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Biotechnol Lett (2010) 32:14131418 DOI 10.

1007/s10529-010-0313-x

ORIGINAL RESEARCH PAPER

Succinic acid production with metabolically engineered E. coli recovered from two-stage fermentation
Jiang-Feng Ma Min Jiang Ke-Quan Chen Bing Xu Shu-Wen Liu Ping Wei Han-Jie Ying

Received: 13 April 2010 / Accepted: 12 May 2010 / Published online: 22 May 2010 Springer Science+Business Media B.V. 2010

Abstract Escherichia coli AFP111 cells recovered from spent two-stage fermentation broth were investigated for additional production of succinic acid under anaerobic conditions. Recovered cells produced succinic acid in an aqueous environment with no nutrient supplementation except for glucose and MgCO3. In addition, initial glucose concentration and cell density had a signicant inuence on succinic acid mass yield and productivity. Although the nal concentration of succinic acid from recovered cells was lower than from two-stage fermentation, an average succinic acid mass yield of 0.85 g/g was achieved with an average productivity of 1.81 g/l h after three rounds of recycling, which was comparable to two-stage fermentation. These results suggested that recovered cells might be reused for the efcient production of succinic acid. Keywords Cell recovery Escherichia coli Succinic acid Two-stage fermentation

Introduction Succinic acid is used in the production of many industrial chemicals, including 1,4-butanediol, tetrahydrofuran, N-methyl pyrrolidinone, 2-pyrrolidinone, c-butyrolactone, and biodegradable polymers such as polyamides (Willke and Vorlop 2004). It is a compound in the tricarboxylic acid cycle, and is produced by obligate or facultative anaerobes, including Anaerobiospirullum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens (Songa and Lee 2006), Corynebacterium glutamicum (Okino et al. 2008) and Escherichia coli (Clark 1989). To improve the efciency of succinic acid production by E. coli, several strategies have been used to diminish co-products, and improve succinic acid production. E. coli NZN111, which is constructed by insertional disruption of fermentative lactate dehydrogenase (encoded by ldhA) and pyruvate:formate lyase (encoded by pB) is a candidate of succinic acid producer (Bunch et al. 1997). However, it fails to grow anaerobically on glucose, which might be due to that enzymes responsible for anaplerosis and NAD? regeneration are not fully induced (Wu et al. 2007). A breakthrough in succinic acid production by E. coli occurred with the isolation of strain AFP111, a spontaneous chromosomal mutation of ptsG gene in strain NZN 111, which grows fermentatively on glucose with succinic acid as the main fermentation product (Chatterjee et al. 2001).

J.-F. Ma M. Jiang (&) K.-Q. Chen B. Xu S.-W. Liu P. Wei H.-J. Ying State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, No. 5 Xinmofan Road, Gulou District, Nanjing 210009, China e-mail: bioengine@njut.edu.cn

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In addition to the genetic manipulation of central metabolic pathways, fermentation conditions have been investigated to improve succinic acid production and reduce formation of by-products. Vemuri et al. (2002) compared growth, substrate consumption, product formation, and the activities of seven key enzymes in E. coli AFP111 fermentation under exclusively anaerobic and two-stage conditions, in which an aerobic growth phase is followed by an anaerobic production phase. A higher mass yield of succinic acid was obtained under two-stage conditions. Lu et al. investigated the effects of CO2 concentration, pH, and different kinds of bases, on succinic acid production by E. coli AFP111 in twostage fermentation (Lu et al. 2009a, b). Jiang et al. (2010) investigated the effects of growth-phase feeding strategies and achieved 4099 g succinic acid/l, with productivities of 1.83.6 g/l h in the anaerobic stage of two-stage fermentation with E. coli AFP111. However, two-stage fermentation with recombinant E. coli requires an aerobic stage for cell growth without succinic acid production. Thus, the productivity and the yield decrease markedly if the substrate and time consumed in the aerobic stage are considered. However, overall productivity and yield would be improved if the anaerobic succinic acid production time could be prolonged. Andersson et al. (2009) adopted a strategy to maintain high succinic acid productivity by resuspending cells in fresh media, increasing the amount of succinic acid produced during a 100 h fermentation by more than 60%. In this study, cells recovered from the spent two-stage succinic acid fermentation broth were evaluated for efcient production of succinic acid.

When the glucose consumption rate decreased below 0.3 g/l h, cells were recovered for further anaerobic production of succinic acid in fresh media. Cells were recovered by centrifugation in a benchtop centrifuge at 4,100 rpm for 10 min at 4C, and resuspended in fresh media. Media and conditions Six fermentation media were investigated for production of succinic acid with recovered cells, including JSM, as described previously (Lu et al. 2009a, b). JSM-P medium was prepared by omitting phosphate from JSM, JSM-N medium was prepared by omitting ammonium salt from JSM, JSM-T medium was prepared by omitting trace elements from JSM, JSMB was prepared by omitting VB1 and biotin from JSM, and BM medium contained only glucose. MgCO3 was added at 80% (w/w) of the glucose concentration for fermentations carried out in sealed serum bottles, and intermittently supplemented to maintain a pH between 6.4 and 6.8 for fermentations carried out in a fermenter. Fermentations in sealed serum bottles were at 37C and 200 rpm. The headspace in the sealed bottles was lled via a gassing manifold with oxygenfree CO2 for at least 2 min. For repeated production of succinic acid in a 3 l fermenter, the initial volume was maintained at approx. 1.5 l, and the dry cell weight (DCW) was approx. 23 g/l. When glucose dropped below 5 g/l, 80 ml sterilized glucose solution (600 g/l) was added. Anaerobic production of succinic acid commenced when the culture was sparged with CO2 at 1 l/min. When the glucose consumption rate decreased below 0.3 g/l h, fermentation was terminated for the next round of recycling. Analytical procedures DCW was computed from the OD600; an OD600 of 1 = 450 mg dry wt per liter. Glucose was measured by a glucose analyzer containing glucose oxidase (Institute of Biology, Shandong, China). Organic acids were quantied by HPLC and the data was analyzed with a Chromeleon data system (Dionex Corporation, USA). The mass yield of succinic acid was dened as the amount of succinic acid from 1 g glucose consumed, and expressed in g/g.

Materials and methods Strain E. coli strain AFP111 [F? k- rpoS396 (Am) rph-1 4 (pAB::Cam) ldhA::Kan ptsG] was used exclusively in this work, and was kindly provided by Professor D.P. Clark (Southern Illinois University). Cell recovery Standard two-stage fermentations were carried out with Escherichia coli AFP111 (Vemuri et al. 2002).

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Results Effects of culture media on succinic acid fermentation with recovered cells The effects of different culture media on succinic acid production with recovered cells were investigated. As shown in Table 1, regardless of media used, mass yields reached a high value of 0.95 0.97 g/g. DCW dropped to 1.38 g/l with JSM-T, and 1.72 g/l with BM media, because of the absence of trace elements. No obvious change in DCW was observed for the other four media. Similarly, cell density did not increase in the anaerobic stage during two-stage fermentation (Vemuri et al. 2002). Thus, omitting complex nutrients in the resuspension media might not have signicant effects on succinic acid productivity and yield. In addition, when BM medium was used for succinic acid production, the amounts of the accumulated co-products acetic acid and pyruvic acid were lower than with the other ve media. Effects of initial glucose concentration on succinic acid fermentation with recovered cells High concentrations of glucose cause severe osmotic stress, which affects carbohydrate transport (Roth et al. 1985), intracellular activities, and distribution of carbon ux (Nanchen et al. 2006). Therefore, the effects of different initial glucose concentrations on succinic acid production were investigated in BM medium. As shown in Table 2, the consumption of glucose was severely inhibited at an initial glucose concentration of 118 g/l, and a productivity of 0.32 g/l h was obtained, which was markedly lower than

when the initial glucose concentration was below 87.4 g/l. E. coli AFP184, a derivative of AFP111, tolerates up to 100 g/l of initial glucose concentration with a productivity of 1.27 g/l h during batch fermentation (Andersson et al. 2007). This indicates that the osmotolerance of the recovered cells declined when production of succinic acid was carried out in BM medium. In addition, mass yield decreased with increasing initial glucose concentration. When the glucose was above 67 g/l, pyruvic acid ([0.73 g/l), and considerable amounts of acetic acid ([6.78 g/l) accumulated. Effects of initial cell density on succinic acid fermentation with recovered cells Higher cell density results in greater volumetric productivity of succinic acid (Andersson et al. 2007), so we conducted experiments with different initial cell concentrations in BM medium with approximately 35 g glucose/l. As shown in Table 3, when compared to an initial cell density of 9.5 g/l, succinic acid productivity increased by almost 4-fold when the DCW was increased by 2.5-fold to 33.5 g/l. Thus, the productivity per cell improved, with an appropriate increase in specic succinic acid productivity of 39%. In contrast, the mass yields of succinic acid decreased with increased initial cell density. This might be attributed to the increased accumulation of acetic acid and pyruvic acid, and consequent decrease in carbon ux to succinic acid. Alternatively, bacteria can expend energy on functions that are not directly growth-related, although this was for a non-growth anaerobic process for succinic acid production (Russell and Cook 1995). The maintenance energy per cell may have increased with increasing cell density,

Table 1 Effects of culture media on succinic acid production with recovered cells of E. coli AFP111 in sealed serum bottles Media DCW (g/l) Initial JSM JSM-P JSM-N JSM-T JSM-B BM
a

Final 8.45 0.33 8.24 0.42 7.13 0.32 7.17 0.34 8.15 0.26 7.80 0.33

Consumed glucosea (g/l) 19.5 0.6 18.3 0.7 19.5 0.7 17.8 0.8 18.6 0.7 16.5 0.5

Succinic acid (g/l) 18.70 0.45 17.41 0.38 18.74 0.36 17.23 0.42 18.00 0.54 15.59 0.47

Acetic acid (g/l) 2.89 0.09 3.19 0.08 2.86 0.13 2.32 0.12 2.57 0.11 1.37 0.12

Pyruvic acid (g/l) 0.39 0.03 0.46 0.05 0.23 0.04 0.44 0.04 0.48 0.05 0. 31 0.01

Mass yield (g/g) 0.96 0.01 0.95 0.01 0.96 0.01 0.97 0.01 0.97 0.01 0.97 0.01

8.71 0.22 8.90 0.31 7.91 0.33 8.55 0.25 8.73 0.34 9.52 0.26

Data are means standard deviations from three replications

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Table 2 Effects of initial glucose concentrations on glucose consumption and product formation with recovered cells of E. coli AFP111 in sealed serum bottles Time (h) 26 48 60 72 84 Glucose (g/l) Initial 31.3 1.1 50.5 1.6 67.5 1.8 87.4 1.1 Final DCW (g/l) Initial Final Succinic acid Acetic acid (g/l) (g/l) Pyruvic acid (g/l) Productivity (g/l h) Mass yield (g/g) 0.96 0.02 0.89 0.03 0.73 0.02 0.68 0.05 0.66 0.02

1.0 0.1 9.80 0.31 9.24 0.32 28.77 0.62 1.5 0.1 9.65 0.45 8.17 0.44 43.22 0.52 0.5 0.1 9.71 0.32 8.05 0.33 48.61 0.52

3.24 0.12 0.41 0.01 1.11 0.02 3.18 0.15 0.46 0.01 0.90 0.04 6.78 0.13 0.73 0.01 0.81 0.02

6.5 0.3 9.95 0.44 8.15 0.56 54.66 0.42 10.00 0.15 0.75 0.02 0.76 0.05

118.0 1.5 76.5 1.5 9.65 0.44 7.05 0.26 27.54 0.62 10.32 0.10 0.88 0.03 0.32 0.07

Data are means standard deviations from three replications Table 3 Effects of initial cell density on succinic acid production with recovered cells of E. coli AFP111 in sealed serum bottles DCW (g/l) Time (h) Consumed glucose (g/l) 21.5 0.9 27 1.0 28 1.3 33 1.5 Succinic acid (g/l) 20.5 0.33 24.9 0.53 24.8 0.32 25.1 0.44 Acetic acid (g/l) 1.74 0.05 2.83 0.08 3.80 0.06 4.52 0.06 Pyruvic acid (g/l) 0.47 0.02 0.63 0.03 0.79 0.02 1.03 0.01 Productivity (g/l h) 1.14 0.01 2.77 0.03 4.13 0.02 5.58 0.03 Mass yield (g/g) 0.95 0.02 0.92 0.03 0.89 0.04 0.76 0.14

9.50 0.36 19.13 0.57 26.52 0.76 33.53 1.05

18 9 6 4.5

Data are means standard deviations from three replications

causing the decline in mass yield. This was supported by results showing that the ratio of total products (succinic acid, acetic acid and pyruvic acid) to substrate (glucose), decreased from 1.05 to 0.93 when the initial cell density increased from 9.5 to 33.5 g/l. Therefore, cell density at the onset of anaerobic bioconversion had a signicant inuence on succinic acid productivity and mass yield. Thus, to balance yield and productivity, an initial cell density around 20 g/l should be appropriate. Repeated recovery of cells for succinic acid production in a 3 l fermenter To conrm the fermentation properties of the recovered cells, production of succinic acid was carried out in a 3 l fermenter with an initial cell density of 23 g/l, and an initial glucose concentration of 35 g/l in BM medium. Two-stage fermentation was conducted, and terminated at 85.5 h, with a nal succinic acid concentration of 101 g/l and an overall mass yield of 0.82 g/g. Cells were recovered and resuspended in BM medium three times, for anaerobic production of succinic acid. The time proles of cell density and concentrations of glucose and organic acids are shown in Fig. 1. The productivity of succinic acid
Fig. 1 Time-course of glucose, products and cell density during the anaerobic production phase with recovered E. coli AFP111 cells in a 3-l stirred bioreactor. Triangles represent DCW; circles represent glucose; squares represent succinic acid; inverted triangles represent acetic acid

decreased from 2.56 to 1.30 g/l h, and the nal concentration of succinic acid decreased from 64 to 33.8 g/l, with increasing recycle times. However, a mass yield of succinic acid of approx. 0.85 g/g was achieved for each recovery. The productivities and mass yields for twostage fermentation and repeated fermentation using

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Table 4 Succinic acid productivities and mass yields for each stage during two-stage fermentation and repeated recovered cells fermentation Time (h) Two-stage fermentation Aerobic stage Anaerobic stage First recovery stage Second recovery stage Third recovery stage 32 53 25 33 26 24.9 98 74.5 61 40 64 52.2 33.8 2.56 1.58 1.3 0.86 0.85 0.84 101 1.19 0.82 Consumed glucose (g/l) Succinic acid (g/l) Productivity (g/l h) Yield (g/g)

recovered cells are summarized in Table 4. The overall productivity was 1.19 g/l h, and the mass yield was 0.82 g/g for two-stage fermentation, when time and substrate consumed in the aerobic stage were included. Andersson et al achieved an average productivity of 1.77 g/l h with an average mass yield of 0.77 g/g over three resuspensions for succinic acid production (Andersson et al. 2009). In comparison, no decrease in succinic acid productivity and mass yield was seen with the recovered cells, which averaged 1.81 g/l h productivity, and 0.85 g/g mass yield over three rounds of recycling.

Conclusions This study demonstrated that E. coli AFP111 cells recovered from spent two-stage fermentation broth could be reused for succinic acid production in an aqueous environment, using only a substrate (glucose) and a neutralizer (MgCO3). The initial glucose concentration and cell density had a signicant inuence on the yield and productivity. During repeated succinic acid production in a fermenter, mass yields of 0.85 g/ g, and productivities above 1.3 g/l h, were achieved at each recycle. Therefore, we found that recovering cells from the spent two-stage fermentation broth for further production of succinic acid was an efcient complement to two-stage fermentation.
Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 20606017), 973 Program of China (No. 2009CB724701).

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