Appl Microbiol Biotechnol (2003) 61:257–260 DOI 10.1007/s00253-003-1258-y SHORT CONTRIBUTION C. R. Squio · C. Marangoni · C. S. De Vecchi · G. M. F. Arag¼o Phosphate feeding strategy during production phase improves poly(3-hydroxybutyrate-co-3-hydroxyvalerate) storage by Ralstonia eutropha Received: 4 November 2002 / Revised: 24 December 2002 / Accepted: 3 January 2003 / Published online: 5 March 2003  Springer-Verlag 2003 Abstract The effect of a phosphate feeding strategy and the optimal rate of biomass production (r x ) during the production phase of P(3HB-co-3HV) in a 6-l fermentor were determined in cultures of Ralstonia eutropha with the goal of enhancing polymer productivity. Rates of biomass production (r x ) between 0.00 and 0.20 gr l 1 h 1 were monitored during the production phase. When a low rate of cell growth was maintained (r x of 0.02 gr l 1 h 1 ), polymer production improved, resulting in a final cell mass, P(3HB-co-3HV) mass, and P(3HB-co-3HV) con- tent of 98.2 g, 62.0 g and 63.1 wt%, respectively, after 27.3 h. The maximum polymer productivity obtained during the production phase was 1.36 g l 1 h 1 . Introduction Polyhydroxyalkanoates (PHAs) are carbon and energy storage materials that accumulate as intracellular granules in a variety of microorganisms during unbalanced growth (Anderson and Dawes 1990). PHAs have received much attention because of their similarity to conventional plastics and their complete biodegradability (Lee 1996). The best-studied polymer is polyhydroxybutyrate [P(3HB)], although copolymers of poly(3-hydroxybu- tyrate-co-3-hydroxyvalerate [P(3HB-co-3HV)], possess potentially more useful thermoplastic properties (Byrom 1987). Ralstonia eutropha is a PHA-producing bacteria that could be used for industrial production of PHAs due to its high yield, rapid production rate and ability to grow from renewable sources (Byrom 1987; Marangoni et al. 2002). P(3HB-co-3HV) is synthesised by R. eutropha when fed with carbon sources and co-substrates such as propionic acid (Byrom 1987). Polymer production in R. eutropha is carried out in two phases: (1) cell growth under non-limiting nutrient conditions, with the aim of biomass generation; (2) nutrient (nitrogen, phosphate, oxygen, etc.) limitation with polymer accumulation according to the source of carbon offered (Dawes and Senior 1973). PHA production processes usually utilize the total exhaustion of an element essential for growth. Under these conditions, residual biomass (all non-polymer cellular material) remains constant during polymer storage (Braunegg et al. 1998). By contrast, supplying the limiting nutrient during the production phase supports constant growth of the cells, enhancing polymer productivity (Suzuki et al. 1986; Bitar and Underhill 1990; Arag¼o et al. 1996). However, the optimal rate of biomass production during the polymer production phase has still not been defined. Nitrogen and phosphate limitation have been most widely applied to polymer production by R. eutropha. When nitrogen limitation is used to induce polymer accumulation, NH 4 OH cannot be used for pH control. Instead, NaOH is added; however, its toxicity can lead to significant cell lysis. Research has shown that phosphate limitation could be advantageous because it is not necessary to substitute NH 4 OH for NaOH for pH control and there is no cell lysis (Ruy et al. 1997). As phosphate does not participate directly in protein composition (Pelczar et al. 1993), its limitation allows residual cell growth for some time. Moreover, this element is involved in the recycling of energetic intermediates (ATP/ADP), leading to an increase in polymer production. In the present work, the effect of a phosphate feeding strategy and the optimal rate of biomass production (r x ) during the production phase of P(3HB-co-3HV) were determined in cultures of R. eutropha with the goal of enhancing polymer productivity. C. R. Squio · C. Marangoni · C. S. De Vecchi · G. M. F. Arag¼o ( ) ) Chemical and Food Engineering Department, Technological Center, Federal University of Santa Catarina, C.P. 476, 88040–900 Florianópolis, Brazil e-mail: glaucia@enq.ufsc.br Tel.: +55-48-3319842 Fax: +55-48-3319687