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.

Ocean acidification and warming are current global challenges that marine diazotrophs must cope with. Little is known about the effects of pH and temperature changes at elevated CO₂ levels in combination with different nutrient regimes on N₂ fixers, especially on heterotrophic bacteria. Here, we selected four culturable diazotrophs, i.e., cyanobacteria and heterotrophic bacteria, found in association with the endemic Mediterranean seagrass Posidonia oceanica. We tested different pH (from pH 4 to 8) and temperature levels (from 12 to 30 °C), under different nutrient concentrations of both phosphorus, P (0.1 µM and 1.5 mM), and iron, Fe (2 nM and 1 µM). We also tested different CO₂ concentrations (410 and 1000 particles per million (ppm)) under different P/Fe and temperature values (12, 18, and 24 °C). Heterotrophic bacteria were more sensitive to changes in pH, temperature, and CO₂ than the cyanobacterial species. Cyanobacteria were resistant to very low pH levels, while cold temperatures stimulated the growth in heterotrophic bacteria but only under nutrient-limited conditions. High CO₂ levels (1000 ppm) reduced heterotrophic growth only when cultures were nutrient-limited, regardless of temperature. In contrast, cyanobacteria were insensitive to elevated CO₂ levels, independently of the nutrient and temperature levels. Changes in N₂ fixation were mainly controlled by changes in growth. In addition, we suggest that alkaline phosphatase activity (APA) and reactive oxidative species (ROS) can be used as biomarkers to assess the plasticity of these communities to climate change factors. Unlike other studies, the novelty of this work lies in the fact that we compared the responses of cyanobacteria vs. heterotrophic bacteria, studying which changes occur at the cell plasticity level. Our results suggest that the responses of diazotrophs to climate change may depend on their P and Fe status and lifestyle, i.e., cyanobacteria or heterotrophic bacteria.