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EPA (eicosapentaenoic acid) producing bacteria were screened from intestines of fresh water fish, e.g. sweet fish, and sea water fish, e.g. mackerel. 15 strains of marine bacteria and 5 stains of fresh water bacteria were found to have EPA producing ability. Strain PL001which was screened from wild sweet fish had the highest EPA yield of 3.08 mg/L by using HCI-methanol extraction method. Its DHA (docosahexaenoic acid) yield is 1.58 mg/L. Strain SC007 which was screened from mackerel showed the EPA yield of 1.30 mg/L. DHA was not found in the starin SC007.
The strains SC007 and PL001 were gram negative and rod shaped. They all had the ability to produce hydrogen sulfide. Both of them showed positive enzyme activities for cytochrome oxidase, catalase, gelatinase, and DNase.They were able to grow in a medium containing 6% (w/v) NaCl at 20℃. A single motile polar flagellum was obsereved by TEM for the both strains. Strain SC007 had the flagellum length of 6.5~10.5□m, while PL001 had the length of 7.5~12.0□m. Strain SC007 was 0.4~0.6□m in diameter and 1.0~2.0□m in length and strain PL001 was 0.4~0.5□m in diameter and 1.2~2.8□m in length. The testing results of physiological characteristics, morphological characteristics (including light microscope and EM observation), and API 20NE system confirmed that both strains SC007 and PL001 were belong to Shewanella putrefaciens species.
Strain SC007 was grown at a peptone-yeast extract medium at 20℃ for 48 hr and harvested by centrifugation. The bacteria were then freeze dried and subjected to oil extraction with different extraction methods.The bacterial cells which were ultrasonificated for 30 min and then extracted by the HCl-methanol extraction method had the highest oil yield and EPA yield. Their values were 16.45% and 2.80mg/L, respectively. Those who were extracted by the supercritical fluid extraction (SFE) method had the second highest values in both the oil yield and EPA yield. They were 16.23% and 2.25 mg/L, respectively. The lowest values were 4.20% and 1.82 mg/L, respectively, which were obtained by using the HCl-methanod extraction method alone, without ultrasonification.
The cultural conditions for obtaining the highest EPA yield in S.putrefaciens SC007 were also investigated. The peptone-yeast extract medium was used as the medium. Various cultural durations, temperatures, pH values, NaCl concentrations, stirring rates, and concentrations of phosphate buffers were studied. The EPA yields rose from 4.88 to 6.77 mg/L when the temperature was lowered from 20 to 8℃. When the initial pH value was 7.0, the cell yield and EPA yield reached to their highest values, as 3.20 g/L and 6.70 mg/L, respectively. The cell yields dropped to 1.01 and 2.28 g/L, respectively, while changed to pH 5.0 and pH 9.0. The EPA yields of the bacteria which were culculated in the peptone-yeast extract medium with 1% (w/v) NaCl at 20℃ for 2 days was found to be 2.81 folds of the yield of which the bacteria were cultured in the medium without adding NaCl. The EPA yields for those cultured at the stirring rates of 100, 120, and 150 rpm were 8.15, 8.38, and 8.34 mg/L, respectively. The PUFA (polyunsaturated fatty acids) and EPA contents rose from 4.81 to 5.44% and 4.58 to 25.26%, respectively, while 10% (v/v) phosphate buffer was added to the medium. The EPA yield reached to a maximum value of 15.17 mg/L when the bacteria were first cultured at 20℃for one day, followed by lowering the temperature to 8℃ for two days and then turn it back to 20℃ for one day.
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