Gerit Tolborg

The growing concern over synthetic colorants in our food has led to a strong interest in natural coloring alternatives. So far, these natural colorants are extracted from fruits or roots and their production has the disadvantage of being dependent on the supply of the extraction source. To overcome this limitation, the production of colorants by microorganisms like filamentous fungi is at the focal point of interest. Fungi are known to naturally synthesize and excrete pigments within an extraordinary range of colors. One known example already used in the Asian food industry are “Monascus pigments”. Monascus pigments belong to of the chemical class of azaphilones and are produced by the fungus Monascus. Unfortunately, their production is associated with undesired mycotoxins, and these pigments are not approved for human consumption in Europe and the USA.

Another Monascus pigment producing fungi is called Talaromyces atroroseus. So far, no mycotoxin production has been detected in T. atroroseus which makes it a promising alternative producer of red azaphilone pigments. Some of the pigments produced by T. atroroseus are known, but the majority has not been structurally characterized.

The major aim of this thesis is to develop a fermentation process using T. atroroseus for production of red azaphilone pigments. For that, different media compositions were tested and it was shown that certain combinations of nutrients supported red pigment production better than others. Also, process parameters of the bioreactor were investigated and it was demonstrated that pigment production of T. atroroseus was heavily influenced by pH and temperature. It was shown that there is a negative correlation between growth of the fungus and red pigment production, since no tested pH value supported both equally well. However, in order to promote both, a cultivation method with a pH switch from pH 3 to pH 4.5 was developed. By employing this method, pigment production by T. atroroseus could be increased by 35%. Furthermore, in collaboration with DTU Natural Product Chemistry the pigments produced by T. atroroseus were investigated. As a result of this collaboration, a novel series of azaphilone pigment were characterized. These have been named atrorosins and have different amino acids as side chains in the pigment core structure.

Finally, a fermentation process tailoring pigment production in T. atroroseus was designed to selectively produce only one atrorosin at a time. This cultivation design made product recovery faster and easier and at the same time also increased the pigment yield. Final pigment titers of 0.9 g/L could be obtained.

Altogether, the work presented in this thesis, has established a stable, quantifiable and high yielding cultivation process for T. atroroseus and sets therewith the cornerstone in implementing pigment production in filamentous fungi.