Natural-abundance I3C NMR spectroscopy has been used to study the metabolism of the L-lysine-producing bacterium, Brevibacteriumflavum. Relationships of biomass formation, precursor uptake, and product excretion, as a function of culture medium, oxygen supply and specific cell membrane permeability, were rapidly determined using 67.89-MHz 13C NMR. The induction of lysine production throughout the growth cycle was studied. Intracellular and extracellular levels of free metabolites and unconsumed precursor were quantitatively measured as a function of growth culture conditions. Limited availability of oxygen resulted in accumulation and excretion of unfavorable products: lactate, succinate, alanine and valine. However, under optimal aeration conditions Llysine was the sole metabolite detected extracellularly. Various important long-lived intermediates and storage compounds were detected in the intact cells (by NMR measurements). Carbon resonances of carbohydrates and amino acids were resolved and easily identified. Of particular interest are those of trehalose carbons, a storage carbohydrate.Natural-abundance 3C NMR spectroscopy seems most suitable for biotechnological processes where high concentrations of intermediates and end-products can be observed. We anticipate that this approach will be employed to screen overproducing bacterial strains.The biosynthesis of amino acids in microorganisms is normally regulated to meet the organism's needs. Pertubations of metabolism can result in the overproduction of specific amino acids. The most direct and general method for overproduction is the genetic removal of feedback control [l, 21. A great variety of bacteria have been reported to excrete excessive amounts of L-lysine, including auxotrophic and regulatory mutants [3]. The objective of our study is to apply 13C and I5N NMR spectroscopy for metabolic studies and to develop methods for large-scale preparation of natural compounds labelled with stable isotopes [4 -81. We have already shown how in vivo 15N NMR spectroscopy of highly enriched "N-labelled substrates unravelled various problems of nitrogen metabolism in a rapid and non-destructive method [4], although this approach is most suitable for monotoring only nitrogenous compounds.Here we apply natural-abundance 13C NMR to study the regulation and overproduction of L-lysine by bacterial fermentation employing Brevibacteriumflavum. The ability of the cells to produce and excrete L-lysine is mainly due to the deregulation of aspartokinase (converting aspartate to 4-phospho-aspartate which is converted by aspartate-semialdehyde dehydrogenase to aspartic semialdehyde) and the defectiveness of homoserine dehydrogenase (transforming aspartic semialdehyde to homoserine) in this mutant (see Fig. 1). Problems such as oxygen supply, alternative metabolic routes, glucose transport rate and activities of the glycolytic and Krebs cycle enzymes were investigated using NMR techniques. Limited oxygen supply results in accumulation and excretion of unfavourable products [9, lo]. T...

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The enzyme-linked immunosorbent assay is suggested as a reliable, sensitive, and highly specific method for the identification and enumeration of Azospirillum brasilense Cd. As few as 105 CFU/ml can be practically identified by this method. At higher bacterial numbers, sensitivity increased linearly up to 5 x 108 CFU/ml, yielding useful standard curves. No cross-reaction was found either with different closely related Azospirillum strains or with other rhizosphere bacteria. The method allows for a specific identification of A. brasilense Cd. both in pure cultures and in mixtures with other bacterial species, even when the colony morphology is variable. The method was successfully applied to assess the degree of root colonization on various cereals by A. brasilense Cd.

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