Physiology and biochemistry of poly 3-hydroxybutyrate production by Alcaligenes eutrophus.

Alcaligenes eutrophus NCIMB 40529 was grown in continuous culture (D=0.1h-1) under glucose, ammonium, potassium and oxygen limitation. The rates of PHB production (qPHB) were highest during ammonium-limited growth and lowest during glucose-limited growth. The PHB content of cells grown under ammonium limitation exhibited an inverse relationship to the dilution rate (D), whilst the rate of substrate (glucose) utilisation (qS) increased with D. It was concluded that glucose is utilised increasingly for the production of non-polymer biomass (NPB) as D increases, with the resulting diminution of PHB synthesis. PHB production from alternative carbon substrates was also studied in ammonium-limited chemostat culture (D=0.1h-1). PHB content, the rate of PHB production (qPHB) and qS varied substantially with the nature of the carbon source in the order lactate pyruvate glucose/gluconate fructose. qPHB was not related to umax in batch culture or to the relative oxidation-reduction states of the substrates, but was directly related to the qSsurplus i.e. the rate of substrate utilisation above that required solely for the production of NPB. The substantially higher qPHB from lactate than from glucose in ammonium limited chemostat culture reflected the inability of the cell to regulate lactate utilisation at D umax, whereas glucose utilisation is partially repressed under these conditions. Washed cells prepared from, glucose- and lactate-limited chemostat cultures (D=0.1h-1) metabolised glucose and lactate respectively at significantly faster rates than the cultures from which they were derived. Both types of washed cells synthesized PHB, indicating that the biosynthetic enzymes are constitutive; this was confirmed by assay of a representative biosynthetic enzyme in broken cells prepared from carbon- and ammonium-limited chemostat cultures. Washed cells also excreted pyruvate. It is suggested that carbon-limited cells are derepressed for carbon substrate uptake and catabolism, and that pyruvate excretion reflects the limited capacity of non-growing cells for pyruvate utilisation. Citrate synthase, a potentially important regulatory enzyme of the tricarboxylic acid cycle in A. eutrophus, was purified. The enzyme consisted of a single type of subunit with a Mr of 47,000, and exhibited a native Mr of 227,000. Citrate synthase activity displayed a hyperbolic dependence on both oxaloacetate (Km=1.2mM) and acetyl Co A (Km= 94uM) concentration, and a sigmoidal dependence of inhibition by NADH (I50=0.62mM). It is concluded that, apart from its insensitivity to AMP, the enzyme is similar to citrate synthases purified from other Gram-negative aerobes.