JOUR~AI. OF FSP.A~STATION AND BIOE~OmEEPdNO Vol. 74, No. 5,288-291. 1992

Accumulation of Polyphosphate and Substrate Gas Utilization Efficiency in PHB Accumulation Phase of Autotrophic Batch

Culture of Alcaligenes eutrophus ATCC17697 "r KENJI TANAKA, ~ AYAAKI ISHIZAKI, ~* AND PETER F. STANBURY 2

Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812, Japan I and Division of Bioscience, Hatfield Polytechnic, Hatfield, Herts.,

AL 10 9AB, United Kingdom 2

Received 18 May 1992/Accepted 19 August 1992

The consumption of phosphate, formation of polyphosphate and the intracellular ATP level were com- pared in exponential phase and PHB-accumulating cells of Alcaligenes eutrophus. The phosphate consump- tion (mg per g dry cell weight) by PHB-accumulating cells was larger than that by exponentially growing cells. The phosphate consumed by the PHB accumulating cells was converted to polyphosphate, which was a storage substance for phosphorus. The intracellular ATP concentration in PHB-accumulating cells (ex- pressed as/anol g-1 protein) was 1.5 times that in exponentially growing cells. The same proportional increase in H2 oxidation was observed. From this result it was observed that the ATP pool size expressed in terms of ATP/g protein per tool H2 product was the same in both culture phases.

Poly-3-hydroxybutyric acid (PHB) is a raw material for the manufacture of biodegradable plastics. In a previous study (1), we reported on the conditions for the produc- tion of PHB from carbon dioxide by autotrophic batch culture of the hydrogen oxidizing bacterium, Alcaligenes eutrophus. We demonstrated that the amount of hydrogen which was required for the fixation of 1 mol CO2 in the PI-IB accumulation phase was about 1.5 times that required during exponential cell growth (1). Furthermore, it was necessary to supply phosphate to the culture medium during cultivation to prolong the fermentation, and the amount of phosphate consumed per g dry cell weight in the PHB accumulation phase was larger than that in the exponential growth phase.

In this paper, phosphorus consumption and metabolism were investigated during the PHB accumulation phase. The efficiency of carbon dioxide fixation during the ex- ponential growth phase and the PHB accumulation phase was also considered from the viewpoint of intracellular ATP level. In addition, the relationship between hydrogen consumption, product formation and the intracellular ATP pool was investigated.

MATERIALS AND METHODS

Microorganism and culture conditions The micro- organism used was Alcaligenes eutrophus ATCC 17697 T. The seed culture was prepared by using a shake flask, and the main culture was performed by using a recycled-gas closed-circuit culture system. The media, substrate gasses, pH, temperature, agitation speed and gas feeding rates were the same as previously reported (2). The working volume was 150 ml. PHB accumulation in the cells was induced by oxygen-limited conditions (1).

Analytical methods The composition and consump- tion of substratc gasscs in the system, the partial pressure of dissolved oxygen in thc culture liquid, and the concen-

* Corresponding author.

trations of biomass, PHB and protein were determined as described previously (1, 2). Phosphate concentration was determined by the method of Allen (3). Cellular polyphos- phate was extracted and determined according to Wiame (4). Soluble polyphosphate was extracted from acetone- dried cells (obtained from 10 ml of culture broth) with cold 0.5 N perchloric acid (PCA; twice for 15 min at 4°C). The insoluble polyphosphate was extracted with hot 0.5 N PCA (twice for 15 min at 70°C) from the above cells after removing fat with ethanol and ethanol-ether treatment. Polyphosphate was determined by precipitation with Ba 2+ at pH 4.5 and measuring the dry weight.

The amount of intracellular ATP was determined by spectrophotometric method (5, 6). Ten milliliters of broth was removed from the jar fermentor and immediately added to cold 0.5 N perchloric acid (at 0°C, final concen- tration 0.5 N), and ATP was extracted for 15 min. The cells were removed from the suspension by centrifuga- tion and the supernatant was neutralized with chilled 5 M KOH containing 1 M triethanolamine. The precipitated KC104 was removed by centrifugation. After the sample was concentrated by the freeze-drying method, the amount of ATP was determined by the spectrophoto- metric method using NADH. The ATP solution (0.1 ml) was added into 0.8 ml of NADH buffer solution which was composed of 10 ml of 1 M triethanolamine-HCl buffer (pH 8.0), 1 M KC1, 1 M MgCI2, 40 ml of distilled water and 0.6ml of 10mg/ml NADH solution. Ten pl of 1 M 3-phosphoglyceric acid solution and 5/A glyceraldehyde- 3-phosphate dehydrogenase (10mg/ml) solution were added into the NADH buffer containing ATP. After that, 5 pl of phosphoglycerate kinase solution was added and the reaction was started. The decrease of optical absorb- ance of NADH was monitored at 340 nm for 2 min with a spectrophotometer (Uvidec-320, Jas. Co., Ltd., Tokyo). The amount of ATP was obtained by calculating from the decrease of NADH.

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VOL. 74, 1992 SUBSTRATE UTILIZATION EFFICIENCY OF A. EUTROPHUS 289

RESULTS

Phosphate c o n s u m p t i o n in hatch culture As re- ported previously (1), the autotrophic batch cultivation of A. eutrophus was prolonged by supplementing concen- trated KH2PO4 to the culture during the fermentation. The phosphate concentration was 0.349 g/l at the start of cultivation and the phosphate was exhausted when the cell concentration reached about 7 g/l. After that, 1 ml aliquots of the concentrated phosphate solution (50g/l of KH2PO4) were manually added through a sterile filter so that phosphate in the culture liquid was not ex- hausted. The phosphate concentration was not main- tained above 0.05 g/I throughout the fermentation. By supplementing with phosphate, the final cell concentra- tion in batch culture increased to 60 g/l, compared with 50 g/! if additional phosphate was not added. The phos- phorus consumption per g dry cell weight in the batch culture was calculated from the amount of supplemented phosphate, and is shown in Fig. 1. The phosphorus con- sumption was constant (about 17.5 mg-P/g-dry cell weight) during the exponential growth phase and accumulation of PHB was not observed. After 16 h cultivation, oxygen- limited conditions were established and PHB accumula- tion in the cell was observed. Phosphorus consumption by the microorganism increased as the PHB content of the cells rose. The phosphorus consumption and the percent- age of PHB at the end of cultivation were 22 mg/g-dry cell weight and 58.2%, respectively. The phosphorus con- sumption by the cells in the PHB accumulation phase was 1.26 times higher than that in the exponential growth phase.

P o l y p h o s p h a t e accumulat ion in batch culture The increase in phosphorus consumption during the PHB accu- mulation phase implied the accumulation of some phos- phorus compounds in the cells. According to Kaltwasser (7), the hydrogen-oxidizing bacterium, Hydrogenomonas strain 20, accumulated acid-insoluble polyphosphate in the ceils under ammonium limitation. Thus, polyphosphate were extracted from A. eutrophus with 0.5 N perchloric acid (PCA) and its concentration was determined by Ba 2+ precipitation. Polyphosphate was only extractable from the cells with hot PCA and not with cold PCA, which indicated that the polyphosphate accumulated in the

3( _160

_ /

2C _

o p 8

~ _ 1 0

I I I I I go o 0 10 20 30 40 50 Cultivation time (h)

FIG. 1. Relationship between phosphorus consumption and PHB accumulation in autotrophic batch culture of A. eutrophus. Symbols: t~, phosphorus consumption (P-rag/dry cell weight-g); O, PHB content (%).

60

5O PI tB

3( 1.5

~2o 1.o 8

10 0.5 ~

o I'o 2'o 'o ,'o 5'0 'o Cultivation time (h}

FIG. 2. Time courses for accumulation of PHB and polyphos- phate in batch culture of A. eutrophus. Symbols: D, polyphos- phate; ©, PHB.

cells was insoluble polyphosphate. Figure 2 shows the accumulation of polyphosphate (as the barium salt) dur- ing the phosphate supplemented batch culture of A. eutrophus. The microorganism accumulated polyphos- phate during the PHB accumulation phase for as long as phosphate was present in the medium. However, the amount of accumulated polyphosphate was not very large (15 mg/g dry cell weight at the end of cultivation) compared with that of PHB. The accumulation of poly- phosphate was closely related to the accumulation of PHB and the percentage of polyphosphate content-in the cells became higher as the percentage of PHB content in the cells increased. A similar result has been reported for the heterotrophic culture ofA. eutrophus by Doi et al. (8). In this case, the uptake of phosphate and accumulation of polyphosphate took place rapidly during PHB accu- mulation when the microorganism had been previously starved of phosphate for a long period (data not shown). Furthermore, it has been shown (9, 10) that in Aerobacter aerogenes, if growth and nucleic acid synthesis are halted due to a nutritional imbalance condition such as starva-

0 D

2 --

1 --

0 I I I I I i0 20 30 40 50

PHB content in cells ( % )

FIG. 3. Relationship between PHB content in cell and intracellu- far level of ATP. Symbols: O, phosphate was not supplemented during cultivation; &, phosphate was supplemented during cultiva- tion.

290 TANAKA ET AL. J. FERmenT. BIOEnO.,

TABLE 1. Intracellular level of ATP and gas consumption in exponential growth phase and PHB accumulation phase of A. eutrophus

H2 consumption Intracellular ATP ATP pool size ~ per tool-CO2 fixed Growth phase (mol/mol product) (pmol/g biomass) (pmol/g protein) (pmol ATP/g protein per tool H2 oxidized)

Exponential 19.6 5.38 6.4 0.082 PHB accumulation 29.5 3.21 10.5 0.089

a These values were estimated by dividing the intracellular ATP (pmol/g protein) by the H2 and CO2 consumption (moles) required for the for- mation of l-tool product.

tion of sulfur or previous starvation of phosphorus, poly- phosphate accumulation in cells is observed. Similarly in our experiment, polyphosphate accumulated in the cells under oxygen limited conditions, which caused PHB accumulation and halted the synthesis of nucleic acid and protein, but it was not accumulated in the exponential growth phase even in presence of excess phosphate.

Intracellular A T P level in the P H B accumula t ion phase The stoichiometry obtained previously (1, 2) for the autotrophic cultivation of A. eutrophus showed that hydrogen consumption for the fixation of 1 mol of CO2 during the PHB accumulation and storage was 1.5 times that required during the exponential growth phase. Thus, we investigated the relationship between the intracellular ATP concentration and PHB content of PHB accumulat- ing cells. The intracellular ATP concentration was deter- mined for several batch cultures in which the percentages of PHB content were different. The results are shown in Fig. 3. The intracellular ATP level per gram dry cell weight decreased as the percentage of PHB content increased, and there was no significant difference in the ATP level between phosphate-supplemented and non-supplemented cultures. However, PHB accounts for a considerable pro- portion of the biomass during the accumulation phase, which makes dry cell weight a poor parameter for the cal- culation of specific ATP levels. Thus, the amount of ATP per g cell protein was calculated. Using this calculation, the specific ATP concentration is seen to increase from 6.4pmol/g protein in exponential phase cells to 10.5 pmol/g protein in cells containing 50% PHB (Fig. 3). Table 1 summarizes the data for intracellular ATP concen- tration and hydrogen consumption for the two cell types. From Table 1, it can be seen that the size of the ATP pool (in terms of pmol ATP/g protein) is in proportion to hydrogen consumption, both the ATP pool and hydrogen consumption during the PHB accumulation phase being approximately 1.5 times the respective values during exponential growth. This is shown in Table 1 by the very similar ATP pool size expressed in terms of ATP/ g protein per tool H2 product in both phases.

DISCUSSION

Our results show that the increased phosphate uptake in PHB-accumulating cells may be partially accounted for by the storage of insoluble polyphosphate. A similar result has been reported by Doi et al. (8) for the hetero- trophic growth of A. eutrophus. In this case, the uptake of phosphate took place rapidly during PHB accumula- tion when the organism had been starved of phosphate for long periods. Furthermore, it has been shown in Aero- bacter aerogenes that polyphosphate accumulation occurs if growth and nucleic acid synthesis is stopped by a nutri- tion imbalance such as sulfur limitation, low pH or pre vious starvation of phosphate (9, 10). The difference in hydrogen consumption between the PHB accumulation phase and exponential growth of A. eutrophus is an

interesting bioenergetic observation. This phenomenon may indicate that ATP generation accompanying hy- drogen oxidation and/or the utilization efficiency of ATP for biosynthesis are different in these two culture phases. It has been speculated that 2 or 3 mol of ATP will be gen- erated by the oxidation of I mol of hydrogen during the autotrophic growth of A. eutrophus (12, 13). However, it is impossible to measure actual amount of ATP generated from substrate oxidation in aerobic fermentation. Thus, in this study, the intracellular ATP concentration (ATP pool) was determined in the two phases. The size of the ATP pool is a result of the balance between ATP synthesis and utilization. It was demonstrated that the size of the ATP pool (in terms of pmol ATP g- l protein) was in pro- portion to hydrogen consumption, and both the ATP pool and hydrogen consumption during the PHB accu- mulation phase were approximately 1.5 times the respec- tive values during exponential growth. This result may pre- dict that the amount of ATP generated by hydrogen oxi- dation was the same in both phases. It is possible that the integration of hydrogen oxidation and ATP consump- tion is not as well controlled under oxygen-limited condi- tions, resulting in a high ATP pool. However, the possi- bility of the conversion of ATP to polyphosphate by poly- phosphate kinase during the accumulation phase should not be ignored. It was also reported that polyphosphate for- mation and the activity of ATP synthesis in production of GMP employing Brevibacterium ammoniagenes was dependent on the concentration of phosphate (14). A fur- ther interesting observation is that considerable heat gener- ation was observed during the PHB accumulation phase. Thus, more light may be shed on the physiology of PHB accumulating cells by investigating the efficiency of energy production and utilization coupled with the phenomenon of heat dissipation.

ACKNOWLEDGMENT

PFS wishes to express his gratitude to The Japanese Ministry of Education, Science and Culture (Monbusho) and the British Council for the funding of a visiting Professorship to Kyushu University.

REFERENCES

1. Ishizaki, A. and Tanaka, K.: Production of poly-~-hydro- xybutyric acid from carbon dioxide by Alcaligenes eutrophus ATCCI769T r. J. Ferment. Bioeng., 71, 254--257 (1991).

2. Ishizald, A. and Tanaka, K.: Batch culture of Alcaligenes eu- trophus ATCC 17697 x using recycled gas closed circuit culture system. J. Ferment. Bioeng., 69, 170-174 (1990).

3. Allen, R. J. L.: The estimation of phosphorus. Biochem. J., 34, 858-865 (1940).

4. Wiame, J. M.: The occurrence and physiological behavior of two metaphosphate fractions in yeast. J. Biol. Chem., 178, 919-929 (1949).

5. Cook, A.M. , Urban, E., and Schlegel, H.G.: Measuring the concentrations of metabolites in bacteria. Anal. Biochem., 72, 191-201 (1976).

6. Slater, E.C.: Measurement of phosphorylation, p. 25-29. In

VoL 74, 1992 SUBSTRATE UTILIZATION EFFICIENCY OF A. EUTROPHUS 291

Estabrook, R. W. (ed.), Methods in enzymology, vol. 10. Aca- demic Press, New York (1967).

7. Kaltwasser, H.: Die rolle der polyphosphate imphosphat- stoffwechsel eines knallgasbakteriums (Hydrogenomonas stamm 20). Arch. Mikrobiol., 41, 282-306 (1962).

8. Doi, Y., Kawaguchi, Y., Nakamura, Y., and Kunioka, M.: Nuclear magnetic resonance studies of poly(3-hydroxybutyrate) and polyphosphate metabolism in Alcaligenes eutrophus. Appl. Environ. Microbiol., 55, 2932-2938 (1989).

9. Harold, F.M.: Accumulation of inorganic polyphosphate in Aerobacter aerogenes. J. Bacteriol., 86, 216-221 (1963).

10. Harold, F. M. and Sylvan, S.: Accumulation of inorganic poly-

phosphate in Aerobacter aerogenes. J. Bacteriol., 86, 222-231 (1963).

II. Harold, F.M.: Depletion and replenishment of the inorganic polyphosphate pool in Neurospora crassa. J. Bacteriol., 83, 1046-I057 (1962).

12. Bongers, L.: Energy generation and utilization in hydrogen bacte- ria. J. Bacteriol., 104, 145-151 (1970).

13. Fang, F. S. and Burris, R. H.: Cytochrome in Hydrogenomonas eutropha. J. Bacteriol., 96, 298-305 (1968).

14. Fujio, T. and Furuya, A.: Production of 5'-guanylic acid. Hakko to Kogyo, 42, 570-580 0984). (in Japanese)