Growing Pichia in Bioflo 110

8/7/2019 Growing Pichia in Bioflo 110

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P. Pastoris Fermentation Using A BioFlo ® 110 BenchtopFermentor

Introduction

This Application Report is part of a series documenting culture growth in the

BioFlo 110. With appropriate vessels and control modules, the BioFlo 110 can

efficiently grow yeast and bacteria, as well as mammalian, plant cells and insect

cells.

Pichia pastoris: Pichia pastoris is a methlotrophic yeast, which provides a unique expression

system for producing high levels of recombinant protein, including enzymes,

proteases, protease inhibitors, receptors, single-chain antibodies, and regulatory

proteins at various different levels. Pichia pastoris is also the only system that

offers the benefits of E. coli (cost effective, high-level expression and easy scale-

up) combined with advantages of expression in a eukaryotic system (protein

processing, folding, and posttranslational modifications).



A standard 7.5L BioFlo 110 Advanced Fermentation Kit was used to grow Pichia

pastoris in a fed batch fermentation. We used BioCommand Plus® supervisory

software to control the feed schedule, achieving 91.0 g/L dry cell weight (DCW).

Next, a BioFlo 110 Gas-Mix Controller was added, and the fermentation repeated

with oxygen supplementation of the sparge gas. This second run, described in the

APPENDIX (page 5), achieved a very high dry cell weight of 177.4 g/L. Neither

run was fully optimized, but the descriptions of procedures and materials, as well

as the data discussion will be useful to operators of similar fermentors.

The Fermentor

VesselThe BioFlo 110 Advanced Fermentation Kit, NBS Catalog Number M1273-

1125, was equipped with a heat-blanketed 7.5 L fermentation vessel with nominal

5.7 L working volume. All BioFlo 110 fermentation vessels are configured with a

4-baffle stainless-steel insert, dual Rushton agitation impellers, and a high-speed,



direct-drive agitation system with mechanical face-seal. Dissolved oxygen and pH

probes (Mettler Toledo) are also included, as are a variety of items such as liquid

addition bottle kits (3), cables, tubing and clamps.

Control System

The four control modules included with the Advanced Fermentation Kit were used

for the first run.

Materials and Methods

Overview

This Pichia fermentation follows a well-established protocol in which glycerol is

the initial carbon source, and after a brief carbon starvation, we switch to a

methanol feed. The switch to methanol produces a metabolite of interest by

triggering the AOX1 promoter in genetically engineered Pichia. These are fed-

batch fermentations, since first glycerol and later methanol is added while the

culture is growing.

Control Program



We created a feed control program using BioCommand Plus software, NBS

Catalog Number M1291-0000. It turned on the glycerol feed-pump when the

dissolved oxygen (DO) level rose above 40%. Each time DO exceeded 40%, the

glycerol pump turned on; each time it fell below 40%, the pump turned off. 40% is

a high DO level, indicative of reduced metabolism due to carbon exhaustion. The

rationale for this strategy is that the DO increases due to reduced growth of the

cells, which is a result of nutrient depletion. Approximately one hour after a rise in

DO which indicated depletion of the supplementary glycerol, the program

automatically turned on the methanol feed-pump.

Inoculum

The inoculum was prepared using Pichia shake-flask growth medium:

Potassium phosphate monobasic (anhydrous) . . .11.5 g/L

Potassium phosphate dibasic (anhydrous) . . . . . . .2.7 g/L

Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 g/L

10X YNB solution. . . . . . . . .. . . . . . . . .. . . . . . . 10% by volume

10x YNB solution consists of 67 g/L YNB without amino acids. The solution is

filter-sterilized and added to other media components after they are heat-sterilized

and cooled.

The inoculum was cultivated for 40 hours at 28°C in a rotary shaker (NBS model

G25) running at 240 rpm. Optical Density at 600 nm (OD600) was 10.94 at

inoculation.

Medium

The initial fermentor medium composition included:

Calcium sulfate dihydrate . . . . . . . . . . . . . . . . .. . . .0.93 g/L

Potassium sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . .18.2 g/L



Magnesium sulfate heptahydrate . . . . . . . . . .. . . . . .14.9 g/L

Potassium hydroxide . . . . . . . . . . . . . . . . . . . . . .. . .4.13 g/L

Phosphoric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . .26.7 ml/L

Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .40.0 g/L

Antifoam (Breox Foam Control Agent FMT 30) . . .1.0 ml/L

To allow space in the 5.7 L (working volume) vessel for components added after

sterilization, the initial medium volume was only 3.5 L. Post-sterilization medium

components included:

Trace Metals solution, PTM1 . . . . . . . . . . .4.6 mL/L

Base, to adjust the initial pH . . . . . . . . . . . .25 mL/LInoculum . . . . . . . . . . . . . . . . . . . . . . . . .. .200 mL

Glycerol* . . . . . . . . . . . . . . . . . . . . . . . . . .< 400 mL

Methanol* . . . . . . . . . . . . . . . . . . . . . . . . .<2 L

Base (to maintain pH at setpoint)* . . . . .. . .< 250 mL

(*) Added, as required

Pichia trace metals solution, PTM1 consisted of :

Cupric sulfate pentahydrate . . . . . . . . . . . . . . .6 g/L

Sodium iodide . . . . . . . . . . . . . . . . . . . . . . . . .0.08 g/L

Manganese sulfate monohydrate . . . . . . . . .. . .3 g/L

Cobalt chloride (anhydrous) . . . . . . . . . . . .. . .0.5 g/L

Zinc chloride (anhydrous) . . . . . . . . . . . . . .. . .20 g/L

Boric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . .0.02 g/L

Sodium molybdate dihydrate . . . . . . . . . . . . . .0.2 g/L

Ferrous sulfate heptahydrate . . . . . . . . . . . . .. .65 g/L

Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..0.2 g/L

6N sulfuric acid . . . . . . . . . . . . . . . . . . . . . . . .30 mL/L



Control Setpoints

Setpoints were keyed into the controller prior to inoculation and, except for DO

which remained high until culture was introduced, the vessel was allowed to

equilibrate prior to inoculation.

Temperature . . . . . . . . . . . . . . . . . . . . . . . 30°C

pH . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 5.0

Dissolved Oxygen . . . . . . . . . . . . . . . . ... . 30%

Agitation . . . . . . . . . . . . . . . . . . . . 300 - 1,200 rpm

(responding automatically to oxygen demand)

Dissolved Oxygen (DO) Control

The DO probe was calibrated at 0%, (obtained by briefly disconnecting the cable),

and at 100% (obtained using 1,000 rpm agitation and 5 L/m (1 vvm) airflow. After

calibration, DO remained at approximately 100% until inoculation.

An agitation cascade was selected in the controller to maintain DO at setpoint

through automatic adjustment of agitation speed. The agitation cascade increases

agitation speed with increasing oxygen demand. To set up the cascade, we used the

DO control display and keypad on the PCU to select:

Cascade: . . . . . . . . . . . . . . . . . . . .Agit

Minimum RPM : . . . . . . . . . . . . . . .300

Maximum RPM: . . . . . . . . . . . . .1,200

Nutrient Feed

Initial feed was 360 mL of 50% glycerol solution with 12 mL/L of PTM1 (trace

metals). BioCommand began this feed automatically when the dissolved oxygen

showed a sudden rise above the setpoint, a well-known carbonexhaustion indicator.

After all the glycerol was consumed, we allowed a brief starvation phase, then



Results and Discussion

The DO and agitation trend graphs (Figures 1 & 2) reveal the fermentation history.

We limited the carbon source in order to restrict growth to levels that non-

oxygenenriched air could support, which resulted in a healthy culture. Temperature

and pH were stable throughout the entire run.



The most important fermentor characteristics for highdensity cultures, such as

Pichia, are the fermentor's maximum oxygen transfer rate (OTR) and maximum

heat transfer rate. In dense robust cultures, the fermentor must:

1) incorporate oxygen at a high rate from the sparge gas into the dissolved oxygen

needed for metabolism. Additionally, OTR depends on agitation-motor power and

impeller design.

2) dissipate the heat of metabolism and agitation without allowing culture

temperature to rise above the growth optimum. Good temperature control depends

on cooling system design and coolant temperature.

Of course factors such as substrate concentration and metabolite build-up can also

be limiting, but these are often more controllable than inherent physical limitations

of the fermentor.

Conclusion

Pichia pastoris growth in the BioFlo 110 was successful. Culture density of 91.0



g/L DCW was achieved, and when oxygen supplementation was added (see

Appendix, page 5), cell density reached 177.4 g/L DCW.

Temperature control was excellent using unchilled (55°F) tap water as the coolant.

Nevertheless, fermentors with large-area stainless-steel heat exchangers, such as

the New Brunswick Scientific’s BioFlo® 3000, have an advantage in temperature

control at high cell density compared to systems with immersed coils (like the

BioFlo 110 used here); or when compared to systems that rely on waterjackets

made of glass. Glass has poorer thermal conductivity than stainless steel, but glass

jackets have larger surface areas than immersed coils. Both immersed coils and

glass jackets can work well, but the advantage of a fermentor like the BioFlo 3000with a large stainless steel heat exchanger becomes significant at higher cell

densities or with higher coolant temperatures.

Overall, our protocol and the BioFlo 110 performed extremely well. The BioFlo

110 Advanced Fermentation Kit is a suitable instrument for culturing Pichia

pastoris. Furthermore, we expect that similar results can be achieved when using

BioFlo 110 fermentation vessels in other sizes (1.3, 3.0 or 14 L liters), as well as

when using a water-jacket configuration.

APPENDIX:

Effect of Gas Mix Controller and Oxygen Supplementation

A second continuous-batch was performed, this time adding the BioFlo 110 Gas

Mix Controller, , M1273- 3104, to demonstrate the impact of oxygen

supplementation on final dry cell weight. To maintain consistency, the media,

nutrients, inoculum, base, and control set-up was the same as the first run, except

for the DO cascade as listed below.



Dissolved Oxygen (DO) Control

Cascade……..Agitation and Oxygen

The cascade first increased agitation and then added oxygen gas as needed to

maintain the DO at setpoint.

Figure 4 shows that the DO declined steadily towards the 30% setpoint during the

first 10 hours, while agitation changed as required, to maintain the DO setpoint.

After reaching 1,200 rpm at ~24 hours, further oxygen demand went

uncompensated, causing dissolved oxygen to drop below the setpoint.

Growing Pichia in Bioflo 110

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