University of Bath PHD Genetic Optimisation of Bacteria-Induced Calcite Precipitation Hoffmann, Timothy Award date: 2021 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 06. Oct. 2021 Genetic Optimisation of Bacteria-Induced Calcite Precipitation Timothy Dennis Hoffmann A thesis submitted for the degree of Doctor of Philosophy University of Bath Department of Biology and Biochemistry September 2020 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with the author and copyright of any previously published materials included may rest with third parties. A copy of this thesis has been supplied on condition that anyone who consults it understands that they must not copy it or use material from it except as licenced, permitted by law or with the consent of the author or other copyright owners, as applicable. Declaration of authorship I am the author of this thesis, and the work described therein was carried out by myself personally, with the exception of Chapter 1 which is presented as a literature review manuscript where Figure 3 was prepared by Bianca J. Reeksting and sections 1.5 and 1.6 were written together between the two of us. Signed Timothy D. Hoffmann 2 Table of Contents Acknowledgments ............................................................................................................................ 11 Abstract ............................................................................................................................................ 12 Chapter 1..................................................................................................................................... 13 1.1 Bacteria-induced mineral precipitation. ............................................................................... 14 1.2 Cell surface: nucleation catalysis, saturation state, and nucleation template. ................... 16 1.3 Cell surface: polymorph ratio, crystal morphology, mineral type, and crystal size. ........... 19 1.4 Cell surface or metabolism: are precipitating bacteria dead or alive? ................................ 21 1.5 Cell metabolism: pH, dissolved inorganic carbon (DIC) and inorganic anions. .................... 22 1.6 Cell metabolism: cation accumulation. ................................................................................. 25 1.7 Prospects. ............................................................................................................................... 26 Chapter 2..................................................................................................................................... 28 2.1 BICP biotechnologies: self-healing concrete. ........................................................................ 29 2.2 Challenges to BICP application. ............................................................................................. 31 2.3 PhD aims and objectives. ....................................................................................................... 34 Chapter 3..................................................................................................................................... 36 3.1 Chemicals and growth media. ............................................................................................... 37 3.2 Growth conditions and storage. ............................................................................................ 37 3.3 Plasmid and strain construction. ........................................................................................... 38 3.3.1 DNA manipulation. .......................................................................................................... 38 3.3.2 Transformations, deletion strains, and phage transduction. ........................................ 39 3.3.3 Conjugations. ................................................................................................................... 41 3.4 Zeta potential measurements. .............................................................................................. 54 3.5 Nisin growth inhibition assay. ............................................................................................... 54 3.6 Cytochrome C binding assay. ................................................................................................. 54 3.7 Calcite precipitation assays. ................................................................................................... 55 3.8 Whole cell urease assays. ...................................................................................................... 55 3.9 Electron microscopy. .............................................................................................................. 56 3.10 Fluorescence measurements. .............................................................................................. 56 Chapter 4..................................................................................................................................... 57 4.1 Introduction. ........................................................................................................................... 58 4.1.1 The ureolytic pathway. ................................................................................................... 58 4.1.2 Bacterial surface charge and biofilms. ........................................................................... 60 4.2 Results. ................................................................................................................................... 63 3 4.2.1 The role of ureolysis in calcite precipitation by Bacillus subtilis W168. ....................... 63 4.2.1.1 Initial trials in establishing heterologous urease gene expression in B. subtilis. .. 63 4.2.1.2 Selection of suitable urease gene cluster for heterologous expression in Bacillus subtilis W168. ....................................................................................................................... 65 4.2.1.3 Semi-quantitative urease activity assay. ................................................................ 68 4.2.1.4 Solid media urease and precipitation assay. .......................................................... 70 4.2.1.5 Calcite precipitation on LB and B4 precipitation media. ........................................ 73 4.2.1.6 SEM imaging of calcite colony precipitates. ........................................................... 77 4.2.2 The role of biofilm on calcite precipitation in Bacillus subtilis W168. .......................... 80 4.2.2.1 Establishing a biofilm precipitation model in Bacillus subtilis. .............................. 80 4.2.3 The effect of modulating biofilm on calcite precipitation in B. subtilis W168. ............ 86 4.2.4 The role of surface charge on calcite precipitation of Bacillus subtilis W168. ............. 92 4.2.4.1 Establishing surface charge changes in Bacillus subtilis W168: Electrophoretic mobility measurements, cytochrome C assay and nisin time dependent kill curves. ...... 92 4.2.4.2 Surface charge: calcite precipitation assays. .......................................................... 98 4.2.4.3 SEM imaging of calcite colony precipitates from strains grown on LBGMC and LBC. ..................................................................................................................................... 101 4.3 Discussion. ............................................................................................................................ 106 4.3.1 Urease discussion.......................................................................................................... 106 4.3.2 Biofilm discussion. ........................................................................................................ 110 4.3.3 Surface charge discussion. ............................................................................................ 114 4.3.4 General summary.......................................................................................................... 117 Chapter 5 .................................................................................................................................. 118 5.1 Introduction. ........................................................................................................................ 119 5.2 Results. ................................................................................................................................. 123 5.2.1 Construction of the mobilisable reporter plasmid pAT28-Pveg-GFPmut1. .................. 123 5.2.2 Establishment of selection and counter-selection