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© 2020 Lee et al.Glycerol is an organic waste material, which can be used for production of microbial biomass and consequently serving valuable biocatalysts promoting generation of electric current in microbial fuel cell (MFC). [NiFe]-Hydrogenases (Hyd) of Escherichia coli and Ralstonia eutropha may be applied as potential anode biocatalysts in MFC. In this study, E. coli K12 whole cells or crude extracts and R. eutropha HF649 synthesizing Strep-tagged membrane-bound Hyd (MBH) were evaluated, as anode enzymes in the bioelectrochemical