Amyloid Based Ceramic Biofilm and Its Applications in Building Materials

Abstract

Biofilm-Integrated Nanofiber Display (BIND) (Nguyen, Botyanszki, Tay, & Joshi, 2014a) is a platform for the engineering of the bacterial extracellular matrix material in amyloid. In this platform an engineered version of the CsgA protein can be functionalized in a variety of ways by fusing different peptide domains to it. We thought to exploit this mechanism by engineering the CsgA protein in order to create a ceramic coating which resample the properties of nacre and can therefore potentially exhibiting corrosion and mold protection, as well as fire resistance. Due to its biological means of production, the resulting coating is emission-free, non-toxic and would require far less caloric input than typical ceramic processes, which are famously energy intensive. Microbiologically induced calcium carbonate precipitation (MICP) is a natural process which can be mimicked and used for a number of applications, such as metal remediation, concrete restoration and carbon sequestration. The carbonate precipitation induced by these microbes can be achieved via Urease or Carbonic Anhydrase (CA). The latter is one of the fastest known catalysts and is the enzyme responsible for the reversible hydration of CO2 and can therefore constitute an interesting tool for CO2 sequestration. For this reason it was decided to fuse CA to CsgA in order to assist the biomineralization process, thereby creating an amyloid based ceramic biofilm. Different sequences of CA from different species: Gallus gallus α-CAII, Bacillus subtilis XF-1 β-CA, e. Coli β-CA, ζ-CA from Thalassiosira weissflogi were successfully cloned using Gibson Assembly into a previously described plasmid (Dorval Courchesne, Duraj-Thatte, Tay, Nguyen, & Joshi, 2016). All four different CAs cloned showed enzymatic activity at pH above 8.5 making these the first set of active enzymes directly fused to CsgA. Furthermore, all the constructs have been showing the ability of retaining the precipitation of calcium carbonate attached to their biofilm even after extensive washing. It is concluded that this is due to the presence of the enzyme, given that in the positive control the precipitation was rinsed away after washing. Altogether these results demonstrate that the enzymes fused to CsgA are capable not only of assisting in the precipitation, but also in retaining the precipitants on the biofilm, confirming thus the importance of the enzyme in the biomineralization process and therefore making it an indispensable element in the creation of a ceramic biofilm.