Novel polyphosphate analytics for the development of biotechnological polyphosphate production

• Neue Polyphosphatanalytik für die Entwicklung biotechnologischer Polyphosphatproduktion

Christ, Jonas Johannes; Blank, Lars M. (Thesis advisor); Schwaneberg, Ulrich (Thesis advisor)

1. Auflage. - Aachen : Apprimus Verlag (2020)
Book, Dissertation / PhD Thesis

In: Applied microbiology 19
Page(s)/Article-Nr.: 1 Online-Ressource (xiii, 229 Seiten) : Illustrationen, Diagramme

Dissertation, RWTH Aachen University, 2020

Abstract

Phosphate (PO43-, Pi) is an irreplaceable nutrient in the human diet and is mined from Pi rock. Strategies for the recycling of Pi from unused Pi waste streams must be developed because global Pi rock reserves will be depleted in a few hundred years. Canola meal, in which Pi is bound as phytic acid, is generated as a side product during the deoiling of canola seed (Brassica napus) and constitutes such an unused Pi waste stream. Four project partners developed the ValuePP process to recycle Pi from canola meal into inorganic polyphosphate (polyP). PolyP is the linear polymer of Pi with a chain length n of 2 to ca. 1,000 P-subunits, is currently only produced chemically, and is used in food products. The ValuePP process began with a phytase treatment to release Pi from the canola meal. Pi was accumulated and intracellularly polymerized into polyP by Saccharomyces cerevisiae. PolyP-rich S. cerevisiae served as the substrate to produce polyP-rich yeast extract or pure biotechnologically synthesized (bio-)polyP. The overall goal of this Ph.D. thesis was to develop the latter three processes and methods for the analysis of polyP. An analytical polyP extraction was developed that extracts 40 % more polyP than the best literature method (5 h, five reaction tubes), takes 30 min, and requires one reaction tube. A colorimetric Pi assay was developed, which involved the addition of one reagent to the sample and took 2 min for color development (best literature method: 2 reagents, 30 min). An enzymatic total polyP assay was developed that fully quantified polyP (the best literature method excludes polyP2). Recently, the average polyP chain length could only be detected quantitatively by 31P nuclear magnetic resonance spectroscopy. Here, an enzymatic assay was developed for the determination of this parameter, which required only commonly available laboratory equipment. A cultivation strategy was designed to produce wild type S. cerevisiae with the so-far highest reported polyP content (28 % polyP as KPO3 in cell dry weight). A yeast extract fabrication process was developed to obtain the novel product polyP-rich yeast extract. A highly scalable process for the preparation of the first water-soluble food-grade bio-polyP (purity on a par with chemically produced polyP) was developed, too. Together with project partners, Pi was, for the first time, recycled from canola meal and upcycled into polyP-rich yeast extract and pure bio-polyP. Both products can be used as food additives. The phytate-reduced canola meal can be used in the Pi-controlled feeding of livestock. The German (2,700 t) and European (11,000 t) demand for polyPn ≥ 4 could be satisfied if one-third of the canola meal (2 * 106 t) that is annually produced in Germany would be subjected to the ValuePP process.