This README_Supplementary_Data.txt file was generated on 2026-04-22 by Kayla Reata Dittmer (kayla.dittmer@tuhh) ------------------- GENERAL INFORMATION ------------------- Name: Kayla Reata Dittmer (ORCID: 0009-0005-4841-9438) Role/Function: Data collector (alternative contact person) Institution: Institute of Technical Biocatalysis, Hamburg University of Technology Address: Denickestraße 15, 21073 Hamburg, Germany Email: kayla.dittmer@tuhh.de Name: Leandros Paschalidis (ORCID: 0000-0003-1578-4154) Role/Function: Data collector (alternative contact person) Institution: Institute of Process Systems Engineering Address: Am Schwarzenberg-Campus 4, 21073 Hamburg Email: leandros.paschalidis@tuhh.de Name: Chloe Ng-Brossard (ORCID: 0009-0008-9665-5955) Role/Function: Data collector Institution: Institute of Technical Biocatalysis, Hamburg University of Technology Address: Denickestraße 15, 21073 Hamburg, Germany Email: chloe.ng-Brossard@tuhh.de Name: Daniel Ohde (ORCID: 0000-0002-5482-3534) Role/Function: Supervision Institution: Institute of Technical Biocatalysis, Hamburg University of Technology Address: Denickestraße 15, 21073 Hamburg, Germany Email: daniel.ohde@tuhh.de Name: Andreas Liese (ORCID: 0000-0003-4503-4039) Role/Function: Principal Investigator (alternative contact person) Institution: Institute of Technical Biocatalysis, Hamburg University of Technology Address: Denickestraße 15, 21073 Hamburg, Germany Email: liese@tuhh.de Name: Mirko Skiborowski (ORCID: 0000-0001-9694-963X) Role/Function: Principal Investigator (main contact person) Institution: Institute of Process Systems Engineering Address: Am Schwarzenberg-Campus 4, 21073 Hamburg Email: mirko.skiborowski@tuhh.de Date of data collection: December 2025 - February 2026 Location of data collection: Institute of Technical Biocatalysis and Institute of , Hamburg University of Technology, Hamburg, Germany Funding: This project is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – SFB 1615 – 503850735. --------------------------- SHARING/ACCESS INFORMATION --------------------------- Title of data set: "CO2-Driven pH Control in the Enzymatic Hydrolysis of Urea: A Coupled Modeling–Experiment Approach" DOI of data set: https://doi.org/10.15480/882.17011 Keywords: pH-control, kinetic modelling, Bayesian-optimization, biocatalysis --------------------- DATA & FILE OVERVIEW --------------------- ### Table_S1.csv (creation date: 22.04.2026, version: 01) - **Description:** Ammonia calibration curve using ammonia test kits. Results are presented as mean ± the standard deviation. - **Columns:** - Concentration (mg/L) - Absorbance at 660 nm ### Table_S2.csv (creation date: 22.04.2026, version: 01) - **Description:** Urea calibration curve in the HPLC at 0.5 mL/min flowrate with double distilled water as eluent. Results are presented as mean ± the standard deviation. Measurements are independent triplicates. - **Columns:** - Concentration (mM) - Peak Area (-) ### Table_S3.csv (creation date: 22.04.2026, version: 01) - **Description:** Values for the linear regression of the HPLC calibration curve. - **Columns:** - Parameter - Value ### Table_S4.csv (creation date: 22.04.2026, version: 01) - **Description:** pH of the reaction solution after 1 h of reaction, measured directly after opening the reactors. Results are presented as mean ± the standard deviation. Measurements are independent triplicates. - **Columns:** - Pressure (bar) - pH (-) ### Table_S5.csv (creation date: 22.04.2026, version: 01) - **Description:** Final urea concentration of the reaction solution after 1 h of reaction, calculated from measuring with ammonia test kits. Results are presented as mean ± the standard deviation. Measurements are independent triplicates. - **Columns:** - Pressure (bar) - Urea (mM) ### Table_S6.csv (creation date: 22.04.2026, version: 01) - **Description:** Final urea concentration of the reaction solution after 1 h of reaction, measured using HPLC. Results are presented as mean ± the standard deviation. Measurements are independent triplicates. - **Columns:** - Pressure (bar) - Urea (mM) ### Table_S7.csv (creation date: 22.04.2026, version: 01) - **Description:** Experimental results for the validation of the Bayesian optimization. - **Columns:** - Initial urea (mM) - Pressure (bar) - Final urea (mM) - pH (-) ### Bayesian_Optimisation.py (creation date: 24.02.2026, version: 01) - **Description:** Performs Bayesian optimization to determine the optimal initial CO₂ pressure or reaction conditions. ### Experimental_data_plotting.py (creation date: 24.02.2026, version: 01) - **Description:** Creates plots and figures from experimental results. ### NLR_for_parameterization.py (creation date: 24.02.2026, version: 01) - **Description:** Performs non-linear regression to fit the mass-transfer coefficient and activity correction factor to experimental data. ### Simulation.py (creation date: 24.02.2026, version: 01) - **Description:** Solves the coupled reactor model including enzymatic urea hydrolysis, pH calculation, and gas–liquid mass transfer. ### Bayesian_Optimisation_w_for_loop.txt (creation date: 24.02.2026, version: 01) - **Description:** Performs Bayesian optimisation for different initial urea concentrations. ### contour_pH.py (creation date: 24.02.2026, version: 01) - **Description:** Creates contour plot for the pH from simulation. ### pH_response_curve.py (creation date: 24.02.2026, version: 01) - **Description:** Creates response curve plot for pH from simulation. ### parity_plot_ph.py (creation date: 24.02.2026, version: 01) - **Description:** Creates parity plot for the pH from simulation and experimental results. ### parity_plot_u.py (creation date: 24.02.2026, version: 01) - **Description:** Creates parity plot for the final urea concentration from simulation and experimental results. --------------------------- METHODOLOGICAL INFORMATION --------------------------- 1. Gel Synthesis: The responsive hydrogels were synthesized by radical polymerization, according to the formulation in Table_S7.csv. In the copolymeric formulations, the amount of itaconic acid corresponded to 10 mol.% of the total monomer content. 2. Swelling Equilibria: After synthesis, the hydrogels were characterized with respect to swelling behavior and mechanical properties. The degree of swelling (DoS) was determined as the ratio of the mass of the equilibrated gel to the dry polymer mass. For this purpose, hydrogels were equilibrated in deionized water for 24 h at 25 °C. The dry mass was obtained after vacuum drying at 40 °C for 48 h. Stimuli-responsive swelling was evaluated under pH- and temperature-varying conditions. For both analyses, monoliths were pre-equilibrated in deionized water (24 h, 25 °C). For pH-responsiveness, samples were equilibrated in 50 mM potassium phosphate buffer at pH 8 (state I), followed by stepwise transfer to buffers of pH 7, 6, and 4 (state II), with equilibration and gravimetric determination at each step. The pH 4 buffer was adjusted using 85 wt.% ortho-phosphoric acid. Temperature-responsiveness was assessed by comparing swelling at 25 °C (state I) and 40 °C (state II) in deionized water. The relative swelling S was calculated as the mass ratio between state I and state II. 3. Mechanical Analysis: The elastic modulus was determined by uniaxial compression under ambient conditions using a universal testing machine (nominal force: 2.5 kN). A flat cylindrical stamp (40 mm diameter) was used to ensure full surface contact with the hydrogel samples. The measurement started at a contact force of 0.02 N and compression was applied at a constant rate of 50 mm*min⁻¹. The test was terminated at 75% strain or upon reaching the load cell limit (50 N). Force and displacement were continuously recorded. The elastic modulus E_C was calculated from the linear elastic region of the force–deformation curve using the slope of elastic deformation and the sample cross-sectional area. 4. Enzyme Immobilization: Adsorptive immobilization was performed by incubating hydrogels in an FDH solution (0.5 U·mL⁻¹ final concentration) at 4 °C for 24 h under gentle shaking. Samples were withdrawn over time to monitor immobilization. Protein concentration in the supernatant was determined using the Bradford assay (595/450 nm) with bovine serum albumin as calibration standard (0–200 µg·mL⁻¹). For covalent immobilization, an EDC/NHS coupling protocol was applied. Hydrogels were pre-equilibrated in water (24 h), activated with EDC (52 mM) and NHS (86.8 mM) in 100 mM MES buffer (pH 5) for 3 h at 25 °C, rinsed, and subsequently incubated in enzyme solution as described for adsorption. Silica beads were used as non-responsive reference material and functionalized by adsorption only. To prevent fracture due to capillary forces, beads were pre-wetted via water vapor exposure at 40 °C before immersion in enzyme solution. 5. FDH Activity Assay: Enzyme activity was determined by monitoring NADH formation at 340 nm. Immobilizates were added to a reaction solution containing 200 mM sodium formate and 1 mM NAD⁺ in 50 mM potassium phosphate buffer (pH 8). Samples were taken over 5 h and analyzed spectrophotometrically. NADH concentration was calculated using the Lambert–Beer law. The effective microplate pathlength (100 µL volume) was determined by comparison with a 1 cm cuvette using absorbance differences at 975 and 900 nm. 6. Immobilization Characterization: Immobilization performance was evaluated using four parameters: Immobilization yield (Y_immo): Percentage of enzyme mass bound relative to the initially added enzyme mass, determined via mass balance of the supernatant. Enzyme loading (E): Mass of immobilized enzyme per dry carrier mass. Activity yield (Y_EA): Specific activity of immobilized enzyme relative to free enzyme under identical conditions. Enzyme leaching: Percentage of enzyme mass released during reaction relative to the initially immobilized enzyme mass. ----------------- RESEARCH CONTEXT ----------------- This study investigates stimuli-responsive hydrogels as smart carrier materials for enzyme immobilization. Three polymer systems were analyzed: pH-responsive poly(HEMA-co-IA), temperature-responsive pNIPAM, and dual-responsive poly(NIPAM-co-IA). The materials were characterized in terms of swelling behavior, mechanical properties, and morphology to confirm their stimulus-dependent responses. Formate dehydrogenase (FDH) from Candida boidinii was immobilized using adsorption and EDC/NHS-mediated covalent binding on two carrier geometries (monoliths and particles). Immobilization yield, enzyme loading, activity yield, and enzyme leaching were systematically evaluated to compare the influence of polymer chemistry, carrier size, and immobilization strategy. ---------------------------------- EXPLANATION OF MEASURED VARIABLES ---------------------------------- Degree of Swelling (Dos) (m_gel / m_dry_polymer) : Ratio of equilibrated gel mass to dry polymer mass. Swelling Ratio (S) (-): Relative mass ratio between two stimulus states (temperature or pH), comparing 40 °C / 25 °C and pH4 / pH8. Immobilization Yield (Y_immo) (%): Percentage of enzyme bound relative to initial enzyme mass. Enzyme Loading (E) (mg_enzyme/g_polymer): Mass of immobilized enzyme per dry mass of polymer carrier. Activity Yield (Y_EA) (%): Specific activity of immobilized enzyme relative to free enzyme. Relative Leaching (%): Percentage of enzyme released during activity assay relative to immobilized enzyme mass.