Schematic Layout of a P. pastoris Fermentation System

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

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) 90-150g/L wet cells expected (approx. 1,500g wet cells)

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 1-1.25 vvm Agitation between 80-100% of maximum RPM. (approx. 1350 RPM) At end of phase II CY of 180-220g/L wet cells

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 350-450g/L of wet cells can be obtained