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Effect of phosphate supply and aeration on poly-β-hydroxybutyrate production in Azotobacter chroococcum
Process Biochemistry 34 (1999) 109 – 114
Effect of phosphate supply and aeration on poly-b-hydroxybutyrate production in Azotobacter chroococcum L. Savenkova *, Z. Gercberga, Z. Kizhlo, E. Stegantseva Institute of Microbiology and Biotechnology, Lat6ian Uni6ersity, 4, Kron6alda Boule6ard, Riga LV-1586, Lat6ia Received 2 February 1998; received in revised form 21 April 1998; accepted 26 April 1998
Abstract The effects of phosphate supply and aeration on cell growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of increasing PHB production. Phosphate limitation favoured PHB formation in Azotobacter chroococcum 23, but inhibited growth. Azotobacter chroococcum 23 cells demonstrated intensive uptake of orthophosphate during exponential growth. At the highest phosphate concentration (1·5 g/litre) and low aeration the amount of intracellular orthophosphate/g residual biomass was highest. Under conditions of fed-batch fermentation the possibility of controlling the PHB production process by the phosphate level in the cultivation medium was demonstrated. A 36 h fed-batch fermentation resulted in a biomass yield of 110 g/litre with a PHB cellular concentration of 75% dry weight, PHB content 82·5 g/litre, PHB yield YP/S = 0·24 g/g and process productivity 2·29 g/litre·h. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Azotobacter chroococcum; Poly-b-hydroxybutyrate; Phosphate; Fed-batch fermentation
1. Introduction Poly-b-hydroxybutyrate (PHB) is a biodegradable thermoplastic polyester produced by a variety of bacteria from different renewable carbon sources under conditions of deprivation of oxygen, nitrogen, phosphate, sulphur, magnesium or potassium [1 – 9]. A mutant strain of Azotobacter 6inelandii UWD, accumulates PHB during exponential growth without nutrient limitation [10,11]. Many reports have appeared in the literature on the effect of oxygen and nitrogen supply on the production of PHB in Azotobacteriaceae [1,10–14]. Nitrogen limitation does not promote PHB production in Azotobacter spp. [11]. Nitrogen-fixation requires a large amount of ATP and reducing power (such as from NAD(P)H), which arc also required for the synthesis of cellular components. including PHB. Ammonium is one of the most important substrates which controls nitrogenase not only by repressing its synthesis but also by inhibiting its activity [15 – 17]. The addition of ammonium increased both growth and PHB accumulation in Azotobacter chroococcum 23 [14,18,19]. * To whom correspondence should be addressed.
PHB accumulation is induced during oxygen limitation in Azotobacter beijerinckii [12,13,20] but oxygen limitation could cause an adverse effect or reduce cell growth. Hine & Lees [21] observed that the growth of Azotobacter chroococcum was significantly enhanced by intensive aeration, both cell growth and PHB accumulation increasing with a sufficient supply of air in fed-batch fermentation of Azotobacter chroococcum 23. It was also noted that maintaining the oxygen level under limiting conditions was critical for the achievement of high PHB productivity. Thus, a high titre of PHB (52 g/litre) with a high cellular content (60%) was obtained after 48 h of fed-batch fermentation by oxygen supply control [14]. There are only a few reports regarding specific effects of phosphate limitation on Azotobacter spp. For instance, phosphate-limitation inhibited growth and decreased viability of Azotobacter chroococcum [22,23], as well as influencing oxygen absorption from the medium [24]. Under conditions of phosphate deficiency, breakage of the cell wall structure and inability to form cysts was observed in Azotobacter 6inelandii [25]. Little has been reported about the effect of phosphate concentrations on PHB product ion by Azotobacter spp. Phosphate-limited cells of Azotobacter
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6inelandii contain elevated amounts of PHB but adenylate nucleotide energy charge values and oxygen consumption activities were lower than those in control cells [25,26]. Since phosphate is essential for the conversion of substrate energy to ATP then ATP regeneration may be restricted due to the lack of inorganic phosphate. The entire substrate load would be channeled in to PHB synthesis and phosphate-limited cells would contain much larger amounts of PHB than phosphatesufficient cells [25]. The synthesis and accumulation of PHB in Azotobacter 23 takes place in the exponential growth phase and this is why limitation by phosphates, like limitation by oxygen leads to a decrease in the total yield of the polymer. In this study the effects of phosphate supply and aeration on growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of attaining high productivity of PHB.
2. Materials and methods
ensure that the glucose level in the culture broth was not limiting (within 1–5 g/litre). pH and temperature were maintained at 7·2 and 30°C, respectively. The air flow rate and agitation speed were controlled within a range of 0·5–2·0 vvm and 300–1000 rpm, respectively.
2.2. Analytical methods Cell growth was monitored by measuring the optical density of the culture broth at 540 nm. The cell concentration was also determined by measuring the dry cell weight. Residual (RB), non-PHB biomass was calculated as total dry weight minus PHB dry weight. Glucose concentration was determined using a DNS reagent [28]. Phosphate was measured by reaction with amidol reagent [29]. PHB concentration was determined by gas chromatography (Chrom-5) with benzoic acid as an internal standard [30]. For calibration, sodium-DLB-hydroxybutyric acid (SIGMA, Deisenhofen, Germany) was used. All experiments were repeated at least three times. Representative results are reported.
2.1. Bacterial strain and culture conditions Azotobacter chroococcum 23 [27] was grown in a basic fermentation medium contained (per litre): 40·0 g glucose, 3·0 g NH4NO3, 0·64 g K2HPO4, 0·2 g KH2PO4, 0·41 g MgSO4 × 7H2O, 0·1 g CaCl2, 10 mg FeSO4 × 7H2O, 0·5 g Na-citrate and 6 mg Na2MoO4 ×2H2O. The content of K2HPO4 and KH2PO4 were varied in order to maintain total PO34 − concentration within 0·15 –1·5 g/litre depending on experimental conditions. Flask experiments were carried out in 750 ml capacity flasks at 30°C and 190 rpm on a rotary shaker. The medium for fed-batch fermentation was the same as above. Aqueous ammonia (28%) was supplied as the nitrogen source at the amount required for pH control. Fed-batch fermentation was carried out in a 2-litre laboratory fermentor MBR (Switzerland) equipped with a pH controller. 200 ml seed culture was cultivated at a temperature of 30°C for 24 h in shake flasks and then transferred to a fermentor containing 1·3 litre of fermentation medium. Feed solution was supplied to
3. Results and discussion The effect of three phosphate concentrations (PO34 − g/litre: 0·5, 1·0, 1·5) at two aeration levels (culture volume: 50 ml, 100 ml) on growth and PHB accumulation (Table 1, Fig. 1) were investigated in preliminary flask experiments. Under both aeration conditions there was rapid uptake of orthophosphate at the beginning of the exponential growth and the amount of orthophosphate uptake per unit RB was higher at the higher initial concentration in the medium (Table 1). At the highest phosphate concentration (1·5 g/litre) and lowest aeration level used the amount of intracellular phosphate per unit RB was highest (Fig. 1) and up to 480 mg PO34 − (or 170 mg P) per unit RB (Table 1). Such high phosphate uptake activities are typical of phosphorus accumulating bacteria. In Acinetobacter johnsonii 120 under carbon limitation, the uptake of phosphorous was highest, ranging up to 66 mg P per
Table 1 Effect of initial phosphate concentration on growth and PHB accumulation in Azobacter chroococcum Aeration (culture volume) (ml)
Time (h)
50
12
100
12
in medium (g/litre) PO3− 4
Glucose in medium (g/litre)
Initial
Current
Current
0·62 1·05 1·59 0·62 1·05 1·59
0·42 0·40 1·26 0·39 0·73 1·26
37·0 34·6 32·2 35·0 32·3 32·0
DCW (g/litre)
PHB (%)
PO3− 4 /RB (g/g)
1·18 1·28 1·23 0·80 1·02 0·96
17·1 17·6 13·8 31·3 32·7 28·5
0·21 0·26 0·31 0·42 0·46 0·48
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Fig. 1. Effects of different phosphate-supply conditions and two aeration levels on residual biomass concentration and phosphate accumulation in Azobacter chroococcum 23 cells. () 50 ml culture () 100 ml culture.
unit dry weight [31]. The ability of A. 6inelandii and A. agile 22 -D to take up exogenic orthophosphate actively and accumulate it as polyphosphates has been investigated in detail by Zaitseva [32]. Polyphosphates had also been found in complexes with low molecular PHB and calcium in membranes of A. 6inelandii [33]. The lowest PHB concentration was observed at the highest phosphate concentration at both aeration levels (Table 1). Aeration increased cell growth and phosphate uptake (PO34 − /RB) was much lower at the higher aeration level (Fig. 1). In this case the amount of orthophosphate uptake was dependent on the concentration of exogenic phosphate to a greater extent than that at a low level of aeration. It is possible that Azotobacter chroococcum 23, like other representatives of the Azotobacter spp., is capable of accumulating phosphate [32]. Since the synthesis and accumulation of PHB increase under conditions of phosphate limitation [22,23,25,26], phosphate accumulation in Azotobacter chroococcum 23 may stimulate inhibition of PHB synthesis. The amount of phosphate accumulated per unit RB in Azotobacter
chroococcum 23 depends not only on the concentration of exogenic phosphate but also on the aeration intensity. The effect of phosphate on PHB production was studied further under conditions of fed-batch fermentation, permitting the control of aeration intensity and orthophosphate concentration. In order to obtain high PHB production in fed-batch fermentation, it was necessary to determine the optimum phosphate concentration, sufficient for cell growth, but which would not lead to increased phosphate uptake. Fed-batch fermentations were conducted at three initial phosphate concentrations (0·15, 0·5 and 1·0 g/litre) (Figs. 2–4). Air flow rate and agitation speed were maintained within the range 0·5–2·0 vvm and 300–1000 rpm, respectively in order that the pO2 level was within 4–6% saturation [14]. Under phosphate-limiting conditions (initial phosphate concentration 0·15 g/litre), the phosphate concentration in the medium, in the exponential phase of growth, was maintained around 0·01 g/litre and the PHB accumulation level reached 90% DCW, but cell
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growth was inhibited (RB 4g/litre) (Fig. 2). This is related to the observation made by Tsai on Azotobacter 6inelandii [25]. With an initial phosphate concentration of 1 g/litre the phosphate following concentration was maintained at a level of 0·5 g/litre during exponential growth. As a result the content of PHB in cells was only 60% dcw and, total polymer yield was 52 g/litre, in spite of the fact that the cell growth was not inhibited (RB 25 g/litre) (Fig. 3). In cultures of the PHB producer Azotobacter 6inelandii UWD the phosphate concentration was maintained at a level of 0·5 g/litre [34], with the PHB accumulating Pseudomonas sp.K, an optimum phosphate concentration in the medium was about 2 g/litre [8]. Assuming that phosphate-limiting conditions promote PHB accumulation in Azotobacter 23 cells, then in non-limiting conditions with high biomass growth, there must be an optimum phosphate concentration that does not inhibit either growth or PHB accumulation. In an attempt to obtain both high PHB cellular concentrations and high total PHB yield, the phosphate concentrations in the basic fermentation
Fig. 3. Time courses of cell growth, PHB content and phosphate content under phosphate-sufficient conditions. Initial phosphate concentration 1·0 g/litre.
Fig. 2. Time courses of cell growth, PHB content and phosphate content under phosphate-limited conditions. Initial phosphate concentration 0·15 g/litre.
medium and in the feed solutions were decreased to 0·5g/litre (PO34 − ), which was the optimum phosphate concentration in the flask experiments. The phosphate concentrations in these fermentations from the exponential growth phase to the end of the process were maintained at 0·05 g/litre (Fig. 4). In a 36 h period, the following results for Azotobacter 23 growth and PHB accumulation were obtained: biomass 110 g/litre, PHB cellular concentration 75% dry weight, PHB content 82·5 g/litre, PHB yield YP/S = 0·24 g/g, process productivity 2·29 g/litre.h. Page & Knosp described Azotobacter 6inelandii UWD, a mutant strain that produced relatively large amount of PHB and did not form a capsule, thus overcoming one of the obstacles that has limited the use of Azotobacter spp. in PHB manufacture [35]. In fed-batch beet molasses culture of Azotobacter 6inelandii UWD up to 31 g/litre cell mass containing up to 71% poly (3HB-co-3HV) was obtained. However, the yield of PHA/g glucose equivalent in the fermentation process was higher (YP/S = 0·38) [36], than the equivalent in the fed-batch cultivation process of Azo-
L. Sa6enko6a et al. / Process Biochemistry 34 (1999) 109–114
tobacter chroococcum 23 (YP/S =0·24). In other basic indices the results obtained in fed-batch cultivation of Azotobacter chroococcum 23 were comparable to literature data on other PHB producers. The production of PHB with a recombinant strain of E.coli XL1 has been investigated. 42 h of a fed-batch process yielded 117 g/litre biomass which contained 76% PHB, the process productivity reaching 2·1 g/litre.h [37]. With Alcaligenes latus in a one-step fed-batch fermentation process 100 g/litre biomass containing 75 – 80% PHB [38] were produced. Cultivation of Alcaligenes eutrophus in a twostep fed-batch fermentation process (during the second step the phosphate source was limited), every phase lasting 48 h resulted in 100g/litre cell dry matter, PHB content 60–70% [39]. We are currently studying ways of optimizing Azotobacter chroococcum 23 fed-batch fermentation using starch hydrolyzates. In preliminary flask experiments PHB yields per glucose equivalent were 0·32g/g for beet molasses and 0·40 g/g for maize starch hydrolyzate. Therefore, we believe that further development of the
Fig. 4. Current phosphate concentration and aeration levels providing high cell and PHB yield. Initial phosphate concentration 0·5 g/litre.
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fed-batch fermentation using complex carbon sources will allow us to reach higher PHB yields.
Acknowledgements This work was supported by a grant from the Latvian Council of Science.
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[16] Gordon, J. K, Shah. V. K. and Brill, W. J., Feedback inhibition of nitrogenase. Journal of Bacteriology 1981, 148, 884–888. [17] Robson, R. L., Kennedy, C. and Postgate, J. R., Progress in comparative genetics of nitrogen fixation. Canadian Journal of Microbiology 1983, 29, 954–967. [18] Savenkova, L.F., Bonartseva, G.A. and Gertsberg, Z.V., Nitrogen fixation and poly-3-hydroxybutyrate accumulation by some Azotobacter genus bacteria strains. Proceedings of the Lat6ian Academy of Science (Section B) 1994, 5/6, 89–92. [19] Oppenheim, J. and Marcus, L., Correlation of ultrastructure in Azotobacter 6inelandii with nitrogen source for growth. Journal of Bacteriology 1970, 101, 286–991. [20] Carter, B. S. and Dawes, E. A.. Effect of oxygen concentration and growth rate on glucose metabolism, poly-b-hydroxybutyrate biosynthesis and respiration of Azotobacter beijerinckii. Journal of General Microbiology 1979, 110, 393–400. [21] Hine, P. W. and Lees, H., The growth of nitrogen-fixing Azotobacter chroococcum in continuous culture under intense aeration. Canadian Journal of Microbiology 1976, 22, 611–618. [22] Dalton, H. and Postgate, J. R., Effect of oxygen on growth of Azotobacter chroococcum in batch and continuous cultures. Journal of General Microbiology 1969, 54, 463–473. [23] Dalton, H. and Postgate, J. R.. Growth and physiology of Azotobacter chroococcum in continuous cultures. Journal of General Microbiology 1969, 56, 307–309. [24] Lees, H. and Postgate, J. R.. The behaviour of Azotobacter chroococcum in oxygen- and phosphate-limited chemostat cultures. Journal of General Microbiology 1973, 75, 161–166. [25] Tsai, J. C., Aladegbami, S. L. and Vela, G. R., Phosphate-limited culture of Azotobacter 6inelandii. Journal of Bacteriology 1979, 2, 639 – 645. [26] Rubia, T., de la Gonsalez-Lopez, J. and Moreno, J. etal.,, Adenine nueleotide content and energy charge of Azotobacter 6inelandii grown at low phosphate concentration. Archi6es of Microbiology 1987, 147, 354–357. [27] Savenkova, L. F., Zagreba, E. D., Gercberga, Z. V., et al., Azotobacter chroococcum strain 23 —production of poly-b-hydroxybutyric acid (in Latvian). Patent LV 5297, P-93-635, 22·06·1993.
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[28] Miller, G. L., Use of the dinitrosalicylic acid reagent for determination of reducing sugars. Analytical Chemistry 1959, 31, 426– 428. [29] Gerhardt, P.(ed.), Manual of Methods for General Bacteriology, 1st edn. American Society for Microbiology, Washington, 1981. p. 341. [30] Braunegg, G., Sonnleitner, B. and Lafferty, R. M., A rapid gas chromatographic method for the determination of poly-b-hydroxybutyric acid in microbial biomass. European Journal of Applied Microbiology and Biotechnology 1978, 6, 29 – 37. [31] Weltin, D., Hoffmeister, D., Dott, W. and Kampfer, P., Studies of polyphosphate and poly-b-hydroxyalkanoate accumulation in Acinetobacter johnsonii 120 and some other bacteria from activated sludge in batch and continuous cultures. Acta biotechnologia 1996, 16, 91 – 102. [32] Zajtseva, G. N., The Biochemistry of Azotobacter (in Russian). Nauka Publishers, Moscow, 1965, pp. 120 – 127. [33] Reusch, R. N. and Sadoff, H. L., D(-)-Poly-b-hydroxybutyrate in membranes of genetically competent bacteria. Journal of Bacteriology 1983, 156, 778 – 788. [34] Page, W. J., Production of poly-b-hydroxyalkanoates by Azotobacter 6inelandii UWD in beet molasses culture. FEMS Microbiology Re6iews 1992, 103. 149 – 158. [35] Byrom, D., Polymer synthesis by microorganisms: technology and economics. TIBTECH 1987, 5, 246 – 250. [36] Page, W. J., Manchak, J. and Rudy, B., Formation of poly (hydroxybutyrate-co-hydroxy-valerate) by Azotobacter 6inelandii UWD. Applied En6ironmental Microbiology 1992, 58, 2866– 2873. [37] Kim. B. S., Lee, S. Y. and Chang, H. N., Production of poly-b-hydroxybutyrate by fed-batch culture of recombinant Eschericia coli. Biotechnology Letters 1992, 14, 811 – 816. [38] Hrabak, O., Industrial production of poly-b-hydroxybutyrate. FEMS Microbiology Re6iews 1992, 103, 251 – 256. [39] Byrom, D., Industrial production of copolymer of Alcaligenes eutrophus. In No6el Biodegradable Microbial Polymers, ed. E. A. Dawes. Kluwer Academic Publishers, Dordrecht, 1990. pp. 113– 117.
Effect of phosphate supply and aeration on poly-b-hydroxybutyrate production in Azotobacter chroococcum L. Savenkova *, Z. Gercberga, Z. Kizhlo, E. Stegantseva Institute of Microbiology and Biotechnology, Lat6ian Uni6ersity, 4, Kron6alda Boule6ard, Riga LV-1586, Lat6ia Received 2 February 1998; received in revised form 21 April 1998; accepted 26 April 1998
Abstract The effects of phosphate supply and aeration on cell growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of increasing PHB production. Phosphate limitation favoured PHB formation in Azotobacter chroococcum 23, but inhibited growth. Azotobacter chroococcum 23 cells demonstrated intensive uptake of orthophosphate during exponential growth. At the highest phosphate concentration (1·5 g/litre) and low aeration the amount of intracellular orthophosphate/g residual biomass was highest. Under conditions of fed-batch fermentation the possibility of controlling the PHB production process by the phosphate level in the cultivation medium was demonstrated. A 36 h fed-batch fermentation resulted in a biomass yield of 110 g/litre with a PHB cellular concentration of 75% dry weight, PHB content 82·5 g/litre, PHB yield YP/S = 0·24 g/g and process productivity 2·29 g/litre·h. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Azotobacter chroococcum; Poly-b-hydroxybutyrate; Phosphate; Fed-batch fermentation
1. Introduction Poly-b-hydroxybutyrate (PHB) is a biodegradable thermoplastic polyester produced by a variety of bacteria from different renewable carbon sources under conditions of deprivation of oxygen, nitrogen, phosphate, sulphur, magnesium or potassium [1 – 9]. A mutant strain of Azotobacter 6inelandii UWD, accumulates PHB during exponential growth without nutrient limitation [10,11]. Many reports have appeared in the literature on the effect of oxygen and nitrogen supply on the production of PHB in Azotobacteriaceae [1,10–14]. Nitrogen limitation does not promote PHB production in Azotobacter spp. [11]. Nitrogen-fixation requires a large amount of ATP and reducing power (such as from NAD(P)H), which arc also required for the synthesis of cellular components. including PHB. Ammonium is one of the most important substrates which controls nitrogenase not only by repressing its synthesis but also by inhibiting its activity [15 – 17]. The addition of ammonium increased both growth and PHB accumulation in Azotobacter chroococcum 23 [14,18,19]. * To whom correspondence should be addressed.
PHB accumulation is induced during oxygen limitation in Azotobacter beijerinckii [12,13,20] but oxygen limitation could cause an adverse effect or reduce cell growth. Hine & Lees [21] observed that the growth of Azotobacter chroococcum was significantly enhanced by intensive aeration, both cell growth and PHB accumulation increasing with a sufficient supply of air in fed-batch fermentation of Azotobacter chroococcum 23. It was also noted that maintaining the oxygen level under limiting conditions was critical for the achievement of high PHB productivity. Thus, a high titre of PHB (52 g/litre) with a high cellular content (60%) was obtained after 48 h of fed-batch fermentation by oxygen supply control [14]. There are only a few reports regarding specific effects of phosphate limitation on Azotobacter spp. For instance, phosphate-limitation inhibited growth and decreased viability of Azotobacter chroococcum [22,23], as well as influencing oxygen absorption from the medium [24]. Under conditions of phosphate deficiency, breakage of the cell wall structure and inability to form cysts was observed in Azotobacter 6inelandii [25]. Little has been reported about the effect of phosphate concentrations on PHB product ion by Azotobacter spp. Phosphate-limited cells of Azotobacter
0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 8 ) 0 0 0 7 0 - 3
L. Sa6enko6a et al. / Process Biochemistry 34 (1999) 109–114
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6inelandii contain elevated amounts of PHB but adenylate nucleotide energy charge values and oxygen consumption activities were lower than those in control cells [25,26]. Since phosphate is essential for the conversion of substrate energy to ATP then ATP regeneration may be restricted due to the lack of inorganic phosphate. The entire substrate load would be channeled in to PHB synthesis and phosphate-limited cells would contain much larger amounts of PHB than phosphatesufficient cells [25]. The synthesis and accumulation of PHB in Azotobacter 23 takes place in the exponential growth phase and this is why limitation by phosphates, like limitation by oxygen leads to a decrease in the total yield of the polymer. In this study the effects of phosphate supply and aeration on growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of attaining high productivity of PHB.
2. Materials and methods
ensure that the glucose level in the culture broth was not limiting (within 1–5 g/litre). pH and temperature were maintained at 7·2 and 30°C, respectively. The air flow rate and agitation speed were controlled within a range of 0·5–2·0 vvm and 300–1000 rpm, respectively.
2.2. Analytical methods Cell growth was monitored by measuring the optical density of the culture broth at 540 nm. The cell concentration was also determined by measuring the dry cell weight. Residual (RB), non-PHB biomass was calculated as total dry weight minus PHB dry weight. Glucose concentration was determined using a DNS reagent [28]. Phosphate was measured by reaction with amidol reagent [29]. PHB concentration was determined by gas chromatography (Chrom-5) with benzoic acid as an internal standard [30]. For calibration, sodium-DLB-hydroxybutyric acid (SIGMA, Deisenhofen, Germany) was used. All experiments were repeated at least three times. Representative results are reported.
2.1. Bacterial strain and culture conditions Azotobacter chroococcum 23 [27] was grown in a basic fermentation medium contained (per litre): 40·0 g glucose, 3·0 g NH4NO3, 0·64 g K2HPO4, 0·2 g KH2PO4, 0·41 g MgSO4 × 7H2O, 0·1 g CaCl2, 10 mg FeSO4 × 7H2O, 0·5 g Na-citrate and 6 mg Na2MoO4 ×2H2O. The content of K2HPO4 and KH2PO4 were varied in order to maintain total PO34 − concentration within 0·15 –1·5 g/litre depending on experimental conditions. Flask experiments were carried out in 750 ml capacity flasks at 30°C and 190 rpm on a rotary shaker. The medium for fed-batch fermentation was the same as above. Aqueous ammonia (28%) was supplied as the nitrogen source at the amount required for pH control. Fed-batch fermentation was carried out in a 2-litre laboratory fermentor MBR (Switzerland) equipped with a pH controller. 200 ml seed culture was cultivated at a temperature of 30°C for 24 h in shake flasks and then transferred to a fermentor containing 1·3 litre of fermentation medium. Feed solution was supplied to
3. Results and discussion The effect of three phosphate concentrations (PO34 − g/litre: 0·5, 1·0, 1·5) at two aeration levels (culture volume: 50 ml, 100 ml) on growth and PHB accumulation (Table 1, Fig. 1) were investigated in preliminary flask experiments. Under both aeration conditions there was rapid uptake of orthophosphate at the beginning of the exponential growth and the amount of orthophosphate uptake per unit RB was higher at the higher initial concentration in the medium (Table 1). At the highest phosphate concentration (1·5 g/litre) and lowest aeration level used the amount of intracellular phosphate per unit RB was highest (Fig. 1) and up to 480 mg PO34 − (or 170 mg P) per unit RB (Table 1). Such high phosphate uptake activities are typical of phosphorus accumulating bacteria. In Acinetobacter johnsonii 120 under carbon limitation, the uptake of phosphorous was highest, ranging up to 66 mg P per
Table 1 Effect of initial phosphate concentration on growth and PHB accumulation in Azobacter chroococcum Aeration (culture volume) (ml)
Time (h)
50
12
100
12
in medium (g/litre) PO3− 4
Glucose in medium (g/litre)
Initial
Current
Current
0·62 1·05 1·59 0·62 1·05 1·59
0·42 0·40 1·26 0·39 0·73 1·26
37·0 34·6 32·2 35·0 32·3 32·0
DCW (g/litre)
PHB (%)
PO3− 4 /RB (g/g)
1·18 1·28 1·23 0·80 1·02 0·96
17·1 17·6 13·8 31·3 32·7 28·5
0·21 0·26 0·31 0·42 0·46 0·48
L. Sa6enko6a et al. / Process Biochemistry 34 (1999) 109–114
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Fig. 1. Effects of different phosphate-supply conditions and two aeration levels on residual biomass concentration and phosphate accumulation in Azobacter chroococcum 23 cells. () 50 ml culture () 100 ml culture.
unit dry weight [31]. The ability of A. 6inelandii and A. agile 22 -D to take up exogenic orthophosphate actively and accumulate it as polyphosphates has been investigated in detail by Zaitseva [32]. Polyphosphates had also been found in complexes with low molecular PHB and calcium in membranes of A. 6inelandii [33]. The lowest PHB concentration was observed at the highest phosphate concentration at both aeration levels (Table 1). Aeration increased cell growth and phosphate uptake (PO34 − /RB) was much lower at the higher aeration level (Fig. 1). In this case the amount of orthophosphate uptake was dependent on the concentration of exogenic phosphate to a greater extent than that at a low level of aeration. It is possible that Azotobacter chroococcum 23, like other representatives of the Azotobacter spp., is capable of accumulating phosphate [32]. Since the synthesis and accumulation of PHB increase under conditions of phosphate limitation [22,23,25,26], phosphate accumulation in Azotobacter chroococcum 23 may stimulate inhibition of PHB synthesis. The amount of phosphate accumulated per unit RB in Azotobacter
chroococcum 23 depends not only on the concentration of exogenic phosphate but also on the aeration intensity. The effect of phosphate on PHB production was studied further under conditions of fed-batch fermentation, permitting the control of aeration intensity and orthophosphate concentration. In order to obtain high PHB production in fed-batch fermentation, it was necessary to determine the optimum phosphate concentration, sufficient for cell growth, but which would not lead to increased phosphate uptake. Fed-batch fermentations were conducted at three initial phosphate concentrations (0·15, 0·5 and 1·0 g/litre) (Figs. 2–4). Air flow rate and agitation speed were maintained within the range 0·5–2·0 vvm and 300–1000 rpm, respectively in order that the pO2 level was within 4–6% saturation [14]. Under phosphate-limiting conditions (initial phosphate concentration 0·15 g/litre), the phosphate concentration in the medium, in the exponential phase of growth, was maintained around 0·01 g/litre and the PHB accumulation level reached 90% DCW, but cell
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growth was inhibited (RB 4g/litre) (Fig. 2). This is related to the observation made by Tsai on Azotobacter 6inelandii [25]. With an initial phosphate concentration of 1 g/litre the phosphate following concentration was maintained at a level of 0·5 g/litre during exponential growth. As a result the content of PHB in cells was only 60% dcw and, total polymer yield was 52 g/litre, in spite of the fact that the cell growth was not inhibited (RB 25 g/litre) (Fig. 3). In cultures of the PHB producer Azotobacter 6inelandii UWD the phosphate concentration was maintained at a level of 0·5 g/litre [34], with the PHB accumulating Pseudomonas sp.K, an optimum phosphate concentration in the medium was about 2 g/litre [8]. Assuming that phosphate-limiting conditions promote PHB accumulation in Azotobacter 23 cells, then in non-limiting conditions with high biomass growth, there must be an optimum phosphate concentration that does not inhibit either growth or PHB accumulation. In an attempt to obtain both high PHB cellular concentrations and high total PHB yield, the phosphate concentrations in the basic fermentation
Fig. 3. Time courses of cell growth, PHB content and phosphate content under phosphate-sufficient conditions. Initial phosphate concentration 1·0 g/litre.
Fig. 2. Time courses of cell growth, PHB content and phosphate content under phosphate-limited conditions. Initial phosphate concentration 0·15 g/litre.
medium and in the feed solutions were decreased to 0·5g/litre (PO34 − ), which was the optimum phosphate concentration in the flask experiments. The phosphate concentrations in these fermentations from the exponential growth phase to the end of the process were maintained at 0·05 g/litre (Fig. 4). In a 36 h period, the following results for Azotobacter 23 growth and PHB accumulation were obtained: biomass 110 g/litre, PHB cellular concentration 75% dry weight, PHB content 82·5 g/litre, PHB yield YP/S = 0·24 g/g, process productivity 2·29 g/litre.h. Page & Knosp described Azotobacter 6inelandii UWD, a mutant strain that produced relatively large amount of PHB and did not form a capsule, thus overcoming one of the obstacles that has limited the use of Azotobacter spp. in PHB manufacture [35]. In fed-batch beet molasses culture of Azotobacter 6inelandii UWD up to 31 g/litre cell mass containing up to 71% poly (3HB-co-3HV) was obtained. However, the yield of PHA/g glucose equivalent in the fermentation process was higher (YP/S = 0·38) [36], than the equivalent in the fed-batch cultivation process of Azo-
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tobacter chroococcum 23 (YP/S =0·24). In other basic indices the results obtained in fed-batch cultivation of Azotobacter chroococcum 23 were comparable to literature data on other PHB producers. The production of PHB with a recombinant strain of E.coli XL1 has been investigated. 42 h of a fed-batch process yielded 117 g/litre biomass which contained 76% PHB, the process productivity reaching 2·1 g/litre.h [37]. With Alcaligenes latus in a one-step fed-batch fermentation process 100 g/litre biomass containing 75 – 80% PHB [38] were produced. Cultivation of Alcaligenes eutrophus in a twostep fed-batch fermentation process (during the second step the phosphate source was limited), every phase lasting 48 h resulted in 100g/litre cell dry matter, PHB content 60–70% [39]. We are currently studying ways of optimizing Azotobacter chroococcum 23 fed-batch fermentation using starch hydrolyzates. In preliminary flask experiments PHB yields per glucose equivalent were 0·32g/g for beet molasses and 0·40 g/g for maize starch hydrolyzate. Therefore, we believe that further development of the
Fig. 4. Current phosphate concentration and aeration levels providing high cell and PHB yield. Initial phosphate concentration 0·5 g/litre.
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fed-batch fermentation using complex carbon sources will allow us to reach higher PHB yields.
Acknowledgements This work was supported by a grant from the Latvian Council of Science.
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