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“Hydrogen Production by Escherichia Coli Genetically Modified”

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“Hydrogen Production by Escherichia Coli Genetically Modified” INSTITUTO POTOSINO DE INVESTIGACIÓN CIENTÍFICA Y TECNOLÓGICA, A.C. POSGRADO EN CIENCIAS EN BIOLOGIA MOLECULAR “Hydrogen production by Escherichia coli genetically modified” Tesis que presenta M. en C. Luis Manuel Rosales Colunga Para obtener el grado de Doctor en Ciencias en Biología Molecular Codirectores de la Tesis: Dr. Antonio De León Rodríguez Dr. Elías Razo Flores San Luis Potosí, S.L.P., Septiembre de 2011 i Constancia de aprobación de la tesis La tesis ―Producción de hidrógeno por Escherichia coli genéticamente modificada‖ presentada para obtener el Grado de de Doctor en Ciencias en Biología Molecular fue elaborada por Luis Manuel Rosales Colunga y aprobada el 27 de septiembre de 2011 por los suscritos, designados por el Colegio de Profesores de la División de Biología Molecular del Instituto Potosino de Investigación Científica y Tecnológica, A.C. ____________________ Dr. Antonio De León Rodríguez (Codirector de la tesis) ____________________ Dr. Elías Razo Flores (Codirector de la tesis) ____________________ Dr. Alfredo Martínez Jiménez (Asesor de la tesis) ___________________ Dr. Felipe Alatriste Mondragón (Asesor de la tesis) ii Créditos Institucionales Esta tesis fue elaborada en el Laboratorio de Biotecnología Molecular de la División de Biología Molecular del Instituto Potosino de Investigación Científica y Tecnológica, A.C., bajo la codirección de los doctores Antonio De León Rodríguez y Elías Razo Flores. Durante la realización del trabajo el autor recibió una beca académica del Consejo Nacional de Ciencia y Tecnología (CONACYT-174494). La investigación de esta tesis fue parcialmente financiada por el proyecto FMSLP-2005-COI-23 del Fondo Mixto San Luis Potosí-CONACYT, asignado al Dr. Elías Razo Flores. iii Dedico esta tesis con cariño para: Mis abuelos Teodoro (†) y Amparo (†) Mi mamá Amparo, Pacho, Enrique. Gracias por su apoyo. v Agradecimientos A los doctores Antonio, Elías, Alfredo y Felipe por su dirección y asesoría. A mis compañeros Arlette, Gaby, Mabel, Gil, Joshua, Lupita, Zazil, Jorge, Falcon, Sandra, Ivon, Astrid y ex compañeros de laboratorio Nahomy, Faby, Ara, Hera, Teresita, Emilio, Victor, Jacinto, Juan Antonio, Sergio, Vania y Ciria por su apoyo, compañerismo, amistad y por compartir tantas experiencias Al M en C. Leandro Gabriel Ordoñez Acevedo y al Dr. Gustavo Dávila Vázquez por sus comentarios y asesoría, a la M en C. Dulce Isela de Fátima Partida Gutiérrez y M en C. Guillermo Vidriales Escobar por su asesoría técnica Al IPICyT Y CONACYT vi Contenido Constancia de aprobación de la tesis ................................................................. ii Créditos Institucionales ....................................................................................... iii Acta de examen .................................................................................................... iv Dedicatorias ........................................................................................................... v Agradecimientos .................................................................................................. vi List of Figures ........................................................................................................ x List of Tables ...................................................................................................... xiii Resumen ............................................................................................................. xiv Abstract ................................................................................................................ xv Chapter 1 General Introduction ............................................................................ 1 1.1 Background. ................................................................................................... 1 1.2 Hydrogen production by Escherichia coli. ...................................................... 2 1.3 Improvement of hydrogen production and yield by genetic manipulations. .... 4 1.4 Scope and structure of the thesis ................................................................... 7 1.5 References ..................................................................................................... 7 Chapter 2 Hydrogen production by Escherichia coli ∆hycA ∆lacI using cheese whey as substrate .................................................................................... 9 Abstract ................................................................................................................ 9 2.1Introduction ................................................................................................... 10 2.2 Experimental procedures ............................................................................. 11 2.2.1 Construction of mutant strains ............................................................... 11 2.2.2 Culture media. ....................................................................................... 13 2.2.3 Comparative growth kinetics of mutant strains using glucose or lactose 14 2.2.4 Comparative hydrogen production ability by the WDH and WDHL mutant strains ............................................................................................................. 14 2.2.5 Experimental design. ............................................................................. 15 2.2.6 Batch cultures on bioreactor. ................................................................. 15 2.2.7 Analytical methods. ................................................................................ 16 vii 2.3 Results and discussion. ............................................................................... 17 2.3.1 Hydrogen production using CW as substrate by WDH and WDHL mutant strains. ............................................................................................................ 17 2.3.2 Hydrogen production by the WDHL strain in bioreactor. ........................ 19 2.3.3 Optimization of the culture conditions to improve the hydrogen production. ...................................................................................................... 21 2.3.4 Hydrogen production under the best conditions. .................................... 25 2.4 Conclusions ................................................................................................. 29 2.5 References: .................................................................................................. 30 Chapter 3 Influence of pH on hydrogen production by Escherichia coli ∆hycA ∆lacI using cheese whey as substrate. ............................................................. 34 Abstract .............................................................................................................. 34 3.1 Introduction .................................................................................................. 35 3.2 Experimental procedures. ............................................................................ 36 3.2.1 Strain and culture media. ....................................................................... 36 3.2.2 Cultures on bioreactor. ........................................................................... 37 3.2.3 Analytical methods ................................................................................. 37 3.3 Results and discussion ................................................................................ 38 3.3.1 Influence of on line pH control on hydrogen production ......................... 38 3.3.2 Effect of pH on substrate consumption. ................................................. 40 3.3.3 Effect of pH on the production of metabolites. ....................................... 42 3.4 Conclusions.................................................................................................. 48 3.5 References. .................................................................................................. 48 Chapter 4 Fermentation of lactose and its constituent sugars by E. coli WDHL: Impact on hydrogen production............................................................ 52 Abstract .............................................................................................................. 52 4.1 Introduction. ................................................................................................. 53 4.2 Materials and methods. ................................................................................ 54 4.2.1 Strain and culture media. ....................................................................... 54 4.2.2 Batch Cultures. ...................................................................................... 55 4.2.3 Analytical methods. ................................................................................ 55 4.3 Results ......................................................................................................... 56 viii 4.3.1 Growth of E. coli using different sugars as substrate. ............................ 56 4.3.2 Fermentation of Lactose. ....................................................................... 56 4.3.3 Fermentation of glucose. ....................................................................... 58 4.3.4 Fermentation of galactose. .................................................................... 60 4.3.5 Fermentation of a mixture of glucose and galactose. ............................. 61 4.4 Discussion. ................................................................................................... 63 4.5 Conclusions.................................................................................................
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