High Cell Density Fermentation of Escherichia coli

High Cell Density Fermentation of Escherichia coli Using the
™
®
New Brunswick BioFlo 115
Bin Li, Stacey Willard, and Ma Sha, Eppendorf, Inc., Enfield, USA
Abstract
Abstract
Results
Materials and Methods
Equipment
Fermentation was performed in a New Brunswick BioFlo 115 benchtop fermentor (Eppendorf) with the configuration outlined in Table 1.
The E. coli K12 strain (ATCC®, 10798™) was grown in a 2 L working
volume New Brunswick BioFlo 115 heat-blanketed glass vessel (Eppendorf). Glucose concentrations were measured using a Cedex® Bio
Analyzer (Roche®). The OD600 was measured with an Epoch™ Microplate Spectrophotometer (BioTek®) using the optional cuvette attachment.
Parameter
Gas Mix
Gas Flow Control
Vessel
Motor
Impeller
Sparger
Configuration
Automatic gas mix option
One thermal mass flow controller
(TMFC) with 0 – 20 SLPM flow range
2 L working volume heat-blanketed
glass vessel with baffle assembly
Direct drive motor
Two rushton impellers
Ring sparger (Macrosparger)
Table 1: New Brunswick BioFlo 115 hardware configuration
The initial fermentation temperature was set to 30 °C. Antifoam 204
(Sigma-Aldrich®, A6426) was added only when needed, since it may
reduce the oxygen transfer rate (OTR) and possibly lower the final cell
density. In this experiment, ~100 µL of 5 g/L antifoam 204 was added
at the beginning of the run to prevent foaming and ~4 mL was added
between 8 – 11 h of fermentation, as foam accumulation warranted.
Pump 3 was assigned as the feeding pump (maximum speed is 24 mL/
min with 0.188 in inner diameter tubing). The feeding strategy included increasing or decreasing the feeding pump speed accordingly,
based on the glucose concentration. To achieve high cell density, the
target glucose concentration was ≤ 2 g/L. Excessively high glucose
concentrations may alter E. coli metabolism and reduce peak cell density. Table 2 illustrates the adjustments made to the pump speed over the
course of the fermentation while trying to maintain glucose concentration at or below 2 g/L.
Cell growth and glucose concentration were monitored offline using
5 mL samples taken according to the following schedule.For OD600
readings, samples were taken every hour and diluted appropriately for
accurate measurement. For the determination of glucose concentration,
samples were taken every hour before the initiation of feeding, and
then every ~30 min after the feeding began. The specific growth rate
(µ) was calculated from the fitted OD600 value in Microsoft® Excel®.
pH Calibration and Control
pH calibration was done outside the vessel using a two-point calibration method and standard buffers. Buffer pH 7.0 was used to set
“ZERO” and pH 4.0 for the “SPAN.” The pH sensor was calibrated prior
to autoclaving the vessel. The pH was automatically maintained at 6.8
by adding 6 N HCl via pump 1 (assigned as “acid”) to lower the pH and
adding 25 % (v/v) NH4OH via pump 2 (assigned as “base”) to raise the
pH. The deadband for pH control was set to 0.02.
Figure 1: The New Brunswick BioFlo 115 benchtop fermentor with water-jacketed (left) and heat-blanketed (right) vessels
Medium
The initial fermentation medium was prepared as
follows: 150 mL 10 X phosphate/citric acid buffer
[133 g/L KH2PO4, 40 g/L (NH4)2HPO4, 17 g/L citric acid]
and 1.35 L deionized (DI) water were added to the vessel before sterilization at 121 °C for 20 min. After the
solution was cooled to room temperature, the following sterile components were added to make the complete fermentation medium: 15 mL of 240 g/L MgSO4,
0.34 mL of 20 g/L thiamine, 15 mL of 100 X trace element solution, and 22 mL of 70 % glucose solution.
The 100 X trace element solution contained: 10 g/L
iron (III) citrate, 0.25 g/L CoCl2•6H2O, 1.5 g/L MnCl2•4H2O, 0.15 g/L CuCl2•6H2O, 0.3 g/L H3BO3, 0.25 g/L
Na2MoO4•2H2O, 1.3 g/L zinc acetate•2H2O, 0.84 g/L
EDTA [1, 2]. The pH was adjusted to 6.8 using 25 %
(v/v) NH4OH or 6 N HCl.
Elapsed Fermentation Time (EFT, hours)
5
6
6.5
7.5
8
8.5
9
9.5
10
10.5
Table 2: Pump output during the fed-batch fermentation
Pump Output
1.4 %
1.7 %
2.4 %
3.5 %
4.8 %
6.3 %
7.5 %
8.5 %
10 %
13 %
A
Dissolved oxygen (DO) sensor calibration and gassing control
DO sensor calibration was performed using a standard two-point calibration method: 0 % (set “ZERO”) was obtained by disconnecting the
sensor from the cabinet and allowing the raw value to stabilize; 100 %
(set “SPAN”) was obtained by running 1200 rpm agitation and 3 SLPM
air flow until the DO value stabilized at maximum.
The New Brunswick BioFlo 115 Reactor Process Controller (RPC) software offers a selection of automatic gassing control cascades that are
dependent upon the configuration of the unit. The New Brunswick
BioFlo 115 used in this study included the automatic gas mix option
and one TMFC with a flow range of 0 – 20 SLPM (Table 1). Operating
in fermentation mode, the automatic DO cascade “Agit/GasFlo/O2” was
selected with a DO setpoint of 30 %. User-defined minimum and maximum limits were populated in the cascade screen and are listed in
Table 3.
Parameter
Agit Casc Low Limit
Agit Casc High Limit
GasFlo Casc Low Limit
GasFlo Casc High Limit
O2 mix Casc Low Limit
O2 mix Casc High Limit
Value
300 rpm
1200 rpm
0 SLPM
3 SLPM
0%
100 %
20
18
16
14
12
10
8
6
4
2
0
140
[Glucose]
120
OD600
100
80
60
40
OD600
E. coli is a Gram-negative bacterium that has had a long history in the world of laboratory and industrial processes due to its ease of manipulation and well understood genome. It is widely cultivated under aerobic conditions. High cell density fermentation of E. coli is a powerful technique for the production of recombinant proteins. In this application note, E. coli cultivation achieved a high OD600 value of 140 at
11 h using fed-batch fermentation with the New Brunswick BioFlo 115 benchtop fermentor (Figure 1).
Inoculum and Fermentation
The inoculum was grown in Terrific Broth (TB) medium, prepared as
described previously [3]. Two 500 mL baffled shake flasks (VWR®,
30623-210) each containing 100 mL of TB medium were inoculated
from a frozen vial of E. coli and incubated at 30 °C, 200 rpm overnight
in a New Brunswick Innova® 40 benchtop incubator shaker (Eppendorf). After the overnight culture, the OD600 value was ~9. The vessel
was inoculated with 75 mL of inoculum (5 % of the initial working volume).
Samples were taken periodically to monitor the cell growth (OD600 value) and glucose concentration.
Feeding was initiated when the glucose concentration dropped below 2 g/L, which occurred at 5 h
of cultivation. After starting the feed, the pump rate was adjusted according to the current glucose
concentration with the end goal of keeping it at or below 2 g/L (Table 2). As shown in Figure 2A, within 11
h, the OD600 value reached 140. The growth curve was also plotted on a log scale to calculate the specific
growth rate (µ = 0.58 h-1, Figure 2B).
[Glucose] (g/L)
Introduction
An additional concentrated feeding medium was prepared separately in
a 1 L glass bottle. 45 mL of 240 g/L MgSO4, 1.66 mL of 20 g/L thiamine
solution, 15 mL of 100 X trace element solution, and 70 % glucose
solution were added to a final volume of 500 mL.
20
0
0
1
2
3
4
5
6
7
8
9
10 11
Time (h)
B
ln (OD600)
This application note presents a successful example of a high density fermentation of Escherichia coli (E. coli) using the New Brunswick
BioFlo 115 benchtop, autoclavable fermentor from Eppendorf. The highest optical density (OD600) achieved in this study was 140 at 11 h
without optimized fermentation medium and conditions.
5.3
4.8
4.3
3.8
3.3
2.8
2.3
1.8
1.3
0.8
0.3
-0.2
-0.7
y = 0.58x - 1.01
R² = 1.00
0
1
2
3
4
5
6
7
8
9
10 11
Time (h)
Figure 2: Fermentation growth curve and growth rate calculation
A: The OD600 and glucose concentration over the course of the 11 h fermentation
B: The growth curve plotted on a log scale; a linear trend line was applied in Microsoft Excel, the slope of which
is equivalent to the specific growth rate, µ (h-1)
Conclusions
High density E. coli growth in the New Brunswick BioFlo 115 was achieved using a fed-batch fermentation
method. An optical density of 140 was reached at 11 h. Although efforts were made to maintain a glucose
concentration below 2 g/L, the fermentation was not optimized for medium, growth conditions, or any
product yield.
Table 3: DO cascade low and high limits
REFERENCES:
[1] Geerlof A. High cell-density fermentation of Escherichia coli. http://www.helmholtz-muenchen.de 2008.
[2] Korz D.J., Rinas U., Hellmuth K., Sanders E.A., Deckwer W.D. Simple fed-batch technique for high cell
density cultivation of Escherichia coli. Journal of Biotechnology 1995; 39(1): 59-65.
[3] Terrific Broth. Cold Spring Harbor Protocols 2006; 2006(1):pdb.rec8620.
ATCC® is a registered trademark of American Type Culture Collection, USA. Cedex® and Roche® are registered trademarks of Roche Diagnostics GmbH, Germany. BioTek® is a registered trademark of BTI Holdings, Inc., USA. Microsoft® and Excel® are registered trademarks of Microsoft Corporation, USA. VWR® is a registered trademark of
VWR International, LLC, USA. Sigma-Aldrich® is a registered trademark of Sigma-Aldrich Co., LLC, USA. Epoch™ is a trademark of BTI Holdings, Inc., USA. 10798™ is a trademark of American Type Culture Collection, USA.
Eppendorf® and the Eppendorf logo are registered trademarks of Eppendorf AG, Germany. New Brunswick™ is a trademark of Eppendorf AG, Germany. BioFlo® and Innova® are registered trademarks of Eppendorf, Inc., USA.
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