1. Schematic Layout of a P. pastoris Fermentation System

2. Schematic Layout of a P. pastoris Fermentation System
Motor
Exhaust condenser
Foam probe
Multiple Ruston impellers
A multi-orifice ring sparger
Sensors for temperature, pH, DO & methanol
Air & O2 added through solenoid valves
Mass flow controllers
Peristaltic pumps which add the liquid substrates – MeOH, glycerol, base (NH4OH) and antifoam.
Substrate addition bottles &/or culture vessel are put on weighing scales

3. Fermentation Protocol & Control strategies
Phase I – Glycerol batch Culture to create biomass
Culture mass 70% of reactor volume (10,500mL)
Max 4% glycerol (600mL)
Dissolved Oxygen (DO) – 1st agitation & then gas flow ratio control (GFRC)
Agitation range approx. 30% of maximum RPM. (450RPM)
When glycerol in reactor is completely exhausted – O2 demand will sharply decline
A cellular yield (CY) g/L wet cells expected (approx. 1,500g wet cells)

4. Fermentation Protocol & Control strategies
Phase II – Glycerol fed batch culture
DO control should be changed at end of Phase I. DO should not drop below 20% of air saturation.
Total gas flow vvm
Agitation between % of maximum RPM. (approx RPM)
At end of phase II CY of g/L wet cells

5. Fermentation Protocol & Control strategies
Phase III – Methanol fed batch culture
Glycerol must be exhausted before methanol feed starts – this can be achieved by turning off glycerol pump & by allowing DO to rise to 100%
It may take 1-3hrs to stabilize back to 30% DO
Methanol levels must be carefully controlled.
Feed rate at 3.5mL/hr/L of culture volume.
Once culture adapts to current methanol levels the feed rate is increased in increments of 1mL/hr/L of culture volume every 1-2hrs until reaching a feed rate of 11-12mL/hr/L
A final CY of g/L of wet cells can be obtained