3.1. Characterization of Glass Fibers
To characterize the glass fibers, we performed a scanning electron microscopy (SEM) analysis, which provides a complex image of the surface topography of the sample at high magnification. The SEM image of the glass fibers shown in
Figure 2 was recorded with the Philips SEM XL series. We can see that a single fiber has a diameter of about 10 mm. We can see that the diameter of the glass fiber is the same over the entire length of the fiber. The length of the fibers is not important because we used the glass fiber sheets for the immobilization of the enzyme and later for the lactose hydrolysis reaction.
The thermogravimetric analysis of the glass fibers was carried out with the TGA 2 from Mettler Toledo. The analysis was performed in the temperature range from 30 to 800 °C with a heating rate of 10 °C/min in an air atmosphere. The results of the thermogravimetric analysis are presented in
Figure 3.
According to thermogravimetric analysis, the total loss of mass was less than 2.9%. Although the mass loss was really low, it was attributed to the loss of water at lower temperatures and probably some impurities at temperatures above 200°C. This is because the characterization was performed with the glass fibers as received without purification.
The N
2 adsorption/desorption technique was used to measure the surface area and pore size of the glass fibers. BET was performed with TriStar II from Micromeritics. The results are shown in
Figure 4.
As we can see from the results of the BET measurement in
Figure 4, the hysteresis loop was not fully completed. This is due to the very small surface area of the glass fibers. The BET surface area was determined to be 0.25 m2/g and the pore size 2.29 nm. Since the available surface area is small, we have to use a larger mass of the support for the immobilization of the enzyme compared to supports with a large surface area.
3.2. Hydrolysis of Lactose
Firstly, we tested the influence of GA and APTES concentration on the yield of immobilization of β-galactosidase and further on the hydrolysis of lactose conversion.
The immobilization procedure was performed with four different GA concentrations (
φGA = 0.5, 1, 2, 5%), while the APTES concentration (
φAPTES = 10%) and the galactosidase enzyme concentrations were kept constant. The dynamic profiles of lactose conversion until steady state was reached are shown in
Figure 5. The hydrolysis of lactose was carried out over a period of 4 hours and samples were taken every hour to analyze the amount of glucose formed. In this step, the experiments were carried out at a temperature of
T = 30 °C and with a substrate flow rate of
qV = 3 mL/min. The lactose solutions were prepared with phosphate buffer at
pH = 7 and a concentration of
c = 0.05 mol/L.
Figure 5 shows that the steady state was reached after 1 hour and that the highest conversion of lactose (30%) was achieved with GA at a concentration of
φGA = 0.5%.
We continued with the experiments by changing the concentrations of APTES using the previously determined optimal concentration of GA (
φGA = 0.5%).
Figure 6 shows that the highest lactose conversion (30%) was achieved with APTES at a concentration of
φAPTES = 10%.
The optimal concentration of APTES for successful β-galactosidase immobilization varies [
30,
31]. Therefore, we tested the immobilization procedure with APTES at concentrations of
φAPTES = 10% and 20%. Since a higher concentration only decreased the immobilization efficiency (
Figure 6), we continued the study with the previously reported value.
After determining the optimal immobilization conditions (φGA = 0.5% and φAPTES = 10%), we investigated the influence of substrate flow rate, reaction temperature and pH on lactose conversion.
The influence of the lactose flow rate (1, 2, 3 mL/min) on the final conversion is shown in
Figure 7. These experiments were performed at a temperature of
T = 20 °C. The reaction of lactose hydrolysis was carried out for 4 hours. As expected, the lowest lactose flow rate,
qV = 1 mL/min, results in the highest conversion of lactose, namely 38.7%. At the two higher flow rates, the conversion was almost the same, 26.8%.
In the next step, we repeated the experiments at 25 °C. Again, we investigated the effects of the flow rate of the lactose solution on the reaction conversion –
Figure 8.
At a temperature of
T = 25 °C and a flow rate of
qV = 1 mL/min, a conversion of up to 50% was reached (
Figure 8). At lower flow rates, the conversions were again similar and around 29%.
Regardless of the temperature, we observed that at low flow rates (qV = 1 mL/min) the shape of the glass fiber roll apparently allowed good contact between substrate and immobilized enzyme, while at higher flow rates some of the substrate flowed through the small hole in the middle of the roll, where there was less contact with the enzyme and consequently conversions was noticeable lower.
Figure 9 shows that at 30 °C the highest conversion of lactose is 56% at the lowest flow rate and more than 30% lower at the two higher flow rates. The difference in conversion of lactose of about 5% was observed between 2 and 3 mL/min only at the highest temperature of 30 °C, while the profiles of lactose conversion at the two highest flow rates are practically the same at the two lower temperatures (20 and 25 °C). The difference is probably more pronounced at higher temperatures, which is due to the higher reaction rate.
If we compare the conversions of lactose at the same flow rate as a function of temperature (
Figure 7,
Figure 8 and
Figure 9), we see that within this temperature range, the conversion of lactose increases with increasing temperature. We could probably expect a higher conversion if we further decreased the flow rate of lactose. However, for our study we only tested the efficiency of immobilization at three selected flow rates. The optimal temperature range for the hydrolysis of lactose is between 25 and 50 °C and depends on whether the enzyme is free or immobilized [
1,
15,
32].
In order to determine the optimal values of the most important process parameters for the lactose hydrolysis reaction, we also investigated the influence of the
pH of the phosphate buffer used for the preparation of the lactose solutions. We performed the hydrolysis of lactose at three
pH values of the substrate: 6, 7 and 8.
Figure 10 shows the conversion profiles during the reaction until steady state was reached for different
pH values of the lactose solution.
From all three conversion profiles in
Figure 10, we can see that a steady state was reached after about 3 hours. The highest conversion of lactose (50%) was obtained at
pH = 7, while the conversion of lactose was slightly lower at
pH = 6 (40%) and less than 10% at
pH = 8. As can be read in the literature, the optimal
pH of lactose hydrolysis using the galactosidase enzyme varies depending on the origin of the b-galactosidase, the type of enzyme (free or immobilized) and the substrate used [
32,
33,
34]. The optimal
pH for the immobilized b -galactosidase on the Cu-trimesic acid support was found at
pH = 7 [
33], ], on the polymer-coated magnetic nanoparticles at pH = 6[
15] and for the free b-galactosidase entrapped in calcium alginate at
pH = 7 [
1].
After obtaining the optimal process conditions for the immobilization and hydrolysis reaction, we investigated the possibilities of reusing the immobilized β-galactosidase enzyme. The immobilized enzyme was used repeatedly for 3 cycles -
Figure 11.
The highest conversion of lactose that was reached after 2 hours was around 20%.
Figure 11 shows that immobilization of the β-galactosidase enzyme on the glass fiber rolls preserves enzyme activity and stability. The same roll was reused for three consecutive lactose hydrolysis reactions with almost unchanged conversion, which certainly indicates high immobilization efficiency and practically no leakage of the enzyme from the support during the reaction and in between the reactions cleaning of the glass fiber rolls.