DOI: 10.34810/data2945
title: Replication Data for: Bioprocess intensification for 3-hydroxypropionic acid production from methanol using engineered Komagataella phaffii

description: Raw and processed data obtained from the bioreactor-scale experiments. Including: Specifications volumes (bioreactor inlets and outlets); Raw_DCW (measurements for dry cell weight calculations); Raw_OD600 (measurement of optical density at 600 nm); Raw_NH4+ quantification (absorbance measurements at 595 nm for the quantification of ammonium ion in the bioreactor culture); IR_Raw_Data_HPLC (measurements with refractive index detector); IR-calibration plots; IR-peaks Integration; IR-HPLC results processing; Overview offline data (Summary of processed offline measurements: 3-HP and metanol concentration, DCW, optical density at 600 nm; ammonium quantification); overview online data (measurements of different process variables: air and pure oxygen flow rates, base addition, pH control, temperature control, methanol, exhaust gases, dissolved oxygen control); BlueSens_Off-Gas Analysis (measurements of exhaust gases concentrations).
description: METHODOLOGICAL INFORMATION

Biomass determination
OD600 measurements were performed using a Lange DR 3900 spectrophotometer (Hach, Loveland, CO, USA). Additionally, biomass concentration was measured by determining the dry cell weight (DCW). 10 mL of distilled water with 9 g/L NaCl were used to wet the pre-weighted glass microfiber filters (APFF04700, Merck Millipore) before filtering 2 mL of culture for each triplicate. After that, the filters were washed using the same volume of the NaCl solution, dried for 24 h at 105ºC and subsequently weighed to calculate the DCW. Both biomass determinations were performed in triplicate. The relative standard deviation (RSD) was always below 5%.

Substrate and product quantification
Culture samples (2 mL) were centrifuged for 5 min at 13,400 rpm using a MiniSpin centrifuge (Eppendorf, Germany). The supernatant was filtered through a 0.2 µm single‑use syringe filter (SLLGX13NK, Merck Millipore, CA, USA) and stored at −20 °C until HPLC analysis. Methanol, formate, and 3‑HP were quantified using a Dionex Ultimate 3000 HPLC system (Thermo Fisher Scientific). Separation was performed with an ICSep ICE‑COREGEL 87H3 ion‑exchange column (Transgenomic, Omaha, NE, USA) using 6 mM sulfuric acid as the mobile phase at 0.6 mL/min. Metabolites were detected via refractive index (RI), with an RSD below 2%.

Ammonium determination
Ammonium concentration was measured by spectrophotometry. Ammonium sulfate served as the standard, and absorbance was recorded at 595 nm using a microplate reader. All measurements were performed in triplicate, with an RSD below 10%.
Reference: JM. Robert, FS. Lattari, AC. Machado, AM. de Castro, RV. Almeida, FAG. Torres, F. Valero, DMG. Freire. Production of recombinant lipase B from Candida antarctica in Pichia pastoris under control of the promoter PGK using crude glycerol from biodiesel production as carbon source. Biochem. Eng. J. 118, pp.123-131 (2017). https://doi.org/10.1016/j.bej.2016.11.018.

Intracellular NADP⁺ and NADPH quantification
Intracellular NADP⁺ and NADPH levels were measured using the NADP⁺/NADPH Assay Kit (Abcam, ab65349) following the manufacturer’s instructions. Approximately 1 mL of culture was rapidly quenched by mixing with an equal volume of −20 °C methanol and centrifuged at 2,000 rpm for 5 min at −9 °C. The pellet was washed with ice‑cold PBS, diluted to an OD600 of 0.5–2.0, and centrifuged again. Cell pellets were resuspended in extraction buffer and lysed using a FastPrep‑24 homogenizer with glass beads. Lysates were clarified and deproteinized using 3‑kDa MWCO centrifugal filters, and the extracts were stored at −80 °C until analysis. Absorbance at 450 nm was recorded in a 96‑well plate, and data were processed according to the kit protocol. All measurements were performed in triplicate, with RSD &lt; 10%.

Methanol on‑line analysis
On‑line methanol concentration was monitored using an immersion methanol sensor (Raven Biotech, Vancouver, BC, Canada), as previously described [21]. Briefly, the probe contains a semi‑permeable membrane that selectively allows volatile compounds to diffuse into a carrier airflow. This airflow continuously transports the volatiles—primarily methanol—to a Figaro TGS‑822 gas sensor (Figaro USA Inc., Glenview, USA) for real‑time detection.

Online CO2 measurement of the bioreactor exhaust gas
CO2 concentration from the bioreactor exhaust gas was monitored on-line using a BlueInOne gas analyzer (BlueSens, Herten, Germany).

Calculation of process parameters
Mean specific rates, yields, productivities, and their time‑dependent profiles were calculated following approaches adapted from the literature. In this study, time derivatives of the main state variables—biomass, substrates, products, and CO₂—were obtained using a moving linear regression (MLR) method, which estimates local slopes by incorporating adjacent time points to reduce noise and improve robustness. The mean biomass within each MLR interval was calculated as the logarithmic mean. Because biomass concentration (X) is measured in the total fermentation broth, whereas substrate (S), product (P), and by‑product (BP) concentrations are measured in the clarified supernatant after centrifugation, their actual concentrations in the bioreactor were corrected using: C=C′⋅(1−Xσ⋅ρ), where C and C′ represent the concentrations in the culture broth and supernatant, respectively; σ is the biomass dry‑matter fraction; and ρ is the biomass density. The values σ=0.304 g/g and ρ=1068 g/L were taken from the literature.
Reference: X. Ponte, JL. Montesinos-Seguí, F. Valero. Bioprocess efficiency in Rhizopus oryzae lipase production by Pichia pastoris under the control of PAOX1 is oxygen tensión dependent. Proc. Biochem. 51: 1954-1963 (2016). https://doi.org/10.1016/j.procbio.2016.08.030.
description: MS Excel, null