Growth on solid media

A basic R2YE media (Section 2.2.1), supplemented with 1% (w/v) soluble starch, was

used to obtain a model of normal growth of S. lividans. The colonies were found to take

2 days before becoming large enough to be visible and turned white and grey at the onset

of sporulation, which occurred 6 - 8 days after inoculation. S. lividans was then grown on

the same medium supplemented with 1% (w/v) of various different lipids. Lipid substrates were inclined to rise to the surface of the agar medium before it set, rather than dispersing properly; however, this did not appear to adversely affect growth. Colonies formed were no different in appearance or morphology to the cells grown with the starch medium and

all were able to sporulate. The range of lipids used were free fatty acids (FFA),

triacylglycerols (TAG) and fatty acid methyl esters (FAME) and were composed of fatty acids of various chain lengths. The FFA were also supplied as a co-carbon source with glycerol, where each substrate had a concentration of 1% (w/v) in the medium. The

C h a p te r 3. E jfects o f lip id s on g ro w th a n d m e ta b o lism o f S. li vidaos

Fatty Acid Fatty acid Fatty acid and Fatty acid Triacyl-

Chain Length glycerol methyl ester glycerol

^ 3 : 0 + + nd nd ^ 4 : 0 - - nd + ^ 6 : 0 - + + nd ^ 8 : 0 - + nd nd ( - 1 0 : 0 - - nd nd ( - 1 1 : 0 - - + + ( - 1 2 : 0 - - nd nd ^ 1 3 : 0 - - nd nd ^ 1 4 : 0 + + nd nd ^ 1 5 : 0 + + nd nd C l 6 : 0 + + + + ( - 1 7 : 0 + + nd + ( - 1 8 : 1 + + + + ( - 2 2 : 0 + + nd nd

Table 3.1.1 Growth o f S. lividans on solid media with lipid (1% w/v), or free fatty acid and glycerol (2% wA, 1:1), as the carbon source: + indicates that growth occurred, - no growth after 14 days, nd indicates that the experiment was not done.

Triacylglycerols and methyl esters are better carbon sources for the growth of S. lividans

than free fatty acids, since at all fatty acid carbon chain lengths they supported viable colony formation. There also seems to be no difference between odd- and even-chain fatty acids and their derivatives as growth substrates. Since glycerol is able to support growth when provided as the sole carbon source, it was added to the free fatty acid media to determine if the FFA that could not support growth were toxic, or simply not suitable

as carbon sources for S. lividans. In the case of Q-g and Cg.g FFA, the glycerol produced

viable cultures, but for C^.g and C^g.g^^^.g FFA no growth was evident even when glycerol was included. The toxicity of the FFA of these chain lengths was not reflected in the other types of lipid. The FAME and TAG of undecanoic acid (Cn.g) and the TAG of butyric acid (C^^g) all produced normal development of the cell colonies.

The low pH of the free fatty acids may have been responsible for the lack of cell growth, so the lipid-supplemented agar was prepared with the pH in the range 7.0-7.5 before inoculation. At the end of 14 days, no colonies had grown, even though the pH of the medium had remained at near-neutral. The toxic effect of the free fatty acids is probably not based on acidity.

The toxic effect of medium-chain fatty acids and their salts to both prokaryotic and eukaryotic cells has been noted previously (Stratford and Anslow, 1996). Medium-chain

C h a p te r 3. E ffects o f lip id s on g ro w th a n d m e ta b o lism o f S. lividans

fatty acids are fungitoxic compounds and can only be metabolised by the few genera of fungi that are capable of producing methyl ketones (Hatton and Kinderlerer, 1991). Fay

and Farias (1981) discovered that E. coli mutants deficient in lipopolysaccaride in the cell

membrane are more susceptible to damage by short- and medium-chain (C4.J1) fatty acids,

but the effect is reversed if the mutants are also constitutive for ^-oxidation activity.

Lactobacillus leichmanii is inhibited by the presence of ImM FFA of chain length Cg.Q_^2:o

in a 1% glucose medium (Nunez de Kairuz et a l, 1983). Shimada et a l (1992)

investigated lipase activity in the fungus Geotricum candidum and found that Cg.g, C]o.Q,

C l2 : 0 f&tty acids, Cg.Q TAG and methyl octanoate all produce very poor growth compared

with other chain-length FFA when provided as a 1% addition to com steep liquor. The

mechanism of toxicity of decanoic acid (Cio:o) to Saccharomyces cerevisiae was

investigated by Stratford and Anslow (1996). Starting with the premise that medium- chain fatty acids acted either as weak acid inhibitors or as respiratory uncouplers, disengaging oxidative phosphorylation from respiration, their research showed that the mechanism is different to both these effects. The decanoic acid causes leakage of amino acids and protons through the membrane and very rapid cell lysis occurs when a critical concentration is exceeded. Growth is inhibited with sub-lethal amounts of decanoate and

the effects are magnified when the yeast is grown in media with a low pH. Lamas et a l

(1995) found that in mammalian neuroglioma cells, octanoic acid reduces the inward flow of the Na+ membrane channel, causing a proton and ion imbalance. The short-chain fatty

acid butyrate is stimulatory to the growth of Nocardia amarae and Rhodococcus spp.,

when supplied as an additive to complex media and is used preferentially over long chain

fatty acids (Iwahora et a l, 1995). However, Thompson and Hinton (1996) found that

butyric and propionic acid are toxic to Salmonella enteriditis and Escherichia coli,

affecting the physiology of the cell. DNA synthesis is also inhibited.

Given the effect on other species, it is unsurprising that the growth of S. lividans is

inhibited by certain short- and medium-chain fatty acids. However, the ability of the strain to grow on the methyl esters and triacylglycerols of the same fatty acids is more unexpected. This indicates that the cell is capable of processing the TAG without the release of large quantities of free fatty acids, which would otherwise prove toxic. The cells must also be able to import medium-chain free fatty acids after they have been hydrolysed from the glycerol backbone by extracellular lipase activity.