Syngas fermentation offers a promising route for biological hydrogen (H2) production, yet industrial application is restrained by low yields and limited understanding of nutrient requirements in natural microbial consortia. This study investigated the macro- and micronutrient roles influencing carbon monoxide (CO)-to-H2 conversion in a thermophilic consortium using a medium for thermophilic, anaerobic, and carboxydotrophic microorganisms as the starting formulation. Yeast extract and trace element solution showed the strongest positive associations with H2 production, supplying essential vitamins, nitrogen, and enzymatic cofactors, including Ni and Fe, for CO dehydrogenase and hydrogenase activity. In contrast, sulfide solution and NH4Cl showed inhibitory trends, likely through redox imbalance and hydrogenase suppression. Optimizing the concentrations of yeast extract (7.5 g/L) and trace element solution (4 mL/L) increased H2 production by 53% (3.59 ± 0.06 mmol), although the elevated volatile fatty acid concentration indicated partial reliance on organic carbon metabolism. Reducing yeast extract to 0.5 g/L and supplementing with Wolin’s vitamin solution maintained equivalent productivity while reducing medium costs by approximately 45% relative to DSMZ 507. These findings suggest that precise nutrient management plays a decisive role in governing CO conversion toward H2, providing critical insights for scalable, economically viable biohydrogen production from syngas fermentation.

Pyrolysis is a widely used method to produce biochar and/or bio-oil. During this process,synthesis gas (syngas) is also formed with a main composition of a mixture of carbon monoxide(CO), hydrogen (H2), and carbon dioxide (CO2). Syngas can be converted by a biologicalwater-gas shift reaction, where CO is converted to H2 without the necessity of hightemperatures and expensive catalysts, as opposed to the traditional water-gas shift reaction.This process not only meets the high demand for biohydrogen in various industrial applications(e.g., chemical, food, and pharmaceutical processes) but also utilizes off-gases from industrialand thermochemical processes as energy and carbon sources for microorganisms. Whilebatch processes have been successfully demonstrated, scaling up to continuous operationsremains challenging. This study aims to identify the key challenges in scaling up from batch tocontinuous processes by applying parameters that were efficient in batch experiments to atrickle bed reactor (TBR). In batch experiments, suitable environmental factors were proposedusing pretreated wastewater treatment granules after evaluating pH values, temperatures, andinoculum concentrations. Using a mixed culture of microorganisms from a wastewatertreatment plant as inoculum and a chemical-free methanogenesis inhibition approach, abiohydrogen yield of up to 0.07 g H2/g CO was achieved. The same parameters, inoculumsource, and growth medium were then applied to a TBR to enhance gas-liquid mass transferand process control. The biohydrogen yield achieved was 0.05 g H2/g CO/h during seven daysin continuous mode. However, process stability and product selectivity were key challenges.Despite using pretreated inoculum and in-situ heat treatments, methane production eventuallyoccurred, causing fluctuations in biohydrogen production. This shift in metabolic activitysuggests that microbial community dynamics in continuous operation differ significantly frombatch conditions, despite using identical environmental parameters. Methanogenic activity inbatch mode may not have been detected due to the shorter duration of the experiments. Thesefindings highlight critical considerations for scaling up biohydrogen production with mixedculture, contributing to our understanding of microbial metabolic activities and the processchallenges in syngas fermentation.

This study evaluates the biological water-gas shift reaction of syngas into hydrogen (H2) using natural microbial consortia from wastewater treatment facilities. Various inoculum pretreatment techniques to inhibit methanogenesis and different operational conditions such as temperature, pH, and inoculum concentrations were tested to improve H2 production. An inoculum heat pretreatment of 85 °C for 30 min followed by a fermentation temperature of 65 °C and an initial pH value of 9, resulted in the maximum CO-to-H2 conversion (2.35 ± 0.00 mmol) in which Firmicutes dominated with a total inhibition of methanogens, such as with the chemical inhibition treatment. Additionally, volatile fatty acids (VFA) production was observed, being influenced by the pre-treatment. This study highlights the potential of natural consortia for efficient, cost-effective and scalable syngas fermentation processes, offering new insights into the bioconversion of H2 without costly chemical inhibitors.

Synthesis gas (syngas) fermentation represents a promising biological method for converting industrial waste gases, particularly carbon monoxide (CO) and carbon dioxide (CO2) from industrial sources (e.g. steel production or municipal waste gasification), into high-value products such as biofuels, chemicals, and animal feed using acetogenic bacteria. This review identifies and addresses key challenges that hinder the large-scale adoption of this technology, including limitations in gas mass transfer, an incomplete understanding of microbial metabolic pathways, and suboptimal bioprocess conditions. Our findings emphasize the critical role of microbial strain selection and bioprocess optimization to enhance productivity and scalability, with a focus on utilizing diverse microbial consortia and efficient reactor systems. By examining recent advancements in microbial conditioning, operational parameters, and reactor design, this study provides actionable insights to improve syngas fermentation efficiency, suggesting pathways towards overcoming current technical barriers for its broader industrial application beyond the production of bulk chemicals.