In Situ Entrapment of Catalase within Macroporous Cryogel Matrix for Ethanol Oxidation: Flow-through Mode versus Batch Reactor

Abstract

In this article, the biocatalytic oxidation of ethanol into acetaldehyde was studied using a catalase entrapped within a monolithic polyampholyte cryogel, p(APTAC-co-AMPS), as catalyst. When an anionic monomer, 2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt (AMPS), was mixed with a cationic monomer, (3-acrylamidopropyl) trimethylammonium chloride (APTAC), under cryo-polymerization conditions at a molar ratio of monomers [APTAC]:[AMPS] = 75:25 mol.% in the presence of 10 mol.% cross-linking agent, N,N-methylenebisacrylamide (MBAA), the macroporous polyampholyte cryogels containing various amounts of catalase were synthesized in situ. The conversion of ethanol into acetaldehyde in good-to-high yields was observed in flow-through and batch-type reactors under optimal conditions: at T = 10–20 °C, pH = 6.9–7.1, [C₂H₅OH]:[H₂O₂] = 50:50 vol.%. According to the SEM images, the pore sizes of the p(AMPS-co-APTAC) cryogel vary from 15 to 55 μm. The catalytic activity of catalase entrapped within a monolithic polyampholyte cryogel in the conversion of ethanol into acetaldehyde was evaluated through the determination of kinetic parameters such as the Michaelis constant (K_m), maximum enzymatic rate (V_max), activation energy (E_a), turnover number (TON) and turnover frequency (TOF). The catalase encapsulated within the monolithic polyampholyte cryogel exhibits a high conversion of ethanol into acetaldehyde. The key parameters of ethanol oxidation in flow and batch reactors in the presence of the cryogel monolith were calculated.

1. Introduction

Acetaldehyde has its main industrial application in the synthesis of different organic compounds, such as acetic acid, acetic anhydride, ethyl acetate, glyoxal, crotonaldehyde, and maleic acid. It is also used as an intermediate in the synthesis of dyes, pharmaceuticals, perfumes, and pesticides. Usually, acetaldehyde is obtained through the aerobic oxidation of ethylene to acetaldehyde catalyzed by an aqueous solution of palladium(II) and copper(II) salts, and is known as Wacker oxidations [1,2]. The copper co-catalyst is a mediator for the reoxidation of palladium(0). The use of CuCl₂, even in catalytic amount, resulted in the production of noxious copper waste, which further lead to the formation of substantial amounts of ecologically hazardous chlorinated by-products. The elimination of CuCl₂ as a co-catalyst would render the Wacker oxidation a completely green process [3].

Later some essential results were obtained in the oxidative conversion of ethanol over noble metal-based catalysts, such as platinum [4,5], palladium [6], ruthenium [7], iridium [8], and gold [9,10] anchored on the surface of various supports. The catalysts revealed high activity in the partial oxidation of ethanol. Additionally, acetaldehyde can be synthesized by hydration of acetylene and ethanol dehydrogenation [11]. The combination of metal catalysts with molecular oxygen and hydrogen peroxide as green and readily available oxidants has practical advantages because of the favorable economics associated with O₂ and the formation of water and hydrogen peroxide as environmentally benign by-products [12,13,14].

Nowadays, biocatalytic oxidation offers practical solutions for the oxidation of alcohols. According to the classification of biocatalytic processes, immobilized-enzyme catalysis has been attracting attention from many industries and researchers, mainly because of their high selectivity, specificity and activity under environmental benign conditions [15]. Entrapment methods can be used to immobilize isolated enzymes and render them more stable and easier to separate and recycle [16]. Keilin and Hartree [17] firstly showed that catalase was also capable of oxidizing a wide variety of compounds including ethanol in the presence of a hydrogen peroxide-generating system. Later, the selective oxidation of primary alcohols to the aldehyde level with non-immobilized [18,19] and with immobilized enzymes [20,21] was successfully realized.

Last year, great attention was paid to macroporous cryogels that can be obtained by conventional radical copolymerization in cryoconditions [22]. On a microscopic level, moderately frozen molecular solutions can be represented as heterophase systems containing the polycrystals of a frozen solvent (for instance, water crystals) and some unfrozen fraction called “unfrozen liquid microphase”, where the monomers are concentrated. The reaction occurs inside of unfrozen regions due to the extremely high local monomer concentration, while the crystals of the frozen solvent play the role of porogens after defrosting. The pore size of cryogels can vary from several microns to several hundred microns. Due to porous structure, cryogels can retain up to 74% of water molecules in large pores, and can be used as a monolithic (stationary) or flow-through catalytic reactor.

The achievements in the creation of polymeric cryogels, mainly for the needs of biotechnology and biomedicine, have been described in reviews and book chapters [23,24,25]. For instance, anionic and cationic cryogels were used for the nanoparticles’ immobilization into the superporous cryogels matrix for sodium borohydride decomposition [26,27,28]. Recently, Demirci et al. [29,30] reported on the employment of neutral, anionic, and cationic superporous cryogels, obtained in situ under polymerization conditions, as support matrices for the immobilization of alpha-Glucosidase. Cryogels with tunable porosity, the range of pore sizes and functionality for the entrapment of other enzymes can be an effective and relevant tool in the development of novel biotechnological uses in real industrial applications [29,30,31].

Three approaches for the preparation of immobilized enzyme microreactors, namely (i) the wall-coated type, (ii) the monolithic type, and (iii) the packed-bed type, were described by Coloma et al. [32]. Nowadays, to avoid mass transfer limitation, significant amounts of wastes, and handling toxic reagents, different continuous flow systems are used for the manufacturing of fine chemicals [33]. The types of reactors used for immobilized enzymes were summarized in [34]. Basically, the same principles as for other processes using heterogeneous catalysis are valid, resulting in well-known reactor configurations.

The present work was inspired by our previous research reports on the aerobic oxidation of alcohols mediated by a biocatalyst composed of catalase immobilized within the polyampholyte cryogel p(APTAC-co-AMPS) [35]. The one-stage immobilization of metal nanoparticles into a matrix of amphoteric cryogels was developed by our research group [36,37,38] and furthermore, was successfully used for the selective hydrogenation of various substrates in a flow-through reactor. The traditional batch-type laboratory reactor was widely used for the liquid-phase oxidative catalysis in the reaction coupling of low-valent phosphorus compounds with alcohols, yellow phosphorus, and octene-1 [39,40,41,42].

The present manuscript deals with the study of the biocatalyst composed of catalase immobilized within a polyampholyte cryogel in ethanol oxidation, because the previous article [35] was focused on the study of iso-propanol and n-butanol oxidation in a flow-through catalytic reactor. The aim of this work is a comparative study of the catalytic properties of catalase immobilized within a monolithic polyampholyte cryogel in oxidation of ethanol to acetaldehyde under mild conditions (ambient temperature, atmospheric pressure) using the flow-through and batch catalytic reactors.

2. Results and Discussion

2.1. Preparation and Characterization of Biocatalyst

The monolithic p(APTAC-co-AMPS) cryogel containing various amounts of catalase was synthesized under cryoconditions (T = −12 °C) according to the literature [35]. For this goal, an initial molar ratio of monomers [APTAC]:[AMPS] was 75:25 mol.%. The synthesis of the p(APTAC-co-AMPS) cryogel was carried out in the presence of 10 mol.% MBAA crosslinker. As an initiator, ammonium persulfate (APS) was used and N,N,N’,N’-tetramethylethylenediamine (TEMED) was employed as an accelerator (Figure 1).

Electrostatic attraction between the negatively charged protein and the excess of cationic groups leads to the stabilization of catalase in the matrix of the p(AMPS-co-APTAC) cryogel [43]. As seen from the SEM images, the p(AMPS-co-APTAC) cryogel samples with immobilized catalase have porous structures, with the range of pore sizes between 15 and 55 μm (see Figure 2). These pore sizes allow the free flow of the mixture of liquid substrate and oxidation agent under gravity and at hydrostatic pressure.

The activity of catalase immobilized within the monolithic cryogel matrix in the decomposition of hydrogen peroxide was calculated in previous work [35]. The initial activities of catalase immobilized in cryogel samples (diameter 10 mm and height 10 mm) were 89.6, 200 and 221 U·mL⁻¹ for 1, 5 and 10 mg of the amount of immobilized catalase that correspond to an immobilization yield of 40.32%, 90.0%, and 99.45%, respectively. The best catalytic activity was found for the catalase sample with an immobilization yield of 99.45%. It was selected for oxidation of ethanol because it becomes inactivated only after the decomposition of 50 mL of H₂O₂.

2.2. Catalytic Oxidation of Ethanol Using Catalase Immobilized within the Monolithic Cryogel p(APTAC-co-AMPS) in a Flow-through Catalytic Reactor

The flow-through catalytic reactor containing the monolithic cryogel sample has an entire volume of 25 mL and is composed of a glass tube with an inner diameter of 10 mm and height of 40 mm. To maintain a constant temperature, the reactor was equipped with a thermostatic jacket. It operates under a low-Reynolds-number flow (0.021), corresponding to laminar flow. The exhibition of high void fractions minimizes pressure drop (ΔP = 21 × 10⁻³ Pa) [44]. Using the equation for a Sherwood number (Sh = $\frac{kd}{D_e}$) of 2 for stagnant solution [34] and a value for D_e found for the ethanol–water binary system equal to 0.5 × 10⁻⁹ m² s⁻¹ [45], the mass transport coefficient (k = 2.86 × 10⁻⁵ kg m⁻² s⁻¹ (kg m⁻³)⁻¹) was calculated as a function of the particle diameter.

It was found that an excess of either ethanol or hydrogen peroxide produces low yields of the acetaldehyde. Figure 3 shows the results of chromatographic analysis of the products after the first run of the optimal mixture of ethanol to hydrogen peroxide equaling 1:1 by volume in the flow-through catalytic reactor at 20 °C. The chromatographic peak 3 in the chromatogram, which appears at t = 3.5 min, corresponds to ethyl acetate. Peak 1 at t = 1.99 min belongs to acetaldehyde, whereas peak 2 at t = 3.0 min matches ethanol itself (Figure 3). Oxidation of the mixture of ethanol-hydrogen peroxide was found to produce acetaldehyde at a yield of 91.8%. Successive oxidation of the ethanol/hydrogen peroxide mixture leads to decreasing yields of acetaldehyde up to 85.2% (2nd cycle) and 14.5% (3rd cycle). This loss in the effectiveness is probably connected with the shrinking of the amphoteric cryogel p(APTAC-co-AMPS) matrix in the water–organic solvent mixture or leaching out of catalase.

The effect of pH, temperature and volume ratios of the alcohol to hydrogen peroxide on the conversion degree of ethanol was investigated.

The medium pH is an important parameter for ethanol oxidation. The studies were carried out at 25 °C in the pH range of 4–10. The pH 6.9 value was found to be the optimum solution pH of the ethanol-hydrogen peroxide mixture for the p(APTAC-co-AMPS) cryogel containing immobilized catalase, demonstrating the maximum conversion of ethanol (Figure 4). In the case of iso-propanol oxidation using catalase@p(APTAC-co-AMPS) cryogel, the studies confirm the pH of the iso-propanol hydrogen peroxide mixture to reach the high conversion of alcohol, i.e. 6.4 [35].

It was observed that the oxidative activity of amphoteric cryogel-immobilized catalase is higher at low temperatures but is lower at high temperatures, as in the case of iso-propanol oxidation [35]. This fact can be explained by the exothermic character of the oxidation of aliphatic alcohols. It is noticeable that the maximum activity of gellan gel-immobilized catalase [46] was demonstrated at a pH of 6.5. For hydrogen peroxide degradation, the p(NIPAM-co-HEMA) hydrogel-immobilized catalase has the same optimum as the free enzyme (around pH 7.0) [47].

The optimal ratio of the ethanol-hydrogen peroxide mixture passed through the sample of the monolithic cryogel with a flow rate of 6–7.5 mL·min⁻¹ and was equal to 50:50 vol.%. Under these conditions, the consumption of ethanol to acetaldehyde reached up to 89.5% (Figure 5).

The yield of acetaldehyde is higher at low temperature (Figure 6). It is probable that at low temperature, the cryogel is preferentially in the swelled state and the pores are more accessible for the substrate and peroxide [47]. The increasing in temperature can cause shrinking of the cryogel matrix, diminishing the effective diffusion coefficient of substrate to surface area, due to higher internal mass transfer resistance. Another reason may be the exothermic character of ethanol oxidation by hydrogen peroxide.

In the course of ethanol oxidation, the consumption of ethanol and the accumulation of acetaldehyde change in the opposite direction (Figure 7). The oxidation of ethanol can be divided into three stages. The first stage is up to 4 min, where the process is slow (induction period). The second stage lasts between 4–10 min, where fast oxidation of ethanol takes place. The third stage, in the range of 15–35 min, is related to a stationary state where the conversion degree is gradually stabilized. Considering that the boiling point of acetaldehyde is 20 °C, it can be assumed that some loss of acetaldehyde will occur at a higher temperature.

Oxidation of ethanol by hydrogen peroxide was performed by the cryogels themselves without immobilized catalase (

Table 1. Oxidation of ethanol by anionic, cationic, and amphoteric monolithic microporous cryogels at 20 °C, [H₂O₂] = 0.06 mol·L⁻¹, [Catalase] = 10 mg, V_EtOH = 5 mL, V_H2O2 = 5 mL.

Cryogels	p(APTAC)	p(AMPS)	p(APTAC-co-AMPS)	p(APTAC-co-AMPS)
Composition (mol.%)	100	100	50:50	25:75
Ethanol conversion (%)	6.7	7.0	7.3	7.4

) and free catalase (Figure 8). In both cases, the conversion is rather low and does not exceed 7.0 ± 0.3%. The conclusion is that the anionic, cationic and amphoteric cryogels without immobilized catalase are not active in the oxidation of ethanol. At the same time, the pristine catalase without the cryogel matrix is also inactive.

In reusability studies, one sample of cryogel was used as mentioned earlier at 25 °C, at pH 6.9, and washed in 10 mL of distilled water to remove the reaction product. Cryogel-immobilized catalase was able to retain its activity for five successive oxidation reactions (

Table 2. Reusability of cryogel-encapsulated catalase in oxidation of ethanol at 20 °C. [catalase] = 10 mg, [H₂O₂] = 0.06 mol·L⁻¹, V_EtOH = 5 mL, V_H2O2 = 5 mL, pH = 7.1.

Runs	1	2	3	4	5
Throughput rate, mL·min−1	5.0	5.0	3.0	2.0	2.0
Contact time, min	2.0	4.0	9.0	20.0	25.0
Conversion, %	91.8	85.2	14.5	0.7	0.3

). The conversion degree of ethanol to acetaldehyde by catalase@p(APTAC-co-AMPS) cryogel decreased under 10% and after five consecutive uses, became extremely low (0.3%). This phenomenon can be explained by the following assumptions. The first assumption is connected to the shrinking of cryogel samples in the mixture of ethanol-hydrogen peroxide (reactant) or acetaldehyde-water (product) that can retard the accessibility of active centers with respect to substrate. Moreover, the presence of organic substances within the cryogel matrix can deactivate or even poison the heme structure of catalase. The leaching out of catalase due to the destruction of cryogel samples should also be taken into account.

The kinetic parameters of ethanol oxidation were determined from the Michaelis–Menten plots in the concentrations range of ethanol 0.0343–0.0857 moles. The Lineweaver–Burk plots were also constructed according to Equation (1) [48].

$$\frac{1}{{\mathrm{V}}_0}=\frac{1}{{\mathrm{V}}_{\mathrm{m}\mathrm{a}\mathrm{x}}}+\frac{{\mathrm{K}}_{\mathrm{m}}}{{\mathrm{V}}_{\mathrm{m}\mathrm{a}\mathrm{x}}}\frac{1}{\left[\mathrm{S}\right]}$$

(1)

where V₀ is the initial rate (μmol·min⁻¹), [S] is the ethanol concentration (mM), K_m is the Michaelis constant (mM), and V_max is the maximum enzymatic rate (μmol·min⁻¹).

The segment cut off by the line on the ordinate axis is equal to $\frac{1}{{\mathrm{V}}_{\mathrm{m}\mathrm{a}\mathrm{x}}}$. The tangent of the slope of the straight line is equal to the ratio $\frac{{\mathrm{K}}_{\mathrm{m}}}{{\mathrm{V}}_{\mathrm{m}\mathrm{a}\mathrm{x}}}$. Using the correlation V_max = k₂[E]₀, the catalytic constant (or number of conversions) k₂ can be determined, where [E]₀ is the initial concentration of the enzyme. The value of the coefficient of correlation (R² = 0.9811) indicates good regression, which can be used to explain 98.11% of the total variation in the response (Figure 9).

The value of the Michaelis constant for immobilized catalase was equal to K_m = 4.0 M. The maximum rate of the enzymatic process is V_max = 5 × 10⁻² mol·L⁻¹·min⁻¹. The catalytic constant (or number of conversions) can be considered as the number of moles of product formed per unit time by one mole of pure enzyme saturated with the substrate, and it was equal to k₂ = 1.1 × 10⁶ min⁻¹. It is known that the conversion number for catalase is 5 × 10⁶ min⁻¹ [34].

The activation energy (E_act, kJ·mol⁻¹) was calculated from the slope of the straight line in Arrhenius coordinates lgk-1/T at a temperature interval of 5–20 °C (Figure 10). The negative value of E_act = −7.31 kJ·mol⁻¹ that is one half of the value of the activation energy calculated for the decomposition of H₂O₂ (E_act = −14 kJ·mol⁻¹) is explained by the exothermic character of ethanol oxidation by hydrogen peroxide [48]. The physical meaning of a negative activation energy is that the energy must be dissipated from the reacting particles rather than being supplied to them in order for the reaction to proceed successfully.

The change in the entropy of activation (ΔS^‡) in the temperature range of 5–20 °C lies in the limit of ΔS^‡ = −250.0 − (−246.01) J·mol⁻¹·grad⁻¹). Negative values for ΔS^‡ indicate that entropy decreases on forming the transition state, which often indicates an associative mechanism in which two reaction compounds form a single activated complex.

The calculated values of turnover number (TON) and turnover frequency (TOF) in oxidation of ethanol are rather high (

Table 3. Calculated values of TON and TOF for oxidation of ethanol in a flow-through catalytic reactor in the presence of cryogel monolith.

[EtOH]:[H2O2], mL	1:9	2:8	3:7	4:6	5:5	6:4	7:3	8:2	9:1
TON·10−7 [a]	0	0.14	0.23	6.75	16.86	3.23	0.45	0.48	0.10
TOF·10−7 [b]	0	0.15	0.28	9.00	22.48	4.31	0.89	1.07	0.51

^[a] TON, mol of product (mol Cat)⁻¹; ^[b] TOF, mol of product mol Cat ⁻¹ min⁻¹.

).

2.3. Oxidation of Ethanol by Cryogel-Immobilized Enzyme—Catalase in Batch-Type Catalytic Reactor

The oxidation of ethanol by catalase entrapped in a cryogel matrix of macroporous polyampholyte was studied in a batch-type reactor at 20 °C with hydrogen peroxide, molecular oxygen (100%) and air (

Table 4. Oxidation of ethanol by catalase, entrapped in a cryogel matrix, in a catalytic “duck” at 20 °C with hydrogen peroxide, molecular oxygen and air ^[a].

Reaction Time, min (Volume of Absorbed O2)	Yield of Acetaldehyde, %						Mass of Sample, mg
	Cryogel Monolith			Cryogel Powder			Cryogel Monolith	Cryogel Powder
	H2O2	O2	Air	H2O2	O2	Air
30	97.0	-	-	95.2	-	-	98.8	86.0
30 (13 mL)	-	97.7	-	-	-	-	95.2	-
30 (11.8 mL)	-	-		-	96.7	-	-	95.6
30	-	-	59.8	-	-	60.5	95.2	93.2
5	96.7	-	-	-	-	-	89.6	-

^[a] Reaction conditions: H₂O₂ = 0.06 mol L⁻¹, V_ethanol = 5 mL, solvent = 5 mL, 20 °C, P_O2 = 1 atm.

). The batch-type reactor consists of an outer and inner jacket made of glass. The constant temperature was kept by a circulated water system. The batch reactor with an entire volume of 150 mL is called a catalytic “duck”.

One of the strategies to reduce mass transport by film diffusion is stirring (or shaking), which can minimize the thickness of the boundary layer [34]. The laboratory batch reactor frequency of about 250–300 swingings per minute can minimize mass transfer resistance.

The experiments in the batch-type reactor demonstrate that the yields of acetaldehyde are high and independent of the reaction time, e.g., 5 or 30 min. The reason for selecting the different times is to show that the yield of the product is independent of the duration of the experiment. Since it was noted that the oxidation of ethanol is an exothermic process, increasing of the oxidation temperature even by 5 °C reduces the product yield and ethanol conversion (Figure 6) and increases the duration of the experiment.

Oxidation of ethanol was carried out in the kinetic mode with intensive shaking, which significantly reduced diffusion inhibition and improved heat and mass transfer. When ethanol was oxidized by molecular oxygen for the duration of 5–30 min, the yield of acetaldehyde was equal to 97–98% regardless of the form of the used cryogel as a monolith or powder. The volume of oxygen absorbed during ethanol oxidation was equal to 11.8 mL in the case of the powdered cryogel with immobilized catalase and 13.0 mL in the case of the monolithic cryogel (Table 4). The yield of acetaldehyde, according to GC analysis, is rather high for both powdered cryogel (96.7%) and monolithic cryogel (97.7%). The reason is that the oxidation process probably takes place on the inner surface of the cryogels, namely in the pores. Therefore, the shape of the used cryogel does not affect the yield of acetaldehyde and ethanol conversion. However, the yield of acetaldehyde and conversion of ethanol decrease by 1.6 times when the experiments were performed in air. This fact can be explained by the low concentration of molecular oxygen in air (23%) in comparison with molecular oxygen.

The reusability of one sample of catalase@p(APTAC-co-AMPS) cryogel in the batch reactor was employed as in the flow reactor at 25 °C, at pH 6.9, without washing by distilled water before use (

Table 5. Reusability of cryogel-encapsulated catalase in oxidation of ethanol at 25 °C. [catalase] = 10 mg, [H₂O₂] = 0.06 mol·L⁻¹, V_EtOH = 5 mL, V_H2O2 = 5 mL, pH = 6.9.

Runs	1	2	3	4	5
Contact time, min	40.0	30.0	30.0	20.0	15.0
Conversion, %	65.8	63.8	60.4	57.9	57.3

). The conversion degree of ethanol to acetaldehyde by the catalase@p(APTAC-co-AMPS) cryogel decreased after 5 successive runs from 65.8% (1st run) to 57.3% (5th run). Gradual decreasing of contact time for each run is probably connected with both swelling of cryogels and changing of the internal structure of pores. During the first run, the cryogel pores should be swelled, filled out by the reaction mixture and contacted with entrapped catalase. In the successive runs, the reaction mixture flows more freely through macropores because most of them are already swelled and occupied by the reaction mixture that decreases the contact time.

The calculated TON and TOF values for oxidation of ethanol in the batch-type reactor are given in

Table 6. Calculated values of TON and TOF for oxidation of ethanol in batch-type reactor in the presence of monolith and powder cryogel ^[a].

Oxidant	H2O2			O2		Air
	Cryogel Monolith		Cryogel Powder	Cryogel Monolith	Cryogel Powder	Cryogel Monolith	Cryogel Powder
TON·10−7 [b]	18.3	18.2	17.9	18.4	18.2	11.3	11.4
TOF·10−7 [c]	0.6	3.7	0.6	0.6	0.6	0.4	0.4

^[a] [EtOH]:[H₂O₂], mL = 5:5; ^[b] TON, mol of product (mol Cat)⁻¹; ^[c] TOF, mol of product mol Cat⁻¹ min⁻¹.

.

The TON and TOF values for the batch-type reactor were calculated for the optimal ratio of the ethanol-hydrogen peroxide mixture of 50:50 vol.%. and reaction time equal to 5 or 30 min with cryogel monolith and powder. Independently from the type of oxidizer, TON values do not differ much and lie in the range 11.3–18.4 mol of product (mol Cat)⁻¹; TOF values in most runs are similar (0.4–0.6 mol of product (mol Cat)⁻¹) and depend mainly on reaction duration.

The TON value for the flow-through catalytic reactor with the cryogel monolith using the optimal ratio of the ethanol-hydrogen peroxide mixture of 50:50 vol.%. (16.86 mol of product (mol Cat)⁻¹) is similar with the TON value for the batch-type reactor (18.3 mol of product (mol Cat)⁻¹). The difference between TOF values for flow and batch reactors in 22.48/0.6 = 37.5 (30 min) and 22.48/3.7 = 6 times (5 min) indicate that the flow reactor is more efficient in the oxidation of ethanol.

The key parameters of ethanol oxidation in flow-through and batch-type reactors for the oxidation of ethanol in the presence of a cryogel monolith are given in

Table 7. Key parameters of ethanol oxidation in flow and batch reactors in the presence of a cryogel monolith.

Parameter	Flow	Batch
Oxidant	H2O2	H2O2	O2	air
[EtOH], M	8.6	8.6	8.6	8.6
Conversion, %	89.5	96.7	97.7	59.8
tres, min	0.75	5	30	30
TON·10−7	16.9	18.2	18.4	11.3
TOF·10−7	22.5	3.7	0.6	0.4
S.T.Y.·10−2, g·L−1·h−1	48.6	8.8	8.9	5.4

. Since batch and continuous flow system setups have a completely different geometry, a direct comparison based on conversion or yield is simply not possible [49]. In contrast, the calculation of the space–time yield (STY) enables a fair comparison between the different systems. This comparison has been made at the same level of conversion since the product formation in batch and flow follow different kinetics.

2.4. Mechanistic Aspects of Ethanol Oxidation

Catalase is an antioxidant enzyme found in most aerobic organisms. The mechanism of ethanol oxidation to acetaldehyde is similar to the one proposed in [35]. The initial alcohol binds to the active center of the enzyme, namely, the iron(III) ion, and is oxidized by deprotonation/hydride transfer to form hydrogen peroxide. Next, hydrogen peroxide H₂O₂ protonates B (the imidazole ring of histidine (75His) with a lone pair of electrons) and converts it to BH^⊕ [50]. Then, there is coordination with the heme HOO^Ɵ, Fe(III) iron, and its oxidation to the Fe(IV) state with a π-cationic porphyrin radical and a water molecule. The BH^⊕ deprotonates to B: after oxidation of Fe(III) to Fe(IV). The regenerated B: is available for the next cycle. Active hydroxyl radicals •OH are generated by the porphyrin radical, which then attack hydrogen atoms and hydroxyl groups of alcohols, transforming them into either ketones or aldehydes.

A simplified mechanism of oxidation of alcohols by catalase in the presence of pure oxygen and air instead of hydrogen peroxide follows a similar mechanism, which is shown in Figure 11 [51]. Here, the molecular oxygen acts as a terminal electron acceptor. As result of a two- or four-electron transfer, hydrogen peroxide (2 e⁻) or water (4 e⁻) as a by-product can be generated [52].

3. Experimental Section

3.1. Materials and Methods

Anionic and cationic monomers, including 2-acrylamido-2-methylpropanesulfonic acid sodium salt (AMPS, 98 wt.%) and(3-acrylamidopropyl)trimethylammonium chloride (APTAC, 75 wt.% in water), crosslinking agent N,N’-methylenbisacrylamide (MBAA, >99% purity), ammonium persulfate (APS, >99% purity), and N,N,N’,N’-tetramethylethylenediamine (TEMED, >99% purity) were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA), and used without further purification. Catalase from bovine liver with catalytic activity of 2000 U·mL⁻¹ was also acquired from Sigma-Aldrich (USA). Commercial ethanol of high purity (99.5–99.9%) (Sigma-Aldrich, USA) and deionized water were used in all experiments. Potassium phosphate monobasic (98–100.5%, Sigma Aldrich), sodium phosphate dibasic (98–100.5%, Sigma Aldrich), sodium hydroxide (NaOH, 99.9%, Sigma Aldrich), and hydrochloric acid (HCl, 36.5%, Sigma Aldrich) were used for the preparation of buffer solutions. Potassium permanganate (KMnO₄, >99% purity), hydrogen peroxide (H₂O₂, 0.06 M), H₂SO₄ (20 wt.%) and phosphate buffer (mixture of 0.2 M of KH₂PO₄ and 0.2 M Na₂HPO₄ water solutions) with pH 7.0 were used for the determination of the activity of catalase utilizing the permanganometric method.

Gas–liquid chromatographic analysis was carried out on a DANI Master GC (Cologno Monzese, Italy). A gas chromatograph mass spectrometer Agilent 6890 N/5973 N (Santa Clara, CA, USA) was used for the identification of the products. The morphology of cryogels was observed utilizing a SEM JSM-6390 LV (JEOL, Tokyo, Japan).

The ethanol conversion was calculated by the difference between inlet and outlet concentrations; the formula was described as follows,

$${\mathrm{X}}_{\mathrm{V}\mathrm{O}\mathrm{C}}=\frac{{\mathrm{C}}_{\mathrm{V}\mathrm{O}\mathrm{C}\left(\mathrm{i}\mathrm{n}\right)}-{\mathrm{C}}_{\mathrm{V}\mathrm{O}\mathrm{C}\left(\mathrm{o}\mathrm{u}\mathrm{t}\right)}}{{\mathrm{C}}_{\mathrm{V}\mathrm{O}\mathrm{C}\left(\mathrm{i}\mathrm{n}\right)}}\times 100\mathrm{\%}$$

(2)

where C_VOC(in) (mg L⁻¹) and C_VOC(out) (mg L⁻¹) are the concentrations of volatile organic compound (VOC) in the inlet and outlet, respectively [53].

3.2. Synthesis of Catalase@p(APTAC-co-AMPS) Cryogel

Synthetic protocol of encapsulation of catalase within the APTAC-co-AMPS cryogel matrix has been already published [35]. The cationic monomer APTAC (0.9 g, 3.27 mmol) and anionic monomer APMS (0.5 g, 1.09 mmol) were mixed and dissolved in 8 mL of deionized water. Then, 10 mg of catalase and 67.1 mg of MBAA crosslinker (0.435 mmol) were added into the solution and stirred for 20 min at ambient temperature. After the addition of 10 mg of APS, the solution was stirred and then purged with argon for 20 min. Next, 0.1 mL of TEMED solution was added as accelerator, and the mixture was vigorously stirred for several minutes. The resulting mixture was placed in a pre-cooled (−12 °C) glass tube (diameter 10 mm, height 10 mm). The glass tube was kept in deep freezer at −12 °C for 24 h to complete simultaneous polymerization and crosslinking of APTAC and AMPS monomers under cryogenic conditions to deliver macroporous p(APTAC-co-AMPS) cryogels. The obtained cryogel sample has the shape of the reaction vessel. Then, it was washed out using deionized water for 72 h and air dried at room temperature overnight. To reach a constant mass, the cryogel sample was placed in a vacuum oven set at room temperature.

3.3. Enzymatic Activity Determination of Catalase

The permanganometric method was used for the titration of hydrogen peroxide excess after the reaction with catalase (Equation (3)).

5H₂O₂ + 2KMnO₄ + 3H₂SO₄ = 2MnSO₄ + K₂SO₄ + 5O₂ +8H₂O

(3)

The reaction was found to be stoichiometric in the H₂SO₄ medium. The method relies on the oxidizing ability of permanganate in the H₂SO₄ medium and the quantifications were achieved by titrimetry. The enzymatic activity of catalase was calculated by the Equation (4).

[Catalase] = 4.17·((V_control − V_tested)·n/V_tested)

(4)

where V_control is the volume of 0.025 M KMnO₄ solution used for titration of solution without addition of the enzyme, V_tested is the volume of 0.025 M KMnO₄ solution used for the tested sample, n is the dilution number of the initial solution of catalase, 4.17 is the conversion coefficient of catalase with arbitrary unit [54].

3.4. Calculation of the Immobilization Yield of Catalase in Cryogel Samples

The yield of the immobilization of catalase entrapped into cryogel samples (D = 10 mm, H = 10 mm) was determined according to the procedure described in [55] and calculated according to Equation (5):

Yield (%) = 100 (immobilized activity/starting activity)

(5)

3.5. Oxidation of Ethanol in Flow-through and Batch-Type Catalytic Reactors

3.5.1. Flow-through Reactor

The oxidation of ethanol in a flow-through catalytic reactor with an entire volume of 25 mL was carried out by hydrogen peroxide in anaerobic conditions (Figure 12). The dried monolithic cryogel sample with a diameter of 10 mm and height of 10 mm was placed into a glass tube (diameter 10 mm, height of 40 mm). To maintain a constant temperature, the reactor was equipped with a thermostatic jacket. Then, 2 mL of deionized water was passed through the cryogel sample for quick swelling of the cryogel. The formed tight seal between the inner wall of the glass tube and swollen sample allows the mixture of alcohol and hydrogen peroxide to flow freely under gravity through the cryogel pores. Additionally, it creates enough contact between the catalase and reaction mixture. The concentration of hydrogen peroxide used in all experiments was equal to 0.06 mol·L⁻¹. The 10 mL of mixture of ethanol and aqueous solution of hydrogen peroxide (1:1 by volume) was passed through the catalytic reactor containing cryogel-immobilized catalase, resulting in an immobilization yield of 99.45%. The total volume of the solution poured into the reactor creates enough hydrostatic pressure of the liquid to flow out through the cryogel [56]. The collected product was extracted utilizing ethyl acetate/or hexane, and then was analyzed using gas–liquid chromatography and gas chromatograph mass spectrometry.

3.5.2. Batch-Type Reactor

The oxidation of ethanol in a thermostated glass batch reactor with an entire volume of 150 mL was carried out (also called a catalytic “duck”) by hydrogen peroxide, molecular oxygen or air in anaerobic and aerobic conditions (Figure 13). The kinetic reaction regime was reached by shaking the reactor with frequency of about 250–300 swingings/min. A dried cylindrical piece of cryogel with immobilized catalase as a monolith with weight of 89.6–98.8 mg or a powder with weight of 86.0–93.2 mg was placed inside of the reactor. The 5 mL of ethanol was added to the reactor and was thoroughly purged with argon for a duration of 5 min. Then, the 5 mL of hydrogen peroxide (0.06 mol·L⁻¹) was added into the reactor against a brisk argon current through the dropping funnel. The mixture of ethanol and hydrogen peroxide was 10 mL (1:1 by volume). The 5 mL hexane was added to the reaction mixture in oxidation of ethanol by gaseous oxidizer as O₂ or air, for two reasons. The first is to keep the total solution volume at 10 mL, and the second is to use it as an extraction solvent preventing the ingress of moisture into the chromatographic column. The total time of the experiment was 5 or 30 min. The product accumulation was monitored by checking their composition by GC analysis.

4. Conclusions

Thus, flow-through and batch-type catalytic reactors in laboratory conditions are suitable for selective oxidation of ethanol into acetaldehyde in good-to-excellent yields under mild conditions using a biocatalytic system based on cryogel-entrapped catalase. The acetaldehyde yields irrespective of cryogel forms, monolith or powder were in the range of ~60–98%. The conversion of ethanol into acetaldehyde in a flow-through catalytic reactor reaches up to 91.8%. In a batch-type reactor, the oxidation degree of ethanol to acetaldehyde by O₂-rich hydrogen peroxide and molecular oxygen is higher (~97–98%) than in air atmosphere (~60%). The following optimal conditions for ethanol oxidation with hydrogen peroxide were found: T = 10–20 °C, pH = 6.9–7.1, [C₂H₅OH]:[H₂O₂] = 50:50 vol.%. Kinetic parameters such as the Michaelis constant K_m (4.0 M) and V_max (5 × 10⁻² mol·L⁻¹·min⁻¹) for the immobilized catalase on the p(APTAC-co-AMPS) monolith cryogel were determined by Lineweaver–Burk plots using ethanol as substrate. The catalytic constant (or number of conversions) was equal to k₂ = 1.1 × 10⁶ min⁻¹. The catalyst after completion of the reaction was separated easily by centrifugation and it was reused in five successive cycles. The space–time yield for the flow-through catalytic reactor was 5.5 times higher than that of the batch laboratory reactor. Thus, the suggested methods might be useful for the oxidation of aliphatic alcohols into their corresponding carbonyl compounds. In spite of the major drawback of cryogel microreactors as weak mechanical properties, the minimal volume of catalyst, high surface-to-volume ratio, energy saving and “green chemistry” aspects, high selectivity and productivity of enzyme-entrapped cryogel samples make them attractive for the development of different organic transformations independently from type of reactor and hydrodynamic mode.