Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: Growth kinetics and production improvement

Wenhui Zhang, Karen J. Hywood Potter, Bradley A. Plantz, Vicki L. Schlegel, Leonard A. Smith, Michael M. Meagher

Research output: Contribution to journalArticlepeer-review

87 Scopus citations


Fed-batch fermentation of a methanol utilization plus (Mut+) Pichia pastoris strain typically has a growth phase followed by a production phase (induction phase). In the growth phase glycerol is usually used as carbon for cell growth while in the production phase methanol serves as both inducer and carbon source for recombinant protein expression. Some researchers employed a mixed glycerol-methanol feeding strategy during the induction phase to improve production, but growth kinetics on glycerol and methanol and the interaction between them were not reported. The objective of this paper is to optimize the mixed feeding strategy based on growth kinetic studies using a Mut+ Pichia strain, which expresses the heavy-chain fragment C of botulinum neurotoxin serotype C [BoNT/C(Hc)] intracellularly, as a model system. Growth models on glycerol and methanol that describe the relationship between specific growth rate (μ) and specific glycerol/methanol consumption rate (Vgly, VMeOH) were established. A mixed feeding strategy with desired μglyMeOH = 1, 2, 3, 4 (desired μMeOH set at 0.015 h-1) was employed to study growth interactions and their effect on production. The results show that the optimal desired μglyMeOH is around 2 for obtaining the highest BoNT/C(Hc) protein content in cells: about 3 mg/g wet cells.

Original languageEnglish (US)
Pages (from-to)210-215
Number of pages6
JournalJournal of Industrial Microbiology and Biotechnology
Issue number4
StatePublished - Apr 1 2003
Externally publishedYes


  • Botulinum neurotoxin
  • Fed-batch fermentation
  • Growth model
  • Mixed feed
  • Pichia pastoris

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


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