*2.2.1. Collection of plant sample and preparation of plants extract*

Plants (*Euphorbia amygdaloides*) were collected from Hasankale town of Erzurum city. They were washed with distilled water several times for cleaning dust and soil on plants. Then, plants were cut into small pieces. Small pieces (50 g) were thoroughly shattered to form a homogeneous mixture in blender using 250 mL, 10 mM sodium phosphate buffer (pH 6.0). Then, it was centrifuged at 5000×*g* for 10 min and the supernatant was used for enzyme purification [23].

#### *2.2.2. Partial purification of the peroxidase enzyme with ammonium sulfate precipitation*

Prepared Euphorbia (*Euphorbia amygdaloides*) plant homogenate was saturated from 60 to 80% with ammonium sulfate, then the peroxidase enzyme was precipitated by centrifuged at 8000×*g*, 10 min. Obtained precipitate was dissolved at 10 mM sodium phosphate buffer (pH 6.0) and was incubated at 4°C for further analysis [23].

#### *2.2.3. Peroxidase enzyme activity test*

Determination of peroxidase activity was made by substrate of 1 mM 2,2′-azino-bis(3-ethylbenzthiazoline-sulfonic acid) diammonium salt (ABST) prepared in 0.1 M phosphate buffer at pH 6. For this purpose, 2.8 mL ABST was transferred to a test tube, and then the reaction mixture was formed by the addition 100 μL of 80% enzyme and 100 μL of 3.2 mM H2 O2 solution into the test tube. The change in absorbance was monitored at 412 nm using UV–Visible spectrophotometer at 1 min intervals for 3 min. Blank test tube was prepared using distilled water instead of enzyme in the reaction mixture.

#### *2.2.4. Synthesis of Fe<sup>3</sup> O4 and ZnO nanoparticles*

100 μL of purified peroxidase enzyme from Euphorbia (*Euphorbia amygdaloides*) plant were added in sample FeCl2 of solution (2.9 mL, 10 mM) and incubated in a closed space for 4 h. The solution was becoming dark red, which indicates the presence of Fe<sup>3</sup> O4 nanoparticles. The same procedures were repeated using 2.9 mL of 10 mM ZnCl2 solution to obtain ZnO nanoparticles. The solution became a white turbid state indicating the presence of ZnO nanoparticles. Then, water was removed by the help of an evaporator, and synthesized iron and zinc oxide nanoparticles were dried at 70°C for 24 h.

curve was prepared in the range 0–40 ng/cm<sup>3</sup>

mg/g) were calculated from the equation:

as a function of dye concentration, where *C*<sup>o</sup>

The amounts of the dyes adsorbed onto LS-pure, LS-ZnO, LS-Fe<sup>3</sup>

the volume of solution (L) and *m* is the mass of adsorbent (g).

were used for the purification of peroxidase enzyme [25–29].

spectrophotometer.

and *Ce*

**2.5. Adsorption isotherms**

**3. Results and discussion**

where *Co*

Plots of ln(*C*/*C*<sup>o</sup>

tions of dye.

of DB15. The reaction mixture was prepared

http://dx.doi.org/10.5772/intechopen.73216

O4

and *C* represent remaining color intensity at the

, and LS-ZnO/Fe<sup>3</sup>

O4 (*q<sup>e</sup>* in 211

by adding membrane forms and in flasks containing 50 mL volume DB15 dye solution. The samples were taken out from the flasks periodically with a micropipette and were centrifuged at 5000 rpm for 10 min. The supernatant solutions were filtered with 0.45 mm filters. Then, the concentration of DB15 was measured with a UV–VIS spectrophotometer at λ = 596 nm. Scanning electron microscopy (SEM) was used to examine the surface of the adsorbents before and after dye adsorption (JEOL JSM-6400 SEM) and FTIR, XRD were performed for dye adsorption. Optimum contact time, pH, temperature, concentration of dye to determine optimal conditions for the decolorization of DB15 azo dye were analyzed using UV-Visible

The Investigation of Removing Direct Blue 15 Dye from Wastewater Using Magnetic *Luffa sponge* NPs

*<sup>q</sup><sup>e</sup>* <sup>=</sup> (*Co* <sup>−</sup> *Ce*) <sup>×</sup> *<sup>V</sup>* \_\_\_\_\_\_\_\_\_ *<sup>m</sup>* (1)

start of the experiment (zero time) and at any time *t*, respectively, for various fixed concentra-

**3.1. Partial obtaining peroxidase enzyme from** *Euphorbia amygdaloides* **plant**

The data obtained in the purification process of peroxidase enzyme are given in **Table 1**.

There are many studies on the purification of peroxidase enzyme in the literature. Plants such as wheat seeds, barley and wheat, soybeans, fava beans, sorghum, watermelon seeds, red beets, cotton, pearl millet seedlings, Asian rice, lettuce, wild radish, and pearl barley hybrids

Peroxidase enzyme was purified by ammonium sulfate precipitation from *Euphorbia amygdaloides* plant with CM-cellulose ion exchange chromatography and Sephacryl S-100 gel filtration chromatography. According to the data obtained in 75% ammonium sulfate precipitation step, the enzyme was purified with purification coefficient of 6.4 and 51.6 for 10 mL volume [23]. In our study, the enzyme was purified with a purification coefficient of 149.5 and a yield of 29.5 for 20 mL volume, according to the order of 60–80% ammonium sulfate precipitation step.

are the initial and equilibrium concentrations of dye in solution (mg/L); *V* is

) and time were drawn to estimate rate constants (k values) for decolorization

#### *2.2.5. Characterization of Fe<sup>3</sup> O4 and ZnO nanoparticles*

Synthesized Fe<sup>3</sup> O4 and ZnO NPs were characterized by scanning at range of 200–1000 nm by using UV-Vis spectrophotometer (Epoch nanodrop spectrophotometer). Determination of topography for Fe<sup>3</sup> O4 and ZnO nanoparticles was performed by SEM (Scanning Electron Microscope). In addition, XRD analysis (X-ray diffraction analysis) and FT-IR (Fourier transform infrared spectroscopy) were performed for Fe<sup>3</sup> O4 and ZnO NPs.

Contact time, pH, temperature, and metal ion concentration were determined for the purpose of optimization synthesized Fe<sup>3</sup> O4 and ZnO NPs. For determination of the optimum contact time, samples were spectrophotometrically measured between 0 and 240 min with 3 min intervals. Synthesis of Fe<sup>3</sup> O4 and ZnO NPs was performed in sodium phosphate buffer at pH 2.0–3.0, sodium acetate buffer at pH 4.0–6.0, sodium phosphate buffer at pH 7.0–8.0 and sodium carbonate buffer at pH 9.0–11.0 and the values of absorbance were measured. pH was adjusted by using 0.1 N HCl and 0.1 N NaOH. Synthesis of NPs was separately carried out from 10° to 90°C, respectively, and changes in absorbants of the samples were measured. Synthesis of NPs was performed by using related solution at 0.5, 1, 3, 5, and 7 mM and the absorbance of samples was measured. All measurements were performed by UV–VIS spectrophotometer and deionized water was used for blank sample.

## **2.3. Preparation of LS material, immobilization of nanoparticles procedure**

Dried LS material was made into small pieces and was autoclaved for 20 min to soften the fibrous structure. Then, it was transformed into dough using blender. It was incubated for 4 h with 1 N NaOH at 80°C. Then, the fibers were collected and were thoroughly washed with distilled water until NaOH is resolved. About 0.1% hypochlorite was used for decoloration of washed fibers and then, they were washed with distilled water. Fibers with the length of 10–50 μm were collected and were dispersed with distilled water to form a suspension form. The suspension was filtered under aseptic conditions using filter paper and obtained LS fibers were dried on filter paper at 40°C for 4 h [24]. Immobilization was performed by treatment solutions containing Fe<sup>3</sup> O4 and ZnO NPs with LS which was pretreated in ultrasonic bath for 1 h. Then, the obtained membrane forms (LS-pure, LS-ZnO, LS-Fe<sup>3</sup> O4 , LS-ZnO/Fe<sup>3</sup> O4 ) was dried in oven for 2 h.

### **2.4. Azo dye remediation**

The prepared membranes were used for decolorization of DB15 solution which was prepared in the laboratory. Synthetic wastewater was prepared by dissolving DB15 dye. A calibration curve was prepared in the range 0–40 ng/cm<sup>3</sup> of DB15. The reaction mixture was prepared by adding membrane forms and in flasks containing 50 mL volume DB15 dye solution. The samples were taken out from the flasks periodically with a micropipette and were centrifuged at 5000 rpm for 10 min. The supernatant solutions were filtered with 0.45 mm filters. Then, the concentration of DB15 was measured with a UV–VIS spectrophotometer at λ = 596 nm. Scanning electron microscopy (SEM) was used to examine the surface of the adsorbents before and after dye adsorption (JEOL JSM-6400 SEM) and FTIR, XRD were performed for dye adsorption. Optimum contact time, pH, temperature, concentration of dye to determine optimal conditions for the decolorization of DB15 azo dye were analyzed using UV-Visible spectrophotometer.

The amounts of the dyes adsorbed onto LS-pure, LS-ZnO, LS-Fe<sup>3</sup> O4 , and LS-ZnO/Fe<sup>3</sup> O4 (*q<sup>e</sup>* in mg/g) were calculated from the equation:

$$q\_{\epsilon} = \frac{(C\_o - C\_o) \times V}{m} \tag{1}$$

where *Co* and *Ce* are the initial and equilibrium concentrations of dye in solution (mg/L); *V* is the volume of solution (L) and *m* is the mass of adsorbent (g).

### **2.5. Adsorption isotherms**

The solution was becoming dark red, which indicates the presence of Fe<sup>3</sup>

 *and ZnO nanoparticles*

O4

**2.3. Preparation of LS material, immobilization of nanoparticles procedure**

O4

photometer and deionized water was used for blank sample.

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1 h. Then, the obtained membrane forms (LS-pure, LS-ZnO, LS-Fe<sup>3</sup>

ticles. The solution became a white turbid state indicating the presence of ZnO nanoparticles. Then, water was removed by the help of an evaporator, and synthesized iron and zinc oxide

by using UV-Vis spectrophotometer (Epoch nanodrop spectrophotometer). Determination

Microscope). In addition, XRD analysis (X-ray diffraction analysis) and FT-IR (Fourier trans-

Contact time, pH, temperature, and metal ion concentration were determined for the pur-

contact time, samples were spectrophotometrically measured between 0 and 240 min with

at pH 2.0–3.0, sodium acetate buffer at pH 4.0–6.0, sodium phosphate buffer at pH 7.0–8.0 and sodium carbonate buffer at pH 9.0–11.0 and the values of absorbance were measured. pH was adjusted by using 0.1 N HCl and 0.1 N NaOH. Synthesis of NPs was separately carried out from 10° to 90°C, respectively, and changes in absorbants of the samples were measured. Synthesis of NPs was performed by using related solution at 0.5, 1, 3, 5, and 7 mM and the absorbance of samples was measured. All measurements were performed by UV–VIS spectro-

Dried LS material was made into small pieces and was autoclaved for 20 min to soften the fibrous structure. Then, it was transformed into dough using blender. It was incubated for 4 h with 1 N NaOH at 80°C. Then, the fibers were collected and were thoroughly washed with distilled water until NaOH is resolved. About 0.1% hypochlorite was used for decoloration of washed fibers and then, they were washed with distilled water. Fibers with the length of 10–50 μm were collected and were dispersed with distilled water to form a suspension form. The suspension was filtered under aseptic conditions using filter paper and obtained LS fibers were dried on filter paper at 40°C for 4 h [24]. Immobilization was performed by treatment

The prepared membranes were used for decolorization of DB15 solution which was prepared in the laboratory. Synthetic wastewater was prepared by dissolving DB15 dye. A calibration

and ZnO NPs were characterized by scanning at range of 200–1000 nm

O4

and ZnO nanoparticles was performed by SEM (Scanning Electron

and ZnO NPs.

and ZnO NPs. For determination of the optimum

and ZnO NPs was performed in sodium phosphate buffer

and ZnO NPs with LS which was pretreated in ultrasonic bath for

O4

, LS-ZnO/Fe<sup>3</sup>

O4 ) was

same procedures were repeated using 2.9 mL of 10 mM ZnCl2

*O4*

nanoparticles were dried at 70°C for 24 h.

O4

form infrared spectroscopy) were performed for Fe<sup>3</sup>

*2.2.5. Characterization of Fe<sup>3</sup>*

O4

pose of optimization synthesized Fe<sup>3</sup>

3 min intervals. Synthesis of Fe<sup>3</sup>

solutions containing Fe<sup>3</sup>

**2.4. Azo dye remediation**

dried in oven for 2 h.

Synthesized Fe<sup>3</sup>

of topography for Fe<sup>3</sup>

210 Iron Ores and Iron Oxide Materials

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solution to obtain ZnO nanopar-

nanoparticles. The

Plots of ln(*C*/*C*<sup>o</sup> ) and time were drawn to estimate rate constants (k values) for decolorization as a function of dye concentration, where *C*<sup>o</sup> and *C* represent remaining color intensity at the start of the experiment (zero time) and at any time *t*, respectively, for various fixed concentrations of dye.
