP
from synthetic wastewater samples. The investigators observed that
when these two pollutants were treated separately, 2,4-DCP was com-
pletely removed within 3 h of treatment while only 70% removal of
phenol within the same treatment time was obtained. However, the co-
presence of these two pollutants has led to an increment in the re-
mediation extent of phenol to 94% within 3 h. It is known that laccase
catalyzes the removal of phenolic components through the poly-
merization into large, water-insoluble, molecules. Thus, the improved
removal of phenol in the presence of 2,4-DCP has been correlated to the
reactions between the free radicals generated from both pollutants.
However, there are some studies reporting negative impacts of the
presence of co-pollutants [113,135]. HRP was also immobilized on
amine-modified magnetic Fe3O4/SiO2 nanoparticles; the immobilized
enzyme was used to remediate 2,4-DCP from synthetic wastewater
samples [147]. A remediation of 80% was obtained within 3 h of
 Table 6
Enzyme immobilization on various nano-structured supports and the reported activity and stability of the immobilized enzymes.
Enzyme Support/Carrier Immobilization Pollutant(s) Maximum Treatment time Storage stability Thermal stability Ref
method removal (%)

HRP Electrospun microfibrous Electrospinning BPA 93% 3h At 4 °C and after 18 days, the immobilized and free Both free and immobilized enzymes kept more than [138]
M. Alshabib and S.A. Onaizi

membranes peroxidases kept ∼95% and 25% of their original 90% of their original activities at temperatures
activities, respectively between 30 and 40 °C within 3 h.
HRP Nanostructu-red graphene Physisorption Phenol 64% 0.5 h At 4 °C, the insoluble enzyme kept 56% of its original At 50 °C and after 2 h, the immobilized and soluble [151]
oxide 4-MEP 69% activity while the mobile enzyme lost 88% of its original enzymes had remaining activities of 50% and 20%,
2-MEP 68% activity after 40 days respectively
3-AP 72.7%
Catechol 87.6%
2-CP 20.4%
2,4-DMEP 34.4%
HRP Nano-spray dried ethyl Covalent 2,4-DCP 97.7% 2h At room temperature, the immobilized enzyme kept more NR [159]
cellulose particles than 50% of its original activity after 4 weeks while the
free enzyme became almost inactive.
HRP Graphene oxide/Fe3O4 Covalent Phenol Individual 3h NR NR [84]
nanoparticles 2,4-DCP 70%
100%
Mixed
94%
100%
HRP Amine-modified magnetic Covalent 2,4-DCP 80% 250 min NR At 60 °C, the immobilized and soluble enzymes [147]
Fe3O4/SiO2 nanoparticles preserved 90% and 60% of their original activities,
respectively
HRP Fe3O4/graph-ene oxide Covalent 2-CP 23% 2h At room temperature, the insoluble enzyme maintained At 70 °C, the mobile and immobilized enzymes [83]

200
nanocompos-ite 4-CP 44% about 84% of its original activity within 15 days, while maintained 9% and 40% of their original activities,
2,4-DCP 83% the mobile enzyme had only 30% of its original activity. respectively
HRP Chitosan-halloysite hybrid- Covalent Phenol 98.8% 0.5 h At 4 °C, the insoluble HRP remained almost fully active NR [154]
nanotubes after 35 days, while the mobile one kept only 27% of its
original activity
Laccase Nano-porous silica beads Covalent 2,4-DNP 91% 12 h NR NR [117]
Laccase Titania nanoparticles Covalent BPA ∼100% 24 h At 25 °C, the immobilized enzyme maintained more than At 70 °C, the immobilized enzyme had a retained [115]
CBZ 10% 75% of its original activity after 30 days whereas the free activity of 20% while the mobile enzyme almost
enzyme almost lost the whole activity after 23 days became inactive after 2 h
Laccase PAN nanofibrous membrane Covalent 2,4,6-TCP 87% 4h At 4 °C, the free enzyme lost 80% of its original activity NR [160]
while the insoluble laccase lost only 8% of its original
activity within 18 days.
Laccase TiO2 sol-gel coated Covalent BPA ∼100% 24 h NR NR [158]
polyvinylid-ene fluoride
membrane
Laccase Chitosan/po-ly(vinyl alcohol) Covalent 2,4-DCP 87.6% 6h At room temperature, the immobilized laccase retained At 55 °C, the mobile laccase lost almost 60% of its [146]
composite nanofibrous 60% of its original activity whereas the free laccase initial activity whereas the immobilized laccase
membranes almost became inactive after 10 days kept 57% of its original activity after 3 h
Laccase Chitosan-halloysite hybrid Physisorption 2,4-DCP 95% 10 h NR At 60 °C, the insoluble enzyme maintained ∼42% [143]
porous microspheres of its initial activity while the mobile laccase
maintained 23% after 280 min
Laccase Metal-ion-chelated magnetic Physisorption BPA Chelated with Cu 12 h At 4 °C, the free enzyme lost 83% of its original activity At 65 °C, the retained activity of the immobilized [16]
microspheres (II): 85% while the insoluble laccase lost only 38% of its original enzyme was over 38% of its original value while the
Chelated with activity within 2 weeks. whole activity of the free enzyme was lost.
Mn(II): 88%
Laccase Magnetic mesoporous silica Physisorption Phenol > 90% 40 h NR NR [123]
nanoparticles
Laccase Bimodal carbon-based Physisorption Phenol 4-CP 78% 12 h NR At 60 °C, the insoluble laccase maintained ∼67% of [150]
mesoporous magnetic 84% its original activity while the mobile enzyme almost
composites became inactive after 4.5 h
Separation and Purification Technology 219 (2019) 186–207
M. Alshabib and S.A. Onaizi
treatment. The immobilized peroxidase maintained 85% of its original
activity after four cycles of use, suggesting a reasonably good enzyme
stability [147].
However, most of the aforementioned covalent immobilization
techniques (whether using conventional or nano-sized supports) are
costly, and not straightforward. In some cases, they might negatively
alter the conformation of the enzyme, leading to a decrease in its ac-
tivity [101,119,161]. Additionally, some strategies utilize toxic che-
micals such as glutaraldehyde to activate the support or the enzyme
[100]. To circumvent these limitations, researchers have proposed di-
rect enzyme immobilization methods. For instance, Dey et al. [162]
synthesised a novel scaffold by direct mixing of a supramolecular hy-
drogel and polyphenol oxidase. This non-toxic hydrogel was prepared
via the combination of 2,4,6-tris(4-pyridyl)pyridine and DNA [163].
UV-absorption analysis revealed that the prepared hydrogel enhanced
the oxidation efficiency of phenolic compounds (i.e., phenol, catechol,
resorcinol and quinol) as a result of the orientation of the enzyme active
site within the hydrogel [162].
Another study examined the potential of Hippospongia communis
spongin scaffolds to directly immobilize laccase from Trametes versicolor
with the aim of treating wastewater polluted with bisphenols [142].
The results indicated that the insoluble enzyme, which was stored at
4 °C in acetate buffer (pH 5), possessed an excellent storage stability as
it preserved about 80% of its original activity after 50 days of storage,
relative to less than 20% in the case of the mobile enzyme. The im-
mobilized enzyme was utilized for the treatment of synthetic waste-
water samples (separately) containing 2 mg/mL BPA, BPF or BPS. The
immobilized enzyme completely removed BPA and BPF from the was-
tewater samples. This positive effect of such immobilization technique
could be attributed to the facilitation of the electron transfer between
the bisphenols and the insoluble laccase, rendering it more effective
[142]. However, the removal of BPS was only about 40%, which is an
obvious limitation.
6.2. Surface active additives
Although immobilization usually improves the stability and activity
of enzymes, there are cases (see for instance [15,84,115]) where sig-
nificant loss in enzyme activity is still encountered even with im-
mobilization. Such undesirable loss in enzyme activity might be caused
by the free radical attack on the enzyme and/or via the formation of
enzyme inhibitory polymeric products, in addition to enzyme dena-
turation due to structural changes induced by temperature and/or pH
[87,164]. Thus, some researchers have investigated the potential of
surface active agents (i.e., polymers, surfactants and biosurfactants) in
enhancing enzymatic treatment of phenolic wastewaters via the pre-
vention or at least the minimization of enzyme deactivation caused by
the above mechanisms.
Polymeric additives have been utilized by a number of researchers
to enhance enzymatic remediation of phenolic wastewaters
[73,75,165]. Owing to its low cost [166] and superior effectiveness at
low concentrations [167], polyethylene glycol (PEG) has been widely
utilized as a polymeric additive to enhance the enzymatic remediation
of phenolic wastewaters [1,73,75,139,168]. In this regard, some re-
searchers revealed that the addition of PEG at a concentration of 4 g/L
has lowered the required amount of HRP by 200-fold [87,169]. Other
researchers reported that a 50-fold reduction in the required amount of
laccase was attained upon the addition of PEG to the wastewater
sample containing bisphenols (i.e. BPA, BPB, BPC, BPE, BPF, BPO, BPT,
BPZ) [141]. Additionally, the presence of PEG has resulted in a 50%
reduction in laccase amount required to remove more than 95% 2,4-
DMP (initial concentration ranged from 1 to 5 mM) from a wastewater
sample. The monumental reduction in enzyme dose is mainly correlated
to the interaction of PEG with the free radicals and the formed poly-
meric products, leading to the elimination/reduction of their inhibitory
interaction with the enzyme molecules [75,170].
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Separation and Purification Technology 219 (2019) 186–207
Other polymeric additives have been also utilized for the enhance-
ment of the enzymatic remediation of phenolic wastewaters [94,165].
However, their performance was lower than that of PEG. Nonetheless,
there are other cases where the addition of PEG was not beneficial
[1,91] or even had a negative effect [107]. Such contradicting ob-
servations on the effect of PEG addition (and polymeric additives in
general) pinpoint to the importance of other factors such as enzyme
type and source, phenolic type, characteristics of polymeric additives,
temperature, pH, and the presence of organic/inorganic in the reaction
medium. These factors will play significant roles in the type and the
level of intermolecular interactions between the components present in
the reaction medium and might totally turn PEG addition from being
highly beneficial to detrimental.
Besides polymers, chemical surfactants such as Triton-X100
[4,164,166] have been also utilized with the aim of improving the re-
moval efficiency of phenolic pollutants from wastewaters using en-
zymes. For example, it was reported that the addition of Triton X-100
(125–645 mg/L) has lowered the required amount of SBP by more than
10 folds for attaining more than 95% phenol removal from wastewater
samples [171]. Another nonionic surfactant (Dynol 604) has been also
reported to preserve the activity of recombinant coprinus cinereus per-
oxidase, leading to a sharp increase (i.e. almost double) in the removal
rate of 1-naphthol from a wastewater sample [172]. Such an im-
provement could be linked to the entrapment of phenol molecules
within the surfactant micelle (when surfactant in the reaction medium
is at or its critical micelle concentration) or/and to the formation of
surfactant-product and/or surfactant-phenol insoluble complexes
[3,4,173]. It must be noted, however, that there are cases where the
addition of surfactants was not beneficial [3,172].
Even though the addition of polymers and chemical surfactants had
superior influence on the enzymatic reaction rate as mentioned above,
most of these materials are found to be toxic and harmful to the en-
vironment [174–176]. Additionally, it has been reported that the ad-
dition of polymeric or surfactant additives has resulted in a higher
toxicity of the treated phenolic wastewater [74]. As an alternative to
these additives, biologically produced surfactants (i.e., biosurfactants)
have been proposed as potential alternatives to polymeric and surfac-
tant additives due to their unique characteristics such as biodegrad-
ability [177–179], biocompatibility [178,180], and effectiveness even
at low concentrations [177]. Accordingly, Tonegawa et al. [181] stu-
died the effectiveness of rhamnolipid, which is a glycolipid biosurfac-
tant, in enhancing the enzymatic remediation of 2,4-DCP from waste-
water samples using minced horseradish from Armoracia rusticana and
reported that the addition of this biosurfactant has resulted in 60%
enzymatic remediation enhancement of this phenolic pollutant [181].
In line with this observation, other researchers reported that the same
biosurfactant led to a 4-fold increase in the removal efficiency of phenol
from wastewater using laccase [3]. The same study also compared the
effectiveness of rhamnolipid with two chemical surfactants, namely
sodium dodecyl sulfonate (SDS) and hexadecyltrimethyl ammonium
bromide (CTAB), and reported that unlike rhamnolipid, SDS and CTAB
adversely affected the activity of laccase. This finding is supported by
the reported observation that biosurfactants usually don not inhibit
enzyme activity [182].
6.3. Enzyme mimics “artificial enzymes”
Despite the benefits, even though with some disagreements, of en-
zyme immobilization and surface active addition, the utilization of
enzymes for the remediation of phenolic wastewaters on an industrial
scale is still economically uncompetitive with other wastewater treat-
ment technologies due to the high cost of enzymes. Thus, enzyme mi-
micking approach has emerged recently with the aim to develop ma-
terials that possess enzyme-like features (i.e., artificial enzymes
[183,184]) but are less sensitivity to severe conditions, have high sta-
bility, and are cheaper than enzymes [185]. Iron oxides [186,187],
M. Alshabib and S.A. Onaizi
biogenic magnetic nanoparticles [185], hemes [188], platinum alloys
[189,190], and nucleotides [191,192] have been successfully exploited
as enzyme mimicking materials with a potential utilization for phenolic
wastewater treatment.
Owing to their low cost, ease of fabrication, and high thermal sta-
bility [187], ferromagnetic nanoparticles have received more attention
for mimicking peroxidases. For instance, Wu et al. [186] synthesised a
composite of graphene quantum dots and Fe3O4 nanoparticles and
tested their ability to remove a mixture of phenol, methoxyphenols,
aminophenols, and chlorolphenols from a synthetic wastewater sample.
Comparing the performance of the composite to that of HRP, the au-
thors reported that the composite was more effective [186]. Similar
observation was also reported by Zhang et al. [187] where the ferro-
magnetic nanoparticles outperformed HRP, particularly at higher pol-
lutant concentrations. For example, at an initial phenol concentration of
12 mM, the composite about three times more active than the enzyme
(i.e., phenol removal extents by HRP and the composite were about
30% and 80%, respectively [187]. The superiority of the composite
might be linked to the generation of reactive oxygen in the presence of
hydrogen peroxide [190], leading to a higher phenol oxidation by the
composite. Unlike the composite, deactivation of the enzyme molecules
by the produced free radicals and/or the reaction products, in addition
to the inhibitory effect of H2O2, might have also contributed to the
lower phenol removal by HRP at higher phenol concentration (note:
both catalysts provided comparable phenol removal when the initial
pollutant concentration was 2 mM).
Mimicking the nature of laccases for the same purpose has been also
studied in the literature. Recently, a facile technique to prepare laccase
mimics with the aid of guanosine monophosphate (GMP) coordinated
copper has been proposed by Liang et al. [191]. The authors revealed
that such a mimic could sustain harsh conditions and maintain excellent
operational and storage stabilities. The superior characteristics of Cu/
GMP rendered it more efficient than laccase at oxidizing various phe-
nols [191]. Similar finding has been reported in another study, where
DNA-stabilized Pt nanoparticles outperformed laccase in terms of cat-
alytic activity and stability [192].
Despite the superior activity and stability of artificial enzymes, their
application for treating industrial wastewaters could be limited by their
toxicity as well as their low selectivity [184]. Therefore, it is imperative
to design smart materials having the potential to circumvent these is-
sues. Additionally, despite the promising results in terms of activity and
stability of the “artificial enzymes”, the limited number of published
studies in this regard makes it difficult to draw a solid conclusion on the
effectiveness of these materials in treating pollutants other than those in
the above-mentioned studies. Furthermore, the presence of organic/
inorganic co-pollutants is likely to negatively impact the performance
of these enzyme mimicking materials. Thus, thorough studies that
tackle these issues and others (i.e., durability, regenerability and re-
cyclability/disposability) are highly needed.
7. Unresolved Challenges, concluding remarks and future
outlooks
Enzymatic treatment of phenolic wastewaters is a promising tech-
nology. One of the attractive characteristics of such a method is the
utilization of sustainable and environmentally friendly materials (i.e.,
enzymes). Another attractiveness of utilizing enzymes for phenolic
pollutants removal from wastewaters is the potential effectiveness of
these biocatalysts even at very low concentrations (on a nano-molar
level). The fact that enzymes are biodegradable eliminates the concern
of secondary pollution encountered with some other treatment
methods. Additionally, the nontoxicity and harmless nature of these
biocatalysts are among the desirable characteristics.
However, despite the potential effectiveness of enzymes at low
concentrations, enzymatic wastewater treatment process is still ex-
pensive to the point that it is still not competitive with the currently
202
Separation and Purification Technology 219 (2019) 186–207
utilized methods. Most of the enzymes reported in the literature for
phenolic wastewater treatment are still not commercially available,
making the adaptation of large scale enzymatic wastewater treatment
processes a real technical and economic challenge. Thus, bulk produc-
tion of enzymes is highly and urgently needed in order to make these
enzymes commercially available and economically affordable.
Although there is a great potential to produce enzymes on a large scale
and at a lower cost using recombinant-DNA technology [71], it is still
not demonstrated industrially for most of the enzymes. Even if re-
combinant-DNA technology (or any other prospect technologies)
eventually proved effective for enzyme production, separation and
purification of produced enzymes will still be a bottleneck unless
breakthroughs in this regard are achieved. Despite that pure enzymes
are not required for enzymatic wastewater treatment from economic
perspectives, a certain level of purity is still required for technical issues
(e.g., crude enzymes with unknown and highly variable compositions
will make the operation and its control a big challenge). Thus, extensive
and carefully designed work for improving enzyme production and
purification is one of the immediate important requirements for making
enzymatic wastewater treatment a viable option in the near future.
One of the other serious limitations of utilizing enzymes for phe-
nolic wastewater treatment is the loss of enzyme activity. In some cases,
particularly at unfavored conditions, incubating the enzyme in an
aqueous medium for less than a day results in a huge reduction in its
activity [78,117]. The presence of inhibitory components in the enzy-
matic reaction medium might render the enzyme ineffective. Real
wastewaters contain many organic and inorganic components that
might affect enzyme activity. For instance, Jiang et al. [193] reported
that kaolin, a suspended solid particle, inhibited HRP. Although the
effect of suspended solid particles might be eliminated through the
removal of these inorganic materials using some pre-treatment methods
such as flocculation [194,195], coagulation [196,197], and/or filtration
[198], as is currently the case with biological wastewater treatment, it
is more desirable to develop enzymes that are tolerant to the presence
of potentially inhibitory components in order to eliminate the cost and
the environmental impact of the pre-treatment process(es). Tolerance to
extreme values of pH is also required in order to enable the enzymatic
treatment of acidic and alkaline phenolic wastewaters without bearing
extra cost for pH adjustment. Enzyme mutation (i.e., engineering)
might be successful in developing enzymes with superior character-
istics. However, such enzyme engineering technology is still expensive
and not very mature. Work has to be done in this regard with focusing
not only on modifying the enzyme structure but also on taking into
consideration the effectiveness of the engineered enzymes under severe
conditions. Besides enzyme mutation (or even microbes mutation),
high-throughput screening for microbial strains that can produce su-
perior enzymes in large quantities should also be extensively explored.
Besides the research work concentrating on developing enzymes
with exceptional properties, well-planned research on enzyme im-
mobilization has to be also approached. Despite that there is extensive
work on enzyme immobilization and utilization for the removal of
phenolic components from wastewaters, the conclusions derived from
such studies on the effect of enzyme immobilization on its stability and
activity are different and even contradictory in some cases. For in-
stance, some researchers demonstrated that heterogeneous are more
efficient than homogeneous enzymatic reaction systems in remediating
phenolic pollutants from wastewaters [16,151] while other researchers
reported the opposite trend [127,148]. Part of the problem might stem
from the fact that almost no two studies are conducted under exactly
the same conditions (e.g., enzyme type and source, enzyme con-
centration, substrate type and concentration, treatment conditions, re-
action time, etc.). It is highly recommended that such a huge variability
is minimized in order to obtain a meaningful benchmarking of the ef-
fectiveness of a given enzyme on a given phenolic substrate. Once the
best source of the enzyme to remove that phenolic pollutant is identi-
fied, a deep look (on a molecular level) into what makes such an
M. Alshabib and S.A. Onaizi
enzyme more effective than others should follow. By doing this, we
might develop a clear understanding of the interaction of a given en-
zyme-pollutant system and, thus, be able to use mimicking approaches
to select the most suitable enzyme(s) for remediating a given phenolic
pollutant on a priori informed basis. With accumulating knowledge in
this regard, it might be eventually possible to develop enzymes with a
very broad and effective catalytic function. It might also be possible to
track the influential enzyme-phenolic pollutant interactions on a mo-
lecular level and build models that are capable of predicting the effects
of external factors (e.g., temperature, pH, the presence of co-pollutants,
the presence of inhibitory components, etc.) on such interactions. Then,
tuning these interactions in a way that enable the effective simulta-
neous removal of several phenolic pollutants would likely be possible.
Another factor that worth investigation in future work is the optimal
distance between the immobilized enzyme molecules on/within a solid
support. Steric hindrance, particularly in the case of large phenolic
substrates, might significantly affect the extent and the rate of phenolics
removal. Thus, careful design of enzyme immobilization with pre-de-
termined distance between the immobilized enzyme molecules could
potentially lead to an effective wastewater treatment process through
not only the elimination of potential steric hindrance but also through
the utilization of the exact amount of the required enzyme (most of the
conducted studies so far dump huge enzyme quantities to the support
without any clear idea of how much of the added enzyme would be
functional). Immobilizing enzyme in this manner with an optimal
spacing distance can be achieved through using supports with reactive
functional groups (used to covalently attach the enzyme to the support)
that are sufficiently spaced-apart using a short, inert molecule [48,199].
Another approach to minimize/eliminate the hindrance effect is
through the use of flexible spacers (e.g., PEG). These spacers are mo-
lecules with a skeleton that is very easy to twist and bend. Attaching
one end of these flexible spacers to the solid support and the other end
to the enzyme molecule will allow a huge freedom in the enzyme mo-
bility while still ensuring the easy recovery and reuse of such im-
mobilized enzyme. Enzyme mobility at interfaces has been reported to
be a crucial factor in heterogeneous biocatalytic reactions [48,49,200].
With a flexible enzyme mobility at the solid support and wastewater,
the anchored enzyme molecules will easily encounter phenolic sub-
strates, overcoming mass transfer limitation (if any). Furthermore, en-
zyme movement will enhance the detachment of molecules that might
bind to the enzyme; such binding might be long-lasting if the enzyme is
confined within the support or bound to the support surface through
stiff spacer molecules such as polymethyl methacrylate and polyvinyl
chloride [70].
In some cases, enzyme immobilization might result in a partial or
complete loss of the enzyme activity. This is because reagents used
during enzyme immobilization might destroy or block the active site of
the enzyme. To avoid such a scenario, harsh immobilization conditions
have to be replaced with mild ones. Reagents that may destroy/block
the enzyme active site have to be replaced with other less damaging
molecules. However, if such replacement is impossible or more ex-
pensive, a reversible blockage of the enzyme active site during the
enzyme immobilization is a good alternative; the blocking component is
released from the enzyme active site afterwards.
Another important consideration is that the enzyme active site
should not be buried upon immobilization. Contrarily, the active site
must be clearly and, without obstacles, exposed to the reaction medium
to facilitate the interaction between the immobilized enzyme molecules
and phenolic substrates. In order to achieve this configuration, directed
enzyme immobilization has to be carefully adopted. In such an im-
mobilization, the reaction between the reactive functional group of the
solid support is guided to take place with a suitable functional group on
the enzyme molecule that is at the longest distance from the active site.
If such a functional group is not available at the desired location on the
enzyme molecule, it might be installed through a suitable chemical
coupling method [137,201,202]. However, care should be taken in
203
Separation and Purification Technology 219 (2019) 186–207
order to make sure that such an enzyme modification will not result in a
negative effect on the enzyme activity and stability. With a clear un-
derstanding of the enzyme-phenolic substrate interactions mentioned
above, it might be possible to easily predict whether such a modifica-
tion will harm the enzyme or not. Additionally, with such under-
standing, it might be possible to design the desired enzyme modifica-
tion without compromising the enzyme stability and activity.
Although the emerging trends (particularly, the addition of surface
active components and the enzyme mimicking approach) in treating
phenolic wastewaters have the potential to improve wastewater treat-
ment, they are still surrounded by a number of challenges that have to
be addressed. Surface active additives should not cause secondary
pollution or add economic burden to the process. Mimicking approach,
on the other hand, should be robust, environmentally friendly, techni-
cally sound, and economically feasible. These desirable characteristics
are yet to be established.
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