The oxygen transfer rate as key parameter for the characterization of Hansenula polymorpha screening cultures

Abstract

Screening cultures are usually non-monitored and non-controlled due to a lack of appropriate measuring techniques. A new device for online measurement of oxygen transfer rate (OTR) in shaking-flask cultures was used for monitoring the screening of Hansenula polymorpha. A shaking frequency of 300 rpm and a filling volume of 20 ml in 250-ml flasks ensured a sufficient oxygen transfer capacity of 0.032 mol (l h)⁻¹ and thus a respiration not limited by oxygen. Medium buffered with 0.01 mol phosphate l⁻¹ (pH 6.0) resulted in pH-inhibited respiration, whereas buffering with 0.12 mol phosphate l⁻¹ (pH 4.1) resulted in respiration that was not inhibited by pH. The ammonium demand was balanced by establishing fixed relations between oxygen, ammonium, and glycerol consumption with 0.245±0.015 mol ammonium per mol glycerol. Plate precultures with complex glucose medium reduced the specific growth rate coefficient to 0.18 h⁻¹ in subsequent cultures with minimal glycerol medium. The specific growth rate coefficient increased to 0.26 h⁻¹ when exponentially growing precultures with minimal glycerol medium were used for inoculation. Changes in biomass, glycerol, ammonium, and pH over time were simulated on the basis of oxygen consumption.

Appendix

Symbols

c :

Concentration [mol l⁻¹], [g l⁻¹]

H⁺ (released):

Released protons [mol]

H₂PO₄ ^- :

Dihydrogen phosphate [mol]

H ₃ PO ₄ :

Phosphoric acid [mol]

NH₃ (assim):

Assimilated ammonium [mol]

NH₄ ⁺ :

Ammonium [mol]

OTR:

Oxygen transfer rate [mol (l h)⁻¹]

OTR _max :

Maximum oxygen transfer capacity [mol (l h)⁻¹]

$pK_{{CO_{2} }}$ :

Dissociation constant of the dissociation pair H₂CO₃/HCO₃ ⁻ [–]

$pK_{{NH_{4} }}$ :

Dissociation constant of the dissociation pair NH₄ ⁺/NH₃ [–]

$pK_{{PO_{4} }}$ :

Dissociation constant of the dissociation pair H₃PO₄/H₂PO₄ ⁻ [–]

$R_{{Glyc/O_{2} }}$ :

Stoichiometric ratio between glycerol and oxygen [mol mol⁻¹]

$R_{{NH_{4} /Glyc}}$ :

Stoichiometric ratio between ammonium and glycerol [mol mol⁻¹]

$R_{{NH_{4} /O_{2} }}$ :

Stoichiometric ratio between ammonium and oxygen [mol mol⁻¹]

R _Uracil/Glyc :

Stoichiometric ratio between uracil and glycerol [mol mol⁻¹]

t ₀ :

Initial time [h]

t :

Point of time [h]

$Y_{{X/O_{2} }}$ :

Oxygen-dependent biomass yield [g mol⁻¹]

Y _X/Glyc :

Glycerol-dependent biomass yield [g mol⁻¹]

Y _Prot/Glyc :

Glycerol-dependent protein yield [g mol⁻¹]

µ _max :

Maximum specific growth rate coefficient [h⁻¹]

Indices

Glyc:

Glycerol

H⁺ (released):

Released protons

H₂PO₄ ⁻ :

Dihydrogen phosphate

H₃PO₄ :

Phosphoric acid

PO₄(total):

Total phosphate

NH₃ (assim):

Assimilated ammonium

NH₄ :

Ammonium

O₂ :

Oxygen

X:

Biomass

0:

Initial point of time