Analysis of Fermentation Broths

The infrared method has the convenience of no weighing being required, nor are any accurate volumes needed. It can therefore be used for very small samples. 

The configuration of alkenes can be determined from the frequency of their C—bending vibrations. Cfs-isomers absorb between 840 and 700cm while trans-isomers absorb between 1000 and 930 cm The latter band can be used as the basis for an analytical method. 

It is normal to hydrolyse the triglyceride mixture to glycerol and a mixture of fatty acids, and the latter are then converted to their methyl esters. It is this methyl ester mixture which is analysed in this case. 

Both of these geometric isomers absorb strongly at 1163 cm which represents the C—O stretching frequency from the ester group. The ds-isomers absorb weakly at 965 cm i, while the traHS-isomers absorb strongly at this frequency. It can be shown that the following relationship applies  

Equation (5.9) represents a straight line of gradient K and with a y-axis intercept of /, if a plot of % trans-cont tii versus the ratio of the absorbances at the two frequencies is produced. You will need to prepfe a series of solutions of known trans-compositions, measure the ratios of the absorbances and then plot the % trans-contonts against these values. 

---

Finally, ion chromatography is sometimes used for process applications, allowing for the tracking of the manufacturing process in order to optimize process variables and to allow for better control of process parameters. One example of this is the application of ion chromatography to the analysis of fermentation broths. Here ion chromatography is used both to measure the level of ionic nutrients in the fermentation broth in order to control the fermentation process and also to measure the level of fermentation by-product ions which may be indicative of problems with the fermentation process. 

W.E. Rich, R.D. Rocklin, and D.G. Gillen, Analysis of Fermentation Broths for Carbohydrates, Metals, Organic Acids and Inorganic Ions by Ion Chromatography , Presentation Pittsburgh Conference 1986. Atlantic City, N.J., USA. 

Traditional methods of pharmaceutical analysis involve a series of multiple steps. For example, the identification of natural products traditionally involves the scale-up of fermentation broths, solvent extraction, liquid/liquid or column fractionation, chromatographic fraction collection, and spectroscopic analysis (usually NMR) of the individual components. Figure 5.2 illustrates the integration of these bench-scale steps into a dedicated LC/MS/MS system (Lee et al., 1997). Integration provides unique and powerful advantages for the on-line identification of natural products (Kerns et al., 1994 Ackermann et al., 1996a). Experiments that once required 2 weeks to perform with traditional approaches are now performed in half a... 

Metabolite analysis of ultrafiltered broth from a high-yielding CPC fermentation showed, in addition to CPC, the accumulation of penicillin, DAOC, and DAC... 

Marko-Varga, G. Buttler, T. Gorton, L. Olsson, L. Durand, G. Barcelo, D. Qualitative and quantitative carbohydrate analysis of fermentation substrates and broths by ligand chromatographic techniques. J. Chromatogr., A 1994, 665, 317 - 332. 

After the batch culture using chloroform-treated granules, 5 mL of fermentation broth was withdrawn for analysis. In addition, 5 mL of 80 g COD/L fresh culture medium with the same concentration of nutrients as the culture medium described above was added to the fermentation broth to start another batch. For the fed-batch culture without pH control, the pH was not adjusted at the beginning of each culture. For the fed-batch culture with pH adjustment, the pH was adjusted to about 7, after 5 mL of fi esh culture medium was added into the serum bottles, and then a new batch was started. 

Figure 1441. Sampling from a fermenter for on-line analysis (after [366]). 1. Direct removal of fermentation broth (analyte A) 2. indirect sampling by ultrafiltration, dialysis, electrodialysis, per-vaporation, providing an analyte A of proportional concentration, normally diluted 3. indirect sampling by extraction of fermentation broth by external buffer 4. in situ measurement by means of an enzyme electrode or using a sterile housing with inserted electrode. F = fermenter, W = waste.

Additionally, a variety of analytical equipment and techniques that allow the examination of small- (and micro-) scale microbial cultures and their products have become available. Examples include near infrared and Fourier transform infrared spectroscopy, which offer the ability for in situ detection of specific compounds in fermentation broth [22]. However, sensitivity and the required sample volumes pose serious obstacles that still have to be overcome. Another alternative is offered by sensitive pyrolysis mass spectroscopy, which was demonstrated to be suitable for quantitative analysis of antibiotics in 5-pl aUquots of fermentation broth when combined with multivariate calibration and artificial neural networks [91]. The authors concluded that a throughput of about 12,000 isolates per month could be expected. Furthermore, standard chromatographic methods such as gas chromatography or high-performance liquid chromatography, possibly in combination with mass spectroscopy (MS) for detection, can provide simultaneous quantitative detection of many metabolic products. 

LC-MS finds wide application in the analysis of compounds that are not amenable to GC-MS, i.e. compounds that are highly polar, ionic and thermo-labile, as well as (bio)macromolecules. In environmental applications, LC-MS is applied, often in combination with off-line or on-line solid-phase extraction, to identify pesticides, herbicides, surfactants and other environmental contaminants. LC-MS plays a role in the confirmation of the presence of antibiotic residues in meat, milk and other food products. Furthermore, there is a substantial role for LC-MS in the detection and identification of new compounds in extracts from natural products and the process control of fermentation broths for industrial production of such compounds, e.g. for medicinal use. LC-MS technology is also widely applied in the characterization of peptides and proteins, e.g. rapid molecular-mass determination, peptide mapping, peptide sequencing and the study of protein conformation and noncovalent interactions of drugs, peptides and other compounds with proteins and DNA. However, the most important application area... 

Many natural products are charged substances, and can be isolated by IEC methods. Dufresne has published a comprehensive review describing various resins and column operating conditions applicable to purification of natural products from fermentation broths or crude extracts.168 Among natural products, antibiotics are of special interest due to their widespread use in humans and animals. Sample cleanup by IEC prior to analysis by other LC methods for quantitative determination of antibiotics in biological fluids is frequent.I69171 Also, IEC followed by TLC appears useful for the quantitation of fumonisin Bl, a mycotoxin found in agricultural products.172... 

Goodacre, R. Trew, S. Wrigley-Jones, C. Saunders, G. Neal, M. J. Porter,N. Kell, D. B. Rapid and quantitative analysis of metabolites in fermentor broths using pyrolysis mass spectrometry with supervised learning Application to the screening of Penicillium chrysogenum fermentations for the overproduction of penicillins. Anal. Chim. Acta 1995,313, 25 43. 

The natural product panepophenanthrin (6/1-170), isolated in 2002 from the fermented broth of the mushroom strain Panus radus IFO 8994 [90], is the first example of an inhibitor of the ubiquitin-activating enzyme [91]. Retrosynthetic analysis based on a biomimetic analysis led to the conjugated diene 6/1-172 by a retro-Diels-Alder reaction via the hemiacetal 6/1-171. Further disconnections of 6/1-172 produces the vinyl stannane 6/1-173 and the vinyl bromide 6/1-174 [92]. 

Recognizing the need for a more economically and environmentally friendly citric acid recovery process, an adsorptive separation process to recover citric acid from fermentation broth was developed by UOP [9-14] using resin adsorbents. No waste gypsum is generated with the adsorption technique. The citric acid product recovered from the Sorbex pilot plant either met or exceeded all specifications, including that for readily carbonizable substances. An analysis of the citric acid product generated from a commercially prepared fermentation broth is shown in Table 6.2, along with typical production specifications. The example sited here is not related to zeolite separation. It is intent to demonstrate the impact of adsorption to other separation processes. 

The Charm Test was initially applied to the analysis of 3-lactam residues in milk although its application to the analysis of body, fluids, meat extracts, and fermentation broths was indicated. There appears to be no rationale why this basic procedure cannot be applied to all types of matrices (water, soil, animal feeds, premixes). ... 

Environmental process analysis requires the characterization of chemical process and waste streams in order to evaluate their environmental abuse potential and treatability characteristics. An integral part of this analysis, as well as environmental fate determinations, is the isolation of organic compounds and metabolic products from very complex matrices such as waste water effluents, process streams, biological reactors, and fermentation broths. Generally, the organics involved are fairly polar, water-soluble compounds that must be ex-... 

The second half of the 1990s saw an increase in the use of dialysis (as a liquid-liquid extraction procedure). Its main advantage is the possibility of operating in an automatic mode by coupling a dialysis unit with an automatic injector, as demonstrated not only in HPLC analysis (17) but also in flow-injection determinations of reducing sugars in wines (18) and alcoholic fermentation broths (19). 

---