Project: Encapsulation of bacteria with biosilica for fabrication of bacterial bioreactors and biosensors

Industrial biotechnology has gained increasing importance for chemical, pharmaceutical, agro and food industry. Biosensors are extensively employed in many fields of industrial biotechnology (process control, fermentation control and analysis), but also for food and drug analysis, clinical diagnosis, and pollution control and monitoring. The chemical and mechanical stability of the applied microorganisms as well as techniques for their immobilization are often critical factors for the applicability of the technology._x000D__x000D_Bioencapsulation is an intriguing way to immobilize biological materials, including cells, in silica, metal-oxides or hybrid sol-gel polymers. Until now only the sol-gel precursor technology has been utilized to immobilize bacteria or yeast cells in silica. The P at the Université Pierre et Marie Curie (J. Livage), is an internationally recognized expert in this field and one of the pioneers who used sol-gel chemistry for encapsulation of living cells. The discovery of silicatein, an enzyme from marine sponges, by one of the participating SMEs - BIOTECmarin GmbH - and the P at the University of COz, makes it now possible to produce silica (“biosilica”) biocatalytically - under physiological (mild) conditions. _x000D__x000D_The aim of this project is the introduction of the technique (developed and patented by BIOTECmarin GmbH and the P at COz University) of enzyme-catalyzed (silicatein-mediated) biosilica synthesis for the fabrication of silica-encapsulated (enclosed in a silica glass cage) bacterial bioreactors and biosensors. The CO interest of BIOTECmarin is the production of a novel type of bacterial bioreactor for the production of industrially relevant recombinant proteins. In addition, a biosensor for environment-relevant applications will be realized. _x000D__x000D_Bacterial cells will be transformed with the silicatein gene from marine or freshwater sponges (cDNAs/genes coding for various isoforms of silicatein are available). The heterologous expression of biocatalytically active silicatein on the surface of the transformed bacteria will be used for the synthesis of porous, biocompatible silica shells from silicic acid. The aim of the proposed project is to increase – through bioencapsulation of the bacteria cells with a silica shell, performed under mild conditions - their mechanical and chemical stability and thus to facilitate their industrial applicability as bacterial bioreactors or biosensors. _x000D__x000D_The second SME P, MicroVacuum Ltd, is a world-wide leader in the R&D and production of optical grating-coupled waveguide (OWLS) based biosensors and instrumentation. This company has developed a label-free technique for quick, reliable detecting of E. coli using the Optical Waveguide Lightmode Spectroscopy (OWLS) sensing technique. This techniques allows the in situ and label free study of surface processes at molecular levels. OWLS technique is based on the measurement of the diffraction of a linearly polarized laser beam (He-Ne laser) on a diffraction grating in a thin waveguide layer (SiO2-TiO2). This technology will now be used - in combination with the technology developed by BIOTECmarin GmbH and COz University and the expertise of the P at the Université Pierre et Marie Curie - for the development of a novel type of bacterial biosensor consisting of silica encapsulated bacterial cells on a sensor chip surface. The particular interest of this P is the application of this biosensor in food industry (detection of contaminants)._x000D__x000D_The specific aims of the proposed 36-month project are:_x000D_1. Transformation of E. coli and heterologous expression of the silicatein genes_x000D_2. Biocatalytic formation of biosilica on the bacterial cell surface and physical chemical characterization of the formed biosilica shells_x000D_3. Determination of biosilica deposition on the bacterial cell surface applying the Optical Waveguide Lightmode Spectroscopy (OWLS) sensing technique_x000D_4. Preparation of bacterial bioreactors and bacterial biosensors consisting of encapsulated bacteria for the production of selected proteins, and the detection of selected stressors/environmental pollutants _x000D_5. Development of a bacterial biosensor based on biosilica encapsulated bacteria attached to sensor chips and OWLS technology_x000D_6. Characterization of the biological, chemical and mechanical properties of the biosilica-encapsulated bioreactors and biosensors _x000D__x000D_This project will markedly increase the position on the market of the two R&D-performing SMEs involved in this project – BIOTECmarin GmbH and MicoVacuum Ltd. – in one of the most exciting fields in nano-biotechnology, the synthesis of inorganic silica by an enzyme, at the cutting edge between inorganic chemistry and biochemistry._x000D_

Acronym SILIBACTS (Reference Number: 4289)
Duration 01/09/2008 - 30/09/2012
Project Topic The aim of this project is the introduction of the technique of enzyme (silicatein)-catalyzed biosilica synthesis, developed and patented by the applicants, for the production of silica-encapsulated bacterial bioreactors/sensors (bacteria in a silica glass cage) for biotechnological applications.
Project Results
(after finalisation)
The objective of this prohtramme was to prepare cell-based optical sensors by immobilization in inorganic materials whose formation was catalyzed by enzymes. In addition to our laboratory, two SMEs, one in charge of enzymes and one of the optical detection, and one academic P, involved in biology, were associated to this work and the project was initially elaborated on the basis of thier preiminary results. As the only chemists of the consortium, we were in charge of developping the fabrication process of the material. The first part of our work consited in reproducing and trying to improve the preliminary data. The reproduction was achieved but did not allow to obtain a robust material. Hence we decided to change our strategy keeping in mind our objective of obtaining stable and transparent materials compatible with cell immobilization. This was the core of our work in this project, and that was succesfully achieved. In particular, we set up a new protocol for multi-layered deposition of entrapped cells, allowing to preserve fluorescent bacteria (as obtained from one P) during several weeks and in different conditions. We could also demontrate for the first time the possibility for cell division inside these materials. After consulting the project Ps, this work was published in scientific journals (two papers with Silibacts reference). Unfortunately, the evaluation of the materials by the industrial P could not be achieved due to lack of time
Network Eurostars
Call Eurostars Cut-Off 1

Project partner

Number Name Role Country
5 Johannes Gutenberg-Universität Partner Germany
5 Université Pierre et Marie Curie Partner France
5 BIOTECmarin GmbH Observer Germany
5 NanotecMARIN GmbH Coordinator Germany
5 MicroVacuum Ltd Partner Hungary