Study On Fluorescence-based Quantification Of Enterobacter Aerogenes And Its NADH Pathway Mechanism For Hydrogen Production

Hydrogen evolution pathway catalyzed by hydrogneases and its metabolic network control are of great importance for biohydrogen production. In this thesis, Enterobacter aerogenes, a rapid hydrogen-producing bacterium, was studied to establish new research platform for fermentative biohydrogen production. Two frontier topics in the field of fermentative biohydrogen production were studied: first, the method for analysis dynamic distribution of constituent bacterial strains in an anaerobic mixed culture system; and second, the molecular mechanism of"NADH pathway"and its control for intracellular metabolic network. The main results were summarized as follows.Green fluorescence protein (GFP) was used as a reporter protein to rapidly quantify the strain of E. aerogenes in anaerobic hydrogen-producing system. As mature GFP requires oxygen for the development of the fluorophore to exhibit fluorescence, GFP expressed under the anaerobic condition can not exhibit fluorescence. A method named Aerobic Fluorescence Recovery (AFR) was established to solve this problem. With the AFR technique, E. aerogenes was quantified in the whole course of anaerobic hydrogen-producing process. Mixed culture of E. aerogenes and Clostridium paraputrificum was studied as model system, and the dynamic distribution of the two participant strains was quantified with the AFR method.The"NADH pathway"in E. aerogenes was found to consist of NADH oxidase module and [NiFe] hydrogenase module which contributed to hydrogen production from NADH. NADH oxidases, which located in the outer membrane of the cells, oxidized NADH to NAD+, accompanying with the formation of electrons. Under anaerobic condition, protons were the final electron acceptors, and electrons formed from NADH oxidation were transfered through the hydrogenases on the inner membrane to protons, to form hydrogen. While under aerobic condition, electrons from NADH were transfered through the complex I in the cell membrane, which had similar ancestor with hydrogenases, and finally reacted with oxygen to form water.According to the features of the NADH oxidase of E. aerogenes, E. aerogenes whole cells were successfully coupled with alcohol dehydrogenase (ADH) to regenerate NAD+ for the transformation of ethanol to acetylaldehyde. Moreover, E. aerogenes whole cells and E. coli (pHLA-READH) whole cells which displayed alcohol dehydrogenase from Rhodococcus erythropolis were coupled to regenerate NAD+ for the transformation of phenylethanol to phenylaldehyde.Based on the molecular mechanism of"NADH pathway", external NADH or NAD+ was used to control the hydrogen metabolism of E. aerogenes. External NADH decreased hydrogen yield from 1.42 to 1.31 mol-H₂·(mol-glucose)^-1; while external enhaced hydrogen yield to 1.52 mol-H₂·(mol-glucose)^-1.Through the metabolic flux analysis of the anaerobic metabolism of E. aerogenes, lactate dehydrogenase and alcohol dehydrogenase were determined as the deletion targets to enhance the"NADH pathway". The ldh and adh genes were acquired successfully through homologous sequence analysis. Red system, a recombinant engineering tool, was successfully applied to the gene knockout of E. aerogenes.