CHARATERISTIC STUDY AND RAPID DETECTION OF HYDROGENASES IN ENTEROBACTER AEROGENES

Hydrogenases play a key role in fermentative bio-hydrogen production. The hydrogenase in Enterobacter aerogenes IAM 1183 was found to have some promising charateristics of NADH dependent and oxygen tolerance, which would be benifical for the enhancing of hydrogen producing processes of this strain.
Rapid hydrogenase assay and evaluation would largely faciliate the process optimization. Different chemicals capable of enhancing the cell-wall permeability such as Triton X-100 and CTAB were used in intact cell assay to establish a simplified hydrogenase assay method. This method is suitable for both H₂ uptake and evolution activities assay. By using this method, the presence of H₂ uptake hydrogenase activity was first found in E. aerogenes.
In addtion, a gfp- and kanamycin-resistance gene-containing plasmid pUCGK was successfully constructed and transformed into E. aerogenes to develop a rapid GFP-based method for quantifying the bacterial concentration under anaerobic conditions. Fluorescence detection was performed by developing a method of aerobic fluorescence recovery (AFR) of the anaerobically expressed GFP. Rapid and non-disruptive cell quantification of E. aerogenes by fluorescence density was achieved for analyzing the hydrogen production process.