This README_Supplementary_Data.txt file was generated on 2026-04-22 by Kayla Reata Dittmer (kayla.dittmer@tuhh)

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GENERAL INFORMATION
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	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
	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: November 2025 - February 2026

Location of data collection: Institute of Technical Biocatalysis and Institute of Process Systems Engineering, Hamburg University of Technology, Hamburg, Germany

Funding: This project is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – SFB 1615 – 503850735. 

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SHARING/ACCESS INFORMATION
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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

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DATA & FILE OVERVIEW
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### 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.
- **Rows:**
  - Standard error
  - Intercept
  - Slope
  - R2
  - LOD
  - LOQ

### 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 (-)

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METHODOLOGICAL INFORMATION
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1. The enzymatic hydrolysis of urea: urease-catalyzed conversion of urea to NH₃ and CO₂; NH₃/NH₄⁺ and CO₂/HCO₃⁻/CO₃²⁻ acid–base equilibria considered; transient ammonium carbamate neglected.

2. Kinetic model: modified Michaelis–Menten kinetics with Arrhenius temperature dependence, pH-dependent enzyme activity, and non-competitive NH₃ product inhibition; kinetic parameters from literature and enzyme-specific activity factor fitted experimentally.

3. Acid–base equilibrium model: pH calculated from electroneutrality using temperature-dependent equilibrium constants for NH₃/NH₄⁺, CO₂/HCO₃⁻/CO₃²⁻, and water autoionization.

4. Gas–liquid mass transfer: NH₃ and CO₂ transfer between liquid and gas phases modeled using Henry’s law and kLa-based mass transfer coefficients.

5. Reactor model: ideally mixed, isothermal batch reactor with fixed liquid and gas volumes; coupled mass balances for urea, TAN, DIC, NH₃(g), and CO₂(g).

6. Numerical implementation: Python model solved with SciPy `solve_ivp` using the stiff BDF integrator; pH solved at each timestep with Brent root-finding.

7. Optimization methodology: Bayesian optimization (`gp_minimize`, scikit-optimize) used to identify initial CO₂ pressure minimizing residual urea after 60 min.

8. Experimental setup: 20 mL pressurizable stirred reactors with 15 mL of 30 mM urea solution and 150 µL urease solution; reactors pressurized with CO₂ between 0–3.5 bar and operated at 35 °C, 500 rpm for 60 min.

9. Sample handling: after reaction, pH measured immediately; samples thermally deactivated at 90 °C for 15 min and analyzed in triplicate.

10. HPLC analysis: urea quantified on an Agilent 1100 HPLC with Prontosil C18 AQ column, Milli-Q water eluent, 0.5 mL/min flow rate, 200 nm detection, and 5.54 min retention time.

11. Ammonia assay: NH₄⁺ quantified using commercial salicylate-based ammonia test kits after 1:25 dilution and absorbance measurement at 660 nm.

12. Calibration and validation: calibration curves generated for HPLC urea and ammonia test kits; HPLC limit of detection = 1.77 mM, limit of quantification = 5.37 mM.

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RESEARCH CONTEXT
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This study examines the use of CO₂ to regulate pH during urease-catalyzed urea hydrolysis. Urea hydrolysis increases pH through ammonia formation, which can reduce enzyme activity and process efficiency. By dissolving CO₂, carbonic acid forms and counteracts this alkalization. Experiments in pressurized reactors were combined with a mechanistic model including kinetics, acid–base equilibria, and gas–liquid mass transfer to identify conditions that maximize urea conversion and maintain favorable pH.