Two-dimensional fluorescence spectroscopy is presented as a new method for bioprocess monitoring. It covers a wide range of excitation and emission wavelengths and is a further development of the fluorescence measurements performed so far, which concentrated mainly on NAD(P)H culture fluorescence. Biogenic fluorophores such as proteins, coenzymes, and vitamins can simultaneously be detected qualitatively and quantitatively inside and outside the cells. This optical method is noninvasive, suitable for in vivo measurements. One whole spectrum (excitation, 250-550 nm; emission, 260-600 nm) with the described parameters is performed within 1 min, which allows an almost continuous monitoring of the bioprocess. The technique is ideal for on-line, in situ measurements via fiber optical systems. Results are presented for cultivations of Claviceps purpurea, Escherichia coli, Saccharomyces cerevisiae, and Sphingomonas yanoikuyae. Cell growth and the metabolism of the cells (changes from aerobic to anaerobic conditions and uncoupling of the oxidative phosphorylation) could be detected.

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The fluorescence properties of one chemically and seven biologically produced polyhydroxyalkanoic acid were investigated as film castings and in living cells respectively after staining with Nile red. All these polyesters show a similar fluorescence behaviour, revealing a clear fluorescence maximum at an excitation wavelength between 540 nm and 560 nm and an emission wavelength between 570 nm and 605 nm. This could be shown by the use of two-dimensional fluorescence spectroscopy and flow cytometry. The examination of native poly(3-hydroxybutyric acid), poly(3HB), granules isolated from cells of Ralstonia eutropha H16 showed that the addition of 6.0 micrograms Nile red is necessary for total staining of 1.0 mg granules. The fluorescence intensity at an excitation wavelength of 550 nm and an emission wavelength of 600 nm showed high correlation to the poly(3HB) concentration of grana suspensions at different grana concentrations. These results and the staining of cell suspensions during cultivation experiments revealed that Nile red has a high potential for the quantitative determination of hydrophobic bacterial polyhydroxyalkanoic acids.

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The production of a mutant green fluorescent protein (S65TGFP), controlled by the maltose inducible glucoamylase promoter, was followed in situ in fed-batch cultures of recombinant Aspergillus niger using multi-wavelength fluorescence spectroscopy. Disturbance of quantitative product analysis by interfering fluorescence signals was resolved by using a set of defined combinations of excitation and emission wavelengths (lambda(ex)/lambda(em)). This technique resulted in excellent linearity between on-line signal and off-line determined S65TGFP concentrations. Spore germination was detectable in situ by monitoring the back scattered light intensity. Moreover, flavin-like fluorophores were identified as the dominating fungal host fluorophores. The time-dependent intensity of this fluorophore, potentially fungal flavin-containing oxidoreductase(s), did not correlate with the biomass concentration but correlated well with the fungal metabolic activity (e.g. respiratory activity). Other fluorophores commonly found in microbial cultures such NADH, pyridoxine and the aromatic amino acids, tryptophan, phenylalanine and tyrosine did not contribute significantly to the culture fluorescence of A. niger. Thus, multi-wavelength fluorescence spectroscopy has proven to be an effective tool for simultaneous on-line monitoring of the most relevant process variables in fungal cultures, e.g. spore germination, metabolic activity, and quantitative product formation.