A custom bioink was developed that would allow bacteria and chemical substrates for materials production to flow through the printhead in liquid state, then rapidly solidify upon contact with the printing surface to form a stably patterned shape. The bioink consists of live bacteria mixed with dissolved alginate. When the bioink is extruded onto a surface containing calcium ions during the printing process, cross-linking of the alginate molecules is triggered, forming a stable, biocompatible aerogel scaffold within seconds. (25, 26) In order to optimize the bioink composition, the alginate and calcium ion concentration were systematically varied (from 0.5% w/v to 6% w/v alginate; and 0.0087 mol/cmto 0.44 mol/cmCaCl) and tested in the printing system. Insufficient concentrations of alginate and calcium ions resulted in poor gelation and low printing resolution, while excessive concentrations led to premature gelation of the bioink, blocking the pipet tip and preventing further printing. The optimal conditions were found to be 1 M CaCland 2.5% w/v alginate. With our printer system, each printed layer can only contain materials that are present in the active syringe; the mixture of materials from two different syringes within one layer is not possible due to the rate of scaffold formation. However, a wide range of different types of materials is compatible with alginate polymerization and may be admixed in the active syringe for inclusion in individual printed layers. (21, 27)

In order to create a straightforward bacterial 3D printer, multiple modifications were made to an inexpensive (300 USD) commercial 3D printer ( Figure 1 A). The extruder of the printer was removed and replaced with a pipet tip ( Figure 1 B, 3a) and system of tubing ( Figure 1 B, 3c). This alteration allows the liquid biological ink (“bioink”) to be transported under ambient temperatures that are amenable to microbes, rather than the elevated temperatures that are applied to melt plastic filament. A secondary pipet tip was affixed to the printhead ( Figure 1 B, 3b) to allow for rapid alternation between the deposition of different types of bioink. A syringe pump ( Figure 1 A, 1) was added to the system to generate continuous but adjustable flows of bioink through the sets of tubing into the pipet tips. Printed shapes are created through the flow of bioink through the movable printhead while it is in motion, the trajectory of which is programmably controlled by an external computer. The shape of printed objects can be createdthrough computer-aided design (CAD) software programs, then converted into printing instructions for the 3D printer using slicing and printer-specific software programs. These adaptations can be performed on all 3D printing systems that employ an accessible and removable extruder.

Figure 4. Internal structure of printed layers. Modified strains of E. coli expressing two different fluorescent proteins were printed one on top of the other in a 2-layered square. After 24 h of incubation, the internal structure of the printed bacterial layers was inspected by confocal microscopy. The bottom layer contained 81% ± 5% blue fluorescent cells, while the top layer contained 93% ± 5% yellow cells.

Since some applications may require the printing of multilayered structures containing spatially separated bacterial strains, the internal structure of multilayered printed bacteria was analyzed. Bilayered structures were printed containing engineered, in which the bacteria in bottom layer of bioink expressed the yellow fluorescent protein mVenus, and the bacteria in the top layer expressed the blue fluorescent protein mCerulean. Each layer was printed using separate tubing and pipet tips to prevent contamination of the top layer by bacteria printed in the previous layer. After 24 h of incubation, the structure was imaged at different depths using confocal microscopy, and the extent of bacterial mixing between the layers was quantified through image analysis. The bottom layer was 81% ± 5% homogeneous, while the top layer was 93% ± 5% homogeneous ( Figure 4 ). This analysis indicates good separation of bacteria between adjacent printed layers, even after extensive periods of incubation. The lower layer may have been less pure due to incomplete solidification before printing of the top layer, which could be improvedan increased waiting time between printing of layers.

To characterize the spatial resolution of 3-dimensional printed structures, 14-layered elliptical structures were printed. The structures’ widths were measured following deposition of each layer. The heights of the structures were measured for only a subset of layers, since each height measurement required removal of the gel from the printing surface, halting the printing process. The average line width increased significantly but incrementally for the first six layers, with an average increase of 0.14 ± 0.01 mm per layer between layers 1 and 6 ( Figure 3 B). Following the sixth layer, the line widths approached a plateau; no significant differences were observed between the line widths of any of the layers between layers 6 and 14 (ANOVA + Tukeytest,-value: 0.995). The height of the printed material was observed to increase continually, by an average of 0.16 ± 0.02 mm per layer. The final 14-layered structures were 2.14 ± 0.11 mm in height, with a width of 2.32 ± 0.37 mm. These measurements indicate that our printing system is capable of fabricating 3-dimensional structures at submillimeter-scale precision in all dimensions. Further improvements in resolution may be possible by rebuilding our system using a commercial 3D printer employing more accurate printhead positioning. (15)

The printer can be directed to deposit bioink directly on top of previously printed material to create multilayered structures. A second aerogel layer can be printed on top of a base layer at a range of different syringe pump extrusion rates, with no modification of the printing commands ( Figure 3 A). Fabrication of structures taller than two layers in height requires an increase of the-position of the printhead by 0.15 mm/layer. Stacked layers of bioink are able to solidify due to interaction with calcium ions that have diffused from the printing surface up through the first printed layer(s). Each additional printed layer resulted in a fractional increase in the width of the final structure, due to the time required for the new layer to gelate ( Figure 3 A, B). No significant change in the final width was observed when the time between printing of successive layers was varied between 40 and 240 s (Student’stest,-value: 0.037, CV: 18.36, CV: 20.82), indicating that multilayered structures can be printed at different paces with no loss of resolution. The total time required to print a 14-layered ellipse with a pause of 40 s between printing successive layers was 15 min.

Figure 3. Printing at millimeter-scale resolution in three dimensions. (A) The distribution of widths of single- and double-layered structures printed at different syringe pump speeds ( n = 4). The printhead movement speed was 200 mm/min in all cases. The tops and bottoms of the boxes represent the 75th and the 25th percentiles, respectively; the lines within the boxes are the median values; and the tops and bottoms of the vertical lines are the maximum and minimum values. (B) The line width (blue) and line height (red) of printed structures containing up to 14 layers ( n = 3). Error bars indicate the standard error.

In order to maximize the printer resolution, a range of printing parameters was tested. The two most critical factors affecting the printed line width were found to be the extrusion rate of the syringe pump and the movement speed of the printhead, in agreement with previous work. (28) A range of printhead movement speeds (100–500 mm/min) and syringe pump extrusion rates (17–50 μL/min) were applied to print straight lines of bioink. In general, increased printing resolution could only be achieved by adjusting both parameters in parallel:, slower printhead movement speeds required slower syringe pump rates. Other less-critical factors contributing to printer resolution were the distance between the printhead and the printing surface, as well as the uniformity of the printing surface. The narrowest line width obtainable was 1.00 ± 0.15 mm, achieved with a printhead movement speed of 200 mm/min and a syringe pump extrusion rate of 33 μL/min ( Figure 3 A).

Figure 2. Reproducible printing of alginate structures. (A) A representative printed elliptical structure. The numbers indicate the locations of the 6 measurement positions. (B) The distribution of measured line widths at 6 different positions within elliptical printed structures ( n = 3). The tops and bottoms of the boxes represent the 75th and the 25th percentiles, respectively; the lines within the boxes are the median values; and the tops and bottoms of the vertical lines are the maximum and minimum values. No significant differences exist between the width distributions of any of the different positions (ANOVA, p -value: 0.964).

The reproducibility and consistency of the printing process was assessed by analyzing patterned monolayers that were printed using the optimized bioink. An elliptical form with two long straight lines on each side was chosen as the printing object in order to test the performance of the printer in fabricating both curved and straight-edged structures ( Figure 2 A, Supporting Information ). Following printing, the width of each printed structure was measured at six different positions, sampling a variety of straight and curved portions. No statistically significant differences were seen among any of the widths measured at the same position in different prints (ANOVA,-value: 0.964, CV%: 11.16) ( Figure 2 B). These data indicate that our printer and bioink can fabricate printed structures of varying shapes in a consistently uniform manner.

Survival and Metabolic Activity of Printed Bacteria

E. coli was incorporated into alginate aerogels, and gels were incubated for varying amounts of time from 0 to 48 h at 37 °C. The gels were then added to a solution of sodium citrate to chelate the calcium ions and dissolve the gel. The samples were grown on LB-agar plates to determine the number of viable cells (colony forming units). An increase in colony forming units observed between the first two data points may indicate that bacterial growth occurs within the alginate gel during the first 24 h after gel production (E. coli by approximately 50% ( In order for our alignate-based printing system to be successfully applied to microbial materials production, bacteria must be able to survive well within the alginate gel. To test this property,was incorporated into alginate aerogels, and gels were incubated for varying amounts of time from 0 to 48 h at 37 °C. The gels were then added to a solution of sodium citrate to chelate the calcium ions and dissolve the gel. The samples were grown on LB-agar plates to determine the number of viable cells (colony forming units). An increase in colony forming units observed between the first two data points may indicate that bacterial growth occurs within the alginate gel during the first 24 h after gel production ( Figure 5 A). Thereafter, colony forming units remained fairly constant for up to 48 h. Comparison with bacteria that were incubated in nonprinted, liquid bioink indicated that the printing process initially reduces the viability ofby approximately 50% ( Figure 5 B). Thereafter, the levels of viable bacteria in the nonprinted bioink remained nearly constant, likely due to nutrient limitation. The dramatic increase in the number of viable bacteria in the printed gel resulted in an overall increase in viability of approximately 200% in comparison to the nonprinted bioink, which may be due to the additional nutrients in the agar printing substrate and the lower bacterial density after printing. Bacteria can thus remain viable within the alginate gel of our bioink for at least 2 days following gel formation, providing sufficient time for microbial-mediated materials production or patterning to occur.

Figure 5 Figure 5. Robust bacterial survival within printed alginate gels and planktonic bioink. The number of colony forming units is shown for E. coli printed within alginate gels (A) or as a planktonic sample (B), incubated for varying amounts of time (n = 6). Error bars indicate the standard error.

E. coli containing the rhamnose-inducible red fluorescent protein RFP were printed onto an agar plate containing the rhamnose inducer. The gel was incubated, and the color of the gel was monitored over 48 h. After 8 h of incubation, the induced bioink showed a noticeable red color, which became very intense after 48 h ( Both survival and metabolic activity of the printed bacteria are key factors to demonstrate the applicability of our printing system. To assess the ability of our printed bacteria to create a product,containing the rhamnose-inducible red fluorescent protein RFP were printed onto an agar plate containing the rhamnose inducer. The gel was incubated, and the color of the gel was monitored over 48 h. After 8 h of incubation, the induced bioink showed a noticeable red color, which became very intense after 48 h ( Figure 6 ). This experiment demonstrates that our printed bioink is able to support the production of bacterially made materials over short periods of time. Our printing system could be readily applied to the patterned production of bacterially created materials in a variety of different formats. Bioink containing both active bacteria and material precursors could be printed onto a neutral surface, to create a three-dimensionally patterned aerogel within which the bacteria chemically convert the precursors to the desired final product. The thorough commingling of bacteria and chemical substrates within the gel in this configuration would lead to high efficiency of material production. Alternately, alginate gel containing only the chemical precursor could be printed and then immersed within a liquid bacterial culture to create a final 3D-patterned material that is largely bacteria-free. In a third scenario, bacteria-containing bioink could be printed onto a surface that is coated with material precursors, which the bacteria could then convert into a two-dimensionally patterned final product. This approach has the appealing feature that the alginate gel could be dissolved away after the fact, leaving behind only the final material.

Figure 6 Figure 6. Metabolic activity within printed alginate gels. E. coli with and without a rhamnose-inducible RFP plasmid were printed onto a substrate containing rhamnose. Printed gels were incubated at 37 °C, and color changes were observed over time.

The work shown here demonstrates the development of macroscopic material printing with millimeter-scale resolution using removable aerogels and bacterial chemistry. This approach enables us also to print precursor or supportive material directly with the bacteria. Our printing technique is inexpensive, straightforward, and can produce bacterial structures of a wide variety of three-dimensional shapes without requiring printing scaffolds, excepting structures that contain internal bridges or enclosed hollow spaces. Our technology is well-suited for the use of wild-type organisms or synthetically modified bacteria, which could be designed to carry out new combinations of microbial reactions to create a great number of different types of materials. Connecting our novel and straightforward bacteria printing techniques with approaches of synthetic biology will further improve its value as a “green” material production process and patterning methodology. The ease and simplicity of our printing approach will allow any interested research group to adapt and improve this process at low cost for multiple possible applications.