High-Throughput Calcium Carbonate Precipitation Assay

Objective

Author(s): Evan Wong, Yuki Guan, Matthew Chan

To identify the most efficient clones and optimal condition (pH, temperature, Ca²⁺⁺ bicarbonate) for CaCO₃ precipitation through carbonic anhydrase (CA).

Phase I: Screen ALL candidates with a colorimetric assay (o-Cresolphthalein chromogenic reagent) ⇒ high-throughput
Phase II: A gravimetric + XRD/SEM validation of the best-performing clones/conditions

PHASE I: High-Throughput Screening via Colorimetric Ca²⁺ Depletion

Materials Required:

o-Cresolphthalein reagent
Alkaline buffer (imidazole or Tris, pH ~10.5)
Calcium calibrator (prepare 2.5 mM CaCl₂)

Calcium Assay Protocol Summary: (Following inoculation, induction, and CO₂ incubation)

Add samples (supernatant) and/or standards to wells
Add buffer, mix gently (no bubbles)
Add chromogenic reagent (o-Cresolphthalein reagent) and incubate at RT for 5 min
Read the OD absorbance at 570nm and 700nm
Analyze with microplate reader

Variable	Levels to Test on Each clone
pH	6.5, 7.0, 7.5, 8.0, 8.5
Temperature	20°C, 30°C, 37°C, 42°C
[CaCl₂]	5, 10, 25, 50 mM
[NaHCO₃]	10, 25, 50, 100 mM
Expression Time	24h, 48h, 72h (optional variable)

PHASE IIa: Gravimetric Validation and Mineral Characterization (Medium Throughput)

Materials Required:

PBS
- 750 mM phosphate buffer, pH 7 (tube)
- 750 mM phosphate buffer, pH 8 (tube)
- 30X 450 mM phosphate buffer, pH 7 (tube)
- 30X 450 mM phosphate buffer, pH 8 (tube)

Gravimetric Analysis Protocol Summary: (Following identification of the top Calcium producing clones and conditions)

Harvest CaCO3 precipitate via centrifugation
Wash each precipitate with:

1 mL PBS (pH 8.0) → removes residual media and salts
1 mL distilled water → removes buffer salts

Drying precipitates at 60 °C overnight (12—16 h)
Determine each pellet’s mass
Store sample in desiccator if not actively in use

PHASE IIb: XRD+SEM Protocols: (Low Throughput)

SEM Protocol FEI Quanta 650 SEM Tools:

Gloves
Tweezers
Small metal spatula
SEM stub
Stub sample box (as a stub holder)
Double sided carbon or copper conductive tape
Sputter Coater Sample prep:

Attach carbon tape to SEM stub
Scoop a small amount of powder and sprinkle a very THIN layer
Slightly spread sample on adhesive
Tilt and tap to get rid of loose particles
Load stub into sputter coater chamber and place on quartz crystal microbalance
Turn small black knob to get the stage rotating for better coverage
Vacuum the chamber and begin deposition roughly 2.0 nm thickness of coating is enough
Remove from sputter coater and place into microscope using gloves and tweezers

XRD:( prepping powdered sample, loaded sample holder)

Ground crystals into fine powder using a mortar and pestle
Take the top of the sample holder, orient it flat-side down and ensure that it clicks in place held by the 3 buttons
Fill well with sample and press down with pressing block