Lysozyme as a flocculant-inducing agent improving the silica removal from aqueous solutions - A turbidimetric study

Journal of Environmental Management 226 (2018) 187–193

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Research article

Lysozyme as a flocculant-inducing agent improving the silica removal from aqueous solutions - A turbidimetric study

T

Katarzyna Szewczuk-Karpisza,∗, Małgorzata Wiśniewskab a b

Institute of Agrophysics, Polish Academy of Sciences, Doświadczalna 4, 20-290 Lublin, Poland Department of Radiochemistry and Colloid Chemistry, Faculty of Chemistry, Maria Curie-Sklodowska University, M. Curie-Sklodowska Sq. 3, 20-031 Lublin, Poland

A R T I C LE I N FO

A B S T R A C T

Keywords: Lysozyme Silica Adsorption Zeta potential Potentiometric titration Stability

In this paper, the lysozyme (LSZ) adsorption impact on the silica suspension stability was established. In other words, the stabilization/destabilization mechanism of the SiO2/LSZ system was explained based on the adsorption, electrokinetic and stability measurement results. Lysozyme adsorbs on the silica surface in the whole pH range. This process contributes to the changes in silica surface charge and zeta potential values. The lysozyme addition influences the system stability too. At pH 7.6 and 9, a large decrease in the silica suspension stability was found. It is connected with the neutralization of solid negative charge by the positively charged macromolecules. As a result, large aggregates can be formed, which is highly desirable in the silica removal procedure.

1. Introduction Lysozyme (LSZ) is an enzymatic protein with antibacterial properties. It hydrolyses the β-1,4-glycosidic linkage between N-acetylglucosamine and N-acetylmuramine acid in the bacteria cell wall. This protein is a component of tears, saliva and other tissue fluids. LSZ is used in many industries, including the food and pharmaceutical ones (Panfil-Kuncewicz, 1988; Wang et al., 2005). Nowadays, lysozyme is an object of various scientific research. Many of them relates to the lysozyme use in drug delivery (Haselberg et al., 2011; Cai and Yao, 2013). The LSZ adsorption on the silica surface is well-documented. Rezwan et al. (2005a) described the influence of the solid surface charge on the protein adsorption mechanism. They clarified the conditions, under those the lysozyme and bovine serum albumin adsorb on the silica particles. Steri et al. (2013) examined the ionic strength effect on the LSZ adsorption/desorption amount on the SBA-15 mesoporous silica. Czeslik and Winter (2011) explained the temperature input on the lysozyme macromolecules conformation adsorbed on the SiO2 particles. Kumar et al. (2014) presented pH-dependent interaction and resultant structure of silica particles and lysozyme macromolecules. Rimola et al. (2013) described SiO2 surface features and their role in the biomolecule adsorption using computational and experimental methods. Bharti et al. (2014) focused on bridging interactions proteinsilica as a function of pH, ionic strength and protein concentration. In turn, Su et al. (1988) studied the silica-water interface with the LSZ macromolecules adsorbed using neutron reflection.

∗

On the other hand, the lysozyme adsorption effect on the suspension stability as a function of pH value is scarcely described in the literature. Therefore, this paper focused on this subject. It presents the probable stabilization/destabilization mechanism of the silica suspension in the LSZ presence. The measurements were performed using a turbidimeter, which determines the system stability precisely. The explanation of the LSZ adsorption effect on the SiO2 suspension stability is not possible without adsorption amount and electrokinetic studies and, due to this fact, this paper described also these experiments. The presented information on SiO2/LSZ system stability may be valuable in the environmental engineering. In water purification procedure proteins may act as flocculant-inducing agents (Santiago et al., 2002). Coagulation is generally the first process in the water and wastewater purification procedure. Including oxidation and sedimentation, it is the most important step in this technology. Coagulation is based on the colloidal system destabilization, which decreases the dispersion degree (Adamski, 2002). This process occurs after the coagulant addition, immediately. It reduces the electrokinetic potential and thereby weakens the repulsive forces acting between colloidal particles. The most effective coagulation is possible when the zeta potential approaches or is equal to zero (Magrel, 2000). Aluminum and iron salts are commonly used coagulates. After their hydrolysis, they form hydroxides neutralizing the impurity charge. However, due to the slow rate of the process, the polyelectrolytes or flocculant-inducing agents must be added to the system (Santiago et al., 2002; Sanchez-Martin et al., 2012). These substances initiate flocculation, i.e. the

Corresponding author. E-mail address: [email protected] (K. Szewczuk-Karpisz).

https://doi.org/10.1016/j.jenvman.2018.08.026 Received 7 February 2018; Received in revised form 1 August 2018; Accepted 6 August 2018 0301-4797/ © 2018 Elsevier Ltd. All rights reserved.

Journal of Environmental Management 226 (2018) 187–193

K. Szewczuk-Karpisz, M. Wiśniewska

silica is a mesoporous material. In turn, the SiO2 specific surface area was 145 m2/g. The crystalline structure of the solid was determined using X-ray diffractometer (Empyrean, PANalytical). The obtained XRD pattern is shown in Fig. 1. The mean particle size was also measured (zetameter Zetasizer 3000, Malvern Instruments); it was equal to 225 nm. Porous and crystalline silica was chosen for experiments due to its wide application in industry. Lysozyme (LSZ), delivered by Sigma-Aldrich (62971, Poland), isolated from chicken egg white, was also used in the study. According to literature, the LSZ molecular weight is 14.3 kDa and the isoelectric point (pI) is about 11. The LSZ macromolecule is composed of 128 amino acids (Watter, 1951). Lysozyme has a high internal stability, i.e. its structure is not highly dependent on the solution pH value. In the pH range from 1 to 8 its structure remains unchanged (Ogasahara and Hamagichi, 1967).

agglomeration of destabilized particles in the macromolecular compound presence (Adamski, 2002; Wiśniewska et al., 2018a). Polyelectrolytes may be also helpful in metal acquisition from geothermal water (Wiśniewska et al., 2018b). Looking for new flocculants is a significant issue due to drinking water lack in many regions. Taking this into account, this paper described lysozyme as potential agent improving flocculation. It defines the pH value, at which the aqueous silica suspension is the most destabilized after the LSZ addition. The silica removal from aqueous solutions is essential because it is a very common mineral on the Earth's sphere that occurs also in surface waters. In addition, silica is widely used in the industry (glass and construction), so its presence in the sewage is inevitable. The silica removal from aqueous solutions is examined by many researchers. Hermosilla et al. (2012) focused on the coagulation and reverse-osmosis of SiO2 particles present in effluents from recovered-paper mills. Miranda et al. (2015) described the silica coagulation with aluminum salt in the suspended solids presence. These researchers claimed that silica is one of the most important substances that accumulate in papermaking water cycles. Den and Wang (2008) stressed that SiO2 makes desalination difficult. Thus, they used electrocoagulation pretreatment in silica removal from brackish water. Emamjomeh and Sivakumar (2009) summarized the pollutants that may be removed by electrocoagulation and electrocoagulation/flotation processes. Therefore, the presented subject may be considered as a very important and actual. Our previous study described another potential flocculant-inducing agent. It was related to the chromium(III) oxide removal in the albumin presence (Szewczuk-Karpisz and Wiśniewska, 2014).

2.2. Methods All measurements were performed at room temperature, i.e. 25 °C. As a supporting electrolyte 0.01 M NaCl was used. The experiments were carried out as a function of pH value (3–9). The lysozyme concentration is expressed in ‘ppm’ (parts per million), which is equivalent to ‘mg/l’ (weight concentration).

2. Experimental

2.2.1. Viscosity measurements Viscosity measurements were used for the hydrodynamic radius (rh) determination of the lysozyme macromolecules at various pH values. The LSZ concentration was in the range of 10–500 ppm. The measured viscosity (η) was converted to the relative viscosity (ηr) and the intrinsic viscosity [η] using the equations (Porejko et al., 1965):

2.1. Materials

ηr =

Silicon(IV) dioxide (silica, SiO2), delivered by Sigma-Aldrich, was used in the experiments. This is a finely crystalline, white solid with porous structure. Using the BET equation (Brunauer et al., 1938) and nitrogen adsorption/desorption isotherm method (ASAP 2405 analyzer. Micrometritics) the SiO2 average pore size and specific surface area were determined. The first parameter was equal to 11.3 nm which means that

η ηs

[η] = lim ⎛ c→0 ⎝

(1)

η ηr − 1 ⎞ = lim ⎛ sp ⎞ c → 0 c ⎠ ⎝ c ⎠

(2)

where: c – the protein concentration, η – the EPS solution viscosity, ηr – the relative viscosity, ηs – the solvent viscosity, ηsp – the specific viscosity, [η] – the intrinsic viscosity.

Counts 90000

SiO2

40000

10000

0

10

20

30

40 50 60 Position [°2Theta] (Copper (Cu))

70

80

Peak List Si O2; 04-008-7651

Fig. 1. Diffractogram of the SiO2 sample with matched phases from the ICDD PDF4 + 2016 diffraction database.

188

90

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measurement. One result was the average of three measurements.

The hydrodynamic radius of the LSZ macromolecules was established using the formula developed by Einstein-Simha:

rh =

3

3[η] M 10πNA

2.2.4. Adsorption measurements Adsorption measurements were performed by a static method. 0.0085 g SiO2 was added to the LSZ solutions of various concentrations (50–500 ppm). Then the pH value was adjusted and the adsorption was conducted for 7 h under continuous shaking conditions, until the equilibrium was reached. The process time was previously established by kinetic measurements. After the adsorption completion, the suspensions were centrifuged and the supernatants obtained were analyzed. The LSZ concentration was determined using a spectrophotometer UV–Vis (Cary 100, Agilent Technology) at 280 nm. At this wavelength the protein solution showed the maximum light absorption. Aromatic aminoacids (tryptophan, tyrosine) present in the macromolecules are mainly responsible for this phenomenon. The adsorption amount (Γ) was calculated based on the equation:

(3)

where: M – the molecular weight, NA – the Avogadro number. Standard deviation of the results obtained did not exceed 5%. 2.2.2. Potentiometric titration Potentiometric titration is a method used for the surface charge determination. In the supporting electrolyte solution, hydroxyl groups interact with the electrolyte and hydrogen ions and as a result a surface charge is formed (Janusz, 1991):

≡ SiOH 0 + H+ ↔ ≡SiOH2+

≡

SiOH 0

≡

SiOH2+Cl−

≡

SiOH 0

≡SiO−

↔

+

↔

+

≡SiOH 0

Na+

↔

(4) (5)

H+ +

H+

≡SiO−Na+

+

Cl−

(6)

+

H+

(7)

Γ=

where: cads – the polymer adsorbed concentration, the difference in the polymer concentration in the system before and after adsorption, V – the suspension volume. The single result was the average of three repetitions. The measurement error did not exceed 5%.

The surface charge is an algebraic sum of the above group charges:

σ0 =

B·{≡SiOH2+

+

≡SiOH2+Cl−

−

≡SiO−Na+

−

≡SiO−}

(8)

where: B – the factor for the conversion of the surface concentration [μmol/m2] into charge density [μC/cm2]. The apparatus used for determination of the silica surface charge density (σ0) in the absence and presence of lysozyme consisted of: teflon thermostated vessel, water thermostat RE 204 (Lauda), glass and calomel electrodes (Beckman Instruments), pH meter PHM 240 (Radiometer), automatic microburette Dosimat 765 (Metrohm), PC and printer. The σ0 value was calculated by the computer program ‘titr_v3’, based on the following equation (Janusz, 1999):

σ0 =

ΔV ·cb·F m · Sw

2.2.5. Stability measurements The silica suspension stability in the absence and presence of lysozyme was measured by the turbidimetric method (Turbiscan TLabExpert with a cooling module TLab Cooling). During the measurement, a light beam (λ = 880 nm) passes through the suspension. It is partially dispersed on solid particles and partially passes through the sample. The intensity of the scattered and passing light is measured by detectors. 0.02 g SiO2 was added to the supporting electrolyte solution and sonicated for 3 min. Then LSZ was added (100 ppm) and the pH value was adjusted. A single measurement lasted 3 h. The results were obtained in the form of graphs of light transmission and backscattering as well as the Turbiscan Stability Index (TSI). This parameter takes a value in the range of 0–100. The higher suspension stability, the lower TSI value is. This index was calculated by the computer program cooperating with a turbidimeter based on the formula:

(9)

where: ΔV – the difference in the base volume added to the suspension and the supporting electrolyte solution that leads to the specific pH value (ΔV = Vs-Ve), cb – the base concentration, F – the Faraday constant, m – the metal oxide mass in the suspension, Sw – the mineral oxide surface area. The SiO2/supporting electrolyte (lysozyme) systems were titrated by the 0.1 NaOH solution. The lysozyme concentration was 50 or 100 ppm. The SiO2 mass was 0.1 g. Every titration started at pH 3.5 and finished at pH 11.

n

TSI =

2.2.3. Electrokinetic potential measurements The zeta potential (ζ) of silica particles in the absence and presence of lysozyme was calculated based on the electrophoretic mobility measured by zetasizer Nano ZS (Malvern Instruments). The computer program used the Henry's formula because the κa parameter for SiO2 was about 34 (Oshima, 1994):

μ=

εζ f (κa) η

cads·V Sw · m

∑i = 1 (x i − xBS )2 n−1

(11)

where: xi – the average backscatter for each minute of measurement, xBS – the average xi value, n – the scans number. 3. Results and discussion 3.1. The internal stability of the LSZ macromolecules

(10)

According to the literature report, the lysozyme structure does not change in the pH range from 1 to 8 (Ogasahara and Hamagichi, 1967). The performed viscosity measurements provided the results consistent with the above paper. The specific viscosity for the examined systems is presented in Fig. 2, whereas the hydrodynamic radius is summarized in Table 1. In the pH range of 3–7.6, no significant changes in the LSZ hydrodynamic radius were observed. It rises minimally, from 2.72 (pH 3) to 2.88 nm (pH 7.6), which confirms the macromolecule stability. At pH 9, the LSZ macromolecule size increases noticeably and rh is equal to 3.17 nm. Under these conditions, the protein macromolecules denature, i.e. their III- and IV-order structure changes due to strongly alkaline pH value. Hydrogen bonds present in polypeptide chains are destroyed and, as a result, the lysozyme biological activity is lost.

where: ε – the dielectric constant, η – the viscosity, μ – the electrophoretic mobility, 1/κ – the thickness of electrical double layer, a – the particle radius. The measurements were performed based on the microelectrophoresis phenomenon. The silica particles were placed in a homogeneous electric field at the stationary level of the electrophoretic cell. Depending on the charge they moved towards the cathode or anode. When the applied voltage compensated their movement, the device read the electrophoretic mobility. The measurements were performed in the pH range from 3 to 9. This parameter was changed automatically by a titrator. The lysozyme concentration was 50 or 100 ppm. The SiO2 mass was 0.01 g. The samples were sonicated for 3 min before the 189

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analysis showed that lysozyme adsorbs on the silica surface at all pH values (3, 4.6, 7.6 and 9). The highest adsorption amounts were observed at pH 7.6 and 9. To explain this relationship, the SiO2 surface charge density must be analyzed. Previously performed potentiometric titration indicated that the point of zero surface charge (pHpzc) of silica is equal to 3. This means that at pH 3 the amounts of positive –SiOH2+ and negative –SiO- groups are identical. What is more, in the pH range 3–8, the SiO2 surface charge density is close to 0. This phenomenon is connected with specific surface properties of silicon(IV) oxide. As the experiments showed, in the aqueous solution the SiO2 surface contains two types of silanol groups (≡SieOH). There are isolated (19%) and vicinal (81%) ones. The first are formed when the silicon atom bind to three Si atoms inside the solid phase and one –OH group. The second – when two isolated groups are bound to two different silicon atoms and connected by a hydrogen bond. The silica surface groups are characterized by different pKa values, i.e. isolated – 4.9, vicinal – 8.5. In the pH range of 3–8, a very small amount of silanol groups is dissociated and, as a result, the SiO2 surface charge is close to 0. In turn, at pH > 8, almost all vicinal groups are dissociated. Due to the fact that these groups are dominating on the silica surface, their dissociation contributes to significant σ0 decrease (Ong et al., 1992; Bergna and Roberts, 2005; Zhuravlev, 2000; Terpiłowski et al., 2015). The isoelectric point of lysozyme is equal to 11. This is equivalent to the positive charge of LSZ macromolecules in the whole examined pH range. Owning to it, lysozyme adsorption on the silica surface is possible. The highest adsorption amounts observed at pH 7.6 and 9 are associated with electrostatic attraction occurring between the positive LSZ macromolecules and the negatively charged solid. At pH 3 and 4.6, the amount of adsorbed protein was much smaller due to the zero SiO2 surface charge.

0.7 pH 3

pH 4.6

pH 6

pH 7.6

pH 9

0.6

ηsp [dm3/g]

0.5 y = 0.1034x + 0.3042

0.4

y = 0.1013x + 0.2238 y = 0.1003x + 0.2139 y = 0.066x + 0.1931 y = 0.0812x + 0.1992

0.3 0.2 0.1 0 0

0.1

0.2

0.3

0.4

0.5

0.6

c [g/dm3]

Fig. 2. Intrinsic viscosity of the LSZ solutions at various pH values.

Table 1 Hydrodynamic radius of LSZ macromolecules at various pH values. pH

Hydrodynamic radius (nm)

3 4.6 6 7.6 9

2.72 2.75 2.81 2.86 3.17

3.2. The LSZ adsorption on the silica surface Lysozyme adsorbs on solid surface only under strictly defined conditions, i.e. when the electrostatic repulsion between protein and particles is not present in the system (Rezwan et al., 2005a,b). The adsorption isotherms for the silica-LSZ system obtained at various pH values are shown in Fig. 3. They were obtained using logarithmic approximation; Ce is an equilibrium protein concentration (the LSZ concentration in the solution after the adsorption process). The result

3.3. The effect of LSZ adsorption on the SiO2 surface charge and the electrokinetic potential The lysozyme adsorption affects the suspension electrokinetic properties, especially silica surface charge and electrokinetic potential.

2.5

pH 3 pH 4.6

[mg/m2]

2

pH 7.6 pH 9

1.5

1

0.5

0 0

100

200

300

400

500

Ce [ppm] Fig. 3. Adsorption isotherms of lysozyme on the silica surface (logarithmic approximation). 190

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5 0 2

3

4

5

6

7

8

9

10

11

12

0

( C/cm2)

-5 -10 -15 -20 silica

-25

silica + LSZ 50 ppm silica + LSZ 100 ppm

-30 -35 -40

pH Fig. 4. Silica surface charge in the absence and presence of lysozyme.

where: σads – the contribution of adsorption to charge density, z – the charge of adsorbed protein macromolecule, F – the Faraday constant, Γmax – the maximum coverage of the surface by the adsorbate, θ – the fractional coverage, pHs – pH at the surface, φs – the potential at the surface, pKai – the pKa value of the N-terminus and the side chains of arginine, histidine and lysine of the adsorbed amino acids, pKaj – the pKa value of the C-terminus and aspartate, glutamate, cysteine and tyrosine amino acid side chains, zAds – the charge of the interfacial groups. These equations indicate that the surface charge density in the protein presence depends, among others, on amino acid type that are the closest to the solid. The amino acid nature and sequence determine the change character in the σ0 parameter. According to the literature, the segments containing many positive groups (eNH3+) located close to the mineral oxide surface contribute to the solid charge decrease. On the other hand, the same groups that are far away from the solid induce the σ0 increase (Janusz et al., 1997; Ostolska and Wiśniewska, 2014, 2015). In the examined system, the changes in the silica surface charge are minimal. At the pH range 3–5, the SiO2 surface is approximately neutral and thus, in the LSZ presence, the σ0 parameter is mainly determined by the bonded protein. They are positively charged due to the amino group protonation and, as an adsorption result, the solid adopt a slight positive charge. Thus most eNH3+ groups must be located at a distance from the surface. At pH higher than 6, when the silica surface becomes negative, protonated amino groups make the SiO2 charge less negative. It is also worth mentioning that at pH > 9 the LSZ macromolecules change their conformation. As a result, other segments begin to interact with silicon(IV) oxide. Identical SiO2 surface charge after the addition of different LSZ concentrations (50 and 100 ppm) is a very interesting phenomenon. The lysozyme adsorption amounts for these concentrations vary, i.e. more macromolecules are bound to the solid in the 100 ppm lysozyme presence. Consequently, the density of adsorption layer is different. As it was mentioned above, the solid surface charge density is the resultant of two effects - the σ0 increase by NH3+ groups located far away from the solid and the σ0 reduction by NH3+ groups present in adsorbed

5 0 2

4

6

8

10

[mV]

-5 -10 -15 silica -20

silica + LSZ 50 ppm silica + LSZ 100 ppm

-25 pH Fig. 5. Zeta potential of silica particles with and without lysozyme.

These changes are presented in Figs. 4 and 5, respectively. The potentiometric titration results showed that there is a slight σ0 increase after the lysozyme addition. It was observed for the both LSZ concentrations (50 and 100 ppm). The silica pHpzc point in the LSZ presence is about 5.8. The changes in the surface charge density after the protein adsorption were described by the formulas developed by Hartvig et al. (2011): (12)

σads = zFΓmax Θ pHs = pHbulk + 0.434 k

z Ads =

Fϕs RT

10 pKai

∑ 10 pHs + 10 pKai

(13) l j

i

m

z=

⎛ 10 pKaj − ⎜∑ 10 pH + 10 pKai ⎝ i

10 pHs

∑ 10 pHs + 10 pKaj

−

n

10 pH

⎞

∑ 10 pH + 10 pKaj ⎟ + z Ads j

(14)

⎠

(15) 191

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pH 9

pH 7.6 TSI

silica silica-LSZ

pH 4.6

pH 3

a) 0

10

20

30

40

50

60

Fig. 6. TSI values for the silica/supporting electrolyte and silica/supporting electrolyte/lysozyme systems.

segments (very close to the surface). In the 50 and 100 ppm LSZ presence, the amounts of groups directly bounded to the surface and located at a distance from the solid are not identical. However, in both cases, their overall effect remains very similar. As a result, no significant changes in the SiO2 surface charge density were observed after 50 and 100 ppm lysozyme addition. The LSZ adsorption is also equivalent with the SiO2 zeta potential increase (Fig. 5). The silica pHiep value is equal to 3. But in the LSZ presence, the above parameter is shifted to 5.8. This means that at pH 5.8, the slipping plane charge of SiO2 particles covered with LSZ macromolecules is equal to 0. The amounts of positive and negative groups in this structure are the same. At pH < 5.8 the positive groups are dominant in the silica slipping plane, whereas at pH > 5.8 – the negative ones. The pHiep shift was noticed for both LSZ concentrations (50 and 100 ppm). During the adsorption, protein macromolecules cover the mineral oxide surface gradually and, as a result, the solid properties become similar to those of protein (Rezwan et al., 2005b). When the whole surface is covered with the protein macromolecules, the solid pHiep value is equal to the protein pI point. Due to the fact that, in the lysozyme presence, the silica pHiep point is different from the LSZ pI value, it was stated that the protein does not cover the whole mineral oxide surface. The lack of differences in SiO2 zeta potential values in the 50 and 100 ppm LSZ presence, in the pH range 1–8, is associated with almost identical position of the slipping plane. Macromolecules adsorbed contribute to the shift of this structure. However, for both concentrations, due to the lack of distinct conformational changes of the lysozyme macromolecules, its position remains the same. In view of the fact that the adsorption amounts is different, the density of the adsorption layer changes. But its thickness maintains identical. Slight differences in the electrokinetic potential, after the 50 and 100 ppm lysozyme addition, were observed at pH 9 and 10. Under these conditions, the lysozyme is denatured and its size increases slightly. Consequently, at a higher lysozyme concentration, more packed and wider adsorption layer shifts the slipping plane a little bit stronger and the zeta potential is more negative. It must be also noticed that, when the lysozyme is adsorbed on the silica surface, the particle zeta potential is relatively low and changes in the range from −7 to 2 mV. These values favour the mineral oxide coagulation.

b) Fig. 7. Transmission and backscatter curves for the silica suspension with and without lysozyme at pH 9.

(the TSI values are higher than 30). This is connected with the zero charge of the surface and slipping plane. Under these conditions, there are no electrostatic forces that hinder the particle contact. Silica forms large aggregates which fall onto the vessel bottom. On the other hand, at pH 7.6 and 9 the SiO2 suspension is stable (the TSI values are lower than 5). There is an electrostatic stabilization in the system. The silica surface charge is negative and every particle is surrounded by chloride ions derived from the supporting electrolyte solution. These sheaths prevent the SiO2 aggregate formation. The LSZ adsorption influences the suspension stability. At pH 3, no significant changes were observed (the difference in the TSI value was only 0.6). Under these conditions, the LSZ adsorption amount is low (about 0.1 mg/m2) and macromolecules are not able to change the interactions between the solid particles significantly. At pH 4.6, the lysozyme addition results in a slight reduction in the silicon(IV) dioxide stability. Since the pKa value of isolated silanol groups is 4.9, at pH 4.6 nearly half of them are dissociated. The positive LSZ macromolecules, adsorbed on the solid surface, neutralize the ≡SiO- group charge partially and thus the system stability decreases slightly. At pH 7.6 and 9, the LSZ adsorption is equivalent to significant reduction in the SiO2 stability. This is reflected in a clear TSI value increase (about 40 and more units). The stability changes observed at pH 9 are presented in Fig. 7, in the form of light transmission and backscattering curves. They are mainly reflected by the distance between the individual curves. In the diagram for silica without protein all curves overlap, which is characteristic of systems with high stability. In turn, in the diagram for the silica suspension containing lysozyme the distance between curves is larger. This indicates less system stability. At pH 7.6 and 9, the particles are negative and the adsorption of the positive LSZ macromolecules causes their charge neutralization. As a result, repulsive interactions become weaker and dynamic processes may occur in the system. That leads to the formation of big aggregates and their sedimentation. Analyzing the above results, it can be concluded that lysozyme can be a potential flocculant-inducing agent in the procedure of silica removal from aqueous solutions, especially at pH 7.6 and 9.

3.4. The LSZ influence on the silica stability The silica suspension stability has been described in the literature (Wiśniewska et al., 2015; Szewczuk-Karpisz et al., 2016). The obtained TSI values are shown in Fig. 6. At pH 3 and 4.6 the system is unstable

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4. Conclusions

stability of lysozyme. J. Biochem. 61 (2), 199–210. Ong, S., Zhao, X., Eisenthal, K.B., 1992. Polarization of water molecules at a charged interface: second harmonic studies of the silica/water interface. Chem. Phys. Lett. 191 (3–4), 327–335. Oshima, H., 1994. A simple expansion for Henry's function for the retardation effect in electrophoresis of spherical colloidal particles. J. Colloid Interface Sci. 168, 269–271. Ostolska, I., Wiśniewska, M., 2014. Comparison of the influence of polyaspartic acid and polylysine functional groups on the mechanism of polymeric film formation at the Cr2O3 - aqueous solution interface. Appl. Surf. Sci. 311, 734–739. Ostolska, I., Wiśniewska, M., 2015. The polymer structure impact on adsorption of the ionic polyamino acid homopolymers and their diblock copolymers on the colloidal chromium (III) oxide. RSC Adv. 5, 28505–28514. Panfil-Kuncewicz, H., 1988. Characteristics of lysozyme and its possible use in the food industry. Zyw. Czlow. Metab. 15, 218–222. Porejko, S., Fejgin, J., Zakrzewski, L., 1965. Chemistry of Macromolecular Compounds. Wydawnictwo Naukowo-Techniczne, Warszawa. Rezwan, K., Meier, L.P., Geuckler, L.J., 2005a. Lysozyme and bovine serum albumin adsorption on uncoated silica and AlOOH-coated silica particles: the influence of positively and negatively charged oxide surface coatings. Biomaterials 26, 4351–4357. Rezwan, K., Studart, A.R., Voros, J., Gauckler, L.J., 2005b. Change of ζ potential of biocompatible colloidal oxide particles upon adsorption of bovine serum albumin and lysozyme. J. Phys. Chem. B 109, 14469–14474. Rimola, A., Costa, D., Sodupe, M., Lambert, J.-F., Ugliengo, P., 2013. Silica surface features and their role in the adsorption of biomolecules: computational modelling and experiments. Chem. Rev. 113 (6), 4216–4313. Sanchez-Martin, J., Beltran-Heredia, J., Peres, J.A., 2012. Improvement of the flocculantion process in water treatment by using Moringa oleifera seeds extract. Braz. J. Chem. Eng. 29 (3), 495–501. Santiago, L.G., Gonzalez, R.J., Fillery-Travis, A., Robins, M., Benaldo, A.G., Carrara, C., 2002. The influence of xantan and λ-carrateenan on the creaming and flocculantion of an oil-in-water emulsion containing soy protein. Braz. J. Chem. Eng. 19 (4), 411–417. Steri, D., Monduzzi, M., Salis, A., 2013. Ionic strength affects lysozyme adsorption and release from SBA-15 mesoporous silica. Microporous Mesoporous Mater. 170, 164–172. Su, T.J., Lu, J.R., Thomas, R.K., Cui, Z.F., Penfold, J., 1988. The effect of solution pH on the structure of lysozyme layers adsorbed at the silica-water interface studied by neutron reflection. Langmuir 14 (2), 438–445. Szewczuk-Karpisz, K., Wiśniewska, M., 2014. Investigation of removal possibilities of chromium(III) oxide from water solution in the presence of albumins. Int. J. Environ. Sci. Technol. 12 (9), 2947–2956. Szewczuk-Karpisz, K., Wiśniewska, M., Pac-Sosińska, M., Choma, A., Komaniecka, I., 2016. Stability mechanism of the silica suspension in the Sinorhizobium meliloti 1021 exopolysaccharide presence. J. Ind. Eng. Chem. 31, 108–114. Terpiłowski, K., Wiśniewska, M., Zarko, V.I., 2015. Influence of solution pH, supporting electrolyte presence and solid content on the stability of aqueous nanosilica suspension. J. Ind. Eng. Chem. 30, 71–76. Wang, S., Ng, T.B., Chen, T., Lin, D., Wu, J., Rao, P., Ye, X., 2005. First report of a novel plant lysozyme with both antifungal and antibacterial activities. Biochem. Biophys. Res. Commun. 327, 820–827. Watter, R.L., 1951. Immunological studies on egg white proteins: IV. Immunochemical and physical studies of lysozyme. J. Biol. Chem. 192, 237–242. Wiśniewska, M., Szewczuk-Karpisz, K., Sternik, D., 2015. Adsorption and thermal properties of the bovine serum albumin – silicon dioxide system. J. Therm. Anal. Calorim. 120, 1355–1364. Wiśniewska, M., Chibowski, S., Urban, T., Fijałkowska, G., Nosal-Wiercińska, A., Saglikoglu, G., Sadikoglu, M., Yilmaz, S., 2018a. Stability of metal oxide suspension in the cationic polyacrylamide presence. J. Sci. Perspect. 2 (2), 13–22. Wiśniewska, M., Fijałkowska, G., Ostolska, I., Franus, W., Nosal-Wiercińska, A., Tomaszewska, B., Goscianska, J., Wójcik, G., 2018b. Investigations of the possibility of lithium acquisition from geothermal water using natural and synthetic zeolites applying poly(acrylic acid). J. Clean. Prod. 195, 821–830. Zhuravlev, L.T., 2000. The surface chemistry of amorphous silica, Zhuravlev model. Colloids Surf. A 173, 1–38.

Lysozyme, as a protein of high internal stability, maintains its conformation over a wide pH range (1–8). It adsorbs on the silica surface when there are no repulsive electrostatic interactions in the system (pH range 3–9). The highest adsorption amounts were noticed at pH 7.6 and 9 (1.5 and 2 mg/m2) because then the positive macromolecules are attracted by the negative silica surface. The LSZ adsorption affects the electrokinetic and stability properties of the system. The changes in the silica surface charge and zeta potential was observed. What is more, lysozyme influences the SiO2 stability. At pH 3 and 4.6, the stability changes were minimally (the TSI growth was up to 7) due to a small adsorption amount. On the other hand, at pH 7.6 and 9, a large drop in the system stability was noticed. The TSI increase was about 40, what means that lysozyme adsorption favours the particle aggregation. References Adamski, W., 2002. Water Purification System Modeling. PWN, Warsaw. Bergna, H.E., Roberts, W.O., 2005. Colloidal Silica: Fundamentals and Applications. CRC Press. Bharti, B., Meissner, J., Klapp, S.H.L., Findenegg, G.H., 2014. Bridging interactions of proteins with silica nanoparticles: the influence of pH, ionic strength and protein concentration. Soft Mater. 10 (5), 718–728. Brunauer, S., Emmett, P.H., Teller, E., 1938. Adsorption of gases in multimolecular layers. J. Am. Chem. Soc. 60 (2), 309–319. Cai, H., Yao, P., 2013. In situ preparation of gold nanoparticle-loaded lysozyme-dextran nanogels and applications for cell imaging and drug delivery. Nanoscale 5, 2892–2900. Czeslik, C., Winter, R., 2011. Effect of temperature on the conformation of lysozyme adsorbed to silica particles. Phys. Chem. Chem. Phys. 3, 235–239. Den, W., Wang, C.-J., 2008. Removal of silica from brackish water by electrocoagulation pretreatment to prevent fouling of reverse osmosis membranes. Separ. Purif. Technol. 59 (3), 318–325. Emamjomeh, M.M., Sivakumar, M., 2009. Review of pollutants removed by electrocoagulation and electrocoagulation/flotation processes. J. Environ. Manag. 90 (5), 1663–1679. Hartvig, R.A., Van de Weert, M., Ostergaard, J., Jorgensen, L., Jensen, H., 2011. Protein adsorption at charged surfaces: the role of electrostatic interactions and interfacial charge regulation. Langmuir 27, 2634–2643. Haselberg, R., Harmsen, S., Dolman, M.E.M., de Jong, G.J., Kok, R.J., Somsen, G.W., 2011. Characterization of drug-lysozyme conjugates by sheathless capillary electrophoresis–time-of-flight mass spectrometry. Anal. Chem. Acta 698, 77–83. Hermosilla, D., Ordonez, R., Blanco, L., de la Fuente, E., Blanco, A., 2012. pH and particle structure effects on silica removal by coagulation. Chem. Eng. Technol. 35, 1632–1640. Janusz, W., 1991. Determination of surface ionization and complexation constants from potentiometric titration data. Pol. J. Chem. 65, 799–807. Janusz, W., 1999. Electrical Double Layer at Metal Oxide-electrolyte Interface in ‘interfacial Forces and Fields Theory and Applications’. M. Dekker, New York. Janusz, W., Kobal, I., Sworska, A., Szczypa, J., 1997. Investigation of the electrical double layer in a metal oxide/monovalent electrolyte solution system. J. Colloid Interface Sci. 187, 381–387. Kumar, S., Vinod, K., Callow, P., 2014. pH-Dependent interaction and resultant structures of silica nanoparticles and lysozyme protein. Langmuir 30 (6), 1588–1598. Magrel, L., 2000. Water and Wastewater Treatment. Devices, Processes, Methods. Wydawnictwo Ekonomia i Środowisko, Białystok. Miranda, R., Latour, I., Blanco, A., 2015. Influence of suspended solids on silica removal by coagulation with aluminum salt. Cellul. Chem. Technol. 49 (5–6), 497–510. Ogasahara, K., Hamagichi, K., 1967. Structure of lysozyme: XII. Effect of pH on the

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