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and considerable increase in culture volume, but the volume
of the workable supernatant (approximately 7 L after
biomass removal) hardly changed during the fermentation
process (Figure 3).
The initial carbon source was glycerol, added first as a
batch, and later (after 18 h) at a constant rate of 2 g L
h
throughout the rest of the fermentation. The residual
glycerol concentration in the culture was between 0.01 and
0.05 g L
-1
, a range that does not affect the AOX1 promoter
[13]. When the culture density reached approximately 150
OD at 600 nm (10% v/v), about 20 h after starting the fermentation,
methanol addition was commenced and its concentration
in the culture was kept at 2 g L
-1
using the
described methanol sensor. Although the methanol controller
displayed oscillatory behavior and was sensitive to the
culture temperature, the methanol level was maintained
successfully at 2 (±0.5) g L
-1
-1
. It was important to begin
induction with methanol at a cell density between 150 and
200 OD (at 600 nm). Inducing the culture at higher cell
densities resulted in significant problems with maintaining
the desired temperature and dissolved oxygen level because
the high amounts of oxygen, needed for methanol oxidation,
generate a large amount of heat during the
exponential growth phase on methanol. Higher temperatures
affect the growth of Pichia, the stability of the product
and the calibration of the methanol sensor. During the fermentation,
1120 g of glycerol, 1.15 L of 7 M ammonium
hydroxide and 3.8 L of methanol were added. After 90 h
of fermentation, of which 70 h was the duration of growth
on methanol, 200 mg of angiostatin and 5 kg of biomass
were produced.
Since a high percentage of the culture is biomass, the
appropriate way to express angiostatin production is in mg
per liter supernatant instead of mg per liter culture, the
average production (based on five consecutive runs) was
20 (±5) mg L
supernatant. A slightly lower concentration
has been claimed in a recent patent application [15]


Original text

and considerable increase in culture volume, but the volume
of the workable supernatant (approximately 7 L after
biomass removal) hardly changed during the fermentation
process (Figure 3).
The initial carbon source was glycerol, added first as a
batch, and later (after 18 h) at a constant rate of 2 g L
h
throughout the rest of the fermentation. The residual
glycerol concentration in the culture was between 0.01 and
0.05 g L
-1
, a range that does not affect the AOX1 promoter
[13]. When the culture density reached approximately 150
OD at 600 nm (10% v/v), about 20 h after starting the fermentation,
methanol addition was commenced and its concentration
in the culture was kept at 2 g L
-1
using the
described methanol sensor. Although the methanol controller
displayed oscillatory behavior and was sensitive to the
culture temperature, the methanol level was maintained
successfully at 2 (±0.5) g L
-1
-1
. It was important to begin
induction with methanol at a cell density between 150 and
200 OD (at 600 nm). Inducing the culture at higher cell
densities resulted in significant problems with maintaining
the desired temperature and dissolved oxygen level because
the high amounts of oxygen, needed for methanol oxidation,
generate a large amount of heat during the
exponential growth phase on methanol. Higher temperatures
affect the growth of Pichia, the stability of the product
and the calibration of the methanol sensor. During the fermentation,
1120 g of glycerol, 1.15 L of 7 M ammonium
hydroxide and 3.8 L of methanol were added. After 90 h
of fermentation, of which 70 h was the duration of growth
on methanol, 200 mg of angiostatin and 5 kg of biomass
were produced.
Since a high percentage of the culture is biomass, the
appropriate way to express angiostatin production is in mg
per liter supernatant instead of mg per liter culture, the
average production (based on five consecutive runs) was
20 (±5) mg L
supernatant. A slightly lower concentration
has been claimed in a recent patent application [15]

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