UNIVERSITI TUN HUSSEIN ONN MALAYSIA

IMMOBILISED ENZYME IN WASTE WATER TREATMENT

ASSIGNMENT
SEMESTER II
SESSION 2018/2019

GROUP MEMBERS NAME : ASWINI PURUSHOTHANAN (AN160118)

JIVASHINI BALASUPUR MANIAM (AN160185)
NURUL AIMI FARHANA BINTI AZHARI
(AN160009)
NUR SYHUADA BT ABD MALIK (DN160306)

COURSE NAME : FERMENTATION ENGINEERING

TECHNOLOGY

COURSE CODE : BNN 30304

PROGRAMME : 3 BNN

SUBMISSION DATE : MAY 2019

DURATION : 4 WEEKS

LECTURER’S NAME : Ts. Dr. SITY AISHAH BINTI MANSUR

TABLE OF CONTENT

CONTENT PAGE

1.0 INTRODUCTION TO IMMOBILIZED ENZYME 3-4

2.0 BACKGROUND OF THE INDUSTRY INDUSTRY 5-6

3.0 CONVENTIONAL FERMENTATION METHOD, PLANT AND

6-12
BIOREACTOR DESIGN AND CHALLENGES

4.0 APPLICATION OF IMMOBILISED ENZYME 12-17

5.0 ADVANTAGES AND LIMITATIONS OF APPLICATION OF

17-18
IMMOBILIZED PEROXIDASE FOR PHENOL

6.0 SUGGESTIONS AND RECOMMENDATIONS 19-20

7.0 CONCLUSION 20

8.0 REFERENCES 21-22

1.0 INTRODUCTION TO IMMOBILISED ENZYME

Generally enzymes are referred as biocatalyst that encourage the alteration of during a
chemical reaction. Catayst does not take part in the reaction instead they help to speed up the
reaction. Thousands of atoms are arranged in a precise manner that allow them to be able to
synthesis of the various chemical reaction in biological cells. Besides that, they have the
ability to synthesize reaction at a very mild condition but with a high degree of specificity of
the substrate. By this, production of by product can be reduced. Some enzymes comes with a
high cost of isolation and relatively unstable. Technically, recovery of active enzyme when
from the solution mixture of reaction after use is hard.
Catalytic reaction of enzyme involves three different states that are, appearing as an
individual molecule, as a aggregation or as attachment to a surface. The attached surface are
also known as immobilization state.
Immobilized enzyme defined by the enzyme that is confined physically and localized in a
specific surface region for the retentivity of their catalytic reaction. Immobilized enzymes are
recyclable and can be used repeatedly. They can be used in a various industrial application
processes and as biosensors. The first usage of immobilized enzyme Aspergillus Cryzae
Aminoacylase was done by Chibata and co-workers in late 1967. The immobilized was
developed from the resolution of synthetic racemic D-L amino acids.

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 At first, only single immobilized enzymes were used however in the late 1970s, the
immobilization development becomes complex that includes co-factor regeneration with two
enzymatic reaction and living cells. The major part of the immobilized enzymes are its matrix
and the mode of attachment.
The enzymes can be attached to the support through interactions ranging from reversible
physical adsorption and ionic linkage to stable covalent bonds. Catalytic reaction and thermal
stability are some of the properties that can be altered as a result of enzyme immobilization.
Stabilization was an important motor for enzyme immobilization.

The performance of immobilized enzyme important for the selection of the reactor and they
depends on the characteristics of the matrix and. The ideal support properties includes
hydrophilicity, resistivity to physical compression, low cost, resistance to microbial attack and
biocompatibility. Support can be divided into organic and inorganic depending on their
chemical composition.
Irreversible and reversible methods are the classification of various approach to
immobilization enzymes. Irreversible method refers to the irreversible detach of biocatalyst to
the support without destroying either the biological activity of the enzyme of the support.
Covalent coupling, cross linking, entrapment and microcapsulation are the most common
procedures irreversible enzyme immobilization. While for reversible method of the
immobilization enzyme, the detachment of the enzyme from the support are reversible and the
most common procedure are disulfite bond, adsorption, ionic binding, chelation and affinity
binding.

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2.0 BACKGROUND OF THE INDUSTRY AND PRODUCT DESCRIPTION
(IMMOBILIZED ENZYME IN WASTE WATER TREATMENT)

With the raising population over the last century, water pollution has becoming an important
issue that need to be addressed soon. The disposal of large amount of toxic waste into the
water bodies is one of the major causes of water pollution.
In this research, we are focusing on synthetic dyes and phenol that are highly toxic and
visible that is one of the main contributor of the environmental problems. The dyes are from
textile, food, pharmaceuticals and cosmetic industries while phenols are from petrol refining,
metal coating, plastic and paper manufacturing indu. With the rapid evolution of this type of
industries, environmental problems seems to be increasing as well. The chemicals in the
synthetic dyes are harmful to he aquatic life as the disturbing the food chain by interrupting
the penetration of light. Hence, the discharge of highly toxic coloured waste water can effect
the biodiversity of aquatic life and ecosystem. As for humans, exposure to synthetic dyes can
cause risky side effects to human like skin irritation, allergy and might as well cause
malfunctioning of organs. On other hand, the phenol even at its lowest concentration posses
significant health impact to animals and humans. Chronic exposure to phenolic compound can
cause damage to nervous system and kidney.
There are various methods of waste water treatment depending on the types of industry.
The most common methods are physio-chemical membrane separation, and biological
treatment. Compared to other methods, biological treatment method can be considered cost
effective, environmentally friendly and executable method. This approach uses microbes,
bcateria by the process of aerobic, anaerobic or combination of both processes. However,
these method has their own disadvantages like production of sludge, failure to treat high
concentrated pollutants, high maintenance, low rate of microbial degradation.

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 Recently, enzymatic treatment in treating waste water has caught attention. It is
considered the simplest and effective method of treating polluted water. In terms of high
specificities and selectivity, environmental friendliness and biodegradability, the enzymatic
approach has better potential than traditional methods. It also works with a wide range of pH,
temperature, enzyme and substratum levels. Contrary to chemical catalysts, enzymes have
high efficiency in the conversion under mild responsiveness of complex chemical structure.

3.0 CONVENTIONAL METHOD OF WASTEWATER TREATMENT AND

CHALLENGES

Wastewater treatment is vital as it determines the cleanliness of our water source and prevents
pollution. As such, many ways are developed in order to treat wastewater especially from
industries in order to improve water quality before discharge. There was always conventional
wastewater treatment prior to the advancement nowadays. They consist of biological,
chemical and physical operations to remove organic matter and solids.

3.1 GENERAL METHOD

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PRELIMINARY TREATMENT

The first phase functions mainly to eliminate coarse solids and other materials that are larger
in size from wastewater. This is vital to improve the effectiveness of operation and
maintenance of involved equipment. Grit chambers, coarse screening and occasionally
reduction of big sized objects are operations included in this phase of treatment.
Grit chambers or bar screens are to prevent settling of organic solids with the water
velocity passing through it kept at a high rate. In some cases, comminutors are used to
enhance coarse screening by minimizing the larger particles in order to enable them to be
removed as sludge later.

PRIMARY TREATMENT

In order to eliminate inorganic and organic solids that are settleable by sedimentation, primary
treatment was carried out. Then, via skimming, the materials need to be removed would float
as scum. Usually, about 70% total suspended solids (TSS), 50% and 65% of biochemical
oxygen demand (BOD) and oil or grease respectively would be eliminated. Besides, certain
organic nitrogen, phosphorus and heavy metals too are eliminated in this phase. The effluent
from this treatment is known as primary effluent.
Primary treatment can be said as the least preapplication treatment level needed for
wastewater irrigation. But some might consider that it is sufficient if only the specific
wastewater was used for watering crops that will not be consumed by human. On the other
hand, some believe that secondary treatment is vital to avert potential harm.
Primary sedimentation tanks or primary clarifiers are often installed in this phase where
solids (sludge) that are settleable, eliminated usually from the tanks’ bottom to a central well
by sludge rakes. The sludge would be later pumped into sludge processing units. In larger
scale, primary sludge is normally processed via anaerobic digestion which is a biological
method, where anaerobic bacteria metabolizes the organic material in sludge and reduce
ultimate volume needed for clearance, making the sludge stable. This part of phase is carried
out in anaerobic digesters where the main challenge is that the residence time in it varies form
minimum 10 days to 60 days. Methanosarcina sp. and Lactobacillus sp. are examples of
bacteria involved.
The pros of anaerobic process is that electrochemical equipment is not needed. However,
production of hydrogen sulfide and methane gas during the process can be hazardous.

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SECONDARY TREATMENT

The main aim is to extra purify the primary treatment effluent to eradicate organics residuals
and suspended solids. It is done via an aerobic biological treatment processes which requires
the presence of oxygen for the microbes involves metabolizing the organic particles in
wastewater. However, this would lead to production of more inorganic end-products and
microorganisms especially, H2O, NH3 and CO2. Microbes that are normally involved in this
step is Amoeba proteus and Vorticella sp.
Biological processes of high rate are dependent on quite small reactor volumes and high
microbe concentration compared with processes of low rate. Thus, the growth rate of new
organisms in the high-rate systems are greater because of the well-controlled environment.
The challenge here is that the microbes used must be parted from wastewater treatment by
sedimentation to produce a clear secondary effluent which is found to be a little difficult
process. Sludge formed here are removed to combine with the primary sludge.

Activated sludge process and trickling filter are examples of a high rate processes.

(i) Activated Sludge Process

Aerobic microbes are used to produce a liquid that is free from suspended solids or organic
materials. The main two instruments involved in this process is:

8
 The boxes below show the activated sludge overall process.

(ii) Trickling Filter

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Trickling biofilter is an equipment that is equipped with support media (Ex: stones).

The general working steps for this biofilter is as follows:

In short, trickling filter is for separation and disposal of the slough. The banes of this
trickling filter are that it requires forced air blowers and a walled environment. For pros, it
saves space in the plant and has a high-performance reliability. Processing parts of this filter
are also durable.
Aerobic bacteria are advantageous as they possess the ability to remove a higher
percentage of organic substances. However, the main challenge is that there must be a
continuous maintenance and oversight of the operator due to the mechanical nature of aerobic
digestion process.

TERTIARY TREATMENT

Tertiary treatment was used to remove specific wastewater content that are hard to be
eliminated after the secondary process. Often, they are carried out collectively with primary
and secondary treatments. As such, in phenol removal for instance, adsorption process is used
which is also known as one of the conventional steps.

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3.2 ADSORPTION (PHENOL)

Adsorption can be known as one of the suitable techniques used to remove phenol from
wastewater due to its own boons. This process basically involves build up of pollutant on
adsorbent’s surface. The adsorbent chosen must be appropriate and porous with bigger surface
area, increased ability to selectively collect pollutants from water onto its surface and
advanced hydrophobicity. (Anku, W. et al. 2017)
Adsorbents play major role in determining the efficiency of this method. Thus, many
researchers are continuously studying various type of adsorbent such as activated carbon that
can be used for phenol removal. Figure 1 below displays the basic steps involved in
adsorption of pollutants from water. (Mohammadi, S., et al. 2014)

Adsorption process also depends on certain properties. As such, phenol adsorption

process depends on pollutant speciation and concentration initially, which sequentially
depends on the solution’s pH. Figure 2 below shows the other relatable properties that
governs it.

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 Each method has its own advantages and disadvantages. As such, adsorption was
considered to be one of the suitable techniques for phenol removal from wastewater as it has a
simple design and operation. It also does not produce toxic wastes. The spent sorbent serves
as a fuel source to provide power. On the other hand, adsorption with high cost activated
carbon can only be used to purify low phenol concentrated wastewater due to their low
adsorptive capability. (Anku, W. et al. 2017)
Even though conventional methods are still used its undesired properties, expensive cost,
low stability and efficiency and incomplete purification causes trouble. Thus, new advanced
method with improved technology should be employed.

4.0 APPLICATION OF IMMOBILISED ENZYME TECHNOLOGY

The ineffective of conventional method lead to the discovering of other alernative that more
effective for the removal of phenol in waste water treatment. The enzymatic treatment been
seen as the potential alternative for the waste water application. This approach has more
advantages in aspect of high specificity and selectivity, biodegradability and environmental
friendly (F. Gholami-Borujeni et. al, 2011). The enzymatic treatment mentioned are also
enhanced due to few factor till the immobilised enzyme treatment are developed. In phenol
removal, there are several reseacher reported the use of immobilised peroxidase in wastewater
treatment. For example, Vasileva et al. has studied the immobilization of Horseradish
peroxidase (HRP) onto modified ultrafiltration membrane of acrylonitrile (AN) co-polymer in
treatment of phenol from the wastewater (Vasileva et al. 2009).

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4.1 Type of Immobilization

Suitable immobilization technique on suitable support material is compulsory so that it can be

fully works towards the goals to remove phenol in wastewater treatment. The selection of
immobilization technique and support material play a major role in term to optimize the
catalytic activity of an enzymes. here are several parameters which can affect the catalytic
activity of an immobilized enzyme as well as the operating conditions
In this study, immobilized HRP are covalently attached on the improved ultrafiltration
membrane of acrylonitrile (AN) co-polymer with the presence of glutaldehyde which create
the covalent bond between the amino groups on the surface of the AN membrane and the
HRP. This technique basically create a strong bonding between enzyme and support or carrier
which increase the resistance toward harsh operating condition. This technique also avoid the
enzyme from leak as well as avoid desorption from occur which allow it to be used for batch
and continuous processes.

Figure 4.1 The illustration of enzyme covalently attached to the support

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4.3 Type of Support

The support used in this study are modified membrane of AN co-polymer that act as an
excellent carrier for immobilized HRP. The modified membrane are prepared from 91.3 % of
acrylonitrile co-polymer, 7.3 % of methylmethacrylate and 1.4 % of sodium vinylsulfonate
(Vasileva et al. 2009). This excellent support evidently enhanced the enzymatic activity in a
shorter time in 45 minutes, have better operational and thermal stability as well as have up to
95.4 % of phenol removal in wastewater.

Table 4.1 Comparison of immobilization of peroxidases on various support for phenol

removal (Jun LY et al. 2019)

Reaction Optimum
Enzyme Optimum temperature Phenol
Support time
sources pH removal (%)
(min) (°C)

Modified AN
Horseradish 45 5.0 30 95.4
copolymer
Poly
Horseradish 120 7.0 25-35 86
GMA-MMA
Horseradish Eupergit® C 240 7.0 20 92
Modified
Horseradish 200 7.0 37 99.9
PAN bead
Magnetic
Horseradish - 7.0 37 85.2
nanofiber
Horseradish PVA-PA.Am 180 7.0 50 29.68

4.4 Type of Enzyme Used

The type of enzyme used in this phenol removal application are peroxidase. Peroxidase have
the potential to remove phenol and the aromatic amines as following reason (Jun LY et al.
2019);
i) The enzymes treat the waste include the diluted waste efficiently as it highly selective.

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ii) The cost involved might be lower if the commercially available enzymes produced in bulk
quantities as well as have lower risk to be inhibited by the toxic substance.
iii) The enzymes also can works in a wide range of concentration and need lower retention
time compared to free enzymes.
Peroxidase can be categorized into two category which animal peroxidase and plant
peroxidase. Under the main category plat peroxidase there are three sub-category which based
on its primary structures and origins. The subcategory are Class I from the intracellular
peroxidases, Class II from the extracellular fungal peroxidase and lastly Class III from the
secretory plant peroxidase such as Soy Bean peroxidase, Turnip peroxidase, Ginger
peroxidase and the one that used in this phenol removal treatment is Horseradish peroxidase
(HRP) (Vineh B. M. et al, 2018).
The HRP used in this phenol treatment are extracted from horseradish roots (Armoracia
rusticana. HRP catalyzed the oxidation reaction of substrates such as aromatic amines and
phenols by using an oxidant agent which commonly H2O2 (Lavery et al. 2010, Zhang et al.
2014, Feng et al. 2015).

HRP
Substrate (reduced) + H2O2 Substrate (oxidized) + H2O

There are some study that state that the optimum pH of free HRP is 7.0, and some study
state the HRP exhibit maximum activity at pH 5.0 and pH 6.0. From Vasileva research, the
optimum pH condition for immobilized HRP are slightly increase to 5.0 compared to free
HRP enzyme which is only 4.2. This increment might occur due charge of carboxyl groups
that present in the AN co-polymer after processed with sodium hydroxide, that do not form
any bond to ethylenediamine and HRP. The hydrogen ion are attracted to the membrane
surface with the present of carboxyl group which generate higher pH in the solution (Vasileva
et al. 2009). The graph below shows that immobilized HRP had the maximum activity at

30 °C and still have higher activity compared to free enzymes at higher temperature, 60 °C.

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 Figure 4.2 the enzyme activity of (1) free HRP and (2) immobilized HRP on improved
membrane of AN co-polymer against temperature.

4.5 Mechanism of The Reaction Process

According to Vasileva studies, the enzyme activity of immoblized HRP only decreases after
20 days. The immobilized enzyme been evaluated in aspect of operational stability by
pressing the model solution that have the concentration of phenol through the matrix of
immobilized HRP using the ultrafiltration cell for 10 days. In that interval time, there are no
significant of any enzyme activity reduced or changed. The volume of model solution in the
operating stability evaluation is around 230 cm3. The inactivation of enzymes activity only
been observed after the 20th day of the evaluation which is up to 50 % of the enzyme activity
(Vasileva et al. 2009). The inactivation might be happen via the stabilization of phenoxy
radical which limit the substrate or phenol diffusion to the active site (Bodalo et al. 2006).

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 Figure 4.3 The degree of phenol oxidation on the minimum phenol concentration with the
presence of (1) free HRP and (2) immobilized HRP on improved AN co-polymer.

The figure 4.3 represent the result of phenol oxidation on different type of enzymes. The
degree of phenol oxidation at the lowest concentration of phenol are much higher than the
degree of phenol oxidation at the higher concentration for enzymes. The degree of phenol
oxidation in was different for different concentration of phenol contained.

5.0 THE ADVANTAGES OF APPLICATION OF IMMOBILIZED PEROXIDASE

FOR PHENOL

The current application has practiced in the immobilization of Horseradish peroxidase

(HRP) onto the improved ultrafiltration membranes of acrylonitrile (AN) copolymer in the
application of phenol removal from waste water treatment. There are several advantages that
create good impact towards efficiency of the related process. According to a research, the
acrylonitrile (AN)copolymer can assist as a good carrier for immobilized HRP as it performs
great operational and thermal stability (Bayramoglu, 2008) . Moreover, it can intensify the
activity of catalytic enzyme with highest phenol removal up to 95.40% (Bayramoglu, 2008).
The immobilize enzyme can work in extensive range of pH and temperature while
maintaining its stability. So, the immobilized HRP can boost up the phenol removal efficiency
up to 86% under best and optimum condition of 35°C, at pH 7 within 2 hours (Bayramoglu,
2008) . In addition, the immobilization of turnip peroxidase (TP) for phenol removal has
demonstrate that two different methods of turnip peroxidase have been visualized and being
differentiate (Duarte-Vázquez, 2008) . So, the first technique is entrapment and the other one
is by covalent bonding. The results show that immobilized TP can reach the removal of

17
phenol up to 90% (Duarte-Vázquez, 2008).For instance, the entrapment method of TP showed
higher phenol removal efficiency rather than the covalent bonding of immobilized TP.
After all, the enzymatic approach has a better prospect because of high specificity and
selectivity, environmental and its biodegradability properties. Besides that, the immobilized
enzyme is a protein that do not need any extensive treatment and it will naturally degrade.
Lastly, immobilized enzymes are immediately available for reuse properties and it allows for
continuous process and production of product is much faster, energy saving and gain more
profit.

5.1 THE DISADVANTAGES OF APPLICATION OF IMMOBILIZED

PEROXIDASE FOR PHENOL

From the economical point of view, the immobilized enzyme required expensive cost to run
the process. The immobilization of turnip peroxidase (TP) for phenol removal by using
covalent bonding method has its limitations too. The chemical modification of enzyme can go
further to functional conformation loss. The covalent binding may alter the conformational
structure and active center of enzyme, resulting in major loss of activity or changes of the
substrate. However, this can be solved through enzyme immobilization with the formation
enzyme substrate or the competitive inhibitor.
Next, the second method for the immobilization of turnip peroxidase (TP) for phenol
removal is called entrapment. This method has its disadvantages too as the enzyme may lead
to leakage and has limited pore diffusion at certain times. Other than that, any contamination
is costly to deal with because the whole system would need to be stopped and new process set
up would take long time to be set again.

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6.0 SUGGESTIONS AND RECOMMENDATIONS

Over the years, immobilized enzyme treatment has been widely used for production of many
products that provides impressive impact in managing the wastewater that enclose with
phenol compounds rather than the traditional conventional methods used before. The enzyme
immobilization technique has gained importance due to their environmental and economic
benefits. The previous technology has been constantly improvised in order to maximize its
productivity and efficiency. So, new technology has been developed to improve the current
waste water treatment for phenol removal.
First of all, nanobiotechnology is one of the advance technologies which has ongoing
rapid growth and it has been continuously improvised in the evolution of nanostructure
materials as a new support material for enzyme immobilization. Nano particles has many
benefits as a promising support material due to their large surface to volume ratios, highly
porous surface and hollow structures (Mubarak, 2019) . Some studies had emphasized on the
enzyme immobilization on Nano-structured materials which includes metal nanoparticles,
graphene oxide, carbon nanomaterials and Nano-sized polymer beads in waste water
treatment applications.
For instance, a study revealed that the immobilization of Horseradish peroxidase
(HRP) on Nano-composite support in removing the Acid Blue 113 and Acid Black 10BX
(Mubarak, 2019) . It gives great impact on efficiency of catalyst, the thermal stability and
operating stability rather than the normal free enzymes. The result of the experiment
illustrates that the maximum removal of Acid Blue 113 and Acid Black 10BX by using
immobilized HRP were 95.4% and 90.3% respectively, at pH 7 and 30°C within 35 minutes
(Mubarak, 2019) . Other than that, the immobilized HRP can be used again and has most
decolorization efficiency of 79.4% for Acid Blue 113 and 71.1% Acid Black 10BX at the 12th
cycle of reuse (Mubarak, 2019). Consequently, nanostructure materials were chosen as a new
support material for enzyme immobilization regarding to its significant cost effective,
increased in enzymatic activity and it can provide intensification in stability against pH
imbalance and temperature.
Furthermore, magnetic nanocomposite has been selected and chosen as a new support
material to carried out the enzyme immobilization in many industry and treatment of waste
water as it can provide easy separation process, great bio-compatibility, secure and smooth

19
recovery and reusable (Mubarak, 2019). Moreover, these types of advanced Nano-biocatalyst
have shown an improvement on the catalytic activity and thermal stability rather than the free
soluble enzyme used before in conventional method.
Some researchers demonstrated the practicability of the Horseradish peroxidase (HRP)
which is covalently immobilized on magnetic iron oxide (Fe3O4) followed by graphene oxide
magnetic composite. It reveals that the highest removal chrono phenol efficiency up to 84%
and can be acquired under optimum conditions which consist of pH 6.4 with temperature of
30°C and H2O2/chrono phenol with molar ratio of 1.0. Next, the recovered immobilized (HRP)
can be reused and hold on up to 66% of its initial activity in repeated cycles. To sum up, the
features of the magnetic nanocomposite can functionally separate the enzyme and
contaminants quickly by put in an external magnetic field (Mubarak, 2019) . After all, the
magnetic nanocomposite support material is acceptable and capable for big scale continuous
practices as it is recyclable and can be reused for many times.

7.0 CONCLUSION

Overall, immobilized enzymes method in of treating waste water containing phenol is reliable
and promising technique. The immobilized enzyme has significantly improved catalytic
efficiency, superior stocking and operating stability as well as the recovery and reusability of
enzymes as compared with free enzymes. The catalytic activity of an immobilized enzyme
was determined by the choice of materials, immobilization techniques, operational conditions,
especially pH, reaction time, temperature, and concentrations of the enzymes and substrates.
Immobilized Horseradish peroxidase in wastewater treatment can be considered as a
breakthrough for industrial waste water treatment. The usage of Horseradish peroxidase (HRP)
immobilized enzyme can work in extensive range of pH and temperature while maintaining
its stability. The main disadvantage is that immobilized enzymes are very expensive.

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8.0 REFERENCES

Anku, W. W., Mamo, M. A., & Govender, P. P. (2017). Phenolic Compounds in Water
Sources, Reactivity, Toxicity and Treatment Methods. Phenolic Compounds - Natural
Sources, Importance and Applications. doi:10.5772/66927

Ayramoglu, G. (2008). Enzymatic removal of phenol and p-chlorophenol in enzyme reactor:

Horseradish peroxidase immobilized on magnetic beads. 148-155.

M. Besharati Vineh, A. A. Saboury, A. A. Poostchi, and L. Mamani, Physical Adsorption of

Horseradish Peroxidase on Reduced Graphene Oxide Nanosheets Functionalized by
Amine: A Good System for Biodegradation of High Phenol Concentration in Wastewater,
International Journal of Environmental Research. 12 (2018) 45-57.

Bodalo A, Gomez JL, Gomez E et al (2006) Comparison of commercial peroxidases for

removing phenol from water solutions. Chemosphere 63:626–632

Duarte-Vázquez, A. (2008). Polyethylene glycol improves phenol removal by immobilized

turnip peroxidase. Bioresource Technology, 8605-8611.

Feng D, Liu T-F, Su J, Bosch M, Wei Z, Wan W, Yung D, Chen YP, Wang X, Wang K, Lian
X, Yuan Gu Z, Zou JPX, Zhou H-C (2015) Stable metal-organic frameworks containing
singlemolecule traps for enzyme encapsulation. Nat Commun 6 Article number: 5979

F. Gholami-Borujeni, A. H. Mahvi, S. Nasseri, M. A. Faramarzi, R. Nabizadeh, and M.

Alimohammadi, Enzymatic Treatment and Detoxification of Acid Orange 7 from Textile
Wastewater, Applied Biochemistry and Biotechnology. 165 (2011) 1274-1284.

Jun LY,Yon LS, Mubarak NM, Bing CH, Pan S, Danquah MK, Abdullah EC, Khalid M, An
Overview of Immobilized Enzyme Technologies for Dye, Phaenolic Removal
fromWastewater, Journal of Environmental Chemical Engineering (2019)

Lavery CB, MacInnis MC, MacDonald MJ, Williams JB, Spencer CA, Burke AA, Irwin DJG,
D’Cunha GB (2010) Purification of peroxidase from horseradish (Armoracia rusticana)
Roots. J Agric Food Chem 58:8471–8476

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Mohammadi, S., Kargari, A., Sanaeepur, H., Abbassian, K., Najafi, A., & Mofarrah, E.
(2014). Phenol removal from industrial wastewaters: a short review. Desalination and
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Removal from Wastewater. 59.

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Vasileva, T. Godjevargova, D. Ivanova, and K. Gabrovska, Application of Immobilized

horseradish peroxidase onto modified acrylonitrile copolymer membrane in removing of
phenol from water, International Journal of Biological Macromolecules. 44 (2009)
190-194.

Zhang T, Xu X-L, Jin Y-N, Wu J, Xu Z-K (2014) Immobilization of horseradish peroxidase

(HRP) on polyimide nanofibers blending with carbon nanotubes. J Mol Catal B Enzyme
106:56–62

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