Global warming and climate change are some of the greatest challenges human society is facing (Rawat et al., 2024, Bevacqua et al., 2025). The massive release of greenhouse gases (GHGs) (main components carbon dioxide, methane and nitrous oxide) has become a key problem worldwide. Reaching the 2015 Paris Agreement goals (Filonchyk et al., 2024), which call for a global average temperature increase of less than 2 °C by the end of the century, requires an enormous reduction in global anthropogenic GHG emissions. In response, the EU is currently targeting an 80–95 % reduction in GHGs by 2050 (European Commission. Directorate General for Climate Action., 2024). This is a very challenging target that will require solutions from numerous sectors (Cifuentes-Faura, 2022). Efficient biotechnological processes to convert CO2 into organic compounds need to be developed and will provide a substantial contribution to this goal by laying the foundation for a C1-bioeconomy (García and Galán, 2022). These processes will have an important impact on the comprehensive strategy to reduce carbon dioxide pollution in the atmosphere. Bioprocesses for commodity production based on CO2 can substantially decrease the use of land-grown organic feedstock as substrates (Bachleitner et al., 2023, Wang et al., 2024).

The original idea of cellular agriculture and single cell protein (SCP) was developed back in the 1960s, and sources included various microbial phyla of bacteria, yeast or other fungi and microalgae, with the protein serving as livestock feed, aquaculture feed additive and food component. However, novel cultivation techniques that yield biomass for SCP production based on microbes that oxidise reduced gas fractions as an energy source for their metabolism are a relatively new development (Nyyssölä et al., 2022). In general, chemolithoautotrophic cultivation of aerobic gas fermenting bacteria involves an oxidizable reduced gas fraction (hydrogen, methane, carbon monoxide) as an energy source (electron donor) in a respiratory metabolism with oxygen as the final electron acceptor (Lambauer and Kratzer, 2022, Meyer and Schlegel, 1983, Schink and Schlegel, 1978). As a carbon source, single-carbon compounds (C1) (e.g.: CO2, methane, CO) are provided in the culture medium. In addition, gaseous ammonia can be applied as nitrogen source and all other macro- and micronutrients such as for instance phosphate, sulphur compounds as well as metal ions and trace elements can all be supplied solely from inorganic sources. The nutrient requirement already shows two of the main advantages of cultivating aerobic gas fermenting bacteria for SCP production: no agricultural-based feedstock is required, and the needed land area for production plants can be kept to a minimum. This contrasts with the cultivation of microalgae or fungi, which require either a large surface area (algae) or an agricultural-based carbon feedstock (fungi) (Ritala et al., 2017).

The use of gaseous substrates enabling chemolithoautotrophic metabolism, such as hydrogen and carbon dioxide, represents an interesting alternative to common agro- or petroleum-based production schemes for the sustainable synthesis of various bulk products. Microorganisms that use gases as the sole source of carbon and energy, such as acetogenic or knallgas bacteria, are thus increasingly coming into focus (Rittmann et al., 2015). C. necator has been studied since the early 1960s because of its special autotrophic metabolism and is one of the best-characterized knallgas bacteria and the workhorse for biotechnological poly-3-hydroxybutyrate (PHB) production from various carbon sources (Zhang et al., 2022). C. necator has long been investigated for its remarkable metabolic flexibility to efficiently convert both autotrophic and heterotrophic substances into valuable products (Alhafiz et al., 2025). The range of products obtained from chemolithoautotrophic cultivation spans from biopolymer production (PHB) (Kim et al., 2022), over Isopropanol (Garrigues et al., 2019, Grousseau et al., 2014) and alkene (Crépin et al., 2016, LoPachin and Gavin, 2014, Panich et al., 2021) to single-cell protein (Chee et al., 2019, Raberg et al., 2018). One application to mention in particular is the use of 13CO2 for the production of labelled amino acids via gas fermentation (Lütte et al., 2012). These labelled amino acids, currently available commercially, are valuable tools for various research and diagnostic applications.

Econutri (https://econutri.com/) is an Austrian startup company that aims at the efficient production of single cell protein applying chemolithoautotrophic conditions. C. necator is well known for the fact that the cultivation conditions can strongly control the carbon metabolism. The bacterium utilizes the Calvin-Benson-Bassham (CBB) cycle to fix carbon dioxide (Jeffke et al., 1999, Peplinski et al., 2010, Schäferjohann et al., 1996). Carbon flux through these pathways can be directed towards either the synthesis of the storage polymer poly 3-hydroxy butyrate (PHB) or biomass production rich in protein (Kim et al., 2022). C. necator exhibits up to 60 % energy conversion efficiency when using hydrogen and carbon dioxide as substrates but is strongly influenced by the gas composition and the availability of oxygen (Yu et al., 2013). Effective supply of the carbon source and limitation of essential components for growth such as nitrogen, oxygen, phosphor or sulphur sources directs the metabolism from biomass (protein) formation to PHB synthesis as a carbon and energy storage mechanism.

A key point in this regulatory network at chemolithoautotrophic growth conditions is the supply of the gaseous substrates (H2, O2, CO2, NH3). Not only the composition of the gas phase is relevant, but also parameters that are changing the solubility of the different gases in the aqueous fermentation broth such as pressure, pH, and temperature influence the metabolic balance and product distribution. Therefore, a deep understanding of the factors influencing the balance between the gaseous substrates is crucial for optimizing bioprocesses towards the desired product.

An additional point in autotrophic fermentations is the seed culture preparation. Usually, reactor precultures are grown on carbohydrates and not chemolithoautotrophically. Since precultures play a crucial role in optimizing microbial growth, this point should not be overlooked to ensure efficient transition to larger scale fermentations. The conventional method with heterotrophic seed culture leads to a long lag phase at the beginning of the fermentation (Crépin et al., 2016, Lambauer and Kratzer, 2022) and thereby reduces the space-time-yield of the reactor.

In this work, we report on the chemolithoautotrophic high cell density cultivation of C. necator in a pilot-scale pressurized gas bioreactor of 300 L total volume for the first time. The bioreactor was designed for high-efficiency gas-liquid transfer and can be operated with explosive gas compositions. Our goal was exploring the potential of this unique pilot scale gas fermenter in terms of cell mass formation and fermentation speed. We compare two different fermentation runs inoculated with heterotrophically or chemolithoautotrophically grown cells in view of protein-rich single cell production. The influence of inoculum source is demonstrated and growth characteristics as well as gas consumption data are shown.