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Process Biochemistry 45 (2010) 1103–1114 Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Microbial succinic acid production: Natural versus metabolic engineered producers Joeri J. Beauprez, Marjan De Mey , Wim K. Soetaert Ghent University, Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Coupure Links 653, B-9000 Ghent, Belgium article info Article history: Received 18 November 2009 Received in revised form 21 March 2010 Accepted 29 March 2010 Keywords: Succinic acid Metabolic engineering abstract The increased consciousness for environmental issues and the depletion of mineral oil reserves led to the search for alternative energy sources but also for alternative biochemical processes. One of these chemicals that is identified to have great economical potential in a biobased economy is succinic acid. This chemical is a precursor for various high value-added derivatives which have application in the detergent/surfactant market, the ion chelator market, the food market and the pharmaceutical market. This review investigates the goals and preconditions to have an economical viable biosuccinic acid process. The different production hosts for biosuccinic acid are examined and the metabolic engineering strategies and possibilities are discussed. Finally, the state of the art of biosuccinic acid production pro- cesses is critically evaluated in function of the production host, media, fermentation strategy, titers and yields. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction The past century, chemical industry has been tightly interwoven with the oil industry. The rising oil prices and the environmental impact of many petrochemical processes are imposing the recon- sideration of many of the current chemical technologies. This trend channel resources towards the development of more economical as well as environmental sound routes and allows increasingly the introduction of biochemical alternatives. The question is, which technologies need to be developed and which biochemicals will be of use in this context? The US Department of Energy (DOE) and the European Commission have confronted this issue by ordering a study around sustainable pro- duction of chemicals from renewable resources [1–3]. These reports indicated several chemicals that can be biochemically produced and can be economical viable, stipulating the necessity for further research in white biotechnology. A chemical building block listed in the top 10 by the US Department of Energy as a potential platform chemical for the production of various high value-added deriva- tives from renewable resources is the C4-molecule succinic acid (1,4-butanedioic acid, SA) [1]. Nowadays, the succinic acid market is about 20 000–30 000 ton per year worldwide and is manufactured on industrial scale by catalytic hydrogenation of petrochemically derived maleic acid or maleic anhydride [4]. Corresponding author. Tel.: +32 9 264 60 26; fax: +32 9 264 62 31. E-mail address: [email protected] (M. De Mey). The four existing succinic acid markets are the deter- gent/surfactant market, the ion chelator market, food market (e.g. acidulants, flavours or antimicrobials) and the pharmaceutical mar- ket (Fig. 1, upper part). These markets have high added value and do not require very cheap feedstock. However, commodity chemicals are mostly low cost bulk chemicals [5]. Three succi- nate derivatives with major applications are obtained through hydrogenation routes. These are gamma-butyrolactone (GBL), 1,4- butanediol (BDO) and tetrahydrofuran (THF). BDO has three main branches—polymers, tetrahydrofuran (THF) derivatives and - butyrolactone (GBL) derivatives [5–7]. A second group of succinic acid derived molecules are the pyrrolidones. Their applications are mainly located in the solvent and polymer industry. Ammonium succinate and succinimides are alternative reactants for pyrroli- done production. Through reductive amination, succinic anhydride or maleic anhydride can be converted in an aqueous environment [6,7]. Fumarate, malate and itaconate form a third group of poten- tial succinic acid derivatives. The chemical conversion of succinate to these three compounds involves high temperatures and pres- sures, in some cases in a multistep process. These high energy consuming processes can be avoided by direct fermentative pro- duction. All three of these compounds are naturally produced by microorganisms and production systems are being developed for industrial production [8–10]. In this review the microbial production of succinic acid is dis- cussed. First, the economic viability of a biosuccinate production process is assessed. Next, the different production hosts are exam- ined. Beside the natural succinate producing strains, metabolic engineering strategies and possibilities are discussed. Finally, a 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.03.035

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Process Biochemistry 45 (2010) 1103–1114

Contents lists available at ScienceDirect

Process Biochemistry

journa l homepage: www.e lsev ier .com/ locate /procbio

icrobial succinic acid production: Natural versus metabolic engineeredroducers

oeri J. Beauprez, Marjan De Mey ∗, Wim K. Soetaerthent University, Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering,oupure Links 653, B-9000 Ghent, Belgium

r t i c l e i n f o

rticle history:eceived 18 November 2009eceived in revised form 21 March 2010ccepted 29 March 2010

a b s t r a c t

The increased consciousness for environmental issues and the depletion of mineral oil reserves led tothe search for alternative energy sources but also for alternative biochemical processes. One of thesechemicals that is identified to have great economical potential in a biobased economy is succinic acid.

eywords:uccinic acidetabolic engineering

This chemical is a precursor for various high value-added derivatives which have application in thedetergent/surfactant market, the ion chelator market, the food market and the pharmaceutical market.

This review investigates the goals and preconditions to have an economical viable biosuccinic acidprocess. The different production hosts for biosuccinic acid are examined and the metabolic engineeringstrategies and possibilities are discussed. Finally, the state of the art of biosuccinic acid production pro-

ted in

cesses is critically evaluayields.

. Introduction

The past century, chemical industry has been tightly interwovenith the oil industry. The rising oil prices and the environmental

mpact of many petrochemical processes are imposing the recon-ideration of many of the current chemical technologies. This trendhannel resources towards the development of more economicals well as environmental sound routes and allows increasingly thentroduction of biochemical alternatives.

The question is, which technologies need to be developednd which biochemicals will be of use in this context? The USepartment of Energy (DOE) and the European Commission haveonfronted this issue by ordering a study around sustainable pro-uction of chemicals from renewable resources [1–3]. These reports

ndicated several chemicals that can be biochemically producednd can be economical viable, stipulating the necessity for furtheresearch in white biotechnology. A chemical building block listed inhe top 10 by the US Department of Energy as a potential platformhemical for the production of various high value-added deriva-ives from renewable resources is the C4-molecule succinic acid1,4-butanedioic acid, SA) [1]. Nowadays, the succinic acid market is

bout 20 000–30 000 ton per year worldwide and is manufacturedn industrial scale by catalytic hydrogenation of petrochemicallyerived maleic acid or maleic anhydride [4].

∗ Corresponding author. Tel.: +32 9 264 60 26; fax: +32 9 264 62 31.E-mail address: [email protected] (M. De Mey).

359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2010.03.035

function of the production host, media, fermentation strategy, titers and

© 2010 Elsevier Ltd. All rights reserved.

The four existing succinic acid markets are the deter-gent/surfactant market, the ion chelator market, food market (e.g.acidulants, flavours or antimicrobials) and the pharmaceutical mar-ket (Fig. 1, upper part). These markets have high added valueand do not require very cheap feedstock. However, commoditychemicals are mostly low cost bulk chemicals [5]. Three succi-nate derivatives with major applications are obtained throughhydrogenation routes. These are gamma-butyrolactone (GBL), 1,4-butanediol (BDO) and tetrahydrofuran (THF). BDO has three mainbranches—polymers, tetrahydrofuran (THF) derivatives and �-butyrolactone (GBL) derivatives [5–7]. A second group of succinicacid derived molecules are the pyrrolidones. Their applications aremainly located in the solvent and polymer industry. Ammoniumsuccinate and succinimides are alternative reactants for pyrroli-done production. Through reductive amination, succinic anhydrideor maleic anhydride can be converted in an aqueous environment[6,7]. Fumarate, malate and itaconate form a third group of poten-tial succinic acid derivatives. The chemical conversion of succinateto these three compounds involves high temperatures and pres-sures, in some cases in a multistep process. These high energyconsuming processes can be avoided by direct fermentative pro-duction. All three of these compounds are naturally produced bymicroorganisms and production systems are being developed forindustrial production [8–10].

In this review the microbial production of succinic acid is dis-cussed. First, the economic viability of a biosuccinate productionprocess is assessed. Next, the different production hosts are exam-ined. Beside the natural succinate producing strains, metabolicengineering strategies and possibilities are discussed. Finally, a

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1104 J.J. Beauprez et al. / Process Biochemistry 45 (2010) 1103–1114

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Fig. 1. Overview of applications and pro

ummary of the many biosuccinate production processes describedn literature are given. Next to the reported strains, media andermentation strategy, also the yields are given. The pre- and down-tream processing of biosuccinic acid were not considered. Moreetails about downstream processing, can be found in [11–17] andeferences therein.

. Microbial succinate production

.1. Economical viability of a biosuccinic acid process

To have an economical viable biosuccinic acid process, it shoulde competitive with the current applied chemical process. Basedn the petrochemical analogue, maleic anhydride, the DOE haveet the targeted production price of biosuccinic acid at 0.45 D /kg.

owadays, with the increasing oil price, this analogue more than

ripled in price [18,19].Three important process parameters determine the economical

iability of a bioprocess: yield, titer and production rate. While theield relates more to the variable cost of raw feedstock and will be

derived from succinic acid [5–7,20,21].

of increasing importance with increasing sugar prices. Productionrates and titers relate more to the fixed cost and total investment.Low rates imply larger energy and labour cost; low titers will resultin larger capital investment to maintain high plant capacities. Basedon these facts Wilke calculated that a yield of 100% (w/w), a rateof 3 g/l/h and titers up to 250 g/l would result in a total productioncost of 0.45 D /kg [23,24].

2.2. Succinate production hosts

Because succinic acid is an intermediate of the Krebs cycle anda fermentative end-product, microorganisms lend themselves per-fectly as production hosts. The choice of production host is verydiverse, although most natural production hosts described in liter-ature are capnophilic microorganisms. The non-natural production

hosts, on the other hand, are chosen on the basis of their geneticaccessibility. The most current used strains are Actinobacillussuccinogenes, Anaerobiospirillum succiniciproducens, Mannheimiasucciniciproducens and Escherichia coli. Corynebacterium sp. andBacteroides fragilis have been introduced very recently as succi-
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ate platform strains [27,28]. The latter is the result of extensivecreening for succinate producing microorganisms in bovine rumen29].

.2.1. Natural succinate producing strainsMost bacteria, which produce succinate naturally in significant

iters, have been isolated in the rumen of ruminants. The anaer-bic conditions, caused by carbon dioxide, methane and traces ofydrogen production, create the unique environment for microbialuccinic acid production [30]. A. succinogenes, A. succiniciproducens,. succiniciproducens and B. fragilis are natural succinate producing

trains, which all have been isolated in the rumen. They produce aixture of volatile organic acids and as capnophiles they can copeith high carbon dioxide and use it as a carbon source together with

ugars [21]. In some cases carbon dioxide is essential for growth anddapted screening methods have to be employed to isolate novelapnophilic strains [31]. Most probably these efforts will lead toany more isolates that efficiently produce succinate.Optimised fungal succinate production systems are rarely

escribed in literature, although Fusarium spp., Aspergillus spp.nd Penicillium simplicissimum are known to excrete the acid [32].. simplicissimum succinate and citrate co-excretion was studiednder anaerobic and aerobic conditions. A strong increase in suc-inate excretion was observed when the respiratory chain wasnhibited, either by sodium azide or anaerobic conditions. Oddlynough, even though the intracellular concentration of citrate anduccinate increased in this case, only succinate excretion increased.robably, the transport mechanisms behind this are different foroth acids. Depolarization of the plasma membrane does not inhibitransport of succinic acid in this filamentous fungus, which, in con-rast, is the case in Saccharomyces cereviseae [33,34]. S. cereviseaeuccinate production is mostly studied in the context of wine andiquor manufacturing [35]. Mutants with elevated succinate pro-uctions profiles are used to modify the taste of rice wine [36,37].

A combined fungal–bacterial two step process with Rhizophusp. and Enterococcus faecalis showed very high production rates andield (respectively 2.2 g/l/h and 0.95 g/g). In the first step the fun-us produces fumarate which is then transferred to a second reactorhere E. faecalis efficiently converts it to succinic acid. However this

nnovative system shifts the problem from succinate to fumarateroduction, which is in this case low in yield (0.5 g/g) and has a

ow rate (0.2 g/l/h) [38]. This process is illustrative for an essen-ial problem that occurs for succinate formation in Eukaryotes,ompartmentalization. Succinate has to cross two borders in ordero be excreted (mitochondrial membrane and cytoplasma mem-rane); fumarate on the other hand does not [8]. This makes it moreavourable to use bacteria instead of fungi or yeasts to produce suc-inic acid. Yeasts and fungi on the other hand grow at rather acidicH, which would make downstream processing of those processesore favourable [39].

.2.2. Metabolic engineering of succinate producing strainsThe past two decades the development of a biotechnological

uccinate production process has drawn a lot of attention. Onef the key technologies for this purpose is metabolic engineeringhich consists of the design, engineering and optimization of aicroorganism to optimize a production process for the production

f chemicals from renewable resources [40]. However, to success-ully perform metabolic engineering, sufficient genetic tools andnformation should be available for the targeted host. This is notlways the case for the natural succinate producing strains. Below,

he possibility of metabolic engineering of these strains is investi-ated. Furthermore, all metabolic engineering strategies to improveuccinate production reported in literature are discussed. This sum-ary is limited to bacteria because they are in favour to yeast and

ungi as production host (as mentioned above).

istry 45 (2010) 1103–1114 1105

2.2.2.1. A. succinogenes. A. succinogenes is a facultative anaero-bic, capnophilic, mesophilic, pleomorphic, Gram-negative rod andmember of the Pasteurellaceae family. A total of 2115 genes havebeen identified of which 1768 have a predicted function. In totalonly 404 genes have been connected to a KEGG pathway, whichmeans a lot of research is still needed to fully understand the bio-chemistry of this organism [41].

Because A. succinogenes is capnophilic, it needs carbon dioxide tosimulate growth and succinate production [42]. A key enzyme forthese organisms is phosphoenolpyruvate carboxykinase (PEPCK),which converts phosphoenolpyruvate with carbon dioxide and ADPinto oxaloacetate and ATP. The increase in growth rate is thus linkedto the increase in substrate level phosphorylation by this reaction.The difference with non-capnophilic bacteria PEPCK, such as E. coliPEPCK, can be found in the kinetic properties of the enzyme. InE. coli PEPCK functions as a part of gluconeogenesis, which meansthat the enzyme has a high affinity for ATP and oxaloacetate, buta low affinity for ADP and carbon dioxide or carbonate [43]. Theintroduction of an A. succinogenes PEPCK in E. coli was described byKim et al. [44] and resulted in an 6.5-fold increase of succinate pro-ductions in anaerobic, CO2-rich conditions, proving its differencein function.

A. succinogenes can ferment a broad spectrum of carbonsources [42,45,46] and these properties allow fermentation of canemolasses, whey and wheat hydrolysates that are much cheapercarbon sources than refined sugar and glucose [47–49]. A dis-advantage of the same environment is the richness of differentsubstrates. A lot of vitamins and amino acids are abundant in therumen, which resulted in the loss of biosynthetic routes mak-ing the addition of these vitamins and amino acids to minimalmedium necessary [50]. The fact that glutamate is an essentialamino acid and alfa-ketoglutarate can be used as a substitute indi-cates that two essential genes from the TCA cycle are missing orinactive during growth on glucose, isocitrate dehydrogenase andalfa-ketoglutarate dehydrogenase. 13C flux analysis of the A. suc-cinogenes metabolism should clarify how the carbon flows throughthe metabolism and which genes are active. Such a study com-bined with comparative genomics can be a powerful tool to identifymetabolic engineering targets. Especially, the presence of the gly-oxylate shunt [51] and the split ratios of the pentose phosphatepathway, Entner-Doudoroff pathway and glycolysis are important.Whereas in Pasteurellaceae the 5% flux via the PPP accounts for only20% of the total needed NADPH in the cell, in E. coli, the flux throughthe PPP (29%) accounts for 50% of the needed NADPH and isoci-trate dehydrogenase for the rest. In the former this gap has to befilled by transhydrogenases [52]. One of the enzymes responsiblefor transhydrogenation in A. succinogenes could be malic enzymecombined with malate dehydrogenase. This enzyme system formsalso the shunt between the C4 and C3 pathways and thus betweensuccinate formation and by-product formation. Through pyruvateoxidation, the major (by)-products of A. succinogenes fermenta-tions are formed: formate, acetate and ethanol [51]. These reactionsform extra reductive power under the form of NADH (except forethanol), which can increase the flux through the reductive TCAbranch towards succinate. Their formation is modulated by thepresence of carbonate and carbon dioxide in the medium [50]. Theaddition of extra reductive power by means of hydrogen reducesthe fumarate and acetate flux and increases succinate and ethanolformation [53].

Clearly the above described observations point at some inter-esting metabolic engineering targets. Logically the genes coding

for ethanol dehydrogenase, acetate kinase and formate dehydro-genase should be knock out to prevent by-product formation. Aformate dehydrogenase mutant A. succinogenes strain FZ6, showedincreased pyruvate production instead of succinate production[54,55]. This indicates that by-product formation is the result of
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problem that occurs more upstream in the metabolism, namelyt the phosphoenolpyruvate–pyruvate–oxaloacetate node. The fluxowards pyruvate should be controlled as such that is allowsnough pyruvate as a biosynthetic precursor, which means a per-ect fine-tuning of this node [56].

Every engineering strategy of course requires the essentialenetic tools to modify the organism accordingly. Not many geneticngineering technologies have been developed yet for Pasteurel-aceae. Only recently a shuttle vector has been constructed for A.uccinogenes which allows the overexpression of exogenous genes57]. The availability of genomic information will very likely speedp the access to more advanced genetic tools for gene expressionne-tuning, which are already available for E. coli [58].

.2.2.2. Mannheimia succiniproducens. M. succiniciproducens is aacultative anaerobic, capnophilic, mesophilic, Gram-negative rodnd similar to A. succinogenes, a member of the Pasteurellaceaeamily. M. succiniciproducens ferments a wide variety of substratesnd hydrolysates [59]. A disadvantage of this strain is the manyuxotrophies it exhibits [60]. In contrast to A. succinogenes, M. suc-iniciproducens has a complete TCA cycle and can efficiently grown aerobic as well as in anaerobic conditions. Such a metabolismequires stringent regulatory systems. This has been elaboratelytudied in E. coli. On a genomic/transcriptomics level, the wholeerobic/anaerobic metabolism of E. coli is regulated by a two com-onent signal transduction system, ArcAB and an oxygen sensitiveystem FNR [61–69]. In comparison to E. coli, M. succiniciproducenslso possesses a FNR and ArcAB system. The FNR protein was anno-ated from the genome with high identity (82%) but has not yet beenhysiologically investigated [70]. The ArcAB system has been char-cterised recently [69]. Sequence analysis showed that, althoughigh homology exists between the ArcB of E. coli and M. succinicipro-ucens (and its family, the Pasteurellaceae), ArcB of the latter lackslmost an entire linker region, corresponding to residues 93 to71, in which the two regulatory cysteines should be present. Thebsence of these regulatory residues has made the protein insen-itive to quinones and fermentative by-products (lactate, acetatend pyruvate), which could imply that in the case of M. succinicipro-ucens, the mode of operation of ArcAB is quite different from the. coli mode and thus the redox state of the cell is controlled in aifferent way [69]. From a metabolic engineering perspective, regu-

atory modification strategies, such as applied for E. coli (see below),ould not be easily transferred.

The genome sequence has lead to a big leap forward in the devel-pment of genetic engineering tools for this succinate producingtrain. With the construction of stable vectors for rumen bacteria,verexpression of certain genes became possible for these bacte-ia [57,71]. Such a vector is also essential for the development ofene knock out strategies. Looking at the Datsenko and Wannerethodology [72], for E. coli, multiple of these vectors are neces-

ary to develop a fast and specific knock out system. Alternatively,single vector could be developed that requires a less efficient dou-le cross-over of the plasmid with genomic DNA. The group of Sangup Lee constructed a plasmid, especially for these purposes [73].

n order to be able to identify the gene deletion, for each knockut an antibiotic resistance gene is introduced into the genome athe location of the target gene. Because a limited amount of antibi-tic markers exist, these marker genes have to be eliminated fromhe strain after modification. In order to do so, two particular DNAequences are added flanking the resistance gene. These sequencesre recognised by specific exogenous recombinases that excise the

enetic marker. The recombinase preferably has to be introducedn a temperature sensitive plasmid, so it can be removed from the

utant strain after excision. This property is mainly determinedy the origin of replication (ori) of the plasmid. A point mutation inhe ori of pMVSC1, a Mannheimia varigena plasmid, resulted in tem-

istry 45 (2010) 1103–1114

perature sensitivity and thus an excellent plasmid for a markerlessgene knock out system [74].

The developed genetic tools have already resulted in severalM. succiniciproducens mutant strains. Within these strains by-product formation was targeted. The target genes were ldhA, pflB,pta and ackA, coding for lactate dehydrogenase, pyruvate–formatelyase, acetylphosphotransferase, and acetate kinase, respectively.The optimal succinate production route for M. succiniciproducensinvolves then PEP carboxykinase as well as malic enzyme (Fig. 2),both will fix one carbon dioxide with the formation of 1 ATP. Sucha route was envisaged in a strain designated LPK7, eliminatingacetate, lactate and formate formation. A fed-batch culture resultedeventually in the formation of about 52 g/l succinate with acetate,malate and pyruvate as by-products. The yield reached 68% of themaximal theoretical yield of 1.12 g/g (Table 1), although the pro-posed route in Fig. 2 only would allow a maximal theoretical yieldof 0.66 g/g due to the lack of sufficient reduce equivalents [73]. Heretwo notes should be made. Firstly, Fig. 2 is a simplification of themetabolism and does not consider the oxidative pentose phosphateroute, which yields two NAD(P)H’s instead of one NADH formedin the glycolysis; secondly, the medium composition should beevaluated in detail. The medium contains on the one hand yeastextract, which can introduce extra reductive equivalents, but moreimportantly, it contains disodium sulphide. Conversion of 1 moleof sulphide to 1 mole sulphate yields 3 NADPH and 1 reduced ferri-doxine. This effect has also been shown with an in silico analysis ofthe M. succiniciproducens metabolism. The calculated flux towardssuccinate increased about 30% in a hydrogen–carbon dioxide envi-ronment [70]. Metabolic flux analysis of different culture conditionsalso showed that glycolysis is the major source for reduced equiv-alents, about 85% of the total, which leads to the lower yields thatwere observed and the accumulation of malate and pyruvate asby-products. It also showed the necessity of carbon dioxide and/orcarbonate as an environmental factor and substrate. A dissolvedcarbon dioxide concentration of 141 mM increased succinate yieldwith 1.5 times in comparison with 8.7 mM. This is the result of theaffinity constants of PEP carboxykinase and malic enzyme for car-bon dioxide and carbonate [25,75]. The latter, malic enzyme, wasoverproduced in strain LPK7 in an attempt to reduce pyruvate andmalate production [76]. In this case malate excretion was about37% reduced but pyruvate excretion increased. This formed pyru-vate was finally metabolised by M. succiniciproducens after glucosedepletion with the formation of acetate [77]. The added time tothe fermentation was about 10 h, which decreased the productivityabout 30%. An efficient cofactor manipulation strategy is thus nec-essary to improve yield, reduce by-product formation and increaseproduction rate.

2.2.2.3. Anaerobiospirillum succiniproducens. A. succiniproducens isa strictly anaerobic, capnophilic, mesophilic, pleomorphic, Gram-negative spiral rod [78] and an opportunistic pathogen [79]. Thebroad spectrum of carbon sources it ferments has allowed succinateproduction of several complex sugar mixtures originating from forinstance wood hydrolysates and whey [80,81].

No direct information on specific auxotrophies is available yet.For the isolation of A. succiniciproducens yeast extract and cysteineare required [82], but for optimal growth a combination of differentcomplex medium components is necessary. The amino acid andvitamin of these medium components is quite different, indicatingthat the strain is auxotrophic for multiple amino acids and vitamins[83]. In most cases only yeast extract in combination with peptone

is used and result in high yields and production rates. Corn steepliquor is also frequently used, although quite high concentrationsare needed to supplement for the auxotrophic phenotype (Table 1).

As typical a capnophilic A. succiniciproducens, requires high car-bon dioxide for efficient growth. CO2 supply affects the production

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Table 1Overview of the yields, rates and titers for different succinate production processes described in literature.

Strain Fermentation strategy/medium Y [g/g glc] qsucc [g/g CDW/h]d rsucc [g/l/h] Titer [g/l]c Time/D [h/h−1] By-productsb Reference

A. succinogenesFZ6 an; B; Csl, Ye, Na2CO3 0.94 N.D. 1.01 63.7 62.8 Fo, Py, Pr, Ac [55]130Z an; B; Csl, Ye, Na2CO3 0.79 N.D. 1.56 29–39 39–79 Fo, Py, Ac [125]FZ53 an; B; Glc, Csl, Ye, MgCO3 0.82 N.D. 1.36 105.8 78 Fo, Py, Pr, Ac [126]130Z an; B; Glc, Csl, Ye, Ac, MgCO3 0.87 N.D. 0.18 17.4 96 Fo, Py, Ac [127]130Z an; rB; Glc, Csl, Ye, Ac, 0.86 N.D. 0.88 33.9 38.5 Fo, Py, Ac [128]130Z an; B; Glc, Def, NaHCO3 0.46 0.47 0.28 4.2 15 Fo, Ac, Et [50]130Z an; B; Glc, Def, NaHCO3 0.5 0.19 0.3 4.1 15 Fo, Ac, Et [51]130Z an; B; Glc, Ye, CO2, NaHCO3 0.62 0.3 1.35 33.8 30 Fo, Ac [129]CGMCC1593 an; B; Mo, Ye, CO2, Na2CO3 0.79 N.D. 0.97 46.4 48 Fo, Ac [48]CGMCC1593 an; F; Mo, Ye, CO2, Na2CO3 0.94 N.D. 1.15 55.2 48 Fo, Ac [48]CGMCC1593 an; F; Glc, Ye, CO2, Na2CO3 0.75 N.D. 1.3 60.2 46.3 Fo, Ac [130]130Z an; B; Wh, Ye, CO2 0.57 N.D. 0.58 50 48 Fo, Ac [49]130Z an; B; Whe, CO2, MgCO3 0.81 N.D. 1.19 64.2 65 Fo, Ac [47]CGMCC1593 an; B; CS, CO2, MgCO3 0.81 N.D. 0.95 45.5 48 Fo, Ac [131]CGMCC1593 an; F; CS, CO2, MgCO3 0.82 N.D. 1.21 53.2 44 Fo, Ac [131]

A. succiniciproducensATCC 29305a an; B; Glc, Csl, CO2, Na2CO3 0.79 N.D. 0.79 15.9 20 Fo, Ac, Et, La [84]ATCC 53488 an; B; Glc, Csl, CO2, Na2CO3 0.91 N.D. 1.93 43.5 22.5 Fo, Ac [14]ATCC 53488 an; B; Glc, P, Ye, CO2, Na2CO3 0.88 N.D. 0.87 33.2 38 Fo, La, Ac [132]ATCC 53488 an; 2sC; Glc, Csl, CO2, Na2CO3 0.85 N.D. 2.03 39.1 D = 0.4–0.1 h−1 Fo, Ac [133]FA-10 an; B; Glc, Csl, CO2, Na2CO3 0.66 N.D. 0.77 34.1 44.5 Fo, Ac, Py [134]FA-10 an; B; Glc, Ye, P, CO2, Na2CO3 0.7 N.D. 0.78 31.6 40.3 Fo, Ac [134]ATCC 53488 an; B; Glc, Ye, P, CO2, Na2CO3 0.99 1.5 1.2 32.2 27 Fo, Ac [135]ATCC 53488 an; B; Glc, Ye, P, CO2/H2, 0.86 0.45 1.8 34.4 19 Ac [136]ATCC 53488 an; B; Wh, Csl, CO2, Na2CO3 0.84 N.D. N.D. 34.3 N.D. Fo, La, Ac [80]ATCC 53488 an; F; Wh, Csl, CO2, Na2CO3 0.91 N.D. 0.96 34.7 36 Fo, La, Ac [80]ATCC 53488 an; C; Wh, Csl, CO2, Na2CO3 0.64 N.D. 3 19.8 D = 0.15 h−1 Fo, La, Ac [80]ATCC 53488 an; B; Glc/Gl, P, Ye, CO2, 0.97 0.29 1.35 29.6 22 Ac [137]ATCC 53488 an; B; Wo, Csl, CO2, Na2CO3 0.88 0.37 0.74 23.8 32 Ac [81]ATCC 53488 an; mC; Glc, P, Ye, CO2, 0.88 1.1 10.4 83 D = 0.93 h−1 Ac [119]ATCC 53488 an; mC; Glc, P, Ye, CO2, 0.71 0.51 3.3 14.3 D = 0.20 h−1 Ac [138]ATCC 53488 an; B; Gal, P, Ye, CO2, Na2CO3 0.87 1.32 1.46 15.3 10.5 Ac [139]ATCC 53488 an; B; Gal/Glc, P, Ye, CO2, 0.87 3 0.97 14.7 15 Ac [139]

M. succiniciproducensMBEL55E an; B; Glc, P, Ye, CO2 0.70 0.54 1.87 14 7.5 Fo, La, Ac [59]MBEL55E an; B; Wh, Csl, Ye, CO2 0.72 0.37 1.22 13.5 11 Fo, La, Ac [140]MBEL55E an; C; Wh, Csl, Ye, CO2 0.69 0.45 1.00 12 D = 0.1 h−1 Fo, Ac [140]MBEL55E an; C; Wh, Csl, Ye, CO2 0.60 1.77 3.90 5 D = 0.7 h−1 Fo, La, Ac [140]MBEL55E an; B; Wo, Ye, CO2 0.56 0.58 1.17 11.7 12 Fo, La, Ac [81]MBEL55E an; C; Wo, Ye, CO2 0.60 1.80 1.40 9.5 D = 0.2 h−1 Fo, La, Ac [81]MBEL55E an; C; Wo, Ye, CO2 0.55 6.38 3.19 8 D = 0.4 h−1 Fo, La, Ac [81]LPK7 an; F; Glc, Ye, CO2 0.76 0.72 1.80 52.4 30 Py, Ma, Ac, La [73]MBEL55E an; B; Glc, Ye, CO2, NaHCO3 0.59 0.52 1.75 10.5 6 Fo, La, Ac [75]LPK7 an; C; Glc (9 g/l), Ye, CO2 0.71 0.64 1.29 12.9 D = 0.1 h−1 Py, Ac, La [141]LPK7 an; C; Glc (9 g/l), Ye, CO2 0.29 0.78 1.56 5.2 D = 0.3 h−1 Py, Ac, La [141]LPK7 an; C; Glc (18 g/l), Ye, CO2 0.28 0.53 1.07 10.7 D = 0.1 h−1 Py, Ac, La [141]LPK7 an; C; Glc (18 g/l), Ye, CO2 0.10 0.52 1.05 3.5 D = 0.3 h−1 Py, Ac, La [141]LPK7 an; B; Glc, Def, CO2 0.54 0.53 1.67 10.1 6 Fo, La, Ac [142]

E. coliJCL1208pPC201 an; B; Glc, Ye, T, MgCO3 0.29 N.D. 0.59 10.7 18 Fo, Ac, Et, La [106]NZN111pMEE1 d; B; Glc, Ye, T, CO2/H2, MgCO3 0.64 N.D. 0.32 12.8 40 Ac, Et [107]MG1655/pUC18 an; B; Glc, Ye, T, CO2 0.42 0.48 0.43 4.2 10 Fo, La [143]AFP111 d; B; Glc, Csl, CO2, Na2CO3 0.54 N.D. 0.52 51 99 Ac [144]JCL1242-pyc an; B; Glc, Ye, T, Na2CO3, CO2 0.15 0.17 0.14 1.5 9.8 Fo, Ac, Et, La [145]NZN111pTrcML d; F; Glc, Ye, T, CO2/H2 0.47 N.D. 0.08 9.4 120 Ma, Ac, Fo, La, [108]AFP400 an; B; Glc, Ye, T, CO2, MgCO3 0.61 N.D. 0.61 6.1 35 Fo, Ac, Et, La [95]AFP111-pyc d; B; Glc, Ye, T, CO2, Na2CO3 1.10 0.13 1.30 99.2 76 Ac, Et [146]NZN111pTrcML d; B; So, Ye, T, CO2 1.10 N.D. 0.13 10 75 Ma, Ac, Et [147]SS373 d; B; Glc, Ye, CO2, Na2CO3 0.73 N.D. 0.32 11 34 Py [148]NZN111pTrcMLFu d; B; Glc, Ye, T, CO2/H2 0.35 N.D. 0.06 7 120 Ac, Et [149]HL27615k ae; B; Glc, Ye, T, Na2CO3 0.45 N.D. 0.16 5 49 Py, Ac [113]HL27659k-pepc ae; F; Glc, Ye, T, NaHCO3 0.62 0.09 0.72 58.3 59 Py, Ac [103]HL51276k-pepc* ae; B; Glc, Ye, T, NaHCO3 0.71 0.05 0.14 8.3 59 Py, Ac [114]HL27659k ae; C; Glc, Ye, T, NaHCO3 0.59 0.20 0.70 7 D = 0.1 h−1 Py, Ac [150]SBS550MG an; F; Glc, Ye, T, NaHCO3 1.06 0.21 0.42 40 95 Fo, Ac [115]

E. coliW3110GFA an; F; Glc, Ye, NaHCO3, CO2 0.2 N.D. 0.03 2.1 80 Fo, Ac, Et, La [151]SBS110MG-pyc an; B; Glc, Ye, T, NaHCO3, CO2 0.9 N.D. 0.65 16 24 Fo, Ac [109]TUQ19/pQZ6 d; B; Glc, Ye, T, MgCO3, CO2 0.8 N.D. 0.79 13 16 Ac, Et, La [152]TUQ2/pQZ6/pQZ5 d; B; Glc, Ye, T, MgCO3, CO2 0.8 1.2 0.59 12 20 Ac, Et, La [98]W3110 d; B; Sucr, Ye, T, CO2, MgCO3 1.2 N.D. 0.81 24 30 N.D. [120]SBS550 pHL314 an; F; Glc, Ye, T, NaHCO3, CO2 1.1 N.D. 0.42 40 95 Fo, Ac [153]AFP184 d; B; Glc, Csl, CO2 0.8 N.D. 1.27 38 30 Py [154]

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Table 1 (Continued )

Strain Fermentation strategy/medium Y [g/g glc] qsucc [g/g CDW/h]d rsucc [g/l/h] Titer [g/l]c Time/D [h/h−1] By-productsb Reference

AFP184 d; B; Fru, Csl, CO2 0.7 N.D. 1.01 30 30 Py [154]AFP184 d; B; Xyl, Csl, CO2 0.5 N.D. 0.78 23 30 Py [154]NZN111 d; F; Glc, Ac, NaHCO3, CO2 0.7 0.2 0.70 28 40 Py, Ac [155]W3110 d; B; Mo, Csl, CO2, MgCO3 0.52e N.D. 0.87 26 30 N.D. [156]KJ060 an; B; Glc, def, NaHCO3, CO2 0.9 N.D. 0.90 87 120 Ma, Ac, La [117]KJ073 an; B; Glc, def, NaHCO3, CO2 0.8 N.D. 0.82 79 96 Ma, Ac, Py [117]KJ060 an; B; Glc, def, NaHCO3, CO2 1.1 N.D. 0.61 73 120 Ma, Ac, La, Py [117]KJ122 an; B; Glc, def, NaHCO3, CO2 0.9 0.4 0.88 83 93 Ma, Ac, Py [118]

C. glutamicumR �ae; rF; Glc, Ye, Cas, NaHCO3 0.19 0.13 3.8 23 6 Ac, La [157]R �ldhA-pCRA717 �ae; rF; Glc, Ye, Cas, NaHCO3 0.92 0.06 3.17 146 46 Ac, Ma, La, Py [28]

Bacteroides fragalisMTCC1045 an; B; Glc, Ye, P, Na2CO3, CO2 0.62 N.D. 0.42 12.5 30 N.D. [158]MTCC1045 an; B; Glc, Ye, P, Na2CO3, CO2 0.57 N.D. 0.83 20 24 N.D. [27]

Fermentation strategy: an: anaerobic, ae: aerobic, d: dual phase; �ae: micro-aerobic; B: batch, rB: repeated-batch, F: fed-batch, rF: fed-batch with cell recycling, C: continuousculture, 2sC: two stage continuous culture (2 different dilution rates), mC: continuous culture with integrated membrane for cell recycling; Medium: Glc: glucose, Gl: glycerol,Sucr: sucrose, Fru: fructose Gal: galactose, So: sorbitol, P: peptone, T: tryptone, Ye: yeast extract, Cas: casamino acids, Csl: corn steep liquor, Wh: Whey, Wo: pretreated woodhydrolysate (extraction of inhibitory compounds), Mo: cane molasse, Whe: wheat hydrolysate, CS: corn stover hydrolysate, Ac: acetate, Def: defined medium; Symbols: D:dilution rate, rsucc: volumetric production rate, qsucc: specific production rate, Y: yield (S = carbon source indicated); By-products: Fo: formic acid, La: lactic acid, Ac: aceticacid, Py: pyruvate, Ma: malate, Pr: propionate, Et: ethanol.

a ATCC 29305 has been redeposited as ATCC 53488 in 1992.b In most cases no carbon balance was reported, in some cases the balance does not close, indicating the presence of more by-products.c Some of the titers are low due to low initial carbon source concentration.d Most studies only report OD values, thus the specific production rate could not be calculated (N.D.).e Approximated value based on an average molasse sugar content.

Fig. 2. The proposed metabolic route to succinate in E. coli and Mannheimia succiniciproducens [73], with pck: PEP carboxykinase, pykA: pyruvate kinase A, pykF: pyruvatekinase F, pps: PEP synthase, ptsG: subunit of the phosphotransferase system, ldhA: lactate dehydrogenase, pflB and tdcE: pyruvate-formate lyase, poxB: pyruvate oxidase,adhCEP: subunits of alcohol dehydrogenases, acs: acetyl-CoA synthase, pta: acetylphosphotransferase, ackA: acetate kinase, maeA and maeB: malic enzyme, mdh: malatedehydrogenas, fumABC: fumarase A, B and C, frdABCD: subunits of fumarate reductase, eda: oxaloacetate decarboxylase, gltA: citrate synthase, pdh: pyruvate dehydrogenase[56]. PEP carboxykinase activity of Mannheimia succiniciproducens is marked in bold.

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f succinic acid significantly, increasing concentrations resulted inncreasing succinate yield and reduced lactate and ethanol con-entrations, indicating a preference for PEP carboxykinase and theeductive TCA pathway over NADH regeneration via lactate andthanol dehydrogenase [84]. Yields do not approach the theoret-cal maximum due to the lack of sufficient reduced equivalentsnd due to the repression of the second enzyme of the pentosehosphate route by carbon dioxide, 6-phosphogluconate dehydro-enase [85]. This altered flux is typical for capnophilic organismsrown in a CO2-rich environment [53]. Addition of hydrogen gaso the sparging gas, increases yields and production rates signifi-antly, which can be explained by the increase in NADH in the cell.ncreased productivity would seem peculiar at first sight, but cane assigned to an increased activity of PEP carboxykinase. Succinateormation results in ATP formation [86], which makes the enzymendispensible for growth coupled succinate production.

.2.2.4. E. coli. Molecular biologist all time favourite is E. coli.ecause of its small doubling time and its abundance in nature, it iserfect to use as a reference organism. Additionally, genetic engi-eering tools are widely available and quite simple to apply, whichakes it very attractive to develop production processes with suchstrain [26,72,87]. In aerobic conditions, at a maximal growth rate,

t mainly produces acetate as a kind of overflow metabolite. Anaer-bically, it will produce a mixture of formate, acetate, lactate andthanol. Some amounts of succinic acid can also be detected in thatase [88–90]. However, through metabolic engineering economicaliable production processes can be developed, e.g. 1,3-propanediolroduction and artemisinin production in E. coli [91].

Succinate seems to be a very challenging molecule to produceith E. coli. The route to succinate is quite complex and is regulated

n all cellular levels. Optimization strategies have thus far focussedn two main pathways, the PEP–pyruvate–oxaloacetate node andhe TCA/glyoxylate route. The PEP–pyruvate–oxaloacetate nodeFig. 2) forms the cellular roundabout that distributes carbonowards the different biomass precursors, such as amino acidsnd fatty acids, and the energy metabolism (anaplerotic, catabolicnd gluconeogenic route). The three metabolites can be directlyonverted into each other via 6 enzymes, PEP carboxylase, PEP car-oxykinase, pyruvate kinase, PEP synthase, the phosphotransferaseystem and oxaloacetate decarboxylase, also known as 2-keto--deoxygluconate 6-phosphate aldolase. The triangle is furtherxpanded with malic enzyme, pyruvate dehydrogenase, malateehydrogenase, citrate synthase and the acetate pathways [56].

A first complication of this triangle is the PTS system whichounds the glucose uptake to the conversion of PEP into pyru-ate [92] and thus for each glucose molecule metabolised, onlyne PEP molecule can be converted into succinate. Pyruvate couldhen be further converted into acetyl-CoA, lactate, acetate or for-

ate, depending on the growth conditions. In principle pyruvateould be phosphorylated by PEP synthase, but here the rigorousontrol of the node starts to play. All gluconeogenic reactions, PEParboxykinase, PEP synthase and malic enzyme, are transcription-lly regulated by the cAMP dependent global regulators, to preventutile cycling. PEP synthase is controlled by Cra, which activatesranscription in glucose depleted conditions [93,94]. For this prob-em multiple solutions have been proposed.

The PTS can be knocked out to avoid the simultaneous con-ersion of PEP to pyruvate with glucose uptake. In this case, thelternative glucose uptake system in E. coli is the galactose perme-se, glucokinase system. This system is however, not very efficient

nd reduces growth rate significantly in E. coli but increases suc-inate yield [95]. The overproduction of these enzymes leads to aearly wild type growth rate and also reduced by-product forma-ion [96,97]. The expression of these genes was done on plasmidsith IPTG induction, which means expression fine-tuning is very

istry 45 (2010) 1103–1114 1109

hard to do and metabolic burden will occur very easily. Such ametabolic burden is very hard to assess and in a large scale pro-duction context this kind of expression is not desirable [98].

Alternatively PEP carboxylase (PPC) and PEP carboxykinase(PCK) were overproduced to draw PEP away from the PTS systemand aiming for permease/kinase system to take over. Overexpres-sion of PPC resulted in a similar phenotype as the PTS knockout, growth reduced when the expression was to steep. Fine-tuning of the expression lead to a system in which no adversegrowth effects were observed anymore but increase in succinateyield was achieved. This would mean a perfect split between thePEP–oxaloacetate conversion and the PEP–pyruvate conversion.Here a second control level has to be considered, enzymatic con-trol through allosteric inhibition [99,100]. Malate, aspartate andto some extend, succinate and citrate bind at the lysine residueslys491, lys620, lys650 and lys773, which reduces the enzyme activ-ity [101]. In order to resolve this problem, mutant PEP carboxylasefrom Sorghum vulgare was introduced [102,103]. When in additionpantothenate kinase (PANK) is overproduced combined with PPCan increase in succinate yields was observed. PANK increases theCoA and acetyl-CoA levels in the cell, which in turn acts as an acti-vator of PPC. A similar result was obtained with the coexpression ofRhizobium etli pyruvate carboxylase and PANK [104,105]. Overex-pression of endogenous PCK did not show an increase in succinateproduction, nor any adverse effects on growth rate [106].

Because the split between pyruvate and oxaloacetate is highlydependent on the PTS system, genes were introduced to convertpyruvate to oxaloacetate or malate. Malic enzyme takes care ofthe latter reaction, although in gluconeogenic conditions. Over-expression of malic enzyme has to be combined with addition ofcarbonate or carbon dioxide to the environment in order to havean increased succinate formation [107,108]. A similar effect hasbeen achieved by introducing Lactococcus lactis or R. etli pyruvatecarboxylase into E. coli. In contrast to malic enzyme, this reactiondoes not yield any NAD(P)+, but has to be combined with malatedehydrogenase to form malate. The enzyme is strongly activatedby acetyl-CoA and thus crucial for the desired effect on succinateyield. Acetyl-CoA formation, however, competes for pyruvate withpyruvate carboxylase. This means that here the crucial step willalso be the fine-tuning of gene expression [104,109].

Further down in the metabolism, carbon enters the tricarboxylicacid cycle and the glyoxylate bypass. The mode of operation of theseroutes depends strongly on environmental conditions. For instanceglucose and oxygen modulate the different reactions on various cel-lular levels [88,110,111]. In a succinate production context choiceshave to be made which route is optimal towards succinate and howto achieve that. The classical TCA cycle depicted in Fig. 3 could yieldthree different routes. First, the oxidative TCA cycle, which results intwo NAD(P)H and the loss of two carbon dioxide molecules; second,the glyoxylate route, which yields succinate directly from isoci-trate; and last, the reductive TCA cycle, which consumes 2 NADH foreach molecule of succinate, but assimilates one carbon dioxide. Theoxidative TCA is active under aerobic conditions, the reductive TCAunder anaerobic conditions and the glyoxylate route is activatedwhen glucose is absent and for instance acetate is the main carbonsource for growth [89,112]. Aerobic conditions are considered tobe most favourable for production rate, ATP is produced most effi-ciently through oxidative phosphorylation, though NADH is drawnaway from the reductive TCA cycle. In order to compensate that, theglyoxylate route has to be opened. This route is primarily controlledby IclR. IclR opens the bypass when fermentative products, such as

lactate and acetate are solely present in the environment, to avoidcarbon loss through the oxidative branch of the TCA. The net carbonassimilation would otherwise be nil, because for each acetyl-CoAthat enters the TCA, two carbon dioxide molecules would be lost[102,113]. By introducing glyoxylate bypass activity and knocking
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Fig. 3. The TCA cycle in E. coli. With mdh: malate dehydrogenase, gltA: citratesynthase, acn: aconitase, icd: isocitrate dehydrogenase, sucAB: alfa-ketoglutaratedsm

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Table 2Overview of the most applied DSP procedures for succinate purification. Eachmethodology is evaluated on the basis of scalability, robustness, separation yieldand cost according to Kurzrock and Weuster-Botz [17]. A method is marked ‘−’when it scores bad in a certain property and ‘+’ when it scores well.

Method Scalability Robustness Separation yield Cost

Ultrafiltration − + + −Precipitation + + − −Electrodialysis − − +/− −

TOo

d

ehydrogenase, lpd: lipoamide dehydrogenase, sucCD: succinly-CoA synthase,dhABCD: succinate dehydrogenase, fumA: fumarase, aceA: isocitrate lyase, aceB:alate synthase.

ut the genes coding for succinate dehydrogenase, isocitrate dehy-rogenase, and by-product formation enzymes next to R. etli PPCverexpression, the theoretical yield was nearly achieved [114].his strategy however has disadvantages. The constructed strains auxotrophic for alfa-ketoglutarate due to the isocitrate knockut, and needs thus complex medium components such as tryp-one and yeast extract for optimal growth [103]. Recalculations ofhe yields, including the carbon from those complex medium com-onents also shows that the yield was artificially increased (seeelow). In a similar way an anaerobic strain was constructed withdditional by-product gene knock outs and the overexpression ofyruvate carboxylase [109,115,116].

Recently, novel knock out strategies have shown an increased

ields and titers in an alternative E. coli strain, E. coli C [114,117].his strain has by nature an active glyoxylate route in the presencef glucose [56] and does not need extensive TCA cycle modifi-ations. The strain is mainly modified in by-product formation

able 3verview of selected recalculated yields, Adapted 1 includes the total amount of carbon tf carbon dioxide and carbonate added to the medium.

Strain Published yield (c-mol/c-mol) Adapted 1

AFP111-pyc 1.12 0.93HL27659k-pepc 0.57 0.41SBS550MGa 1.07 0.25AFP184 0.48 0.38SBS110MG-pyc 0.83 0.48NZ111pTrcML 0.65 0.39JCL1208pPC201 0.29 0.22HL51276k-pepC 0.73 0.35SBS880MG-pyc 0.63 0.22KJ060b 0.94 0.88KJ073b 0.80 0.80KJ060b 1.07 0.75W3110 1.17 0.96

a Calculations include the biomass production step that preceded the bioconversion.b In these cases a defined medium was used; the fact that the recalculated yields are dif

ata and the reported yields.c The composition of complex medium components were obtained from BDTM and are

Liquid–liquid extraction + + + −Sorption and ion exchange − − + −

pathways, such as ethanol, acetate, lactate and formate. In orderto increase the titers above 58.3 g/l, betaine, a natural osmopro-tectant, was added to the medium. The highest titer, yield andrate obtained with this strain were 86.5 g/l, 0.83 g/g, and 0.9 g/l/h,respectively [117,118], which is so far the best E. coli productionprocess described. The main by-products are still pyruvate, malateand acetate, which points at a redox deficiency in the cell.

2.3. Succinate production processes

Table 1 gives a comprehensive overview of the many succinateproduction processes described in literature. Apart from the strainchoice, also medium and fermentation strategy are shown. Mostlybatch cultures are employed as a production system, althoughhigher titers and rates are obtained by fed-batch and continuousculture systems. The highest volumetric production rate, 10.4 g/l/hwas obtained in a continuous culture of A. succiniciproducens withintegrated membrane for cell recycling at a dilution rate of 0.98 h−1

[119]. Titers up to 146 g/l were obtained in a cell recycling fed batchculture of Corynebacterium glutamicum [28]. In an E. coli dual phasebatch fermentation the highest described yield of 1.2 g/g substrate(in this case sucrose) was obtained [120]. None of the describedproduction systems are homo-fermentative succinate productionsystems. All of them are performed around neutral pH. Down-stream processing (DSP) remains thus a major issue in microbialproduction of succinic acid. Most DSP approaches for succinic acidare principally functional, however industrial realisation dependsgreatly on scalability, robustness, overall separation yield and costs[17,22]. In Table 2 these properties are summarized and evaluated

for the most commonly used succinic acid DSP principles. A goodDSP method should score well on all 4 properties to be industriallyapplicable which is, as can be derived from this table, not yet thecase.

hat originates from complex medium components, Adapted 2 includes the amount

c (c-mol/c-mol) Adapted 2 (c-mol/c-mol) Reference

0.83 [146]0.41 [103]0.24 [115]0.38 [154]0.47 [109]0.38 [108]0.22 [106]0.34 [114]0.22 [159]0.84 [117]0.77 [117]0.72 [117]0.93 [120]

ferent from the reported yields is in this case due to discrepancies between the raw

averages from different batches.

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The overview in Table 1 also shows all obtained yields in gramer gram substrate. In few instances the maximal theoretical yield

s obtained, only in one the theoretical yield has been reached,.21 g/g sucrose. In order to reach such yields, carbon dioxide addi-ion is essential. In principle, a yield should be calculated on theotal amount of carbon put into the system, and then its maximalields can only be equal to 1 c-mol/c-mol. Some of the yields on glu-ose or sucrose are of course higher due to carbon dioxide input,ut also due to the complex medium components added. Table 3hows a short overview of some recalculated E. coli fermentationields. Reported yields to yields corrected for the total amount ofetabolizable carbon that has been added to the culture via com-

lex medium components. In some cases this carbon fraction isather low or non-existent (e.g. when defined media are used) inther cases this fraction is high and should not be ignored. In thease of strain SBS550MG a long biomass accumulation phase pre-eded the actual production phase. During that biomass productionhase large quantities of yeast extract and tryptone were used. Theroduction phase eventually lead to a very high yields of 1.07 c-ol/c-mol glucose, but by incorporating the biomass production

hase in the yield calculation, the yield drops to 0.25 [115]. It is indi-atory though for the influence of complex medium components onuccinate production yields as such.

In a similar way the effect of carbon dioxide and carbonate cane evaluated. In this case it can be concluded that the amount ofarbonate and carbon dioxide added to the medium will not con-ribute significantly to the acquired yield. The carbon loss throughiomass synthesis compensates most of the carbon fixation routesor succinate production. Carbon dioxide and carbonate as mediumr environmental components function as activators of carbon fix-tion routes rather than as substrate for production.

. Conclusion

The optimization of a biosuccinate production process is beenhe topic of many research in the past two decades. Although somef the process performances are very promising, many problemsave been uncovered over the years. Natural succinate produc-

ng strains cope with a number of auxotrophies, which increaseubstrate requirements and price. Furthermore, yields seem to beeaching maximal theoretical yield in such a complex medium,ut only when additional sugar carbon is taken into account. Theroduction rates are rarely reaching the by the US department ofnergy set target of 2.5 g/l/h.

In order to avoid the first problem, auxotrophy, an alterna-ive strategy with E. coli was tried. E. coli can easily grow on a

inimal medium and many known genetic tools exist to alter itsetabolism. Extensive engineering has lead to very nice increases

n succinate yield, but also required again complex medium com-onents. Only recently a strain was developed which does notequire auxotrophy complementation. This strain showed similarields but also showed – as is the case for all succinate pro-uction processes – a large number of by-products. From this

iterature study can be concluded that production rate and by-roduct formation should be the points of attention. Novel wayso increase the production rate should be developed and poten-ial by-product formation pathways should be investigated. A morehorough knowledge of the control mechanisms in the cell will alsoe crucial to identify the right targets for further process optimiza-ion.

Although from literature no strain (and hence bioprocess) is

nown to be economically competitive to the chemical process sev-ral companies has announced to commercial produce biosucciniccid in the near future. Royal DSM N.V. and Roquette have createdstrategic alliance in an effort to commercialize a fermentation-

ased process to produce biosuccinic acid from renewable sources

istry 45 (2010) 1103–1114 1111

(glucose and carbon dioxide). The current target of the coopera-tion is to produce on industrial scale by 2011. Since end 2009 abiosuccinic acid demonstration plant in Lestrem (France) is opera-tional [4,121]. On the other hand, a combined effort of DNP GreenTechnology and ARD, BioAmber, resulted in a succinic acid fermen-tation demonstration unit, integrated into an existing bio-refinerylocated in Pomacle (France), with an annual production capacity of2000 metric tons. They prospected to move into the out-licensingphase for the years 2010–2011 [122]. A third alliance is been formedbetween Myriant Technologies LLC and Uhde for the engineering,procurement and construction (EPC) of Myriant’s biobased succinicacid plants based on renewable feedstocks. Recently, Myriant hasbegun production of ton-sized samples for its customers to ver-ify commercial product specifications and quality. The companyexpects to begin commercial production of biosuccinic acid sec-ond half of 2010 [123]. Finally, BASF AG and Purac Ltd. announcedthat they have joined to produce a commercial biobased succinicacid in 2010. This biosuccinic acid will be used as an input materialfor biodegradable polyester. Meanwhile, Mitsubishi Chemical Corp.and PTT PLC undertake a study on manufacturing a biodegradablepolymer, polybutylene succinate, from biosuccinic acid [124].

Acknowledgment

The authors wish to thank the Institute for the Promotion ofInnovation through Science and Technology in Flanders (IWT-Vlaanderen) for support via the MEMORE project (IWT040125).

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