Porous hydrogel nanocomposites with embedded bacteria for biotechnological applications

Abstract

Technological processes based on microorganisms such as bacteria offer great potential for an environmentally friendly as well as resource-efficient production and purification of, for example, pharmaceuticals and water. Immobilizing microorganism inside semi-permeable substrates such as hydrogels is a suitable strategy to simplify the process of separation and purification in bioreactors, reducing costs and processing time of biotechnological processes. To meet the mechanical requirements, nanoparticles can be integrated into the hydrogel matrix to form a nanocomposite. However, the integration of particles can be detrimental to cells due to toxicity. Furthermore, the processing of hydrogel nanocomposites can be harmful to the cell due to high shear forces in the feedstock. Moreover, nutrient accessibility might be influenced by the particles and can hinder nutrient diffusion into the material and cells. Beyond that, high material porosity and a range of pore sizes are necessary to ensure proper nutrient diffusion. Thus, the processing of hydrogels with suitable rigidity to maintain the porosity and biocompatibility is still a challenge. This work aims to develop new processing strategies for microorganism immobilization into rigid porous materials for bioprocessing applications using only biocompatible materials. Microorganism behavior and accessibility in the different immobilization strategies as well as the influence of porosity on the overall biotransformation performance were investigated. In the first part of this work, a straightforward one-pot processing route based on the reinforcement of an alginate hydrogel with alumina nanoparticles, followed by the addition of bacteria Escherichia coli or Bacillus subtilis and subsequent internal/external ionotropic gelation steps was established. The developed bionanocomposite showed minimal shrinkage, increased structural and mechanical stability, as well as excellent biocompatibility. The immobilized bacteria maintained high viability and similar metabolic activity as non-immobilized cells and were able to consume glucose after several cycles as well as after 60 days of storage. The method could be adapted to each specific cell, such as bacteria, yeast, fungus, or even animal cells, by adding the proper nutrients into the suspension and it can be used with various shaping strategies to produce macroscopic materials like casting, extruding or 3D printing. Yet, this method showed some nutrient transport limitations as well as low material stability in specific solutions such as phosphate-buffered saline (PBS), which resulted in material dissolution after some days. To overcome restricted nutrient diffusion/permeability, a feedstock for 3D bioprinting structures with hierarchical porosity was developed. The feedstock is based on a modified highly particle-filled alumina/alginate nanocomposite gel with immobilized E. coli bacteria and the protein ovalbumin acting as a foaming agent. The foamed nanocomposite was shaped into a porous mesh structure by 3D printing. The influence of albumin on rheological properties, total porosity, water diffusion coefficient, and bacterial viability was characterized by bulk and interfacial rheology, X-ray microtomography (μCT), nuclear magnetic resonance (NMR) tomography, and resazurin assay respectively. The addition of albumin was an effective method to stabilize foams even after the printing procedure, resulting in enhanced material porosity. Albumin addition also increased the printability of the feedstocks as well as induced bacterial growth. The experimental results demonstrated a higher water content when porosity increases and consequently higher effective cell viability. The third part of the work aimed to increase material stability and avoid its dissolution in specific solutions. Therefore, a strategy for synthesizing a feedstock suitable for 3D bioprinting and covalent crosslinking of mechanically robust materials with embedded living bacteria were developed. The processing route is based on a highly particle-filled alumina/chitosan nanocomposite gel which is crosslinked by electrostatic interactions and covalent bonding using alginate and genipin, respectively. Feedstock’s properties and crosslinking time were analyzed by means of bulk rheology while crosslinked material stability in different solutions was analyzed by incubating crosslinked material in different solutions for 60 days. The addition of alginate was essential for printability and effective bacterial viability since samples without alginate showed no bacterial viability. The covalently crosslinked material successfully did not dissolve in phosphate-buffered saline (PBS), hydrochloric acid (HCl), sodium hydroxide (NaOH), or water. The experimental results obtained within this work demonstrate the potential of the described approaches for producing porous macroscopic bioactive materials with complex 3D geometries using only natural materials as a platform for novel applications in bioprocessing.