Comparison of enrichment methods for efficient nitrogen fixation on a biocathode

The production of nitrogen fertilizers in modern agriculture is mostly based on the Haber-Bosch process, representing nearly 2% of the total energy consumed in the world. Low-energy bioelectrochemical fixation of N 2 to microbial biomass was previously observed but the mechanisms of microbial interactions in N 2 -fixing electroactive biofilms are still poorly understood. The present study aims to develop a new method of enrichment of autotrophic and diazotrophic bacteria from soil samples with a better electron source availability than using H 2 alone. The enrichment method was based on a multi-stage procedure. The first enrichment step was specifically designed for the selection of N 2 -fixing bacteria from soil samples with organic C as electron and carbon source. Then, a polarized cathode was used for the enrichment of autotrophic bacteria using H 2 (hydrogenotrophic) or the cathode as electron source. This enrichment was compared with an enrichment culture of pure diazotrophic hydrogenotrophic bacteria without the use of a microbial electrochemical system. Interestingly, both methods showed comparable results for N 2 fixation rates at day 340 of the enrichment with an estimated average of approximately 0.2 mgN fixed /L.d. Current densities up to -15 A/m² were observed in the polarized cathode enrichments and a significant increase of the microbial biomass on the cathode was shown between 132 and 214 days of enrichment.These results confirmed an enrichment in autotrophic and diazotrophic bacteria on the polarized cathode. It was hypothesied that autotrophic bacteria were able to use either the H 2 produced at the cathode or directly the cathode through direct electron transfer (DET) as more biomass was produced than with H 2 alone. Finally, the analysis of the enriched communities suggested that Desulforamulus ruminis mediated microbial interactions between autotrophic anaerobic and heterotrophic aerobic bacteria in polarized cathode enrichment. These interactions could play a key role in the development of biomass in these systems and on N 2 fixation. Based on these findings, a conceptual model on the functioning of mixed cultures N 2 -fixing electroactive biofilms was proposed.