ORIGINAL RESEARCH PAPER

Succinic acid production with metabolically engineeredE. 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 inves-

tigated for additional production of succinic acid

under anaerobic conditions. Recovered cells pro-

duced 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 significant influence on succinic

acid mass yield and productivity. Although the final

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 compara-

ble to two-stage fermentation. These results sug-

gested that recovered cells might be reused for the

efficient 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, tetra-

hydrofuran, N-methyl pyrrolidinone, 2-pyrrolidinone,

c-butyrolactone, and biodegradable polymers such aspolyamides (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 succinici-

producens (Songa and Lee 2006), Corynebacterium

glutamicum (Okino et al. 2008) and Escherichia coli

(Clark 1989). To improve the efficiency 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 pyru-

vate:formate lyase (encoded by pflB) 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. YingState 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

123

Biotechnol Lett (2010) 32:14131418

DOI 10.1007/s10529-010-0313-x

In addition to the genetic manipulation of central

metabolic pathways, fermentation conditions have

been investigated to improve succinic acid produc-

tion and reduce formation of by-products. Vemuri

et al. (2002) compared growth, substrate consump-

tion, 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 condi-

tions. Lu et al. investigated the effects of CO2concentration, pH, and different kinds of bases, on

succinic acid production by E. coli AFP111 in two-

stage 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 efficient production of succinic acid.

Materials and methods

Strain

E. coli strain AFP111 [F? k- rpoS396 (Am) rph-14(pflAB::Cam) ldhA::Kan ptsG] was used exclusivelyin 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).

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 infresh media.

Media and conditions

Six fermentation media were investigated for pro-

duction of succinic acid with recovered cells, includ-

ing 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, JSM-

B 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 sealedbottles was filled via a gassing manifold with oxygen-

free 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 (Insti-

tute of Biology, Shandong, China). Organic acids were

quantified by HPLC and the data was analyzed with a

Chromeleon data system (Dionex Corporation, USA).

The mass yield of succinic acid was defined as the

amount of succinic acid from 1 g glucose consumed,

and expressed in g/g.

1414 Biotechnol Lett (2010) 32:14131418

123

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 investi-

gated. 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 significant 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 five

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 flux (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 approxi-

mately 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 specific 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

flux 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 (Rus-

sell 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) Consumed

glucosea (g/l)

Succinic

acid (g/l)

Acetic

acid (g/l)

Pyruvic

acid (g/l)

Mass

yield (g/g)Initial Final

JSM 8.71 0.22 8.45 0.33 19.5 0.6 18.70 0.45 2.89 0.09 0.39 0.03 0.96 0.01

JSM-P 8.90 0.31 8.24 0.42 18.3 0.7 17.41 0.38 3.19 0.08 0.46 0.05 0.95 0.01

JSM-N 7.91 0.33 7.13 0.32 19.5 0.7 18.74 0.36 2.86 0.13 0.23 0.04 0.96 0.01

JSM-T 8.55 0.25 7.17 0.34 17.8 0.8 17.23 0.42 2.32 0.12 0.44 0.04 0.97 0.01

JSM-B 8.73 0.34 8.15 0.26 18.6 0.7 18.00 0.54 2.57 0.11 0.48 0.05 0.97 0.01

BM 9.52 0.26 7.80 0.33 16.5 0.5 15.59 0.47 1.37 0.12 0. 31 0.01 0.97 0.01

a Data are means standard deviations from three replications

Biotechnol Lett (2010) 32:14131418 1415

123

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 significant influence

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 confirm the fermentation properties of the recov-

ered 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 final 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 profiles of cell density and

concentrations of glucose and organic acids are

shown in Fig. 1. The productivity of succinic acid

decreased from 2.56 to 1.30 g/l h, and the final

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 two-

stage fermentation and repeated fermentation using

Table 2 Effects of initial glucose concentrations on glucose consumption and product formation with recovered cells of E. coliAFP111 in sealed serum bottles

Time

(h)

Glucose (g/l) DCW (g/l) Succinic acid

(g/l)

Acetic acid

(g/l)

Pyruvic

acid (g/l)

Productivity

(g/l h)

Mass yield

(g/g)Initial Final Initial Final

26 31.3 1.1 1.0 0.1 9.80 0.31 9.24 0.32 28.77 0.62 3.24 0.12 0.41 0.01 1.11 0.02 0.96 0.02

48 50.5 1.6 1.5 0.1 9.65 0.45 8.17 0.44 43.22 0.52 3.18 0.15 0.46 0.01 0.90 0.04 0.89 0.03

60 67.5 1.8 0.5 0.1 9.71 0.32 8.05 0.33 48.61 0.52 6.78 0.13 0.73 0.01 0.81 0.02 0.73 0.02

72 87.4 1.1 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 0.68 0.05

84 118.0 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 0.66 0.02

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)

Succinic acid

(g/l)

Acetic acid

(g/l)

Pyruvic acid

(g/l)

Productivity

(g/l h)

Mass yield

(g/g)

9.50 0.36 18 21.5 0.9 20.5 0.33 1.74 0.05 0.47 0.02 1.14 0.01 0.95 0.02

19.13 0.57 9 27 1.0 24.9 0.53 2.83 0.08 0.63 0.03 2.77 0.03 0.92 0.03

26.52 0.76 6 28 1.3 24.8 0.32 3.80 0.06 0.79 0.02 4.13 0.02 0.89 0.04

33.53 1.05 4.5 33 1.5 25.1 0.44 4.52 0.06 1.03 0.01 5.58 0.03 0.76 0.14

Data are means standard deviations from three replications

Fig. 1 Time-course of glucose, products and cell densityduring the anaerobic production phase with recovered E. coliAFP111 cells in a 3-l stirred bioreactor. Triangles representDCW; circles represent glucose; squares represent succinicacid; inverted triangles represent acetic acid

1416 Biotechnol Lett (2010) 32:14131418

123

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 con-

centration and cell density had a significant influence

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 efficient comple-

ment to two-stage fermentation.

Acknowledgment This work was supported by the NationalNatural Science Foundation of China (No. 20606017), 973

Program of China (No. 2009CB724701).

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Table 4 Succinic acid productivities and mass yields for each stage during two-stage fermentation and repeated recovered cellsfermentation

Time (h) Consumed glucose (g/l) Succinic acid (g/l) Productivity (g/l h) Yield (g/g)

Two-stage fermentation

Aerobic stage 32 24.9 101 1.19 0.82

Anaerobic stage 53 98

First recovery stage 25 74.5 64 2.56 0.86

Second recovery stage 33 61 52.2 1.58 0.85

Third recovery stage 26 40 33.8 1.3 0.84

Biotechnol Lett (2010) 32:14131418 1417

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Willke T, Vorlop KD (2004) Industrial bioconversion of

renewable resources as an alternative to conventional

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Wu H, Li Z, Zhou L, Ye Q (2007) Improved succinic acid

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Succinic acid production with metabolically engineered E. coli recovered from two-stage fermentationAbstractIntroductionMaterials and methodsStrainCell recoveryMedia and conditionsAnalytical procedures

ResultsEffects of culture media on succinic acid fermentation with recovered cellsEffects of initial glucose concentration on succinic acid fermentation with recovered cellsEffects of initial cell density on succinic acid fermentation with recovered cellsRepeated recovery of cells for succinic acid production in a 3 l fermenter

ConclusionsAcknowledgmentReferences

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