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The effect of starvation on citrate metabolism by fully grown cells of S/wp~o(~occus lactis subsp. diace/>,/ar/is was studied by three different procedures. An 8-hour starvation period in fermented medium decreased citrate uptake rate but diacetyl and acetoin levels remained constant over a fermentation without starvation period: about 19 mg I-' diacetyl and 130 mg 1-I acetoin were produced after utilization of 8.8 m~ citrate. When fermentation products were discarded from the medium before starvation, diacetyl and acetoin levels reached 30 mg I-' and 240 mg I-*, respectively.When the cells were allowed to rest on an isotonic diluent, citrate uptake rate slowed down, but the diacetyl plus acctoin yield remained higher sincc the cells accumulated as much acetoin (1 20 mg I ~ ') and more diacetyl (50 mg 1-1). Peak production of diacetyl still coincided with citrate exhaustion whereas a delay in acetoin production was observed.Stwprocotcus lactis subsp. cliucetylactis produces little diacetyl and acetoin from carbohydrate mctabolisrn, unless an additional source of pyruvate (e.g. citrate) is present (DRINAN P / a/. 1976).Excess pyruvate, not needed for coenzyme regeneration, becomes toxic towards bacteria, which turn it into neutral compounds such as diacetyl (2-3 butane dione) or acetoin (3 hydroxy 2-butanone) (HARVEY and COLLINS 1963).The biosynthesis of acetoin from pyruvate in bacteria proceeds by two mechanisms. De carboxylation of alpha-acetolactate following its formation by condensation ofthe acetaldehyde TPP complex with another molecule of pyruvate is one mechanism (SPECKMAN and COLLINS 1968). The other method used by some bacteria consists of reducing part or all of the diacetyl that is formed from condensation of the same complex with acetyl CoA ( SPECKMAN and COLLINS, 1968, SEITZ et al. 1963). Diacetyl and acetoin formation proceeds for as long as citrate is present. Once citrate is exhausted, reduction of both compounds may occur (WALSH and COGAN 1973, leading in some bacteria to the production of 2-3 butane diol (THORNILL and COGAN 1984).In Strepcoccus Iuctis subsp. diacrtylactis, strain DRC1, the enzymes involved in these pathways are constitutively present. Diacetyl and acetoin reductases are partly repressed and acetolactate synthase partly induced by growth on a citrate-containing medium (COGAN 198 I). There is also evidence that citrate permease is a plasmid-linked enzyme (KEMPLER and MCKAY 1979). Diacetyl is produced at a much lower concentration than acetoin but it is of greater interest as regards the flavour of several fermented dairy products. For these reasons, our main aim was to enhance diacetyl production by changing physiological cell conditions during citrate metabolism.19 J B m c Microbiol 30 (1990) 8