Toxicity of unsaturated fatty acids to the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens

Abstract

Background: Health-promoting polyunsaturated fatty acids (PUFA) are abundant in forages grazed by ruminants and in vegetable and fish oils used as dietary supplements, but only a small proportion of PUFA finds its way into meat and milk, because of biohydrogenation in the rumen. Butyrivibrio fibrisolvens plays a major role in this activity. The aim of this study was to investigate the mechanisms by which PUFA affect the growth of B. fibrisolvens, how PUFA are metabolized and the metabolic response to growth in the presence of PUFA.

Results: Linoleic acid (LA; cis-9, cis-12-18:2) and alpha-linolenic acid (LNA; cis-9, cis-12, cis-15-18:3) increased the lag phase of B. fibrisolvens JW11, LNA having the greater effect. Growth was initiated only when the PUFA had been converted to vaccenic acid (VA; trans-11-18:1). The major fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n-3)) and docosahexaenoic acid (DHA; 22:6(n-3)), were not metabolized and prevented growth. Cellular integrity, as determined fluorimetrically by propidium iodide (PI) ingression, was affected as much by 18:1 fatty acids, including VA, as 18:2 fatty acids. The methyl esters of LNA, LA, EPA and DHA had no effect on growth or other measurements. The ATP pool decreased by 2/3 when LA was added to growing bacteria, whereas most acyl CoA pools decreased by >96%.

Conclusions: It was concluded that biohydrogenation occurs to enable B. fibrisolvens to survive the bacteriostatic effects of PUFA, and that the toxicity of PUFA is probably mediated via a metabolic effect rather than disruption of membrane integrity.

Figure 1

Concentration of fatty acids in the medium following inoculation of B. fibrisolvens JW11 into M2 medium containing 50 μg ml^-1 linoleic acid (LA; cis-9, cis-12-18:2). Growth (open circle, OD₆₅₀), LA (square), cis-9, trans-11-18:2 (black circle), trans-11-18:1 (triangle). Results are means and SD from three cultures.

Figure 2

Concentration of fatty acids in the medium following inoculation of B. fibrisolvens JW11 into M2 medium containing 50 μg ml^-1 α-linolenic acid (LNA; cis-9, cis-12, cis-15-18:3). Growth (open circle, OD₆₅₀), LNA (black circle), cis-9, trans-11, cis-15-18:3 (open square), trans-9, trans-11, cis-15-18:3 (black square), trans-11, cis-15-18:2 (open triangle), cis-9, trans-11-18:2 (black triangle), trans-11-18:1 (diamond). Results are means and SD from three cultures.

Figure 3

Influence of different fatty acids and fatty acid methyl esters on cell integrity of B. fibrisolvens JW11. Loss of cell integrity was determined fluorimetrically by propidium iodide fluorescence. LNA, cis-9, cis-12, cis-15-18:3; γLNA, cis-6, cis-9, cis-12-18:3; LA, cis-9, cis-12-18:2; CLA, a mixture of cis-9, trans-11-18:2 and trans-10, cis-12-18:2; VA, trans-11-18:1; OA, cis-9-18:1; SA, 18:0. In order of increasing shading density: 50 μg fatty acid ml^-1, 200 μg fatty acid ml^-1, 50 μg fatty acid methyl ester ml^-1, 200 μg fatty acid methyl ester ml^-1. Results are means and SD from three determinations.

Figure 4

Influence of different fatty acids on PI fluorescence of B. fibrisolvens JW11 by flow cytometry. Black - live cells; green - heat-killed cells; pink - 50 μg ml^-1 LA; turquoise - 50 μg ml^-1 LNA; orange - 50 μg ml^-1 CLA; blue - 50 μg ml^-1 VA; yellow - 50 μg ml^-1 SA.

Figure 5

Influence of sodium lactate (70 mM) on the loss of cell integrity of B. fibrisolvens JW11 following incubation with LA (50 μg ml^-1). Loss of cell integrity was determined by fluorescence in the presence of propidium iodide. Sodium lactate + LA (open bar), LA alone (black bar). Results are means and SD from three cultures, each of which was subject to 8 replicate measurements (n = 24).

Figure 6

Influence of LA on ATP pools of B. fibrisolvens JW11 after 50% inoculation into fresh medium. LA (black circle), no LA (open circle). Results are means and SD from three separate cultures.