Riboflavin-meditated interspecies electron transfer in a H2-based denitrifying biofilm

Abstract

H2-based membrane biofilm reactors (MBfR) are effective for nitrogen removal, yet the interactions of microorganisms within the MBfR biofilms have received limited attention. In this study, we explored a flavin-mediated electron transport mechanism occurring between hydrogenotrophic microorganisms and non‑hydrogenotrophic denitrifying microorganisms in H2-based MBfR biofilms. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and electrochemical analysis confirmed riboflavin act as the sole potential redox mediator present in the system. Batch experiments demonstrated that riboflavin accelerates the denitrification process and that microorganisms can use reduced flavins as the sole electron donor to reduce nitrate. Metagenomic and metatranscriptomic analyses revealed increased expression of genes related to riboflavin biosynthesis and transport in hydrogenotrophic microorganisms, indicating endogenous riboflavin secretion into the medium. Our findings reveal the potential existence of a novel flavin-mediated electron transfer mechanism in the H₂-based denitrification process, enriching the understanding of interspecies interactions within MBfR biofilms. This study also provides a theoretical basis for efficient nitrogen removal through regulating interspecies electron transfer.