3D printing of bacteria into functional complex materials

Abstract

Despite recent advances to control the spatial composition and dynamic functionalities of bacteria embedded in materials, bacterial localization into complex three-dimensional (3D) geometries remains a major challenge. We demonstrate a 3D printing approach to create bacteria-derived functional materials by combining the natural diverse metabolism of bacteria with the shape design freedom of additive manufacturing. To achieve this, we embedded bacteria in a biocompatible and functionalized 3D printing ink and printed two types of "living materials" capable of degrading pollutants and of producing medically relevant bacterial cellulose. With this versatile bacteria-printing platform, complex materials displaying spatially specific compositions, geometry, and properties not accessed by standard technologies can be assembled from bottom up for new biotechnological and biomedical applications.

Fig. 1. Schematics of the 3D bacteria-printing platform for the creation of functional living materials.

Multifunctional bacteria are embedded in a bioink consisting of biocompatible HA, κ-CA, and FS in bacterial medium. 3D printing of bacteria-containing hydrogels enables the creation of structures in arbitrary shape and added functionality due to the manifold products of bacterial metabolism. The inclusion of specific bacterial strains leads to a living and responsive hydrogel, a novel class of material named Flink. For example, the inclusion of P. putida and A. xylinum yields 3D-printed materials capable of degrading environmental pollutants and forming bacterial cellulose in situ for biomedical applications, respectively.

Fig. 2. Rheology and shape retention properties of bacteria-laden Flink used for 3D printing.

(A and B) Rheological properties of the individual components (1 wt % κ-CA, 1 wt % HA, and 1 wt % FS) in LB bacterial medium (A) and in combination as Flink at concentrations of 1, 2, and 3 wt % for each individual constituent (hereafter called 3, 6, and 9 wt % Flinks, respectively) (B). The steady-state flow behavior of the inks is measured by viscosity curves at increasing and decreasing shear rates. The elastic (G′) and viscous (G″) moduli are measured by oscillatory amplitude sweeps (strain, 0.01 to 1000%; angular frequency, 1 rad/s). (C) Left: To simulate printing conditions, alternating high (70 s⁻¹) and low (0.1 s⁻¹) shear rates in steady-state rotation mode were applied to 3, 6, and 9 wt % Flinks. Right: Instantaneous recovery of the viscoelastic network of 6 wt % Flink is shown by a sudden shear process in a steady state (70 s⁻¹), followed by an oscillatory time sweep. (D) Left: Dynamic yield stresses of different Flink concentrations measured in strain-controlled measurements. Right: The effect of yield stress and elasticity on structure retention in printed filaments for different Flinks.

Fig. 3. 3D printing accuracy, swelling, and mechanical properties after cross-linking and bacterial growth using a 4.5 wt % Flink and Flink-GMHA.

(A to C) Various shapes of Flink hydrogels containing localized bacteria with high precision in 3D. (D) Multimaterial 3D printing with spatial segregation of two bacterial strains. P. putida is labeled with DAPI (blue) and localized in the horizontal lines, whereas B. subtilis is colored with Nile Red (green) and embedded in the vertical lines. (E) Flink (6 wt %; without bacteria) was modified with GMHA to form a UV–cross-linked water-insoluble hydrogel after UV light exposure (365 nm for 60 s at 90 mW). The hydrogels are shown after printing, after 1 hour in bacterial medium and after 1hour in DI water, respectively. (F) Amplitude oscillatory sweeps of the modified Flink-GMHA (4.5 wt %) before and after cross-linking. Strains in the range from 0.01 to 100% at an angular frequency of 1 rads⁻¹ were applied in these measurements. All scaffolds are self-sustaining and resilient.

Fig. 4. 3D-printed bacteria-functionalized structures with complex shapes for bioremediation and biomedical applications.

(A) A photo–cross-linked grid structure printed using a 4.5 wt % Flink-GMHA loaded with P. putida, a known phenol degrader, was incubated in an MM with phenol as the only carbon source. Phenol concentration and bacterial optical density (OD₆₀₀) are shown as a function of time, as indicators of phenol degradation and bacterial growth. As a control, an equivalent bacteria concentration was incubated as a free-floating culture. (B) The preincubated grid was inoculated for a second time in a phenol containing MM. This time, phenol degradation happened as fast as with the free-floating control due to the higher bacterial concentration now present inside the grid. (C) Staining of bacterial DNA within the grid by ethidium bromide before (top) and after (bottom) incubation in the phenol-containing medium (343 nm). (D) In situ formation of bacterial cellulose by A. xylinum is used to generate a 3D-printed scaffold with a 4.5 wt % Flink in the shape of a T-shirt. Bacterial cellulose is visualized with a specific fluorescent dye at 365 nm. (E) Bacterial cellulose nanofibril network under SEM printed with a 3 wt % Flink. (F) Growth of bacterial cellulose depends on oxygen availability and the viscosity of the Flink. A dense cellulose network is only present in regions of high oxygen levels and low to medium viscosities. Images show, from top to bottom, circular prints using 3, 6, and 9 wt % Flinks. (G) A doll face was scanned, and a 4.5 wt % Flink containing A. xylinum was deposited onto the face using a custom-built 3D printer. In situ cellulose growth leads to the formation of a cellulose-reinforced hydrogel that, after removal of all biological residues, can serve as a skin transplant.