Conclusion

Comparison of Real Time Off-Gas Analysis Equipment for DASGIP® Bioreactors: The DASGIP® GA4 Exhaust Analyzing Module vs. the Thermo-Scientific® (Prima Pro) Mass SpectrometerJacob Ladd1; Keerthi Venkataramanan1, Bill Throndset1 , Robert Glaser2 1Novozymes® North America, Morrisville, NC2Eppendorf AG Bioprocess Center, Jülich, Germany contact: glaser.r@eppendorf.com

Introduction

Real time off-gas analysis is an important tool for gaining insights into a fermentation

process by allowing the user to examine carbon evolution, oxygen utilization, and transfer

during the fermentation. This information can be used to find changes in metabolism, spot

potential problems, or trigger changes in the fermentation process.

We compared two technologies for off-gas analysis, namely a Prima Pro mass

spectrometer (Thermo Fisher Scientific) and a DASGIP GA4 gas analyzer module

(Eppendorf). The latter uses electrochemical and infrared sensors for the quantification of

O2 and CO2, respectively.

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ATCC® is a registered trademark of the American Type Culture Collection, USA. Novozymes® is a registered trademark of Novozymes A/S, Denmark. Thermer-Scientific® is a registered trademark of Thermo Fisher Scientific, Inc., USA. Eppendorf® and the Eppendorf Brand Design and BioBLU® are registered trademarks of Eppendorf AG, Germany. DASGIP® is a registered trademark of DASGIP Information and Process Technology GmbH, Germany. All rights reserved, including graphics and images. Copyright © 2019 by Eppendorf AG.

Objectives

>Compare real time measuring capabilities of both the DASGIP GA4 and Prima Pro Mass Spectrometer

> Test both dry and humid (through a bioreactor) to observe impact of moisture on measurement readings

> Utilize both standard gasses and biological samples to compare the equipment> Ensure two measuring devices in series would not interfere with one another

Materials and Methods

The DASGIP Parallel Bioreactor System (Eppendorf) was utilized for all experiments. Gas

was supplied to the DASGIP MX gas mixing module which regulated air flow where it was

either pushed through a BioBLU® 1f Single-Use Vessel (Eppendorf), or fed directly into

the DASGIP GA4 off gas analyzer, which was followed in the chain by the Prima Pro mass

spectrometer.

Standard calibration gasses were supplied by Airgas (+/- 0.2%) and were fed both directly

to the GA4 and through a BioBLU 1f Single-Use Vessel containing 1L of sterile phosphate

buffer. The gas was supplied at a steady flow rate of 40sL/h for 7 hours to monitor the

stability of the reading over time.

Biological tests for two ATCC® organisms, Bacillus subtilis (ATCC 6051) and Pseudomonas

vancouverensis (ATCC 700688) were conducted in the BioBLU Single-Use Vessel using the

ATCC recommended media and temperature, 1 VVM air flow, agitation based DO control

(30% min), and acid/base pH control (7.0, phosphoric acid, NaOH).

Gas Supply

or or

DASGIP MX4

DASGIP GA4

DASGIP Bioblock

Exhaust

Equipment orientation and gas flow diagram

Prima Pro Mass Spectrometer

Results

Gas

Flo

w [s

L/h

]

GA4

Gas flow measurements from both units demonstrate little interference when two instruments are in the same flow

20,4

20,6

20,8

21,0

21,2

21,4

21,6

O2

[%]

GA4

0,00

0,02

0,04

0,06

0,08

0,10

CO

2 [%

]

GA4

O2 [

%]

GA4

CO

2 [%

]

GA4

0,0

2,0

4,0

6,0

8,0

10,0

12,0

0 1 2 3 4 5

O2 &

CO

2 [%

]

Mass Spec. CO2 %

Evol

utio

n [%

]

Evol

utio

n [%

]

GA4 CO2

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

1,8

2,0

19,0

19,4

19,8

20,2

20,6

21,0

21,4

21,8

22,2

22,6

23,0

0 0,2 0,4 0,6 0,8

Evol

utio

n [%

]

Evol

utio

n [%

]

Elapsed Fermentation Time [HH:mm:ss]

Mass.Spec. O2 GA4 O2 Mass.Spec. CO2 GA4 CO2

The addition of the mass spectrometer to the gas stream did introduce some variation in the off gas measurements of the GA4. This is attributed to the increased back pressure caused by the mass spectrometer’s sampling apparatus. However measurements were still less than ± 0.5%. When measuring with multiple channels, the results were similar, indicating no interference when multiple units were measured at once

Dry Air Measurements of a Single and Two Channels Concurrently -

Averaged Values Over 7hrs (21% O2 & 0.04% CO2)

Humid Air Measurements of a Single and Two Channels Concurrently (through BioBLU with 1L PBS) -

Averaged Values Over 7hrs (21% O2 & 0.04% CO2)

When the gas supply was routed through a bioreactor containing 1L of PBS, the GA4 measures lower amount of oxygen, caused by the increased water vapor which will be compensated for the later OTR and CTR calculations. Variation between GA4’s values and the mass spectrometer were still present (< ± 0.5%)

Measurements of three different standard gasses all showed high correlation between the GA4 and the mass spectrometer (0% O2 with 20% CO2, and 15% O2 with 5% CO2 results not shown)

Measurement of Standard Calibration GasAveraged Values Over 7hrs (21% O2 & 0.04% CO2)

GA4 Flow Reading vs. Mass Spec Flow Reading

> Off gas values and trends from the DASGIP GA4 and Thermo-Fisher Prima Pro were comparable for Air, standard gasses, and biological experiments.> Having the GA4 and the Mass Spec. in the measuring stream leads to interference with the O2 and CO2 measurements of the GA4 due to the MS’ systems backpressure.> The GA4 and mass spectrometer can be used in conjunction with each other, if additional volatile compounds, such as ethanol wished to be measured using the mass spectrometer.

> The GA4’s integration into the DASGIP system allows for automatic, real time calculation of oxygen transfer rate, carbon evolution rate, and the respiratory quotient, due to its compensation strategies with regards bioreactor volume, exhaust gas humidity and exhaust gas flow. These calculations must be done manually for the mass spectrometer.

> Manual calculations can be done DASware control’s scripting opportunity.

B. subtilis (ATCC 6051) 100x phase contrast microscopy – 48hrs EFT

P. vancouverensis (ATCC 700688) 100x phase contrast microscopy – 48hrs EFT

B. subtilis (ATCC 6051) O2 and CO2 Evolution Traces

OT

R [

mM

ol/h

]

Mas.Spec. OTR

Mass.Spec. OTR recalculated using Fout = Fin * X(N2in) / X(N2out) GA4 OTR recalculated using smoothed XO2 signal due to interference with Mass.Spec.

Bacillus subtilis Oxygen Transfer Rate Bacillus subtilis Carbon Transfer Rate

P. vancouverensis (ATCC 700688) O2 and CO2 Evolution Traces

-100

1020304050

OT

R [

mM

ol/h

]Elapsed Fermentation Time [HH:mm:ss]

Mas.Spec. OTR GA4 OTR

0102030405060

0 0,25 0,5 0,75

CT

R [

mM

ol/h

]

Elapsed Fermentation Time [HH:mm:ss]

Mas.Spec. CTR GA4 CTR

Fermentations of two model strains showed consistent O2, CO2 and OTR, CTR values (< ± 0.5%), while also closely matching one another during all phases of growth including lag, exponential, and stationary. In addition, smaller changes in metabolism indicated by changing O2 and CO2 evolution were captured by both units.

P. vancouverensis Oxygen Transfer Rate P. vancouverensis Carbon Transfer Rate

0:00:00 1:12:00 2:24:00 3:36:00 4:48:00 6:00:00 7:12:00

45,0

40,0

35,0

30,0

25,0

20,0

15,0

10,0

5,0

0,0

Gas

Flo

w [

sL/h

]

Duration [hh:mm:ss]GA4 MX4 Mass Spec.

GA4 Mass Spec.

21,6

21,4

21,2

21,0

20,8

20,6

20,4

0,10

0,08

0,06

0,04

0,02

0,00Single-1 GA4+MS-1 GA4+MS-2 Single-2 Single-1 GA4+MS-1 GA4+MS-2 Single-2

GA4 Mass Spec.

O2

[%]

CO

2 [%

]

O2

[%]

21,6

21,4

21,2

21,0

20,8

20,6

20,4

0,10

0,08

0,06

0,04

0,02

0,00

CO

2 [%

]

GA4 Mass Spec.

Single-1 GA4+MS-1 GA4+MS-2 Single-2 Single-1 GA4+MS-1 GA4+MS-2 Single-2

GA4 Mass Spec.

Mass Spec. O2 %

Mass Spec. CO2 % GA4 CO2 %

GA4 O2 %

12,0

10,0

8,0

6,0

4,0

2,0

0,00 1 2 3 4 5

23,0

22,6

22.2

21,8

21,4

21,0

20,6

20,2

19,8

19,4

19,0

3,0

2,7

2,4

2,1

1,8

1,5

1,2

0,9

0,6

0,3

0,0

O2

Evo

luti

on [

%]

CO

2 E

volu

tion

[%

]

Elapsed Fermentation Time [hh:mm:ss]

Mass Spec. O2 Mass Spec. CO2 GA4 CO2GA4 O2

60 50 40 30 20 10 0-10O

TR

[m

Mol

/h]

Mass Spec. OTR

70605040302010 0

Mass Spec. CTRGA4 OTR GA4 CTR

0 0,25 0,5 0,75 0 0,25 0,5 0,75

Elapsed Fermentation Time [hh:mm:ss] Elapsed Fermentation Time [hh:mm:ss]

CT

R [

mM

ol/h

]

23,0

22,6

22.2

21,8

21,4

21,0

20,6

20,2

19,8

19,4

19,0

2,0

1,8

1,6

1,4

1,2

1,0

0,8

0,6

0,4

0,2

0,0

O2

Evo

luti

on [

%]

CO

2 E

volu

tion

[%

]

Elapsed Fermentation Time [hh:mm:ss]Mass Spec. O2 Mass Spec. CO2 GA4 CO2GA4 O2

0 0,2 0,4 0,6 0,8

Mass Spec. OTR Mass Spec. CTRGA4 OTR GA4 CTR

Elapsed Fermentation Time [hh:mm:ss] Elapsed Fermentation Time [hh:mm:ss]

50 40 30 20 10 0-10O

TR

[m

Mol

/h] 60

5040302010 0

0 0,25 0,5 0,75

CT

R [

mM

ol/h

]

0 0,25 0,5 0,75