Factors

If good control of a fermentatation is to be obtained, then all chemical factors which can influence growth and product formation ought to be continuously monitored. This ideal situation has not yet been achieved but a number of techniques are currently being developed.

Ion-specific sensors

Ion-specific sensors have been developed by Orion Research and Radiometer to measure NHJ, Ca2+, K+, Mg2+, PO3 , SO2", etc. (Orion Research Inc., 11 Blackstone Street, Cambridge, MA, U.S.A.; Radiometer — Aagard Nielson and Schroder, Emdrupvej 72, Copenhagen-NV, Denmark). The response time of these electrodes varies from 10 seconds to several minutes depending on the concentration of the ion species, the composition of the sample, etc. However, none of these probes is steam sterilizable.

Enzyme and microbial electrodes

Enzyme or microbial cell electrodes can be used in some analyses. A suitable enzyme or microbial cell which produces a change in pH or forms oxygen in the enzyme reaction is chosen and immobilized on a membrane held in close contact to a pH or oxygen elec trode. Unfortunately, the oxygen demand of an enzyme may restrict the maximum substrate concentration which might be detected in a medium. Enfors (1981) overcame this problem for glucose determination by co-immobilizing glucose oxidase and catalase. More recently, it has been possible to use a ferrocene derivative as an artificial redox carrier to shuttle electrons from glucose oxidase to a carbon electrode, thus making the device largely independent of oxygen concentrations (Higgins and Boldot, 1992). Enzyme electrodes are also commercially available to monitor cholesterol, triglycerides, lactate, acetate, oxalate, methanol, ethanol, creatine, ammonia, urea, amino acids, carbohydrates and penicillin (Higgins et al., 1987; Luong et al., 1988; Higgins and Boldot, 1992).

Ideally, an electrode that can be inserted into a fermenter and steam sterilized is required, but none is yet available which can be used in this manner, even when using enzymes which are stable at high temperatures which can be obtained from thermophilic microorganisms. Hewetson et al. (1979) prepared sterilized penicillinase electrodes by assembling sterile components or by standing assembled components in chloroform before placing in a fermenter.

Near infra-red spectroscopy

Hammond and Brookes (1992) have described the development of near infra-red spectroscopy (NIR; 460-1200 nm) for rapid, continuous and batch analysis of components of fermentation broths. In samples from an antibiotic fermentation, they used NIR absorbance bands to simultaneously estimate fat (in the medium), techoic acid (biomass) and antibiotic (the product). Fat analysis has been made possible with a fibre optic sensor placed in situ through a port in the fermenter wall. The assay time for an antibiotic has been reduced from 2 hours to 2 minutes. A method has also been developed to measure alkaline protease production in broths. Vaccari et al. (1994) have used this technique to measure glucose, lactic acid and biomass in a lactic acid fermentation.

Mass spectrometers

The mass spectrometer can be used for on-line analysis since it is very versatile and has a response time of less than 5 seconds for full-scale response and taking about 12 seconds for a sample stream. It allows for monitoring of gas partial pressures (02, COz, CH4, etc.), dissolved gases (02, C02, CH4, etc.) and volatiles (methanol, ethanol, acetone, simple organic acids, etc.). Heinzle et al. (1981) combined a data processor with a mass spectrometer equipped with a capillary gas inlet to measure gas partial pressures and a membrane inlet to detect dissolved gases and volatiles. It is therefore possible to multiplex this type of system to analyse several fermenters sequentially. Merck and Company Inc. have installed an integrated gas- and liquid-phase analysis system incorporating a mass spectrometer to sample a number of fermenters (Omstead and Greasham, 1989). The availability of low cost mass spectrometers at approximately $15,000 does make their use in a laboratory or pilot plant financially feasible as an alternative to oxygen and carbon dioxide analysers, as well as analysing for a range of other gaseous and volatile compounds.

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