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E. coli Fermentation Using a BioFlo 110 Bench-Top Fermentor


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 be used to efficiently grow mammalian cells, plant cells, insect cells, yeast, and bacteria.

For this report, a standard 7.5L BioFlo 110 Advanced Fermentation Kit, NBS Catalog Number M1273-1125 was first used for an E. coli fed-batch fermentation.

Next, a BioFlo 110 Gas-Mix Controller, M1273-3104, was added, and the fermentation repeated with oxygen supplementation of the sparge gas. This second culture, described in the APPENDIX, achieved a very high dry cell weight of 57.2 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

Vessel
The BioFlo 110 Advanced Fermentation Kit
used for this work was equipped with a heatblanketed 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 with these fermentors. We expect similar results from BioFlo 110 fermentation vessels from 1 through 14 L in volume, and in both heat blanket and water jacket configurations.


Control System
The four control modules included with the Advanced Fermentation Kit were used for the first run. (See Chart)


Small Items
Liquid addition bottles (3), cables, tubing, clamps and other small items were as supplied with the BioFlo 110 fermentor.


Materials and Methods

Inoculum

The inoculum was prepared using LB broth at a 25-g/L concentration as the shake flask medium. The inoculum was cultivated overnight at 28C on a rotary shaker (NBS model G25) at 240 rpm. OD 600 nm was 5.50 at the time of inoculation. Inoculum volume was 5% of the 5 L working volume, or 250 ml.

Medium
Four and one-half liters of E. coli K12 medium was prepared and poured into the vessel for a 5L run. One-half liter of vessel volume was reserved for components, including inoculum, to be added after sterilization.

Initial Medium Composition:
Potassium phosphate monobasic (anhydrous) . . .2 g/L
Potassium phosphate dibasic (anhydrous) . . . . . .3 g/L
Ammonium phosphate dibasic (anhydrous) .. . . . 5 g/L
Tastone 900AG . . . . . . . . . . . . . . . . . . . . . . . . .5 g/L
Breox FMT 30 (International Specialty Chemicals,
Southampton, UK). (antifoam agent) . . .. . . . . . .0.35-0.4 ml/L

After autoclaving and cooling the vessel, we added:

Glucose, . . . . . . . . . . . . . . . . . . . . . . .. . . . .25 g/L
(50 ml/L of 50% sterile glucose solution)
Magnesium sulfate heptahydrate . . . . . . . . . .0.5 g/L
Thiamine . . . . . . . . . . . . . . . . . . . . . . . . . . .1 mg/L
K12 trace metals solution (1) . . . . . . . . . . . .5 ml/L

Control Setpoints
Setpoints were keyed into the controller prior to inoculation, and, except for DO which remained high, the vessel was allowed to equilibrate prior to inoculation.

Temperature . . . . . . . . . . . . . . . . . . . . .. . .37C
pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .7.0
Dissolved Oxygen . . . . . . . . . . . . . . . . . . .35%
Agitation . . . . . . . . . . . . . . . . . . . . .. .. ... .300-1200 rpm
(responds 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 1,200 rpm agitation and 5L/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. An 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

Note that 1200 rpm is a high maximum, and could lead to excessive foam generation were it not for the presence of antifoam agent in the media. Note too, that smoother control occurs when the maximum rpm is limited to about 3 times the minimum rpm. Therefore, using 900 rpm as the agitation maximum would have allowed tighter control and the use of less antifoam in the media. The trade-off is reduced oxygen transfer rate, with possibly lower final cell density.

PH Control
We used liquid base to maintain pH at setpoint, relying on the acid-producing culture to lower pH if needed. The pH control parameters were:

Base . . . . . . . . . . . .. . .Sodium hydroxide, 10% solution
Pump . . . . . . . . . . .. . .Pump 1 of the 4-Pump Module
Transfer tubing . . . . . Narrow bore silicone tubing, as supplied
Vessel inlet . . . . . . . . .Triport adapter in the vessel headplate.
Probe calibration . . . .4.0 and 7.0 buffers

Controller Setup:
1) Pump 1 plugged into "BASE" power-outlet of the Power Controller
2) pH Control Selections: Multiplier = 50%
Dead-band = 0
PID values: factory defaults


Results and Discussion
The DO and agitation trend graphs reveal the fermentation history. Figure 1 shows that the DO declined rapidly to the 35% setpoint during the first hour. Figure 2 shows the early stage more clearly. Once the DO setpoint was reached, the control cascade varied agitation speed to meet increasing oxygen demand.



Agitation reached the preset limit of 1,200 rpm at Elapsed Fermentation Time (EFT) 3.75 hours. Further culture growth over the next 3/4 hour resulted in dissolved oxygen decreasing to well below setpoint.

A decline in agitation and rise in dissolved oxygen began at EFT 4.5 hours, indicating a reduced oxygen demand. This was at least partially due to exhaustion of the carbon source. One hundred grams of glucose were added (200 ml of a 50% solution) at EFT 4.75 hours, which slowed the decline in agitation but did not restore exponential growth, suggesting that other factors were limiting.

Figures 5 and 6 show increasing OD600nm and dry cell weight (DCW) from the time of inoculation through EFT 4.5-5 hours, consistent with the DO graphs. This run entered exponential growth phase upon inoculation. The short to non-existent lag phase results from the vigor of the inoculum, and the nurturing growth environment within the vessel.

The OD600nm and DCW of the culture were fairly stable over the last 3 hours of the run. The run was conducted for a total of 8 hours. A final OD of 26.0 and a final DCW of 10.3 g/L were obtained.

The pH trend graph in Figure 3 shows an initial decline from a slightly high value towards the 7.0 setpoint. The slight undershoot to 6.95 is normal control behavior.





Enhancements
1. DO deprivation over an extended time resulted in a loss of culture vigor. Oxygen supplementation of the sparge was investigated and described in the appendix.

2. Implementing an automated feed program through BioCommand Plus software, NBS Catalog Number M1291-0000, would have accomplished a more timely addition of nutrient, possibly increasing the final cell density.


Conclusion
E. coli growth in the BioFlo 110 was successful. A culture density of 57.2 g/L DCW was achieved in eight hours using oxygen supplementation of the sparge gas (see APPENDIX), and 10.3g/L DCW in eight hours was obtained without oxygen supplementation. Neither run was optimized for controller set points. A slightly modified medium was used to yield a dry cell weight of 57.2 g/L. The BioFlo 110 fermentor is well suited for E. coli work.





APPENDIX:
Effect of Gas Mix Controller and Oxygen Supplementation

A second fed-batch run was performed, using the B ioFlo 110 Gas Mix Controller to demonstrate the impact of oxygen supplementation on final dry cell weight. A slightly modified media was used, and the feed schedule was increased in anticipation of the effects of oxygen supplementation.

Dissolved Oxygen (DO) Control
Cascade : . . . . .Agitation and Oxygen
The cascade first increased agitation, and then added oxygen gas as needed to maintain DO at setpoint.

(1) K12 Trace Metals Solution consists of: sodium chloride 5 g/L, zinc sulfate heptahydrate 1 g/L, manganese chloride tetrahydrate 4 g/L, ferric chloride hexahydrate 4.75 g/L, cupric sulfate pentahydrate 0.4 g/L, boric acid 0.575 g/L, sodium molybdate dihydrate 0.5 g/L and 6N sulfuric acid 12.5 ml/L. Note that the quantity of sulfuric acid can vary as required to dissolve the other components properly, the usual range is between 8 20 ml/L.



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