Phenol removal from wastewater using waste products

Journal Pre-proof Phenol Removal from Wastewater using Waste Products Deyala M. Naguib, Nahla M. Badawy

PII:

S2213-3437(19)30715-8

DOI:

https://doi.org/10.1016/j.jece.2019.103592

Reference:

JECE 103592

To appear in:

Journal of Environmental Chemical Engineering

Received Date:

25 September 2019

Revised Date:

30 November 2019

Accepted Date:

3 December 2019

Please cite this article as: Naguib DM, Badawy NM, Phenol Removal from Wastewater using Waste Products, Journal of Environmental Chemical Engineering (2019), doi: https://doi.org/10.1016/j.jece.2019.103592

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Phenol Removal from Wastewater using Waste Products

By Deyala M. Naguib1,2 and Nahla M. Badawy3,4 1- Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig, Egypt 2- Biology Department, Faculty of Science and Arts in Qilwah, Baha University,

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Qelwah, KSA

3- Chemistry Department, Faculty of Engineering, Ain Shams University, Cairo, Egypt.

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4- Chemistry Department, Faculty of Science and Arts in Qilwah, Baha University,

E-mail: [email protected]

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Tel. +201113632143

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Qelwah, KSA

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Abstract:

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ORCID: 0000-0001-7164-7838

Phenols are very dangerous contaminants found in wastewater. Discharge of these

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compounds without treatment is highly serious risks to living organisms. The present study offers new effective immobilized peroxidase for phenol removal from wastewater. Nano peroxidase particles were prepared through immobilization of peroxidase enzyme extracted from onion dry scales (one of the wastes in food industry) on Silica nano particles prepared from rice straw (one of the most spread agriculture wastes). Immobilization improved the physiochemical properties of the 1

enzyme which allowed the immobilized enzyme to be more resistant and stable under various hard conditions such as high temperature, pH changes or presence of metal ions. The nano peroxidase retained about 77.8% of its activity after incubation at 80ºC. The affinity between the nano peroxidase and its substrate increased about 2.3 fold more than that of the free one. Peroxidase nanoparticle retained 90% of residual activity till 50th reuse cycle. Thus, the method applied in this study increased stability and reusability of peroxidase, and therefore, it can be used for efficient phenol

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removal from wastewater.

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Keywords: Immobilization; Nano-Peroxidase; Nano-silica; Phenols

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1. Introduction:

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Water shortage problem has been increased all over the world. The reuse of the wastewater has become an urgent solution to overcome this problem. Phenols are one of the most dangerous pollutants in the wastewater because of its high toxicity even at

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low concentrations. Phenols reach to surface and ground water through the continuous release of these compounds from petrochemical, coal conversion and phenol

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producing industries. Therefore, the wastewaters containing phenolic compounds

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must be treated before their discharging into the water streams [1]. The amount of phenol present in various industrial effluents is given in “Table 1”. The phenol removal from waste water is highly useful not only for removal its toxicity, but also its removal decrease the biological oxygen demand (BOD) and the chemical oxygen demand (COD). The COD and BOD concentrations play an important role in the re-

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use of these waste effluents, so there is urgent need to minimize these factors in waste water before discharging it into a water stream [2]. Many different treatment technologies for the removal of phenol from the wastewater such as flocculation, adsorption, chemical oxidation, biological process, etc. are available. One of these methods is enzymatic methods considered to be effective solution for removal of phenolic compounds from wastewater. One of the most important advantages for using enzymatic methods is its higher catalytic efficiency

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and lower cost than that in the traditional chemical methods. However, there are some problems facing the usage of the enzymatic methods, such as the non- reusability of the enzymes and their instability in harsh environment of the wastewater [14].

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Immobilization can be a solution to overcome these problems.

Enzyme immobilization is fixing enzymes to or within solid supports stabilizing their

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structure; hence become more resistant to environmental changes [15].

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The most important part in immobilization is identifying new matrix materials with suitable structural characteristics, such as morphology and surface functionality. Using nano-particles as matrix materials for enzyme immobilization has a great

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attention now days. Recently, carbon nanotubes, nano sized polymer beads, metal and metal oxides nanoparticles are used in enzyme immobilization [16]. Immobilization of

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enzyme on nano materials protects enzyme from leaching [15], loss its 3D structure

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[17], and gives the enzyme strong diffusion resistance. Nano materials have large surface area which results in more efficient immobilization than that on macromaterials [18]. The use of nano materials not only offers advantages such as large surface area, increased mechanical strength, and effective enzyme loading, but also exhibits high catalytic efficiency [19-21].

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Peroxidases are a heme-containing enzymes. It possesses significant applications in life sciences, including bioassays, DNA-probes, biosensors, bioremediation of phenol and some of its derivatives are used in wastewater treatment [22]. However, its industrial application is greatly limited due to its low thermostability and low reactivity in hard environment [23]. Peroxidase immobilization can improve its physiochemical characters so it enhances its industrial application [24]. The present study aims to remove phenols from wastewater using highly efficient and

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very low costs way. The treatment methods which utilize agricultural and industrial wastes in a simple manner have become an urgent need [25]. Many studies were conducted trying to test the removal and filtration capacity of waste and natural

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indigenous materials as treatment mediums e.g., shell, limestone, waste paper mixed with refuse concrete, refuse cement, also processed nitrolite, charcoal-bio and

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charcoal [26]. Chakraborty et al. [27] showed a novel low cost proton exchange

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membrane (PEM) synthesized, using biochar derived from food wastes. Similarly Das et al [28] used orange and banana peels to treat anaerobic sludge. From this point of view we achieved our aim in this study. We remove phenols from the wastewater

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using peroxidase extracted from onion dry scale (one of the most available wastes in food industry) and for enhancement of the onion dry scale peroxidase activity, it is

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immobilized on nano-silica particles prepared from rice straw (one of the most

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agriculture wastes).

2. Material and Methods: 2.1. Onion dry scales peroxidase and Silica nanoparticles (Si2O3): Onion dry scales peroxidase (ODSP) was purified from Onion dry scales as reported in Mohamed et al. [29]. Onion dry scales (100g) were juiced with 10ml 50mM Tris–HCl buffer, pH 7.2 then filtered through four layers of polyamide tissue.

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The filtrate was centrifuged at 12000g for 10min, and then the supernatant was precipitated by solid ammonium sulphate up to 75% saturation. The precipitate was collected by centrifugation at 12,000g for 20min and dissolved in a 5ml of 50mM Tris–HCl buffer, pH 7.2, and dialyzed against the same buffer overnight. The dialysate was centrifuged at 15,000g for 20min. The supernatant was purified on a DEAE-Sepharose column (with 100ml of resin), using 20mM Tris–HCl (pH 8.0), at a flow rate of 60ml/h for elution. The eluted fraction was used as the peroxidase

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source.

Silica nanoparticles were prepared from rice straw according to the procedure of

Adam and Fua [30] as following: A sample of dried rice straw (30g) was weighed and

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transferred into a plastic container, then 600mL of 1.0M HNO3 was added into the

container and stirred for 24h to remove unwanted metal. The acid treated rice straw

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was filtered and washed with distilled water to reach a constant pH (4.5–5.0) and

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dried in oven at 60°C overnight. The cleaned rice straw was stirred in 500mL of 1.0M NaOH for 24h. The mixture was filtered to obtain sodium silicate. The prepared

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sodium silicate was titrated slowly with 3.0M HNO3 till pH 9.0. The yellowish gel produced was left in a covered container for 2days. The gel was then centrifuged at

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1500g and followed by washing with copious amount of distilled water. It was dried in an oven at 60°C for 24h. Finally, the product was ground into fine powder for

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characterization.

2.2. Peroxidase assay Peroxidase activity was determined according to Upadhyaya et al. [30]. The assay mixture (5ml) containing 300µM of phosphate buffer (pH 6.8), 50µM pyrogallol, 50µM H2O2 and 1mL of prepared enzyme extract, then incubation at 25ºC for 5min, the reaction was stopped by the addition of 1mL 10% H2SO4. The absorbance of the

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produced color was measured at 430nm and the specific enzyme activity was expressed as the change in the optical density/mg protein/min. 2.3. Immobilization procedure Enzyme immobilization was performed by adsorption on nano-silica. Onion dry scale peroxidase solution was added to the prepared nano-silica at room temperature for different incubation periods. Aliquots of the supernatant were drawn, and the silica nanoparticles were dried at room temperature to verify the progress of immobilization

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and called peroxidase nanoparticles. We tested the effect of different concentration of ODSP, pH and incubation period on the immobilization process

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Immobilization yield of peroxidase nanoparticle for each peroxidase concentration was calculated by Huang et al. [32] according to the following equation:

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IY % = PB / PU ×100

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where IY is immobilization yield, PU is Protein used for immobilization, and PB is Protein bound to a nanoparticle; the amount of bound proteins were evaluated indirectly by measuring the quantity of protein remaining in the supernatant by

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Lowry’s method [33].

The immobilization efficiency % was calculated from the following formula:

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Immobilization efficiency % = Activity of immobilized enzyme/Initial activity of

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soluble enzyme×100

2.4. Structure characterization The average particle size, size distribution and morphology of nano silica and nano peroxidase were examined using transmission electron microscope (JEM-1400 TEM, JEOL Inc., USA). The Fourier-transform infrared spectroscopy (FTIR) spectra of

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samples (Nano-silica and immobilized peroxidase) were obtained on a PerkinElmer spectrum 100 FT-IR spectrometer. 2.5. Physicochemical characterization of the enzyme Kinetic studies were performed using 20 different concentrations (0.005-0.1µM the difference between each concentration is 0.005µM) of pyrogallol and H2O2 as substrates. The Km was calculated from the Lineweaver–Burk double reciprocal plots. The optimal temperature and pH for soluble and immobilized ODSP were determined

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by incubating the enzyme in pH ranging from 3.0 to 10.0 and temperature ranging from 20°C to 95°C. The thermal stability was determined by incubating the enzymes at 6 different temperatures (50-100°C) for 15min before performing the enzyme

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assay. The activity of the non-thermal treated was taken as 100%. The effect of

different metal ions was determined by incubating the enzymes at 5mM metal ion

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treated enzyme was taken as 100%.

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solution for 15min before performing the enzyme assay. The activity of the non-

The relative activity (%) was calculated according to the following equation:

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Activity of the treated enzyme × 100 Activity of non_treated enzyme 2.6. Application of the free and immobilized peroxidase for Phenol removal from

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wastewater:

Separating funnel (22L capacity) was half filled with the prepared nano-peroxidase

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after closing the separating funnel with cotton piece to retain the nano-peroxidase. Wastewater (10L of real effluent from dye factory (Mustafa For Master Batch in Abo Helal Industrial Complex, Qalyub, Qalyubia, Egypt), with pH 8.9, was loaded on the funnel with H2O2 and then funnel was opened to let the wastewater pass through the funnel in velocity 5ml/ min. the phenol concentration was measured in the initial wastewater and in the treated wastewater by the folin Ciocalteu assay [34]. The 7

loaded funnel with nano peroxidase was reused for 50 times to test the reusability of the immobilized peroxidase. Soluble onion dry scales peroxidase was tested for removal of phenol from the wastewater as 20mL of the assay mixture containing 5ml 50µM H2O2 and 5ml of prepared enzyme extract and 10ml wastewater. After incubation at 25 ºC for 10min, the reaction was stopped by the addition of 1mL 10% H2SO4. Then the amount of phenols in the solution was measured.

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Enzyme efficiency for phenol removal (%) = Phenol content in wastewater after enzymetreatment × 100 Phenol content in wastewater before enzyme treatment Statistical analysis

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All results were analyzed by SPSS software (version 14). Data was expressed as mean

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± SD. Comparison of mean values of the soluble and the immobilized ODSP was

3. Results and discussion:

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done using paired T test. P<0.05 was considered to be significant [35].

3.1. Peroxidase immobilization on nano-silica optimization:

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The results of the present study indicated that optimum peroxidase concentration for the most effective immobilization was about 100µg/ml. The optimum pH for

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maximum immobilization efficiency was about 7, and incubated for 18 hrs in room temperature “Figure 1”.

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As illustrated in “Figure 2”, TEM analysis revealed that the synthesized particle ranged between 50 and 70 nm in size with overall spherical shape. The TEM image of the immobilized peroxidase on the nano silica (nano peroxidase) ensured that the anosilica particles are loaded with the peroxidase enzyme. Surface chemistry of the nano-silica and the immobilized peroxidase on nano-silica was studied by FTIR (Fourier-transform infrared spectroscopy) “Figure 3”. The 8

results indicated that the characteristic peaks for nano-silica were at 1070, 960 and 540 cm-1. These peaks are attributed to ν (Si–O–Si), ν (Si–OH) [36]. The nanoperoxidase showed other different peaks. The peak at 3400 cm-1 may be to the asymmetric stretching modes of -NH2 groups [37]. The peak at 3100 cm-1 is due to ν(C–H). The peak at 1650 cm-1 may correspond to Amide I, the most intense absorption band in proteins. It is primarily governed by the stretching vibrations of the C = O and C-N groups [38]. The band at 1400 cm-1 is attributed to δ(C–H) which can

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be attributed to any organic residues, Si–OR or Si–R) [36]. This spectroscopic analysis ensured the effective immobilization of peroxidase onto the surface of nanosilica support.

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3.2. Effect of immobilization on physicochemical characterization of the enzyme: The Michaelis–Menten constant (Km) of free and immobilized peroxidase for H2O2

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were 0.04 and 0.0154 mM, respectively and for pyrogallol were 0.01735 and 0.0076

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mM, respectively. This indicated that immobilization improved the enzyme affinity toward the H2O2 by about 2.6-fold and the enzyme affinity toward the pyrogallol by about 2.3-fold more than that of the free enzyme “Figure 4”. This improvement is

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mostly due to high mass transport of reactants and products within the carrier which is a unique property of the nano-particles as immobilization matrix, beside that nano-

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particles are characterized by the Brownian movement which enhances substrate-

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enzyme interaction [39].

The maximum velocity (Vmax) of immobilized peroxidase for catalytic reaction on H2O2 and pyrogallol was increased by about 2.09 and 2.5-fold, respectively more than that of the free one. The explanation for this increase in the immobilized peroxidase is that nano peroxidase has more efficient conformation than that of free enzyme. The

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high affinity toward substrate and maximum velocity of nano-peroxidase ensured the efficiency and effectiveness of using nano-silica as immobilization support [40]. Results in “Figure 5” indicate that immobilized peroxidase activity was significantly higher than the free peroxidase both in acidic or alkaline pH. Immobilization shifted the optimum pH towards the alkalinity as it increased from 7 in the free enzyme to be 7.5 in the immobilized enzyme. This shift in pH as a result of absorbing the carrier (nano-silica) the H+ from the reactive solution to its surface so pH in the surrounding

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of the immobilized enzyme decreased than that of free enzyme and to protect the high activity of the enzyme, this area pH should be increased to a certain limit [41,42].

Similar results were obtained by Mohamed et al. [24] who reported that the pH shifted

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from 7.0 for soluble horse radish peroxidase to 7.5 for the peroxidase immobilized on magnetic nanoparticles, which retained significantly higher enzyme activity both in

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acidic and alkaline pH compared to the soluble enzyme.

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The present study showed that immobilized peroxidase has higher optimum temperature than that of the free one. This shift in the optimum temperature of the immobilized enzyme towards higher temperatures is explained as immobilization

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decreases the free movement of the enzyme molecules at higher temperatures which protects the amino acids at the active site as well as on the surface of the enzyme at

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higher temperatures. Beside that with temperature increases, the kinetic energy of the

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substrate molecules increase and so can reach the active site of the immobilized enzyme rapidly [43]. This shift in the optimum temperature of immobilized enzymes towards higher temperatures is well documented in previous works [17, 18, 21, 24, 42, 44]. Beside that immobilization improved the thermal stability of peroxidase. The relative activity of immobilized peroxidase was about 77.8% after incubation at 80ºC, while

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the relative activity of the free peroxidase was 0% after incubation at the same temperature “Figure 5”. The thermal stability of the immobilized enzyme is attributed to the multipoint covalent attachment of enzyme to the immobilized matrix which protects the tertiary structure of the enzyme at high temperature [42, 44]. The present study showed that immobilization on nano-silica protected the peroxidase from the inhibitory effect of the metal ions “Table 2”. Protection of immobilized peroxidase against such inhibitors could be explained in two different ways: (i) In

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case of allosteric inhibitor, immobilization of the enzyme leads to distortion of the allosteric site and subsequently causes significant reduction of the inhibition; (ii) If

the inhibitor interacts with the active center, it was found that enzyme immobilization

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may cause a slight change in the enzyme active site configuration that affects the

inhibitor binding to enzyme with less effect on the substrate-enzyme binding [45].

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3.3. Application of the free and immobilized peroxidase for Phenol removal from

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wastewater:

Peroxidase can be used to remove phenols, which have been labeled as “priority pollutants” by the US Environmental Protection Agency, from contaminated water.

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The immobilized enzymes can be used repeatedly in a variety of reactors [46]. The results of the present study showed that onion dry scale peroxidase immobilized on

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nano-silica was more efficient in phenol removal from the industrial wastewater than

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the free one. The efficiency of the nano-peroxidase was about 99.92%, while that of the free peroxidase was about 66.4%. Beside that the immobilized peroxidase can be reused for many cycles with high efficiency about 90% in the 50th cycle “Table 3”. In comparison with other previous works the current study can be considered the highest efficient method in the phenol removal from wastewater till now “supplementary Table”.

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Conclusion: This study investigated the efficiency of new prepared nano peroxidase in phenol removal from wastewater. The nano peroxidase was prepared by the immobilization of peroxidase (extracted from onion dry scale extract) on nano silica particles (prepared from rice straw). The immobilization enhanced the physiochemical properties of the enzyme. Immobilization gave the enzyme high stability under harsh conditions such high temperature, change in the pH, or the presence of toxic metals.

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The nano peroxidase was highly efficient in phenol removal from waste water and its reusability was highly efficient as it retained 90% of its efficiency for phenol removal from wastewater after 50 reuse cycles. The present research article offered cheap and

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effective solution for getting rid of many environmental pollutants (Rice straw; one of the agricultural wastes, onion dry scale; one of the food industry wastes and phenol in

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wastewater).

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Declarations Statements:

Ethics approval and consent to participate Not applicable

Not applicable

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Consent for publication

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Competing interests

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We declare that we have no competing interests. Funding

This paper is self-funding and we did not take any fund from any organization or person. Acknowledgements

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Dr. Deyala M Naguib would like to show her great gratitude to her supervisor Professor Hegazy Sadik Hegazy, professor of physiology, for his effort, time and

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patience giving to her in her life and work.

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References: [1] B.K. Sonawane, S.R. Korake, Review on Removal of Phenol from Wastewater Using Low Cost Adsorbent. Inter. J. Sci. Eng. Tech. Res. 5(2016) 2249-2253. http://ijsetr.org/wp-content/uploads/2016/06/IJSETR-VOL-5-ISSUE-6-22492253.pdf. [2] A. S. Al-Jlil, COD and BOD Reduction of Domestic Wastewater using Activated

473-477. DOI: 10.3923/biotech.2009.473.477

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Sludge, Sand Filters and Activated Carbon in Saudi Arabia. Biotech. 8 (2009)

[3] D.A.D.A. Aljuboury, P. Palaniandy, H.B. Abdul Aziz, S. Feroz, Treatment of

-p

petroleum wastewater by conventional and new technologies - A review. Global NEST J. 19 (2017) 439-452. DOI: https://doi.org/10.30955/gnj.002239

re

[4] N. Ghimire, Sh. Wang, Biological Treatment of Petrochemical Wastewater, in: M.

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Zoveidavianpoor (Eds.), Petroleum Chemicals - Recent Insight, IntechOpen, 2018, pp. 55-74. http://dx.doi.org/10.5772/intechopen.79655. [5] D.A. Yaseen, M. Scholz, Textile dye wastewater characteristics and constituents

na

of synthetic effluents: a critical review. Int. J. Environ. Sci. Tech. 16 (2019) 11931226. https://doi.org/10.1007/s13762-018-2130-z.

ur

[6] N.M. Sivaram, D. Barik, Toxic Waste from Leather Industries, in: D. Barik (Eds.),

Jo

Energy from Toxic Organic Waste for Heat and Power Generation. Wood head Publishing, 2019, pp. 55-67. https://doi.org/10.1016/B978-0-08-102528-4.00005-5.

[7] Y.N. Dhoble, S. Ahmed, Treatment of wastewater generated from coke oven by adsorption on steelmaking slag and its effect on cementitious properties. Cur. Sci. 116 (2019) 134-1355. https://www.currentscience.ac.in/Volumes/116/08/1314.pdf

14

[8] H. Gai, X. Zhang, Sh. Chen, M. Xiao, T. Huang, J. Wang, H. Song (In press) An improved tar–water separation process of low–rank coal conversion wastewater for increasing the tar yield and reducing the oil content in wastewater. Chem. Eng. J. 123229. https://doi.org/10.1016/j.cej.2019.123229 [9] M. Massoudinejad, M. Mehdipour-Rabori, M.H. Dehghani, Treatment of Natural Rubber Industry Wastewater through a Combination of Physicochemical and Ozonation Processes. J. Adv. Environ. Health Res. 3(2015) 242-9.

ro of

[10] N.K. Swamy, P. Singh, I.P. Sarethy, Precipitation of phenols from paper industry wastewater using ferric chloride. Rasayan J. Chem. 4 (2011) 452-456. https://rasayanjournal.co.in/vol-4/issue-2/34.pdf

-p

[11] A. Akyol, B. Samuk, M. Kobya, E. Demirbas (In Press) Treatment of phenol

formaldehyde production wastewater by electrooxidation-electrofenton successive

re

processes. Sep Sci Tech. DOI:10.1080/01496395.2019.1645173.

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[12] N.V. Pradeep, S. Anupama, K. Navya, H.N. Shalini, M. Idris, U.S. Hampannavar, Biological removal of phenol from wastewaters: a mini review. Appl. Water Sci. 5 (2015) 105. https://doi.org/10.1007/s13201-014-0176-8.

na

[13] M.A. Aboulhassan, S. Souabi, A. Yaacoubi, M. Baudu, Treatment of Paint Manufacturing Wastewater by The Combination of Chemical and Biological

ur

Processes. Inter. J. Sci. Env. Tech. 3 (2014) 1747 – 1758.

Jo

http://www.ijset.net/journal/416.pdf. [14] L.A. Dahili, T. Feczkó, Cross-linking of Horseradish Peroxidase Enzyme to Fine Particles Generated by Nano Spray Dryer B-90 Period. Polytech. Chem. Eng. 59 (2015) 209–214. https://doi.org/10.3311/PPch.7590

15

[15] S. Avvakumova, M. Colombo, P. Tortora, D. Prosperi, Biotechnological approaches toward nanoparticle biofunctionalization. Trends Biotech. 32 (2014) 11–20. doi: 10.1016/j.tibtech.2013.09.006. [16] E.P. Cipolatti, M.J.A. Silva, M. Klein, V. Feddern, M.M.C. Feltes, J.V. Oliveira, Ninow J.L., D. de Oliveira, Current status and trends in enzymatic nanoimmobilization. J. Mol. Catal. B Enzy. 99 (2014) 56–67. https://doi.org/10.1016/j.molcatb.2013.10.019

ro of

[17] A. Mukhopadhyay, A.K. Dasgupta, K. Chakrabarti, Enhanced functionality and stabilization of a cold active laccase using nanotechnology based activation immobilization. Bioresour. Tech. 179 (2015) 573–584.

-p

https://doi.org/10.1016/j.biortech.2014.12.070

[18] V. Lettera, C. Pezzella, P. Cicatiello, A. Piscitelli, V.G. Giacobelli, E. Galano, A.

re

Amoresano, G. Sannia, Efficient immobilization of a fungal laccase and its

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exploitation in fruit juice clarification. Food Chem. 196 (2016) 1272–1278. https://doi.org/10.1016/j.foodchem.2015.10.074 [19] J.P. da Costa, R. Oliveira-Silva, A.L. Daniel-da-Silva, R. Vitorino,

na

Bionanoconjugation for proteomics applications-an overview. Biotechnol. Adv. 32 (2014) 952–970. https://doi.org/10.1016/j.biotechadv.2014.04.013

ur

[20] K. Min, Y.J. Yoo, Recent progress in nanobiocatalysis for enzyme

Jo

immobilization and its application. Biotech. Bioproc. E. 19 (2014) 553–567. https://doi.org/10.1007/s12257-014-0173-7.

[21] M. Gahlout, D.M. Rudakiya, S. Gupte, A. Gupte, Laccase-conjugated aminofunctionalized nanosilica for efficient degradation of Reactive Violet 1 dye. Int. Nano Lett. 7 (2017) 195–208. https://doi.org/10.1007/s40089-017-0215-1.

16

[22] A. Khosravi, M. Vossoughi, S. Shahrokhian, I. Alemzadeh, Nano reengineering of horseradish peroxidase with dendritic macromolecules for stability enhancement. Enzy. Micr. Techn. 50 (2012) 10–16. https://doi.org/10.1016/j.enzmictec.2011.09.004 [23] S. Asad, S.F. Torabi, M. Fathi-Roudsari, Phosphate buffer effects on thermal stability and H2O2- resistance of horseradish peroxidase. Int. J. Biol. Macromol. 48 (2011) 566–570. https://doi.org/10.1016/j.ijbiomac.2011.01.021

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[24] S.A. Mohamed, M.H. Al-Harbi, Y.Q. Almulaiky, H.I. Ibrahim, R.M. ElShishtawy, Immobilization of horseradish peroxidase on Fe3O4 magnetic nanoparticles. Elect. J. Biotech. 27 (2017) 84–90.

-p

https://doi.org/10.1016/j.ejbt.2017.03.010

[25] C. De Goes Sampaio, J.G.A.E. Silva, E.S. De Brito, H. Becker, M.T.S. Trevisan,

re

R.W. Owen, Chromium (VI) remediation in aqueous solution by waste products

lP

(peel and seed) of mango (Mangifera indica L.) cultivars. Environ. Sci. Pollut. Res. 26 (2019): 5588. https://doi.org/10.1007/s11356-018-3851-8. [26] Sh. Ahsan,S. Kaneco, K. Ohta, T. Mizuno, K. Kani, Use of some natural and

na

waste materials for waste water treatment. Water Res. 35 (2002) 3738-3742. https://doi.org/10.1016/S0043-1354(01)00047-1.

ur

[27] I. Chakraborty, S. Das, B.K.Dubey, M.M.GhangrekarNovel low cost proton

Jo

exchange membrane made from sulphonated biochar for application in microbial fuel cells. Mat. Chem. Phys. 239 (2020) 122025. https://doi.org/10.1016/j.matchemphys.2019.122025.

[28] S. Das, S. Das, M.M.Ghangrekar, Quorum-sensing mediated signals: A promising multi-functional modulators for separately enhancing algal yield and

17

power generation in microbial fuel cell. Bioresource Tech. 294 (2019) 122138. https://doi.org/10.1016/j.biortech.2019.122138. [29] S.A. Mohamed, K.O. Abulnaja, A.S. Ads, J.A. Khan, T.A. Kumosani, Characterization of an anionic peroxidase from horseradish cv. Balady. Food Chem. 128 (2011) 725–30. https://doi.org/10.1016/j.foodchem.2011.03.096 [30] F. Adam, H.K. Fua, Production of silica from biogenic material. Malaysian Patent (2008) No.: MY-136715-A.

ro of

[31] A. Upadhyaya, D. Sankhla, T.D. Davis, N. Sankhla, B.N. Smith, Effect of paclobutrazol on the activities of some enzymes of activated oxygen metabolism and lipid per oxidation in senescing soybean leaves. J. Plant Physiol. 121(1985)

-p

453–461. https://doi.org/10.1016/S0176-1617(85)80081-X

[32] J. Huang, H. Xiao, B. Li, J. Wang, D. Jiang, Immobilization of Pycnoporus

re

sanguineus laccase on copper tetra-aminophthalocyanine–Fe3O4 nanoparticle

lP

composite. Biotech. Appl. Bioc. 44 (2010) 93–100. https://doi.org/10.1042/BA20050213

[33] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement

na

with the Folin phenol reagent. J. Biol. Chem. 193 (1951) 265–275. http://www.jbc.org/content/193/1/265.short.pdf+html

ur

[34] O. Folin, V. Ciocalteu, On Tyrosine and Tryptophane Determinations in Proteins.

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Biol. Chem. 73 (1927) 627–650. [35] R. Levesque, SPSS Programming and Data Management: Guide for SPSS and SAS Users, Fourth Edition, SPSS Inc., Chicago, IL 60606-6412. (2007).

[36] S.K. Giri, Synthesis of Mesoporous Silicon Nanoparticles and Enzyme Immobilization. GJRA 4 (2015) 225–226. https://www.doi.org/10.36106/gjra.

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[37] M Bragaru, M Simion, M Miu, T Ignat, I Kleps, V Schiopu, A Avram, V Craciunoiu, Study of the nanostructurated silicon chemical functionalization. Rom. J. Inform. Sci. Technol. 11 (2008) 397–407. https://www.romjist.ro/content/pdf/08-bragaru.pdf [38] A. Adochitei, G. Drochioiu, Rapid characterization of peptide secondary structure by FT-IR spectroscopy. Rev. Roum. Chim. 56 (2011) 783–791. http://revroum.lew.ro/wp-content/uploads/2011/RRCh_8_2011/Art%2004.pdf

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[39] S.A. Ansari, Q. Husain, Potential applications of enzymes immobilized on/in nano materials: A review. Biotechnol Adv 30 (2011) 512–523. https://doi.org/10.1016/j.biotechadv.2011.09.005

-p

[40] R. Su, P. Shi, M. Zhu, F. Hong, D. Li, Studies on the properties of graphene

oxide-alkaline protease bio-composites. Bioresour Technol 115 (2012) 136–140.

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https://doi.org/10.1016/j.biortech.2011.12.085

lP

[41] S. Zhang, S. Gao, G. Gao, Immobilization of β-Galactosidase onto magnetic beads. Appl. Biochem. Biotech. 160 (2010) 1386–1393. https://doi.org/10.1007/s12010-009-8600-5.

na

[42] Q. Chang, H. Tang, Immobilization of Horseradish Peroxidase on NH2-Modified Magnetic Fe3O4/SiO2 Particles and Its Application in Removal of 2,4-

ur

Dichlorophenol. Molecul. 19 (2014) 15768–15782.

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https://doi.org/10.3390/molecules191015768. [43] A. Dwevedi, Enzyme Immobilization, Springer International Publishing Switzerland. (2016). DOI 10.1007/978-3-319-41418-8_2.

[44] E. Ranjbakhsh, A.K. Bordbar, M. Abbasi, A.R. Khosropour, E. Shams, Enhancement of stability and catalytic activity of immobilized lipase on silica

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coated modified magnetite nanoparticles. Chem. Eng. J. 179 (2012) 272–276. https://doi.org/10.1016/j.cej.2011.10.097. [45] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, L.R. Fernandez, Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzy. Microb. Tech. 40 (2007) 1451–1463. https://doi.org/10.1016/j.enzmictec.2007.01.018 [46] J. Jia, S. Zhang, P. Wang, H. Wang, Degradation of high concentration 2,4

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Dichlorophenol by simultaneous photocatalytic-enzymatic process using TiO2/UV and laccase. J. Hazard Mater. 205 (2012) 150–155.

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https://doi.org/10.1016/j.jhazmat.2011.12.052.

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Tables

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Table (1) Concentration of Phenols in various industrial wastewater Phenol Concentration

Industrial Source

Reference

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(mg/L)

40 - 185

[3]

Petrochemical

200 - 1220

[4]

Textile

100- 150

[5]

Leather

4.4 - 5.5

[6]

Coke ovens

600 - 3900

[7]

Coal conversion

1700 - 7000

[8]

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Petroleum refineries

20

3 - 10

[9]

Pulp and paper industry

20-80

[10]

Phenolic resin

1200 - 1600

[11]

Fiberglass manufacturing

40 - 2564

[12]

Paint manufacturing

1.1

[13]

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Rubber industry

Table (2): Relative activity of immobilized peroxidase on nano-silica (nano-POX) and

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free peroxidase in presence of 5mM of different metal ion solution.

Al

56.11±1.265

85.91±0.984*

38.55±1.002

Co

93.38±1.326*

Cu

125.93±1.028* 111.67±0.792

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% free POX

95.50±1.00*

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Ca

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Metal % nano-POX

50.50±0.834

Fe

120.86±1.102* 106.45±1.293

Hg

80.04±1.117*

7.40±0.773

K

80.57±0.929*

25.42±1.029

Mg

83.77±0.991*

36.70±1.113

Mn

96.05±0.839*

56.11±1.293

Na

88.58±1.923*

42.31±1.832

21

Ni

91.24±1.000*

49.15±1.110

Pb

78.97±1.029*

5.29±0.772

Zn

122.73±0.839* 112.17±0.958

Values are mean of 5 replicates ± standard error. Means followed by asterisks are

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significantly different from the free enzyme according to paired-samples t test.

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Table (3): Enzyme efficiency in phenol removal from industrial wastewater (%). Treatment

Nano-peroxidase

cycle

Phenol after

Efficiency

treatment (mg/ml)

(%)

treatment (mg/ml)

(%)

0.309±0.065

99.92±0.080

130.178±0.654

66.39±0.992

1.937±0.839

99.5 ±0.982

-

-

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Phenol after

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2

Efficiency

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1

Soluble peroxidase

3

9.799±0.992

97.47±1.02

-

-

4

10.728±0.783

97.23±0.931

-

-

5

10.225±0.758

97.36±0.926

-

-

6

16.848±1.293

95.65±0.891

-

-

7

16.345±1.025

95.78±1.721

-

-

22

8

18.901±0.543

95.12±0.672

-

-

9

19.288±0.932

95.02±0.997

-

-

10

19.366±1.923

95±0.931

-

-

20

19.366±1.002

95±1.381

-

-

30

25.369±0.783

93.45±0.562

-

-

40

26.222±0.873

93.23±0.982

-

-

50

38.73±0.491

90±0.532

-

-

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Total phenols in the used wastewater = 387.32 ± 1.424mg/ml Total phenols in the used wastewater after nano-silica treatment= 387.02± 1.31mg/ml

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Values are mean of 5 replicates ± standard deviation

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Figures

23

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Figure 1. Effect of peroxidase concentration (a), pH (b) and Incubation period (c) on Immobilization yeild and efficiency. Points are means of 5 replicates. The bars represent the standard error.

24

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Figure 2. Transmission electron microscopy picture of nano-silica and nano-

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na

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peroxidase

25

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na

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Figure 3. FTIR Spectra of free nano-silica and nano-silica immobilized peroxidase

26

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Figure 4. Lineweaver–Burk plot relating soluble peroxidase (a) and Nano-peroxidase

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(b) reaction velocity to H2O2 (a1, b1) and pyrogallol concentrations (a2, b2).

27

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Figure 5. Effect of Nano-silica immobilization on optimum pH (a), optimum temperature (b) and thermal stability of peroxidase (c). Points are means of 5 replicates. The bars represent the standard error. Points followed by asterisks are significantly different according to paired-samples t test.

28