succiniciproducens for the production of succinic acid from whey

Transcription

1 Appl Microbiol Biotechnol (2000) 54: 23±27 Ó Springer-Verlag 2000 ORIGINAL PAPER P. C. Lee á W. G. Lee á S. Kwon á S. Y. Lee H. N. Chang Batch and continuous cultivation of Anaerobiospirillum succiniciproducens for the production of succinic acid from whey Received: 23 July 1999 / Received revision: 17 November 1999 / Accepted: 24 December 1999 Abstract Batch and continuous cultivation of Anaerobiospirillum succiniciproducens were systematically studied for the production of succinic acid from whey. Addition of 2.5 g l )1 yeast extract and 2.5 g l )1 polypeptone per 10 g l )1 whey was most e ective for succinic acid production from both treated and nontreated whey. When 20 g l )1 nontreated whey and 7 g l )1 glucose were used as cosubstrates, the yield and productivity of succinic acid reached at the end of fermentation were 95% and 0.46 g (l h) )1, respectively. These values were higher than those obtained using nontreated whey alone [93% and 0.24 g (l h) )1 for 20 g l )1 whey]. Continuous fermentation of A. succiniciproducens at an optimal dilution rate resulted in the production of succinic acid with high productivity [1.35 g (l h) )1 ], high conversion yield (93%), and higher ratio of succinic acid to acetic acid (5.1:1) from nontreated whey. Introduction P. C. Lee á W. G. Lee á S. Kwon á S. Y. Lee (&) H. N. Chang Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, Kusong-dong, Yusong-gu, Taejon , Korea leesy@sorak.kaist.ac.kr Tel.: Fax: Succinic acid is a dicarboxylic acid produced as an intermediate of the tricarboxylic acid cycle and also as one of the fermentation products of anaerobic metabolism (Gottschalk 1986). It can be used for the manufacture of synthetic resins and biodegradable polymers and as an intermediate for chemical synthesis (Zeikus 1980). To date, succinic acid has mostly been produced by chemical processes. Recently, however, fermentative production of succinic acid from renewable biomass by anaerobic bacteria has attracted great interest (Landucci et al. 1994). An anaerobic bacterium Anaerobiospirillum succiniciproducens has been considered as one of the best succinic acid producers because it can produce a significant amount of succinic acid from glucose (Datta 1992; Davis et al. 1976; Glassner and Datta 1992; Lee et al. 1999a, b; Samuelov et al. 1991). Whey is produced in large quantities as a by-product of the cheese and milk industry (Kosikowski 1979). Whey contains approximately 4.5% (w/v) lactose, 0.8% (w/v) protein, 1.0% (w/v) salts, and 0.1%± 0.8% (w/v) lactic acid (Yang et al. 1994). About half of the whey produced in the United States of America is used for animal feed. The rest is disposed of, which can cause considerable environmental problems due to its high biological oxygen demand. For this reason, several environmentally friendly processes based on microbial fermentation have been proposed for whey utilization. Several useful chemicals or materials have been produced from whey by fermentation: lactic acid (Chiarini et al. 1992), ethanol (Porro et al. 1992), propionic acid (Yang et al. 1994), poly(3-hydroxybutyrate) (Wong and Lee 1998), and 2,3-butylene glycol (Barrett et al. 1983). Recently, we have demonstrated that A. succiniciproducens could produce succinic acid from lactose (Lee et al. 1999b). Therefore, it was reasoned that succinic acid might be produced economically from whey. In this article, we report on the production of succinic acid from whey by A. succiniciproducens. Production of succinic acid from whey with high productivity by continuous fermentation is also reported. Materials and methods Organism and growth conditions Anaerobiospirillum succiniciproducens (ATCC 29305) was obtained from the American Type Culture Collection (Rockville, Md.). Cells

2 24 were grown in sealed anaerobic bottles containing 100 ml minimal salts medium 1 (AnS1) containing 5 g l )1 lactose, 2.5 g l )1 polypeptone and 2.5 g l )1 yeast extract with CO 2 as the gas phase. The AnS1 medium contains per liter: 3 g K 2 HPO 4, 1 g NaCl, 1 g (NH 4 ) 2 SO 4, 0.2 g CaCl 2 á 2H 2 O, 0.2 g MgCl 2 á 6H 2 O, and 1 g Na 2 CO 3. The medium was heat sterilized (15 min at 121 C) in an anaerobic bottle with nitrogen headspace. To the sterile medium, concentrated H 2 SO 4 was added to adjust the ph to 6.5. The nitrogen headspace was replaced by CO 2, and Na 2 S á 9H 2 O ( nal concentration: 1 mg l )1 ) was added to ensure strict anaerobic condition. After 15 min, the reduced medium was inoculated with 2.5 ml glycerol stock culture and incubated at 39 C for 24 h. Batch cultures were carried out at 39 C in a jar fermenter (2.5 l; Korea Fermenter Company, Incheon, Korea) containing 1 l minimal salts medium 2 (AnS2) containing 20 g l )1 whey (Sigma, St. Louis, Mo.) or lactose with 5 g polypeptone (Difco, Detroit, Mich.) and 5 g yeast extract (Difco). For cofermentation, the AnS2 medium containing whey or lactose was supplemented with glucose. The AnS2 medium contains per liter: 3 g K 2 HPO 4,1g NaCl, 5 g (NH 4 ) 2 SO 4, 0.2 g CaCl 2 á 2H 2 O, 0.4 g MgCl 2 á 6H 2 O, 5 mg FeSO 4 á 7H 2 O, and 5 g Na 2 CO 3. The ph was controlled at 6.5 using 1.5 M Na 2 CO 3. Foam was controlled by adding Antifoam 289 (Sigma). CO 2 gas sparging rate and agitation speed were controlled at 0.25 vvm and 200 rpm, respectively. All chemicals used were of reagent grade and were obtained from either Junsei Chemical Co. (Tokyo, Japan) or Sigma. Gas was scrubbed free of oxygen by passing through a gas puri er (Cobert, St. Louis, Mo.). Continuous cultures were carried out with a working volume of 500 ml in a 2.5-l jar fermenter, and the AnS2 medium containing 20 g l )1 whey, 5 g l )1 polypeptone, and 5 g l )1 yeast extract was used as a feeding solution. Feeding solution was purged with oxygen-free CO 2 gas for 24 h in order to establish anaerobic condition before use. Other fermentation conditions were the same as those described for batch cultures. After 56 h batch operation, feeding solution was added at various dilution rates while an equal volume of spent medium was removed from the fermenter. The new steady states were con rmed by constant volumetric b-galactosidase activity and constant concentrations of lactose and succinic acid in the medium for three consecutive samples taken at 8-h intervals after at least ve turnovers. Bovine whey powder containing 11% (w/w) proteins and 65% (w/w) lactose was purchased from Sigma. When indicated, proteins present in whey were precipitated by heating the whey solution at 90 C for 20 min, and were removed by centrifugation at 8000g for 20 min. The supernatant was heat-sterilized at 121 C for 30 min (hereafter ``treated whey''). Results Supplementation of complex nitrogen sources in treated and nontreated whey When a minimal salt medium containing whey without any complex nitrogen source was used, A. succiniciproducens did not grow, probably owing to de ciency of essential nutritional components (data not shown). Therefore, to supply these unknown nutritional components, the whey-based medium was supplemented with two di erent concentrations (2.5 and 5 g l )1 ) of yeast extract or polypeptone per 10 g l )1 whey. Cells grew well and produced similar amounts of succinic acid (0.8 g l )1 at 24 h) in these media. Addition of both yeast extract and polypeptone was slightly better for succinic acid production (0.9 g l )1 ). Succinic acid production in media containing either treated or nontreated whey was similar (data not shown). Therefore, a medium containing 2.5 g l )1 yeast extract and 2.5 g l )1 polypeptone per 10 g l )1 nontreated whey was used in the following experiments. Batch fermentation First, batch cultivation was carried out using lactose as a carbon source (Fig. 1). After a long lag period (33 h), cells grew rapidly to reach a maximum cell concentration of 760 mg l )1 at the end of fermentation. The volumetric b-galactosidase activity increased to 3003 units proportionally to the cell concentration. The lactose Analytical methods The concentrations of sugars (glucose, lactose, and galactose), succinic acid, and acetic acid were measured by high-performance liquid chromatography (Hitachi L-3300 RI monitor, L-4200 UV- VIS detector, D2500 chromato-integrator, Tokyo, Japan) equipped with an ion exchange column (Aminex HPX-87H, 300 mm 7.8 mm, Hercules, Calif.) using N H 2 SO 4 as mobile phase. Cell growth was monitored by measuring the absorbance at 660 nm (OD 660 ) using a spectrophotometer (Ultrospec3000, Pharmacia Biotech, Sweden). For the batch and continuous cultures using whey powder, dry cell weights were estimated by subtracting the weight of insoluble particles in the whey powder (Wong and Lee 1998). Cell concentration was de ned as dry cell weight per liter of culture broth. b-galactosidase activity was measured with o-nitrophenyl-b-d-galactopyranoside as a substrate in Z-bu er as described by Miller (1972). The unit for the volumetric b-galactosidase activity was de ned as micromoles of o-nitrophenol formed per milliliter of culture per minute at 28 C, ph 7.0. All experiments were carried out in triplicate. The succinic acid yield was de ned as the amount of succinic acid produced from one gram carbohydrate (or equivalent), and was expressed as a percentage. Fig. 1 Batch fermentation of lactose. Cells precultured on lactose were inoculated into a jar fermenter containing lactose as a sole carbon and energy source. Volumetric b-galactosidase activity (m), cell concentration (d), lactose (j), galactose (,), succinic acid (s), acetic acid (e)

3 25 consumption rate changed from 0.08 to 1.02 g h )1.A small amount of galactose was detected at 36 h, mainly due to the hydrolysis of lactose by the b-galactosidase released from cells. However, the galactose was completely consumed by the end of fermentation. The concentration, yield, and productivity of succinic acid were 16.5 g l )1, 87%, and 0.33 g (l h) )1, respectively. Figure 2 shows the fermentation of A. succiniciproducens in a medium containing 20 g l )1 nontreated whey. The lactose present in the nontreated whey was consumed steadily until 46 h, after which it was consumed rapidly, coinciding with the rapid increase in volumetric b-galactosidase activity. The volumetric b-galactosidase activity reached at the end of fermentation was 3200 units. Again, a small amount of galactose was detected and completely consumed as lactose was depleted. At the end of the fermentation, the concentration, yield, and productivity of succinic acid were 15.5 g l )1, 93%, and 0.24 g (l h) )1, respectively. Cofermentation of glucose/lactose and glucose/whey As shown in Figs. 1 and 2, fermentation time was rather long, which reduced productivity. As a strategy to reduce the fermentation time, cofermentations of glucose/ lactose and glucose/whey were carried out. During the cofermentation of glucose and lactose, glucose was rapidly utilized, followed by lactose (Fig. 3A). Volumetric b-galactosidase activity increased rapidly after the glucose was almost depleted, and it stayed at a similar level during the fermentation. The lactose consumption rate increased from 0.19 to 0.35 g h )1 after the glucose was almost depleted. The concentration, yield, Fig. 3A, B Batch fermentation of glucose/lactose (A) and glucose/ whey (B). Cells precultured on lactose were inoculated into a jar fermenter containing glucose/lactose or glucose/whey. Volumetric b-galactosidase activity (m), cell concentration (d), lactose (j), glucose (.), succinic acid (s), acetic acid (e) Fig. 2 Batch fermentation of whey. Cells precultured on lactose were inoculated into a jar fermenter containing 20 g l )1 nontreated whey. Volumetric b-galactosidase activity (m), cell concentration (d), lactose (j), galactose (,), succinic acid (s), acetic acid (e) and productivity of succinic acid were 16 g l )1, 86%, and 0.62 g (l h) )1, respectively. When glucose and nontreated whey were cofermented, the glucose was consumed followed by the lactose present in the whey (Fig. 3B), which was similar to the above. After the glucose was depleted, the lactose consumption rate was almost constant (0.4 g h )1 ), but was higher than that obtained by the fermentation of glucose and lactose (0.35 g h )1 ). The succinic acid yield (95%) based on glucose and lactose consumed was higher than that obtained using nontreated whey alone (93%). However, the succinic acid productivity [0.46 g (l h) )1 ] was in between those obtained using nontreated whey

4 26 alone [0.24 g (l h) )1 ] and using glucose and lactose together [0.62 g (l h) )1 ]. Continuous fermentation of whey Continuous fermentations were carried out anaerobically to increase the succinic acid productivity. Fresh medium was supplied into the reactor under anaerobic condition at dilution rates of 0.03±0.14 h )1. At new steady states for each dilution rate, the concentrations of succinic acid, acetic acid, lactose, succinic acid productivity, cell concentration, and volumetric b-galactosidase activity were determined and are shown in Fig. 4A. Succinic acid productivity increased from 0.4 g (l h) )1 at the dilution rate of 0.03 h )1 to 1.35 g (l h) )1 at 0.11 h )1, which was 5.6 times higher than that [0.24 g (l h) )1 ] obtained by batch fermentation. However, the productivity deceased to 1.20 g (l h) )1 at the higher dilution of 0.14 h )1. It was found that lactose could not be fully utilized due to the relatively low cell density by increasing the dilution rate above 0.11 h )1. For the continuous cultures with dilution rates up to 0.11 h )1, the succinic acid yield was almost constant at 93% (Fig. 4B). The volumetric b-galactosidase activity was also almost constant at 3300 units for dilution rates up to 0.11 h )1. It decreased to 1790 units at the dilution rate of 0.14 h )1. During the continuous culture, the ratios of succinic acid to acetic acid (g g )1 ) decreased from 5.8:1 to 5.1:1 with increasing dilution rate (Fig. 4B). Discussion Batch and continuous cultivation of A. succiniciproducens were systematically studied for the production of succinic acid from whey. It was possible to convert whey, a waste product of the dairy industry, into succinic acid with a high yield and high productivity. Although whey contains several useful nutritional components for the growth of microorganisms, it is often necessary to add complex nitrogen sources for the growth of microorganisms (Chiarini et al. 1992; Porro et al. 1992). This was also true for A. succiniciproducens, which did not grow in media containing either treated or nontreated whey without complex nitrogen sources. However, when yeast extract and/or polypeptone were added as supplements, it could grow on both treated and nontreated whey. In particular, direct utilization of nontreated whey supplemented with yeast extract and polypeptone is bene cial because the pretreatment steps for whey such as ultra ltration or hydrolysis of lactose are not required. When 20 g l )1 nontreated whey was used, succinic acid yield (93%) was higher than that obtained by fermentation of lactose (85%). However, the slow fermentation of whey resulted in low succinic acid productivity [0.24 g (l h) )1 ]. Even though lactose-grown Fig. 4A, B Continuous fermentation of 20 g l )1 whey (A) and e ect of dilution rates on succinic acid yield and a ratio of succinic acid to acetic acid (B). Cells precultured on lactose were inoculated into a jar fermenter containing 20 g l )1 whey. Continuous fermentation was started after a batch fermentation of 20 g l )1. Fresh medium was supplied under anaerobic condition at dilution rates of 0.03 to h )1. Volumetric b-galactosidase activity (m), cell concentration (d), productivity (.), lactose (j), succinic acid (s), acetic acid (e), succinic acid yield (h), ratio of succinic acid to acetic acid (4) cells were used as inoculum, there was a relatively long lag period in batch cultures using lactose or whey. However, when glucose-grown cells were used as inoculum, cells did not grow in media containing whey or lactose even after 100 h cultivation (data not shown). This means that the state of the precultivation is very important for the growth of A. succiniciproducens. The regulatory mechanism of lactose utilization is not fully elucidated and needs to be investigated. As one possible method of increasing the productivity, cofermentation of glucose and whey was carried out. It was interesting to observe that the lag period was now considerably shortened. Succinic acid could be produced

5 27 with higher productivity [0.46 g (l h) )1 ] and higher yield (95%). These results indicate that cofermentation of glucose and whey is one way to increase succinic acid productivity. However, the productivity was still relatively low compared with that obtained with batch fermentation using glucose [1.0 g (l h) )1 per 20 g l )1 glucose] (Lee et al. 1999a). Therefore, continuous cultivation was employed to increase the productivity of succinic acid. In general, continuous fermentation allows higher productivity than does batch fermentation. Several continuous fermentation processes have been developed for the production of organic acids from biomass. Among the continuous processes, whey permeates have been used as a substrate for the production of lactic acid and propionic acid (Colomban et al. 1993; Timmer and Kfromkamp 1994; Yang et al. 1994). When A. succiniciproducens was grown in continuous culture, a signi cant increase in succinic acid productivity was obtained with the same succinic acid yield compared with that of batch fermentation of whey (5.6 times). The productivity and yield of succinic acid obtained by continuous culture were higher than those obtained by batch fermentation using glucose (Lee et al. 1999a). A productivity as high as 1.35 g succinic acid (l h) )1 could be obtained. Also, continuous fermentation was stably operated for 16 days without contamination or operational problems such as tube clogging by whey particles. This means that nontreated whey can be directly utilized for the succinic acid production. Interestingly, during the continuous fermentation, the ratio of succinic acid to acetic acid (g g )1 ), 5.1:1 ± 5.8:1, was higher than that obtained by batch fermentation of whey or glucose (ca. 4:1) (Lee et al. 1999a). Due to the altered electron ow and unknown components in complex media such as rumen uid or whey, the formation and distribution of end-products can be changed during anaerobic fermentation (Rao et al. 1987; Rao and Mutharasan 1987; Strobel 1992). Therefore, the change in the ratio of succinic acid to acetic acid obtained by batch and continuous fermentation of whey can be partially explained by the contribution of some nutritional compounds present in fresh nontreated whey medium to the metabolism of A. succiniciproducens. In view of the above results obtained by continuous culture, the operation needs to be done at a dilution rate of 0.10±0.11 h )1 to obtain the maximum yield and productivity of succinic acid, and a high ratio of succinic acid to acetic acid. In conclusion, this study shows that succinic acid can be produced e ciently by A. succiniciproducens from whey, a waste product of the dairy industry, by batch and continuous fermentation. Furthermore, the results obtained in this study suggest that nontreated whey can be directly used for succinic acid production, which allows savings in whey preparation. However, relatively expensive yeast extract and polypeptone were used in relatively large amounts. We are currently examining various low-cost complex nitrogen sources that might replace the yeast extract and polypeptone. Acknowledgement This work was supported by the Korean Ministry of Science and Technology and by the Brain Korea 21 project. References Barrett EL, Collins EB, Hall BJ, Matoi SH (1983) Production of 2,3-butylene glycol from whey by Klebsiella pneumoniae and Enterobacter aerogenes. J Dairy Sci 66: 2507±2514 Chiarini L, Mara L, Tabacchioni S (1992) In uence of growth supplements on lactic acid production in whey ultra ltrate by Lactobacillus helveticus. 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