A comprehensive approach to probe the role of hydrogen producing enzymes in Clostridium acetobutylicum

Bio-hydrogen production by dark fermentation relies on complex metabolic pathways that must be further elucidated for this technology to improve and be economically viable. Here, hydrogen production in Clostridium acetobutylicum is investigated through a comprehensive approach consisting of mapping the function and gene expression of four potential hydrogen-producing enzymes onto the organism's metabolic phases. The data show that nitrogenase and NiFe hydrogenase do not produce hydrogen under the conditions of this study, while FeFe hydrogenases are the main hydrogen producers in C. acetobutylicum . Group A FeFe hydrogenase (CaHydA1) is likely the key contributor to hydrogen production, while group B FeFe hydrogenase (CaHydA2) may contribute to hydrogen production due to its high gene expression. Additionally, CaHydA2's bias towards hydrogen evolution may enable hydrogen production at high partial pressure. These findings advance the understanding of hydrogen metabolism and underpin future optimisation of bio-hydrogen production that may reduce the cost of bio-hydrogen.