Assessing the temperature effect on the kinetics of a hydrogenotrophic methanogenic culture using a novel headspace-free methodology

• Headspace-free methodology enables continuous monitoring of dissolved H₂ kinetics. • H₂ uptake rate increases with temperature, reaching 773 mmol H₂·gVSS⁻¹·d⁻¹ at 65°C. • PHF-HUR avoids gas-liquid transfer limitations. Time is reduced compared to SBT. • Temperature dependent half-saturation constant ranges from 4.27·10⁻³ to 6.87·10⁻² mM. • Moser model fits microbial kinetics better than Monod across tested conditions. Accurately determining the biological activity of hydrogenotrophic microbial cultures independently of potential gas-liquid mass transfer limitations for hydrogen is important but is challenging. In this work, a novel methodology called Pressurized Headspace-Free Hydrogen Uptake Rate (PHF-HUR) is presented and validated. The methodology consists on first saturating a microbial culture bubbling pure H 2 under selected pressures in a 120 mL stirred batch bioreactor equipped with a dissolved H 2 probe. Secondly, the decrease of the dissolved H 2 concentration under headspace-free conditions is monitored in real-time. Hydrogen profiles under headspace-free conditions are used to calculate the HUR and to assess the microbial activity and related kinetics. A mixed culture was characterized at different temperatures ranging from 27 °C to 70 °C using this methodology. Modelling of hydrogen uptake rate was conducted using Monod and Moser kinetic equations, the latter providing a better fit of the experimental data. In the novel PHF-HUR methodology, hydrogen uptake increased with temperature reaching 773 mmol H2 ·g VSS −1 ·d −1 at 65 °C, but was nil at 70 °C. The half-saturation concentration for the methanogens was also temperature dependent, ranging from 4.3·10 −3 mM H 2 at 27 °C to 6.9·10 −2 mM at 65 °C. Results indicate the potential of the novel methodology to accurately determine the biological kinetics of hydrogenotrophic methanogens without gas-liquid mass transfer limitations.