3.4 Biosynthesis of 2-PE by using Meyerozyma sp. strain YLG18 with ISPR technology
As mentioned, 2-PE is toxic to microbial cells, as it can make the lipid membrane structure a preferential binding target, resulting in the collapse of transmembrane gradients and the loss of cell viability (Sikkema et al., 1995). To further increase the final 2-PE production and productivity, more robust strains or novel extraction technology should be developed. In situ product recovery (ISPR) techniques can simultaneously remove 2-PE from the fermentation broth while it is produced. Thereby, 2-PE concentration could maintain below the inhibitory level, and microbes are able to continuously produce 2-PE. Suitable extractants including fatty acid methyl ester (FAME), oleic acid and ethyl acetate were first identified. As seen from Fig. 4A, the partition coefficients of these organic solvents were similar for 2-PE. However, different organic solvents had different impacts on the dissolved oxygen level, which would affect microbial growth and lead to significant difference in the final 2-PE titer (Weber & Bont, 1996). Among these organic solvents, FAME gave the highest 2.53 g/L of 2-PE. The volume ratio of medium and extractant phases will also affect the distribution and mass transfer of products. Low ratio of FAME could not extract 2-PE effectively, affecting the bioconversion rate. High ratio of FAME may increase toxicity to cells. Results showed that the highest 2.48 g/L of 2-PE was obtained when FAME : water ratio was maintained at 1:1 (Fig. 4B). It should be noticed that when the ratio of FAME to water was 1:2, the production of 2-PE was 2.45 g/L, which is close to the highest one. Accordingly, the optimal ratio of 1:2 was chosen for following experiments.
Generally, microbes would show a log phase after inoculated into the fresh medium. The presence of FAME in the initial stage may prolong this period and affect the transformation activity of yeasts. Therefore, a proper delay of FAME addition may help to improve the metabolic activity of yeasts and ultimately increase 2-PE production. From Fig. 4C, it can be seen that the earlier extractants were added, the more obvious the strain growth inhibition was. Compared with the experimental results of groups 4, 5 and 6, when FAME was supplemented when strain growth reached exponential phase, the death rate would be decreased significantly. In addition, it can be seen from Fig. 4D that when the strain death rate decreases, the 2-PE production in the group 4 was also the highest, and it can reach 3.20 g/L, which was improved by 25.49% compared to single-phase biotransformation.