Nuclear Magnetic Resonance Studies of Poly(3-Hydroxybutyrate) and Polyphosphate Metabolism in Alcaligenes eutrophus

Abstract

The metabolic pathways of poly(3-hydroxybutyrate) (PHB) and polyphosphate in the microorganism Alcaligenes eutrophus H16 were studied by 'H, '3C, and 31P nuclear magnetic resonance (NMR) spectroscopy and by conventional analytical techniques. A. eutrophus cells accumulated two storage polymers of PHB and polyphosphate in the presence of carbon and phosphate sources under aerobic conditions after exhaustion of nitrogen sources. The solid-state cross-polarization/magic-angle spinning 13C NMR spectroscopy was used to study the biosynthetic pathways of PHB and other cellular biomass components from 13C-labeled acetate. The solid-state 13C NMR analysis of lyophilized intact cells grown on [1-_3C]acetate indicated that the carbonyl carbon of acetate was selectively incorporated both into the carbonyl and methine carbons of PHB and into the carbonyl carbons of proteins. The 31P NMR analysis of A. eutrophus cells in suspension showed that the synthesis of intracellular polyphosphate was closely related to the synthesis of PHB. The roles of PHB and polyphosphate in the cells were studied under conditions of carbon, phosphorus, and nitrogen source starvation. Under both aerobic and anaerobic conditions PHB was degraded, whereas little polyphosphate was degraded. The rate of PHB degradation under anaerobic conditions was faster than that under aerobic conditions. Under anaerobic conditions, acetate and 3-hydroxybutyrate were produced as the major extracellular metabolites. The implications of this observation are discussed in connection with the regulation of PHB and polyphosphate metabolism in A. eutrophus.

Methods for Detection and Quantification of Polyphosphate and Polyphosphate Accumulating Microorganisms in Aquatic Sediments

“…The 31 P-NMR spectroscopic technique was used for the detection and study of Poly-P in different organisms, including bacteria, yeasts, fungi, and algae (e.g., NAVON et al, 1977;SIANOUDIS et al, 1986;ROBERTS, 1987;DOI et al, 1989;BONTING et al, 1993b). Furthermore, 31 P-NMR has been widely used to study the Poly-P storage in activated sludge (e.g., FLORENTZ et al, 1984;UHLMANN et al, 1990;JING et al, 1992;RÖSKE and SCHÖNBORN, 1994) and phytoplankton samples (FEUILLADE et al, 1995).…”

Fungal degradation of polyhydroxyalkanoates and a semiquantitative assay for screening their degradation by terrestrial fungi

“…organisms [25][26][27][28][29]. Principally the following approaches were used to show bacterial PHB degradation: (1) as weight loss of PHB strips incubated in natural environments [1,30]; (2) as loss in turbidity on PHB-containing 'polymer overlayer plates' [9]; (3) as loss in turbidity on PHB-containing simple mineral agar plates [31,32]; (4) quantitatively as extracellular PHB depolymerase activity causing a decrease in turbidity of insoluble PHB [6,[33][34][35]; (5) spectrophotometrically by quantitative determination of crotonic acid as the product of PHB degradation [36,37]; (6) scanning electron microscopically by recording changes of material exposed to microbial enzymatic attack [11,20,21,38]; and (7) by more sophisticated biochemical methods such as gas chromatography (GC) and high pressure liquid chromatography (HPLC) [39,40], and nuclear magnetic resonance spectroscopy (NMR) [41].…”

Application of cybernetic models to metabolic engineering: Investigation of storage pathways

“…Low values of this ratio are indicative of S 2 limitation. It is shown through simulation of this model framework, and inferred by experimental investigations (Brandl et al, 1988;Doi et al, 1989) that these two characteristics have a direct bearing on pathway performance. Figure 2 (lower plate) shows a situation in which the system is supplemental nutrient rich at various values of max 1 / max 2 .…”