Nitrogenase is a complex oxidoreductase that is classified into three different types based on the heterometal cofactor present in the active site of the enzyme complex 5, 6. These findings suggest the effectiveness of humin as a solid-phase extracellular electron mediator for the promotion of anaerobic BNF activity, potentially to serve for the basis for a sustainable technology. The next generation sequencing (NGS) of partial 16S rRNA genes showed the predominance of Clostridiales (Firmicutes) in the consortia. The promotion of anaerobic BNF activity by only reduced humin without any other electron donor suggested that humin did not serve as organic carbon source but as extracellular electron mediator, for electron donation to the nitrogen-fixing microorganisms. The promotion effected by humin differed from the effects of chemical reducing agents and the effects of supplemental micronutrients and vitamins. Various humins from different origins promoted anaerobic BNF activity, although the degree of promotion differed. This was evidenced by increased acetylene reduction activity and total nitrogen content of the consortia. This study reports the promotion of BNF activity of anaerobic microbial consortia by humin, a solid-phase humic substance, at any pH, functioning as an extracellular electron mediator, to levels of 5.7–11.8 times under nitrogen-deficient conditions. One sustainable alternative technology is the electrochemical promotion of biological nitrogen fixation (BNF). Nitrogen fertiliser is manufactured using the industrial Haber–Bosch process, although it is extremely energy-consuming.
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