Insights into the global regulation of anaerobic metabolism for improved biohydrogen production.

To improve the biohydrogen yield in bacterial dark fermentation, a new approach of global anaerobic regulation was introduced. Two cellular global regulators FNR and NarP were overexpressed in two model organisms: facultatively anaerobic Enterobacter aerogenes (Ea) and strictly anaerobic Clostridium paraputrificum (Cp). The overexpression of FNR and NarP greatly altered anaerobic metabolism and increased the hydrogen yield by 40%. Metabolic analysis showed that the global regulation caused more reducing environment inside the cell. To get a thorough understanding of the global metabolic regulation, more genes (fdhF, fhlA, ppk, Cb-fdh1, and Sc-fdh1) were overexpressed in different Ea and Cp mutants. For the first time, it demonstrated that there were approximately linear relationships between the relative change of hydrogen yield and the relative change of NADH yield or ATP yield. It implied that cellular reducing power and energy level played vital roles in the biohydrogen production.

[1]  R. Milo,et al.  Central carbon metabolism as a minimal biochemical walk between precursors for biomass and energy. , 2010, Molecular cell.

[2]  S. Brar,et al.  Biohydrogen production by co-fermentation of crude glycerol and apple pomace hydrolysate using co-culture of Enterobacter aerogenes and Clostridium butyricum. , 2015, Bioresource Technology.

[3]  M. Seeger,et al.  New alkane-responsive expression vectors for Escherichia coli and pseudomonas. , 2001, Plasmid.

[4]  Chong Zhang,et al.  Disruption of lactate dehydrogenase and alcohol dehydrogenase for increased hydrogen production and its effect on metabolic flux in Enterobacter aerogenes. , 2015, Bioresource technology.

[5]  Yu-an Jing.  Biological Hydrogen Production and Hydrogen Generation , 2010 .

[6]  X. Xing,et al.  Regulation of hydrogen production by Enterobacter aerogenes by external NADH and NAD , 2009 .

[7]  Chong Zhang,et al.  States and challenges for high-value biohythane production from waste biomass by dark fermentation technology. , 2013, Bioresource technology.

[8]  S. Evans,et al.  Enhanced Oxygen-Tolerance of the Full Heterotrimeric Membrane-Bound [NiFe]-Hydrogenase of Ralstonia eutropha , 2014, Journal of the American Chemical Society.

[9]  T. Wood,et al.  Metabolic engineering of Escherichia coli to enhance acetol production from glycerol , 2014, Applied Microbiology and Biotechnology.

[10]  M. R. Evans,et al.  FNR Is a Global Regulator of Virulence and Anaerobic Metabolism in Salmonella enterica Serovar Typhimurium (ATCC 14028s) , 2007, Journal of bacteriology.

[11]  Dipankar Ghosh,et al.  Strategies for improving biological hydrogen production. , 2012, Bioresource technology.

[12]  T. Wood,et al.  Metabolic engineering of Escherichia coli to enhance hydrogen production from glycerol , 2014, Applied Microbiology and Biotechnology.

[13]  X. Xing,et al.  A simplified method for assay of hydrogenase activities of H2 evolution and uptake in Enterobacter aerogenes , 2005, Biotechnology Letters.

[14]  Yuan Lu,et al.  Perturbation of formate pathway for hydrogen production by expressions of formate hydrogen lyase and its transcriptional activator in wild Enterobacter aerogenes and its mutants , 2009 .

[15]  G. L. Miller.  Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar , 1959 .

[16]  A. Gedanken,et al.  Escherichia coli and Pseudomonas aeruginosa eradication by nano-penicillin G. , 2016, Nanomedicine : nanotechnology, biology, and medicine.

[17]  G. Unden,et al.  Alternative respiratory pathways of Escherichia coli: energetics and transcriptional regulation in response to electron acceptors. , 1997, Biochimica et biophysica acta.

[18]  Yuan Lu,et al.  Characteristics of hydrogen and methane production from cornstalks by an augmented two- or three-stage anaerobic fermentation process. , 2009, Bioresource technology.

[19]  Dong-Woo Lee,et al.  Biohydrogen Production: Strategies to Improve Process Efficiency through Microbial Routes , 2015, International journal of molecular sciences.

[20]  T. Wood,et al.  Hydrogen production by recombinant Escherichia coli strains , 2012, Microbial biotechnology.

[21]  X. Xing,et al.  Alteration of energy metabolism in Enterobacter aerogenes by external addition of pyrophosphates and overexpression of polyphosphate kinase for enhanced hydrogen production , 2012 .

[22]  X. Xing,et al.  Improvement of Hydrogen Productivity by Introduction of NADH Regeneration Pathway in Clostridium paraputrificum , 2012, Applied Biochemistry and Biotechnology.

[23]  X. Xing,et al.  Improved hydrogen production under microaerophilic conditions by overexpression of polyphosphate kinase in Enterobacter aerogenes. , 2011, Enzyme and microbial technology.

[24]  X. Xing,et al.  Alteration of anaerobic metabolism in Escherichia coli for enhanced hydrogen production by heterologous expression of hydrogenase genes originating from Synechocystis sp , 2012 .

[25]  J. Nielsen,et al.  Bioreaction Engineering Principles , 1994, Springer US.

[26]  X. Xing,et al.  Bioengineering of the Enterobacter aerogenes strain for biohydrogen production. , 2011, Bioresource technology.

[27]  Y. Waché,et al.  Extracellular Oxidoreduction Potential Modifies Carbon and Electron Flow in Escherichia coli , 2000, Journal of bacteriology.

[28]  J. Šimůnek,et al.  Identification and characterization ofClostridium paraputrificum, a chitinolytic bacterium of human digestive tract , 2008, Folia Microbiologica.

[29]  Frederick R. Blattner,et al.  Genome-Wide Expression Analysis Indicates that FNR of Escherichia coli K-12 Regulates a Large Number of Genes of Unknown Function , 2005, Journal of bacteriology.

[30]  X. Xing,et al.  Expression of NAD+-dependent formate dehydrogenase in Enterobacter aerogenes and its involvement in anaerobic metabolism and H2 production , 2009, Biotechnology Letters.

[31]  W. Lubitz,et al.  Hydrogens detected by subatomic resolution protein crystallography in a [NiFe] hydrogenase , 2015, Nature.

[32]  Dong-Hoon Kim,et al.  Hydrogenases for biological hydrogen production. , 2011, Bioresource technology.

[33]  Duu-Jong Lee,et al.  Dark fermentation on biohydrogen production: Pure culture. , 2011, Bioresource technology.

[34]  K. Dill,et al.  Bacterial growth laws reflect the evolutionary importance of energy efficiency , 2014, Proceedings of the National Academy of Sciences.

[35]  X. Xing,et al.  Alteration of hydrogen metabolism of ldh-deleted Enterobacter aerogenes by overexpression of NAD(+)-dependent formate dehydrogenase , 2010, Applied Microbiology and Biotechnology.