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supporting electrolyte interface – The impact on solid aggregation
Journal of Molecular Liquids 284 (2019) 117–123
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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq
Adsorption layer structure at soil mineral/biopolymer/supporting electrolyte interface – The impact on solid aggregation Katarzyna Szewczuk-Karpisz a,⁎, Małgorzata Wiśniewska b 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 l e
i n f o
Article history: Received 11 March 2019 Received in revised form 27 March 2019 Accepted 29 March 2019 Available online 31 March 2019 Keywords: Protein Exopolysaccharide Biopolymer conformation System turbidity Zeta potential
a b s t r a c t This paper focused on the protein and polysaccharide adsorption mechanism on the surface of common soil mineral – silica (SiO2), as well as the biopolymer adsorption effect on the solid aggregation. For the experiments, the proteins of various internal stability were selected, i.e. bovine serum albumin (BSA), ovalbumin (OVA) and lysozyme (LSZ). Moreover, the study involved exopolysaccharide (EPS) synthesized by soil bacteria Sinorhizobium meliloti. The adsorption layer structure on the silica surface and the solid aggregation tendency in the biopolymer presence were established and explained based on the turbidimetric, adsorption and zeta potential measurements. The measured biopolymer adsorbed amounts, biopolymer conformation on the SiO2 surface and solid aggregation were strongly dependent on the pH conditions. The stimulating effect on SiO2 aggregate formation was stated at pH 4.6 for BSA, OVA and LSZ, whereas at pH 7.6 – for LSZ and EPS. The strongest impact was noticed at pH 7.6 in the lysozyme presence (the TSI value increased by 40 units). The presented results can contribute to a better understanding of mineral-organic associations formation under selected soil conditions. Moreover, they may be helpful in developing innovative substances limiting destructive erosion effect. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Soil structure is one of its morphological features, which defines the spatial distribution of solid phase particles as well as their shape and size. Soil structure affects other soil properties, mainly water, air and thermal ones. Proper aggregation accelerates the water filtration into the soil profile and limits its evaporation [1]. Formation of soil aggregates is a very complex process. During the last fifty years, many theories about this process have been developed. The first papers concerned the relationship between soil aggregation and thermodynamic function – entropy [2,3]. Then, the scientists focused on the role of organic-mineral associations in the soil microaggregation. Tisdall and Oades [4] stated that primary soil microaggregates are formed by surrounding organic debris with fine minerals. These structures are stabilized with various substances synthesized by microorganisms and fungi [5]. Chorover et al. [6] found that the mineral-organic connections are formed by electrostatic interactions, in turn Christensen [7] considered them as basic units for larger soil aggregates. Mineral-organic associations occur between the organic and inorganic matter present in the soil [8]. Their formation is determined by various processes. Among physicochemical phenomena, adsorption is the most important one. It occurs shortly after the first contact of soil compounds ⁎ Corresponding author. E-mail address: [email protected] (K. Szewczuk-Karpisz).
https://doi.org/10.1016/j.molliq.2019.03.172 0167-7322/© 2019 Elsevier B.V. All rights reserved.
and affects the microaggregate stabilization significantly [9]. Mineralorganic matter associations are examined by various methods. Chen et al. [10] used a X-ray spectromicroscopy to examine the associations present in pasture soil clay fractions. Mikutta et al. [11] determined the stability of the connections between soil organic matter and minerals using isotopes. In turn, Newcomb et al. [12] measured the binding between organic substances and soil minerals in aqueous environment using a force spectroscopy. Taking into account the key role of organic matter adsorption on the mineral surface in the soil structure and aggregate stability, this paper focused on the biopolymer adsorption on the surface of widespread soil mineral – silica (SiO2) and its impact on the SiO2 particle aggregation. For the experiments, two biopolymer types were selected. The first was exopolysaccharide (EPS) synthesized by soil bacteria Sinorhizobium meliloti, the second – proteins characterized by different structural properties: ovalbumin (OVA), bovine serum albumin (BSA), lysozyme (LSZ). The study included the adsorbed amount measurements, zeta potential determination and turbidity measurements. They induced the biopolymer amount involved in silica-organic associations and, owing to it, immobilized by the mineral soil compound. What is more, they provided the information about silica tendency to aggregation when the proteins or exopolysaccharide are present in the system. All measurements were performed as a function of the solution pH value, i.e. at pH 4.6 and 7.6 (corresponding with common soil environment conditions). Based on the obtained results, the structure
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of adsorption layer formed on the SiO2 surface was proposed. Moreover, the organic substance whose influence on the SiO2 aggregation is the strongest was indicated. Research on the adsorption mechanism of natural and synthetic polymers on the inorganic material surface is a current issue. Some scientists have conducted studies on other mineral oxides, e.g. hydroxyapatite [13] and mixed oxides [14]. Janusz et al. [15] examined the BSA adsorption process on the modified silica surface, in turn Klepka et al. [16,17] are working on specific polymer layers. In our previous work, we focused on adsorptive, electrokinetic and stability properties of chromium(III) oxide and zirconia suspensions in the absence and presence of various macromolecular compounds [18,19]. For this study, we selected silica because it is involved in the soil structure formation. The results presented in this paper are of high scientific value and may find practical application in agriculture. They can be very helpful in developing of innovative additives that improve the soil structure and resistance to erosion. What is more, the paper may contribute to better explanation of the interactions between soil minerals and organic matter occurring in soil environment. 2. Experimental
Table 2 Proteins characteristics: Mw – molecular weight, pI – protein isoelectric point, rh – hydrodynamic radius [24,26,27,46–49,51–54]. Name
Bovine serum albumin Ovalbumin Lysozyme
Abbreviation Mw pI [kDa]
Structural stability
rh [nm] pH 4.6
pH 7.6 3.57
BSA
66.43 4.7–4.9 low
3.32
OVA LSZ
42.7 14.3
2.95 3.39 1.85–1.89 1.85–1.89
4.4–4.9 low 11 high
established using a potentiometric titration and graphic method. Based on this parameter, the EPS dissociation degree (α) at pH 4.6 and 7.6 was also calculated [32,33]. The exopolysaccharide molecule size was determined based on the viscosity of EPS solutions measured under various pH conditions. It was expressed through a parameter called ‘root-meanpffiffiffiffiffi square chain end-to-end distance’ ( r 2 ) [34,35]. The exopolysaccharide used is a succinoglycan; its monomer is composed of seven glucose molecules and one galactose molecule connected by various glycosidic bonds. Such EPS chain is substituted by acetyl, pyruvyl and succinyl groups [36,37]. 2.2. Methods
2.1. Materials Silica (silicon(IV) oxide, SiO2, CAS 7631-86-9) delivered by Sigma Aldrich was used in the study. The solid was characterized by several methods. The SiO2 average pore diameter (Dp) and specific surface area (SBET) were determined using a nitrogen adsorption/desorption isotherm method (analyzer ASAP 2405, Micrometritics) and BET equation [20]. The pHpzc parameter (point of zero charge) and surface charge density were established by a potentiometric titration method [21]. The SiO2 average particle size (d) was measured using a zetameter (Zetasizer 3000, Malvern Instruments), whereas its isoelectric point (pHiep) was determined using a zetameter equipped with a titrator (Zetasizer NanoZS, Malvern Instruments). The obtained parameters are summarized in Table 1. Before the experiment start, the mineral oxide was washed with double distilled water until the supernatant conductivity was below 3 μS/cm. Bovine serum albumin (BSA, CAS 9048-46-8), ovalbumin (OVA, CAS 9006-59-1) and lysozyme (LSZ, CAS 12650–88-3), delivered by Sigma Aldrich, were also used in the experiments. Their parameters (e.g. molecular mass, isoelectric points) are summarized in Table 2. The used proteins were characterized by different internal stability. BSA and OVA are ‘soft proteins’ (of low structural stability), which means that their molecule size differs under various pH conditions [22,23]. The hydrodynamic radius (rh) of BSA and OVA molecules at pH 4.6 is about 3.32 nm and 2.94 nm, whereas at pH 7.6–3.57 nm and 3.39 nm, respectively [24]. Lysozyme is a ‘hard protein’ (of high internal stability) and, as a result, contains a specific conformation in a wide pH range [25]. Within pH range 4–7, the value of rh parameter of the LSZ macromolecules is about 1.85 nm [26] or 1.89 nm [27]. All proteins used are components of soil organic matter (organic debris) [28–30]. Exopolysaccharide synthesized by soil bacteria (Sinorhizobium meliloti) was isolated according to the defined procedure [31]. EPS parameters are summarized in Table 3. The pKa value of the polysaccharide was Table 1 Silica characteristics: Dp – average pore diameter, SBET – specific surface area, d – average particle size, pHiep – isoelectric point, pHpzc – point of zero charge. Parameter
Value
Dp [nm] SBET [m2/g] pHiep pHpzc d [nm]
11.3 145 3 3 225
All experiments were carried out at 25 °C, using 0.01 M NaCl as a supporting electrolyte. The measurements were performed at pH values, i.e. pH 4.6 and 7.6. The pH value was established using 0.1 M HCl, 0.1 M NaOH and a pHmeter Beckman. Biopolymer adsorbed amounts on the silica surface were measured based on the difference in the protein concentration before and after the adsorption process according to the formula [38]: Γ¼
cads ∙V SBET ∙m
ð1Þ
where cads – the polymer adsorbed concentration, the difference in the polymer concentration in the system before and after its adsorption, V – the suspension volume, m – the solid weight. At the beginning, the suspensions were prepared by adding appropriate solid weight to the solution containing supporting electrolyte and selected biopolymer (10–500 ppm). The solid weights in the samples (10 ml) were as follows: 0.0085 g for the protein adsorption and 0.0035 g for the EPS adsorption. These amounts were determined based on the silica specific surface area and the biopolymer properties. After the suspension preparation, the pH value was adjusted and the adsorption was started. The process was performed under shaking conditions (shaking bath Unimax 1010, Heidolph). Its time was determined based on previous kinetic measurements – 7 h for SiO2-protein, 20 h for SiO2EPS. When the adsorption finished, the probes were centrifuged (223e, MPW Med. Instruments; 10,000 rpm) and the supernatants were taken for the biopolymer quantification. The protein/polysaccharide concentration was determined by a spectrophotometric method (spectrophotometer UV–Vis Cary 100, Agilent Technology). The protein concentration was measured at the wavelength, at which the maximum UV absorbance for the protein solution was observed (280 nm – for lysozyme, 279 nm – for bovine serum albumin, 278 nm – for ovalbumin [39]. In turn, the exopolysaccharide concentration was determined using the method developed by Dubois et al. [40]. One adsorption result was the repetition of three measurements. The error (standard deviation) did not exceed 5%. Electrophoretic mobility (μ) of the silica particles, in the absence and presence of biopolymers, was determined using a zetameter (Zetasizer Nano ZS, Malvern Instruments) based on the microelectrophoresis phenomenon. During the measurement, the particles moved in the electrophoretic cell, towards the cathode or anode (depending on the sign of their charge). When the applied voltage compensated their movement, the device read the electrophoretic mobility value. The κa parameter for
K. Szewczuk-Karpisz, M. Wiśniewska / Journal of Molecular Liquids 284 (2019) 117–123 Table 3 Exopolysaccharide characteristics:
pffiffiffiffiffi r 2 – root-mean-square chain end-to-end distance [35,55,56].
Name
Abbreviation
Mw [kDa]
pKa
Exopolysaccharide S. meliloti
EPS
103–104
3.8
εζ f ðκaÞ η
ð2Þ
where: ε – the dielectric constant, η – the viscosity, 1/κ – the thickness of electrical double layer, a – the particle radius. The samples for electrophoretic mobility measurements were prepared by adding 0.01 g of SiO2 to the supporting electrolyte solution (20 ml). After the 3-min sonification, the initial pH value was adjusted. The measurements were carried out using a titrator, which changed the suspension pH value automatically. The examined pH range was 3–9. The biopolymer was added to the system just before the measurement start. Its concentration equals 100 ppm. A single result of electrophoretic mobility was the repetition of three measurements. The error did not exceed 5%. The silica aggregation, in the absence and presence of macromolecular compounds, was established based on the turbidimetric measurements (Turbiscan TLabExpert with a cooling module TLab Cooling). The apparatus measured the transmission and backscattering of the light beam (λ = 880 nm) passing through the sample and determined the suspension stability by the Turbiscan Stability Index (TSI), which was calculated using the following equation: sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi n ∑i¼1 ðxi −xBS Þ2 TSI ¼ n−1
ð3Þ
where: xi – the average backscattering for each minute of measurement, xBS – the average xi value, n – the scans number. This parameter changes from 0 (highly stable systems) to 100 (extremely unstable ones). The probes (20 ml) were prepared by adding 0.02 g of SiO2 to the supporting electrolyte solution. After the 3-min sonification, the biopolymer was added to the suspensions and the pH value was adjusted. The protein/polysaccharide concentration was equal to 100 ppm. Suspension stability was measured two times. The obtained results were almost identical (the error did not exceed 1.5%). 3. Results and discussion Structure of protein(polysaccharide) adsorption layer on the silica surface. The obtained adsorbed amounts of the examined biopolymers on the SiO2 surface are presented in Fig. 1. These amounts were strongly dependent on the solution pH value. What is more, they were associated with the protein internal stability. The results observed for soft and hard proteins were different. At pH 4.6, the adsorption amounts of ovalbumin and bovine serum albumin (albumins, soft proteins) were higher than at pH 7.6. In turn, more lysozyme macromolecules bound to the SiO2 surface at pH 7.6 than at pH 4.6. The EPS adsorbed amount was also dependent on the solution pH value, but the noted differences were smaller than those observed for proteins. The silica surface has unique properties. In aqueous solutions it contains two types of groups: (1) isolated silanol groups – formed when one Si atom bind to three Si atoms inside the solid phase and one hydroxyl group, and (2) vicinal silanol groups – formed through the connection of two isolated silanol groups bind to two different Si atoms by a hydrogen bond. The isolated groups account for 19% and the vicinal ones – for 81% of all surface moieties. Silica groups are characterized by
pffiffiffiffiffi r 2 [nm] pH 4.6
pH 7.6
71.1
79.1
various tendencies to dissociation. The pKa value of isolated moieties was equal to 4.9, whereas of vicinal ones – 8.5. As a result, the SiO2 surface charge is very close to 0 in a large pH range (3–8). Under these pH conditions, a very small amount of silanol groups is dissociated. On the other hand, at basic pH values (pH N 8) almost all vicinal moieties (prevailing on the solid surface) dissociate and make the SiO2 surface charge clearly negative [42–44]. Using potentiometric titration method, the silica surface charge density (σ0) under various pH conditions and its point of zero charge (pHpzc) were determined. The SiO2 pHpzc parameter was about 3, which indicates that at pH 3 the amounts of positive (\\SiOH+ 2 ) and negative (\\SiO−) groups on the solid surface are identical. The σ0 value at pH 4.6 is about 0.05 μC/cm2, in turn at pH 7.6 it is about −2.5 μC/cm2 [45]. The adsorbed amount of soft proteins on the silica surface was higher at pH 4.6 than at pH 7.6. This difference for BSA was 0.43 mg/m2 and for OVA – 0.29 mg/m2. The pH 4.6 value is very close to the isoelectric points (pI) of albumins, which are as follows: 4.7–4.9 for BSA [46,47] and 4.43–4.9 for OVA [48,49]. Due to this fact, at pH 4.6 the total charge of proteins is close to zero (the concentrations of positively and negatively charged groups in their peptide chains are the same). The proteins have a very packed structure because the electrostatic forces within macromolecules, that may influence the spatial arrangement of the peptide chain, do not occur. At pH 4.6 the hydrodynamic radius (rh) of bovine serum albumin is equal to 3.32 nm, whereas that of ovalbumin – 2.95 nm [24]. Such small protein sizes make the adsorption of many macromolecules on the unit sorbent surface possible. At pH 7.6 the observed albumin adsorbed amounts were lower than at pH 4.6. It was mainly connected with the protein conformation changes. At pH 7.6 the total charge of macromolecules is negative, which means that negatively charged segments (containing mainly dissociated carboxylic groups) prevail in the peptide chain. These biopolymer fragments may repel each other and, as a result, the BSA/OVA size increases. At pH 7.6 the rh parameter for BSA/OVA equals 3.57 nm, in turn for OVA – 3.39 nm [24]. The macromolecules of bigger sizes occupy a larger part of the SiO2 surface and the adsorbed amount is reduced consequently. It is also worth mentioning that at pH 7.6 the solid surface has a slight negative charge (−2.5 μC/cm2). Due to this fact, there is a weak electrostatic repulsion between the albumin macromolecules and silica particles. This interaction hinders the contact of compounds and contributes to the lower adsorption level. Under electrostatic repulsion conditions, the albumin adsorption on the SiO2 surface is probably based on the hydrogen bond formation. They are formed between silica silanol groups and ionized groups present in protein macromolecules (carboxylic (\\COO−) and amino (\\NH+ 3 )) groups).
0.9 0.8 adsorpon [mg/m2]
silica was equal to 34 and, due to this fact, the Henry's formula was taken to the zeta potential (ζ) calculations [41]: μ¼
119
0.7 0.6 0.5
pH 4.6
0.4
pH 7.6
0.3 0.2 0.1 0 BSA
OVA
LSZ
EPS
Fig. 1. Adsorbed amount of proteins and exopolysaccharide on the silica surface.
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The adsorbed amount of hard protein, i.e. lysozyme, on the silica particles was higher at pH 7.6 than at pH 4.6. The difference equals 0.59 mg/m2. This observation was connected with various character of electrostatic interactions occurring between LSZ and SiO2 at two examined pH values. The lysozyme conformation does not change under examined conditions significantly because the LSZ macromolecules, thanks to their ‘hard’ properties, have a constant structure in a wide pH range. They do not denaturate in the pH range 1–8 [25]. At pH 4.6 the surface charge density is very close to 0 and, as a consequence, the electrostatic forces do not affect the adsorbed amount. It probably occurs based on the hydrogen bond formation between the macromolecules and the solid surface. In turn, at pH 7.6 negatively charged surface attracts the positive LSZ macromolecules (lysozyme is positively charged at both examined pH values because its isoelectric point is equal to 11). This electrostatic attraction favours the LSZ and silica contact and, thanks to it, the adsorbed amount is higher. Regardless of pH value, the adsorbed amounts observed for lysozyme were always higher than those noted for albumins. This was related to smaller sizes of LSZ macromolecules compared to the BSA/OVA ones. Smaller protein macromolecules (LSZ rh value is 1.95–1.98 nm [26,27] may adsorb on the solid surface in larger amounts because they penetrate the adsorbent pores more freely and their greater number is packed on the unit of solid surface. The results observed for exopolysaccharide were somewhat similar to those noticed for lysozyme. The EPS adsorption amount on the silica surface was also higher at pH 7.6 than at pH 4.6, but the noted differences were not so large (about 0.1 mg/m2). This is related to a small change in the polymer chain size at two examined pH values and different character of electrostatic forces occurring during pffiffiffiffiffi the adsorption process. Due to carboxylic group dissociation, the r 2 parameter of EPS macromolecules increases by 9 nm at higher pH value, but it is not a big change for the polymer chains characterized by the mass equal to 103–104 kDa. At pH 7.6 more carboxylic groups in the EPS chains are dissociated. The dissociation degree (α) of EPS at pH 4.6 is about 0.86, whereas at pH 7.6–0.99 [35]. It results in a slightly stronger electrostatic repulsion between the negative segments within the macromolecules at pH 7.6 and a bit bigger sizes of EPS chains. In the EPS case, larger macromolecule size is not equivalent with the lower adsorption amount as it was observed for albumins. Exopolysaccharide forms long ‘loops’ and ‘tails’ on the silica surface (perpendicular to the solid surface) and the biopolymer interacts with the solid surface by a few segments. Thanks to such biopolymer structure, expanded macromolecules may be adsorbed in a great quantity. The higher adsorbed amount observed at pH 7.6 is mainly associated with the electrostatic repulsion occurring between negative exopolysaccharide chains and negatively charged silica particles (at pH 4.6 it is not present due to the solid surface charge close to 0). This interaction hinders the contact of compounds and reduces the number of EPS segments bound to the surface. Owing to this, ‘loops’ and ‘tails’ become longer and a single macromolecule takes even smaller fragment of SiO2 surface. More biopolymer chains may be adsorbed. Under electrostatic repulsion conditions, hydrogen bonds are present in the system. They are formed between SiO2 silanol groups and EPS carboxylic groups. The adsorption layer formed by biopolymers on the silica surface at pH 7.6 is schematically shown in Fig. 2. Protein and polysaccharide effect on the zeta potential values of silica particles. Performed measurements showed that the isoelectric point (pHiep) of silica is about 3. This means that at pH 3 the charge of the slipping plane is equal to 0 (the quantities of positive and negative groups in the slipping plane area are the same). The determined pHiep value is consistent with the literature reports [45,50]. At pH 4.6 and 7.6 the SiO2 zeta potential is negative. It is about −2.5 mV and −17 mV, respectively. The silica ζ potential values measured in the absence and presence of biopolymers are presented in Fig. 3.
The protein adsorption affects the SiO2 zeta potential value. At pH 4.6, despite the fact that the total charge of albumins is close to 0, the BSA/OVA adsorption makes the ζ potential value more positive. This phenomenon is associated with a specific conformation of albumin macromolecules on the silica surface, in which positively charged fragments of peptide chains (containing mainly protonated amino groups) are located in the slipping plane area. The SiO2 zeta potential in the BSA presence is about 14 mV, whereas in the OVA presence – 13 mV. On the other hand, at pH 7.6, due to a negative charge of albumin macromolecules, their adsorption contributes to more negative values of silica electrokinetic potential. BSA makes the ζ value equal to −23 mV, in turn OVA – −26 mV. Under these conditions, negatively charged groups (mainly dissociated carboxylic ones) are located in the slipping plane and reduce the SiO2 electrokinetic potential. Lysozyme is positively charged at two examined pH values and, as a result, its addition to the system makes the silica zeta potential higher. At pH 4.6 it is about 1 mV, whereas at pH 7.6 – −2 mV. It is also worth emphasized that the protein influence on zeta potential values is stronger in such conditions, in which the protein adsorbed amount on the silica surface is higher. It is connected with larger amount of positive/negative groups present in the adsorbed BSA/OVA/LSZ macromolecules that may locate in the slipping plane area. The exopolysaccharide adsorption also changes the SiO2 zeta potential value. At pH 4.6 and 7.6, the adsorbed EPS chains are negatively charged due to the dissociation of almost all carboxylic groups present in the macromolecules. These moieties are located close to the slipping plane and contribute to lower values of SiO2 zeta potential. What is more, the EPS conformation in adsorption layer is characterized by long ‘loops’ and ‘tails’ perpendicular to the silica surface. These structures move the slipping plane towards the solution, which causes the ζ value reduction, additionally. This effect is not observed in the systems containing examined proteins due to a small size of their macromolecules. The protein/exopolysaccharide effect on the slipping plane position and charge is schematically presented in Fig. 2. The aggregation of silica particles in the absence and presence of biopolymers. The silica aggregation was established based on the turbidity measurements. The TSI values noticed for the examined systems are presented in Fig. 4. The silica suspension stability is strongly dependent on the pH conditions. At pH 4.6 the system turbidity becomes lower and lower over time, i.e. the suspension is of low stability (TSI is about 32). This phenomenon is mainly associated with the SiO2 surface charge density, which under these conditions is close to 0. There are no forces in the system that may hinder the particle contact. At pH 7.6 the silica suspension has high turbidity, which does not change over time, i.e. the system is relatively stable (TSI is about 4). Under these conditions the silica surface charge is about −2.5 μC/cm2 and, as a consequence, there is electrostatic stabilization in the system. The forces of electrostatic repulsion occur between the particles and the solid aggregation is considerably limited. At pH 7.6, high stability of the system is also guaranteed by very high negative values of SiO2 electrokinetic potential. The albumin influence on the SiO2 aggregation is different at pH 4.6 and 7.6. In the first case, the BSA/OVA adsorption contributes to the reduction of the system stability. At pH 4.6 the silica aggregation in the albumin presence is faster. The TSI value increases by 11 units for BSA and 9.5 units for OVA. This phenomenon is dictated by the zero total charge of adsorbed macromolecules. In the second case – at pH 7.6, the BSA/ OVA adsorption causes the minimal increase in the system stability. In the BSA/OVA presence TSI is lower by about 1.5 units. The negative charges of adsorbed macromolecules make the particles repulsion a bit stronger and, due to it, the solid aggregation does not occur. The system turbidity maintained at a constant level all the time. Lysozyme reduces the system stability at two examined pH values. This is associated with the neutralization of silica surface charge by positive LSZ macromolecules. At pH 4.6 this effect is weaker due to the smaller
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121
Fig. 2. Biopolymer macromolecules: bovine serum albumin (b), lysozyme (c), exopolysaccharide (d), adsorbed on the silica surface (a) and their effect on the slipping plane position and charge (pH 7.6).
SiO2 surface charge density. Under these conditions, the silica σ0 parameter is close to 0, but a small amount of isolated silanol groups is dissociated (their pKa point equals 4.9). The adsorbed macromolecules neutralize that charge and make the silica aggregation a bit easier. At pH 4.6 in the LSZ presence, the TSI value is higher by 7 units than those noted without the biopolymer. At pH 7.6 the SiO2 surface charge neutralization by lysozyme is very strong. In this case the TSI growth is the highest of all examined systems (about 40 units). As a result, after the LSZ addition, the silica suspension stability lowers quickly – big aggregates are formed. The neutralization of negative solid surface charge (−2.5 μC/cm2) weakens the repulsive forces acting between the particles significantly. It must be also mentioned that the silica aggregation in the lysozyme presence, at pH 4.6 and 7.6, is favoured by SiO2 zeta potential values, which under these conditions are about 1 and −2 mV, respectively. Exopolysaccharide adsorption also changes the silica suspension turbidity. At pH 4.6, the stability increase was observed (TSI was lower
by 9.5 units), which indicates that the silica aggregation was limited. It is probably dictated by electrosteric interactions based on the electrostatic repulsion between charges present in the adsorbed exopolysaccharide chains as well as steric repulsion of formed adsorption layers. On the other hand, at pH 7.6 a small decrease in the SiO2 suspension stability was noted (the TSI growth equals 4 units). This is caused by the formation of polymer bridges between the particles. A single EPS chain of large molecular mass may be adsorbed on two or more particles simultaneously, which stimulates its aggregation. To sum up, under acidic pH conditions (e.g. pH 4.6) the silica aggregation occurs, but this process is faster when albumins or lysozyme macromolecules are adsorbed on the SiO2 surface. In turn, under neutral pH conditions (i.e. pH 7.6) the silica aggregation is possible only in the lysozyme or exopolysaccharide presence. The structure of silica aggregates that may be formed in the biopolymer presence at two examined pH values are shown in Fig. 5.
20
50
15
45
10
40 35 30
0 5 10
silica
silica BSA
silica OVA
silica LSZ
silica EPS
pH 4.6 pH 7.6
15 20 25 30
Fig. 3. Zeta potential values of the silica particles in the absence and presence of macromolecular compounds (100 ppm).
TSI
[mV]
5
25
pH 4.6
20
pH 7.6
15 10 5 0 silica
silica BSA
silica OVA
silica LSZ
silica EPS
Fig. 4. Turbiscan Stability Index (TSI) values for the silica suspensions without and with biopolymers (100 ppm).
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4. Conclusions The performed experiments allowed to formulate the following conclusions: (1) protein adsorbed amount on the silica surface is strictly dependent on the solution pH value; this parameter determines the character of electrostatic interactions occurring within the macromolecules and between the solid particles and biopolymer, (2) at pH 4.6
more albumin macromolecules are bound with the SiO2 surface than at pH 7.6, (3) the lysozyme and exopolysaccharide macromolecules adsorb on the silica surface in greater amount at pH 7.6 compared to pH 4.6, (3) biopolymer adsorption affects the silica zeta potential values, (4) the zeta potential change is associated with the macromolecule charge and conformation under selected conditions, (5) the SiO2 particles aggregate easily at pH 4.6, (6) at pH 4.6 BSA, OVA and LSZ accelerate
Fig. 5. Structure of silica aggregates that may be formed in the biopolymer presence based on the electrostatic interactions, hydrogen bonding and/or polymer bridges formation at pH 4.6 (a) and 7.6 (b).
K. Szewczuk-Karpisz, M. Wiśniewska / Journal of Molecular Liquids 284 (2019) 117–123
the SiO2 aggregate formation, in turn at pH 7.6 a similar dependency was observed for LSZ and EPS, (7) the strongest positive effect on the silica aggregation was observed at pH 7.6 in the lysozyme presence (the highest TSI value growth). References [1] S. Uziak, Z. Klimowicz, Elementy geografii gleb i gleboznawstwa, Wydawnictwo UMCS, Lublin, 2002. [2] A.R. Dexter, A statistical measure of the structure of tilled soil, J. Agric. Eng. Res. 22 (1977) 101–104. [3] C. Andraud, A. Beghdadi, E. Haslund, R. Hilfer, J. Lafait, B. Virgin, Local entropy characterization of correlated random microstructures, Physica A 235 (1997) 307–318. [4] J.M. Tisdall, J.M. Oades, Organic matter and water-stable aggregates in soils, J. Soil Sci. 33 (1982) 141–163. [5] J.M. Oades, A.G. Waters, Aggregate hierarchy in soils, Aust. J. Soil Res. 29 (1991) 815–828. [6] J. Chorover, M.K. Amistadi, O.A. Chadwick, Surface charge evolution of mineral organic complexes during pedogenesis in Hawaiian basalt, Geochim. Cosmochim. Acta 68 (2004) 4859–4876. [7] B.T. Christensen, Physical fractionation of soil and structural and functional complexity in organic matter turnover, Eur. J. Soil Sci. 52 (2001) 345–353. [8] K.U. Totsche, W. Amelung, M.H. Gerzabek, G. Guggenberger, E. Klumpp, C. Knief, E. Lehndorff, R. Mikutta, S. Peth, A. Prechtel, N. Ray, I. Kogel-Knabner, Microaggregates in soils, J. Plant Nutr. Soil Sci. 181 (2018) 104–136. [9] K. Keiser, G. Guggenberger, Mineral surfaces and soil organic matter, Eur. J. Soil Sci. 54 (2003) 219–236. [10] Ch. Chen, J. Dynes, J. Wang, Ch. Karunakaran, D.L. Sparks, Soft X-ray spectromicroscopy study of mineral-organic matter associations in pasture soil clay fractions, Environ. Sci. Technol. 48 (12) (2014) 6678–6686. [11] R. Mikutta, M. Kleber, M.S. Torn, R. Jahn, Stabilization of soil organic matter: association with minerals or chemical recalcitrance? Biogeochemistry 77 (1) (2006) 25–56. [12] C.J. Newcomb, N.P. Qafoku, J.W. Grate, V.L. Bailey, J.J. De Yoreo, J.J., Developing a molecular picture of soil organic matter-mineral interactions by quantifying organomineral binding, Natura Commun. 8, Article number 396 (2017). [13] E. Skwarek, W. Janusz, D. Sternik, The inflence of the hydroxyapatite syntesis method on the electrochemical, surface and adsorption properties of hydroxyapatite, Adsorpt. Sci. Technol. (2017)https://doi.org/10.1177/0263617417698966. [14] E. Skwarek, S. Khalameida, W. Janusz, V. Sydorchuk, N. Konovalova, J. SkubiszewskaZięba, R. Leboda, Influence of mechanochemical activation on structure and some properties of mixed vanadium-molydbenum oxides, J. Therm. Anal. Calorim. 106 (3) (2011) 881–894. [15] W. Janusz, N.Y. Klymenko, E. Skwarek, S. Chibowski, J. Skubiszewska-Zięba, N.P. Galagan, K.N. Jurewnoy, V. Zarko, Structure of electrical double layer at the metal oxide with proteins/NaCl electrolyte solution interface, Adsorpt. Sci. Technol. (2015)https://doi.org/10.1260/0263-6174.33.6-8.567. [16] T. Klepka, Effectiveness of forming of polymeric special sliding layers, Polimery 47 (9) (2002) 649–653. [17] T. Klepka, R. Jeziorska, A. Szadkowska, Thin wall products made of modified high density polyethylene, Przemysł Chemiczny 94 (8) (2015) 1352–1355. [18] K. Szewczuk-Karpisz, M. Wiśniewska, Lysozyme adsorption on the colloidal chromium(III) oxide surface: its impact on the system stability, Appl. Surf. Sci. 347 (2015) 492–498. [19] K. Szewczuk-Karpisz, M. Wiśniewska, D. Myśliwiec, Albumin adsorption influence on the stability oft he mesoporous zirconia suspension, J. Ind. Eng. Chem. 32 (2015) 113–119. [20] S. Brauner, P.H. Emmett, E. Teller, Adsorption of gases in multimolecular layers, J. Am. Chem. Soc. 60 (2) (1938) 309–319. [21] W. Janusz, Determination of surface ionization and complexation constants from potentiometic titration data, Pol. J. Chem. 65 (1991) 799–807. [22] D.C. Carter, J.X. Ho, Structure of serum albumin, Adv. Protein Chem. 45 (1994) 153–203. [23] F.W. Putnam, The Plasma Proteins: Structure, Function and Genetic Control, 2nd ed. Academic Press, New York, 1975. [24] K. Szewczuk-Karpisz, M. Wiśniewska, Adsorption properties of the albuminchromium(III) oxide system – effect of solution pH and ionic strength, Soft Mater. 12 (3) (2014) 268–276. [25] K. Ogasahara, K. Hamagichi, Structure of lysozyme: XII. Effect of pH on the stability of lysozyme, J. Biochem. 61 (2) (1966) 199–210. [26] D.E. Kuehner, C. Heyer, C. Ramsch, U.M. Fornefeld, H.W. Blanch, J.M. Prausnitz, Interactions of lysozyme in concentrated electrolyte solutions from dynamic lightscaterring measurements, Biophys. J. 73 (1997) 3211–3224. [27] S.A. Parmar, M. Muschol, Hydratation and hydrodynamic interactions of lysozyme: effects of chaotropic and kosmotropic ions, Biophys. J. 97 (2) (2009) 590–598.
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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq
Adsorption layer structure at soil mineral/biopolymer/supporting electrolyte interface – The impact on solid aggregation Katarzyna Szewczuk-Karpisz a,⁎, Małgorzata Wiśniewska b 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 l e
i n f o
Article history: Received 11 March 2019 Received in revised form 27 March 2019 Accepted 29 March 2019 Available online 31 March 2019 Keywords: Protein Exopolysaccharide Biopolymer conformation System turbidity Zeta potential
a b s t r a c t This paper focused on the protein and polysaccharide adsorption mechanism on the surface of common soil mineral – silica (SiO2), as well as the biopolymer adsorption effect on the solid aggregation. For the experiments, the proteins of various internal stability were selected, i.e. bovine serum albumin (BSA), ovalbumin (OVA) and lysozyme (LSZ). Moreover, the study involved exopolysaccharide (EPS) synthesized by soil bacteria Sinorhizobium meliloti. The adsorption layer structure on the silica surface and the solid aggregation tendency in the biopolymer presence were established and explained based on the turbidimetric, adsorption and zeta potential measurements. The measured biopolymer adsorbed amounts, biopolymer conformation on the SiO2 surface and solid aggregation were strongly dependent on the pH conditions. The stimulating effect on SiO2 aggregate formation was stated at pH 4.6 for BSA, OVA and LSZ, whereas at pH 7.6 – for LSZ and EPS. The strongest impact was noticed at pH 7.6 in the lysozyme presence (the TSI value increased by 40 units). The presented results can contribute to a better understanding of mineral-organic associations formation under selected soil conditions. Moreover, they may be helpful in developing innovative substances limiting destructive erosion effect. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Soil structure is one of its morphological features, which defines the spatial distribution of solid phase particles as well as their shape and size. Soil structure affects other soil properties, mainly water, air and thermal ones. Proper aggregation accelerates the water filtration into the soil profile and limits its evaporation [1]. Formation of soil aggregates is a very complex process. During the last fifty years, many theories about this process have been developed. The first papers concerned the relationship between soil aggregation and thermodynamic function – entropy [2,3]. Then, the scientists focused on the role of organic-mineral associations in the soil microaggregation. Tisdall and Oades [4] stated that primary soil microaggregates are formed by surrounding organic debris with fine minerals. These structures are stabilized with various substances synthesized by microorganisms and fungi [5]. Chorover et al. [6] found that the mineral-organic connections are formed by electrostatic interactions, in turn Christensen [7] considered them as basic units for larger soil aggregates. Mineral-organic associations occur between the organic and inorganic matter present in the soil [8]. Their formation is determined by various processes. Among physicochemical phenomena, adsorption is the most important one. It occurs shortly after the first contact of soil compounds ⁎ Corresponding author. E-mail address: [email protected] (K. Szewczuk-Karpisz).
https://doi.org/10.1016/j.molliq.2019.03.172 0167-7322/© 2019 Elsevier B.V. All rights reserved.
and affects the microaggregate stabilization significantly [9]. Mineralorganic matter associations are examined by various methods. Chen et al. [10] used a X-ray spectromicroscopy to examine the associations present in pasture soil clay fractions. Mikutta et al. [11] determined the stability of the connections between soil organic matter and minerals using isotopes. In turn, Newcomb et al. [12] measured the binding between organic substances and soil minerals in aqueous environment using a force spectroscopy. Taking into account the key role of organic matter adsorption on the mineral surface in the soil structure and aggregate stability, this paper focused on the biopolymer adsorption on the surface of widespread soil mineral – silica (SiO2) and its impact on the SiO2 particle aggregation. For the experiments, two biopolymer types were selected. The first was exopolysaccharide (EPS) synthesized by soil bacteria Sinorhizobium meliloti, the second – proteins characterized by different structural properties: ovalbumin (OVA), bovine serum albumin (BSA), lysozyme (LSZ). The study included the adsorbed amount measurements, zeta potential determination and turbidity measurements. They induced the biopolymer amount involved in silica-organic associations and, owing to it, immobilized by the mineral soil compound. What is more, they provided the information about silica tendency to aggregation when the proteins or exopolysaccharide are present in the system. All measurements were performed as a function of the solution pH value, i.e. at pH 4.6 and 7.6 (corresponding with common soil environment conditions). Based on the obtained results, the structure
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of adsorption layer formed on the SiO2 surface was proposed. Moreover, the organic substance whose influence on the SiO2 aggregation is the strongest was indicated. Research on the adsorption mechanism of natural and synthetic polymers on the inorganic material surface is a current issue. Some scientists have conducted studies on other mineral oxides, e.g. hydroxyapatite [13] and mixed oxides [14]. Janusz et al. [15] examined the BSA adsorption process on the modified silica surface, in turn Klepka et al. [16,17] are working on specific polymer layers. In our previous work, we focused on adsorptive, electrokinetic and stability properties of chromium(III) oxide and zirconia suspensions in the absence and presence of various macromolecular compounds [18,19]. For this study, we selected silica because it is involved in the soil structure formation. The results presented in this paper are of high scientific value and may find practical application in agriculture. They can be very helpful in developing of innovative additives that improve the soil structure and resistance to erosion. What is more, the paper may contribute to better explanation of the interactions between soil minerals and organic matter occurring in soil environment. 2. Experimental
Table 2 Proteins characteristics: Mw – molecular weight, pI – protein isoelectric point, rh – hydrodynamic radius [24,26,27,46–49,51–54]. Name
Bovine serum albumin Ovalbumin Lysozyme
Abbreviation Mw pI [kDa]
Structural stability
rh [nm] pH 4.6
pH 7.6 3.57
BSA
66.43 4.7–4.9 low
3.32
OVA LSZ
42.7 14.3
2.95 3.39 1.85–1.89 1.85–1.89
4.4–4.9 low 11 high
established using a potentiometric titration and graphic method. Based on this parameter, the EPS dissociation degree (α) at pH 4.6 and 7.6 was also calculated [32,33]. The exopolysaccharide molecule size was determined based on the viscosity of EPS solutions measured under various pH conditions. It was expressed through a parameter called ‘root-meanpffiffiffiffiffi square chain end-to-end distance’ ( r 2 ) [34,35]. The exopolysaccharide used is a succinoglycan; its monomer is composed of seven glucose molecules and one galactose molecule connected by various glycosidic bonds. Such EPS chain is substituted by acetyl, pyruvyl and succinyl groups [36,37]. 2.2. Methods
2.1. Materials Silica (silicon(IV) oxide, SiO2, CAS 7631-86-9) delivered by Sigma Aldrich was used in the study. The solid was characterized by several methods. The SiO2 average pore diameter (Dp) and specific surface area (SBET) were determined using a nitrogen adsorption/desorption isotherm method (analyzer ASAP 2405, Micrometritics) and BET equation [20]. The pHpzc parameter (point of zero charge) and surface charge density were established by a potentiometric titration method [21]. The SiO2 average particle size (d) was measured using a zetameter (Zetasizer 3000, Malvern Instruments), whereas its isoelectric point (pHiep) was determined using a zetameter equipped with a titrator (Zetasizer NanoZS, Malvern Instruments). The obtained parameters are summarized in Table 1. Before the experiment start, the mineral oxide was washed with double distilled water until the supernatant conductivity was below 3 μS/cm. Bovine serum albumin (BSA, CAS 9048-46-8), ovalbumin (OVA, CAS 9006-59-1) and lysozyme (LSZ, CAS 12650–88-3), delivered by Sigma Aldrich, were also used in the experiments. Their parameters (e.g. molecular mass, isoelectric points) are summarized in Table 2. The used proteins were characterized by different internal stability. BSA and OVA are ‘soft proteins’ (of low structural stability), which means that their molecule size differs under various pH conditions [22,23]. The hydrodynamic radius (rh) of BSA and OVA molecules at pH 4.6 is about 3.32 nm and 2.94 nm, whereas at pH 7.6–3.57 nm and 3.39 nm, respectively [24]. Lysozyme is a ‘hard protein’ (of high internal stability) and, as a result, contains a specific conformation in a wide pH range [25]. Within pH range 4–7, the value of rh parameter of the LSZ macromolecules is about 1.85 nm [26] or 1.89 nm [27]. All proteins used are components of soil organic matter (organic debris) [28–30]. Exopolysaccharide synthesized by soil bacteria (Sinorhizobium meliloti) was isolated according to the defined procedure [31]. EPS parameters are summarized in Table 3. The pKa value of the polysaccharide was Table 1 Silica characteristics: Dp – average pore diameter, SBET – specific surface area, d – average particle size, pHiep – isoelectric point, pHpzc – point of zero charge. Parameter
Value
Dp [nm] SBET [m2/g] pHiep pHpzc d [nm]
11.3 145 3 3 225
All experiments were carried out at 25 °C, using 0.01 M NaCl as a supporting electrolyte. The measurements were performed at pH values, i.e. pH 4.6 and 7.6. The pH value was established using 0.1 M HCl, 0.1 M NaOH and a pHmeter Beckman. Biopolymer adsorbed amounts on the silica surface were measured based on the difference in the protein concentration before and after the adsorption process according to the formula [38]: Γ¼
cads ∙V SBET ∙m
ð1Þ
where cads – the polymer adsorbed concentration, the difference in the polymer concentration in the system before and after its adsorption, V – the suspension volume, m – the solid weight. At the beginning, the suspensions were prepared by adding appropriate solid weight to the solution containing supporting electrolyte and selected biopolymer (10–500 ppm). The solid weights in the samples (10 ml) were as follows: 0.0085 g for the protein adsorption and 0.0035 g for the EPS adsorption. These amounts were determined based on the silica specific surface area and the biopolymer properties. After the suspension preparation, the pH value was adjusted and the adsorption was started. The process was performed under shaking conditions (shaking bath Unimax 1010, Heidolph). Its time was determined based on previous kinetic measurements – 7 h for SiO2-protein, 20 h for SiO2EPS. When the adsorption finished, the probes were centrifuged (223e, MPW Med. Instruments; 10,000 rpm) and the supernatants were taken for the biopolymer quantification. The protein/polysaccharide concentration was determined by a spectrophotometric method (spectrophotometer UV–Vis Cary 100, Agilent Technology). The protein concentration was measured at the wavelength, at which the maximum UV absorbance for the protein solution was observed (280 nm – for lysozyme, 279 nm – for bovine serum albumin, 278 nm – for ovalbumin [39]. In turn, the exopolysaccharide concentration was determined using the method developed by Dubois et al. [40]. One adsorption result was the repetition of three measurements. The error (standard deviation) did not exceed 5%. Electrophoretic mobility (μ) of the silica particles, in the absence and presence of biopolymers, was determined using a zetameter (Zetasizer Nano ZS, Malvern Instruments) based on the microelectrophoresis phenomenon. During the measurement, the particles moved in the electrophoretic cell, towards the cathode or anode (depending on the sign of their charge). When the applied voltage compensated their movement, the device read the electrophoretic mobility value. The κa parameter for
K. Szewczuk-Karpisz, M. Wiśniewska / Journal of Molecular Liquids 284 (2019) 117–123 Table 3 Exopolysaccharide characteristics:
pffiffiffiffiffi r 2 – root-mean-square chain end-to-end distance [35,55,56].
Name
Abbreviation
Mw [kDa]
pKa
Exopolysaccharide S. meliloti
EPS
103–104
3.8
εζ f ðκaÞ η
ð2Þ
where: ε – the dielectric constant, η – the viscosity, 1/κ – the thickness of electrical double layer, a – the particle radius. The samples for electrophoretic mobility measurements were prepared by adding 0.01 g of SiO2 to the supporting electrolyte solution (20 ml). After the 3-min sonification, the initial pH value was adjusted. The measurements were carried out using a titrator, which changed the suspension pH value automatically. The examined pH range was 3–9. The biopolymer was added to the system just before the measurement start. Its concentration equals 100 ppm. A single result of electrophoretic mobility was the repetition of three measurements. The error did not exceed 5%. The silica aggregation, in the absence and presence of macromolecular compounds, was established based on the turbidimetric measurements (Turbiscan TLabExpert with a cooling module TLab Cooling). The apparatus measured the transmission and backscattering of the light beam (λ = 880 nm) passing through the sample and determined the suspension stability by the Turbiscan Stability Index (TSI), which was calculated using the following equation: sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi n ∑i¼1 ðxi −xBS Þ2 TSI ¼ n−1
ð3Þ
where: xi – the average backscattering for each minute of measurement, xBS – the average xi value, n – the scans number. This parameter changes from 0 (highly stable systems) to 100 (extremely unstable ones). The probes (20 ml) were prepared by adding 0.02 g of SiO2 to the supporting electrolyte solution. After the 3-min sonification, the biopolymer was added to the suspensions and the pH value was adjusted. The protein/polysaccharide concentration was equal to 100 ppm. Suspension stability was measured two times. The obtained results were almost identical (the error did not exceed 1.5%). 3. Results and discussion Structure of protein(polysaccharide) adsorption layer on the silica surface. The obtained adsorbed amounts of the examined biopolymers on the SiO2 surface are presented in Fig. 1. These amounts were strongly dependent on the solution pH value. What is more, they were associated with the protein internal stability. The results observed for soft and hard proteins were different. At pH 4.6, the adsorption amounts of ovalbumin and bovine serum albumin (albumins, soft proteins) were higher than at pH 7.6. In turn, more lysozyme macromolecules bound to the SiO2 surface at pH 7.6 than at pH 4.6. The EPS adsorbed amount was also dependent on the solution pH value, but the noted differences were smaller than those observed for proteins. The silica surface has unique properties. In aqueous solutions it contains two types of groups: (1) isolated silanol groups – formed when one Si atom bind to three Si atoms inside the solid phase and one hydroxyl group, and (2) vicinal silanol groups – formed through the connection of two isolated silanol groups bind to two different Si atoms by a hydrogen bond. The isolated groups account for 19% and the vicinal ones – for 81% of all surface moieties. Silica groups are characterized by
pffiffiffiffiffi r 2 [nm] pH 4.6
pH 7.6
71.1
79.1
various tendencies to dissociation. The pKa value of isolated moieties was equal to 4.9, whereas of vicinal ones – 8.5. As a result, the SiO2 surface charge is very close to 0 in a large pH range (3–8). Under these pH conditions, a very small amount of silanol groups is dissociated. On the other hand, at basic pH values (pH N 8) almost all vicinal moieties (prevailing on the solid surface) dissociate and make the SiO2 surface charge clearly negative [42–44]. Using potentiometric titration method, the silica surface charge density (σ0) under various pH conditions and its point of zero charge (pHpzc) were determined. The SiO2 pHpzc parameter was about 3, which indicates that at pH 3 the amounts of positive (\\SiOH+ 2 ) and negative (\\SiO−) groups on the solid surface are identical. The σ0 value at pH 4.6 is about 0.05 μC/cm2, in turn at pH 7.6 it is about −2.5 μC/cm2 [45]. The adsorbed amount of soft proteins on the silica surface was higher at pH 4.6 than at pH 7.6. This difference for BSA was 0.43 mg/m2 and for OVA – 0.29 mg/m2. The pH 4.6 value is very close to the isoelectric points (pI) of albumins, which are as follows: 4.7–4.9 for BSA [46,47] and 4.43–4.9 for OVA [48,49]. Due to this fact, at pH 4.6 the total charge of proteins is close to zero (the concentrations of positively and negatively charged groups in their peptide chains are the same). The proteins have a very packed structure because the electrostatic forces within macromolecules, that may influence the spatial arrangement of the peptide chain, do not occur. At pH 4.6 the hydrodynamic radius (rh) of bovine serum albumin is equal to 3.32 nm, whereas that of ovalbumin – 2.95 nm [24]. Such small protein sizes make the adsorption of many macromolecules on the unit sorbent surface possible. At pH 7.6 the observed albumin adsorbed amounts were lower than at pH 4.6. It was mainly connected with the protein conformation changes. At pH 7.6 the total charge of macromolecules is negative, which means that negatively charged segments (containing mainly dissociated carboxylic groups) prevail in the peptide chain. These biopolymer fragments may repel each other and, as a result, the BSA/OVA size increases. At pH 7.6 the rh parameter for BSA/OVA equals 3.57 nm, in turn for OVA – 3.39 nm [24]. The macromolecules of bigger sizes occupy a larger part of the SiO2 surface and the adsorbed amount is reduced consequently. It is also worth mentioning that at pH 7.6 the solid surface has a slight negative charge (−2.5 μC/cm2). Due to this fact, there is a weak electrostatic repulsion between the albumin macromolecules and silica particles. This interaction hinders the contact of compounds and contributes to the lower adsorption level. Under electrostatic repulsion conditions, the albumin adsorption on the SiO2 surface is probably based on the hydrogen bond formation. They are formed between silica silanol groups and ionized groups present in protein macromolecules (carboxylic (\\COO−) and amino (\\NH+ 3 )) groups).
0.9 0.8 adsorpon [mg/m2]
silica was equal to 34 and, due to this fact, the Henry's formula was taken to the zeta potential (ζ) calculations [41]: μ¼
119
0.7 0.6 0.5
pH 4.6
0.4
pH 7.6
0.3 0.2 0.1 0 BSA
OVA
LSZ
EPS
Fig. 1. Adsorbed amount of proteins and exopolysaccharide on the silica surface.
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The adsorbed amount of hard protein, i.e. lysozyme, on the silica particles was higher at pH 7.6 than at pH 4.6. The difference equals 0.59 mg/m2. This observation was connected with various character of electrostatic interactions occurring between LSZ and SiO2 at two examined pH values. The lysozyme conformation does not change under examined conditions significantly because the LSZ macromolecules, thanks to their ‘hard’ properties, have a constant structure in a wide pH range. They do not denaturate in the pH range 1–8 [25]. At pH 4.6 the surface charge density is very close to 0 and, as a consequence, the electrostatic forces do not affect the adsorbed amount. It probably occurs based on the hydrogen bond formation between the macromolecules and the solid surface. In turn, at pH 7.6 negatively charged surface attracts the positive LSZ macromolecules (lysozyme is positively charged at both examined pH values because its isoelectric point is equal to 11). This electrostatic attraction favours the LSZ and silica contact and, thanks to it, the adsorbed amount is higher. Regardless of pH value, the adsorbed amounts observed for lysozyme were always higher than those noted for albumins. This was related to smaller sizes of LSZ macromolecules compared to the BSA/OVA ones. Smaller protein macromolecules (LSZ rh value is 1.95–1.98 nm [26,27] may adsorb on the solid surface in larger amounts because they penetrate the adsorbent pores more freely and their greater number is packed on the unit of solid surface. The results observed for exopolysaccharide were somewhat similar to those noticed for lysozyme. The EPS adsorption amount on the silica surface was also higher at pH 7.6 than at pH 4.6, but the noted differences were not so large (about 0.1 mg/m2). This is related to a small change in the polymer chain size at two examined pH values and different character of electrostatic forces occurring during pffiffiffiffiffi the adsorption process. Due to carboxylic group dissociation, the r 2 parameter of EPS macromolecules increases by 9 nm at higher pH value, but it is not a big change for the polymer chains characterized by the mass equal to 103–104 kDa. At pH 7.6 more carboxylic groups in the EPS chains are dissociated. The dissociation degree (α) of EPS at pH 4.6 is about 0.86, whereas at pH 7.6–0.99 [35]. It results in a slightly stronger electrostatic repulsion between the negative segments within the macromolecules at pH 7.6 and a bit bigger sizes of EPS chains. In the EPS case, larger macromolecule size is not equivalent with the lower adsorption amount as it was observed for albumins. Exopolysaccharide forms long ‘loops’ and ‘tails’ on the silica surface (perpendicular to the solid surface) and the biopolymer interacts with the solid surface by a few segments. Thanks to such biopolymer structure, expanded macromolecules may be adsorbed in a great quantity. The higher adsorbed amount observed at pH 7.6 is mainly associated with the electrostatic repulsion occurring between negative exopolysaccharide chains and negatively charged silica particles (at pH 4.6 it is not present due to the solid surface charge close to 0). This interaction hinders the contact of compounds and reduces the number of EPS segments bound to the surface. Owing to this, ‘loops’ and ‘tails’ become longer and a single macromolecule takes even smaller fragment of SiO2 surface. More biopolymer chains may be adsorbed. Under electrostatic repulsion conditions, hydrogen bonds are present in the system. They are formed between SiO2 silanol groups and EPS carboxylic groups. The adsorption layer formed by biopolymers on the silica surface at pH 7.6 is schematically shown in Fig. 2. Protein and polysaccharide effect on the zeta potential values of silica particles. Performed measurements showed that the isoelectric point (pHiep) of silica is about 3. This means that at pH 3 the charge of the slipping plane is equal to 0 (the quantities of positive and negative groups in the slipping plane area are the same). The determined pHiep value is consistent with the literature reports [45,50]. At pH 4.6 and 7.6 the SiO2 zeta potential is negative. It is about −2.5 mV and −17 mV, respectively. The silica ζ potential values measured in the absence and presence of biopolymers are presented in Fig. 3.
The protein adsorption affects the SiO2 zeta potential value. At pH 4.6, despite the fact that the total charge of albumins is close to 0, the BSA/OVA adsorption makes the ζ potential value more positive. This phenomenon is associated with a specific conformation of albumin macromolecules on the silica surface, in which positively charged fragments of peptide chains (containing mainly protonated amino groups) are located in the slipping plane area. The SiO2 zeta potential in the BSA presence is about 14 mV, whereas in the OVA presence – 13 mV. On the other hand, at pH 7.6, due to a negative charge of albumin macromolecules, their adsorption contributes to more negative values of silica electrokinetic potential. BSA makes the ζ value equal to −23 mV, in turn OVA – −26 mV. Under these conditions, negatively charged groups (mainly dissociated carboxylic ones) are located in the slipping plane and reduce the SiO2 electrokinetic potential. Lysozyme is positively charged at two examined pH values and, as a result, its addition to the system makes the silica zeta potential higher. At pH 4.6 it is about 1 mV, whereas at pH 7.6 – −2 mV. It is also worth emphasized that the protein influence on zeta potential values is stronger in such conditions, in which the protein adsorbed amount on the silica surface is higher. It is connected with larger amount of positive/negative groups present in the adsorbed BSA/OVA/LSZ macromolecules that may locate in the slipping plane area. The exopolysaccharide adsorption also changes the SiO2 zeta potential value. At pH 4.6 and 7.6, the adsorbed EPS chains are negatively charged due to the dissociation of almost all carboxylic groups present in the macromolecules. These moieties are located close to the slipping plane and contribute to lower values of SiO2 zeta potential. What is more, the EPS conformation in adsorption layer is characterized by long ‘loops’ and ‘tails’ perpendicular to the silica surface. These structures move the slipping plane towards the solution, which causes the ζ value reduction, additionally. This effect is not observed in the systems containing examined proteins due to a small size of their macromolecules. The protein/exopolysaccharide effect on the slipping plane position and charge is schematically presented in Fig. 2. The aggregation of silica particles in the absence and presence of biopolymers. The silica aggregation was established based on the turbidity measurements. The TSI values noticed for the examined systems are presented in Fig. 4. The silica suspension stability is strongly dependent on the pH conditions. At pH 4.6 the system turbidity becomes lower and lower over time, i.e. the suspension is of low stability (TSI is about 32). This phenomenon is mainly associated with the SiO2 surface charge density, which under these conditions is close to 0. There are no forces in the system that may hinder the particle contact. At pH 7.6 the silica suspension has high turbidity, which does not change over time, i.e. the system is relatively stable (TSI is about 4). Under these conditions the silica surface charge is about −2.5 μC/cm2 and, as a consequence, there is electrostatic stabilization in the system. The forces of electrostatic repulsion occur between the particles and the solid aggregation is considerably limited. At pH 7.6, high stability of the system is also guaranteed by very high negative values of SiO2 electrokinetic potential. The albumin influence on the SiO2 aggregation is different at pH 4.6 and 7.6. In the first case, the BSA/OVA adsorption contributes to the reduction of the system stability. At pH 4.6 the silica aggregation in the albumin presence is faster. The TSI value increases by 11 units for BSA and 9.5 units for OVA. This phenomenon is dictated by the zero total charge of adsorbed macromolecules. In the second case – at pH 7.6, the BSA/ OVA adsorption causes the minimal increase in the system stability. In the BSA/OVA presence TSI is lower by about 1.5 units. The negative charges of adsorbed macromolecules make the particles repulsion a bit stronger and, due to it, the solid aggregation does not occur. The system turbidity maintained at a constant level all the time. Lysozyme reduces the system stability at two examined pH values. This is associated with the neutralization of silica surface charge by positive LSZ macromolecules. At pH 4.6 this effect is weaker due to the smaller
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Fig. 2. Biopolymer macromolecules: bovine serum albumin (b), lysozyme (c), exopolysaccharide (d), adsorbed on the silica surface (a) and their effect on the slipping plane position and charge (pH 7.6).
SiO2 surface charge density. Under these conditions, the silica σ0 parameter is close to 0, but a small amount of isolated silanol groups is dissociated (their pKa point equals 4.9). The adsorbed macromolecules neutralize that charge and make the silica aggregation a bit easier. At pH 4.6 in the LSZ presence, the TSI value is higher by 7 units than those noted without the biopolymer. At pH 7.6 the SiO2 surface charge neutralization by lysozyme is very strong. In this case the TSI growth is the highest of all examined systems (about 40 units). As a result, after the LSZ addition, the silica suspension stability lowers quickly – big aggregates are formed. The neutralization of negative solid surface charge (−2.5 μC/cm2) weakens the repulsive forces acting between the particles significantly. It must be also mentioned that the silica aggregation in the lysozyme presence, at pH 4.6 and 7.6, is favoured by SiO2 zeta potential values, which under these conditions are about 1 and −2 mV, respectively. Exopolysaccharide adsorption also changes the silica suspension turbidity. At pH 4.6, the stability increase was observed (TSI was lower
by 9.5 units), which indicates that the silica aggregation was limited. It is probably dictated by electrosteric interactions based on the electrostatic repulsion between charges present in the adsorbed exopolysaccharide chains as well as steric repulsion of formed adsorption layers. On the other hand, at pH 7.6 a small decrease in the SiO2 suspension stability was noted (the TSI growth equals 4 units). This is caused by the formation of polymer bridges between the particles. A single EPS chain of large molecular mass may be adsorbed on two or more particles simultaneously, which stimulates its aggregation. To sum up, under acidic pH conditions (e.g. pH 4.6) the silica aggregation occurs, but this process is faster when albumins or lysozyme macromolecules are adsorbed on the SiO2 surface. In turn, under neutral pH conditions (i.e. pH 7.6) the silica aggregation is possible only in the lysozyme or exopolysaccharide presence. The structure of silica aggregates that may be formed in the biopolymer presence at two examined pH values are shown in Fig. 5.
20
50
15
45
10
40 35 30
0 5 10
silica
silica BSA
silica OVA
silica LSZ
silica EPS
pH 4.6 pH 7.6
15 20 25 30
Fig. 3. Zeta potential values of the silica particles in the absence and presence of macromolecular compounds (100 ppm).
TSI
[mV]
5
25
pH 4.6
20
pH 7.6
15 10 5 0 silica
silica BSA
silica OVA
silica LSZ
silica EPS
Fig. 4. Turbiscan Stability Index (TSI) values for the silica suspensions without and with biopolymers (100 ppm).
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4. Conclusions The performed experiments allowed to formulate the following conclusions: (1) protein adsorbed amount on the silica surface is strictly dependent on the solution pH value; this parameter determines the character of electrostatic interactions occurring within the macromolecules and between the solid particles and biopolymer, (2) at pH 4.6
more albumin macromolecules are bound with the SiO2 surface than at pH 7.6, (3) the lysozyme and exopolysaccharide macromolecules adsorb on the silica surface in greater amount at pH 7.6 compared to pH 4.6, (3) biopolymer adsorption affects the silica zeta potential values, (4) the zeta potential change is associated with the macromolecule charge and conformation under selected conditions, (5) the SiO2 particles aggregate easily at pH 4.6, (6) at pH 4.6 BSA, OVA and LSZ accelerate
Fig. 5. Structure of silica aggregates that may be formed in the biopolymer presence based on the electrostatic interactions, hydrogen bonding and/or polymer bridges formation at pH 4.6 (a) and 7.6 (b).
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