VG Prima dB Fermentation Gas Analysis Application Summary

Introduction

Fermentation is where chemical changes are brought about using microorganisms, e.g., for production of pharmaceuticals, food additives and animal feedstuffs. Respiration is the process whereby an organism oxidizes food to produce energy. An important parameter is the respiratory quotient RQ.

RQ = Carbon dioxide Evolution rate (CER) / Oxygen Uptake Rate (OUR)
CER = %vol of CO_2out x FLOW_out - %vol CO_2in x FLOW_in
OUR = %vol of O_2in x FLOW_in - %vol O_2out x FLOW_out

Therefore RQ =   (%vol of CO_2out x FLOW_out - %vol CO_2in x FLOW_in) / 
                       (%vol of O_2in x FLOW_in - %vol O_2out x FLOW_out)

The accurate determination of RQ relies on determination of the ratio of the flows in and out of a fermentor (ref 1). This ratio is easily determined with the VG Prima dB Process Mass Spectrometer, which measures Nitrogen and Argon in addition to Oxygen and Carbon Dioxide - the ratio of Nitrogen in the feed gas to Nitrogen in the off-gas can be used to make this very important flow correction. Argon can be used instead of nitrogen for fermentations involving Nitrogen Fixation.

Note the above flow correction cannot be done just using discrete analyzers (NDIR for CO₂, paramagnetic detector for O₂), and as pointed out by van der Aar, et al (ref 1), the possible errors in RQ due to lack of flow correction can be very large (e.g. greater than 10 %).

RQ can be determined with the Prima GasWorks software using derived value calculations.

VG Prima dB Performance Specification

It is extremely important to measure the changes in gas concentrations precisely. The Prima magnetic sector mass spectrometer analyzer is ideally suited to the analysis, and the unit has established itself as pre-eminent gas analyzer for this application.

Analysis precision is shown below:

Precision is the standard deviation observed over 24 hours.

Analysis time is 5 seconds for measurement of N₂, O₂, Ar and CO₂ plus 5 seconds flushing time. An additional 3 seconds is required for measurement of Methanol and Ethanol. This yields a total analysis time per stream of 10 seconds (or 13 seconds including Methanol and Ethanol).

Typical recalibration interval is one month.

For performance estimate for other components (e.g., sulfur compounds, ammonia, water, other alcohols, acids) please consult Thermo ONIX.

System Configuration

The vast majority of fermentation applications require off gas analysis from multiple fermentors, together with the air feed to these fermentors. The VG Prima dB uses our unique RMS Rapid Multistream Sampler, which has been field proven for over 10 years. Available in 32-way or 64-way versions, it is covered by a 3-year warranty.

Sample Conditioning

The VG Prima dB requires a sample flow of 0.1 to 1 l/min at atmospheric pressure, which should be verified by rotameter. The maximum and minimum pressures that should be applied at the RMS are 2 and 0.8 bar absolute respectively.

Where analysis of alcohols is required it is recommended that heated (80° C) PTFE tubing be used between the fermentor and the RMS.

To protect the Prima analyzer against blockage by liquids and dust, a two-filter system is used. Typically this comprises a 1 micron glass filter followed by a membrane filter.

Summary

The VG Prima dB provides precise determination of RQ plus the measurement and computation of a number of other important metabolic parameters.

• Verify absence of contamination pre-inoculation      
• Verify absence of contamination during sterility hold tests (using low aeration rates to increase sensitivity)      
• Periodically carry out a full mass scan to ?fingerprint? normal, abnormal and contaminated fermentations      
• Identify new molecular species that can be used for information and for control feedback      
• Produce complex measurement outputs suitable for training neural nets.      
• Allow for the computation of biomass levels, growth rate, product formation rate and product concentration at unsurpassed accuracies      
• Directly quantify volatile substrate or product molecules such as ethanol, ethyl acetate, methanol and diacetyl      
• Detect and quantify earlier than by any other method the tiny amounts of oxygen uptake and CO₂ evolution during the fermentation lag phase      
• Detect and quantify the tiny amount of CO₂ uptake during the germination of fungal spores      
• Quantify carbon dioxide concentrations and bicarbonate buffer status, in conjunction with pressure measurements (especially important when cells of multicellular organisms such as plants, animals and insects are fermented)      
• Calculate mass aeration flow without need for extra meters. (Derived from the dilution rate of a very accurate inert gas injection)      
• Identify species that might lead to environmental problems when the process is scaled up (hydrogen sulfide, for example)      
• Do all of the above with a rugged, plug-in-and-go, software programmable package.

References

1. P.C. van der Aar, A.H. Stouthamer and H.W. van Verseveld, Possible misconceptions about O2 consumption and CO2 production measurements in stirred microbial cultures, Journal of Microbiological methods 9, (1989) 281-286.