2025.09.21 Testing MGS-1 Alginate Gels With Carbonic Anhydrase
Purpose
To test whether adding commercial carbonic anhydrase (CA) to a 3 wt% alginate / 3.5% CMC bioink containing 20 wt% MGS-1 (Martian regolith simulant) accelerates carbonate mineralization (CaCO₃) and increases gel cross-linking/mechanical strength when exposed to Ca²⁺ and CO₂. This will serve as a positive control and demonstrate that we can produce bricks using this bioink.
Materials & Methods
Materials
bioink (3% alginate, 3.5%CMC, 30wt% MGS-1(425um))
3 wt% alginate solution
CMC
425 µm sieved MGS-1
Sigma Aldrich Carbonic Anhydrase from bovine erythrocytes
Add CA to the dry mix and add to the alginate solution
Extrude three shapes of each condition in a 6-well plate
straight lines
grid shape
hollow rectangle
crosslink with 100 mM calcium chloride solution for 10 minutes
connect the experimental setup to the carbon dioxide pump
switch to more concentrated calcium chloride solution if needed
place in a ziploc bag to ensure minimal carbon dioxide escapes
ensure the tubes are submerged in the solution and that carbon dioxide is directly bubbled in
let sit for up to 24 hours or when crystallization is observed
Results
Figure 1. Scaffolds submerged in 100 mM calcium chloride in 6-well plate (left). Plate connected to the carbon dioxide pump, was difficult to get individual tubes bubbling carbon dioxide separately into the wells (middle). Final experimental setup connected to the carbon dioxide pump, with all scaffolds submerged in 1M calcium chloride solution in a flask (right).
Figure 2. Scaffolds removed from carbon dioxide pump and calcium chloride solution after incubating for 24 hours (left). Scaffolds air dried for 24 hours in 37C (right). The middle row consists of MGS-1 gels containing carbonic anhydrase, while the bottom row does not contain any carbonic anhydrase.
Figure 3. Light microscope image of the alginate gel containing carbonic anhydrase after 24 hours incubation in carbon dioxide pump and calcium chloride solution. Freckles and granular view on the bottom half may be calcium carbonate precipitate.
Observations
only a singular tube was on the carbon dioxide pump → put all scaffolds into a singular flask to better ensure sufficient carbon dioxide delivery
the 3 wt% alginate and CMC control could not hold its shape and could not form the hollow or grid structure (collapsed and filled the entire well)
MGS-1 alginate gel could be picked up by a tweezer and not fall apart after 10 minutes crosslinking in 100 mM calcium chloride
Used 1M calcium chloride solution in the flask
the 3 wt% alginate and CMC control became cloudy and could see microbubbles after 30 minutes in the bubbler and 1M calcium chloride solution
scaffolds were removed from the solution after ~24 hrs and left out to air dry for another 24 hrs
scaffolds were like a stiff gel
Dried MGS-1 alginate samples were qualitatively comparable in strength to non-CA samples from previous experiments
though this is not conclusive as the non-CA samples were left out for very long
The alginate control scaffolds were opaque and rough to touch
Summary
qualitatively, there is a difference in touch and transparency in the alginate gels when incorporating carbonic anhydrase
opaque and granular view on the microscope may be indicating the precipitation of calcium carbonate
need a more quantifiable method in comparing the compressibility between gels containing CA, and those than do not
