Online-Ethanol and Online-MethanolMeasurements in Fermentation Processes

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Application Note

1. IntroductionEthanol and methanol are products of various microorganisms. Depending on the process they can be main product, by-product, or can be used as inducer. In a lot of processes it is important to monitor and control the concentration of ethanol or methanol in order to know the end point of the fermentation. Thus, avoiding a toxic concentration inside the fermentation broth. For some organisms methanol is used as an inducer, where the concentra-tion must be controlled in a very tight range. BioPAT® Trace is a new online analyzer for measuring methanol and ethanol in fermentation processes.

2. BioPAT® Trace on-line analyzerBioPAT® Trace is an on-line analyzer for measuring ethanol or methanol (Figure 1). Both substances can be detected continuously without operator influence.

Figure 1: BioPAT® Trace on-line analyzer

The range of methanol concentration is 0.5 – 20 g/L and the range of ethanol concentration is 1 – 40 g/L . The measure-ment frequency is about 25 per hour. Because of the internal temperature correction coefficient a temperature range from 15°C to 25°C is possible.

Stuart Tindal1 Michael Hartlep and Wolfgang Künnecke2)

Holger Penders 3)

1) Sartorius Stedim Biotech GmbH August-Spindler-Strasse 11 37079 Goettingen

2) TRACE Analytics GmbH Richard-Wagner-Straße 1-2 38106 Braunschweig (Germany)

3) Sanofi-Aventis Deutschland GmbH Industriepark Hoechst 65926 Frankfurt am Main (Germany)

3. Enzymatic reaction and detectionIn the presence of oxygen the immobilized enzyme alcoholoxidase (AOD) catalyses the transformation of alcohol (e.g. methanol or ethanol) to the corresponding aldehyde and hydrogen peroxide. The alcohol content is measured indirectly on a plati-num electrode via the formed hydrogen peroxide, which is oxidized to water andoxygen. In an amperometric measurement the electrical current is proportional to the amount of oxidized alcohol.

– Safe and economic operations

– Low installation size and less maintenance effort

– Dual methanol and ethanol assay

– Disposable consumables (tubing set, sensor, reactors and transport solution)

– Enzyme reactors with long-term stability and low drift

– No sample volume required

– Connection to Biostat® vessels

4. Enzyme reactorThe enzymatic bioreactor has life time of 5000 measurements or 14 days. The reactor is very small, reusable, and easy to connect with a Luer connector (Figure 2).

Figure 2: The BioPAT® Trace enzyme reactor

5. Analytical performanceThe linearity of the measurements was proven bychanging the concentrations of methanol in the rangebetween 0 and 20 g/L (Figure 3) and by changing theconcentrations of ethanol in the range between 0 and40 g/L (Figure 4).

y = 0,9713x + 0,0448 R² = 0,9984

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0 5 10 15 20

actu

al v

alue

[g/

L]

reference value [g/L]

Methanol

Data pointsgraph of reference valuesLinear (Data points)

Figure 3: Linearity of methanol measurements

y = 0,9833x + 0,1658 R² = 0,9988

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0 5 10 15 20 25 30 35 40

actu

al v

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Ethanol

Data pointsgraph of reference valuesLinear (Data points)

Figure 4: Linearity of ethanol measurements

6. Ethanol monitoring in an industrial fermentation processA fed-batch fermentation of a recombinant yeast was performed. This process run over 70 hours. The yeast was cultivated on a glucose medium. During the first 20 hours ethanol was produced and consumed. After this period the ethanol concentration stayed at a very low level near 0 g/L. With the BioPAT® Trace it was possible to monitor this trend (dark green line in Figure 5).

Figure 5: Ethanol concentration in a fermentation run with a recombiant yeast.

The ethanol concentration data from the BioPAT® Tracewere correlated with offline data (yellow points in Figure 5)and with online data from another alcohol analyzer (brownline in Figure 5).

It was possible to show the robustness of the process andof the BioPAT® Trace in three replicate fermentation runs (Figure 6).

Ethanol concentrations can be reliably monitored infermentation processes with the BioPAT® Trace.

Figure 6: Ethanol profiles of three fermentation runs.

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