Continuous production of poly-3-hydroxybutyrate by Ralstonia eutropha in a two-stage culture system

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1 Journal of Biotechnology 88 (2001) Continuous production of poly-3-hydroxybutyrate by Ralstonia eutropha in a two-stage culture system Guocheng Du a, Jian Chen a, Jian Yu b, *, Shiyi Lun a a School of Biotechnology, Wuxi Uni ersity of Light Industry, Wuxi, Jiangsu Pro ince, People s Republic of China b Department of Chemical Engineering, The Hong Kong Uni ersity of Science and Technology, Clear Water Bay, Kowloon, Hong Kong Received 27 June 2000; received in revised form 11 January 2001; accepted 7 March 2001 Abstract Poly-3-hydroxybutyrate (PHB) is mainly accumulated by Ralstonia eutropha under unbalanced growth conditions, which limits its production in batch or fed-batch modes. The continuous production of PHB was investigated in a two-stage continuous culture system. The first-stage produced cell mass giving the maximal cell dry weight of 27.1 g l 1 at 0.21 h 1 of dilution rate. High specific cell growth rate results in the decrease of PHB synthesis under glucose-limited and nitrogen-rich conditions in the first-stage. The second-stage produced PHB giving the maximal PHB concentration of 47.6 g l 1 at 0.14 h 1 of dilution rate. Specific PHB synthetic rate reached highest value at low dilution rate under nitrogen-limited condition in the second-stage, and decreased with the increase of ammonium concentration in the culture. In the continuous culture system, the maximal PHB productivity could reach 1.43 g l 1 h 1 at a dilution rate of 0.12 h 1, but with relatively low PHB content of 47.6%. Maximal yield of PHB on glucose could reach 0.36 g g 1 glucose at h 1 of dilution rate with relatively high PHB productivity of 1.23 g l 1 h 1 and PHB content of 72.1%, respectively Elsevier Science B.V. All rights reserved. Keywords: Ralstonia eutropha; Poly-3-hydroxybutyrate; Two-stage continuous culture system; PHB continuous production; Dilution rate; Nitrogen-limited condition 1. Introduction Poly-3-hydroxybutyrate (PHB) is an energy and carbon storage material accumulated intracellularly by numerous micro-organisms under unfavorable growth conditions in the presence of * Corresponding author. Tel.: ; fax: address: kejianyu@ust.hk (J. Yu). excess carbon source (Anderson and Dawes, 1990; Steinbuchel, 1991; Byrom, 1994; Page, 1995; Lee, 1996). Under normal growth conditions, PHB content in the cells is usually not very high. Depending on bacterial strains, it is 2 10% weight of the dry cell mass. However, PHB content can reach up to 80% of the dry cell mass if growth is limited by the depletion of an essential nutritional compound such as nitrogen, phosphorus, sulfur, or magnesium. Because nitrogen is /01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S (01)

2 60 G. Du et al. / Journal of Biotechnology 88 (2001) easy to control and essential to cell growth, it is usually chosen as a limiting nutrient in PHB formation (Suzuki et al., 1986). On the other hand, nitrogen limitation is more efficient in the stimulation of PHB accumulation in Ralstonia eutropha (Du et al., 2000). PHB and other poly-hydroxyalkanoates (PHAs) have attracted much attention as candidates for biodegradable polymers, because they possess material properties similar to the common petrochemical-based synthetic thermoplastics and elastomers currently in use. They can be completely degraded to carbon dioxide and water (and methane under anaerobic conditions) by micro-organisms in the environment (Holmes, 1988; Lee, 1996). However, the production cost of PHB is quite high compared with that of chemical synthetic non-biodegradable plastics. Efforts have been devoted to make this process economically more feasible by further understanding PHB accumulation, improving PHB productivity and developing an economical recovery process (Kim et al., 1996; Wang and Lee, 1998; Choi and Lee, 1999). High PHB productivity can be achieved in several ways, including high cell density, high PHB concentration and short cultivation time. A continuous production process is of greater commercial interest due to its high productivity, especially for strains with a high maximum specific growth rate (Ramsay et al., 1991; Braunegg et al., 1995; Koyama and Doi, 1995). R. eutropha (formerly Alcaligenes eutrophus) is the most widely employed bacterium for the production of PHB since it grows fast and accumulates a large amount of PHB in a relatively simple medium (Kim et al., 1994; Lee and Chang, 1995). By using R. eutropha, conventional PHB production is a two-step fed-batch process in which a high biomass concentration is first produced followed by PHB accumulation. A two-stage continuous culture system is investigated in this work, in which cells grow substantially in the first-stage under balanced growth conditions and PHB is synthesized in the second-stage under nitrogen limiting conditions. On the basis of kinetic studies on continuous culture in R. eutropha (Du et al., 1998), we also use this two-stage continuous culture system to further understand the mechanism of PHB accumulation at different dilution rates and to maximize the PHB production. 2. Materials and methods 2.1. Micro-organism and growth conditions R. eutropha WSH3 (lab collection) was used in this study. The strain was maintained by monthly subculture on 2.0%-agar slants containing (per liter): yeast extract 10 g, peptone 10 g, beef extract 10 g, ammonium sulfate 5 g and ph 7.0. The same medium without agar was used for seed cultivation. The seed was prepared in 500 ml flasks containing 100 ml of seed medium, and incubated in a rotary shaker at 200 rev min 1 with a temperature of 30 C for 24 h. The initial medium for set up of the first-stage culture of the continuous system was a mineral solution containing 10 g l 1 of glucose as carbon source and 2.5gl 1 of (NH 4 ) 2 SO 4 as a nitrogen source. One liter mineral solution contained 0.2 g of MgSO 4, 10 mg of CaCl 2 2H 2 O, 6 mg of ferrous ammonium citrate, 9.92 g of Na 2 HPO 4 7H 2 O, 0.83 g of KH 2 PO 4, 0.1 ml of concentrated H 2 SO 4,and1ml of trace element solution. The trace element solution contained (per liter) 0.3 g of H 3 BO 3, 0.2 g of CoCl 2 6H 2 O, 0.1 g of ZnSO 4 7H 2 O, 30 mg of MnCl 2 4H 2 O, 30 mg of NaMoO 4 2H 2 O, 20 mg of NiCl 2 6H 2 O, and 10 mg of CuSO 4 5H 2 O. The feeding solution (F 1 ) for the first-stage culture had 50 g l 1 of glucose in the mineral solution. The feeding solution (F 2 ) for the second-stage culture had 500 g l 1 of glucose in the mineral solution Two-stage continuous culture system The two-stage continuous culture system consisting of two 2 l INFOR jar fermentors is shown in Fig. 1. The first-stage continuous culture was carried out in a 2 l jar fermentor equipped with two six-bladed disk turbines. Ten percent of seed culture was used to inoculate the fermentor, and the volume of the first-stage was 1.2 l. Aeration rate was 1.5 v v 1 min 1. Dissolved oxygen (DO) concentration was controlled above 20% of air saturation by adjusting the agitation speed from

3 G. Du et al. / Journal of Biotechnology 88 (2001) to 1000 rev min 1 to avoid DO limitation. ph was maintained at 7.0 by adding 28% (v v 1 ) of ammonia water or 3 mol l 1 of HCl solution. The cultivation temperature was set at 30 C. Glucose was the growth-limiting component in the first-stage continuous culture. Different dilution rates were controlled by feeding F 1 continuously via peristaltic pump. The broth from the first-stage reactor was pumped into the second-stage reactor continuously. The ammonium ion in the first-stage was brought into the second-stage, and more ammonium ion was carried into the second-stage at high dilution rate. In order to keep a high C/N ratio, a fresh medium (F 2 ) was also fed to maintain glucose concentration of the second-stage culture at around 15 g l 1. The volume of the second-stage was 1.4 l. Aeration rate was 1.5 v v 1 min 1. Agitation speed was controlled from 800 to 1000 rev min 1 to maintain DO at above 20% of air saturation. The temperature of cultivation was 30 C. ph was controlled at 7.0 by the feeding of 3 mol l 1 NaOH instead of ammonia water in this stage culture Analytical methods Dry cell weight (DCW) was determined by measuring cell concentration as follows. Five milliliters of culture broth was centrifuged, washed twice with distilled water, and dried at 80 C to constant weight. Glucose and ammonium ions were measured as described by Haywood et al. (1990). PHB concentration was determined by gas chromatography with benzoic acid as an internal standard (Riis and Mai, 1988). All chemicals were of analytical grade. Residual biomass was defined as the mass difference between the dry cell mass and PHB mass. The PHB content (wt.%) was defined as the percentage of PHB mass in the dry cell mass. DO concentration was monitored with a polarographic oxygen-sensing probe and DO monitor. 3. Results 3.1. Effects of dilution rates on DCW, PHB content and glucose concentration in the first-stage Fig. 2 shows the influence of dilution rate on DCW, PHB content and residual glucose concentration in the first-stage continuous culture. DCW increased from 22.0 to 27.1 g l 1 with dilution rate in the range of h 1, but decreased when dilution rate was above 0.21 h 1. It dropped to 8.8 g l 1 at 0.40 h 1 of dilution rate. Residual glucose concentration increased rapidly when dilution rate approached 0.40 h 1, which indicated that this dilution rate was approximate to wash-out point of the continuous culture system. With the increase of dilution rate, i.e. the increase of specific cell growth rate, intracellular PHB content decreased to a minimum level around 3.0%. Fig. 1. Experimental setup of two-stage continuous culture system. F 1,50gl 1 of glucose solution; F 2, 500 g l 1 of glucose solution; F 3, outlet.

4 62 G. Du et al. / Journal of Biotechnology 88 (2001) Fig. 2. Relation between DCW, PHB content, glucose and dilution rates in the first-stage: ( ) DCW, ( ) glucose and ( ) PHB content. In the first-stage continuous culture, cells grew faster at higher dilution rate, and the glucose was mainly used for biosynthetic reactions under nitrogen-rich conditions. Intracellular NAD(P)H was oxidized quickly, resulting in a low concentration of the cofactors in the cells, and the activities of enzymes in the tricarboxylic acid (TCA) cycle remained high. Acetyl-CoA mainly entered TCA cycle for energy generation and formation of aspartate, glutamate and other amino acids, which are essential for the synthesis of cell constituents. The citrate synthase reaction results in the liberation of free CoASH as shown in Fig. 3. Under such growth conditions one would expect the intracellular concentration of acetyl-coa to be low and consequently that of free CoASH to be high. The high CoASH concentration results in an inhibition of the 3-ketothiolase condensation reaction and of PHB synthesis (Fig. 3). Therefore, the high specific cell growth rate results in the decrease of PHB synthesis. reactor working volume, the dilution rates of the two-stages were different as shown in Table 1. DCW and PHB content of the inlet of the secondstage were equal to the values of the first-stage at corresponding dilution rates as shown in Fig. 2. When dilution rate was increased from to 0.14 h 1, DCW was increased from 40.1 g l 1 to its maximal level 47.6 g l 1 correspondingly (Fig. 4). However, DCW was decreased to 16.8 g l 1 at 0.35 h 1 of dilution rate with further increase in dilution rate. PHB concentration reached its maximal value of 30.5 g l 1 at h 1 of dilution rate, but dropped to 0.59 g l 1 at dilution rate of 0.35 h 1. PHB content reached its maximal value of 75% at dilution rate of h 1, and decreased rapidly with the increase of dilution rate. R. eutropha can only accumulate large amounts of PHB under the conditions of excessive carbon source (glucose) and limited nitrogen source. So glucose concentration in the second-stage should be controlled around 15 g l 1 by the continuous feeding of concentrated glucose solution Effects of dilution rate on DCW, PHB concentration, PHB content and glucose concentration in the second-stage The culture broth of the first-stage fermentor was pumped continuously into the second-stage fermentor by a peristaltic pump. Due to the continuous feeding (F 2 ) of concentrated glucose solution in the second-stage culture and the different Fig. 3. Regulation of PHB metabolism in R. eutropha. Modified and reproduced from Lee et al. (1999).

5 G. Du et al. / Journal of Biotechnology 88 (2001) Table 1 Experimental dilution rate of the first-stage and the second-stage cultures D in 1st stage (h 1 ) D in 2nd stage (h 1 ) Effects of dilution rates on residual biomass, ammonium ion in the second-stage culture In the first-stage, glucose was the only cell growth limiting factor while nitrogen was rich. In the second-stage, however, ammonium ion was close to zero at lower dilution rate, and increased with dilution rate as shown in Fig. 5. Consequently, the cells could accumulate substantial amounts of PHB at low dilution rates due to the deficient of nitrogen source. In the second-stage of the continuous culture system, residual biomass increased with the dilution rate from to h 1, which means more cell growth rather than PHB accumulation, but decreased quickly with the further increase of dilution rate, and remained only 16.0 g l 1 at 0.35 h 1 of dilution rate. By comparing the residual biomass in the first-stage culture with that in the second-stage culture, we found that the residual biomass of the second-stage was less than the inlet value from the first-stage when dilution rate was below 0.12 h 1 (Fig. 5). The possible reason was the limited ammonium ion in the broth, resulting in the lysis of R. eutropha cells. However, with the increase of dilution rate more ammonium in the effluent of the first reactor was brought into the second reactor which favored cell growth, and hence increase in residual biomass in the secondstage. With the increase of dilution rate, ammonium ion was accumulated gradually in the secondstage culture, which favored the cell growth of R. eutropha, but was unfavorable to the synthesis of PHB. A close relationship could be observed between the specific PHB synthetic rate (q PHB )and ammonium concentration. q PHB reached its highest value only when ammonium ion was almost close to zero, i.e. at very low dilution rate. q PHB decreased linearly with the increase of ammonium concentration at low dilution rates, which indicates that substantial amounts of PHB can only be synthesized when cell growth is hampered under nitrogen limited conditions. The enzymatic activities of PHB synthetic pathway were inhibited by high ammonium concentration, resulting in the decrease of q PHB. On the other hand, q PHB did not drop to zero even when abundant ammonium ion existed in the broth (Fig. 6). It suggests that R. eutropha still accumulates small amounts of PHB even under nitrogen-rich growth conditions. In the second-stage continuous culture, cell growth was limited due to the deficiency of nitrogen at low dilution rate. If protein synthesis is impaired or inhibited, pyruvate and the intermediates of the TCA cycle do not flow into anabolic pathways. As a consequence the concentration of acetyl-coa will be high and the concentration of free CoASH will be low. Consequently, 3-ketothiolase will not be inhibited and acetoacetyl-coa synthesis will proceed uninhibited, leading to the 3.4. Specific PHB synthetic rate in the second-stage culture Fig. 4. Relation between DCW, PHB concentration, PHB content and glucose with dilution rates in the second-stage: ( ) DCW, ( ) PHB concentration, ( ) glucose and ( ) PHB content.

6 64 G. Du et al. / Journal of Biotechnology 88 (2001) Fig. 5. Relation between residual biomass, ammonium ion and dilution rates in the second-stage: ( ) residual biomass in the first-stage, ( ) residual biomass in the second-stage and ( ) ammonium ion. substantial synthesis of PHB (Fig. 3). In contrast, under nitrogen-rich (i.e. at high dilution rate), the enzymatic activities in TCA cycle increased and the concentration of acetyl-coa decreased. Consequently, the concentration of CoASH increased, which inhibited the activity of 3-ketothiolase, resulting in the low intracellular PHB content at high dilution rate. Under nitrogen-rich conditions, cells can still accumulate a small amount of PHB. Mansfield et al. (1995) found that the level of acetyl-coa remained constant during the growth phase but that of CoASH increased and reached a maximum towards the end of exponential growth phase. The accumulation of some PHB ( 6 Fig. 6. Relationship between specific PHB synthetic rate and ammonium ion in the second-stage. Fig. 7. Relationship of PHB productivity and yield of PHB to glucose with dilution rates in the continuous culture system: ( ) PHB productivity and ( ) yield of PHB to glucose. wt.%) during exponential growth phase, despite the high level of CoASH at this time, shows that the 3-ketothiolase cannot be completely inhibited by high CoASH. This enzyme has been found to be only 95% inhibited in vitro in the presence of 0.5 mm CoASH (Oeding and Schlegel, 1973) Effects of dilution rate on PHB producti ity, ratio of PHB to residual biomass and yield of PHB to glucose in the continuous culture system PHB productivity is the amount of PHB produced per hour per reactor volume. The ratio of PHB to residual biomass is the amount of PHB produced per unit cell biomass. The yield of PHB to glucose is the amount of PHB produced per unit glucose consumed. They are the key parameters in the production of PHB, and the important factors in the PHB production cost. Both PHB productivity and yield of PHB to glucose refer to the values of the whole continuous culture system. In the two-stage continuous culture system, PHB productivity increased with dilution rate in the range of h 1, and the value increased from 0.87 g l 1 h 1 at h 1 to 1.43 g l 1 h 1 at 0.12 h 1 (Fig. 7). With the further increase of dilution rate, however, PHB productivity dropped rapidly to 0.13 g l 1 h 1 at 0.35 h 1 of dilution rate. Despite PHB productivity reached its highest value of 1.43 g l 1 h 1 at 0.12 h 1 of dilution rate, the PHB content obtained

7 G. Du et al. / Journal of Biotechnology 88 (2001) was relatively low (47.6% of DCW), which would lead to difficulty and high cost of PHB recovery. Thus, this dilution rate is not regarded as the optimal working point of the continuous culture system. The yield of PHB to glucose reached its maximal value of 0.36 g g 1 at the dilution rate of h 1, then decreased with the further increase of dilution rate. At dilution rate of h 1, from an overall point of view, PHB productivity, yield of PHB to glucose and PHB content reached 1.23 g l 1 h 1, 0.36 g g 1 and 72.1%, respectively. That is to say, high PHB productivity, yield of PHB to glucose and PHB content can be achieved simultaneously at dilution rate of h 1. References Anderson, A.J., Dawes, E.A., Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54, Braunegg, G., Lefebvre, G., Renner, G., Zeiser, A., Haage, G., Loidl-Lanthaler, K., Kinetics as a tool for polyhydroxyalkanoate production optimization. Can. J. Microbiol. 41 (Suppl. 1), Byrom, D., Polyhydroxyalkanoates. In: Mobley, D.P. (Ed.), Plastics from Microbes: Microbial Synthesis of Polymers and Polymer Precursors. Hanser, Munich, pp Choi, J.I., Lee, S.Y., Efficient and economical recovery of poly(3-hydroxybutyrate) from recombinant Escherichia coli by simple digestion with chemicals. Biotechnol. Bioeng. 62 (5), Du, G.C., Chen, J., Gao, H.J., Chen, Y.G., Lun, S.Y., Effects of environmental conditions on cell growth and poly- -hydroxybutyrate accumulation in Alcaligenes eutrophus. World J. Microbiol. Biotechnol. 16, Du, G.C., Chen, J., Gao, H.J., Chen, Y.G., Lun, S.Y., Studies on the kinetics of Alcaligenes eutrophus continuous culture. Chem. Reaction Eng. Technol. 14, in Chinese. Haywood, G.W., Anderson, A.J., Ewing, D.F., Dawes, E.A., Accumulation of a polyhydroxyalkanoate containing primly 3-hydroxydecanoate from simple carbohydrate substrates by Pseudomonas sp. Strain NCIMB Appl. Environ. Microbiol. 56, Holmes, P.A., Biologically produces PHA polymers and copolymers. In: Bassett, D.C. (Ed.), Developments in Crystalline Polymers, vol. 2. Elsevier, London, pp Kim, B.S., Lee, S.C., Lee, S.Y., Chang, H.N., Chang, Y.K., Woo, S.I., Production of poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control. Biotechnol. Bioeng. 43, Kim, S.Y., Kim, P., Lee, H.S., Kim, J.H., High production of poly- -hydroxybutyrate (PHB) from Methylobacterium organophilum under potassium limitation. Biotechnol. Lett. 18, Koyama, N., Doi, Y., Continuous production of poly( hydroxybutyrate-co- -hydroxyvalerate) by Alcaligenes eutrophus. Biotechnol. Lett. 17, Lee, S.Y., Bacterial polyhydroxyalkanoates. Biotechnol. Bioeng. 43, Lee, S.Y., Chang, H.N., 1995a. Production of poly(hydroxyalkanoic acid). Adv. Biochem. Eng. Biotechnol. 52, Lee, S.Y., Lee, Y., Wang, F., Chiral compounds from bacterial polyesters: sugars to plastics to fine chemicals. Biotechnol. Bioeng. 65, Mansfield, D.A., Anderson, A.J., Naylor, L.A., Regulation of PHB metabolism in Alcaligenes eutrophus. Can. J. Microbiol. 41 (Suppl. 1), Oeding, V., Schlegel, H.G., Ketothiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly- -hydroxybutyrate metabolism. Biochem. J. 134, Page, W.J., Bacterial polyhydroxyalkanoates, natural biodegradable plastics with a great future. Can. J. Microbiol. 41 (Suppl. 1), 1 3. Ramsay, B.A., Saracovan, I., Ramsay, J.A., Marchessalt, R.H., Continuous production of long-side-chain poly- -hydroxyalkanoates by Pseudomonas oleo orans. Appl. Environ. Microbiol. 57, Riis, V., Mai, W., Gas chromatographic determination of poly- -hydroxybutyric acid in microbiol biomass after hydrochloric acid propanolysis. J. Chromatogr. 445, Steinbuchel, A., Polyhydroxyalkanoic acids. In: Byrom, D. (Ed.), Biomaterials: Novel Materials from Biological Sources. Stockton, New York, pp Suzuki, T., Yamane, T., Shimizu, S., Mass production of poly- -hydroxybutyric acid by fully automatic fed-batch culture of methylotroph. Appl. Microbiol. Biotechnol. 23, Wang, F., Lee, S.Y., High cell density culture of metabolically engineered E. coli for the production of poly(3-hydroxybutyrate) in a defined medium. Biotechnol. Bioeng. 58,