KGBSM Media Preparation Protocol

Overview

This protocol describes the preparation of Keoni Gandall Basal Salts Medium (KGBSM) for high-density Pichia pastoris cultivation. It includes 200 mL trace mineral and vitamin stocks, and basal media formulas for both 4 L and 200 mL batches. It was adapted from the MBSM media by changing from ammonium sulfate to urea, so that batch bioreactors can be used, rather than monitoring the pH.

1. Trace Mineral Stock (200 mL)

Ingredients:

• FeSO₄·7H₂O – 13.0 g
• CuSO₄·5H₂O – 1.2 g
• ZnSO₄·7H₂O – 4.0 g
• MnSO₄·H₂O – 0.6 g
• KI – 0.084 g
• H₃BO₃ – 0.02 g
• Na₂MoO₄·2H₂O – 0.2 g
• H₂SO₄ (98%) – 2 mL or 37 mL of 1M
• Distilled water to 200 mL

Instructions:

1. Dissolve salts in 150 mL distilled water.
2. Add acid carefully (use gloves and eye protection).
3. Bring to 200 mL with distilled water.
4. Filter sterilize (if possible) or freeze immediately in small aliquots.

2. Vitamin Stock (200 mL)

Ingredients:

• Biotin – 0.16 g
• Thiamine HCl – 0.16 g
• Pyridoxine HCl – 0.16 g
• Calcium pantothenate – 0.16 g
• Nicotinic acid – 0.04 g
• Myo-inositol – 1.6 g
• K₂HPO₄ – 0.8 g (optional)
• Distilled water to 200 mL

Instructions:

1. Dissolve all ingredients in 150 mL distilled water.
2. Bring to 200 mL total volume.
3. Filter sterilize if possible; otherwise freeze sterile aliquots.

3. KGBSM Basal Media

A. 4 L Batch

• Glycerol – 200 g
• KH₂PO₄ – 40 g
• MgSO₄·7H₂O – 12.8 g
• CaCl₂·2H₂O – 1.4 g
• Urea or (NH₄)₂SO₄ – 50 g
• Distilled water to 4 L

Post-autoclave additions:

• Trace Mineral Stock – 17.2 mL
• Vitamin Stock – 10 mL

B. 200 mL Test Batch

• Glycerol – 10 g
• KH₂PO₄ – 2 g
• MgSO₄·7H₂O – 0.64 g
• CaCl₂·2H₂O – 0.07 g
• Urea – 2.5 g
• Distilled water to 200 mL

Post-autoclave additions:

• Trace Mineral Stock – 850 uL
• Vitamin Stock – 500 uL

Methanol Induction

Once the initial sugar source (glycerol or glucose) is exhausted, begin induction:

• Add 0.5–1.0% methanol (v/v) to culture
• Repeat every 12–24 hours to maintain 0.5–1.0% concentration
• Continue for 2–5 days depending on target protein

Notes on pH

• Check and adjust final media pH to ~5.0 after all additions.
• Urea: breaks down slowly, releasing ammonium without strong acidification. Preferred when no pH control is available.
• Ammonium sulfate: assimilated ammonium leads to proton release → medium acidification. May require stronger buffering or pH monitoring.
• MBSM depends on adding nitrogen in the form of ammonium sulfate during growth to prevent medium acidification. Because of this, less is used in normal MBSM. In our modified procedure, we add all the nitrogen up-front as urea.
• Glycerol (~$1) and methanol (~$1) are largest cost, while monopotassium phosphate (~$0.50), urea (~0.15), and trace (~trivial) follow. In total, at bulk amazon prices, it costs ~$3 to ~$4 per 4L.