See all notebook entries: biocementing bacteria , bioprinter & bioink

2025.07.29 Incorporating UTEX 2973 into Earth Sand Bioink

Purpose

Testing the protocol for incorporating wild type UTEX 2973 into 50 wt% 425 µm earth sand, 9 wt% CMC, and 3 wt% alginate gel, as well as curing it in mineralization medium to test for calcium carbonate formation.

Materials & Methods

Materials

UTEX 2973 liquid culture (‘Passage 5’)
Cuvettes for OD check
1 mL syringes
50 wt%, 425 µm earth sand alginate gel
35 mm petri dishes
BG-11 + 100 mM calcium chloride + 100 mM sodium bicarbonate mineralization medium
BG-11 growth medium
BG-11 + 100 mM calcium chloride + 100 mM sodium carbonate mineralization medium
Falcon/microcentrifuge tubes
Centrifuge Methods

Take OD750 reading of ‘Passage 5’ Culture using the spectrophotometer
Determine cell concentration (cells/mL) using the calibration equation (x = OD)
$y=\left(10^{6.34}\right)\left(x\right)^{\left(0.9\right)}$
Determine how many mL (y) of culture needed for 1g of bioink ([1])
$\frac{7\cdot10^{7}}{y}$
Transfer the amount of culture needed for 1g of bioink into an appropriately sized falcon tube
Centrifuge at 3700g for 10 minutes (until bacterial pellet is prominent)
Discard supernatant and resuspend pellet in 100 µL BG-11 growth medium
Load 1 mL of gel into a sterile syringe
Transfer bacteria resuspension into a sterile microcentrifuge tube and add the 1 mL of gel
Mix well by aspirating up and down with a syringe
Load the bioink into the syringe and extrude into a petri dish:
1. hollow square shape
2. 3x3 grid shape
Submerge scaffolds in sterile 100 mM calcium chloride solution and allow it to crosslink for 10 minutes
Remove calcium chloride solution with a pipette
Submerge scaffolds in 2 mL mineralization medium
Parafilm the opening of the petri dish and place into the light incubator (maintained between 30-38ºC)

Results

Observations

4 samples
- Grid shape: BG11 only and BG11 + 100mM CaCl2 + 100mM Na2CO3
- Rectangle shape: BG11 only and BG11 + 100mM CaCl2 + 100mM NaHCO3
1 mL of bioink weight was approximately 1.31 g
2 x 40 mL Passage 5 culture was loaded in a 50 mL falcon tube (OD750 was 0.80, ~38 mL needed for 70 million cells)
Bacterial pellet contained a top green layer and white/yellow bottom layer
- bottom layer may be contamination and/or dead cells
Final resuspension was ~ 300 µL
1 mL of bioink made 2 shapes
Tried to use an 18G needle to extrude the shape, but was clogged by the sand
Precipitate began accumulating around the scaffold when submerged in the two mineralization mediums
After 1 day, the grid scaffold in BG-11 + calcium chloride + sodium carbonate dissolved/disintegrated
On day 4, grids began to bleach and lose its green colour

Summary

a lot of bacteria / suspension volume is required for 1 mL of bioink → consider lowering seeding density
mixing bacteria suspension with the bioink was quite inefficient, consider using syringe adaptors to mix better
need to determine viability of cells prior to incorporation and after certain incubation times in the media
- consider doing a live/dead stain

Hand off

4 small petri dishes of 3 different medium conditions stored in the light incubator

1. Reinhardt O, Ihmann S, Ahlhelm M, Gelinsky M. 3D bioprinting of mineralizing cyanobacteria as novel approach for the fabrication of living building materials. Front Bioeng Biotechnol [Internet]. 2023 Apr 3 [cited 2025 Mar 7];11. Available from: https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1145177/full