# Aerobic autotrophic isopropanol production under low H2 atmosphere in Hollow Fiber Membrane Bioreactor

Federico Di Bisceglie, Eric Lombard, R. Kratzer, N. Gorret, O. Lorain, S.E.

Guillouet

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Federico Di Bisceglie, Eric Lombard, R. Kratzer, N. Gorret, O. Lorain, et al. Aerobic autotrophic isopropanol production under low H2 atmosphere in Hollow Fiber Membrane Bioreactor. Bioresource Technology, 2026, 440, pp.133407. 10.1016/j.biortech.2025.133407. hal-05362695

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# Aerobic autotrophic isopropanol production under low H₂ atmosphere in Hollow Fiber Membrane Bioreactor

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F.Di Bisceglie<sup>a,b,c</sup>, E. Lombard<sup>a</sup>, R. Kratzer<sup>b,c</sup>, N. Gorret<sup>a</sup>, O. Lorain<sup>d</sup>, S.E. Guillouet<sup>a,*</sup>

## HIGHLIGHTS

* Membrane contactor for carrying out gaseous fermentation outside explosive conditions.

* Membrane contactor to transfer dissolved H₂ reducing its content in the headspace.

* HFMC coupled to gas reactor to produce Biomass & Isopropanol under low H₂ environment.

## GRAPHICAL ABSTRACT

<figure><img src="image_4.png" /></figure>

## ABSTRACT

One of the challenges of aerobic gas fermentation for the production of biomolecules is to control the gas environment in order to operate outside the explosive zone, i.e., with low O₂ (< 6%) and/or H₂ (< 4%) contents. In this study, a bioreactor was to a hollow fiber membrane contactor to supply hydrogen to the microbial suspension. This technology allows the transfer of H₂ directly in soluble form into the culture medium without generating bubbles, responsible for the enrichment of the gas mixture with hydrogen by bursting in the gas headspace.

Coupling a Hollow Fiber Membrane Contactor to the gas bioreactor in *C. necator* cultures resulted in either 10 g/L of biomass or 1.3 g/L of isopropanol, while minimizing the H₂ content in the gas headspace to a level very close to, or even below, the safety threshold of 4 mol% H₂. Isopropanol production performance was compared with that obtained in a gas reactor without HFMC (6.8 g/L of isopropanol). Although the production performance was lower, the coupling still achieved it under low H₂ atmosphere, providing proof of concept that it is possible to perform aerobic autotrophic process under low hydrogen atmosphere, thus allowing the exit from the explosive zone regardless of the oxygen content of the reactor.

This advancement should help make aerobic gas fermentation much safer and extend these processes on a larger scale.

<footnote><sup>a</sup> Toulouse Biotechnology Institute (TBI), Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France</footnote>

<footnote><sup>b</sup> Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/II A-8010 Graz, Austria</footnote>

<footnote><sup>c</sup> Austrian Centre of Industrial Biotechnology (ACIB), Krenngasse 37 A-8010 Graz, Austria</footnote>

<footnote><sup>d</sup> Polymem, Castanet-Tolosan, France</footnote>

<footnote>* Corresponding author at: Toulouse Biotechnology Institute (TBI), Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France.</footnote>

<footnote>E-mail addresses: dibisceg@insa-toulouse.fr (F.Di Bisceglie), elombard@insa-toulouse.fr (E. Lombard), regina.kratzer@tugraz.at (R. Kratzer), ngorret@insa-toulouse.fr (N. Gorret), o.lorain@polymem.fr (O. Lorain), guillouet@insa-toulouse.fr (S.E. Guillouet).</footnote>
# 1. Introduction

Global greenhouse gas (GHG) emissions are a major cause of global warming, and have serious impacts on human and animal life (Frölicher et al., 2018; Stott, 2016). One solution to reduce these emissions is to develop systems to harness GHGs to lower their atmospheric concentrations, such as the production of fuels and chemicals by microorganisms using C1 gases as carbon sources (F. M. Liew et al., 2016; Staples et al., 2018).

Cupriavidus necator is a versatile organism capable of doing so. It can use CO₂ as a carbon source, H₂ as a source of reducing power and O₂ as electron acceptor. It metabolizes the Carbon source through the Calvin-Benson Bassham (CBB) pathway and, via hydrogenases, catalyzes the conversion of H₂ to 2H⁺ and 2e⁻, providing it with energy (Panich et al., 2021; Sillero et al., 2006). Its metabolic versatility and efficient Polyhydroxy Alkanoates (PHA) production make it an attractive host for metabolic engineering. C. necator is indeed able of synthesizing PHAs with titers exceeding 70% of its total Cell Dry Weight (CDW) even on CO₂ (Ishizaki et al., 2001), highlighting a strong capacity to redirect carbon flow into value-added products. This ability has been used to genetically modify new strains capable of producing heterologous products such as isopropanol (Garrigues et al., 2020), alkanes (Crépin et al., 2018), terpenes (Sydow et al., 2023) among others on CO₂ as the sole source of C. Isopropanol is among the most widely used disinfectants worldwide due to its strong antimicrobial activity (Rosenberg et al., 2013). Its high energy density and elevated octane rating also make it a promising additive for biofuels. In addition, it is frequently employed as a solvent in various sectors, such as the cement industry, cosmetics, and healthcare products. Within the chemical industry, isopropanol serves as a precursor for the synthesis of acetone, polypropylene, acrylates, and isopropylamines (Logsdon and Loke, 2000; Rosenberg et al., 2013). Conventionally, it is produced through the direct or indirect hydration of propene, a compound derived from fossil resources (Logsdon and Loke, 2000). Various strain engineering and process optimization approaches have successfully enabled heterologous isopropanol production in C. necator (Grousseau et al., 2014; Subagyo et al., 2021). Under autotrophic conditions, the highest reported titers to date are 7.7 g L⁻¹ with a genome-integrated variant (Bommareddy et al., 2020). In addition, initial bioelectrochemical systems for isopropanol synthesis with engineered C. necator have shown encouraging outcomes, with the most effective setup achieving 1 g L⁻¹ isopropanol (Torella, 2015; Liu, 2016; Schoenmaker, 2025).

One of the main challenges for aerobic autotrophic bioprocesses lies in the explosive nature of the gas mixture composed of hydrogen and oxygen. The flammability of hydrogen in a mixture with oxygen occurs within specific concentration ranges, depending on the nature of the diluent gas. For example, for hydrogen in an air mixture, the Lower and Upper Flammability Limits (LFL and UFL) are 4% and 75% (v/v). Similarly the LFL for oxygen in a mixture containing more than 4% Hydrogen is between 4 and 6% (v/v). However it also depends on temperature, pressure, container geometry and ignition energy (Ring et al., 2008; Hu et al., 2023). Thus, to avoid the occurrence of explosive conditions, different strategies have been reported in the literature for aerobic autotrophic fermentation such as managing the O₂ level in the reactor headspace rich in H₂ below 6% (v/v) via air flow management and/or inerting with nitrogen flow (Garrigues et al., 2020; Lambauer and Kratzer, 2022). Another strategy would be to manage the H₂ level in the bioreactor headspace below the LFL of 4% (v/v), without further concern for the oxygen level, but to some extent due to the inhibition of O₂ on hydrogenases (Siegel and Ollis, 1984). In the case of H₂, management of its content in the headspace by H₂ gas flow or nitrogen inerting would not be possible due to the high demand of H₂ for growth and its low solubility in the liquid phase (Morinaga et al., 1978; Ishizaki and Tanaka, 1991). Thus, the strategy applied in this study was to use membrane technology to supply hydrogen to the culture, allowing the gas to permeate through the membrane in contact with the liquid phase.

Dissolving H₂ directly into the liquid phase would reduce the amount of hydrogen reaching the culture broth in the form of bubbles, thus decreasing the amount of hydrogen gas in the bioreactor headspace. Here only hydrogen was supplied to the culture via a membrane contactor, while O₂ and CO₂ were directly bubbled into the culture medium. This choice was made because hydrogen is the least soluble of the three gases in liquid media, and, since the membrane contactor supply is also expected to promote solubility by permeation of the gas into the membrane polymer, hydrogen was the best candidate. (Lambauer and Kratzer, 2022)

Membrane bioreactors have been developed as an efficient option for syngas fermentation, offering advantages such as large surface area-to-volume ratio, an important factor for gas transfer, high selectivity and low energy consumption (Shen et al., 2014; Bakonyi et al., 2017; Orgill et al., 2013). Among existing membrane technologies, hollow fiber membranes (HFM) stand out for their high surface area to volume ratio due to their packing density, which optimizes gas transfer efficiency within compact modules. (Wang et al., 2018). C. necator has already been cultivated using membrane contactors, either to provide oxygen in a heterotrophic culture (Akkoyunlu et al., 2024) or to achieve autotrophic culture providing the complete gas mixture (CO₂/H₂/O₂) across the membrane (Przybylski et al., 2012, 2015).

The novelty of this study lies in the use of a composite HFM-contactor to supply only hydrogen rather than the full gas mixture to specifically address the explosion hazard while ensuring the growth and isopropanol production of C. necator under non-limiting conditions in gaseous substrates.

# 2. Materials and Methods

## 2.1. Strain

Two strains were used in this study. C. necator Re2133, derived from C. necator strain H16 by deletion of the genes encoding the acetoacetyl-CoA reductases *phaB1*, *phaB2*, *phaB3* and for the PHA synthase *phaC1*, and is therefore unable to produce PHA (Budde et al., 2011). C. necator Re2133/pEG7c was obtained through the plasmid-based expression of the genes encoding for the enzymes β-ketothiolase A (THL), acetoacetyl-CoA transferase (CTF), acetoacetate decarboxylase (ADC) and alcohol dehydrogenase (ADH), the latter two being codon-optimized *Clostridium* genes, while the former two being are additional copies of native C. necator genes (Grousseau et al., 2014). The four genes were organized in an operon under the control of the arabinose inducible promoter PBAD (Grousseau et al., 2014).

## 2.2. Media

### 2.2.1. Rich Medium

The rich medium for cell revival contained 27.5 g L⁻¹ Bacto™ Tryptic Soy Broth, 0.01 g L⁻¹ gentamycin sulfate and 0.1 g L⁻¹ kanamycin sulfate. Gentamycin was added to maintain sterility, as the strain is naturally resistant to it. Kanamycin was added as a source of selective pressure, as all recombinant genes are encoded on a plasmid that also encodes kanamycin resistance gene. The solid medium used for colony isolation plates contained the same concentrations of Bacto™ Tryptic Soy Broth, gentamycin and kanamycin, but it also contained 20 g L⁻¹ agar to solidify the medium.

## 2.3. Minimal Media

### 2.3.1. MIT Medium

Heterotrophic precultures were performed in MIT medium, a minimal medium whose composition was adapted to the growth of C. necator's in flasks. It contains 5.2 g L⁻¹ NaH₂PO₄·2H₂O, 11.6 g L⁻¹ Na₂HPO₄·12 H₂O, 0.45 g L⁻¹ K₂SO₄, 0.04 g L⁻¹ NaOH, 0.8 g L⁻¹ MgSO₄·7H₂O, 0.08 g L⁻¹ CaCl₂·2H₂O, 0.015 g L⁻¹ FeSO₄·7H₂O, 0.0024 g
$L^{-1}$ $MnSO_{4} \cdot H_{2}O$, 0.0024 $g \cdot L^{-1}$ $ZnSO_{4} \cdot 7H_{2}O$, 0.00048 $g \cdot L^{-1}$ $CuSO_{4} \cdot 5H_{2}O$. D-fructose was the only carbon source in heterotrophic precultures, at a concentration of 10 $g \cdot L^{-1}$. $NH_{4}Cl$ was used as a nitrogen source and added at a concentration of 1 $g \cdot L^{-1}$. In this medium too, 0.01 $g \cdot L^{-1}$ gentamycin sulfate was added to maintain sterility and 0.1 $g \cdot L^{-1}$ kanamycin sulfate for selective pressure. The medium is buffered to a pH value of 6.8 through its phosphate buffer ($NaH_{2}PO_{4}/Na_{2}HPO_{4}$). Due to the nitrogen concentration in the medium, a maximum biomass concentration of $2 g_{CDW} \cdot L^{-1}$ can be reached.

### 2.3.2. FAME Medium

The medium for the autotrophic cultivation was the FAME medium, containing 0.75 $g \cdot L^{-1}$ $MgSO_{4} - 7 H_{2}O$, 2.8 $g \cdot L^{-1}$ ($NH_{4})_{2}SO_{4}$, 0.285 $g \cdot L^{-1}$ NitriloTriacetic acid (NTA, metal chelator), 0.09 $g \cdot L^{-1}$ Ammonium iron (III) citrate, 4.50E-04 $g \cdot L^{-1}$ $H_{3}BO_{3}$, 3.00E-04 $g \cdot L^{-1}$ $CoCl_{2} - 6 H_{2}O$, 1.50E-04 $g \cdot L^{-1}$ $ZnSO_{4} - 7 H_{2}O$, 4.50E-05 $g \cdot L^{-1}$ $MnCl_{2} - 4 H_{2}O$, 4.50E-05 $g \cdot L^{-1}$ $Na_{2}MoO_{4} - 2 H_{2}O$, 1.50E-05 $g \cdot L^{-1}$ $CuSO_{4} - 5 H_{2}O$, 9.72E-05 $g \cdot L^{-1}$ $NiCl_{2} - 6 H_{2}O$, 0.015 $g \cdot L^{-1}$ $CaCl_{2} - 2 H_{2}O$, 1.57 $g \cdot L^{-1}$ $Na_{2}HPO_{4} - 2 H_{2}O$ and 0.26 $g \cdot L^{-1}$ $KH_{2}PO_{4}$. This medium was adapted to the cultivation of *C. necator* under bioreactor conditions. As the bioreactor cultures in this work were always carried out autotrophically, the sole carbon source was $CO_{2}$. Gentamycin sulfate was not added because, without fructose, the risk of contamination was low. However, 0.1 $g \cdot L^{-1}$ of kanamycin sulfate was always present to maintain selective pressure on the strain. The medium was not buffered, as the pH is expected to be controlled at 7.0 by the bioreactor systems, and the amount of biomass that the medium could support due to its nitrogen content was $4.3 g_{CDW}/L$.

### 2.3.3. Seed Train

The strain from the glycerol stock was streaked onto a rich agar plate and incubated at 30°C for 3 days, then, a single colony was picked and incubated in 5 mL of liquid TSB medium at 140 rpm and 30°C for 12h. Next, the culture was used to inoculate 50 mL of MIT medium supplemented with 10 $g \cdot L^{-1}$ of fructose also incubated at 140 rpm and 30°C for 12h. This preculture was then used to inoculate another heterotrophic preculture consisting of 75 mL of MIT medium supplemented with 4.3 $g \cdot L^{-1}$ of fructose, to ensure that after 17h all the fructose would have been consumed, so that none would be transferred to the autotrophic bioreactor cultivation. This preculture was also incubated at 140 rpm and 30°C, but, as planned, for 17 hours. The preculture was then added to 630 mL of fructose-free FAME medium in the bioreactor, to initiate autotrophic cultivation. The cultures were carried out at a constant temperature of 30°C. The stirring speed and gas flow rates were varied according to the metabolic needs of the microorganism.

### 2.3.4. Cultivation Systems

The bioreactors were custom-built systems, suitable for autotrophic cultivations. The 7.5 L Bioreactor (Solaris Biotech, Porto Mantovano, Italy) was equipped with a 6-bladed Rushton stirrer and three separate spargers, used for air, carbon dioxide, and hydrogen supply. A fourth inlet gas was added to the headspace for pure $N_{2}$ supply as a safety measure. The reactor was equipped with a temperature sensor (dtrans T100, Jumo, Fulda, Germany), a pH sensor (EasyFerm Bio HB Arc 120, Hamilton, Switzerland) and a foam sensor (Solaris, Ostiglia, Italy). Solaris software (V4.3.0) was used for data logging and process control. The reactor was equipped with mass flow controllers for each gas (Bronkhorst High-Tech B.V., Ruurlo, The Netherlands). Dissolved oxygen concentration and oxygen concentration in the headspace were measured using VisiFerm dissolved oxygen sensors (Hamilton, Nevada, USA). Gaseous $CO_{2}$ and $O_{2}$ concentrations were measured using a BlueInOne gas analyzer (BlueSens GmbH, Herten, Germany), while gaseous $H_{2}$ concentration was measured using a BlueVary gas analyzer (BlueSens GmbH, Herten, Germany). The gas volume exiting the bioreactor was measured using a gas Volumeter (Ritter Apparatebau GmbH & Co., Bochum, Germany).

A dedicated 1-liter gas bioreactor (H.E.L. Group, Hemel Hempstead,

United Kingdom) was also used in this study. The system included four 1-L stainless steel bioreactors, capable of withstanding up to 10 bar overpressure, and three mass flow controllers for each bioreactor to individually monitor and regulate the flow rates of $CO_{2}$, $H_{2}$ and $O_{2}$. A fourth inlet gas was added in the headspace to feed pure $N_{2}$ as a safety measure. The bioreactor was equipped with pH, dissolved oxygen (DO), temperature and pressure controllers. Xplorer® software managed the reactor's online monitoring and control systems of the reactor. $CO_{2}$ and $O_{2}$ gas concentrations were measured using a Tandem Pro gas analyzer (Magellan Biotech, Borehamwood, United Kingdom), while the gaseous $H_{2}$ concentration was measured using a BlueVary gas analyzer (BlueSens GmbH, Herten, Germany).

The bioreactors were safely controlled by the $O_{2}$ concentration in the headspace to avoid any risk of explosion. The headspace was purged with pure $N_{2}$ (flow rates of 2-3 L $h^{-1}$ in Solaris reactor, 1-1.2 L $h^{-1}$ in HEL reactor) if the $O_{2}$ exceeded 5% in the headspace. The temperature was maintained at 30°C. The pH was maintained at 7.0 by the addition of a 14% (v/v) ammonia solution.

The Silmem™ membrane contactors contained PDMS-coated composite fibers with a total surface area of 0.2 $m^{2}$, potted using polyurethane resin (Polymem, Castanet-Tolosan, France) (Fig. S1). The modules were connected to the bioreactors by a loop, equipped with liquid-side and gas-side pressure probes, as well as $H_{2}$ probes to measure the dissolved hydrogen concentration in the medium (Unisense A/S, Aarhus, Denmark) (Fig. 1). The cultivation medium was recycled in the loop using a peristaltic pump Watson

At the end of the experiments, the biomass was discarded after autoclave treatment.

## 2.4. Analytical Methods

### 2.4.1. Hollow Fiber Membrane characterization : Gas/Gas Permeance

The custom-made membranes used in this study (Polymem, Castanet, France) rely on PDMS for the gas transfer, as it is one of the most gas permeable dense polymer materials available. Specifically, the permeability coefficient of PDMS to hydrogen is 650 barr. After production, the custom-made Hollow Fiber Membranes (HFM) were potted in membrane contactors of various sizes for testing or application in autotrophic cultures. A composite membrane is composed of a support layer and a coating layer. The support layer is usually porous, supports the membrane structure and allows undifferentiated permeation based only on particle size. The coating layer, on the other hand, is usually dense and made of a polymer that, thanks to its structural properties, ensures the selectivity of the entire membrane. The support layer, which is generally non-selective, should be thicker, as its main function is to provide structural support. Thanks to this support, the coating layer, which determines selectivity and permeance, can be thinner, increasing the permeance, defined as $P=P/l$, where P is the gas permeance through the membrane (GPU), P is the membrane permeability (barrer) and l is the membrane layer thickness (m). In the case of the Silmem membrane, the support layer was made of Polysulfone (PSU), while the coating layer was made of Polydimethylsiloxane (PDMS).

Fig. S1 shows the general scheme of a membrane contactor, identifying the lumen and the shell side of the fibers. During cultures, hydrogen flowed through the lumen side, permeating through the membrane towards the shell side, where the culture medium circulated.

In gas/gas permeance tests, gases flowed from both sides, and the direction of permeation varied depending on the test being performed. Outside/in mode meant permeation was from the shell towards the lumen, while inside/out mode meant permeation was from the lumen towards the shell. To measure the $CO_{2}$ and nitrogen permeance of the Silmem membrane, the test was performed in outside/in mode, meaning that the inlet of the shell side of the fibers was connected to a gas cylinder containing $CO_{2}$ and the gas was purged to remove all air, before connecting the outlet to a manometer.

The lumen side was connected to a mass flow controller and the gas
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Schematic representation of the installation coupling an HFMC to the gas bioreactor. V: valves; SA, SB: dissolved H₂ sensors; P: pressure sensors; HFMC: Hollow Fiber Membrane Contactor</figcaption></figure>

was allowed to flow through the membrane for 20 minutes. The same was performed for N₂. The permeance (P) to both gases was then obtained by dividing the flow rate obtained by the applied pressure and the membrane surface. The H₂ permeance, unlike what was done for CO₂ and N₂, was measured in inside/out mode, meaning that the gas this time flowed from the lumen of the membranes to the outside, after purging all the air from the system.

**3. Estimation of the gas bubbling permeation rate through the membrane.**

Gas bubbles were observed passing through the membrane in the HFMC module. The flow of dissolved hydrogen ($\emptyset_{H_2}^{diss}$) through the membrane was calculated from the dissolved H₂ values measured before ($H_{2\text{diss}}^{SB}$) and just after the membrane contactor ($H_{2\text{diss}}^{SA}$) (assuming that the dissolved H₂ was not too much impacted by microbial consumption at this location) as follows: $\emptyset_{H_2}^{diss} = Q_{liq} \times (H_{2\text{diss}}^{SA} - H_{2\text{diss}}^{SB})$ expressed in $\mu\text{mol}\_H_2\text{h}^{-1}$ with $Q_{liq}$ the liquid flow rate passing through the HFMC

The total H₂ flow delivered by the HFMC was calculated as: $\emptyset_{H_2}^{total} = Q_{H_2} \times (H_2^{in})$ expressed in $\mu\text{mol}\_H_2\text{h}^{-1}$ with $Q_{H_2}$ and $H_2^{in}$ the H₂ gas flow rate and the H₂ content in the gas applied at the entrance of the HFMC, respectively. Considering that the total H₂ flux is the sum of the fluxes of dissolved H₂ and H₂ bubbles, the permeation rate of the H₂ bubbles can be estimated from the other two calculated fluxes.

**3.1. Biomass Calculation**

The microorganism's growth was monitored by measuring the optical density at 600 nm using a Hach Lange Spectrophotometer DR3900 (Hach Lange, Berlin, Germany) and 2mm path length glass cuvettes. The OD600nm value was correlated to the biomass concentration against Cell Dry Weight (CDW) measurements calibration, according to the correlation, verified unchanged throughout the study that $2 g_{CDW}L^{-1}$ corresponds to 1 unit of OD600nm. All biomass values will subsequently be expressed as CDW values.

**3.2. Determination of Metabolites Concentrations**

Metabolites (isopropanol, acetone, organic acids) were quantified by High-Performance Liquid Chromatography as described in Boy et al., 2023.

**3.3. Single cell analysis**

**3.3.1. Flow Cytometry Equipment**

An BD Accuri C6® flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used for measurements of cellular morphology and membrane integrity via Propidium Iodide (PI) staining. The instrument was equipped with blue (488 nm) and red (640 nm) excitation lasers. Light scatter was collected from two angles: forward (FSC: 0° ± 13°) and side (SSC: 90° ± 13°) scatter. FSC and SSC signals correlate to cell size and granularity, respectively. Fluorescence intensity was measured by
four detectors (Photomultipliers/PMT): FL1 (533 ± 30 nm), FL2 (585 ± 40 nm), FL3 (>670 nm), and FL4 (675 ± 25 nm) bandpass/long pass optical filter/photomultiplier detector systems.

## 3.4. Cellular Membrane Permeability Measuring Procedure

Propidium iodide (Molecular probes, Invitrogen, USA) was used to evaluate membrane permeability as a cellular indicator. The commercial solution is composed of 20 mM PI in anhydrous dimethyl sulfoxide solvent, and was diluted at a concentration of 20 µM before use. First, the culture samples were diluted in NaCl 0.9% to achieve a cell concentration of approximately $10^6$ cells/mL. Then, the cells were stained with 2 µL of PI working solution and incubated for 20 min at room temperature in the dark. A 100 % dead-cell control was prepared by incubating cells in 70 % isopropanol for at least 1 h at room temperature. Sample analyses were performed at low flow-rate (14 µL/min) using Milli-Q grade water (18.2 mΩ-cm resistance) as the sheath fluid. For all sample analysis, FSC signal (threshold: 12, 000) and SSC signal (threshold: 2, 000) were used as trigger channels. PI-permeable cells were quantified at the FL3 channel.

## 4. Results and discussion

The objective of this study was to evaluate the potential of an HFM contactor to transfer H₂ in a dissolved form limiting the presence of H₂ bas bubbles while promoting unlimited autotrophic culture for the production of molecules such as isopropanol. Supplying hydrogen to the liquid medium through the contactor could reduce the fraction of the gas reaching the headspace, thus making the process safer.

### 1. Hollow Fiber Membrane characterization

The HFM-Silmem contactor produced for the study had a total external surface of 0.25 m². In the absence of a dedicated hydrogen testing laboratory, the first test performed on the module was the selectivity test between CO₂ and N₂. In a composite membrane, this test is classically used to evaluate the integrity and uniformity of the dense coating layer. Since the permeability coefficient of CO₂ through PDMS is 11.6 times higher than that of N₂, the permeances of the two gases through a uniform coating layer will be accordingly different. In case the coating was not homogeneous or fractured though, the permeances would be more similar, because they would only be discriminated by the support layer, whose selectivity for the two gases is much lower. This is why the selectivity test between CO₂ and N₂ is a good indicator of the integrity of a coating layer.

To measure the permeance to CO₂ and nitrogen of the Silmem membrane the test was performed in outside/in mode. These tests were repeated at different pressures, until the flow rate was too high to be read by the flowmeter. The average of the permeances obtained was then taken as the permeance value for this membrane. The *P* CO₂ to *P* N₂ ratio gave the membrane selectivity, from which the integrity of the coating layer was assessed. The values of permeance were 397 GPU and 47 GPU, respectively for CO₂ and N₂, respectively, giving a selectivity of 8.5, of the same order of magnitude as the theoretical value of 11.6 for PDMS, confirming the integrity and uniformity of the dense coating layer of the membrane contactor.

Hydrogen permeance, unlike what was done for CO₂ and N₂, was measured in inside/out mode This was done because the inside/out mode was the one chosen to perform the membrane during the microbial cultivations in our study. The culture medium could flow outside the fibers, unrestrained by the smaller volume of the lumen and also possibly reducing the risk of fouling the porous support layer of the Silmem HFM. The permeation for Hydrogen was found to be 98 GPU. By multiplying the permeance in GPU by the thickness of the membrane in µm, the permeability in barrer units can be obtained. Due to its small thickness, the hydrogen permeability of the Silmem HFM was 291

barrer.

### 2. Reference bubbled gas fermentation for isopropanol production

Before the start of membrane cultures, the isopropanol-producing strain Re2133/pEG7c was grown under autotrophic conditions in the bioreactor without membrane contactor, as a reference to evaluate the growth and production obtained subsequently with the membrane. In this, the three gases were supplied by bubbling through three independent spargers.

The hydrogen and CO₂ flow rates were kept constant for most of the culture, as both were in excess compared to the microorganism's needs (Fig. 2). It was not possible to do the same for the air flow rate, as a dissolved oxygen concentration above 0.05 mmol/L would have inhibited *C. necator* hydrogenases, thus hindering its growth (Siegel and Hollis, 1984). For this reason, the concentration was set to 0.024 mmol/L (10mol% of saturation) and the air flow rate, as well as the stirring speed and pressure, were regulated by the bioreactor software to achieve this goal. This is the reason for the uneven increase in air flow rate between 8 and 30 hours of culture. During this period, *C. necator* experienced exponential growth with its highest value µmax of 0.13 h⁻¹. After 32 hours, 1 g L⁻¹ arabinose was added in the reactor to induce the expression of the isopropanol pathway under the control of the PBAD promoter. At this stage gas transfer control was set by increasing the pressure in the reactor at fixed gas flow rates as previously used in Garrigues et al. (2020). At the end of the culture (50 hours) the overpressure reached 4.6 bars. This strategy allowed maintaining the dissolved oxygen concentration at a stable and low value around 2% saturation while maintaining a rich environment in H₂ (around 20% of saturation) during the remainder of the culture. Cell viability was analyzed by flow cytometry after staining with propidium iodide. Cells not permeable to PI are generally considered viable. Their percentage exceeded 97 % throughout the culture, even when the overpressure reached 4.6 bars, confirming their robustness to high pressures as previously reported (Garrigues et al., 2020).

Isopropanol production started earlier than the induction point, due to the leakiness of the promoter PBAD controlling the expression of the isopropanol-producing genes in heterotrophy. However, the highest increase in its production occurred after induction, with a specific production rate at a value of 0.04 g gDCWh⁻¹. After 40 hours of culture, biomass growth decreased sharply, while isopropanol concentration continued to increase.

The final biomass concentration reached 9.9 g L⁻¹, while the isopropanol concentration reached a final value of 6.2 g L⁻¹. This implies a yield of isopropanol over biomass of 0.65 gIPA gDCW, indicating a good trade-off of metabolic resources towards the production of the desired product. Comparatively, Garrigues et al. (2020) achieved a yield of 1.2 gIPA gDCW with a constitutive isopropanol producing strain Re2133/pEG7b.

Acetone, the main by-product of isopropanol synthesis, was present in very low concentrations (Fig. 2) ultimately reaching 0.28 g L⁻¹, 22 times lower than that of isopropanol one. The acetone-to-isopropanol ratio was 0.05 g g⁻¹ much lower than that reported by Garrigues et al. (2020) and Bommareddy et al. (2020).

### 4.1. HFMC gas bioreactor fermentations

### 3. Autotrophic growth in HFMC gas bioreactor

The first membrane cultivation trial was performed by connecting the HFM-Silmem contactor to the gas bioreactor to efficiently perform autotrophic cultures. The contactor contained hollow fibers for a total surface area of 0.25 m², while the working volume of the bioreactor was 4.5 L. The cultivation was performed with the *C. necator* reference strain Re2133 which does not carry the plasmid for isopropanol production in order to study only the presence of the membrane without that of
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Autotrophic cultivation of *C. necator* Re2133/pEG7c in gas bioreactor. Evolution of (A) gas flow rates and overpressure, (B) dissolved oxygen concentration, (C) gas composition in the headspace and outlet, (D) biomass concentration (●), % of non PI permeable cells (■) and isopropanol (▲), acetone (◆) concentrations in the bioreactor liquid phase. The time of addition of arabinose is marked by the green vertical dashed line.</figcaption></figure>

isopropanol (Fig. 3). The hydrogen flow, was maintained constant at 10 L h⁻¹ throughout the culture to provide enough gas to be able to freely regulate its transmembrane flow by only acting on the TMP, while the flows of air and CO₂ were varied during the culture to provide enough carbon and oxygen to the strain through the spargers. The effect of the membrane becomes evident by observing the relative gas concentration profiles in the headspace. Despite a higher hydrogen flux than CO₂ and lower solubility in aqueous media, its percentage in the headspace was lower than that of carbon dioxide for most of the culture, thus achieving the goal of remaining below 4 mol%. (Fig. 3).

In the contactor, the $Q_{LIQ}$ had to be quickly increased to its maximum value, 64.6 L h⁻¹, because its increase systematically favored the hydrogen flow through the membrane by removing the hydrogen-saturated medium from the surface of the membrane fibers. The TMP, on the other hand, was gradually increased to avoid hydrogen limitation. The dissolved hydrogen concentration was indeed close to zero from the beginning of the culture, and continuous pressure increases were necessary to maintain it above this value. Once the TMP reached 4 bars, the increases were stopped to avoid membrane rupture. This quickly led to the onset of hydrogen-limiting conditions after 15 hours of cultivation, which persisted for the rest of the culture and resulted in a linear growth profile, reaching a final concentration of 10 g L⁻¹. The decrease in the specific biomass growth rate, which reached a maximum at 0.18 h⁻¹ and then declined sharply throughout the culture, is further

evidence that the growth was limited by the $H_2$.

Our results are very promising and demonstrate the possibility of efficiently performing gas fermentation by controlling the $H_2$ content in the headspace below the 4% limit, i.e. under non explosive conditions while producing high amount of biomass (10 g L⁻¹) which could be a safer bioprocess in the future for the application of single-cell proteins.

Due to the short period without $H_2$ limitation (15 hours), it was decided to change the ratio between the volume of the HFM-contactor and that of the bioreactor to improve the capacity to provide sufficient $H_2$. Therefore the following experiments were carried out in a 1L bioreactor instead of 7L reactor, with the same HFM surface.

## 4. Autotrophic Isopropanol production in HFMC gas bioreactor

Isopropanol production was then evaluated with the strain C. necator Re2133/pEG7c in a 1L gas bioreactor connected to the HFM-Silmem contactor placed on the external loop as previously (Fig. 4). This time the connection of the membrane contactor was set after 25 hours of cultivation (Fig. 4). The $Q_{LIQ}$, on the other hand, was set at 5 L h⁻¹ at the connection time of the contactor, and then gradually increased to a maximum value of 18.6 L h⁻¹ at the end. The hydrogen flow rate was finally maintained at approximately 0.9 L h⁻¹ throughout the culture.

The CO₂ flow rate was increased to a final value of 0.54 L h⁻¹, to ensure sufficient carbon supply to the culture. The air flow rate, once
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Autotrophic cultivation of *C. necator* Re2133 in gas bioreactor connected to the HFMC. Evolution of (A) gas and liquid flow rates and Trans Membrane Pressure in the HFMC, (B) gas flow rates in the reactor, (C) dissolved oxygen and hydrogen concentrations, (D) corrected gas composition in the headspace and outlet (corrected to account for N₂ flushing to determine the gas content that could have been achieved without N₂ flushing), (E) biomass concentration (●), % of non PI permeable cells (■).</figcaption></figure>

again controlled by the bioreactor software to maintain the dissolved oxygen concentration around 0.023 mmol L⁻¹, presented some problems immediately after membrane connection, until we switched to manually adjusting the air flow rates. The air flow rate was then gradually increased to 1.2 L h⁻¹, only to decrease towards the end of the trial due to a decrease in the required dissolved oxygen concentration (Fig. 4). At 17 hours, H₂ was supplied by the HFM-contactor set at a TMP of 0.18 bar, then gradually increased to 0.55 bar. However the transfer was not sufficient as the dissolved H₂ concentration remained zero for the rest of the culture. This time nitrogen was flushed to the bioreactor headspace at a constant flow rate of 1.2 L h⁻¹ throughout the culture, helping to keep the headspace gas concentration lower as safety measure (but was also taken into account to calculate the corrected gas % in the headspace). The concentration of H₂ in the headspace remained below the safety threshold for almost the entire culture, and even taking into account the N₂ dilution flush it remained around 4-5%. A fully safe membrane culture was then successfully achieved, but under H₂ limited conditions. These H₂ limited conditions strongly impacted the growth and production performances. The biomass specific growth rate peaked at 0.1 h⁻¹ before the connection of the contactor when growth was exponential, then declined and remained around 0.03 h⁻¹ for the

majority of the membrane-based culture. The final biomass concentration reached 3.7 g L⁻¹, and very little isopropanol production was observed with the highest concentration of 0.12 g L⁻¹. Adding 1 g L⁻¹ arabinose to induce the PBAD promoter had no significant effect on isopropanol production. Acetone was not produced at all. The cells' viability was considerably high in this culture, approaching 99% of non-PI permeable cells both before and after membrane connection.

The following fermentation was carried out at a higher TMP in order to reach a non-H₂ limited culture (Fig. 5). After connecting the membrane contactor (after 17 hours of cultivation), the TMP was rapidly increased over 1 bar, to then reach 1.7 bar by the end of the trial. A peculiarity of this culture was that, to achieve a TMP value of 1.7 bar, the H₂ outlet of the contactor was closed from 50 hours of cultivation, transforming the system into closed-end mode. The QLIQ was also considerably increased compared to the previous culture, reaching 23.1 L h⁻¹ immediately after connecting the contactor, and the maximum value of 25.3 L h⁻¹ by the end. The hydrogen flow rate, instead, was maintained at approximately 0.9 L h⁻¹ throughout the cultivation. This combination of operational configurations allowed the culture to be maintained without H₂ depletion, with the dissolved H₂ concentration always above 0.05 mmol L⁻¹, i.e. above 7 mol% of saturation (Fig. 5).
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Autotrophic cultivation of *C. necator* Re2133/pEG7c in gas bioreactor connected to the HFMC. Evolution of (A) gas and liquid flow rates and Trans Membrane Pressure in the HFMC, (B) gas flow rates in the reactor, (C) dissolved oxygen and hydrogen concentrations, (D) corrected gas composition in the headspace and outlet (corrected to account for N₂ flushing to determine the gas content that could have been achieved without N₂ flushing), (E) biomass concentration (●), % of non PI permeable cells (■) and isopropanol (▲), acetone (◆) concentrations in the bioreactor liquid phase. The time of addition of arabinose is marked by the green vertical dashed line, the connection of the HFMC by the black vertical dashed line.</figcaption></figure>

This time, the CO₂ flow rate had to be increased, to a final value of 1.1 L h⁻¹, to ensure sufficient carbon supply to the culture. The air flow, once again controlled by the bioreactor software, displayed the usual problems at the moment of contactor connection and reached a maximum level of 1.2 L h⁻¹. The dissolved oxygen concentration remained below approximately 0.019 mmol L⁻¹, i.e. 8.5 mol% of saturation.

The nitrogen flow rate into the bioreactor headspace was kept constant at 1.2 L h⁻¹ throughout the cultivation, helping to keep the headspace gas concentration low. The headspace gas content values presented in Fig. 5 were corrected to account for N₂ flushing to determine the gas content that could have been achieved without N₂ flushing. This time, however, with the higher hydrogen flow rate through the membrane due to the higher TMP, the corrected H₂ content fluctuated from 18% at the connection of the HFM-contactor and decreased

steadily during the cultivation to reach the lowest value of 6% (Fig. 5).

At the same time, the corrected O₂ content never exceeded 8.5% in the headspace. Even if the corrected H₂ content (i.e. hypothetically without N₂ flushing) had been above the target limit of 4%, the combination of the corrected values of H₂ (6-18%) and O₂ (6-8.5%) contents in the headspace would have resulted in safer operating conditions under low H₂ and low O₂. However, better management of gas flow rates in the bioreactor and HFMC by an automated system should allow maintaining performance below the target limits of 4% and 6%, for H₂ and O₂, respectively.

The final biomass concentration was 2.85 g L⁻¹. Due to the lack of hydrogen limitation, isopropanol and acetone production was efficient. After the first induction, which occurred after 21 hours, the isopropanol production rate reached a peak of 0.04 g g⁻¹DCwh⁻¹ at around 28 hours of
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Autotrophic cultivation of *C. necator* Re2133/pEG7c in gas bioreactor connected to the HFMC. Evolution of (A) gas and liquid flow rates and Trans Membrane Pressure in the HFMC, (B) gas flow rates in the reactor, (C) dissolved oxygen and hydrogen concentrations, (D) corrected gas composition in the headspace and outlet (corrected to account for N₂ flushing to determine the gas content that could have been achieved without N₂ flushing), (E) biomass concentration (●), % of non PI permeable cells (■) and isopropanol (▲), acetone (◆) concentrations in the bioreactor liquid phase. The times of addition of arabinose are marked by the green vertical dashed line, the connection of the HFMC by the black vertical dashed line.</figcaption></figure>

cultivation and appeared to slow down after 40 hours. A second in-
duction at 48 hours effectively increased its production rate and allowed
for additional synthesis, bringing the final concentration of isopropanol
to 1.3 g L⁻¹ (Fig. 5). The final acetone concentration was 0.05 g L⁻¹, with
a peak at 0.24 g L⁻¹ reached after its production rate slightly increased
after the second induction. The final selectivity of acetone over iso-
propanol was 0.04 g g⁻¹, meaning that the final acetone concentration
was only 4% of the final isopropanol one. This was the same as that
obtained in the previous cultivation, without the use of a membrane
contactor, proving that the strain’s production capabilities were not
hampered by the membrane contactor, provided that sufficient
hydrogen was added to the culture.

During cultivation, bubbles were observed in the membrane con-
tactor appearing to originate through the membranes. We compared the
hydrogen flow across the membrane, calculated from the dissolved H₂
values measured just after the membrane contactor (assuming that the
dissolved H₂ was not too much impacted by microbial consumption at
this location) with the hydrogen flow to the membrane contactor

measured by the flowmeter. After the outlet was completely closed, the
difference between the two flows was only 20%, meaning that 80% of
the hydrogen sent to the contactor ends up permeating the membrane
fibers in dissolved form, and only 20% in the form of bubbles. Consid-
ering that when hydrogen is fed to a culture through a conventional
sparger, it reaches the culture medium in the form of bubbles, this
represents an 80% reduction in the amount of bubbles formed. This also
explains why, while the hydrogen in the headspace was still close to the
safe concentration achieved in the previous culture, a non-limiting dis-
solved hydrogen concentration was reached in the bioreactor. With the
vast majority of hydrogen permeating the membrane in dissolved form,
significant progress has been made towards a completely safe and effi-
cient culture.

As observed in previous gas fermentations, the percentage of cells
not permeable to PI was consistently above 95%, meaning that no shear
stress effect was observed on the cell's membrane permeability
following their recirculation through the membrane contactor and the
loop. However, to further investigate the possible impact of microbial
suspension circulation in the loop and HFMC on cells, single cell analysis was performed on cell morphology and viability by flow cytometry during all cultures. (Fig. 6). Analysis of the fluorescence intensity distribution of cells after incubation with IP showed homogeneity regardless of the conditions encountered, in the presence or absence of external loop/HFMC and in the presence or absence of isopropanol. However, differences in the distribution on the Forward Scatter signal of the population were observed depending on the culture conditions. In the “classical” gas bioreactor (without HFMC), the distribution of FSCs of the Re2133/pEG7c strain did not show differences during culture with an increasing isopropanol concentration to 6.8 g L⁻¹ (Fig. 6A). Conversely, the distribution on the FSC increased when Re2133/pEG7c was grown in the reactor connected to the loop, and even more so when the HFMC was connected (Figs. 6 C and D) and this regardless of the isopropanol concentration to 1.3 g L⁻¹. Comparatively, the non-isopropanol-producing Re2133 strain grown in the reactor connected to the HFMC showed a higher FSC than Re2133/pEG7c in the “classical” reactor (Fig. 6B), but a similar distribution to that of Re2133/pEG7c grown in the reactor with circulation in the loop but not connected to the HFMC (Fig. 6 C and D, green areas). Since the FSC signal is correlated with cell size, circulation of the microbial suspension in the external

loop tends to increase the size and size distribution within the bacterial population. Passage of the microbial suspension in the HFMC led to an even greater increase in cell size and size heterogeneity within the population. Conversely, the presence of isopropanol does not appear to be responsible for a change in cell morphology at least up to 1.3 g L⁻¹. Changes in the size of *C necator* have been previously reported in the literature in response to PHA accumulation, (Ismail et al., 2024; Lee et al., 1993; Vadlja et al., 2016; Volova et al., 2013) and substrate availability and associated growth rates (Sakarika et al., 2023; Wiacek et al., 2006). In our case, the change in cell morphology was mainly related to the passage through the external loop, and even more so in HFMCs, rather than to the presence of isopropanol. The very short residence time of the cells in the loop (less than a minute, which eliminates a nutritional limitation effect) could rather indicate a stress effect related to the shearing of the cells in contact with the hollow fiber membrane.

The cultivation in HFMC gas bioreactor achieved a maximum final isopropanol concentration of 1.3 g L⁻¹ in 68 hours. This performance is not the best reported in the literature with conventional gas bioreactors in terms of final isopropanol concentration (currently up to 7.7 g L⁻¹ (Bommareddy et al., 2020)) and IPA over biomass Yield (0.45 g gDCW compared to 1.2 g gDCW (Garrigues et al., 2020; Weickardt et al., 2025)), but similar in terms of maximal IPA specific production rate (0.04 g gDCW h⁻¹) and cell's viability (Garrigues et al., 2020) or better in term of final IPA-to-acetone ratio (0.04 g g⁻¹ compared to 0.26-0.27 g g⁻¹ (Bommareddy et al., 2020; Garrigues et al., 2020), highlighting that the kinetic characteristics of the strain were not altered by the use of HFMC. However, all productions reported to date have been carried out under pressure and/or by controlling the oxygen level below the 4% limit for exiting the explosive zone. This study provides proof of concept that it is possible to achieve similar production performances with the use of HFMC but in a low hydrogen environment, thus allowing exit from the explosive zone regardless of the reactor's oxygen content.

Supplementing oxygen and hydrogen via separate HFMCs to promote dissolution and reduce the headspace concentration of both is an avenue to surely explore in the future.

Beyond the biomass growth and isopropanol production performances, the cultivation systems connected to the HFMC significantly reduced the hydrogen concentration in the headspace, compared to cultivations performed without a membrane contactor. The HFMC was able to fully support biomass growth and isopropanol production without dissolved hydrogen becoming limiting, while maintaining the hydrogen gas concentration between 6% and 18%, bringing it down from the 50% it was at just before the membrane connection. Furthermore, it was calculated that only 20 % of the hydrogen reaching the contactor permeated the membrane fibers in the form of bubbles. This represents an 80% reduction compared to what happens in the absence of a membrane contactor, where all the gas reaches the culture medium in the form of bubbles. This is a considerable increase in operational safety, as evidenced by the low hydrogen concentration in the headspace.

## 5. Conclusion

Autotrophic cultivation of *C. necator* in the HFMC bioreactor was efficient, reaching growth rates similar to those without a membrane contactor. It enabled control of hydrogen in the headspace near or below the 4 mol% safety threshold, serving as proof of concept. Isopropanol performance (1.3 g L⁻¹, 0.45 g gDCW⁻¹) was lower than in conventional gas bioreactors, but biomass production (10 g L⁻¹) approached realistic levels for large-scale SCP production. Further optimization is needed to improve growth and isopropanol yields, requiring larger membrane surface area, refined hollow fiber packing, and fermentation strategies tailored to the membrane reactor.

<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Box plot representation of distribution of FSC (top) and fluorescence intensity of IP-permeable cell in the FL3 channel (bottom) during the different gas fermentations: (A) Gas fermentation of Re2133/pEG7c without HFMC, (B) Gas fermentation of Re2133 with HFMC, (C) Gas fermentation of Re2133/pEG7c with HFMC with low Isopropanol production, (D) Gas fermentation of Re2133/pEG7c with HFMC with Isopropanol production. The blue areas indicate samples collected during Isopropanol production (intensity of blue relates to the level of isopropanol concentration). The green areas indicate samples collected during the periods where HFMC was not yet connected to the reactor.</figcaption></figure>
Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This work was supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 955740 named ConCO2rde project. F. DiBisceglie benefited from a French Government grant managed by the French National Research Agency under the "Investissements d'Avenir" program with the reference ANR-18-EURE-0021.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biortech.2025.133407.

Data availability

Data will be made available on request.

References

Akkoyunlu, B., Daly, S., Syron, E., Casey, E., 2024. Fermentation strategies for PHB production in a novel membrane bioreactor: Investigating batch and fed-batch operations. Biochem Eng J 204, 109239. https://doi.org/10.1016/j.bej.2024.109239.

Bakonyi, P., Buitrón, G., Valdez-Vazquez, I., Nemestóthy, N., Bélafi- Bakó, K., 2017. A novel gas separation integrated membrane bioreactor to evaluate the impact of self-generated biogas recycling on continuous hydrogen fermentation. Appl Energy 190, 813–823. https://doi.org/10.1016/j.apenergy.2016.12.151.

Bommareddy, R.R., Wang, Y., Pearcy, N., Hayes, M., Lester, E., Minton, N.P., Conradie, A.V., 2020. A Sustainable Chemicals Manufacturing Paradigm Using CO2 and Renewable H2. IsScience 23 (6), 101218. https://doi.org/10.1016/j.isci.2020.101218.

Boy, C., Lesage, J., Alfenore, S., Gorret, N., Guillouet, S.E., 2023. Comparison of plasmid stabilization systems during heterologous isopropanol production in fed-batch bioreactor. J Biotechnol 366, 25–34. https://doi.org/10.1016/j.jbiotec.2023.02.011.

Budde, C.F., Riedel, S.L., Willis, L.B., Rha, C.K., Sinskey, A.J., 2011. Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains. Appl. Env. Microbiol. 77 (9), 2847–2854. https://doi.org/10.1128/AEM.02429-10.

Crépin, L., Barthe, M., Leray, F., Guillouet, S.E., 2018. Alka(e)ne synthesis in *Cupriavidus necator* boosted by the expression of endogenous and heterologous ferredoxin-ferredoxin reductase systems. Biotechnol.Ll. Bioeng. 115 (10), 2576–2584. https://doi.org/10.1002/bit.26805.

Frölicher, T.L., Fischer, E.M., Gruber, N., 2018. Marine heatwaves under global warming. Nature 560 (7718), 360–364. https://doi.org/10.1038/s41586-018-0383-9.

Garrigues, L., Maignien, L., Lombard, E., Singh, J., Guillouet, S.E., 2020. Isopropanol production from carbon dioxide in *Cupriavidus necator* in a pressurized bioreactor. New Biotechnol 56, 16–20. https://doi.org/10.1016/j.nbt.2019.11.005.

Grousseau, E., Lu, J., Gorret, N., Guillouet, S.E., Sinskey, A.J., 2014. Isopropanol production with engineered *Cupriavidus necator* as bioproduction platform. Appl Microbiol Biotechnol 98 (9), 4277–4290. https://doi.org/10.1007/s00253-014-5591-0.

Hu, Q., Zhang, X., Hao, H. 2023. A review of hydrogen-air cloud explosions:The fundamentals, overpressure prediction methods, and influencing factor. Intl J Hydrogen Energy 48, 13705-1373. https://doi.org/10.1016/j.ijhydene.2022.11.302.

Ishizaki, A., Tanaka, K., 1991. Production of poly-β-hydroxybutyric acid from carbon dioxide by *Alcaligenes eutrophus* ATCC 17697T. J. Ferm. Bioeng. 71 (4), 254–257. https://doi.org/10.1016/0922-338X(91)90277-N.

Ishizaki, A., Tanaka, K., Taga, N., 2001. Microbial production of poly-D-3-hydroxybutyrate from CO2. Appl Microbiol Biotechnol 57 (1-2), 6–12. https://doi.org/10.1007/s002530100775.

Ismail, S., Giacinti, G., Raynaud, C.D., Cameleyre, X., Alfenore, S., Guillouet, S., Gorret, N., 2024. Impact of the environmental parameters on single cell protein production and composition by *Cupriavidus necator*. J Biotechnol 388, 83–95. https://doi.org/10.1016/j.jbiotec.2024.04.009.

Lambauer, V., Kratzer, R., 2022. Lab-Scale Cultivation of *Cupriavidus necator* on Explosive Gas Mixtures: Carbon Dioxide Fixation into Polyhydroxybutyrate. Bioeng. 9 (5), 1–18. https://doi.org/10.3390/bioengineering9050204.

Lee, I.Y., Nam, S.W., Choi, E.S., Chang, H.N., Park, Y.H., 1993. Production of poly-β-hydroxybutyrate and measurement of related enzyme activities in *Alcaligenes*

*eutrophus*. J Ferment Bioeng 76, 416–418. https://doi.org/10.1016/0922-338X(93) 90037-9.

Liew, F.M., Martin, M.E., Tappel, R.C., Heijstra, B.D., Mihalcea, C., Köpke, M., 2016. Gas Fermentation-A flexible platform for commercial scale production of low-carbon-fuels and chemicals from waste and renewable feedstocks. Front Microbiol 7. https://doi.org/10.3389/fmicb.2016.00694.

Liu, C., Colón, B.C., Ziesack, M., Silver, P.A., Nocera, D.G., 2016. Water splitting-biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis. Science. 352 (6290), 1210–1213. https://doi.org/10.1126/science.aaf5039.

Logsdon, J.E., Loke, R.A., 2000. Isopropyl Alcohol. Kirk-Othmer Encyclopedia of Chemical Technology, John Wiley Sons Ltd.

Morinaga, Y., Yamanaka, S., Ishizaki, A., Hirose, Y., 1978. Growth characteristics and cell composition of *Alcaligenes eutrophus* in chemostat culture. Agric Biol Chem 42 (2), 439–444. https://doi.org/10.1080/00021369.1978.10862993.

Orgill, J.J., Atiyeh, H.K., Devarapalli, M., Phillips, J.R., Lewis, R.S., Huhnke, R.L., 2013. A comparison of mass transfer coefficients between trickle-bed, Hollow fiber membrane and stirred tank reactors. Biores. Technol. 133, 340–346. https://doi.org/10.1016/j.biortech.2013.01.124.

Panich, J., Fong, B., Singer, S.W., 2021. Metabolic Engineering of *Cupriavidus necator* H16 for Sustainable Biofuels from CO2. Trends Biotechnol 39 (4), 412–424. https://doi.org/10.1016/j.tibtech.2021.01.001.

Przybylski, D., Rohwerder, T., Harms, H., Mueller, R.H., 2012. Third-generation feed stocks for the clean and sustainable biotechnological production of bulk chemicals: Synthesis of 2-hydroxyisobutyric acid. Energy Sustain. Soc. 2 (1), 1–9. https://doi.org/10.1186/2192-0567-2-11.

Przybylski, D., Rohwerder, T., Dilßner, C., Maskow, T., Harms, H., Müller, R.H., 2015. Exploiting mixtures of H2, CO2, and O2 for improved production of methacrylate precursor 2-hydroxyisobutyric acid by engineered Cupriavidus necator strains. Appl Microbiol Biotechnol 99 (5), 2131–2145. https://doi.org/10.1007/s00253-014-6266-6.

Ring, R., Stott, J., Hales, C., 2008. Modeling the risk of fire/explosion due to oxidizer/ fuel leaks in the Ares I interstage. 3rd IAASS Conference paper.

Rosenberg, E., DeLong, E.F., Lory, S., Stackebrandt, E., Thompson, F. (Eds.), 2013. The prokaryotes: Applied bacteriology and biotechnology, 4. ed. ed. SpringerLink Bücher. Springer, Berlin, Heidelberg, 394 pp.

Sakarika, M., Kerckhof, F.M., Van Peteghem, L., Pereira, A., Van Den Bossch, T., Bouwmeester, R., Gabriels, R., Van Haver, D., Ulcar, B., Martens, L., Impens, F., Boon, N., Ganigué, R., Rabaey, K., 2023. The nutritional composition and cell size of microbial biomass for food applications are defined by the growth conditions. Microb Cell Fact 22, 254. https://doi.org/10.1186/s12934-023-02265-1.

Schoenmakers, P., Rad, R., Ihl, A., Weickardt, I., Guillouet, S.E., Apfel, U.P., Lauterbach, L., 2025. Isopropanol production from carbon dioxide by Cupriavidus necator using a zero-gap cell with culture broth as catholyte. iScience. 28, 113018. https://doi.org/10.1016/j.isci.2025.113018.

Shen, Y., Brown, R., Wen, Z., 2014. Syngas fermentation of *Clostridium carboxidivorans* P7 in a hollow fiber membrane biofilm reactor: Evaluating the mass transfer coefficient and ethanol production performance. Biochem Eng J 85, 21–29. https://doi.org/10.1016/j.bej.2014.01.010.

Siegel, R.S., Ollis, D.F., 1984. Kinetics of Growth of the Hydrogen-Oxidizing Bacterium *Alcaligenes eutrophus* (ATCC 17707) in Chemostat Culture. Biotechnol Bioeng 26, 764–770. https://doi.org/10.1007/s00253-007-0983-z.

Sillero, A., Selivanov, V.A., Cascante, M., 2006. Pentose phosphate and Calvin cycles: Similarities and three-dimensional views. Biochem. Mol. Biol. Edu. 34 (4), 275–277. https://doi.org/10.1002/bmb.2006.49403404267.

Staples, M.D., Malina, R., Suresh, P., Hileman, J.I., Barrett, S.R.H., 2018. Aviation CO2 emissions reductions from the use of alternative jet fuels. Energy Policy 114, 342–354. https://doi.org/10.1016/j.enpol.2017.12.007.

Stott, P., 2016. How climate change affects extreme weather events. Science 352 (6293), 1517–1518. https://doi.org/10.1126/science.aaf7271.

Subagyo, D.C.H., Shimizu, R., Orita, I., Fukui, T., 2021. Isopropanol production with reutilization of glucose-derived CO 2 by engineered Ralstonia eutropha. J. Biosc. Bioeng. 132 (5), 479–486. https://doi.org/10.1016/j.jbiosc.2021.08.004.

Sydow, A., Becker, L., Lombard, E., Ulber, R., Guillouet, S.E., Holtmann, D., 2023. Autotrophic Production of the Sesquiterpene α-Humulene with Cupriavidus necator in a Controlled Bioreactor. Bioengineering 10, 1194. https://doi.org/10.3390/bioengineering10101194.

Torella, J.P., Gagliardi, C.J., Chen, J.S., Bediako, D.K., Colón, B., Way, J.C., Silver, P.A., Nocera, D.G., 2015. Efficient solar-to-fuels production from a hybrid microbial-water-splitting catalyst system. PNAS. 112 (8), 2337–2342. https://doi.org/10.1073/pnas.1424872112.

Vadlja, D., Koller, M., Novak, M., Braunegg, G., Horvat, P., 2016. Footprint area analysis of binary imaged *Cupriavidus necator* cells to study PHB production at balanced, transient, and limited growth conditions in a cascade process. Appl Microbiol Biotechnol 100, 10065–10080. https://doi.org/10.1007/s00253-016-7844-6.

Volova, T.G., Zhila, N.O., Kalacheva, G.S., Brigham, C.J., Sinskey, A.J., 2013. Effects of intracellular poly(3-hydroxybutyrate) reserves on physiological-biochemical properties and growth of Ralstonia eutropha. Res Microbiol 164, 164–171. https://doi.org/10.1016/j.resmic.2012.10.008.
Wang, H.-J., Dai, K., Wang, Y.-Q., Wang, H.-F., Zhang, F., Zeng, R.J., 2018. Mixed culture 483 fermentation of synthesis gas in the microfiltration and ultrafiltration hollow-fiber membrane biofilm reactors. *Biores. Technol.* **267**, 650–656.

Weickardt, I., Lombard, E., Zhang, A., Blank, L., Guillouet, S.E., 2025. Comparative characterisation of autotrophic and heterotrophic isopropanol formation by

Cupriavidus necator in shake flasks. J Biotechnol **403**, 1–8. https://doi.org/10.1016/j.jbiotec.2025.03.011.

Wiacek, C., Müller, S., Benndorf, D., 2006.A. cytomic approach reveals population heterogeneity of Cupriavidus necator in response to harmful phenol concentrations. *Proteomics* **6**, 5983–5994. https://doi.org/10.1002/pmic.200600244.