May 12, 1992 - and Golden Sea Produce Limited, Hunterston, West Kilbride, Ayrshire,3 Scotland ... when grown aerobically in lipid-free TSBgs medium was V.
N
Vol. 58, No. 11
APPLIED AND ENVIRoNMENTAL MICROBIOLOGY, Nov. 1992, p. 3777-3778
0099-2240/92/113777-02$02.00/0 Copyright © 1992, American Society for Microbiology
Production of Eicosapentaenoic Acid (20:5 n-3) by Vibio pelagius Isolated from Turbot (Scophthalmus maximus (L.)) Larvae EINAR RING0,1t* PAULINE D. SINCLAIR,2 HARRY BIRKBECK,2 AND ANDREW BARBOUR3 The Foundation ofApplied Research at the University of Troms0, P. O. Box 2806, Elverh0y, N-9001 Troms0, Norway,1 and Department of Microbiology, University of Glasgow, Glasgow G12 8QQ,2 and Golden Sea Produce Limited, Hunterston, West Kilbride, Ayrshire, 3 Scotland Received 12 May 1992/Accepted 26 August 1992
Fourteen bacterial strains isolated from turbot, Scophthalmus mximus (L.), larvae were screened for eicosapentaenoic acid (20:5 n-3) (EPA) production. Gas chromatography analysis revealed that one bacterial species, Vibrio pelagius, contained a high proportion of EPA in cellular lipid. This finding was confiruted by gas chromatography-mass spectrometry analysis. A higher concentration of EPA was detected when the bacterium was cultured at 4°C. Until recently, it was generally considered that procaryotes do not contain eicosapentaenoic acid (20:5 n-3) (EPA). However, recent studies have shown that both marine bacteria (4, 10, 11) and freshwater bacteria (8) may contain a high proportion of cellular EPA in their total lipid. These findings indicate that microorganisms, especially members of the family Vibrionaceae, contain more EPA than has been suggested. In this study, the fatty acid compositions of 14 bacterial isolates from turbot, Scophthalmus maximus (L.), larvae were screened for EPA production. The results showed that Vibrio pelagius was the only bacterial species able to produce EPA. From 420 bacterial isolates obtained from larval turbot grown under different conditions at hatcheries at Hunterston, Scotland (Golden Sea Produce Ltd.), or Mowi, Norway (Norsk Hydro Ltd.), and identified to the genus or species level by standard biochemical tests (3, 6, 7), a representative sample of 14 different isolates was selected for further study (Table 1). Bacteria were cultured in a lipid-free medium (TSBgs) containing (per liter) 40 g of tryptic soy broth, 5 g of glucose, and 15 g of NaCl to anA600 of approximately 1.0 at incubation temperatures of 23, 12, and 4°C. Bacterial lipid was extracted as described elsewhere (8). The production of EPA by the 14 bacterial isolates from turbot larvae was tested at 23, 12, and 4°C. The only microorganism examined that produced EPA in cellular lipid when grown aerobically in lipid-free TSBgs medium was V. pelagius. The highest production of EPA, approximately 9% of total fatty acids, was detected when the bacterium was grown at 4°C (Table 2). The large quantity of EPA was of bacterial origin, because only saturated fatty acids (12:0, 14:0, 15:0, 16:0, 17:0, and 18:0) and 16:1 were detected in the growth medium. EPA has been detected in deep-sea isolates (4, 10) and in microorganisms isolated from the gastrointestinal tract of
different fish species (8, 11). The proportion of EPA detected in V. pelagius (9% of total fatty acids) is similar to the proportion found in freshwater Vibrio spp. (8) but considerably lower than that (ca. 30%) reported for several deep-sea bacteria (4) and Shewanella (Alteromonas)putrefaciens (11). Wilkinson (9) reported that 16:1 is the most abundant fatty acid, followed by 16:0 and 18:1, in members of the family Vibrionaceae. However, the fatty acid content of V. pelagius decreased in the order 16:1 > 18:1 > 16:0 when the bacterium was grown at various temperatures (Table 2). Nearly all Vibrio spp. seem to contain small or moderate proportions of fatty acids with branched chains or odd numbers of carbon atoms (5). Branched-chain fatty acids were not detected in V. pelagius, but a small proportion of 17:0 was found (Table 2). Some psychrophilic marine species of the family Vibrionaceae seem to respond in different ways to a reduction in the growth temperature, by increasing the content of either branched-chain fatty acids or odd-numbered fatty acids (2). However, other marine species of the family Vibrionaceae, e.g., Vibrio parahaemolyticus (1) and Vibrio marinus (4), show an increase in the proportion of monoene or polyene fatty acids as the growth temperature is reduced. The results of this study with V. pelagius indicated that increases in the
TABLE 1. Origins of the bacterial isolates screened for the production of EPA Bacterial isolates
Source of turbot larvae
Golden Sea Produce Ltd.; larvae fed on
Vibrio pelagius, Vibno splendidus, Flavobacterium sp., and Moraxella sp.
Golden Sea Produce Ltd.; larvae fed on rotifers ....................
Vibrio anguillarum, Vibrio alginolyticus, Vibno fluvialis, Vibrio natriegens, Vibrio nereis,
copepods ....................
Acinetobacter sp., and Aeromonas caviae
*
Corresponding author.
Norsk Hydro Ltd.; larvae fed on copepods
t Present address: Institute of Biology and Geology, University of Troms0, Dramsveien 201, N-9037 Troms0, Norway. 3777
......
Vibrio campbellii, Vibrio phenon 21, and Vibrio phenon 36
3778
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 2. Composition of the major fatty acids in cellular lipid of V. pelagius after growth in TSBgs medium at 23, 12, and 4'C % Fatty acid at a cultivation temp ('C) of: Fatty acid
12:0 14:0 14:1 16:0 16:1 17:0 18:0 18:1 20:1 20:5 (n-3)b
23
12
4
1.0 2.6 3.5 16.5 36.5 1.0 1.1 24.6 NDa 4.5
0.9 1.7 2.9 10.8 43.4 0.6 0.6 23.5 0.6 6.5
0.4 1.0 3.0 8.8 48.7 0.5 0.5 20.5 0.8 8.7
a ND, not detected. b Identified by gas chromatography-mass spectrometry.
contents of EPA and 16:1 (Table 2) might be essential for the bacterium to maintain membrane fluidity at lower tempera-
2.
3. 4.
5.
6. 7.
8.
tures.
E.R. thanks E. Jensen (University of Troms0) for help during the identification of EPA by gas chromatography-mass spectrometry. This work was supported by grants from NTNF and Norsk Hydro Ltd. REFERENCES 1. Beuchat, L. R, and R. E. Worthington. 1976. Relationships between heat resistance and phospholipid fatty acid composi-
9.
10. 11.
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