Batch culture of Alcaligenes latus, American Type Culture Collection 29713, was investigated for producing the intracellular bioplastic poly(␤-hydroxybutyric acid) (PHB). A central, composite experimental design was used to optimize the composition of the culture medium for maximizing the productivity of PHB. Investigated were the effects of temperature, the initial culture pH, the ionic strength of the medium, the concentration of trace elements, the type of nitrogen source, and the carbon-to-nitrogen ratio. The optimal temperature for growth and PHB synthesis appeared to be 33°C; however, over the 25-37°C range, the effect of temperature was negligible. An initial pH value of 6.5 gave the best results; pH values that differed even slightly from the optimum reduced the culture performance. Typical culture characteristics were: 0.075/h maximum specific growth rate, 0.38 g/l h maximum specific sucrose consumption rate, and 0.15 g/l h maximum specific PHB production rate. PHB was lost because of hydrolysis in the stationary phase, suggesting critical importance of timing the harvest. Under the best conditions, PHB constituted up to 63% of dry cell mass after 93 h of culture. The average biomass yield coefficient on sucrose was about 0.4 kg/kg. Of the four nitrogen sources-ammonium chloride, ammonium sulfate, ammonium nitrate, and urea-used, only the first two supported the culture satisfactorily. The biomass and PHB showed clear yield maxima at 1.5 g/l ammonium chloride (C:N ratio ϭ 21.5) and 1.4 g/l ammonium sulfate (C:N ratio ϭ 28.3). The yields were higher with ammonium sulfate and were relatively more sensitive to changes in its concentration. Ionic strength had a strong negative effect on PHB productivity. The highest PHB yield occurred at 4 g/l phosphate buffer concentration. Iron appeared to have the potential to enhance the proportion of PHB in the cells.

Fed-batch culture of Alcaligenes latus, ATCC 29713, was investigated for producing the intracellular bioplastic poly(b±hydroxybutyric acid), PHB. Constant rate feeding, exponentially increasing feeding rate, and pH-stat fed batch methods were evaluated. pH-stat fed batch culture reduced or delayed accumulation of the substrate in the broth and led to signi®cantly enhanced PHB productivity relative to the other modes of feeding. Presence of excessive substrate appeared to inhibit PHB synthesis, but not the production of cells. In fed-batch culture, the maximum speci®c growth rate (0.265 h )1 ) greatly exceeded the value (0.075 h )1 ) previously observed in batch culture of the same strain. Similarly, the maximum PHB production rate (up to 1.15 g á l )1 á h )1 ) was nearly 8-fold greater than values observed in batch operations. Fed-batch operation was clearly superior to batch fermentation for producing PHB. A low growth rate was not a prerequisite for PHB accumulation, but a reduced or delayed accumulation of substrate appeared to enhance PHB accumulation. Under the best conditions, PHB constituted up to 63% of dry cell mass after 12 h of culture. The average biomass yield coef®cient on sucrose was about 0.35, or a little less than in batch fermentations. The highest PHB concentrations attained were about 18 g á l )1 . List of Symbolsconcentration of ammonium ion (mg á l )1 ) C C mass fraction of sucrose in the feed ()) C N mass fraction of ammonia in pH control solution ()) C T mass fraction of trace elements in feed solution ()) E element F C mass¯ow rate of the sucrose solution (g á h )1 ) F N mass¯ow rate of the ammonia solution (g á h )1 ) F s feed mass¯ow rate (g á h )1 ) F T mass¯ow rate of the trace element solution (g á h )1 ) m maintenance coef®cient ()) N concentration of nitrogen source (g á l )1 ) PHA polyhydroxyalkanoate PHB poly(b-hydroxybutyric acid) q S maximum speci®c sucrose consumption rate (g á l )1 á h )1 ) q P speci®c product formation rate (g á l )1 á h )1 ) q P max maximum speci®c product formation rate (g á l )1 á h )1 ) S total amount of substrate in fermenter at time t (g) SDS sodium dodecyl sulfate S 0 mass fraction of the substrate in feed ()) t time (h) t 0x-intercept in Eq.(1) (h) TES trace element solution or its amount in the medium (ml á l )1 ) X 0 total initial biomass in fermenter (g) X t total biomass in fermenter at time t (g) Y P/S product yield coef®cient on sucrose ()) Y P/X speci®c product yield coef®cient ()) Y R/N PHB-free biomass yield coef®cient on nitrogen source ()) Y R/S PHB-free biomass yield coef®cient on carbon source ()) Y R/T PHB-free biomass yield coef®cient on trace elements ()) Y X/S biomass yield coef®cient on sucrose ())Greek symbols d mass fraction of PHB in cells ()) l speci®ed growth rate (h )1 ) l max maximum speci®c growth rate (h )1 )

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