Copolymeric Hydrogel-Based Immobilization of Yeast Cells for Continuous Biotransformation of Fumaric Acid in a Microreactor

Although enzymatic microbioreactors have recently gained lots of attention, reports on the use of whole cells as biocatalysts in microreactors have been rather modest. In this work, an efficient microreactor with permeabilized Saccharomyces cerevisiae cells was developed and used for continuous biotransformation of fumaric into industrially relevant L-malic acid. The immobilization of yeast cells was achieved by entrapment in a porous structure of various hydrogels. Copolymers based on different ratios of sodium alginate (SA) and polyvinyl alcohol (PVA) were used for hydrogel formation, while calcium chloride and boric or phenylboronic acid were tested as crosslinking agents for SA and PVA, respectively. The influence of hydrogel composition on physico-chemical properties of hydrogels prepared in the form of thin films was evaluated. Immobilization of permeabilized S. cerevisiae cells in the selected copolymeric hydrogel resulted in up to 72% retained fumarase activity. The continuous biotransformation process using two layers of hydrogels integrated into a two-plate microreactor revealed high space time yield of 2.86 g/(L·h) while no activity loss was recorded during 7 days of continuous operation.


Introduction
Microreactor technology has recently been attracting increasing attention due to many advantages such as better heat and mass transfer, as well as more efficient mixing and overall better process control [1,2]. The integration of biocatalysts within microstructured devices opens the opportunity for the efficient valorization of biomass and sustainable transformation of chemical compounds [3]. Therefore, it is expected that the implementation of micro(bio)reactor technology in chemical and pharmaceutical industry will increase in the near future [4][5][6][7].
Immobilization of enzymes or whole cells has been an essential part of many biocatalytic process developments since it enables the reusability of the often-expensive biocatalyst and, thereby, prolongs its lifetime. Advantages of the immobilized biocatalyst compared with the free one also includes protection from harsh environmental conditions such as pH, temperature, organic solvent, and toxic compounds, as well as relative ease of product separation [8,9]. The selection of a suitable matrix for cell immobilization is essential for successful application of the immobilized cells for the bioprocess. Various techniques have been developed for biocatalyst immobilization, including adsorption or covalent linking to insoluble materials, entrapment in polymeric hydrogels and encapsulation in membranes [10][11][12][13].
150 rpm in a rotary shaker [31]. After harvesting by centrifugation, cells were washed thrice with water, permeabilized with 0.2% CTAB for 6 min and then again washed twice with water. Cells were then resuspended in 0.1 M HEPES buffer (pH 7) in a final concentration of about 10 9 cells/mL.

Cell Immobilization in Sodium Alginate-Polyvinyl Alcohol (SA-PVA) Copolymeric Hydrogel
For copolymeric-hydrogel preparation, the aqueous suspension of SA and PVA was mixed and heated to 60 • C in order to completely dissolve both polymers and then cooled to 35 • C. In the case of biocatalyst immobilization, the suspension of permeabilized S. cerevisiae cells was added at this stage to yield final cell concentration of 1.64 × 10 8 cells/mL, followed by a thorough mixing. Separately, CaCl 2 and boric/phenylboronic acid were mixed in demineralized water to obtain the crosslinking solution. A volume of 1364 mL of the copolymer solution was then poured in a small Petri dish and the crosslinking agent was poured on top of it to start the crosslinking process. After 60 min, the layer of formed hydrogel was washed with demineralized water prior to use.
Hydrogel characterization was performed for different compositions of copolymers combining 2% (w/v) of SA and PVA in concentrations from 4-12% (w/v) as shown in Table 1. Besides, two crosslinking solutions were tested; one consisting of 2% (w/v) CaCl 2 and 0.5% (w/v) BA, and the second one consisting of 2% (w/v) CaCl 2 and 2% (w/v) PBA, which is reflected from the hydrogel denomination evident in Table 1.

Characterization of SA-PVA Copolymeric Hydrogel
The effect of PVA concentration and crosslinking reagents on swelling and rheological properties of the hydrogels was determined. Hydrogels were first carefully dried by wiping off the surface liquid using a tissue, after which the sample was weighed. Samples were then immersed in 0.5 L of water and at specific times they were wiped in the same manner as before and weighed. Equation (1) was used to determine the swelling: where W t presents sample weight after the certain incubation time in water, while W 0 states for the sample weight before the immersion in the water. The hydrogel rheology was measured with an Anton Paar Physica MCR302 rheometer in a parallel plate configuration. Each sample was analyzed with a frequency sweep from 0.1 to 100 s −1 . All samples were prepared in 1 mm thickness and with 40 mm diameter. As a measure of the ratio of energy lost to energy stored during cyclic deformation, the loss tangent (tan δ) was calculated: where the loss modulus (G") relates to the energy dissipated from the sample as heat when shear is applied, representing the viscous characteristics of the sample. The storage modulus (G ) relates to the energy elastically stored in the sample when shear is applied, representing the elastic characteristics of the sample [33].

Microreactor Assembly
Microreactor (schematic presentation in Figure 1a and realistic picture in Figure 1b) was assembled using two poly(methyl methacrylate) (PMMA) plates where the upper one had inlet and outlet holes connected to perfluoroalkoxy (PFA) tubes (1.59 mm OD; 0.5 mm ID) via high-pressure polyetheretherketone (PEEK) tube fittings (Vici AG International, Schenkon, Switzerland). A channel for the single-layer microreactor was carved out of 0.5 mm non-compressible polytetrafluoroethylene (PTFE) film (DASTAFLON ® 1620, Dastaflon, Medvode, Slovenia) using a scalpel [12], while for a double-layered hydrogel-based microreactor, 0.7 mm PTFE film was used in order to obtain the same working volume and channel depth of 300 µm. A 5 cm long and 2 cm wide hydrogel layer covered the rectangular part of the hexagon-shaped channel carved from the PTFE film. The microreactor channel, therefore, consisted of the hydrogel filling the rectangular part between two empty triangular chambers, which provided unobstructed inlet and outlet of the fluid through the microreactor. One or two layers of various hydrogels with immobilized yeast cells were inserted in the rectangular part of the channels and glued on inner walls of the PMMA plates with 45 µm thick polypropylene double-sided adhesive tape donated by Adhesives Research (Glen Rock, PA, USA) in a way that inlet solution would flow between both hydrogel layers or between the single hydrogel layer and the PMMA plate through the reactor. Each hydrogel layer was about 200 µm thick. In the case of a single or double hydrogel layer, a microbioreactor had a working volume of 350 (±5) µL, as assured by the PTFE gasket. Each hydrogel layer contained 164 (±1.3) mg of wet yeast cells which corresponds to 33 (±0.8) mg of dry yeast cells with specific enzyme activity of 15.66 U/g, yielding final biocatalyst concentration of 94.25 (±3.7) and 188.5 (±4.5) mg dw of cells /mL of reactor volume in the case of a single or double-layered hydrogel-based microbioreactor, respectively. percentage of active yeast cells inside the hydrogel layers was calculated from the biocatalyst specific activity obtained in a batch process using free permeabilized S. cerevisiae cells, and the biocatalyst productivity of the cells immobilized within the microbioreactor.

The Effect of Temperature on Biotransformation in a Microreactor
Biotransformation of fumaric to L-malic acid was performed with immobilized permeabilized cells in a hydrogel consisting of 8% (w/v) of PVA and 2% (w/v) of SA, crosslinked with PBA for 60 min. Several runs of biotransformation were accomplished by pumping 5 mM fumaric acid dissolved in 0.1 M HEPES at the flow rate of 12 μL/min. A microbioreactor was set in a thermostatic water bath at temperatures between 22 and 60 °C and the samples were taken at the outlet after reaching a steady state at least three times at each tested conditions.

Determination of the Immobilization Effectiveness Factor
In order to determine the immobilization effectiveness factor, the activity of free and immobilized biocatalyst had to be measured. For free biocatalyst activity determination, a batch bioprocess in a flask with 10 mL of 5 mM fumaric acid in 0.1 M HEPES buffer with 15 mg/mL concentration of free permeabilized yeast cells was performed. Activity was measured after 5 min which was within the period of the initial (constant) reaction rate. For the estimation of the immobilized biocatalyst activity, the process parameters stayed the same except that the same concentration of permeabilized yeast cells has been entrapped in a 2 mL of the hydrogel in the form of the thin layer prior to reaction. The batch biotransformations were performed at 30 • C with at 100 min −1 shaking. Immobilization effectiveness factor, η, was calculated using Equation (3): Immobilized enzyme activity Free enzyme activity (3)

Biotransformation in a Microreactor
A microbioreactor with immobilized permeabilized S. cerevisiae cells was prepared by implementation of a porous layer of the hydrogel with cells between two plates as shown in Figure 1c, or with a single hydrogel layer on the bottom as described in Section 2.5. The reaction was performed by pumping 5 mM solution of fumaric acid in 0,1 M HEPES buffer (pH 7) through the microreactor between the hydrogel. The reactions were carried out at different temperatures ranging from 22 to 60 • C, while flow rates ranged from 2 to 20 µL/min. Flow was controlled by the high-pressure syringe pumps (PHD 4400 Syringe Pump Series) from Harvard Apparatus (Holliston, MA, USA). After reaching steady-state conditions, outflows from microreactors were collected and analyzed as specified below. Conversions were calculated based on the inflow and outflow substrate concentrations. The volumetric productivities were calculated from analyzed fumaric acid concentrations assuming the formation of an equimolar amount of product and considering the retention time within the microbioreactor [6]. The biocatalyst productivities were calculated from the outlet fumaric acid concentration and cell dry weight within the given microreactor volume [6]. The percentage of active yeast cells inside the hydrogel layers was calculated from the biocatalyst specific activity obtained in a batch process using free permeabilized S. cerevisiae cells, and the biocatalyst productivity of the cells immobilized within the microbioreactor.

The Effect of Temperature on Biotransformation in a Microreactor
Biotransformation of fumaric to L-malic acid was performed with immobilized permeabilized cells in a hydrogel consisting of 8% (w/v) of PVA and 2% (w/v) of SA, crosslinked with PBA for 60 min. Several runs of biotransformation were accomplished by pumping 5 mM fumaric acid dissolved in 0.1 M HEPES at the flow rate of 12 µL/min. A microbioreactor was set in a thermostatic water bath at temperatures between 22 and 60 • C and the samples were taken at the outlet after reaching a steady state at least three times at each tested conditions.

Determination of Biotransformation System Stability
Operational stability of the microreactor system was assessed by performing the biotransformation continuously for several days at room temperature. The flow of 5 mM fumaric acid aqueous solution was set at 5 µL/min and samples were taken at 24 h intervals; the conversion of fumaric acid was calculated from substrate concentrations analyzed at the outlet of the microreactor and the inlet concentration. The relative productivities were calculated from the results obtained during the long-term continuous process as compared to the ones obtained at the beginning of the process/initial productivity.

High-Performance Liquid Chromatography (HPLC) Analysis of Fumaric Acid Concentration
Biotransformation products were analyzed by an HPLC (Shimadzu, Tokyo, Japan) equipped with a Gemini-NX reverse phase C18 column (Phenomenex, Torrance, CA, USA) and ultraviolet/visible (UV/Vis) detector. The separation was performed at room temperature with 0.1 M phosphoric acid aqueous solution (pH 2.9) as a mobile phase. At a flow rate of 0.5 mL/min, the residence time of fumaric acid, detected at 226 nm, was 2.4 min.

Characterization of SA-PVA Copolymer Hydrogel
In order to develop an effective microreactor system between plates and with a biocatalyst immobilized in the hydrogel layer attached on the wall, it was of great importance to obtain as thin as possible film of a hydrogel. This would prevent diffusional limitations and enable efficient contact between the substrate molecules and the permeabilized yeast cells in the hydrogel. The thickness of the hydrogel layer was decreasing with increasing PVA concentration in the copolymeric hydrogel, with 200 µm being the thinnest layer obtained. Furthermore, the prepared hydrogel should be stable under operational conditions and would need to have a low swelling ratio because of a small reactor volume.
The results of the swelling behavior of the hydrogel samples are shown in Figure 2. It is evident that by raising the PVA concentration in the copolymer solution from 4% to 12% (w/v), the swelling ratio decreases from 25% to 3% after 120 h incubation time. This might be a consequence of increased PVA-borate crosslinking at higher PVA concentrations. The mechanism of PVA-borate crosslinking is believed to include a didiol complex, in which two diol units of PVA chain with one borate ion to form a crosslinked hydrogel [19]. Also, the swelling rate is smaller in samples that were crosslinked with PBA instead of BA which indicates that hydrogel samples crosslinked with PBA and with higher PVA concentrations would be favorable for microreactor application.

Characterization of SA-PVA Copolymer Hydrogel
In order to develop an effective microreactor system between plates and with a biocatalyst immobilized in the hydrogel layer attached on the wall, it was of great importance to obtain as thin as possible film of a hydrogel. This would prevent diffusional limitations and enable efficient contact between the substrate molecules and the permeabilized yeast cells in the hydrogel. The thickness of the hydrogel layer was decreasing with increasing PVA concentration in the copolymeric hydrogel, with 200 μm being the thinnest layer obtained. Furthermore, the prepared hydrogel should be stable under operational conditions and would need to have a low swelling ratio because of a small reactor volume.
The results of the swelling behavior of the hydrogel samples are shown in Figure 2. It is evident that by raising the PVA concentration in the copolymer solution from 4% to 12% (w/v), the swelling ratio decreases from 25% to 3% after 120 h incubation time. This might be a consequence of increased PVA-borate crosslinking at higher PVA concentrations. The mechanism of PVA-borate crosslinking is believed to include a didiol complex, in which two diol units of PVA chain with one borate ion to form a crosslinked hydrogel [19]. Also, the swelling rate is smaller in samples that were crosslinked with PBA instead of BA which indicates that hydrogel samples crosslinked with PBA and with higher PVA concentrations would be favorable for microreactor application.  Table 1  Rheological measurements revealed that all tested SA-PVA samples had average loss tangent parameter tan δ considerably lower than 1. This implies more solid-like structure since it has a bigger storage to loss moduli ratio [33]. From Figure 3 it is clear that BA hydrogel samples have bigger loss tangent value which indicates weaker and softer gel. On the other hand, with increasing PVA concentration, the tan δ value is decreasing, which indicates the creation of stronger gels. This is not the case with PBA samples however, where 12/2-PBA hydrogel is weaker than 8/2-PBA, as evident also from their handling. Namely, the 12/2-PBA hydrogel was more brittle, and it could break more easily. Since 8/2-PBA hydrogel displayed not only the best mechanical properties, but also good swelling ratio, it was selected for further use in the biotransformation studies. Rheological measurements revealed that all tested SA-PVA samples had average loss tangent parameter tan δ considerably lower than 1. This implies more solid-like structure since it has a bigger storage to loss moduli ratio [33]. From Figure 3 it is clear that BA hydrogel samples have bigger loss tangent value which indicates weaker and softer gel. On the other hand, with increasing PVA concentration, the tan δ value is decreasing, which indicates the creation of stronger gels. This is not the case with PBA samples however, where 12/2-PBA hydrogel is weaker than 8/2-PBA, as evident also from their handling. Namely, the 12/2-PBA hydrogel was more brittle, and it could break more easily. Since 8/2-PBA hydrogel displayed not only the best mechanical properties, but also good swelling ratio, it was selected for further use in the biotransformation studies. Micromachines 2019, 10, x 7 of 12

Determination of the Immobilization Effectiveness Factor
Each immobilization technique affects the biocatalyst activity in some way, either with enzyme deformation or in our case by increased mass transfer of substrate to biocatalyst, which consequently affects the reaction rate. From the series of batch processes with free and immobilized enzyme and by using Equation (3), the effect of immobilization on biocatalyst activity was calculated for hydrogels of various compositions (Figure 4). In all developed hydrogels, the immobilization effectiveness factor (η) was above 0.65, which is more than satisfying as compared to the literature [34]. PBA hydrogels with immobilized yeast cells had higher η which is probably due to toxicity of boric acid toward S. cerevisiae cells that in turn lowered their catalytic activity [19]. The effectiveness factors varied from 0.65 to 0.72 for 4/2-BA and 8/2-PBA, respectively.

Determination of the Immobilization Effectiveness Factor
Each immobilization technique affects the biocatalyst activity in some way, either with enzyme deformation or in our case by increased mass transfer of substrate to biocatalyst, which consequently affects the reaction rate. From the series of batch processes with free and immobilized enzyme and by using Equation (3), the effect of immobilization on biocatalyst activity was calculated for hydrogels of various compositions (Figure 4). In all developed hydrogels, the immobilization effectiveness factor (η) was above 0.65, which is more than satisfying as compared to the literature [34]. PBA hydrogels with immobilized yeast cells had higher η which is probably due to toxicity of boric acid toward S. cerevisiae cells that in turn lowered their catalytic activity [19]. The effectiveness factors varied from 0.65 to 0.72 for 4/2-BA and 8/2-PBA, respectively.

Determination of the Immobilization Effectiveness Factor
Each immobilization technique affects the biocatalyst activity in some way, either with enzyme deformation or in our case by increased mass transfer of substrate to biocatalyst, which consequently affects the reaction rate. From the series of batch processes with free and immobilized enzyme and by using Equation (3), the effect of immobilization on biocatalyst activity was calculated for hydrogels of various compositions (Figure 4). In all developed hydrogels, the immobilization effectiveness factor (η) was above 0.65, which is more than satisfying as compared to the literature [34]. PBA hydrogels with immobilized yeast cells had higher η which is probably due to toxicity of boric acid toward S. cerevisiae cells that in turn lowered their catalytic activity [19]. The effectiveness factors varied from 0.65 to 0.72 for 4/2-BA and 8/2-PBA, respectively.

Biotransformation within a Microbioreactor
Conversion of fumaric to L-malic acid was further studied in a microbioreactor using one or two layers of hydrogel between two plates (Figure 1). A microbioreactor with two layers of cell-loaded hydrogel enabled to achieve a biocatalyst load of 188.5 (±4.5) mg dw of cells /mL, which is much higher when compared with the tubular microreactor containing surface-immobilized S. cerevisiae cells with the load of only 5.2 mg dw of cells /mL [6].
As anticipated, two layers of hydrogel offering twice as much of the immobilized biocatalyst than a single hydrogel layer significantly improved the conversion at the outlet of the microbioreactor, as summarized in Figure 5. The highest outlet conversion obtained at 40 min residence time increased from 60% to 82% when using one or two-layered microreactor, respectively, providing the same process conditions. It shows that the conversion increase is not linear with the biocatalyst load, which indicates deeper substrate diffusion within a single hydrogel layer. Space time yield (volumetric productivity) obtained with 5 mM inlet substrate concentration and at retention time of 10 min for a double-layered microbioreactor was calculated to be 2.86 g/(L·h) (512 mM/day). At the same residence time and inlet substrate concentration, a single-layered microbioreactor achieved space time yield (STY) or volumetric productivity of 1.71 g/(L·h) (305 mM/day). Both results are lower than in the case of fumaric acid biotransformation using surface-immobilized permeabilized S. cerevisiae cells in a tubular microreactor of 24.5 µL volume with volumetric productivity of 616 mM/day [6], but substantially higher than that obtained in a membrane bioreactor of 1 dm 3 volume with other strain of S. cerevisiae cells, where 174 mM/day was reported [28]. Conversion of fumaric to L-malic acid was further studied in a microbioreactor using one or two layers of hydrogel between two plates (Figure 1). A microbioreactor with two layers of cell-loaded hydrogel enabled to achieve a biocatalyst load of 188.5 (±4.5) mgdw of cells/mL, which is much higher when compared with the tubular microreactor containing surface-immobilized S. cerevisiae cells with the load of only 5.2 mgdw of cells/mL [6].
As anticipated, two layers of hydrogel offering twice as much of the immobilized biocatalyst than a single hydrogel layer significantly improved the conversion at the outlet of the microbioreactor, as summarized in Figure 5. The highest outlet conversion obtained at 40 min residence time increased from 60% to 82% when using one or two-layered microreactor, respectively, providing the same process conditions. It shows that the conversion increase is not linear with the biocatalyst load, which indicates deeper substrate diffusion within a single hydrogel layer. Space time yield (volumetric productivity) obtained with 5 mM inlet substrate concentration and at retention time of 10 min for a double-layered microbioreactor was calculated to be 2.86 g/(L·h) (512 mM/day). At the same residence time and inlet substrate concentration, a single-layered microbioreactor achieved space time yield (STY) or volumetric productivity of 1.71 g/(L·h) (305 mM/day). Both results are lower than in the case of fumaric acid biotransformation using surfaceimmobilized permeabilized S. cerevisiae cells in a tubular microreactor of 24.5 μL volume with volumetric productivity of 616 mM/day [6], but substantially higher than that obtained in a membrane bioreactor of 1 dm 3 volume with other strain of S. cerevisiae cells, where 174 mM/day was reported [28]. When comparing biocatalyst productivity, a 350 μL-volume microbioreactor with two hydrogel layers resulted in 2.08 mmol/gww of cells/day. The comparison with other reactors and immobilization techniques used for the same biotransformation revealed that the above stated 24.5 μL-volume tubular microbioreactor and a 1 dm 3 -volume membrane bioreactor with S. cerevisiae cells, where biocatalyst productivities of 11.8 and 3.49 mmol/gww of cells/day were achieved, respectively [6,28], could more efficiently make good use of the yeast cells. This indicates lower accessibility of the biocatalyst in deeper layers of the hydrogel in the microbioreactor between two plates, confirmed also with the calculation of the portion of active yeast cells inside the hydrogel layers revealing that only 8.5% of cells were active. Since the hydrogel layer is 200 μm thick and the average diameter of the S. cerevisiae cell is 2-15 μm [35], the solution lies in achieving thinner layers of the hydrogel enabling the exploitation of all immobilized cells. Further optimization of biocatalyst load and When comparing biocatalyst productivity, a 350 µL-volume microbioreactor with two hydrogel layers resulted in 2.08 mmol/g ww of cells /day. The comparison with other reactors and immobilization techniques used for the same biotransformation revealed that the above stated 24.5 µL-volume tubular microbioreactor and a 1 dm 3 -volume membrane bioreactor with S. cerevisiae cells, where biocatalyst productivities of 11.8 and 3.49 mmol/g ww of cells /day were achieved, respectively [6,28], could more efficiently make good use of the yeast cells. This indicates lower accessibility of the biocatalyst in deeper layers of the hydrogel in the microbioreactor between two plates, confirmed also with the calculation of the portion of active yeast cells inside the hydrogel layers revealing that only 8.5% of cells were active. Since the hydrogel layer is 200 µm thick and the average diameter of the S. cerevisiae cell is 2-15 µm [35], the solution lies in achieving thinner layers of the hydrogel enabling the exploitation of all immobilized cells. Further optimization of biocatalyst load and hydrogel layer thickness is, therefore, envisaged. On the other hand, the immobilization procedure used in this work is much more user-friendly, cheaper and less time consuming than the one yielding surface-immobilized cells as a biocatalyst. Also, we intend to further increase the STY and productivity (capacity) using also model-based design of the reactor/reaction set-up.
A microreactor with permeabilized S. cerevisiae cells in 8/2-PBA hydrogel in the layers on both sides of the plates was further used to study the temperature effect on the selected biotransformation process. Hydration of fumaric acid using fumarase in yeast cells is well-investigated reaction, but optimal temperature might shift due to the immobilization technique applied [19]. For permeabilized S. cerevisiae MZKI K86 in the free form the optimal temperature was found to be 30 • C [6]. From Figure 6 it is evident that the same cells immobilized in the copolymeric hydrogel layer performed at 30 • C, but were also very efficient at 40 • C. By contrast, the activity decreased by more than 10% at room temperature and at 50 • C, while further temperature increase resulted in substantial activity loss. With this we have confirmed that developed microbioreactor enabled very efficient in-operando process parameters evaluation in a short time with low material consumption, which is extremely beneficial as compared to conventional systems [3,7].
Micromachines 2019, 10, x 9 of 12 hydrogel layer thickness is, therefore, envisaged. On the other hand, the immobilization procedure used in this work is much more user-friendly, cheaper and less time consuming than the one yielding surface-immobilized cells as a biocatalyst. Also, we intend to further increase the STY and productivity (capacity) using also model-based design of the reactor/reaction set-up. A microreactor with permeabilized S. cerevisiae cells in 8/2-PBA hydrogel in the layers on both sides of the plates was further used to study the temperature effect on the selected biotransformation process. Hydration of fumaric acid using fumarase in yeast cells is well-investigated reaction, but optimal temperature might shift due to the immobilization technique applied [19]. For permeabilized S. cerevisiae MZKI K86 in the free form the optimal temperature was found to be 30 °C [6]. From Figure 6 it is evident that the same cells immobilized in the copolymeric hydrogel layer performed at 30 °C, but were also very efficient at 40 °C. By contrast, the activity decreased by more than 10% at room temperature and at 50 °C, while further temperature increase resulted in substantial activity loss. With this we have confirmed that developed microbioreactor enabled very efficient in-operando process parameters evaluation in a short time with low material consumption, which is extremely beneficial as compared to conventional systems [3,7].

Determination of System Stability
To evaluate the operational stability of the system, the microreactor was tested over a period of 7 days by continuous operation at 5 μL/min flow rate of the 5 mM fumaric acid. A microreactor with immobilized permeabilized yeast cells in a 8/2-PBA hydrogel with 200 μm thickness was assembled as previously described. As evident from Figure 7, the relative productivity of the microreactor with immobilized yeast cells in 8/2-PBA hydrogel was constant and no changes in physical or mechanical characteristics of the hydrogel were observed. In contrast, the relative productivity of 8/2-BA hydrogel with permeabilized S. cerevisiae cells decreased to 65% after 3 days while also showing fractures and cracks and final breaking after 4 days of continuous operation. The deactivation constant for 8/2-BA hydrogel productivity was calculated to be kD = −0.475 h −1 and the half-life (t1/2) was 107.6 h. The stability was therefore significantly improved as compared to surface-immobilized yeast cell in a 24.5-μL tubular microbioreactor where relative productivity dropped to 10% after 4 days [6].

Determination of System Stability
To evaluate the operational stability of the system, the microreactor was tested over a period of 7 days by continuous operation at 5 µL/min flow rate of the 5 mM fumaric acid. A microreactor with immobilized permeabilized yeast cells in a 8/2-PBA hydrogel with 200 µm thickness was assembled as previously described. As evident from Figure 7, the relative productivity of the microreactor with immobilized yeast cells in 8/2-PBA hydrogel was constant and no changes in physical or mechanical characteristics of the hydrogel were observed. In contrast, the relative productivity of 8/2-BA hydrogel with permeabilized S. cerevisiae cells decreased to 65% after 3 days while also showing fractures and cracks and final breaking after 4 days of continuous operation. The deactivation constant for 8/2-BA hydrogel productivity was calculated to be k D = −0.475 h −1 and the half-life (t 1/2 ) was 107.6 h. The stability was therefore significantly improved as compared to surface-immobilized yeast cell in a 24.5-µL tubular microbioreactor where relative productivity dropped to 10% after 4 days [6].

Conclusions
The immobilization of S. cerevisiae cells through entrapment in a porous structure of copolymeric hydrogel was demonstrated to be very efficient. Physico-chemical characterization of the hydrogels of various compositions prepared by different crosslinking agents revealed the best results for the hydrogel composed of 2% of SA and 8% of PVA, crosslinked with PBA. Furthermore, the retained activity was higher using PBA as a crosslinking agent. It was possible to integrate two layers of selected hydrogel with immobilized permeabilized S. cerevisiae cells in a microbioreactor between two plates enabling very high biocatalyst load. The results of continuous fumaric acid biotransformation in this system revealed high volumetric productivities and heavily improved stability as compared to other flow systems for L-malic acid production. Considering the ease of the immobilized biocatalyst preparation and stability, such hydrogel structures used within microreactors present huge potential for implementation in biocatalytic processes.

Conclusions
The immobilization of S. cerevisiae cells through entrapment in a porous structure of copolymeric hydrogel was demonstrated to be very efficient. Physico-chemical characterization of the hydrogels of various compositions prepared by different crosslinking agents revealed the best results for the hydrogel composed of 2% of SA and 8% of PVA, crosslinked with PBA. Furthermore, the retained activity was higher using PBA as a crosslinking agent. It was possible to integrate two layers of selected hydrogel with immobilized permeabilized S. cerevisiae cells in a microbioreactor between two plates enabling very high biocatalyst load. The results of continuous fumaric acid biotransformation in this system revealed high volumetric productivities and heavily improved stability as compared to other flow systems for L-malic acid production. Considering the ease of the immobilized biocatalyst preparation and stability, such hydrogel structures used within microreactors present huge potential for implementation in biocatalytic processes.