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| | <div class="headline" id="Chapter 1"> | | <div class="headline" id="Chapter 1"> |
| − | <h1>
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| − | <i>Bacillus subtilis</i> and biofilm formation
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| − | </h1>
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| − | <div class="headline" >
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| − | <h4>
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| − | <i>Bacillus subtilis</i>
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| − | </div>
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| − | <p>
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| − | We aim to convert micropollutants
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| − | in wastewater treatment plants using an
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| − | enzyme-producing biofilm and came to the
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| − | decision that Bacillus subtilis is the best
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| − | choice as our biofilm forming organism.
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| − | </p>
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| − |
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| − | <p>
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| − | <i>Bacillus subtilis</i> is a gram-positive bacterium which is usually found in soil.
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| − | Gram-positive bacteria only have one cell membrane and are thus often used for protein secretion<sup id="cite_ref-1"><a href="#cite_note-1">[1]</a></sup>.
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| − | On the one hand, secreting proteins is beneficial in protein production because no cell disruption is
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| − | necessary to collect the protein and the formation of inclusion bodies can be prevented<sup id="cite_ref-2"><a href="#cite_note-2">[2]</a></sup>. On the
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| − | other hand, and in our case specifically, we avoid the issue of bringing enzyme substrates across
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| − | the membrane in the cell as well as potential cell toxicity of the substrate and degradation product.
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| − | We want to leverage the ability of <i>B. subtilis</i> to secret proteins to immobilize our target enzymes
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| − | in the biofilm matrix by fusing them to a matrix protein. This is easier to realize with
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| − | <i>B. subtilis</i> as gram-positive model organism than with the gram-negative bacteria,
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| − | e.g. <i>Escherichia coli</i>. In addition, <i>B. subtilis</i> is a facultative anaerobic
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| − | organism which means it can grow in oxygenic, as well as anaerobic conditions,
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| − | which can be of advantage when using the biofilm underwater in the clarifier<sup id="cite_ref-3"><a href="#cite_note-3">[3]</a></sup>.
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| − | </p>
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| − |
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| − | <p>
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| − | In nature <i>B. subtilis</i> forms biofilms on plant roots as it needs a nutrient source. The
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| − | advantage for the plants is that <i>B. subtilis</i> protects it from pathogenic infections by funghi<sup id="cite_ref-4"><a href="#cite_note-4">[4]</a></sup>.
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| − | Since <i>B. subtilis</i> is a natural biofilm former, it has been used as model organism
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| − | for studying biofilms in the last years<sup id="cite_ref-4"><a href="#cite_note-4">[4]</a></sup>. By using this natural biofilm former, we do not have to
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| − | worry about how our organism will form a biofilm. Furthermore, <i>B. subtilis</i> is classified as
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| − | generally recognized as safe (GRAS) by the American Food and Drug Administration (FDA)<sup id="cite_ref-5"><a href="#cite_note-5">[5]</a></sup>, unlike other
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| − | bacteria which are often pathogenic like <i>E. coli</i> or <i>Staphylococcus aureus</i><sup id="cite_ref-6"><a href="#cite_note-6">[6]</a></sup>. It is very
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| − | important for us to use an organism which is generally safe given the fact that we want to bring it
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| − | into a wastewater treatment plant.
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| − | </p>
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| − |
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| − | <p>
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| − | Another characteristic of <i>B. subtilis</i> is its ability to form endospores
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| − | when exposed to extreme environmental conditions<sup id="cite_ref-7"><a href="#cite_note-7">[7]</a></sup>. Spores are highly resistant
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| − | towards extreme temperature, desiccation, radiation and chemicals. This
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| − | resistance makes spores interesting for industrial processes, e.g. by displaying
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| − | enzymes on the surface of spores which can then be used in harsh reaction conditions. Furthermore,
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| − | they are metabolic inactive but can germinate once nutrients are sufficiently available or when they
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| − | experience high pressure<sup id="cite_ref-8"><a href="#cite_note-8">[8]</a></sup>.
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| − | </p>
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| − | <div class="headline" >
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| − | <h4>
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| − | Biofilm formation
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| − | </h4>
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| − | </div>
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| − |
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| − | <div class="headline" id="Chapter 2">
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| | <h1> | | <h1> |
| | Biofilm engineering | | Biofilm engineering |
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| − | <div class="headline" id="Chapter 3"> | + | <div class="headline" id="Chapter 2"> |
| | <h1> | | <h1> |
| | Testing | | Testing |
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| | </p> | | </p> |
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| − | <div class="referencestd">
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| − | <h4 style="text-align: left"> References</h4>
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| − | <a class="anchor" id="cite_note-1"></a>
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| − | <a class="referencestd" href="https://doi.org/10.1007/s00253-013-4960-4"
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| − | target="_blank">1.Liu, L.; Liu, Y.; Shin, H. D. Developing Bacillus Spp. as a Cell Factory for Production of Microbial Enzymes and Industrially Important Biochemicals in the Context of Systems and Synthetic Biology. Applied Microbiology and Biotechnology. Springer July 11, 2013, pp 6113–6127 Doi:10.1007/s00253-013-4960-4 </a>
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| − |
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| − | <a class="anchor" id="cite_note-2"></a>
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| − | <a class="referencestd" href="https://doi.org/10.1016/j.bbamcr.2004.02.011"
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| − | target="_blank">2.Westers, L.; Westers, H.; Quax, W. J. Bacillus Subtilis as Cell Factory for Pharmaceutical Proteins: A Biotechnological Approach to Optimize the Host Organism. Biochimica et Biophysica Acta - Molecular Cell Research. Elsevier November 11, 2004, pp 299–310 Doi:10.1016/j.bbamcr.2004.02.011 </a>
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| − |
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| − | <a class="anchor" id="cite_note-3"></a>
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| − | <a class="referencestd" href="https://doi.org/10.1078/0723-2020-00108"
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| − | target="_blank">3.Clements, L. D.; Miller, B. S.; Streips, U. N. Comparative Growth Analysis of the Facultative Anaerobes Bacillus Subtilis, Bacillus Licheniformis, and Escherichia Coli. Syst. Appl. Microbiol. 2002, 25 (2), 284–286 Doi:10.1078/0723-2020-00108</a>
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| − |
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| − | <a class="anchor" id="cite_note-4"></a>
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| − | <a class="referencestd" href="https://doi.org/10.1038/nrmicro2960"
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| − | target="_blank">4. Vlamakis, H.; Chai, Y.; Beauregard, P. Sticking Together: Building a Biofilm the Bacillus Subtilis Way. Nature Reviews Microbiology. NIH Public Access March 2013, pp 157–168 Doi:10.1038/nrmicro2960 </a>
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| − |
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| − | <a class="anchor" id="cite_note-5"></a>
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| − | <a class="referencestd" href="https://www.fda.gov/food/generally-recognized-safe-gras/microorganisms-microbial-derived-ingredients-used-food-partial-list"
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| − | target="_blank">5. Microorganisms & Microbial-Derived Ingredients Used in Food (Partial List) | FDA https://www.fda.gov/food/generally-recognized-safe-gras/microorganisms-microbial-derived-ingredients-used-food-partial-list (accessed Aug 27, 2020) </a>
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| − |
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| − | <a class="anchor" id="cite_note-6"></a>
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| − | <a class="referencestd" href="https://doi.org/10.1093/femsre/fuv015"
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| − | target="_blank">6.Hobley, L.; Harkins, C.; MacPhee, C. E. Giving Structure to the Biofilm Matrix: An Overview of Individual Strategies and Emerging Common Themes. FEMS Microbiology Reviews. Oxford University Press June 8, 2015, pp 649–669 Doi:10.1093/femsre/fuv015 </a>
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| − |
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| − | <a class="anchor" id="cite_note-7"></a>
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| − | <a class="referencestd" href="https://doi.org/10.1101/2020.08.30.273821"
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| − | target="_blank">7. Karava, M.; Gockel, P.; Kabisch, J. Bacillus Subtilis Spore Surface Display of Photodecarboxylase for the Transformation of Lipids to Hydrocarbons. bioRxiv 2020, 2020.08.30.273821 Doi:10.1101/2020.08.30.273821 </a>
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| − |
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| − | <a class="anchor" id="cite_note-8"></a>
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| − | <a class="referencestd" href="https://doi.org/10.1128/AEM.71.10.5879-5887.2005"
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| − | target="_blank">8.Black, E. P.; Koziol-Dube, K.; Guan, D. Factors Influencing Germination of Bacillus Subtilis Spores via Activation of Nutrient Receptors by High Pressure. Appl. Environ. Microbiol. 2005, 71 (10), 5879–5887 Doi:10.1128/AEM.71.10.5879-5887.2005 </a>
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| − | </div>
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