high cell density fermentation via a metabolically engineered escherichia coli for the enhanced...

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Research ArticleReceived: 11 August 2010 Revised: 19 October 2010 Accepted: 19 October 2010 Published online in Wiley Online Library: 24 December 2010

(wileyonlinelibrary.com) DOI 10.1002/jctb.2543

High cell density fermentation via ametabolically engineered Escherichia colifor the enhanced production of succinic acidDan Wang,a,b Qiang Li,a,b Ziyu Song,a,b Wei Zhou,a,b Zhiguo Sua

and Jianmin Xinga∗

Abstract

BACKGROUND: Succinic acid is a valuable four-carbon organic chemical with applications in many fields. It was found that cellmass was an important factor in succinic acid production by metabolically engineered Escherichia coli strains. In this work, highcell density fermentation was investigated for succinic acid production by a metabolically engineered strain SD121 with ldhA,pflB, ptsG mutation and heterogenous cyanobacterial ppc overexpression.

RESULTS: Under two-stage cultivation, the controlled DO feeding strategy during the aerobic growth phase facilitated biomassup to a dry cell weight of 19.6 g L−1, and enhanced succinic acid production in the following anaerobic fermentation phase to aconcentration of 116.2 g L−1. A near theoretical maximum succinic acid yield of 1.73 mol mol−1 glucose was achieved with anaverage productivity of 1.55 g L−1 h−1.

CONCLUSION: The results indicated the potential advantage of high cell density fermentation for improvement of succinic acidproduction by E. coli.c© 2010 Society of Chemical Industry

Keywords: succinic acid; Escherichia coli; phosphoenolpyruvate carboxylase (PEPC); high cell density fermentation

INTRODUCTIONSuccinic acid, an important four carbon dicarboxylic acid,is listed as one of the 12 building block chemicals frombiomass.1 It has drawn worldwide interest for its industrial marketopportunities in biobased thermosets, bio-butanediol, plasticizers,renewable solvents, deicers and renewable thermoplastics.2

Traditional petrochemical production of succinic acid requireshigh temperature, high pressure and costly catalysts. Therefore,recently much attention has been paid to the bio-conversion ofrenewable feedstocks to succinic acid.3,4

Succinic acid could be produced by naturally selected mi-croorganisms, such as Anaerobiospirillum succiniciproducens,5 Acti-nobacillus succinogenes6 and Mannheimia succiniciproducens7,8

with impressive titers (35.4–106.2 g L−1) and high yields (>1.1 molsuccinate mol−1 glucose). However, these natural producers re-quire complex media ingredients, which add cost associated withproduction, purification, and waste disposal. Although Escherichiacoli produces succinic acid as a minor product of mixed-acidfermentation,9 it has the advantages of fast growth, simple nu-trients requirements, and can be metabolically engineered todecrease by-products formation and improve succinic acid yield.The production of succinate by metabolically engineered E. colistrains has been studied by several groups. Strain NZN111 wasengineered by inactivating two genes, pflB encoding pyruvate-formate lyase and ldhA encoding lactate dehydrogenase. Thisstrain could not ferment glucose under anaerobic conditions. How-ever, when two E. coli genes were overexpressed, mdh encoding

malate dehydrogenase and ppc encoding phosphoenolpyruvatecarboxylase, its glucose-fermentation ability was resumed and thestrain could produce 12.74 g L−1 of succinate with a molar yield of0.98 mol mol−1 glucose.10,11 AFP111 was a spontaneous glucosetransporter (ptsG) mutation of NZN111, which produced succinatefrom glucose with a yield of nearly 0.9 mol mol−1 glucose con-taining only native genes.12,13 Strain HL27659k was engineered bymutating succinate dehydrogenase (sdhAB), phosphotransacety-lase (pta), acetate kinase (ackA), pyruvate oxidase (poxB), ptsG,and the isocitrate lyase repressor (iclR).14 This strain producedsuccinate at less than 60 g L−1 with a yield of 0.94 mol mol−1

glucose. SBS550MG was constructed by inactivating the iclR, al-cohol dehydrogenase (adhE), ldhA, and ackA, and overexpressingBacillus subtilis citrate synthase (citZ) and Rhizobium etli pyruvatecarboxylase (pyc) from a multicopy plasmid to achieve a veryhigh yield of 1.6 mol succinate mol−1 glucose.15 Andersson et al.reported a mutant strain AFP184 with highest succinate produc-tivity of 2.9 g L−1 h−1 with a yield of 1.27 mol mol−1 glucose.16

∗ Correspondence to: Jianmin Xing, P. O. box 353#, Zhongguancun Bei-er-tiao 1,Haidian District, Institute of Process Engineering, Chinese Academy of Sciences,Beijing, 100190, China. E-mail: [email protected]

a National Key Laboratory of Biochemical Engineering, Institute of ProcessEngineering, Chinese Academy of Sciences, Beijing 100190, China

b Graduate university of Chinese Academy of Sciences, Beijing 100049, China

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High cell density fermentation for succinic acid production by E. coli www.soci.org

E. coli KJ060 was constructed using the method of metabolic evo-lution combined with inactivation of the ldhA, adhE, ackA, formatetransporter (focA) and pflB genes. This strain produced a succi-nate concentration of 86.49 g L−1 with a yield of 1.41 mol mol−1

glucose.17

Concerning succinate fermentation by metabolically engi-neered E. coli strains, cell mass was an important index tocharacterize the process. Furthermore, it has been found thatto achieve high concentration of succinate and high specific pro-ductivity in the fermentation process, a high level of cell masswas required.14,16,18 Since simple anaerobic culture did not fa-cilitate cell growth of E. coli, dual-phase fermentation processes,with aerobic cell growth followed by anaerobic conditions, havebeen explored to improve the cell mass accumulation and subse-quently, improve succinate production.16,19 – 21 Under two-stagefermentation, the strain AFP184 produced 25–40 g L−1 succinatewith a productivity of 1.5–2.9 g L−1 h−1. The recombinant AFP111harboring the plasmid pTrc99A-pyc achieved a final succinate con-centration of 99.2 g L−1 with a productivity of 1.3 g L−1 h−1.16,19

However, in Andersson et al.’s study, the concentration of glucosein the medium was 100 g L−1, which added osmotic pressure tothe strains and the final succinate concentration was lower forindustrial application.16 In Vemuri et al.’s research, the time oftransition from aerobic to anaerobic phases was chosen based onthe activities of the key enzymes, which added inconvenience totransform this technology into industrial production.18 In theseprocesses of succinic acid production using E. coli, cell mass ac-cumulated was still lower than the cell density reported for othermetabolites expresssed by E. coli.22 – 24 Thus, new fermentationprocess should be carried out to increase the cell mass at hightiters. This process should be optimized to achieve high concen-tration of succinate and high specific productivity for large-scaleproduction purposes, and the aerobic/anaerobic transition pointshould be easily identified.

In this study, a new fermentation process for a metabolicallyengineered E. coli strain SD121 with ldhA, pflB, ptsG mutationand heterogenous cyanobacterial ppc overexpression was carriedout, in which the time of transition from aerobic to anaerobicphase was simply chosen based on the cell mass. High cell densityfermentation based on the DO control strategy was adopted toincrease the cell mass during the aerobic stage. Then anaerobicfed-batch fermentation was carried out and the distribution of themetabolites was examined.

EXPERIMENTALStrains, plasmids and growth conditionsCyanobacterium Anabaena sp. 7120 was obtained from the Pas-teur Institute in France. E. coli DC1515 [pflB::Cam ldhA::Tn10ptsG400::Kan in W1485] was kindly donated by ProfessorClark, Southern Illinois University. Manipulation of DNA wasdone as previously described.25 The ppc gene was ampli-fied with primers cppc1 and cppc2 using cyanobacteriumAnabaena sp. 7120 genomic DNA as the template. Thesequences of these primers were as follows: cppc1, 5′-AGAGCCGAATTCATGGGTTCTGTTT-3′ (EcoRI restriction site un-derlined); cppc2, 5′-GCCAAGCTTTCAACCTGTATTTCTC-3′ (HindIIIrestriction site underlined). The PCR was performed for 30 cyclesof denaturation for 30 s at 94 ◦C, annealed for 60 s at 48 ◦C, andextended for 180 s at 72 ◦C. The single product was isolated, puri-fied and ligated into the expression plasmid pTrchisB (Invitrogen,USA), with EcoRI and HindIII excision to give a plasmid pTrc-cppc.

Then the plasmid was introduced into DC1515 to form a recom-binant designated as SD121. Parental strain DC1515 carrying thecorresponding backbone plasmid pTrchisB was constructed as thecontrol strain SD120. Each PCR fragment was verified by DNAsequencing at the Shanghai Sangon Biological Engineering Tech-nology & Service Co., Ltd. E. coli Top 10 was used for propagationand amplification of plasmids used in this work.

During strain construction, cultures were grown aerobicallyat 37 ◦C in Luria–Bertani (LB) medium (10 g L−1 Difco tryptone,5 g L−1 Difco yeast extract, and 5 g L−1 NaCl) with 10 g L−1

glucose. Solid media for plates contained 15 g L−1 Difco BactoAgar. Antibiotics were included as necessary at the followingconcentrations: 50 µg mL−1 ampicillin; 50 µg mL−1 kanamycin;10 µg mL−1 tetracycline and 30 µg mL−1 chloramphenicol. Themedium for fermentation in flasks contained (per liter): 20 gglucose, 20 g tryptone, 10 g yeast extract, 0.3 g MgSO4·7H2O,0.45 g Na2HPO4·12H2O, 6 g NaH2PO4·2H2O, 3 g (NH4)2SO4·7H2O,0.2 g CaCl2. The medium for dual-phase fermentation in a 3 Lfermenter was the same as in the flask, except that the initialconcentration of glucose was changed to 15 g L−1. IPTG wasadded at 0.1 mmol L−1 to the medium to induce gene expressionof ppc for plasmid pTrc-cppc.

Fermentation in flasksThe two-stage culture technique was adopted in cultures carriedout in flasks. The transformed colonies were selected from LBplates containing the appropriate antibiotic(s). A seed inoculum of200 µL from an overnight 5 mL culture was added to a 500 mL flaskcontaining 100 mL of fermentation medium with the appropriateantibiotic(s). The strain was cultured at 220 rpm, 37 ◦C to a certaincell density aerobically, specifically, to 1 OD600 nm, 2 OD600 nm,4 OD600 nm, and 8 OD600 nm. Then 50 mL of the cell suspensionwere shifted to 100 mL bottles (Schott, Germany) with 1 g MgCO3

(added to the bottle before autoclaving to control the fermentationpH at 6.7 and provide CO2), and additional glucose was added togive an initial concentration of 20 g L−1. The headspace was filledwith CO2 to start the anaerobic culture at 150 rpm and 37 ◦C for48 h.

Fermentation in bioreactorsDual-phase fermentation was conducted with an initial mediumvolume of 1.2 L in a 3.0 L New Brunswick Scientific (Edison, NJ)Bioflo 110 fermenter. A 5% (v/v) inoculum was used from anovernight culture grown from a single colony for 12 h. In aerobicphase, the pH was controlled at 7.0 with 10 mol L−1 NaOH solutionand 10% H2SO4 (v/v), and the temperature was maintained at 37 ◦C.Oxygen-enriched air as necessary was sparged at 4 vvm with anagitation of 300–1000 rpm to maintain the dissolved oxygen (DO)above 10% as measured with an online probe (Mettler-ToledoProcess Analytical Instruments, Wilmington, MA). When the initialglucose was exhausted, a sterile 80 g L−1 glucose solution wasfed to keep the strains growing to the desired cell density. Thenthe anaerobic period was started after addition of glucose to aconcentration of 40 g L−1 and NaHCO3 to 5 g L−1. The broth wassparged with 2 vvm oxygen-free CO2 and mixed at a 150 rpmagitation rate. The pH was maintained between 6.4 and 6.8 withintermittent supplementation of solid MgCO3. When the glucoseconcentration decreased to near 10 g L−1, a sterile concentratedglucose solution (800 g L−1) was fed into the media.

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www.soci.org D Wang et al.

Enzyme activity assayTo analyze enzyme activities, cell extracts of the E. coli strainswere prepared by washing the cell pellets with appropriate buffer(50 mmol L−1 Tris-HCl (pH 8.0), 1 mmol L−1 EDTA, 1 mmol L−1

EGTA, 0.05% v/v NP40 and 0.1 mmol L−1 DTT) and disrupting thesuspended cells by sonicating at 100 W for 10 min at 1 s intervalson ice. Cell debris was removed by centrifugation at 10 000 gfor 10 min at 4 ◦C. The supernatant was further centrifuged at10 000 g at 4 ◦C for 20 min and the resulting supernatant wasused for the assay of enzyme activity. The PEP carboxylase (PEPC)activity was assayed by monitoring the decrease in absorbanceof NADH at 340 nm using malate dehydrogenase as a couplingenzyme, modification of Kodaki’s method.26 The 1 mL reactionmixture for PEPC analysis consisted of 50 mmol L−1 HEPES (pH7.3), 5 mmol L−1 PEP, 10 mmol L−1 MgCl2, 5 mmol L−1 NaHCO3, 4units of malate dehydrogenase, 0.2 mmol L−1 NADH and 25 µL ofcell extract. The extinction coefficient for NADH at 340 nm was 6.22per cm mmol L−1. 1 U of PEPC activity was defined as the activityof oxidizing 1 µmol L−1 NADH min−1 at 30 ◦C. Enzyme assays wereperformed in triplicate, and if the discrepancy was greater than10%, another pair of assays was performed.

Analytical methodsBiomass concentrations were estimated from the optical density(OD) of the fermentation broth with a spectrophotometer(723N, Shanghai Precision & Scientific Instrument Co. Ltd,China) at a wavelength of 600 nm. The concentrations ofglucose were measured by SBA 40C bio-sense analyzer (BiologyInstitute, Shangdong Academy of Sciences, China). The organicacids formation during fermentation were analyzed by high-performance liquid chromatography, Agilent 1200 (Agilent, Co. LtdUSA) equipped with UV absorbance and refractive index detectorsand a Bio-Rad Aminex HPX-87H ion-exchange column (7.8 by300 mm). A mobile phase of 5 mmol L−1 H2SO4 at 0.6 mL min−1

flow rate was used and the column was operated at 55 ◦C. Samplesof culture were removed anoxically and centrifuged at 10 000 rpmfor 10 min. Each supernatant was diluted with 10 volumes of5 mmol L−1 H2SO4, and 20 µL of the diluted sample was injected.

RESULTSConstruction and characterization of the recombinant strainSD121The recombinant strain SD121 was constructed by introducingthe plasmid pTrc-cppc into the mutant strain DC1515 deficient inldhA, pflB and ptsG. DC1515 was similar to AFP111, which producednearly no formate and lactate, and could produce 0.9 mol succinatemol−1 glucose under anaerobic conditions. The plasmid pTrc-cppc,carrying the cyanobacterial ppc gene, was used to produce highlevels of PEPC in DC1515, and improve the succinate-producingability. First, the positive clone of the plasmid pTrc-cppc wasidentified by gel electrophoresis of the EcoRI and HindIII digestingsamples. The approximately 2950 bp small segment was thesame length of ppc in cyanobacterium Anabaena sp.7120 (referto NCBI sequence NC 003 272.1) as expected (data not shown).The integrity of the cloned ppc and open reading frame wasfurther checked by sequencing. SDS-PAGE was carried out onSD121 induced by 0.1 mmol L−1 IPTG. Soluble crude extracts wereprepared from the fermentative medium without MgCO3 underaerobic conditions every 2 h. A band of increasing intensity around117 KDa size range was observed on the gel upon Commassie

94KD67KD

43KD

30KD

20KD

14KD

1 2 4 5

PEPC, 117KD

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Figure 1. Time course of PEPC expression in SD121 induced by0.1 mmol L−1 IPTG. 1, 6 h after induction; 2, 4 h after induction; 3, 2 hafter induction; 4, protein Marker; 5, 0 h. Arrow indicates the PEPC bond.

Table 1. PEPC activities in U mg−1 of total protein from aerobic oranaerobic cultures of strains 6 h after induced by 0.1 mmol L−1 IPTG

Activity∗ (U mg−1)

Strain Aerobic Anaerobic

SD120 0.22 ± 0.02 0.21 ± 0.03

SD121 5.18 ± 0.35 4.72 ± 0.19

∗ Calculated as µmol NADH used per milligrams of protein per minutewhen coupled with malate dehydrogense.All experiments were performed a minimum of three independent sets,and data present mean ± SD.

Brilliant Blue staining, which was consistent with the molecularweight of PEPC from its nucleotide sequence by software DNAman(Version 5.2.2, Lynnon Biosoft) (Fig. 1). The results showed that theheterogenous ppc gene could be expressed in E. coli successfully.

Second, the strain SD121 was further characterized by deter-mining the activities of PEPC under both aerobic and anaerobicconditions. From Table 1, it can be seen that the PEPC activity ofstrain SD121 was 5.18 U mg−1 protein under aerobic conditions.While under anaerobic conditions, it decreased to 4.72 U mg−1

protein. Control strain DC1515 harboring the plasmid pTrchisBshowed PEPC activities of just 0.22 U mg−1 protein under aerobicconditions, and 0.21 U mg−1 protein under anaerobic conditions.Increased PEPC activity could enhance the carboxylation of PEP tooxaloacetate (OAA). OAA was sequentially reduced to malate, andeventually converted to succinate.

Third, batch fermentations were performed in flasks withstrain SD121 to explore the effect of ppc overexpression on themetabolites distributions. Due to the buffering of MgCO3, the pHvalues at the end of the fermentations were maintained above6.0. When an initial OD600 of 2 was used to start the anaerobicfermentation, the succinate yield of SD121 reached 1.38 mol mol−1

glucose. It was 1.47-fold higher than that of the control strain,which was just 0.94 mol mol−1 glucose. The C4 metabolism ratio(C4 product per sum of C2 + C3 + C4) of SD121 was 70.9%, whichwas 47.0% for SD120. The succinate productivity calculated basedon the final succinate concentration in the medium of SD121 was0.535 g L−1 h−1, while it was 0.422 g L−1 h−1 for the control strainSD120 (Table 2). These results indicated that the recombinantstrain with cyanobacterial ppc overexpression could significantly

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Table 2. Effect of cyanobacterial ppc overexpression on the production of fermentation products

Yield (mol product mol−1 glucose)C4 metabolism Succinate productivity

Straina Succinate Lactate Formate Acetate Ethanol ratio(%)b (g L−1 h−1)c

SD120 0.942 ± 0.016 0.035 ± 0.008 0.002 ± 0.001 0.535 ± 0.047 0.492 ± 0.023 47.0 0.422 ± 0.009

SD121 1.380 ± 0.052 0.021 ± 0.004 0.002 ± 0.001 0.218 ± 0.021 0.326 ± 0.016 70.9 0.535 ± 0.013

All experiments were performed a minimum of three times, and data shown are mean ± SD.a Medium contained glucose approximately 20 g L−1 and magnesium carbonate 20 g L−1 in anaerobic bottles.b C4 metabolism ratio represents the C4 molar yield per sum of C4 + C3 + C2 molar yield.c Succinate productivity is calculated by the final concentration of succinate in the medium divided by the fermentation time.

Table 3. Comparison of different cell densities at the transition point of succinate production in flasks

Succinate yielda

(mol mol−1 glucose)Calculated cell density at Final succinate Succinate Anaerobic

OD600 nm at thetransition time

the transition time(DCW L−1)

concentrationa

(g L−1) First 8 h The overallproductivitya

(g L−1 h−1)fermentation

time (h)

1 0.38 17.52 ± 0.52 1.42 ± 0.02 1.33 ± 0.04 0.417 ± 0.012 42

4 1.52 18.89 ± 0.25 1.48 ± 0.03 1.44 ± 0.02 0.674 ± 0.008 28

8 3.04 19.33 ± 0.13 1.52 ± 0.01 1.47 ± 0.01 1.381 ± 0.011 14

All experiments were performed a minimum of three times, and data shown are mean ± SD.a The data were calculated only for the anaerobic stage.

shift the metabolic flux to succinate production, showing potentialability for industrial scale-up.

By overexpressing of native PEPC in wild E. coli, strainshave shown an increase in the production of succinate.10,27

Overexpression of a mutant Sorghum vulgare PEPC that wasresistant to malate feedback inhibition in an E. coli mutantHL27659k has also been shown to increase succinate production.14

In this work, PEPC enzyme of cyanobacterium was chosenfor its highly evolution adaptively for CO2 assimilation in aphotosynthetic organism. It was also chosen for the relative easeof genetic manipulation of prokaryotic organism, compared withhigher plants.28 Cyanobacteria as a group are characterized bypossessing an incomplete tricarboxylic acid (TCA) cycle lackingboth α-ketoglutarate dehydrogenase and NADH oxidase.29 As aresult of these deficiencies, metabolic energy cannot be derivedfrom this pathway, and the cycle is incapable of regeneration.PEPC functions powerfully in cyanobacteria to synthesize C4 acids,which could be used to replenish the TCA cycle intermediatesand for biosynthesis. The naturally strong carboxylation abilityof cyanobacterial PEPC made it an ideal target for metabolicallyengineering of a succinate formation pathway in E. coli. The resultsin our study have also confirmed the hypothesis.

Comparison of different cell densities at the transition pointon succinate production in flasksThe cell densities were chosen as the key points to mark thetime of transition from aerobic to anaerobic phase. Here theexperiments carried out in flasks were based on three differentcell densities of 1 OD600 nm, 4 OD600 nm, and 8 OD600 nm as themilestones, respectively (Table 3). The relative lower cell density of1 OD600 nm at the transition point resulted in a starting anaerobicphase of 0.38 g DCW L−1, the middle cell density of 4 OD600 nm

at the transition point resulted in a starting anaerobic phase of1.52 g DCW L−1, the relative higher cell density of 8 OD600 nm

at the transition point resulted in a starting anaerobic phase of3.04 g DCW L−1. Results showed that when the cell density at thetransition time varied, the final succinate concentrations, yieldsand overall productivities changed. The higher the cell densitychosen to start the anaerobic stage, the higher the succinateconcentration, yield and overall productivities achieved. Culturesof strain SD121 starting the anaerobic phase at 1 OD generateda succinate yield of 1.42 mol mol−1 glucose through the first8 h, then the yield decreased to 1.33 after 42 h of fermentation.The succinate concentration obtained at the end of fermentationwas 17.5 g L−1. Anaerobic culture starting at 4 OD generated asuccinate yield of 1.48 mol mol−1 glucose for the first 8 h. Thenthe succinate yield decreased to 1.44 after 28 h of fermentationand the succinate concentration obtained was 18.9 g L−1. Whenanaerobic culture of strain SD121 was started at 8 OD to increasethe productivity, the initial succinate yield was 1.52 mol mol−1

glucose, and the yield decreased to 1.47 after 14 h of fermentation.The succinate concentration obtained was 19.3 g L−1 (Table 3).

The high cell density at transition time (starting OD of 8)did achieve high succinate productivity of 2.56 g L−1 h−1 at thebeginning of fermentation (data not shown), and then decreasedto 1.38 g L−1 h−1 at the end of the 14 h fermentation (Table 3).This succinate productivity is higher than that of the two lower celldensities (starting OD of 1 and 4). Succinate productivity of themedium cell density culture (starting OD of 4) was 0.68 g L−1 h−1 atthe end of a 28 h fermentation. Succinate productivity of the smallcell density culture (starting OD of 1) was 0.43 g L−1 h−1 at the endof 42 h fermentation (Table 3). The high succinate productivityof cultivation with high cell density at the transition point wasobtainable apparently due to higher biomass and faster glucoseconsumption rate.

Results showed a significant increase in succinate yield andproductivity when higher cell density was chosen to start theanaerobic fermentation. This indicated that cell mass could be


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Figure 2. The effect of different cell density at the transition point on succinate production in the bioreactor. (a) 8 OD at the time of transition; (b) 20 ODat the time of transition; (c): 50 OD at the time of transition. �: glucose; •: succinic acid; �: lactate; ◦: acetate; �: ethanol; : OD600.


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Table 4. Comparison of different cell densities at the transition point of succinate production in the bioreactora

OD600 at thetransition time

Calculated celldensity at the

transition time(DCW L−1)

Final succinateconcentration

(g L−1)Succinate yield

(mol mol−1 glucose)

Succinateproductivitya

(g L−1 h−1)

The ratio ofsuccinate tobyproducts

Final celldensity

(DCW L−1)

Anaerobicfermentation

time (h)

8 3.04 61.9 1.42 1.03 4.86 6.38 64

20 7.60 97.6 1.67 1.27 6.83 7.08 84

50 19.6 116.2 1.73 1.55 7.00 13.97 86

a The data were calculated from Fig. 2 only for the anaerobic stage.

chosen to mark the transition point, which resulted in variablesuccinate production ability of the strain SD121.

Comparison of different cell densities at the transition pointon succinate production in bioreactorBecause of insufficient O2 supply and non-strict control of pH andsubstrate concentration, it was found that 8 OD was almost theultimate cell density that could be achieved in flasks by SD121.Thus, further experiments were conducted in a 3 L fermenter.Preliminary experiments showed that an initial 15 g L−1 glucosecould sustain the cell growth to a density of 20 OD, and thenbatch-feeding of a glucose solution of 80 g L−1 could promotecell density. Based on the flexibility of DO control in a bioreactor,cell masses of OD 8, OD 20 and OD 50 were chosen to start theanaerobic fermentation. The sterile 800 g L−1 glucose was thenfed to sustain sufficient substrate for succinate synthesis. The fed-batch experiments were performed three times and the resultsshowed that high-level succinate production could be achievedwith high-level cell mass at the transition point. Only one set ofdata is presented.

The results of the bioreactor experiments are shown in Fig. 2.The batch growth of SD121 reached a cell density of OD 8 in4 h, and then fed-batch fermentation could last for 48 h until theglucose consumption rate dropped to 1 g L−1 h−1. By the end ofthe fermentation, 61.9 g L−1 succinate was produced. Throughoutthe entire anaerobic fermentation phase, the average succinateyield was 1.42 mol mol−1 glucose with an overall productivityof 1.03 g L−1 h−1. The final OD after 64 h was 16.8 (Table 4). Noformate was detected under this condition. The concentration oflactate was less than 2 g L−1. The concentration of acetate andethanol was 9.21 g L−1 and 1.87 g L−1, respectively (Fig. 2(a)).

If the aerobic batch culture lasted for 7 h, the strain SD121 couldreach a cell density of 20 OD. Then the anaerobic fermentation wasstarted, and the results showed that by the end of the fermentation,97.6 g L−1 succinate was produced. The accumulative succinateyield was 1.67 mol mol−1 glucose and the overall productivitywas 1.27 g L−1 h−1. The final OD after 84 h was 18.6 (Table 4).No formate was accumulated at the end of the fermentation.The concentrations of ethanol and lactate were less than 1 g L−1

under this condition. The concentration of acetate was 12.95 g L−1

(Fig. 2(b)).When the cell density reached OD 20, the residual glucose was

less than 1 g L−1. If a glucose solution of 80 g L−1 was fed to thefermenter, the cells could continue growing to an OD of 50 in4 h. The feed rate was controlled to maintain the DO above 10%during the 4 h of batch-feeding, which resulted in a fast growthrate of 3.8 g L−1 h−1. In this case, the accumulation of glucose andacetate that affected the cell growth was avoided.23 116.2 g L−1

of succinate was produced at the end of the fermentation by

E. coli for the first time. The final OD after 86 h was 35.75. No formatewas detected as shown in Fig. 2(c). The concentration of acetate,lactate and ethanol was 11.37 g L−1, 1.82 g L−1 and 3.42 g L−1,respectively. Throughout the entire anaerobic fermentation theaverage succinate yield was 1.73 mol mol−1 glucose, the averageproductivity was 1.55 g L−1 h−1 (Table 4).

DISCUSSIONThe high cell density fermentation based on the DO controlstrategy in the aerobic phase facilitated the following anaerobicfermentation for succinic acid production by a recombinant E. colistrain SD121. In this research, the time of transition from aerobic toanaerobic phases was simply chosen based on cell mass, a processthat could be easily controlled for industrial commercialization.

Under optimized fed-batch aerobic conditions, the strainreached a cell density of 50. Then the anaerobic fermentation phasewas carried out for 75 h and this strain produced 116.2 g L−1 ofsuccinate at the end of fermentation, which was the highest valuereported to date when employing recombinant E. coli. Throughoutthe entire anaerobic fermentation the average succinate yieldwas 1.73 mol mol−1 glucose, the average productivity was1.55 g L−1 h−1. Based on the redox-balance of E. coli, Wang et al.proposed the maximum yield of succinic acid to be 1.71 mol mol−1

when both PTS and glyoxylate shunt were active.30 In this study, thesuccinic acid yield obtained under high cell density at the transitionpoint reached 1.73 mol mol−1, which was a little higher than thetheoretical value. This may be attributed to the increased PEPCactivity and active glyoxylate shunt under dual-phase conditions.The tryptone and yeast extract added to the medium could beused as carbon source in the anaerobic fermentation phase forsuccinic acid production, which also contributed to the highersuccinate yield. Besides the advantages of high succinic acidconcentration, yield and productivity, another virtue of highcell density fermentation was the high ratio of succinic acid tobyproducts in the broth (Table 4). Thus, an effective separationprocess could be achieved with economic benefits.

The results of the fed-batch experiment with strain SD121showed that substantial succinate production could be achievedwith high cell density at the time of transition from aerobic toanaerobic phase. More importantly, this was the first time that themilestone to mark the time of transition from aerobic phase toanaerobic conditions was chosen based on rational comparison ofthe effects of different cell densities at the transition point ratherthan testing the key enzyme activities;19 the result was betterthan when choosing the transition point randomly.20,21 This dual-phase process, combining high cell density culture aerobicallyand fed-batch fermentation anaerobically, was also more practicalthan a single-stage anaerobic production system, because the


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high cell mass was accumulated in the aerobic phase. It was alsosuperior to those systems constructed under aerobic conditions.The latter maximum theoretical yield was just 1 mol mol−1 glucose,which decreased the economy of the substrate.14 Conventionaldual-phase succinate production systems were conducted withcomplex manipulation to identify the time of transition, relativelower cell density at the transition point or higher concentrationof glucose exerting osmotic pressure on cell growth in the aerobicphase.16,19 – 21 The dual-phase succinate production system withaerobic high cell density fermentation under a DO-controlled fed-batch strategy in this study is a practical, efficient, and viablealternative to the conventional method. The high cell densityfermentation strategy seems promising for scaling up in thesuccinate fermentation industry.

ACKNOWLEDGEMENTThis research was supported in part by grants from the KnowledgeInnovation Program of Chinese Academy of Sciences (Grant NO.KSCX2-YW-G-021), and State Major Basic Research DevelopmentProgram of China (Grant 2007CB714305). We thank Professor Clark(Southern Illinois University) for strain DC1515.

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