640 J. Jpn. Oil Chem. Soc. (YUKAGAKU) ORIGINAL Effect of ph Shift in the Stationary Phase of Growth on the Incorporation of Exogenous Docosahexaenoic Acid into Euglena gracilis Masahiro HAYASHI, Reiko KOMATSU, and Shozaburo KITAOKA Tsukuba Research Laboratory, Harima Chemicals, Inc. (Tokodai 5-9-3, Tsukuba-shi, Ibaraki-ken, 300-26) Department of Agricultural Chemistry, University of Osaka Prefecture (Gakuencho 1-1, Sakai-shi, Osaka-fu, 593) The effects of ph shift in the stationary phase of growth on the incorporation of exogenous docosahexaenoic acid (DHA) into Euglena cells and lipid classes were examined. At ph 6.5 `7.5 the incorporation of DHA was maximum, and content of DHA in the cells at ph 6.5 (35.7 % in the dry cells) was 2.5 times as much as that at ph 2.5. The percentage of DHA in total fatty acid in the cells was 70% at ph 4.5 `7.5, but decreased at ph 2.5 `3.5 and 8.5. The abundant component of total lipids in Euglena cells, incorporated exogenous DHA, was triacylglycerols (TG). But TG in the cells decreased and free fatty acids increased at ph 8.5. DHA in the cells was mainly incorporated into TG. DHA was partially incorporated into polar lipids and the acyl moiety of wax esters. Only a small amount of docosahexaenoyl alcohol could be detected in the alkoxy moiety of wax esters. It thus follows that, with sufficient shift of ph in the stationary phase, Euglena cells progressively incorporate DHA, to promote the production of more useful fishery feed from Euglena. 1 Introduction Incorporation of exogenous fatty acids into microorganisms was investigated in several bacteria and fungi. And it was known that exogenous oleic and docosahexaenoic (DHA) acids were incorporated into phospholipids of bacteria cells1)-4). Mortierella remanniana var. angulispora incorporatedexogenous linoleic acid into phospholipids and triacylglycerols (TG) and accumulated it in TG5),6). However, the effects of cultural conditions on the incorporation of exogenous highly unsaturated fatty acids (HU- FA) into lipids of microorganisms have not been reported. It was reported that Euglena gracilis incorporated exogenous propionic, butylic, hexanoic, octanoic, oleic, linoleic, linolenic, Corresponding author : Masahiro HAYASHI and ricinoleic acids into wax esters (WE)7) `9). As for the HUFA in Euglena, effects of cultural conditions on the contents of HUFA in the cells have been reported1 ). However, incorporation and distribution of HUFA in lipid classes of Euglena have not been reported heretofore. In the previous paper, Euglena in the stationary phase of growth was shown to incorporate and accumulate exogenous icosapentaenoic acid (EPA) and DHA in the cellsn). It was also shown that shifting ph in the stationary phase greatly affected the lipid content and lipid composition of Euglena 12). Therefore, it is suggested that ph greatly affects lipid metabolism in Euglena. In the present studies, the effect of ph shift in the stationary phase on the incorporation of exogenous DHA into the cells and lipid classes was examined. 18
Vol. 44. No. 9 (1995) 641 2 Experimental 2.1 Cultivation and ph shifting E. gracilis strain Z NIES 48 was grown in a medium reported in the previous paper for 72 h in the dark at 28 Ž in a 2 L jar-fermentor12). The ph of the culture was kept at 4.5 nia and by using a ph controller for 48 h of the cultivation until the cells reached the stationary phase of growth. Then ph of the culture was varied to different values in the range of 2.58.5 by the addition of 2 M KOH or 1 M sulfuric acid, and the cultures were continued for another 24 h keeping the specified value of ph. And 0.5 % of DHA purified by the silver nitrate method 13) to 86.6 % was added to the culture with the shifting ph. The DHA sample added contained 6.5 % of docosapentaenoic acid and 6.3 % of EPA as impurity. Cells were harvested by centrifugation, washed 3 times with distilled water, and kept at -20 Ž until analysis. 2.2 Assays Total lipids were extracted by the Folch method and weighed14). Polar lipids (PL) in the total lipids were separated by using a silica cartridge, 25 mm } 10 mm I.D., (Sep- Pack, Waters Co. Ltd.) following weighing15). Each neutral lipid class was separated and measured by the TLC-FID method16). For the determination of fatty acid composition, the WE and TG in the total lipids were separated by silica gel column chromatography9). Fatty acid and fatty alcohol compositions were examined by Shifting ph of the culture in the stationary phase of growth greatly affected the incorporation of exogenous DHA into the cells (Fig.-1). Higher ph in the range of ph 2.5 `6.5 gave a higher DHA content in the cells. The DHA content in the cells increased from 14.0 % at ph 2.5 to 35.7 % at ph 6.5, but it decreased at ph 7.5-8.5. The percentage of DHA in the total fatty acid of the cells showed a similar tendency to the DHA content in the cells (Table-1), being about 70 % in the range of ph 4.5 ` 7.5, and the maximum percentage was 73.9 % in the total fatty acid. As for other fatty acids, saturated fatty acids, such as C14:0, appeared in a relatively high percentage at ph 2.5 `3.5 and 8.5, showing an inverse tendency to that of DHA. The percentage of EPA was not affected by shifted ph, being 7 `8 % throughout the ph range studied, but it slightly decreased at ph 8.5. 3.2 Effects of ph shifting in the stationary phase on the lipid content and lipid class composition of the cells The lipid content of Euglena cells was not affected by shifting ph in the range of ph 2.5 `5.5, being 33 `34 % of the cells. However, it increased at ph 6.5, being 48.3 %, and it also showed more than 40 % of the cells at ph 7.5 `8.5 (Fig.-2). As for the lipid class composition in the gas liquid chromatography after methanolysis of TG-12) or saponification and methanolysis of WE9). Dry cell weight was measured after cells had been dried at 105 C for 6 h, and the contents of the lipids and DHA were expressed as percentage on dry matter basis. 3 Results 3.1 Effects of ph shift in the stationary phase of growth on the DHA content and fatty acid composition in the cells Fig.-1 The cellular DHA content of E. gracilis after incorporation of DHA at ph 2.5-43.5 in the stationary phase for 24 h. 19
642 J. Jpn. Oil Chem. Soc. (YUKAGAKU) Table - 1 Fatty acid compositions of total lipids in Euglena after incorporation of DHA at ph 2.5 `8.5 in the stationary phase. Fig. - 2 The cellular lipid class contents of E. gracilis after incorporation of DHA at ph 2.5 `8.5 in the stationary phase for 24 h. total lipids, higher ph gave higher TG content in the cells in the range of ph 2.5 `6.5, and it increased from 15.6 % at ph 2.5 to 22.2 % at ph 6.5. But it decreased at alkaline ph, being 2.7 % at ph 8.5. The WE content of the cells was 11-14 % at ph 2.5 `3.5, and it was constant at ph 4.5 `8.5, being 4 `6 %. At ph 6.5 `8.5, free fatty acids increased in the cells, and were abundant at ph 8.5, being 15.3 % of the cells. The contents of other lipids, polar lipids and diacylglycerols, were not affected by ph shifting, being 3 `4 % and 1 `3 % throughout, respectively. 3.3 Effect of ph shifting in the stationary phase on the incorporation of exogenous DHA into lipid classes The percentage of DHA in the fatty acid composition of TG was similarly affected, as was the DHA content of the cells by ph shifting (Table-2). It was 75 `86 % at ph 4.5 `7.5, and ph 6.5 gave a maximum of 86.4 %. On the other hand, it was much lower at ph 2.5 `3.5 being 51 `55 % than those at ph 4.5 `7.5. Little exogenous DHA was incorporated into TG at ph 8.5, being only 5.9 %. As for other fatty acids, saturated fatty acids, such as C14:0, increased at ph 2.5 `3.5. At ph 8.5, oleic and linoleic acids 20
Vol. 44. No. 9 (1995) 643 Table - 2 Fatty acid compositions of triacylglycerols in Euglena after incorporation of DHA at ph 2.5 `8.5 in the stationary phase. %, GC area. Table-3 Fatty acid and alcohol compositions of wax esters in Euglena after incorporation of DHA at ph 2.5-8.5 in the stationary phase. %, GC area increased, as did saturated fatty acids. The percentage of DHA in the fatty acid composition of WE was also higher at ph 4.5 `7.5, being 75 `53 %, than those at ph 2.5 `3.5 and at 8.5 (Table-3). But those values in WE were slightly lower than those in TG. ph 4.5 gave a maximum percentage of 75.8 %. It was suggested that the optimum ph for the incorporation of exogenous DHA into WE was different from that into TG. A small amount (8.9 % in the total fatty alcohol of WE) of docosahexaenoyl alcohol that is supposed to be produced from DHA was detected in WE. Its percentage was 21
644 J. Jpn. Oil Chem. Soc. (YUKAGAKU) slightly higher at ph 4.5 `5.5 similarly to that of DHA in WE than at other ph. At ph 4.5 `5.5 and 8.5, the percentage of docosapentaenoyl alcohol was 11 `22 %, and it was much higher than that of C22:5 acid in the fatty acid composition of WE. The percentage of DHA in the fatty acids of PL was 26 `35 % throughout the ph studied except at ph 8.5 that gave 77.3 % (Table-4). Incorporation of exogenous DHA into PL was less than that into TG and WE, and the effect of shifting ph on the incorporation into PL was much smaller than those into TG and WE. It would be an interesting subject in future studies that the content of DHA in PL greatly increased while it decreased in TG and WE at ph 8.5. More studies are required for the elucidation of the mechanism of the drastic changes of DHA content in PL, WE, and TG. 4 Discussion According to reports of the incorporation of fatty acids into bacterial phospholipids, the amount of fatty acids incorporated depends on the permeability of cell wall and cell membrane, the ability of acyl-coa synthesis in the cells, and the substrate selectivity and the affinity of the acyltransfera- Se2),3),17),18). In the present studies, the increase of DHA incorporation into Euglena cells at ph 6.5 `7.5 might be due to the change of the permeability of pellicle, the outer membrane of the protozoans. However, it appears important to know that the degree of dissociation and solubility of DHA depend on ph of the culture. It was reported that fatty acids such as DHA, EPA, linoleic, and y-linolenic acids, were easily and stably emulsified in the range of ph 6.5 ` 7.519), This finding is consistent with the result that ph 6.5-7.5 gave the maximum content of DHA in Euglena cells in the present studies. It is suggested that the emulsifying condition and the degree of dissociation of DHA affect the incorporation of DHA into the cells. But in the present studies, the change of the cellular lipid content does not parallel that of the DHA content. More investigations will be needed for understanding of ph effects on the fatty acid incorporation into Euglena. Table - 4 Fatty acid compositions of polar lipidsin Euglena after incorporation of DHA at ph 2.5 `8.5 in the stationary phase. %, GC area. 22
Vol. 44. No. 9 (1995) 645 In WE of Euglena, the de novo synthesized fatty acids and fatty alcohols are only C1018 (mainly C14) saturated fatty acids and alcohols20),21). In anaerobic condition, exogenous unsaturated fatty acids, such as oleic, linoleic, linolenic, and ricinoleic acids are mainly incorporated into WE, and part of the fatty acids is reduced to the corresponding fatty alcohols, except for ricinoleic acid9). In the present studies, exogenous DHA was incorporated also into acyl moiety of WE, but only a small amount of DHA was found in the alkoxy moiety of WE. However, a significant amount of docosapentaenoyl alcohol was found in alkoxy moiety of WE. The C22 5 alcohol might be produced via conversion of DHA to docosapentaenoic acid. But since the DHA sample added into the culture contained docosapentaenoic acid as an impurity, involvement of substrate specificity of acyl-coa reductase of Euglena may also be possible. Whereas bacteria was reported to incorporate and accumulate fatty acids mainly into PL1) `4), the DHA incorporated into PL of Euglena was less than that in WE and TG. In mammals and higher plants, it was reported that the changes of activities of phosphatidic acid phosphatase and diacylglycerol acyltransferase are related with the regulation of glycerolipids synthesis22) `24). However, the glycerolipid synthesis in Euglena has not been reported, except for the glycerophosphate acyltransferase which catalyzes the first step of the TG synthe- SiS25) `28). More studies are required for the discussion of the glycerolipid metabolism in Euglena. In the present studies, it has been shown that the ph in the stationary phase greatly affects the incorporation of exogenous DHA into Euglena cells, and ph 6.5 `7.5 gave the maximum incorporation of DHA. The incorporation of DHA into each lipid. class was also affected by the ph of the culture, and incorporated DHA was mainly accumulated in TG in the cells. It was reported that the DHA in TG, DHA methyl ester, and DHA ethyl ester were more suitable for fishery feed than free DHA 29). Therefore, the results reported in the present studies should contribute profitably to the production of better fishery feed by using DHA-enriched Euglena. (Received April 28, 1995) References 1) S. Ando, K. Nakajima, and M. Hatano, J. Ferment. Bioeng., 73, 169 (1992). 2) J.E. Cronan, Jr., J. Bacteriol., 159, 773 (1984). 3) C.O. Rock and S. Jackowski, J. Biol. Chem., 260, 12720 (1985). 4) K. Watanabe, C. Ishikawa, H. Inoue, D. Cenhua, K. Yazawa, and K. Kondo, J. Am. 5) Y. Kamisaka, T. Yokochi, T. Nakahara, and O. Suzuki, Lipids, 25, 54 (1990). 6) Y. Kamisaka, T. Yokochi, T. Nakahara, and O. Suzuki, Lipids, 25, 787 (1990). 7) J. Nagai, T. Ohta, and E. Saito, Biochem. Biophys. Res. Commun., 42, 523 (1971). 8) P. Pohl, Z. Naturforsch., 28 c, 270 (1973). 9) Y. Tani, M. Okumura, and S. Ii, Agric. Biol. Chem., 51, 225 (1987). 10) K. Miyatake, M. Minamikawa, Y. Nakano, and S. Kitaoka, Nippon Eiyo Shokuryo Gakkaishi, 38, 117 (1985). 11) M. Hayashi, K. Toda, and S. Kitaoka, Biosci. Biotech. Biochem., 57, 352 (1993). 12) M. Hayashi, K. Toda, H. Ishiko, R. Komatsu, and S. Kitaoka, Biosci. Biotech. Biochem., 58, 1964 (1994). 13) Y. Misawa, H. Kondo, K. Yazawa, and K. Kondo, Abstracts of the Annual Meeting of the Chemical Society of Japan, (Yokohama), p. 1245 (1991). 14) J. Folch, M. Lees, and G.H. Sloane-Stanley, J. Biol. Chem., 226, 497 (1957). 15) P. Juaneda and G. Rocquelin, Lipids, 20, 40 (1985). 16) R.G. Ackman, Methods in Enzymol., 72, 205 (1981). 17) P.N. Black, S.F. Kianian, C.C. DiRusso, and W.D. Nunn, J. Biol. Chem., 260, 1780 (1985). 18) H. Okuyama, Seikagaku, 47, 999 (1975). 19) T. Inoue and M. Ishiguro, Jpn. Kokai, 93-253464. 20) P.F. Guehler, L. Peterson, H.M. Tsuchiya, and R.M. Dodson, Arch. Biochem. Biophys., 106, 294 (1964). 23
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