3.1. Effects of Different Types of Inorganic Salt in Protein Recovery Using LBF
The type of salts used for LBF is a key for protein extraction in this system as different salts induce different interactions with the protein, causing the separation efficiency of the proteins to alter. This is because salt solutions at the bottom phase are responsible of manipulating the surface tension of water thus changing the hydrophobic interactions between proteins and water at bottom phase [
14]. As a result, when the protein solubility is reduced, proteins will start to migrate to the top phase when aided by flotation. It is reported that the Gibbs free energy of hydration of salt was the key to the formation of a biphasic system between salt and alcohol solution [
15].
This experiment was conducted by firstly determining the most suitable type of salt to be used to obtain the best results and then changing the type of alcohol used. The types of salts used include ammonium sulphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium sulphate, and magnesium sulphate. The results are illustrated in
Figure 2. Dipotassium hydrogen phosphate and disodium hydrogen phosphate show a higher efficiency followed by ammonium sulphate and sodium sulphate in the descending order of 85.18%, 68.99%, 58.85%, and 53.81%, respectively. Magnesium sulphate showed the lowest separation efficiency which is 50.10%. For disodium hydrogen phosphate, the bottom phase forms salt crystals after flotation has been completed. This is due to the volume of the bottom phase being reduced; thus, the salt is unable to be fully dissolved in the remaining solution. Higher maintenance cost of the system is required if disodium hydrogen phosphate were to be used as a medium to create the biphasic conditions of this system.
As for the protein recovery yield, dipotassium hydrogen phosphate exhibits a higher protein recovery yield than all other salts, with a recovery yield of 29.997%. The lowest recovery yield of all salt tested was ammonium sulphate, which has a recovery yield of 7.74%. The other salts, which are disodium hydrogen phosphate, magnesium sulphate, and sodium sulphate, each has a recovery yield of 20.33%, 16.04%, and 10.81% respectively. The recovery yield for dipotassium hydrogen phosphate is significantly higher than that of disodium hydrogen phosphate. Taking into consideration the fact that when disodium hydrogen phosphate is used, the bottom phase will form salt crystals after flotation, and the two results obtained, dipotassium hydrogen phosphate was chosen to be the inorganic salt used in the following tests.
3.2. Effect of Different Types of Alcohols for Protein Recovery
The type of alcohol used for LBF plays an important role in the system as different types of alcohol have different levels of interactions with the proteins, which will determine how much protein can the system extract. The type of alcohol used also affects the formation of biphasic system with salt solution. Many proteins are found to be non-compatible with the alcohol-rich top phase in the LBF process [
16]. Some alcohols such as methanol will form triphasic solutions rather than biphasic solutions when mixed with salt solution. The selection of the type of alcohol used is very crucial as it will affect the overall performance of the system. In this study, ethanol, 1-propanol and 2-propanol of 100% were selected to form a biphasic system with dipotassium hydrogen phosphate at a concentration of 20 g/L.
All three alcohols were found to successfully form a biphasic system with the dipotassium hydrogen phosphate solution and the protein recovery yield for ethanol, 1-propanol and 2-propanol found to be 42.00%, 13.23%, and 23.35%, respectively. Based on
Figure 3, ethanol has outperformed both 1-propanol and 2-propanol in terms of recovery yield, being almost two times the yield of 2-propanol and almost three times the yield of 1-propanol. In terms of separation efficiency, ethanol also outperforms both 1-propanol and 2-propanol. The separation efficiency of ethanol, 1-propanol, and 2-propanol is found to be 92.20%, 87.54%, and 90.56%, respectively. Alcohols usually contain a carbon chain and a functional group (-OH); the difference between ethanol and the other two alcohols is that ethanol has a shorter carbon chain, resulting in it having more ethanol molecules at the same volume. This can be proven by dividing the density of the respective alcohol with its molar mass. For example, the density of ethanol is 0.789 g/cm
3 at 20 °C [
17] and ethanol has a molar mass of 46.07 g/mol [
18], this will result in ethanol a volume of 0.01715 mol/cm
3. This is higher than 1-propanol and 2-propanol as they have volumes of 0.01336 mol/cm
3 and 0.01306 mol/cm
3, respectively. The density of 1-propanol and 2-propanol is 0.803 g/cm
3 [
19] and 0.785 g/cm
3 [
20], respectively at 20 °C while their molar mass is 60.096 g/mol [
21] and 60.1 g/mol [
22], respectively. This difference will result in the protein molecules being able to interact more with the alcohol molecules and not settle back to the bottom phase after flotation. Additionally, the high recovery yield and separation efficiency in ethanol could be because of high polarity of the alcohol compared to the other two alcohols used. High hydroxyl group in ethanol could allow more protein to be accumulated at the top phase thus, giving high recovery yield [
23]. Due to the above reasons, ethanol is chosen to carry out the following tests.
3.4. Effect of Different Concentrations of Ethanol on the Recovery of Proteins
The concentration of alcohol used will also affect the overall performance of the LBF system. Thus, the next parameter to be optimized is the concentration of alcohol. Various concentrations from 60% to 100% of ethanol were tested by using 15% (g/L) dipotassium hydrogen phosphate. As shown in
Figure 5, 80% (
W/
V) shows the best recovery yield of 77.30%, followed by 70% of ethanol concentration which shows a yield of 54.50%. Concentrations of 60%, 90%, and 100% show 45.88%, 46.30%, and 41.48% yield, respectively. The protein recovery yield increases when the ethanol concentration increases, however when the alcohol concentration exceeds a certain point, the protein recovery yield starts to reduce. This is because the formation of the biphasic layers is weak when the concentration of alcohol is low. This result follows the trend in the previous study on protein recovery of wet microalgae using LBF where the highest protein recovery was obtained at 60% of 1-propanol, and the recovery yield decreased when the concentration of alcohol reduced below 40% [
25]. These phenomena are due to the concentration of alcohol decreasing; more hydrophilic proteins can be dissolved into the top phase when the proteins are brought up by flotation air bubbles. However, when the concentration of alcohol decreases, the water at the top phase tends to migrate down to the bottom phase of the LBF system [
16].
In terms of separation efficiency, ethanol solution with 80% concentration also has the highest separation efficiency, being at a value of 93.82%. The separation efficiency is followed by a pure ethanol solution which has a value of 90.59%. Values for 60%, 70%, and 90% are 69.16%, 54.17%, and 84.60%, respectively. The high value obtained at 80% of ethanol could be contributed due to cluster formation of ethanol. The number and size of the clusters strongly depend on the number of hydrogen bonds, and at higher concentration of ethanol, the cluster size is higher, which contributed to higher recovery of protein. Generally, ethanol has maximum viscosity of 75% to 80%, thus, this supports the high recovery of protein and separation efficiency. This indicates that at 80% of ethanol concentration, more proteins can be separated from the bottom phase which is the main point of this study. Therefore, 80% concentration ethanol solutions will be used in the following optimizations.
3.5. Effect of Various Concentrations of Milk
In this section, the effect of milk concentration of the bottom phase of the LBF system was studied. The concentration of milk may pose a potential effect on protein extraction by affecting the formation of the biphasic system, indicating that the concentration of milk used for extraction will have an impact on the yield of proteins recovered [
26]. Milk with concentrations of 5% (
w/
v), 10% (
w/
v), 15% (
w/
v), 20% (
w/
v), and 25% (
w/
v) mixed along with dipotassium hydrogen phosphate solution with 150 g/L concentration were tested to investigate the effects of milk concentration on protein recovery. The highest protein yield obtained is at 10% (
w/
v), closely followed by 5% (
w/
v), which has the values of 93.96% and 93.75%, respectively. At 25% (
w/
v), the recovery yield of proteins is the lowest, having a value of 50.98%. At 15% (
w/
v) and 20% (
w/
v), it has values of 89.77% and 64.74%, respectively. According to
Figure 6, it was observed that the concentration of milk has increased from 5% (
w/
v) to 10% (
w/
v), the recovery yield has only increased slightly; however, when the concentration is further increased, the yield starts to drop significantly, especially from 15% (
w/
v) to 20% (
w/
v), with a total drop of more than 25% protein yield. This is due to the fact that when a high concentration of milk is used, the salt solution mixture tends to form a liquid of which its high viscosity will result in the formation of flotation bubbles to be too difficult to control. By increasing the concentration of milk used, the performance of the LBF would be reduced as the level of impurities within the solution will also increase. The overall composition of the bottom phase will be altered as there is a lot of impurities in the milk [
16,
27]. In terms of separation efficiency, however, the highest value obtained is 94.02%, which is achieved by 25% (
w/
v). The separation efficiency gradually increases as the concentration is increased, starting from 5% (
w/
v), the separation efficiency was found to be 72.62%, 87.14%, 90.96%, and 92.59%, respectively. Due to the higher separation efficiency, 10% (
w/
v) of milk concentration was selected to carry on the following tests.
3.6. Effect of pH on the Recovery of Proteins
Impact of pH partitioning of proteins and enzymes to the phases in the LBF system depends on their isoelectric points. The pH of the system, however, affects the charge of target protein molecules and ionic composition, as well as introduces differential partitioning into the two phases. Most of the biomolecules, especially proteins and enzymes, are stable at neutral pH, which is a favorable condition to conduct the LBF partitioning. However, an increase in pH of the LBF from 7.0 to 9.0 reduced the protein recovery and activity recovered. Enzyme stability is slightly reduced in the acidic area except at the lowest pH and was dramatically lost at pH above 9.0. This dependence on pH for optimal protein recovery can be explained in terms of the charge in the protein. The protein in the LBF is predominantly casein. From the literature, isoelectric point of casein is 4.6, and since the pH of milk is 6.6, casein molecules are positively charged due to the protons provided by the milk medium. Given that the formula for pH is
where
is concentration of hydrogen ions, a pH of 7 to 8 in the system (see
Figure 7 corresponds to a negatively charged medium. With a positive charge, the casein molecules are thus hydrophobic, making them less soluble in water. Given that bubbles are used to push the particles up to the top phase and that the casein molecules are positively charged, the surface charge on the bubbles plays a vital role in the protein extraction efficiency. In particular, the charge of the casein and the surface charge of the bubble will be responsible for the adsorption of the protein molecule to the bubble surface. In order for the protein molecules to be attached to the bubble surface, the charge of the bubble surface must therefore be negative.
To understand the mechanics behind the adsorption of the bubbles, we have developed a mathematical model with the use of partial differential equations (PDEs).
The region of interaction in our model is constructed using the same approach as the Lamm equations (Lamm O., 1929), which is to divide the volume of the container into sector-shaped cells. With reference to
Figure 8 consider a region
in a sector-shaped cell within the chamber of the flotation system. Let
,
, and
be the mass of solute inside
, the mass flow into
, and the mass flow out of
, respectively. By the principle of conservation of mass,
Flux
is defined as the number of bubbles passing though an area
per unit time. Assuming no diffusion, i.e., only convection,
where
and
are the density and velocity of the bubbles respectively.
Putting Equation (2) into (1) gives
The bubbles are assumed to move into the region R, resulting in a negative , and the negative sign is to make it positive.
Integrating,
where
and
are density and magnitude of flux of bubbles respectively,
is the area in the top and bottom surfaces of the region
(see
Figure 1), and cylindrical coordinates are used:
.
From Sminov and Berry (Smirnov et al., 2015), the velocity of the bubble is given by
where
is the free fall acceleration,
is the density difference for liquid and air inside bubbles or the liquid density,
is the radius of bubble,
is the liquid viscosity.
From previous work (Lin YK, 2015), the number of bubbles that can be adsorbed to the surface of a bubble, , is given by where is the radius of the particle.
For maximum adsorption, the number of particles in the region must be times the number of bubbles in the same region R, and assuming a very small , all bubbles that go into the will collide with all particles that go into R.
From Equation (4), substituting
and rearranging gives
Putting
into Equation (7) and integrated w.r.t.
gives
Putting
into Equation (8) yields
Given that
, we can take
. We know that
where
is pressure of fluid, which is equal to the pressure in the bubble,
is density of fluid,
is acceleration due to gravity, and
is height. From Sminov and Berry,
where
is exteral pressure acting on liquid,
is the surface tension and is a constant, and
is the radius of the bubble. Combining Equations (10) and (11) gives:
where
is the pressure of air in the room (most likely atmospheric pressure, 1 atm).
Putting Equation (12) into Equation (6) gives:
where,
.
Putting (13) into (9) gives:
where,
.
To solve Equation (16), Laplace transform is applied to Equation (16), giving
Since
, Equation (17) becomes
To solve Equation (18), we divide Equation (18) by
to give
Multiplying Equation (19) with integrating factor
yields
where,
Integrating Equation (20) gives
where
is a constant, and
. Transforming Equation (22) from the
domain back to the
domain yields
where
L is Laplace transform.
From Equation (23), for the bubbles to hit all protein molecules, a sufficient amount of time is required. In particular, the Dirac delta function in Equation (23) indicates that for maximum protein extraction,
. Thus, with a uniform distribution of bubble holes at the bottom of the chamber, the time it takes to collect all molecules increases as the square of the height of the chamber. This explains the increasing amount of time required for higher protein collection in
Figure 9, which shows both the theoretical and experimental data. It can be seen from this figure that the experimental data is in agreement with the theoretical data for flotation time less than 8 min. The reduction in yield after 8 min can be attributed to the lack of protein in the system after prolonged extraction.
Besides allowing for a better understanding of the mechanism behind the system, this model also serves to provide some insights on design of efficient flotation systems at industrial scale. In particular, since it has been mathematically shown that an increase in height of the chamber drastically increases the flotation time, future chambers of flotation should preferably be as low as industrially viable.
The effect of pH has been studied by altering the pH of the bottom phase by using 1M hydrochloric acid. The initial pH of the bottom phase is 9.15, the tested pH is 6.5, 7.0, 7.5, and 8.0. The acid is added drop by drop until the bottom phase reached the desired pH with the aid of a pH meter. The highest recovery yield of proteins is obtained by the solution to which hydrochloric acid has not been added, which is the solution with a pH of 9.15. The recovery yield of the solution with this pH is at 93.96%, surpassing the second highest, which is a pH of 8.0 with a value of 65.71%, by more than 28%. The other three pH, being 6.5, 7.0, and 7.5, each has a recovery yield of 61.45%, 60.37%, and 62.33%, respectively. The big difference in yield is caused by the hydrochloric acid used to alter the pH having denatured the proteins, thus greatly reducing the yield of the LBF [
24]. As the acid was added drop by drop, each time a drop of acid hits the surface of the bottom phase, the extreme pH of the acid will denature some of the proteins at the bottom phase before being diluted by the rest of the solution at this phase. This results in the big difference in the recovery yield when comparing between a bottom phase of pH 9.15 and 8.0; this is also why the difference in recovery yield between pH 8 and pH 6.5 does not show a big difference when compared with 9.15 and 8.0. The separation efficiency increases when the pH is changed from neutral to 6.5, which is from 85.59% to 86.21%. At a pH of 7.5, the separation efficiency is the highest which is at 97.18%. The separation efficiency of pH 8.0 and 9.15 is 94.33% and 87.14%, respectively. Due to the above reasons, the following test was conducted without altering the pH system.