Stoichiometry and kinetics of single and mixed substrate uptake in Aspergillus niger.

In its natural environment, the filamentous fungus Aspergillus niger grows on decaying fruits and plant material, thereby enzymatically degrading the lignocellulosic constituents (lignin, cellulose, hemicellulose, and pectin) into a mixture of mono- and oligosaccharides. To investigate the kinetics and stoichiometry of growth of this fungus on lignocellulosic sugars, we carried out batch cultivations on six representative monosaccharides (glucose, xylose, mannose, rhamnose, arabinose, and galacturonic acid) and a mixture of these. Growth on these substrates was characterized in terms of biomass yields, oxygen/biomass ratios, and specific conversion rates. Interestingly, in combination, some of the carbon sources were consumed simultaneously and some sequentially. With a previously developed protocol, a sequential chemostat cultivation experiment was performed on a feed mixture of the six substrates. We found that the uptake of glucose, xylose, and mannose could be described with a Michaelis-Menten-type kinetics; however, these carbon sources seem to be competing for the same transport systems, while the uptake of arabinose, galacturonic acid, and rhamnose appeared to be repressed by the presence of other substrates.

Development of tools for quantitative intracellular metabolomics of Aspergillus niger chemostat cultures.

In view of the high citric acid production capacity of Aspergillus niger, it should be well suited as a cell factory for the production of other relevant acids as succinic, fumaric, itaconic and malic. Quantitative metabolomics is an important omics tool in a synthetic biology approach to develop A. niger for the production of these acids. Such studies require well defined and tightly controlled cultivation conditions and proper rapid sampling, sample processing and analysis methods. In this study we present the development of a chemostat for homogeneous steady state cultivation of A. niger, equipped with a new dedicated rapid sampling device. A quenching method for quantitative metabolomics in A. niger based on cold methanol was evaluated using balances and optimized with the aim of avoiding metabolite leakage during sample processing. The optimization was based on measurements of the intermediates of the glycolysis, TCA and PPP pathways and amino acids, using a balance approach. Leakage was found to be absent at -20 °C for a 40 % (v/v) methanol concentration in water. Under these conditions the average metabolite recovery was close to 100 %. When comparing A. niger and Penicillium chrysogenum metabolomes, under the same cultivation conditions, similar metabolite fingerprints were found in both fungi, except for the intracellular citrate level which is higher for A. niger.

Production of 2-butanol through meso-2,3-butanediol consumption in lactic acid bacteria.

2-Butanol has been an issue of industries in many areas, for example, biofuel production (as an advanced alternate fuel), fermented beverages, and food (as taste-altering component). Thus, its source of production, the biological pathway, and the enzymes involved are of high interest. In this study, 42 different isolates of lactic acid bacteria from nine different species were screened for their capability to consume meso-2,3-butanediol and produce 2-butanol. Lactobacillus brevis was the only species that showed any production of 2-butanol. Five of ten tested isolates of L. brevis were able to convert meso-2,3-butanediol to 2-butanol in a synthetic medium (SM2). However, none of them showed the same capability in a complex medium such as MRS indicating that the ability to produce 2-butanol is subject to some kind of repression mechanism. Furthermore, by evaluating the performance of the enzymes required to convert meso-2,3-butanediol to 2-butanol, that is, the secondary alcohol dehydrogenase and the diol dehydratase, it was shown that the latter needed the presence of a substrate to be expressed.