Elevated production of the aromatic fragrance molecule, 2-phenylethanol, using Metschnikowia pulcherrima through both de novo and ex novo conversion in batch and continuous modes

Abstract

Background: 2-phenylethanol (2PE) is a fragrance molecule predominantly used in perfumes and the food industry. It can be made from petrochemicals inexpensively, however, this is unsuitable for most food applications. Currently, the main method of production for the bio-derived compound is to extract the trace amounts found in rose petals, which is extremely costly. Potentially fermentation could provide an inexpensive, naturally sourced, alternative.

Results: In this investigation, 2PE was produced from the yeast Metschnikowia pulcherrima, optimised in flasks before scaling to 2 L batch and continuous operation. 2PE can be produced in high titres under de novo process conditions with up to 1500 mg L^-1 achieved in a 2 L stirred bioreactor. This is the highest reported de novo titre to date, and achieved through high sugar loadings coupled with low nitrogen conditions. The process successfully ran in continuous mode also, with a concentration of 650 mg L^-1 of 2PE being maintained. The 2PE production was further increased by the ex novo conversion of phenylalanine and semi-continuous solid phase extraction from the supernatant. Under optimal conditions 14 000 mg L^-1 of 2PE was produced.

Conclusions: The work presented here offers a novel route to naturally sourced 2PE through a scalable fermentation with a robust yeast highly suited to industrial biotechnology. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Keywords: Adsorption; Biochemical engineering; Bioprocesses; Engineering; Green Engineering/Products; Industrial biotechnology.

Figure 1

Effect of pH on: (a) yeast biomass production; (b) glucose catabolism; (c) fructose consumption; and (d) 2‐phenylethanol in M. pulcherrima cultured in SGJ media at 20 °C, error bars are given as one standard deviation (n = 3).

Figure 2

Fermentation profiles of M. pulcherrima culture in shake flasks in SGJ media pH 4 at 15 °C, 20 °C and 28 °C (n = 3, error bars represent one s.d.).

Figure 3

Effect of glucose concentration on: (a) biomass production; (b) glucose uptake; (c) 2‐phenyl ethanol production; and (d) arabitol production for M. pulcherrima cultured at 20 °C, pH 4, in modified SGJ, with no fructose and varying glucose concentrations of 25 g L⁻¹ (Glu25), 50 g L⁻¹ (Glu50) and 75 g L⁻¹ (Glu75). The C/N ratio was kept constant for all glucose loadings.

Figure 4

Effect of N/C ratio on growth and production of 2PE and in SGJ media with no fructose at 20 °C, pH 4 over 7 days, error bars are given as one standard deviation (n = 3).

Figure 5

Growth, 2PE and sugar utilisation in semi‐continuous shake flask culture of M. pulcherrima with mean retention time (MRT) 3, 6, 9, 12 days (dilution rate 1/3, 1/6, 1/9 and 1/12 day^‐1, respectively). Semi‐continuous process started on day 3 (n = 3, error bars represent one s.d.).

Figure 6

Bioconversion of phenylalanine to 2PE with (a) 10 g L⁻¹ phenylalanine, (b) 20 g L⁻¹ phenylalanine, (c) 30 g L⁻¹ phenylalanine. (d) Liquid extraction of 2PE from cultures using oleyl alcohol, error bars are given as one standard deviation (n = 3).

Figure 7

Solid extraction of 2PE using activated carbon: (a) the sizing; (b) Langmuir isotherm plot of 2PE adsorption on powdered activated carbon (PAC). (c) 2PE production by biosynthesis from L‐phenylalanine with powdered activated carbon (PAC) as an in situ extractant in M. pulcherrima culture 25 mL in SGJ media pH 4 at 20 °C after 7 days (n = 3). (d) 2PE concentrations at equilibrium in different solvents with 2PE 10 mg and PAC 10 mg in 10 mL of solvents in leachability study.

Figure 8

Effect of aeration on the yeast biomass, 2PE and glucose catabolism for M. pulcherrima culture grown in SGJ media at pH 4, 20 °C in 2 L bioreactors.

Figure 9

Continuous de novo M. pulcherrima fermentation with (a) dilution ratio 1/3 d^‐1 (HRT 3 days) at 20 °C, pH 4, (b) dilution ratio 1/6 d^‐1 (HRT 6 days) at 20 °C, pH 4. The bioreactor was run in batch mode from start to day 7 and continuous mode from day 8.

Figure 10

Semi‐continuous ex novo M. pulcherrima fermentation: (a) system set‐up; (b) glucose consumption and biomass loading; and (c) 2PE concentration. The culture was run with a dilution ratio of ½ d^‐1 (HRT 2 days) after 20 days.