| Summary: | 博士 === 國立臺灣大學 === 農業化學研究所 === 88 === In this study, cultivation of microorganism and plant cell was compared on their capability of producing polyunsaturated fatty acids (PUFA) in heterotrophic growth. Two filamentous fungi Mortierella alpina K 14-25 and Mortierella sp. L9-22, isolated from soils of Taiwan area, were selected to compare with the callus of liverwort--Marchantia polymorpha, that was able to grow rapidly, on their PUFA productivity.
PUFA was growth-associated intracellular product of Marchantia polymorpha. It contained C18:3（alpha）, C18:2, ARA and EPA in descending order. The cell contained 5% lipid in dry weight of biomass, of which 35~45% were PUFA. Cells were cultivated in MSK-2 medium. Highest PUFA production was achieved using 2,4-dichlorophenoxyacetic acid as growth regulator. Inoculum size, illumination intensity, organic carbon source, and ferrous ion are the major factors affecting PUFA productivity. Maximum PUFA productivity is attained under low light intensity, with a photon flux density ca. 20 mol/m2/s. Optimal inoculum size and glucose concentration for PUFA production are 12% and 20~30 g/L, respectively. Ferrous ion can promote PUFA productivity by increasing the intracellular lipid content. In shake flask, highest productivity for PUFA and C20 PUFA in batch culture were 35.02.1 mg/L/d and 13.30.7 mg/L/d, respectively. The productivity of PUFA and C20 PUFA in shake-flask fed-batch attained 59.54.5 and 19.01.9 mg/L/d, respectively. When cultivated in a stirred-bioreactor, PUFA productivity in the M. polymorpha suspension culture increases from 22.6 mg/L/d in batch, to 45.2 and 32.1 mg/L/d in fed-batch and perfusion culture, respectively, due mainly to increased biomass production. With DO stat culture that agitation (or substrate feeding rate) was controlled, PUFA productivity was further increased to 52 mg/L/d. ARA was mainly present in phospholipid. EPA and C18:3(alpha) were presented in neutrolipid and glycolipid, respectively. The amount of PUFA was ca. about 1~2% in dry biomass.
ARA was the major PUFA in microbial lipid. Mortierella alpina K14-25 and Mortierella sp. L9-22 contained 30~40% lipid in their dry weight. It was present as triglyceride. About 20~30% of fatty acid in triglyceride for Mortierella alpina K14-25, and 40~60% for Mortierella sp. L9-22 was presented as ARA. ARA was growth associated intracellular metabolite for both producing strains. The productivity of ARA was promoted by factors such as initial pH value and addition of C18:2. The highest ARA concentration in fatty acid was achieved in prolonged culture and addition of cupric ion. It was found that cultivated in darkness might result in better ARA productivity.
An ARA productivity of 333 mg/L/d with ARA/TFA ratio 34.2% was obtained for cultivation of Mortierella alpina K14-25 in 1.5 fold concentration of regular (HD) medium for 10 days. When using soy flour to replace yeast extract as nitrogen source in regular medium, an ARA productivity of 262 mg/L/d, with ARA/TFA ratio 24.0% was obtained from a cultivation of 5 days. Highest ARA/TFA ratio--56%, was obtained when cultivated in medium containing 1 mg/L CuSO45H2O for 15 days.
For Mortierella sp. L9-22, cultivation in HD medium in darkness for 8 days or addition of 0.064% lecithin to regular medium and cultivated for 8 days resulted in the highest ARA productivity. The productivity of ARA was 273 mg/L/d with an ARA/TFA ratio of 49.9%, or 267 mg/L/d with an ARA/TFA ratio 69.9%, respectively, for the above two culture conditions. When using soy flour to replace yeast extract as nitrogen source in regular medium, a productivity of 270 mg/L/d (ARA/TFA ratio was 40.9%) was obtained for 8 days cultivation. Highest ARA relative concentration was obtained as 77.4%, in cultivation with HD medium containing 1 mg/L CuSO45H2O for 15 days.
In comparison on the productivity and relative concentration of fatty acids, cultivation of filamentous fungi resulted in better performance than of the plant cell Marchantia polymorpha. Between the two microorganisms using in this study, Mortierella alpina K14-25 resulted in larger ARA productivity than Mortierella sp. L9-22, however, the oil quality of Mortierella sp. L9-22 was better. In consideration on the cost and procedure of PUFA purification, Mortierella sp. L9-22 was the better choice for the production of high concentration ARA containing oil.
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