Process Analytical Technology (PAT) in Fermentation

In Situ Monitoring of Biomass, Cell Growth and Cell Agglomeration

Real-time monitoring of the cell population within a bioreactor provides invaluable information regarding biomass concentration and growth kinetics that can be essential for detailed characterization and control of the fermentation process. Additionally, real-time information related to cell morphology, aggregation, and floc size have also been documented and shown to aid in the understanding and optimization of bioprocesses.

Process Analytical Technology (PAT) in FermentationDirect correlation to biomass concentration

The quantity of biomass in the fermenter directly relates to the productivity of the reactor – whether the product is the biomass itself or a specific enzyme or metabolite. Focused Beam Reflectance Measurement (FBRM®) provides a precise measurement of the particle population that is sensitive to changes in count, dimension and shape, which has been used effectively for direct correlation to biomass (McDonald et al, 2001; Pearson et al, 2004; Ge at al, 2005).

In complex cell systems, where flocculation and aggregation result in changes in both biomass concentration and changes in the dimensions of flocs and aggregates – simplistic measurements such as turbidity and optical density cannot capture the entire picture. FBRM® measurement – being sensitive to both number and dimension of cells and cell structures – can be used to elucidate the triggers and mechanisms of changes in morphology, as well as quantify the kinetics of these changes.

Real-time measurement and control of cell population
Recent papers [Ge et al (2005), Lei et al (2007)] have explored the fermentation of a self-flocculating yeast in efforts to maximize ethanol production.

Floc size is a critical parameter in controlling the ethanol yield. Ethanol yield was measured as a function of controlled floc size at 100, 200, 300 and 400 microns (Ge et al, 2005). A clear maximum in ethanol yield was reported when floc size was carefully controlled at 300 microns.

Control of the floc size is therefore an important part of ongoing investigation to optimize the process, maximize ethanol production, and improve tolerance of the yeast flocs to ethanol and temperature fluctuations.

Real-time monitoring and control of the floc size by FBRM® and manipulation of the agitation speed, was instrumental in maintaining a consistent floc size and enabling rapid optimization of additional bioprocessing parameters.

The application of advanced Process Analytical Technologies for fermentation and bioprocessing provides a clearer understanding of cell growth and cell morphology changes.

The fast and sensitive response of PAT tools provides real-time monitoring of physical characteristics of the cell population for optimization and control of fermentation processes to maximize yields and improve batch-to-batch consistency.

Monitoring the Kinetics of Fermentation
Optimization of fermentation requires real-time monitoring and control of critical process parameters – including the concentration of limiting nutrients and inhibiting metabolites. Within a bioreactor, most of the action is occurring within the cell itself. However, as the rate of transfer of key nutrients through the cell membrane is usually directly related to the generation of the desired products, monitoring the concentrations of nutrients and extracellular metabolites in the fermentation broth can directly track progress of the batch – from inoculation to an optimal harvest.

A wide range of spectroscopic techniques have been investigated – including near infrared (NIR), mid-infrared (Mid-IR), Raman, and UV-Vis. Mid-IR technology, in the form of FTIR-ATR (Fourier Transform Infrared detection using Attenuated Total Reflection), has been shown to have significant advantages for real-time monitoring of multiple key analytes in complex fermentation media.

The ease of use, high level of specificity and sensitivity, and the availability of robust probes that can be sterilized in place have generated considerable interest in Mid-IR for in situ bioprocess monitoring. The following examples highlight some of the areas in which Mid-IR has been successfully applied:

Provide in situ analysis of key components in the fermentation medium
Offline analysis of fermentation medium requires a battery of tests to determine the concentrations of nutrients, metabolites and biomass, performed at a high cost of time and resources and is not easily automated.  Advanced Process Analytical Technologies (PAT) involving measurements such as Mid-IR offer the ability to carry out the analyses of many of these key components inline and in real time.

Track the critical parameters in the fermentation process from inoculation to final yield
With Mid-IR, multiple analytes can be tracked in real time throughout a fermentation process. Real-time monitoring allows the detection of critical information without delay – such as the transition from lag to exponential growth, the accumulation of inhibiting metabolites, or the deficiency of key nutrients.

In an example of the advanced application of Process Analytical Technology, Kornmann et al. (2004) used in situ ReactIR™ FTIR to simultaneously monitor six analytes, and applied an adaptive control strategy to maximize yield of the desired product and reduce batch-to-batch variability.

Measurements of key nutrients and metabolites – ethanol, acetate, fructose, ammonium, and phosphate – were monitored along with the desired product – gluconacetan. The process was designed with consecutive fed-batch fermentations of Gluconacetobacter xylinus, in which the bacteria was first cultivated on a feed of ethanol, fed at a controlled rate to maintain a constant level of acetate in the bioreactor. Ethanol feed increased exponentially with increasing biomass, maximizing biomass growth while avoiding ethanol inhibition. Once dissolved oxygen became a limiting factor, a fructose feed was used to maximize gluconacetan production.

The ability to measure the concentration of multiple key analytes simultaneously, using in situ Mid-IR, has been described in terms of providing a “metabolic snapshot”[ Sivakesava et al, 2001]. Tracking this metabolic fingerprint in real time throughout the batch provides an insight and a route to bioprocess optimization that cannot be realized with offline sample analysis.

The fast and sensitive response of Mid-IR is capable of providing real-time monitoring of multiple analytes for more efficient characterization and elucidation of the fermentation process. In situ monitoring of critical process variables will also enable real-time optimization and control of fermentation processes for maximized yield and reduced batch-to-batch variability.

See this and more recently reported applications in fermentation and bioprocessing in the new white paper: Process Analytical Technology (PAT) for Biotech.