As a renewable energy carrier, dark fermentative biohydrogen (H₂) represents a promising future alternative to fossil fuels. Recently, the limited H₂ yield of 4 moles of H₂ per mole glucose, the so-called “Thauer limit”, was surpassed by a defined artificial consortium. In this article, we demonstrate the upscaling of this drawing board design, from serum bottles to laboratory scale bioreactors. Our results illustrate that this designed microbial co-culture can be successfully implemented in batch mode, with maximum H₂ yields of 6.18 and 4.45 mol mol⁻¹ substrate. Furthermore, we report volumetric H₂ productivities of 105.6 and 80.8 mmol H₂ L⁻¹ h⁻¹_. These rates are higher than for any other dark fermentative H₂ production system using a synthetic microbial co-culture applied in batch mode on a defined medium. Our study is an important step forward for the application of artificial microbial consortia in future biotechnology and energy production systems.

Formate is one of the key compounds of the microbial carbon and/or energy metabolism. It owes a significant contribution to various anaerobic syntrophic associations, and may become one of the energy storage compounds of modern energy biotechnology. Microbial growth on formate was demonstrated for different bacteria and archaea, but not yet for species of the archaeal phylum Crenarchaeota. Here, we show that Desulfurococcus amylolyticus DSM 16532, an anaerobic and hyperthermophilic Crenarchaeon, metabolises formate without the production of molecular hydrogen. Growth, substrate uptake, and production kinetics on formate, glucose, and glucose/formate mixtures exhibited similar specific growth rates and similar final cell densities. A whole cell conversion experiment on formate revealed that D. amylolyticus converts formate into carbon dioxide, acetate, citrate, and ethanol. Using bioinformatic analysis, we examined whether one of the currently known and postulated formate utilisation pathways could be operative in D. amylolyticus. This analysis indicated the possibility that D. amylolyticus uses formaldehyde producing enzymes for the assimilation of formate. Therefore, we propose that formate might be assimilated into biomass through formaldehyde dehydrogenase and the oxidative pentose phosphate pathway. These findings shed new light on the metabolic versatility of the archaeal phylum Crenarchaeota.