enzymatic decolorization of dyes by laccase immobilized on hybrid carriers · enzymatic...

Spaska Yaneva, Nadezhda Rangelova, Lyubomir Aleksandrov, Dancho Danalev

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ENZYMATIC DECOLORIZATION OF DYES BY LACCASE IMMOBILIZED ON HYBRID CARRIERS

Spaska Yaneva1, Nadezhda Rangelova1, Lyubomir Aleksandrov2, Dancho Danalev3

ABSTRACT

The study focused on sol-gel synthesis of biopolymer-silica hybrids further used as materials for laccase im-mobilization. Silica was obtained from tetraethyl orthosilicate (TEOS), while pectin, derived from citrus fruits, was used as a biopolymer. The structure of the hybrids obtained was investigated by X-ray diffraction, Fourier transform infrared spectroscopy, Scanning electron microscopy and BET analysis. The results provided to conclude that the interaction between SiO2 network and the polysaccharide proceeding via H-bonds formation was successful.

Laccase was immobilized by cross-linking, while the degradation ability was tested against several dyes – Rho-damine B, Methyl orange and Malachite green. The most effective decolorization was reached for Malachite green and Methyl orange.

Keywords: hazardous materials, dyes decolorization, laccase immobilization, sol-gel method, silica hybrid materials.

Received 20 December 2017Accepted 20 June 2018

Journal of Chemical Technology and Metallurgy, 53, 6, 2018, 1203-1210

1 University of Chemical Technology and Metallurgy Department of Fundamentals of Chemical Technology 8 Kl. Ohridski Blvd., Sofia 1756, Bulgaria E-mail: [email protected] Institute of General and Inorganic Chemistry, Bulgarian Academy of Sciences Acad. G. Bonchev Str., bl. 11, Sofia 1113, Bulgaria3 University of Chemical Technology and Metallurgy, Department of Biotechnology 8 Kliment Ohridski, Sofia 1756, Bulgaria

INTRODUCTION

Nowadays, one of the main environmental prob-lems is the contamination of water by residual dyes as products of various industries: textile, pulp and paper, pharmacy, leather industry, etc. Another major problem refers to the presence of persistent organic pollutants in natural water resources because of poorly functioning wastewater treatment systems. All these are extremely toxic and harmful for the living organisms, and which is why their purification is required. There are is a variety of techniques for organic pollutants decomposition [1, 2]. Biocatalysis is the one of the most effective and promis-ing technology in this respect. Microbial or enzymatic decolorization and degradation is an eco-friendly cost-

competitive alternative to the chemical decomposition process helping to reduce water consumption compared to that of physicochemical treatment methods [2]. The dyes Remazol Turquoise Blue G 133, Lanaset Blue 2R are effectively degraded by using not only laccase but also the enzyme horseradish peroxidase (HRP) [3]. It is possible to directly use lignin degrading white rot fungi for the decolourization of azo dyes such as Congo red, Rhodamine 6G, Malachite green. They are also used as a textile industry effluent. The azo dyes removal on the fifth day from an aqueous solution by Poria sp. (of 50 μM concentration) amounts to 93 % in case of Congo red, 64.4% in case of Rhodamine 6G and 94.8 % in case of Malachite green. Ganoderma sp. decolourize Congo red by 77 %, Rhodamine 6G by 65.3 % and Malachite

Journal of Chemical Technology and Metallurgy, 53, 6, 2018

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green by 75.2 % at (at 50 μM concentration) on fifth day. Trametes sp. decolourize Congo red by 89%, Rhodamine 6G by 54.5 % and Malachite green by 76.1 % at (at 50 μM concentration) also on the fifth day [4].

Enzymes immobilization is a common practice mainly because it minimizes the process costs by provid-ing multiple enzyme usage. This means that the enzyme is physically confined, often in a polymer matrix in the form of beads or membranes, in a way that it cannot be transferred to the solution. The use of an immobilized enzyme also generally facilitates the downstream pro-cessing because it can simply be removed by sieving, whereas a considerable effort and money would have to be invested in removing a soluble enzyme from a reactor stream [5].

Laccase, obtained from different sources, is suc-cessfully used for immobilization by entrapment in an alginate-gelatin gel [6] or a polymer matrix [7]. It is also immobilized by cross-linking with glutaraldehyde onto porous silica [8] and chitosan beads [9]. The pH and temperature dependences of laccase activity are investigated in case of decolorization / degradation of different types of dyes such as Congo red, methyl violet, acid blue, acid orange, malachite green and others [6-9]. This enzyme decolorizes some azo dyes without direct cleavage of the azo bonds through a highly non-specific free radical mechanism, thereby avoiding the forma-tion of toxic aromatic amines. Azo dyes can be cleaved symmetrically and asymmetrically, with an active site available for the enzyme to excite the molecule [10, 11]. Zille et al. [12] propose the most probable mechanism of degradation of azo dyes by laccase.

Our previous investigations have used different types of biopolymers and silica sources to prepare sol-gel hybrid materials. The materials obtained are used as matrices for immobilization of bacterial or yeast cells as well as an enzyme aiming degradation or toxic substances bio-accumulation [13-15]. Based on the lit-erature and our previous experience we are motivated to synthesize SiO2/pectin hybrid materials by the sol-gel method, to examine their structural characteristics and to immobilize laccase in order to clarify the decolorization proceeding for several dyes.

EXPERIMENTAL

MaterialsCitrus fruits pectin (CP) with methoxy groups con-

tent of 6.7 %, C2H5OH, 2,2′-Azino-bis(3-ethylbenzothia-zoline-6-sulfonic acid (ABTS, ɛ = 36 000 M-1 cm-1), and laccase (Е.С. 1.10.3.2.) from Trametes versicolor were obtained from Sigma-Aldrich. Rhodamine B, Methyl orange and Malachite green oxalate were also purchased from Sigma-Aldrich. TEOS (98 %), hydrochloric acid (HCl, 36 %) and glutaraldehyde were purchased from Merck. Phosphate buffer for microbiology, APHA, pH 7.2 was provided from Fluka.

Sample preparationThe sol–gel synthesis of SiO2/CP hybrids was car-

ried out by a multi-step method at a room temperature using acid hydrolysis as described previously [13, 16]. The pectin was dissolved in hot water. The silica pre-cursor was pre-hydrolyzed with H2O, ethanol and HCl. After TEOS hydrolysis the pH was adjusted to 7 with a phosphate buffer. The obtained sols were mixed and homogenized on a magnetic stirrer. The biopolymer quantities amounted to 5 mass %, 20 mass % and 50 mass %.

Enzyme immobilization The immobilization of laccase onto different hy-

brids was carried out by cross-linking. The procedure of immobilization, the determination the bonded protein amount and the specific activity of the immobilized en-zyme was described in our previous investigation [15]. The laccase activity was determined by measuring the oxidation of ABTS. The unit was defined as the enzyme amount oxidizing 1 μmol of ABTS per minute under the reaction conditions assayed.

Dyes dеcolorization The ability of the immobilized enzyme to decolorize

several dyes present in aqueous solutions was studied. The dye concentration was as follows: Malachite green (5 ppm), Methyl orange (15 ppm) and Rhodamine B (10 ppm). The reaction mixture contained 2 mL of a


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dye, 2 mL of 0.05M phosphate buffer providing pH of 4.0 and immobilized enzyme carriers (100 mg). The mixture was slowly stirred. An aliquot from the reaction mixture (3mL) was taken to record the corresponding absorbance spectrum at the 1-st h, the 48-th h and the 72-nd h. Quartz cuvettes were used.

All determinations referring to the specific activity of the immobilized enzyme, the amount of the bonded protein and the decolorization of the dyes were per-formed three times in duplicate sets and the average values were taken into account.

Sample characterizationТhe crystalline and amorphous states were identified

by XRD analysis (Bruker D8 Advance diffractometer, Cu K α radiation). The IR spectra were measured using KBr pellet technique on a Nicolet 320 FTIR spectrom-eter with a resolution of ±1 cm-1 by collecting 64 scans in the range of 4000 cm-1- 400 cm-1. SEM images were taken with a Microscope Tescan FIB-SEM Lyra. The obtained materials were covered with gold and the im-ages were taken at an accelerating voltage of 20 kV, while the magnification was 6.9 kx. The specific surface areas of the synthesized materials were measured at 77K using a Gemini 2370 (Micromeritics) surface and porosimetry instrument. The surface area was calculated on the ground of the Brunauer-Emmett-Teller (BET)

N2 adsorption isotherm. The specific activity of the im-mobilized enzyme and the dyes absorption spectra were measured by VWR UV-1600 PC spectrophotometer.

RESULTS AND DISCUSSION

The X-ray diffraction patterns of pectin and the hybrid materials used are shown in Fig. 1. The dif-fractogram of pectin shows a semi-crystalline nature [17, 18] with diffraction peaks at 2θ = 130 and 2θ = 210, respectively. The XRD analysis results show that all hybrids studied are amorphous. This indicates a success-ful hybrids formation. Some authors [19, 20] support the conclusion that these materials amorphous nature is determined by H-bonds formation.

Fig. 2 shows the infrared spectra of pectin and SiO2/CP hybrid materials. The bands at 2940 cm-1 refer to C-H absorption where CH-, CH2- and CH3-stretching and bending vibrations are included. The bands related to C=O stretching of the ester and free carboxyl group are observed at 1740 cm-1 and 1630 cm-1, respectively, in the IR spectra of CP [21, 22] . On the other hand, the bands at 1630 cm-1 can be attributed to the vibration of absorbed water. The characteristic bands of the silica network and the “fingerprint” region of polysaccharides are found in the range from 1300cm-1 to 600cm-1 [21 - 23]. The bands at ca 1089 cm-1 and 797 cm-1 can be

Fig. 1. XRD patterns of pectin from citrus fruits and obtained SiO2 hybrid materials.


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assigned to the asymmetric and symmetric vibrations of the siloxane linkage (Si-O-Si) [23-25]. The bands at 950 cm-1 can be attributed to silanol (Si-OH) groups [23]. The shoulder at ca 1200 cm-1 is typical for the asymmetric vibration of Si-O-Si bridges [23 - 25]. The vibrations of the skeletal (C-O-C and C-C) glycosidic bonds as well as the deformation and valence vibrations of the C-OH bonds in the pyran ring are observed in the pectin spec-trum [21, 22]. The bands at 960 cm-1 and ca 830 cm-1 are due to ν(О–СН3) stretching vibrations and out-of-plane deformational vibrations of pyran ring. Low frequency vibrations of pyran ring can be observed below 690 cm-1

[21, 22]. The bands in the range of 3000 cm-1 -3800 cm-1 can be attributed to Si-OH groups or absorbed water on the surface [23]. The free OH groups in the biopolymer macromolecule, the intramolecular hydrogen bonds and

the intermolecular hydrogen bond are also observed in this range [26, 27]. On the other hand, the bands at 3440 cm-1 can be assigned to OH stretching vibration of H-bonded water or intra H-bonding in the hybrids between Si–OH and OH groups from pectin. These results provide to conclude that the interaction between SiO2 network and the polysaccharide proceeding via H-bonding is successful.

The SEM images of the hybrids are presented in Fig. 3. As indicated the surface of all samples is homogenous without any immiscibility fractions. This corresponds to successful hybrids formation.

The BET analysis shows that the specific surface area is between 385 m2/g and 274 m2/g (Table 1). It is seen that the increase of the pectin content in the hybrids results in decrease of the specific surface. On the other

Fig. 2. FTIR spectra of pectin from citrus fruits and obtained SiO2 hybrid materials.


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Fig. 3. SEM images of obtained SiO2 hybrid materials with 5 mass % (a), 20 mass % (b), and 50 mass % (c) pectin from citrus fruits.

Fig. 4. Absorption spectra of used dyes after treatment with laccase immobilized onto different carriers.


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hand, the nano-dimensional pore size increases from 1.53 nm to 1.86 nm with the specific surface decrease.

The obtained hybrids were used as carriers for lac-case immobilization. Table 1 presents the values of the protein amount and the specific activity. The results show that after the immobilization process, the bonded protein amounts are 3.27 mg/g, 2.06 mg/g, and 1.97 mg/g abs. dry weight corresponding to silica hybrids with 5 mass %, 20 mass %, and 50 mass % pectin, respectively. The higher values of the bonded enzyme correspond to a larger specific surface of the carrier and a smallest amount of pectin. The same results are observed in our previous investigation [15].

The next step of this study refers to the investigation of the possibility of immobilized enzymes to degrade water soluble azo dyes – methyl orange, malachite green and rhodamine B. It is well known that the indicators possess the ability of varying their color in dependence of media pH. Therefore, a buffer of a constant pH value is used. The latter is found an optimal one in respect to an enzymatic activity. The enzymatic treatment results in the dye color change due to the process of the parent molecule decomposition.

The absorption spectra of the enzymatically treated dyes are presented in Fig. 4. The most effective deg-radation is achieved in case of Malachite green with a carrier containing 5 % of a biopolymer. It is seen that analogical results are observed in case of Rhodamine B decolorization by laccase immobilized onto SiO2/5

mass % CP hybrid. The absorption spectrum of methyl orange shows batho shifts of the absorption maximum at λmax = 472 nm. The absorption spectrum results referring to methyl orange subjected to an enzymatic treatment suggest that the structural changes occur in the parent azo dye molecule. It is assumed that the bathochromic shift in the spectrum is indicative of the changes in the dye molecule without breaking the double bond between the nitrogen atoms.

The degree of decolorization is calculated according to Wang et al. [28]. The results are presented in Fig. 5. The value referring to malachite green amounts to 84 % at 72-nd h. The maximum degree of rhodamine B degradation reaches a value of 27 % in case of a carrier with immobilized enzyme containing 5 % of pectin.

The value of the degree of degradation of malachite green indicates that the highest decolorization rate is achieved at the72-nd h for all carriers. The highest decol-orization rate is obtained with an enzyme immobilized on SiO2/5 mass % CP hybrid material. The results of malachite green decolorization on laccase immobilized on carriers containing 20 % and 50 % pectin are identi-cal. A similar tendency of rhodamine B decolorization is observed. Again, the best results are obtained for the enzyme immobilized onto carrier containing 5 % of a polysaccharide. This is most likely due to the fact that the hybrid material with the lowest pectin content has the highest specific surface area. Furthermore, it is character-ized by the highest specific activity and a bonded protein

Fig. 5. Decolorization of MG and RhB solutions over laccase immobilized onto different carriers.


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amount (Table 1). Our future studies will focus on the determination of the residual products in the solution after the enzyme treatment of the dyes and the number of the immobilized system working cycles.

CONCLUSIONS

Amorphous silica hybrids materials based on TEOS and pectin from citrus fruits were successfully synthe-sized via a sol-gel routе. Laccase was immobilized onto the hybrid carriers by cross-linking with glutar-aldehyde. The dyes degradation was performed under optimal conditions for the enzyme. Malachite green (5 ppm), Rhodamine B (10 ppm) and Methyl orange (15 ppm) were used. The highest degree of degradation was achievement for Malachite green. It amounted to 84 % for duration of 72 h using a carrier containing 5 mass % of a polysaccharide.

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Table 1. BET analysis and catalytic properties of immobilized laccase onto hybrid carriers.

Hybrid carrier Specific surface area,

m2/g

Pore diameter,

nm

Protein amount, mg/g abs. dry weight

Specific activity, U/mg

SiO2 5mass %CP 385 1.53 3.27 80

SiO2 20 mass%CP 321 1.61 2.06 63

SiO2 50 mass%CP 274 1.86 1.97 58


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