Fermentation Monitoring

Fermentation. A large brewery.
Image by Vladimir Zapletin, c/o www.123rf.com

Fermentation should proceed without issue if it is controlled and managed properly. A fermentation relies on a microorganism, a bacteria, yeast or plant/animal cell being able to grow and produce the desired product. It may be the cell itself or a chemical which produced extracellularly or intracellularly. The environment for production must be optimal and feasible from a scaled-up and economic point of view.

We are probably very familiar with a number of fermentations. One classic version is the production of ethanol using the yeast Saccharomyces cerevisiae. Another fermentation commonly examined is industrial ethanol production using Zymomonas mobilis. We have also come across the production of lactic acid which gives the tartness in yogurt. That is a fermentation of dairy foods by Lactobacillus bulgaricus

Fermentation processes need to be carefully controlled otherwise the wrong type of product could be produced, the culture begin to die for example. Monitoring is essential to control productivity and ensure high product quality.  Indeed there are a host of issues that need checking throughout the course of fermentation. Detection usually relies on probes which are often electrodes extending into the fermentation milieu so that measures can be made. 

Probe Detectors

Probe detectors often measure pH, brix (sugar concentration using refractometry), dissolved oxygen, measures of salt levels using ion-electrodes and even biosensors which can rely on cells and enzymes. More sophisticated methods use NIR (Near Infra Red) and fluorescence spectroscopy.

During the preparation of a batch of biological products, different forms of probe detection may be used to monitor the process.

Cleaning Your Probes

Upon the completion of the preparation of one batch of agents or pharmaceuticals, it is necessary to sterilize the system in which they were prepared in order that a new batch may be prepared without contamination. Spores and bacteria that may remain from a completed batch, if not killed and removed from the system, can grow and contaminate a subsequent batch. 

A probe detector needs to enter the fermentation vessel. To avoid leakages seals are used including the infamous O-ring. The seal is often at the inner bore of the probe detector support means near its end within the fermentation tank. Such O-ring seals were carried within an O-ring groove located a fraction of an inch inwardly from its end. This has been a weakness in the past because it allows bacteria and virus to contaminate spaces which may not then be accessed by cleaning materials and heat even.

In-Process Monitoring

In-process Monitoring of a fermentation can be at-line, on-line or in-line. Ideally, monitoring a variable inside the fermenter is the ideal situation. It can mean exploiting a unique variable or measuring an important difference.

On-Line monitoring is often linked to a controlling system to manage a fermentation of a microorganism. One example is the application of an on-line HPLC system to analyse glucose and ethanol in a brewing operation. A sample is collected using an auto-sampling device. Pipetting, filtration and dilution of the sample is required before final injection using automation based on a programmed procedure. It requires A/D (analogue/digital) and D/A interfaces to process the signals from the electrodes and from the detector of the HPLC. These then direct the feed pumps, stirrer and gas flow-rate controller.

An example of a simple system using in-process monitoring directed from a simple PC is that of fermentation of Zymomonas mobilis which was operated in batch and fed-batch modes (Liu et al., 2001). Substrate (glucose) and product (ethanol) concentrations  were monitored using on-line HPLC as was biomass concentration was estimated on-line using pH control and a proprietary software sensor. The substrate concentration profile in the fed-back fermentation followed well the set point profile due to the fed-back action of feed flow-rate control.

In the dairy industry, yogurt is fermented by a mixture of Lactobacillus bulgaricus and Streptococcus thermophilus (Guo et al., 2018). Most dairy fermentations rely on monitoring pH and texture through a viscocity measure. A very recent online monitoring system used the dielectric properties of cows milk and yogurt which was measured with an open-ended coaxial-line probe and impedance analyzer. Changes in the dielectric loss factor makes this a possible indicator for online monitoring.

Wine fermentations are good places to see various monitoring methods in action. Here is a classic off-line monitoring example with potential.  Attenuated total reflectance infrared spectroscopy (ATR-FTIR) combined with soft independent modeling of class analogy (SIMCA) to examine the physiological state of some strains of  Saccharomyces cerevisiae (Puxue et al., 2015).

Off-Gas Analysis

The most accurate monitoring is often off-line using mass spectrometry. It is expensive but extremely accurate. Cheaper but less accurate gas analysis is conducted using TanDem, EGAS-L and FerMac analysers for example. These analysers measure both oxygen and carbon dioxide either using electrochemical galvanic action for oxygen and infrared absorption of carbon dioxide.

Most devices will be accurate between the ranges of 0 to 30% for oxygen and 0 to 5% for carbon dioxide. the gas must be dried otherwise inaccurate measurements are made because of condensation in the carbon dioxide sensor.

Use Of Near-Infrared Spectroscopy (NIR)

NIR absorption spectroscopy, ranging from 12 820 to 4000 cm−1, is increasingly used for monitoring fermentation processes to measure the concentration of biologically important bonds (aliphatic C–H, aromatic or alkene C–H, amine N–H and O–H) that absorb in the NIR range (Hills, 2010). Each chemical structure contributes to a characteristic position, shape, and size of the analyte’s absorption bands. Process-related changes can be captured in the NIR spectra of complex culture media, to simultaneously monitor concentrations of nutrients, metabolites, product formation, or biomass. Combination of NIR spectra with chemometrics methods is being used to determine critical parameters in processing that are important for product quality.  

Software

Program software is designed to supervise the control of various factors such as stirring speed, gas flow-rate, pH value, feed flow-rate of medium, and the on-line measurement of substrates and products like glucose and ethanol for example.

Simple programs using proprietary software can be used. For example Microsoft Windows uses Microsoft Visual basic. Signals for chromatographic peaks from the on-line HPLC is captured and processed using an RC filter and a smoothing algorithm.

Data Structures

All the methods of measurement rely on producing different data structures. These can be zero-, first- and second order.

Multivariate data analysis is often required and this can take the form of Principle Component Analysis (PCA) and PARAFAC. These methods are applied to various data sets. The different measurement signals or derivatives are combined using a multiblock strategy.

References

Guo, C., Xin, L., Dong, Y. et al. Dielectric Properties of Yogurt for Online Monitoring of Fermentation Process. Food Bioprocess Technol 11, 1096–1100 (2018) (Article).

Hills, A.E. (2010) Spectroscopy in Biotechnoogy Research and Development. In: Encyclopedia of Spectroscopy and Spectrometry (2nd edt.) Academic Press.  pp. 2662-2667 (Article) . 

Liu, Y. C., Wang, F. S., & Lee, W. C. (2001). On-line monitoring and controlling system for fermentation processes. Biochemical engineering journal7(1), pp. 17-25.

Puxeu, M., Andorra, I. & De Lamo-Castellví, S. (2015) Monitoring Saccharomyces cerevisiae Grape Must Fermentation Process by Attenuated Total Reflectance Spectroscopy. Food Bioprocess Technol 8, pp. 637–646 (Article

 

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