Acid mine drainage (AMD) generated by mining and metallurgical industries creates serious environmental problems. Treatment of AMD using sulfate-reducing bioreactors with rice bran is promising; however, this approach is limited by the biofilm formation at the top of the bioreactor, which can cause a significant reduction in water permeability. To assess biofilm microbiomes, both 16S and 18S rRNA gene amplicon sequencing of 10 biofilm samples were performed. Although the top five dominant microbial operational taxonomic units (OTUs) in the respective biofilms were diverse, common bacterial OTUs were found within the biofilms from the same bioreactor. In prokaryotes, OTUs related to Acinetobacter junii and Pseudomonas palleroniana made up the dominant species. As for eukaryotes, OTUs associated with Mortierella sp. and Chytriomyces sp. were detected, especially from acidic biofilm samples. The addition of anti-biofilm and anti-microbial reagents to the bioreactors may restrict biofilm formation. However, considering implementation costs and environmental influences, the operational management of bioreactors should also be considered; e.g., change of substrate from rice bran to low molecular weight organic compounds under acidic conditions.

To realize a hydrogen society, methods to produce hydrogen in an environmentally friendly manner like hydrogen fermentation using biomass waste are required. In this study, we developed a new approach for producing bio-hydrogen at a lower CO₂ emission using two types of bacteria. To improve hydrogen yield and NaOH consumption, which causes CO₂ emission according to a life cycle assessment of process, a co-culture system of Clostridium perfringens HN001, which produces lactate as a by-product of hydrogen, and Megasphaera elsdenii JCM1772, which produces hydrogen from lactate, was designed based on lactate metabolism. In a pure culture of M. elsdenii, lactate was preferentially consumed over glucose, and the production of hydrogen from lactate was confirmed. In a pure culture of C. perfringens, the production of lactate as a by-product of hydrogen was observed. In the co-culture system of C. perfringens and M. elsdenii, an increase of 131% in hydrogen yield and a reduction of 58% in NaOH consumption were achieved compared to those in the pure culture of C. perfringens due to the conversion of lactate to hydrogen by M. elsdenii. This result demonstrates that the co-culture system contributes improvement of hydrogen yield and reduction of CO₂ emission from fermentation process.