Regulation of PHB metabolism in Alcaligenes eutrophus

Canadian Journal of Microbiology (Impact Factor: 1.18). 02/2011; 41(13):44-49. DOI: 10.1139/m95-166

ABSTRACT Metabolites associated with the poly(3-hydroxybutyrate) (PHB) biosynthetic pathway in Alcaligenes eutrophus were measured to gain an insight into the regulation of PHB synthesis in vivo. Alcaligenes eutrophus was grown in carbon-limited chemostat culture to provide bacteria producing negligible PHB, and in nitrogen-limited chemostat culture to yield PHB-synthesizing bacteria. 3-Hydroxybutyryl-CoA (3HBCoA) was detected only in polymer-accumulating bacteria. The level of coenzyme A (CoASH) was approximately three times higher in the absence of PHB synthesis, in accord with the putative role of this metabolite in the regulation of 3-ketothiolase. The level of acetoacetyl-CoA was, however, similar in PHB-accumulating and nonaccumulating bacteria, suggesting that NADPH-acetoacetyl-CoA reductase may regulate PHB synthesis in bacteria grown under carbon limitation. Immediately after nitrogen exhaustion in batch culture of A. eutrophus, there was an initial large decrease in the weight-average molecular weight, which corresponded to the rapid disappearance of CoASH and the maximum level of 3HBCoA. The decrease in the rate of PHB synthesis in batch culture was consistent with regulation involving NADPH-acetoacetyl-CoA reductase. The disappearance of 3HBCoA coincided with the cessation of PHB synthesis and the maximum level of acetyl-CoA.Key words: metabolites, PHB biosynthesis, regulation, Alcaligenes eutrophus, molecular weight.

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    ABSTRACT: Batch and two-stage batch culture of Ralstonia eutropha ATCC 17697 (Alcaligenes eutrophus) and Alcaligenes latus ATCC 29712, were investigated for producing the intracellular bioplastic poly-â-hydroxybutyric acid (PHB) using shake flasks technique. The highest growth and PHB production of Ralstonia eutropha ATCC 17697 and Alcaligenes latus ATCC 29712 were recorded on (Kim et al 1994a) medium containing glucose or sucrose (as a carbon source), respectively. Ammonium sulfate was the best nitrogen source for PHB production by both strains. The productive medium which contain carbon source and ammonium sulfate in C/N ratio of 12.57 gave the highest PHB either by Ralstonia eutropha ATCC 17697 and Alcaligenes latus ATCC 29712. Washed cells of Alcaligenes strains produced PHB concentration and content higher than crude cells at different limiting nutritional treatments. Applying the two stage batch fermentation with nitrogen limitation increased the PHB content (%) of R.eutropha ATCC 17697 and A.latus ATCC 29712 cells about 48.43 % and 14.29 %, respectively, as compared with that obtained in batch culture using shake flasks technique. INTDRODUCTION Poly-â-hydroxybutyrate (PHB) is an intracellular microbial thermoplastic that is widely produced by many bacteria (Braunegg et al., 1998). In terms of molecular weight, brittleness, stiffness, melting point and glass transition temperature, the PHB homopolymer is comparable to some of the more common petrochemical-derived thermoplastics such as polypropylene. Therefore in certain application, PHB can directly replace some more traditional, nonbiodegradable polymers (Poirier et al., 1995). Alcaligenes eutrophus, non-growth-associated PHB producer, require the limitation of an essential nutritional element such as N, P, Mg, K, O or S in the presence of an excess of carbon source for the efficient synthesis of PHB. Alcaligenes latus, a-growth-associated PHB producer, accumulates PHB up to 80 % of dry cell weight without limitation of any nutrient. Various carbohydrates in the growth media including glucose, sucrose, lactic acid, butyric acid, valeric acid and various combinations of butyric and valeric acids are utilized as carbon sources for the production of bioplastic by some bacterial strains (Kim et al., 1994b;Yu et al., 1998 and Mitomo et al., 1999). The presence of inorganic chemicals such as ammonia or ammonium salts as a source of nitrogen is an important requirement during the growth phase in order to maximize the concentration of biomass responsible for accumulation of PHB. The best growth of A.eutrophus and PHB production was obtained with ammonium sulfate in a synthetic medium containing 3 % glucose at pH 7 (Beaulieu et al., 1995). Yu et al. (1998) reported that, higher C/N ratio (deficiency of nitrogen in the medium) would promote the production of the polymer by microorganisms. Grothe et al. (1999) reported that, the C/N ratio in A.latus medium is wide (21.5) and probably explained by the large amount of PHB in the cells which does not contain nitrogen. The production cost of PHB is quite as compared with that of synthetic nondegradable plastic, and much effort has recently been devoted to make this process economically more feasible by improving the productivity and developing a new separation process (Hankermeyer and Tjeerdema, 1999). So, the present investigation was designed to determined the optimum carbon & nitrogen sources, C/N ratio as well as optimum nutrient limitation for maximizing the accumulation of PHB by Ralstonia eutropha ATCC 17697 and Alcaligenes latus ATCC 29712 using shake flask as a batch and two-stage batch culture.