Anaerobic, solvent-producing bacteria: Molecular ... polysaccharolytic activity and agroindustrial...

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  • Lehrstuhl fr Mikrobiologie der Technischen Universitt Mnchen

    Anaerobic, solvent-producing bacteria: Molecular characterisation, polysaccharolytic activity and

    agroindustrial waste degradation

    Dolly Montoya Castao

    Vollstndiger Abdruck der von der Fakultt Wissenschaftszentrum Weihenstephan fr Ernhrung, Landnutzung und Umwelt der Technischen

    Universitt Mnchen zur Erlangung des akademischen Grades eines

    Doktors der Naturwissenschaften

    genehmigten Dissertation.

    Vorsitzender: Univ.- Prof. Dr. W. Hll

    Prfer der Dissertation: 1. Univ.- Prof. Dr. W. Staudenbauer 2. Univ.- Prof. Dr. H. Parlar

    Die Dissertation wurde am 25.06.2003 bei der Technischen Universitt Mnchen eingereicht und durch die Fakultt Wissenschaftszentrum

    Weihenstephan fr Ernhrung, Landnutzung und Umwelt am 21.10.2003 angenommen.

  • Acknowledgements

    I would like to thank the following for their help and support during the five years spent

    on my thesis.

    Professor Walter Staudenbauer for accepting me in his group and encouraging me to

    start writing my PhD thesis, his advice once I had started to write and bringing the many

    strands of this work together.

    Doctor Wolfgang Schwarz for all his valuable help, encouragement, suggestions and

    patience with reading and re-reading initial drafts of the many documents which have

    gone towards making up this thesis. His help with experimental design and analysis was

    invaluable, especially in terms of what he could explain and offer from the German

    point of view, but done in a most supportive and friendly way.

    Doctor Vladimir Zverlov for all his help and advice with the laboratory techniques

    and, furthermore, for the friendly way in which he offered such help, making these tasks

    much more agreeable when difficulties arose.

    My Research Group at the Biotechnology Institute in Bogot D.C, especially

    Catalina Arevalo for her help in the many experiments, Fabio Aristizabal PhD for his

    help with the molecular methodology and all the students, here in the Institute, for their

    tireless work in the laboratory on those thankless tasks which usually never get

    mentioned or recognised.

    The Universidad Nacional de Colombia, Colciencias, the Volkswagen Foundation

    and the Technischen Universitt Mnchen for providing me with time, resources, an

    international agreement (which is more than just that) and the chance to write my thesis,

    respectively.

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    SUBJECTS

    INTRODUCTION ........................................................................................................... 9 Biochemistry and physiology ....................................................................................................11 Carbon and electron flow regulation ..........................................................................................13 Gene and enzyme organisation ..................................................................................................14 The low-solvent-production phenomena in ABE fermentation .....................................................16 Polysaccharolytic enzymes........................................................................................................16 Project objectives: ....................................................................................................................19 I. MATERIALS AND METHODS...................................................................................... 22 1. MICRO-ORGANISMS.....................................................................................22 2. CULTURE MEDIUM..............................................................................................23 2.1 RCM medium ......................................................................................................23 2.2 T6 broth medium..................................................................................................24 2.3 TYG....................................................................................................................24 2.4 TYS ....................................................................................................................25 2.5 Milk medium for riboflavin production ..................................................................25 2.6 TES Broth............................................................................................................25 2.7 Basal Clostridium medium (CBM) ........................................................................26 2.8 BHI medium for Staphylococcus epidermidis 850 H95 growth ................................26 2.9 LB Luria Bertani medium.....................................................................................26 2.10 Sporulation medium .............................................................................................27 2.11. Industrial medium ...............................................................................................27 2.12. POME (Palm Oil Mill Effluent) Medium. .............................................................28 3. MICRO-ORGANISM GROWTH CONDITIONS...........................................................29 3.1 Anaerobic indicator composition ...........................................................................29 4. MICRO-ORGANISM ISOLATION FROM SOIL ..........................................................30 4.1 Sample collection from soil...................................................................................30 4.2 Strain isolation from soil samples ..........................................................................31 4.3 Isolate conservation ..............................................................................................31 5. PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERISATION ..................................32 5.1 Isolate solvent-production profiles.........................................................................32 5.1.1 Internal standard preparation: (n-propanol) .............................................................33 5.2 Physiological test .................................................................................................33 5.2.1 Rifampicin sensitivity ...........................................................................................34 5.2.2 Riboflavin production ...........................................................................................34 5.2.3 Curd formation before 24 h...................................................................................34 5.3 Selected strain ......................................................................................................35 5.4. Physiological differentiation of Clostridium species................................................35 5.4.1 Tests for glycerol, ribose and gelatin ......................................................................35 5.4.2. Indol....................................................................................................................36 5.4.3. Catalase...............................................................................................................36 6. MOLECULAR CHARACTERIS ATION ......................................................................37 6.1 Genomic DNA isolation protocol for 16SrRNA and DNA-DNA hybridisation .........37 6.2. Plasmids ..............................................................................................................39 6.2.1 DNA isolation ......................................................................................................39 6.2.2. Micro-organism cultures and procedure for growing Clostridium: ...........................40 6.2.3. Plasmid isolation procedure ..................................................................................41

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    6.2.4. Electrophoresis.....................................................................................................43 6.2.5. Plasmid DNA digestion with restriction enzymes ...................................................44 6.3 Pulse-field gel electrophoresis (PFGE)...................................................................45 6.3.1 DNA preparation procedure ..................................................................................48 6.3.2. Pretaq digestion....................................................................................................49 6.3.2 Running gel .........................................................................................................50 6.4 16S rRNA sequencing ..........................................................................................50 6.4.1 Fragment amplification .........................................................................................50 6.4.2 Amplification verification.....................................................................................51 6.4.3 PCR product recovery...........................................................................................51 6.4.4. PCR fragment sequencing .....................................................................................51 6.4.5. Sequence analysis .................................................................................................52 6.5. PCR identification of strains using a Clostridium butyricum specific 16S rRNA

    targeted oligonucleotide........................................................................................52 6.6 DNA-DNA hybridisation......................................................................................53 6.6.1 Chromosomal DNA preparation ............................................................................53 6.6.2 Dot Blot and hybridisation ..............................