Vol. 55, No. 11

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

YOSHIHARU DOI,* YASUSHI KAWAGUCHI, YOSHIYUKI NAKAMURA, AND MASAO KUNIOKAResearch Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta, Midori-ku,

Yokohama 227, Japan

Received 5 June 1989/Accepted 25 August 1989

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

A variety of bacteria produce an optically active form ofpoly(3-hydroxybutyrate) (PHB) as an intracellular storagepolymer (6), and PHB is accumulated within cells as distinctgranules surrounded by membranes (9, 10). Recently, sev-eral bacterial species, such as Alcaligenes eutrophus (8, 21),Bacillus megaterium (11), Pseudomonas oleovorans (1, 26),and Rhodospirillum rubrum (2), have been shown to producepoly(3-hydroxyalkanoates) (PHA) incorporating 3-hydroxy-alkanoate units other than 3-hydroxybutyrate. These bacte-rial polyesters have attracted much attention as enviromen-tally degradable thermoplastics for a wide range ofagricultural, marine, and medical applications, since thepolyesters are hydrolytically (20, 29; Y. Doi, Y. Kanesawa,Y. Kawaguchi, and M. Kunioka, Makromol. Chem., inpress) and enzymatically (27, 30, 38, 40, 44) degradablepolymers. PHB homopolyester and copolyesters of 3-hy-droxybutyrate and 3-hydroxyvalerate are industrially pro-duced by ICI Ltd. in a controlled fermentation process bythe use of A. eutrophus (3, 21).The PHB synthetic pathway and its regulation have been

studied extensively in A. eutrophus (15-17, 33, 39, 41) andZooloea ramigera (4, 5, 12, 13, 31, 34, 35). The pathway ofPHB synthesis in the bacteria is well established. PHB issynthesized from acetyl coenzyme A (acetyl-CoA) by a

sequence of three enzymatic reactions. 3-Ketothiolase cata-lyzes the reversible condensation of acetyl-CoA to ace-

toacetyl-CoA. The intermediate is reduced to D-(-)-3-hy-droxybutyryl-CoA by NADPH-dependent acetoacetyl-CoAreductase, and PHB is then produced by the polymerizationof D-(-)-3-hydroxybutyryl-CoA by the action of PHB syn-

thase. In general, PHB is accumulated in cells under stressedgrowth conditions, such as nitrogen, phosphorus, or oxygen

* Corresponding author.

limitation, and its quantities decrease when limiting condi-tions are relaxed (6).

Despite intensive biochemical studies on PHB synthesis,relatively little is known about the regulation of PHB metab-olism. In addition, we have little knowledge about thecorrelation between PHB and other metabolic pathways. Inthis study, we investigated PHB and polyphosphate meta-bolic pathways in the industrially important organism A.eutrophus. A. eutrophus is known to accumulate polyphos-phate as a storage compound in cells (25). The PHB meta-bolic pathway and its regulation were studied by analysis ofsolid-state '3C nuclear magnetic resonance (NMR) spectra oflyophilized intact cells. Recently, we demonstrated theutility of the solid-state cross-polarization/magic-angle spin-ning (CP/MAS) '3C NMR technique for the quantitativedetermination of PHB in cells (7). The regulation of poly-phosphate metabolism was studied by analysis of high-resolution 31P NMR analysis of intact cells in suspension.The correlation between PHB and polyphosphate metabo-lism is discussed.

MATERIALS AND METHODS

Bacterial strain and stock cultures. A. eutrophus H16(ATCC 17699) was used in this study. A. eutrophus cellswere grown at 30°C for 24 h on a reciprocal shaker in 500-mlSakaguchi flasks containing 100 ml of a nutrient-rich me-dium. The nutrient-rich medium contained 10 g of fructose,10 g of polypeptone, 10 g of yeast extract, 5 g of meatextract, and 5 g of NaCl per liter of distilled water. Stockcultures of A. eutrophus were made by aseptically transfer-ring about 0.5 ml of a culture of cells on 0.5 ml of 20%glycerol aqueous solution into a sterile vial. The contents ofthe vial were then frozen and stored at -20°C until needed.Growth conditions and PHB synthesis. PHB synthesis was

carried out by a two-step batch cultivation of A. eutrophus.

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A. eutrophus cells were first grown at 30°C on a reciprocalshaker in 10 500-ml Sakaguchi flasks containing 100 ml ofnutrient-rich medium. The medium contained 10 g of poly-peptone, 10 g of yeast extract, 5 g of meat extract, and 5 g of(NH4)2SO4 per liter of distilled water. The cells were har-vested by centrifugation at 5,000 x g for 15 min. Under theseculture conditions, accumulation of PHB in the cells was notobserved. To promote PHB synthesis, about 4 g (dry weight)of asepitically centrifuged cells was transferred into a 2.6-liter jar fermentor (equipped with six conventional turbineimpellers and three baffles) in 1.0 liter of nitrogen-freemineral medium containing 20 g of butyric acid as the solecarbon source. The mineral medium contained 3.8 g ofNa2HPO4, 2.65 g of KH2PO4, and 0.2 g of MgSO4 per liter ofdistilled water. In addition, 1 ml of a microelement solutionwas added into the medium. The microelement solutioncontained the following (per liter of 0.1 N HCl): 9.7 g ofFeCl3, 7.8 g of CaCl2, 0.156 g of CUSO4 5H20, 0.119 g ofCoCl2, 0.118 g of NiCl2 - 6H20, and 0.062 g of CrCl2.Temperature and pH were automatically controlled at 30°Cand 7.0, respectively. Concentration of dissolved oxygenwas controlled in the range of 4 to 6 ppm. The cells werecultivated in the nitrogen-free medium and harvested bycentrifugation. The cells were washed once with 0.05 Mphosphate buffer (pH 7.0), centrifuged again, and thenlyophilized. When required, PHB was extracted from thelyophilized cells with hot chloroform in a Soxhlet apparatusand purified by reprecipitation with hexane.CP/MAS 13C NMR analysis. Solid-state 13C CP/MAS NMR

spectra of lyophilized cells were recorded at 67.8 MHz on aJEOL GX-270 spectrometer equipped with a CP/MAS ac-cessory at room temperature. A decoupling field of ca. 12 Gand a spinning rate of ca. 3.4 kHz were used. Free inductiondecays were generated by cross-polarization using a contacttime of 2.0 ms and a recycle time of 2.0 s. 13C NMR chemicalshifts were referenced to external tetramethylsilane.

31P NMR analysis. 31P NMR measurements of A. eutro-phus cells in suspension were made as follows. A. eutrophuscells were cultivated under different conditions and har-vested by centrifugation. The cells were washed and sus-pended in a buffer (pH 7.0) which contained 0.75 g of KCI,0.2 g of MgSO4, 34.6 g of PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)], and 9.7 g of MES [2-(N-morpholi-no)ethanesulfonic acid] per liter of distilled water. The cellsuspension was placed in 10-mm NMR sample tubes andkept at 4°C. 31P NMR spectra of cells in suspension wererecorded at 202 MHz on a JEOL GX-500 spectrometer in theFourier transform mode. The sample tubes were kept at 5°Cduring the NMR measurement. The 202-MHz 31P NMRspectra of cell suspensions were obtained with a repetitionrate of 1.0 s, a 450 pulse, 64,000 datum points, and 4,500accumulations. 31P NMR chemical shifts were referenced toexternal 85% phosphoric acid.The concentration of P043- in the culture solution was

determined by the 202-MHz 31P NMR analysis of the super-natant separated by centrifugation of the suspension of A.eutrophus cells.

'H NMR analysis. 'H NMR analysis of extracellularmetabolites from A. eutrophus cells was performed as fol-lows. The supernatants were separated by centrifugation ofthe cell suspensions, which were incubated under differentconditions. The 'H NMR spectra of supernatants wererecorded at 500 MHz on a JEOL GX-500 spectrometer. Tosuppress the 'H signal of water, gated homonuclear decou-pling with selective saturation of the water protons wasused. Typically, 100 scans were accumulated for 5 min for

quantitative determination of the metabolites. 'H NMRchemical shifts were referenced to internal sodium 4,4-dimethyl-4-silapentanesulfonate. The assignments of metab-olite peaks were confirmed by adding authentic samples ofeach of the metabolites to the medium.GPC analysis. Molecular weight data for PHB were ob-

tained at 40°C by using a Shimadzu 6A gel permeationchromatography system equipped with a 6A refractive indexdetector and a Shodex 80M column. Chloroform was used asthe eluant at a flow rate of 0.5 ml/min, and a sampleconcentration of 1.0 mg/ml was used. Polystyrene standardswith a low polydispersity were used to make a calibrationcurve.GC analysis. The gas chromatography analysis of butyric

acid in culture solutions was performed on a ShimadzuGC-8A system equipped with a flame ionization detector anda Unisole F-200 glass column (2 m). The culture solution wasseparated by centrifugation, and hexanoic acid was added asan internal standard in the supernatants. The pHs of thesupernatants were adjusted at 1.0 by addition of acid (5 NHCl), and samples (2 ,ul) were injected for analysis.

RESULTS

Accumulation and degradation of PHB. The growth char-acteristics of A. eutrophus and conditions of PHB synthesishave been extensively investigated (18, 36, 42). PHB isaccumulated within cells in the form of granules in thepresence of appropriate carbon sources after a growth-limiting nutrient, such as nitrogen, has been exhausted. Inthis work, we carried out a two-step batch cultivation of A.eutrophus. The microorganism was first grown for 24 h at30°C under aerobic conditions in nutrient-rich medium con-taining 10 g of yeast extract, 10 g of polypeptone, 5 g of meatextract, and 5 g of (NH4)2SO4 per liter of water. Under theseculture conditions, accumulation of PHB in cells was notobserved. The whole culture was centrifuged, and about 4.0g (dry weight) of cells was obtained. To promote PHBsynthesis, the cells were transferred into a nitrogen-freemedium (1 liter) containing 20 g of butyric acid as the solecarbon source. The cells were cultivated in a 2.6-liter jarfermentor at 30°C under aerobic conditions (dissolved oxy-gen concentration, 4 to 6 ppm) in the nitrogen-free culture ata pH of 7.0.

Figure 1 shows the time course of PHB accumulation in A.eutrophus cells during batch cultivation in the absence of anitrogen source. The concentration of butyric acid in theculture decreased with time and almost reached zero ataround 60 h of incubation. PHB was accumulated in A.eutrophus cells while butyric acid was present. After 48 h ofincubation, 5.2 g of PHB was produced per liter and 17.6 g ofbutyric acid was consumed per liter. The conversion ofbutyric acid into PHB by A. eutrophus was about 30% inselectivity. After butyric acid was exhausted, PHB contentin cells decreased with time, indicating that PHB is utilizedas a source of energy during starvation. The residual bio-mass, calculated as the difference between total dry cellweight and PHB content, increased slightly during thecourse of PHB accumulation (Fig. 1B). On the other hand,the residual biomass of cells decreased during the course ofPHB degradation. The weight-average molecular weight(Mw) of PHB was almost constant (930,000 + 30,000) duringthe batch cultivation, and its polydispersity (M/lMn) was asnarrow as 2.0 (Fig. 1C).The concentration of phosphate (PO43-) in the culture

decreased gradually under the growth-limiting conditions,

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APPL. ENVIRON. MICROBIOL.2934 DOI ET AL.

,-%1 4 =

-3 -

_ 2 -S_ 1 X

(B)

(C) 4 _

3 ic_) Nt

(A)C,H3 00-3CH-2CH2,C I

3 2w

3 L

SSB

4

20 40 60 80 100 120

Time (h)FIG. 1. Time course of accumulation and degradation of PHB in

A. eutrophus cells during batch cultivation in nitrogen-free medium(1 liter, pH 7.0) under aerobic conditions (dissolved oxygen, 4 to 6ppm) at 30°C. (A) Concentrations of butyric acid (0) and P043- (0).(B) Weights of PHB (0) and the residual biomass (0) in whole drycells. (C) Weight-average molecular weight (Mj) (0) and polydis-persity (MwIM,) (0) of PHB.

indicative of the uptake of phosphate by A. eutrophus (Fig.1A). As described below, A. eutrophus accumulated poly-phosphate in the cells together with PHB.

Solid-state CP/MAS 13C NMR analysis. Figure 2 shows thesolid-state CP/MAS 13C NMR spectra at 67.8 MHz oflyophilized intact cells of A. eutrophus without PHB (Fig.2A) and with PHB (Fig. 2B), together with the spectrum ofisolated PHB in powder form (Fig. 2C). In the spectrum ofthe cells without PHB, a number of broad lines were

observed. The peaks at 15 to 20 ppm were assigned to themethyl carbon resonances of proteins and fatty acids, andthe peaks at 25 to 30 ppm were assignable to the methylenecarbons of proteins, fatty acids, and cell wall components(23, 24, 32). The peaks at 50 to 60 ppm were assigned to theCa carbon resonances of the peptide backbones of proteins,and the resonances in the range of 170 to 180 ppm are due tothe carbonyl carbons of proteins (23, 24, 32). The broadeningof carbonyl carbon resonances of proteins arises from 13C-14N dipolar interaction of the peptide bonds (19).

Figure 2B shows the spectrum of lyophilized cells withPHB (44% of the cellular dry weight). Four narrow linesappeared at very strong intensities in addition to the weaklines associated with bacterial cellular material. These fourpeaks were assignable to the methyl (21.3 ppm; carbon 4 inFig. 2A and 3A), methylene (42.8 ppm; carbon 2 in Fig. 2Aand 3A); methine (68.5 ppm; carbon 3 in Fig. 2A and 3A) andcarbonyl (169.7 ppm; carbon 1 in Fig. 2A and 3A) carbonresonances of PHB (7, 22).

In order to study the synthetic pathways of PHB andbacterial cellular material in A. eutrophus, 13C-labeled ace-

sse

200 100ppm

50

(C)

6

FIG. 2. Solid-state CP/MAS 13C NMR spectra at 67.8 MHz oflyophilized A. eutrophus cells without PHB (A), lyophilized cellswith PHB (44% of the cellular dry weight) (B), and isolated PHB inpowder form (C). SSB, Spinning side band of CO resonances. "Cchemical shift assignments are given in the text.

tate was used as the sole carbon source. In this experiment,A. eutrophus cells were first grown in a nutrient-rich mediumand then transferred into two nitrogen-free culture mediacontaining 20 g of sodium [1-'3C]acetate per liter (6.0% '3C)

and 20 g of sodium [2-13C]acetate per liter (6.0% '3C),

respectively. The cells were cultivated in these media for 10h at 30°C, harvested by centrifugation, and finally lyophi-lized.

Figure 3 shows the solid-state CP/MAS 13C NMR spectraof lyophilized intact cells with 5% (wt/wt) PHB content. ThePHB from [1-13C]acetate exhibited specific enhancements inthe intensities of methine (peak 3) and carbonyl (peak 1)carbon resonances (Fig. 3A). In contrast, the PHB from[2-13C]acetate displayed specific enhancements in the inten-sities of methyl (peak 4) and methylene (peak 2) carbonresonances (see Fig. 3B). When the intensities of peaks frombacterial cellular material in spectra A and B of Fig. 3 are

compared, one may note that spectrum A of the cells from[1-'3C]acetate shows relatively strong intensity for the car-

N

-

IA-t M

Emm

-6 12

C: 8

13: 4

l

16

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PHB AND POLYPHOSPHATE IN A. EUTROPHUS 2935

CH3 0t 0-3-CH -CH2-7C

3

SSB

(A)

4

Suga r

(B)

in

(A)Inorganicphosphate

(B)

Pol Yphosphate

PyrophosphateNTPc,NDPoQ

SSB

260 1 0 i00ppm

50 -6FIG. 3. Solid-state CP/MAS "3C NMR spectra at 67.8 MHz of

lyophilized A. eutrophus cells grown on [1-'3C]CH3COONa (6%13C) (A) or [2-'3C]CH3COONa (6% 13C) (B). PHB in A. eutrophuscells was 5% of the cellular dry weight.

bonyl carbon resonance of protein at 170 to 180 ppm. Thisresult suggests that the synthesis of proteins takes place untilall the ammonia residue in cells has been consumed. Thisprotein synthesis is partially responsible for a slight increasein the residual biomass of cells during the course of PHBaccumulation, as pointed out in Fig. 1B.

High-resolution 31P NMR analysis. A. eutrophus is knownto accumulate polyphosphate as a storage compound in cells(6). In order to study the correlation between PHB andpolyphosphate synthesis, high-resolution 31P NMR spectraof A. eutrophus cells were recorded in suspension at dif-ferent time periods after butyric acid was added as the solecarbon source into a nitrogen-free medium. Figure 4A showsthe 202-MHz 31P NMR spectrum of the cells without PHBbefore the addition of butyric acid, and Fig. 4B shows thespectrum of cells with PHB (54% of the cellular dry weight)cultivated at 48 h after the addition of butyric acid. Thechemical shift assignments of 31P resonances were madealong those reported previously (14, 45). The broad 31presonance at -23 ppm characteristic of polyphosphate was

10 0 -10ppm- I -30.

-20 -30

FIG. 4. 202-MHz 31P NMR spectra of A. eutrophus cells insuspension at 5°C. (A) Cells without PHB. (B) Cells with PHB (54%of the cellular dry weight). NTP and NDP, Nucleotide tri- anddiphosphates, respectively. Chemical shifts are in parts per millionfrom external 85% phosphoric acid.

not observed in the cells without PHB (Fig. 4A). However,the polyphosphate peak at -23 ppm was detected in thespectra of cells with PHB (Fig. 4B). It was found that theintensity of the polyphosphate peak at -23 ppm increasedwith an increase in the PHB content in cells. Thus, it may beconcluded that the accumulation of intracellular polyphos-phate is closely correlated with the accumulation of PHB inA. eutrophus.Carbon and phosphorus starvation. In order to elucidate

the roles of PHB and polyphosphate as storage compoundsof A. eutrophus, the cells with 53% (wt/wt) PHB weretransferred into a carbon, phosphorus, and nitrogen-freemineral solution which contained 0.75 g of KCl, 0.2 g ofMgSO4, 34.6 g of PIPES, and 9.7 g of MES per liter andincubated at 30°C for 48 and 72 h under aerobic andanaerobic conditions, respectively. The PHB content in thecells decreased during the carbon and phosphorus starva-tion. The PHB contents in the aerobic cells were 44 and 38%of the cellular dry weight after 48 and 72 h, respectively. Incontrast, the PHB contents in the anaerobic cells were 20and 8% after 48 and 72 h, respectively. Thus, the rate ofPHB

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3-Hyd roxy-butY rate

3-Hyd roxy-butyrate

DSS

Acetate

3-Hyd roxy-buty rate

ButY rate

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

ppm from DSSFIG. 5. 500-MHz 'H NMR spectrum of the supernatant separated by centrifugation of the suspensions of anaerobic A. eutrophus cells

under the carbon and nitrogen starvation conditions. DSS, 4,4-Dimethyl-4-silapentanesulfonate.

degradation under anaerobic conditions was much fasterthan that under aerobic conditions. Under both aerobic andanaerobic conditions, however, no appreciable degradationof polyphosphate was observed in the 31P NMR spectra ofA.eutrophus cells in suspension.The excretion of several metabolites by A. eutrophus was

observed under anaerobic conditions in the carbon- andnitrogen-free solution containing 3.8 g of Na2HPO4, 2.65 g ofKH2PO4, and 0.2 g of MgSO4 per liter. Figure 5 shows the500-MHz 'H NMR spectrum of the supernatant separated bycentrifugation of the suspensions ofA. eutrophus cells whichwere incubated for 72 h under anaerobic conditions. Themajor components of extracellular metabolites were 3-hy-droxybutyrate and acetate. Butyrate, lactate, and ethanolwere detected as minor metabolites. In contrast, the produc-tion of extracellular metabolites by A. eutrophus was notobserved under aerobic conditions.

DISCUSSION

The data reported here suggest that A. eutrophus cellsaccumulate PHB and polyphosphate simultaneously whengrowth is limited by the exhaustion of the nitrogen source. InA. eutrophus, PHB is synthesized via a three-step metabolicpathway from acetyl-CoA, as represented by the scheme in

Fig. 6. The first step is catalyzed by the enzyme 3-ketothio-lase, which condenses acetyl-CoA to acetoacetyl-CoA (15).The acetoacetyl-CoA is reduced to D-(-)-3-hydroxybutyryl-CoA by an NADPH-dependent acetoacetyl-CoA reductase(16). In the last step, the enzyme PHB synthase catalyzes thehead-to-tail polymerization of the monomer to PHB (17).The solid-state 13C NMR spectra (Fig. 3) of the lyophilizedintact cells with PHB from 13C-labeled acetate indicated thatthe carbonyl carbon of acetate is selectively incorporatedinto the carbonyl and methine carbons of PHB, while themethyl carbons of acetate are incorporated into the methyl-ene and methyl carbons of PHB. The selective incorporationof each 13C-labeled carbon of acetate into PHB confirms theformation of acetoacetyl-CoA by the head-to-tail condensa-tion of two molecules of acetyl-CoA.Only 30% of the butyric acid consumed was converted

into PHB by A. eutrophus even when growth was limited(Fig. 1). The relatively low selectivity of PHB conversionfrom butyric acid may be caused by the concomitant forma-tion of polyphosphate under aerobic conditions. Polyphos-phate is accumulated as an intracellular reserve compound ina variety of microorganisms, and it is synthesized by theaction of polyphosphate kinase, which catalyses the transferof the terminal phosphoryl group of ATP to polyphosphate

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PHB AND POLYPHOSPHATE IN A. EUTROPHUS 2937

C02 4--TCA

ATP

h(9hPolypho sphal

AcetateATP, CoA-SH

(1)AMP, PPi

ADP

CoA-SH

-w D(-)-3-HydroxNADPH+H

te (3) (5)(NADP

D(-)-3-Hydroxybutyryl-CoA (4) o PPHBCoA-SH

FIG. 6. Schematic pathways of PHB and polyphosphate metabolism in A. eutrophus. Enzymes are denoted as follows: (1), acetyl-CoAsynthetase; (2), 3-ketothiolase; (3), NADPH-linked acetoacetyl-CoA reductase; (4), PHB synthase; (5), PHB depolymerase; (6), dimerhydrolase; (7), phosphate acetyltransferase; (8), acetate kinase; (9), polyphosphate kinase.

(6). When growth of A. eutrophus cells is limited underaerobic conditions, acetyl-CoA is utilized to form ATP aswell as PHB. An excess amount of ATP molecules isgenerated via the common pathway of respiration and con-verted into polyphosphate as a storage compound (Fig. 6).The roles ofPHB and polyphosphate in A. eutrophus cells

were investigated under conditions of carbon, phosphorus,and nitrogen source starvation. Under both aerobic andanaerobic conditions, PHB was degraded, whereas littlepolyphosphate was degraded. This result indicates that PHBserves as the major endogenous substrate. Kaltwasser (25)reported that polyphosphate in A. eutrophus cells functionsas a phosphorus reserve rather than as an energy reserve.The rate of PHB degradation under anaerobic condition

was much faster than that under aerobic conditions. PHB isknown to be decomposed into acetyl-CoA via D-(-)-3-hydroxybutyrate (28, 43). Under aerobic conditions, acetyl-CoA is oxidized completely to CO2 and H20 for energygeneration, resulting in the effective formation of ATP.Under an aerobic conditions, acetate and 3-hydroxybutyratewere produced as the major extracellular metabolites fromA. eutrophus cells (Fig. 5). The anaerobic conversion ofacetyl-CoA to acetate is likely to be coupled with thephosphorylation of ADP to generate ATP (Fig. 6). As aconsequence, relatively large amounts of PHB are degradedunder anaerobic conditions.

ACKNOWLEDGMENT

This study was supported in part by a grant-in-aid for ScientificResearch from the Japanese Ministry of Education, Science andCulture.

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