A | Controlled Parameters - micro- Matrix

The Figure 1 depicts the parameters temperature, dO2, pH and the volume of NH4OH added during the fermentation.

The parameters were successfully and individually controlled by the micro- Matrix in the 24 wells during the batch

fermentation.

B | pH control by the addition of NH4OH

During the first 15 minutes of experiment pH control was disabled prolonging sensors time immersed in the culture media

and improving the accuracy of the measurements. After this period pH offset correction was performed with the pH offline

measurement (pH= 7.17, measured with culture media). This calibration leads to the step signaled as 1 in Figure 1.

The liquid dosage lines (purple) show the volume of NH4OH being delivered per pulse. These lines are not linear but

composed of small steps that correspond to each pulse of liquid delivered.

The system started the addition of NH4OH at 3h 38min of experiment marked by point 2. It is also possible to visualize that

each delivered pulse is followed by a momentarily pH increase creating the pH spikes that are observed in point 3.

These spikes refer to the moment the pH sensor gets in direct contact and detects the concentrated NH4OH solution.

After a few seconds and thanks to the continuous shaking, the NH4OH is dilute in the media and the pH value returns to the

set point.

At 7h 51min the liquid dosage stopped (point 4) as the pH went slightly above the set point. It is possible to conclude that at

this point most of the cells were dead and fermentation was ending conclusion also supported by the erratic dO2 profile (red

lines) at the same time point.

The initial pH deviation observed in the wells A5 and C4 (green lines with pH above 7) is explained by a small leak of ammonia

from the valves that caused an increase in the pH value. During fermentation (around 6h 40min) the pH of these wells

decreased to the set point due to the acidic products being produced during the bacterial cells growth. This fact is consistent

with the pattern observed in the dO2 lines indicating oxygen consumption and therefore, cellular growth (red lines).

C | dO2 control with air and pure O2 in cascade

The experiment started with an initial dO2 value around 15% due to the oxygen consumption that occurred between the time of

inoculation and the beginning of the experiment (red lines). Once the orbital shaking speed began it was possible to observe an

increase of this value up to 70%. After the initial increase, the dO2 values started to drop due to bacterial growth and consequent

oxygen consumption. In the point 5 marked in the Figure 1, it is possible to observe the oscillation and a slight increase in the

dO2 meaning cell adaptation to the new environment with higher cell density and produced by-products. After cells adaptation

the dO2 continued to decrease indicating cell growth. The dO2 went below the set point of 30% at 3h 09min and the system

started the dO2 control. The PID default values for this parameter were not suitable for the strain’s growing rate hence, those

were changed (point 6) to the values optimized to a high dO2 demanding bacterial cell culture and dO2 was kept at 40% until

5h 30min.

Around 5h 50min of experiment the oscillation in the dO2 becomes erratic (point 7) which was expected as exponential growth

phase started. The end of batch fermentation occurred at 8h 53 when the carbon source was completely depleted. This fact was

confirmed by the dO2 profile that started to increase (point 8) indicating that the oxygen consumption and cell’s growth has stopped.

D | Temperature

Temperature took c.a. 8 minutes (point 9) to reach the set point of 30°C, after which temperature remained stable during the

entire fermentation.

E | Biomass concentration (OD600)

Biomass achieved in the micro-Matrix was determined by measuring the OD600 in the 21 working wells at the end of the batch

fermentation. The final OD600 reached was 22.6 ± 6.2%, which allows to conclude that the micro-Matrix’s results are precise and

reproducible. In the shake flask OD600 was measured to follow-up the cells growth. The final biomass obtained in both systems

it is within the same range supporting the conclusion that the micro-Matrix’s results are reproducible.

• InthepresentworkanE. coli batch fermentation with high reproducible results

has been performed in the micro-Matrix since the final biomass obtained was

22.6 ± 6.2% (average of 21 bioreactors). The same fermentation was reproduced

in a shake flask resulting in a biomass of 21.

• Resultsprovedthatthemicro-Matrixisabletoefficientlycontroltheparameters

pH, dO2 and temperature at the set points during the time of experiment.

• Biomassresultsshowedthatthemicro-Matrixisadevicethatprovidesreliable

and reproducible results and suitable for small scale bacterial cell fermentation.

Results and discussion

Materials and methods

Conclusion

micro- MatrixSmall scale Escherichia coli batch fermentation in micro- Matrix

E. coli batch fermentation was performed in collaboration with Dompé biopharmaceutical (L’Aquila, Italy) who kindly offered their

installations, technical support and supplied the materials here used, including their micro-Matrix device.

1 | Pre- culture

•ThestrainE. coli K12 genetically modified was grown in defined media to a final OD600

(optical density – wavelength 600nm) of 10.92.

•Pre-culturewascultivatedinashakingincubatorat30°C,shakingat200rpm.

2 | Batch fermentation conditions

2.1 Micro- Matrix

•Allthe24wellsofthecassettewerefilledwith5mLofinoculatedmediawiththepre-culturetoaninitialOD600 = 1.46.

•ThepHofthedefinedmediawasmeasuredofflineforfurtherpHoffsetcorrection.

•ThepHwascontrolledbytheuseoftheLDMatthesetpointof7duringfermentationwiththeadditionofNH4OH (12.5% v/v)

with the previous calibration of the Liquid addition valves for 3ms valve opening time.

•Thesetpointoftemperaturewas30°CanddO2 was controlled at 40% by using air and O2 in cascade.

•Orbitalshakingspeedwaskeptat300rpmduringtheexperiment.

•AttheendofbatchphasebiomassconcentrationwasdeterminedbyOD600 measurement.

2.2 Shake flask

•Pre-culturewasdilutedwithdefinedmediatoaninitialOD600 of 1.46.

•Theshakeflaskwaskeptinashakingincubatorat200rpmprovidingasimilaraerationastheonefoundinthemicro-Matrix

(previously optimized by Dompé).

•Temperaturewaskeptat30°CandpHwasnotcontrolled.

•TheOD600 was measured during batch fermentation.

Escherichia coli is used in multiple applications in biotechnology such as the synthesis of DNA and proteins.

These processes require rigorous control of culture conditions. The optimization of these conditions is ideally

performed at small scale reducing time, cost and resource requirements [1].

A suitable control of oxygen, pH and temperature in E.coli fermentation is necessary to obtain a robust process and

achieve optimal yields of biological products. As the oxygen is consumed while cells are replicating it must be supplied

to the culture as much as necessary. Furthermore, the metabolic by-products that are released during the fermentation

obligate the correction of the pH, which requires the presence of a precise pH controller [2, 3, 4].

The micro-Matrix is a small scale 24 micro bioreactors platform that fulfills the demand for a narrow control over the

dissolved oxygen (dO2), pH and temperature during E. coli fermentation. This device allows 24 parallel fermentations

with individual and simultaneously controlled parameters.

In the micro-Matrix, the parameter pH can be controlled by liquid addition using the Liquid Delivery Module (LDM) or

by gas addition. Micro-Matrix system permits the addition of 4 different gases simultaneously hence, the dO2 can be

controlled by addition of air and/or O2 in cascade and/or direct control, independently of the pH control.

The aim of the present work was to perform a typical E. coli batch fermentation using the micro-Matrix. The pH was

controlled with the LDM (alkali control) and CO2 gas (acid control). The dO2 was controlled with air and O2 in cascade.

The bacterial growth was monitored and compared with the growth achieved in a shake flask.

References• [1]Zhang,Z.,Perozziello,G.,Boccazzi,P.,Sinskey,A.,Geschke,andJensen,K.(2007).

J.Assoc.Lab.Autom.,12,143–151.

• [2]Yohannes,E.,Barnhart,D.andSlonczewski,J.(2004).J.Bacteriol.,186,192–199.

• [3]Salmon,K.,Hung,Steffen,S.,Krupp,N.,BaldiR.,Hatfield,P.andGunsalus,R.(2005).

J.Biol.Chem.,280,15084–15096.

• [4]Collins,T.,Azevedo-Silva,J.,Costa,A.,Branca,F.,Machado,R.andCasal,A.(2013).

M. Microb. Cell Fact., 12, 21.

Figure 1 | Profile of the controlled parameters during 8h 53min of E. coli batch fermentation in the micro-Matrix device: temperature (in blue), pH (in green), dissolved oxygen (%) and volume of NH4OH added (μL)

for the pH control. Well D6 was filled with water and used as control (results not showed). In wells A1 and D4 technical problems were detected so the results were discarded.

Introduction

Fernandes, C., MSc¹, Scarabeo, A., PhD² ³, Walvoort, T., MSc¹, Riscaldati, E., PhD², Di Pietro, L. PhD², Bernal, C., PhD¹¹ Applikon Biotechnology - Heertjeslaan 2, 2629 JG Delft The Netherlands ² Dompé, L’Aquila (Italy) ³ Department of Pharmacy, University of ‘G. D’Annunzio’ 66100 Chieti (Italy)