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.