Online analytics of pectic compound degradation in small-scale using $\textit Ustilago Maydis$
- Online-Analytik des Abbaus von Pektinbestandteilen in Kleinkultur mittels $\textit Ustilago Maydis$
Müller, Markus Jan; Büchs, Jochen (Thesis advisor); Feldbrügge, Michael (Thesis advisor)
Aachen (2019, 2020)
Dissertation / PhD Thesis
Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2019
In the spirit of a circular bioeconomy, pectin-rich biomass waste streams represent a valuable substrate for a biorefinery process. Particularly rich in pectin are, e.g., citrus peel, apple pomace or sugar beet pulp. For efficient degradation, the applied organism should produce a diverse mixture of cellulases, hemicellulases and pectinases. This is essential for efficient and economic valorization of the pectin fraction. Pectin is mainly composed of galacturonic acid monomers. It is not metabolized by well-established organisms, such as <i>Saccharomyces cerevisiae</i>. Thus, a microbial production system capable of metabolizing pectin and producing high-value products is required. However, due to its complexity, analysis of pectin is challenging. Thus, new online monitoring tools for bioprocesses are required.A method for the online determination of residual substrate was developed for the growth of <i>Ustilago maydis</i> on pectic compounds. Cultivations were carried out in shake flasks with online monitoring of the respiration activity. The residual galacturonic acid concentration during cultivation was predicted based on the overall oxygen consumption and verified by offline sampling. Afterwards, this method was extended towards polygalacturonic acid, pectin and sugar beet pulp, which are challenging to quantify. The influence of heterologous expression of an exo-polygalacturonase on <i>U. maydis</i> growth on polygalacturonic acid was investigated. Co-cultivation of <i>U. maydis</i> strains expressing endo- and exo-polygalacturonases were conducted on polygalacturonic acid and compared to axenic cultivation. Co-cultivations on pectin and sugar beet pulp were supplemented with external cellulases and pectinases.Prediction of the residual substrate concentration based on the overall oxygen consumption was established and validated in small-scale. Based on this method, a co-culture of <i>U. maydis</i> strains expressing fungal endo- and exo-polygalacturonases was identified to be highly efficient in the metabolization of polygalacturonic acid. On pectin or sugar beet pulp, cultures required supplementation with external enzymes to convert half of the complex substrates. The introduced method can be applied to estimate the amount of consumed complex substrates which are currently challenging to quantify. This will support the development of consolidated bioprocesses.