Bacterial cellulose (BC) is a versatile material that could potentially be used as a scaffold for tissue engineering. Therefore, our team is optimizing Komagataeibacter for the production of BC and testing the development of molds that would allow the growth of BC sheets in the correct format for tissue culture and subsequent 3D organ assembly. To that end, we converted the surface of a complex object into a 2D shape, printed the perimeter as a “cookie cutter”, and used it as a template for bacterial cellulose sheet production.
Unfolding the shape of a sphere into a 2D object
The surface of 3D objects can be readily unfolded into 2D objects provided that the object is subdivided into segments with single curvatures. For example, the surface of a cylinder can be unfolded into a rectangle. Objects with curvature in multiple directions, such as spheres, pose a greater challenge. However, there is a great number of published works that suggest 2D shapes that reasonably capture the surface of a sphere.
Demaine and colleagues (Computational Geometry 42 (2009) 748-757) defined a number of 2D shapes to represent the area of a 3D sphere, minimizing area (compatible with small printing beds) or minimizing the perimeter (fewer cuts and joints). Due to the constraints of a small 3D printer the Unicamp_Brazil team had available, we selected a 2D k-petal model with 6 petals for this project (Figure 1, extracted from Demaine et al, 2009).
Delimiting the growth of BC for assays in 24-well plates
Initial experiments for the growth of animal cells using cellulose as a scaffold require cutting the sheets in appropriate proportions to fit them into plates of cell culture medium. The growth of cellulosic fibers in different formats, for later cutting of what would be used, causes destandardization in the final product, in other words, we obtain a portion of cellulose with variable thickness and irregular surface that makes cell cultivation difficult. To minimize the effects of these variables on the culture, we built a mold that allows the production of cellulose in standardized formats ideal for fitting into 24-well culture plates, avoiding variations in the cellulosic surface.
The project design aims to engineer strains with a bimodal growth pattern, divided in biomass accumulation stage and Bc production stage. To achieve this we are engineering a K. rhaeticus AF1 where cellulose synthase genes are off under dark and expressed by the use of blue light. Therefore, we devised a bioreactor to grow Komagataeibacter in an environment where there is no light and switch to an inducible environment when needed.
For growth optimization, some criteria were considered in the design of the bioreactor. As was seen in the math models for growth the Komagataeibacter is obligately aerobic, so the oxygen supply is very important. Another point discussed was the cost of construction. Fitting the bioreactor with an impeller would significantly increase the cost, therefore, to allow for mixing and oxygen supply, it was decided to build an air lift bioreactor.
We investigated different induction alternatives that would allow the expression of cellulose synthate (bcs) when we desired, however, the costs of inducers for the characterized Komagataeibacter promotors are prohibitive. Hence, we explored the scientific literature and encountered a recent paper describing a single-component light-sensitive transcriptional repressor. By using a light-sensitive promoter (LexRO) the production of BC can become cheaper. However, to induce the reaction efficiently, a bright place for the reaction is needed.
To unite the two needs the team developed two pieces of hardware. The first part of the growth will be done in a low-cost reactor. The first part of the growth will be done in a low-cost reactor, made out of common laboratory materials. For the fermentation vessel, a 1-liter reagent bottle was chosen. All inlets were located in the lid, following the design of commercial bioreactors. For the air inlet, a small aquarium compressor was used (4 L/min). For prototyping purposes, this reactor was tested with E. coli, due to its fast growth rate.
The first tests were successful, opening opportunities for improvement in some parts. Our goal is to couple both pieces of hardware: after leaving the bioreactor (biomass accumulation), the biomass will be directed to the second stage, the photobioreactor, which will activate cellulose production in defined molds.
With this integration of the bioreactor with the photobioreactor using peristaltic pumps. Thus making cellulose production a continuous process increasing the productivity.
