2-phenylethanol (2PE) is a valuable fragrance compound which gives a rose-like aroma. As such 2PE is one the highest used fragrances globally. While 2PE is predominantly produced from petrochemical resources, there is a growing market for a naturally derived alternative for food products. 2PE from natural sources is priced so highly due to limited supply from rose petals. Recently, a few reports have demonstrated the production in yeasts through both the de novo production from glucose and ex novo biosynthesis with L-phenylalanine as a precursor. While these are promising most of the yeasts used can only produce low titres under optimal conditions, and the fermentation still appears to be too expensive. In this investigation the wine yeast M. pulcherrima was selected to be assessed for 2PE production. M. pulcherrima is known to produce 2PE in small titres in wine production though has yet to be explored as a platform for this product. M. pulcherrima has several advantages as a yeast platform, in that it produces a range of antimicrobials which can ward off invasive species, allowing for less sterile control in any large scale fermentation. M. pulcherrima was demonstrated to be able to produce 2PE in high titres in the batch mode through de novo synthesis of glucose, producing up to 1 g/L in shake flasks on the lab scale. Arabitol was also observed in the fermentation broth and was produced up to 20 g/L. The fermentation was then scaled up to 2L in batch mode. From these experiments, up to 700 mg/L of 2PE was produced. This is substantially more than any other yeast in the literature to date. Though when xylose or glycerol was present then 2PE production was severely limited. M. pulcherrima was also demonstrated to be able to produce 2PE by bioconversion from phenylalanine up to 1.5 g/L. This 2PE concentration is suggested to be threshold of toxicity to M. pulcherrima by the toxicity study. The production of 2PE could be increased substantially by introducing an absorbent into the process.Liquid solvents and solid adsorbents were assessed to increase 2PE production, used as in-situ 2PE adsorbents. Oleyl alcohol was found to be a good solvent for in-situ extractive solvent in M. pulcherrima culture and increase the production to 3.3 g/L which is higher than 2PE tolerance threshold of the yeast. Activated carbon was also found to be an excellent 2PE adsorbents, with maximum Langmuir adsorption capacity up to 0.807 g/g. 2PE synthesis with activated carbon as an in-situ adsorbent can increase 2PE production to 14 g 2PE/L. Finally, the process was scaled to 2L and run in batch, continuous and semi-continuous modes. This study demonstrates that not only is M. pulcherrima a viable organism to produce 2PE but it has the potential to be scaled up and run in a more cost effective semi-continuous mode when coupled to a continuous extraction technique.
|Date of Award||11 Jan 2017|
|Sponsors||Government of Thailand|
|Supervisor||Chris Chuck (Supervisor) & Roderick Scott (Supervisor)|