It is widely admitted that hydrocarbon combustion is a major source of greenhouse gas emissions, contributing to climate change, and research efforts must be focused on alternative energy sources, with lower environmental impacts. From this point of view, gaseous hydrogen (H2) seems to be a promising energy source due to its very high energy content (142 kJ/g) (Hay et al., 2013). Nowadays, more than 100 methods of hydrogen production have been reported using chemical or biological processes (Worku et al., 2024). However, the vast majority of the world’s hydrogen supply relies on methods that use fossil resources, such as oil refining (16 %), coal gasification (21 %), or methane steam reforming (62 %), resulting in high CO2 emissions. Water electrolysis using renewable electricity is often seen as a potential sustainable way to produce molecular hydrogen. However, the availability of such electricity faces several discontinuities due to variations in meteorological conditions, and the yield of this conversion remains quite low (Droessler and Leach, 2024, Remme, 2024).

Therefore, new ways to produce hydrogen with lower greenhouse gas emissions are needed. Biological routes through fermentation processes are some of the solutions in the sense that they can be based on a wide range of biomass resources, which are often considered as by-products. Among the existing biohydrogen production processes, biophotolysis, photo-fermentation, and dark fermentation are deeply investigated, and a growing number of publications are reported (Fatima et al., 2018, Teke et al., 2023). Light-dependent processes exhibit high theoretical hydrogen yields, but generally low productivities (Worku et al., 2024). However, recent advances relying on the addition of photo-nanocatalysts to improve charge transferring during the fermentation resulted in higher hydrogen production (Liu et al., 2023, Ramzan et al., 2024).

Dark fermentation is still the most common approach for biohydrogen production, since it is more efficient in terms of hydrogen production rate and does not require providing light energy, resulting in cheaper processes (Teke et al., 2023, Argun and Kargi, 2011). Several microbial genera, such as Enterobacter, Enterococcus, Clostridium, Bacillus, or Thermoanaerobacterium are known to produce significant amounts of hydrogen by anaerobic digestion of organic matter through pure cultures (Baeyens et al., 2020, Guo et al., 2010). Comparative analyses on fermentation capabilities were reported. In particular, Clostridium species such as Clostridium acetobutylicum, Clostridium butyricum, and Clostridium beijerinckii tend to show 3–7 times higher cumulative hydrogen productions, 3–10 times higher specific hydrogen production rates, and 2–31 times higher yields (molH2/molhexose) than Enterobacter species (Hu et al., 2013, Hiligsmann et al., 2011, Wang and Yin, 2021). The most interesting species for H2 bioproduction in pure culture therefore, belong to the Clostridium genus.

Clostridia are Gram-positive, strictly anaerobic, and endospore-forming bacteria, whose genus comprises 163 validated species according to the List of Prokaryotic names with Standing in Nomenclature from DSMZ (Dürre, 2014). Several Clostridium species were particularly investigated and numerous information, knowledges, and applications are available. They are capable of degrading organic matter such as hexoses, starch or cellulose through an acid-solvent fermentation, known as dark fermentation. The final products are valuable chemicals, such as organic acids, alcohols, or solvents, along with H2 and CO2 as gaseous emissions (Qu et al., 2022, Lee et al., 1985). Whereas alcohol and solvent production was investigated during the early 20th century before the oil-based mass production (Sauer, 2016), researchers are now focusing on the biogas fraction. Hydrogen production with Clostridium cultivations for further uses is particularly explored, emphasizing the importance of elucidating the mechanisms involved during the life cycle of these bacteria. Clostridium acetobutylicum ATCC 824 remains a reference strain for this purpose, due to its capability to produce acetone and butanol in addition to other products (ethanol, acetate, and butyrate). However, all metabolisms of Clostridium species share similar characteristics, with specific branched pathways varying by species (Fig. 1). Based on reaction stoichiometry, the maximum theoretical yield for hydrogen production from direct glucose consumption is 4 moles of H2 per mole of glucose. This 4:1 ratio is often referred to as the “Thauer limit” and is associated with the theoretical production of acetate alone (Ergal et al., 2020, Thauer et al., 1977). Interestingly, co-cultures with different species can lead to yields higher than 4 molH2/molhexose, which is higher than the theoretical threshold with a pure culture, since the metabolites from one species can be the substrate of the other (Ergal et al., 2020, Thauer et al., 1977).

However, considering data on hydrogen production by Clostridia species only, significant differences on in yields (YH2/S) are reported in the literature, even with the same species, with values varying from 1 to 30 times higher (Wang and Yin, 2021). This could be due to strong differences in culture conditions used (Rittmann and Herwig, 2012). Most of clostridial cultures (77 %) were carried out in batch mode, which remains the most commonly used culture method for obtaining data quickly. The remaining studies were performed in continuous cultures. Moreover, among batch cultures, 51 % were performed in a closed container (Rittmann and Herwig, 2012). However, this condition is critical for hydrogen production, since hydrogenase activities can be inhibited even at low hydrogen partial pressures, which is typically the case when working in a closed vessel (Morra, 2022, Klein et al., 2010). This wide variation in culture conditions makes the results and bacterial performances difficult to compare.

This present study focuses on the relevant parameters influencing biohydrogen production by Clostridium species and proposes to evaluate the production performances of known hydrogen-producing strains. For that purpose, four Clostridium species were studied and compared in a standardized manner based on their capabilities to support high hydrogen productivity. Such productivity is usually associated with acid production, mostly acetate and butyrate. Fermentation studies were all performed in controlled bioreactors with gas outlets and at a regulated pH in order to maintain acidogenesis. Comparative analyses were based on objective parameters. The most important one was the maximum volume productivity of gases produced associated with the H2/CO2 ratio (RH2/CO2), allowing the estimation of the hydrogen outflow. Other parameters, such as the maximum specific growth rate (µmax), the hydrogen to substrate yield (YH2/S), and the acetate to butyrate ratio (RAc/Bu) were also investigated in order to establish a rational comparison of biohydrogen production using bacteria from the Clostridium genus.