Effect of Different Carbon Sources on the Production of Succinic Acid UsingMetabolically Engineered Escherichia coli

Christian Andersson, David Hodge, Kris A. Berglund, and Ulrika Rova*Division of Biochemical and Chemical Process Engineering, Lule University of Technology, SE-971 87 Lule, Sweden

Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodityand specialty chemicals. In the present work, dual-phase batch fermentations with the E. colistrain AFP184 were performed using a medium suited for large-scale industrial production ofSA. The ability of the strain to ferment different sugars was investigated. The sugars studiedwere sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucoseand xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize allsugars and sugar combinations except sucrose for biomass generation and succinate production.For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized.The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumedanaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as acarbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no cataboliterepression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1)than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 gg-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production.The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrationswere in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detectedand accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic aciddecreased when organic acid concentrations reached approximately 30 g L-1. This studydemonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost mediumfrom a variety of sugars with only small amounts of byproducts formed.

IntroductionSustainable production of fuels and chemicals is driven by

the need to control atmospheric concentrations of greenhousegases such as carbon dioxide, depletion of existing oil reserves,and increasing oil prices. In a report from the U.S. Departmentof Energy (USDOE), succinic acid was considered as one of12 top chemical building blocks manufactured from biomass(1). Succinic acid has a wide range of applications and can bea platform for deriving many commodity and specialty chemi-cals, for example, diesel fuel additives, deicers, biodegradablepolymers and solvents, and detergent builders (2).Succinic acid can be produced by a number of organisms

including Bacteroides ruminicola and Bacteroides amylophilus,Anaerobiospirillum succiniciproducens, Actinobacillus succi-nogenes, Mannheimia succiniciproducens, and Escherichia coli(3-9). Corynebacterium glutamicum has also been shown toproduce mixtures of lactic and succinic acid (10). The workdone so far have all used media supplemented to some degreewith high-cost nutrient sources such as tryptone, peptone, andyeast extract.In order to develop a bio-based industrial production of

succinic acid, three main issues need to be addressed. First, alow cost medium must be used. Second, the producing organismmust be able to utilize a wide range of sugar feedstocks,producing succinic acid in good yields in order to make use of

the cheapest available raw material. Glucose and xylose are themost abundant sugars in lignocellulosic biomass, while fructoseand glucose are the components of sucrose, a widely availabledisaccharide. The third and most important factor for thesuccinic acid economy, as described in the USDOE report, isthe volumetric productivity. To make succinic acid productionfeasible, volumetric productivities above 2.5 g L-1 h-1 will benecessary (1).In 1949 Stokes demonstrated that E. coli under anaerobic

conditions produces a mixture of organic acids and ethanol (11).Typical yields from such fermentations are 0.8 mol ethanol,1.2 mol formic acid, 0.1-0.2 mol lactic acid, and 0.3-0.4 molsuccinic acid per mole glucose consumed. If the objective is toproduce succinic acid, the yield of succinic acid relative theother acids must be increased. The USDOE initiated in the late1990s the Alternative Feedstock Program (AFP) with the aimto develop a number of metabolically engineered E. coil strainswith increased succinic acid production. The two most interest-ing mutants developed by the program were AFP111 andAFP184 (9, 12, 13). AFP111 is a spontaneous mutant withmutations in the glucose specific phosphotransferase system(ptsG), the pyruvate formate lyase system (pfl), and thefermentative lactate dehydrogenase system (ldh) (12). Themutations resulted in increased succinic acid yields (1 molsuccinic acid per mole glucose) (13). AFP184 is a metabolicallyengineered strain where the three mutations described abovewere deliberately inserted into the E. coli strain C600 (ATCC23724), which can ferment both five- and six-carbon sugars andhas strong growth characteristics (9). Beside these organisms,

* To whom correspondence should be addressed. Ph: +46920491315.Fax: +46920491199. Email: ulrika.rova@ltu.se.

381Biotechnol. Prog. 2007, 23, 381388

10.1021/bp060301y CCC: $37.00 2007 American Chemical Society and American Institute of Chemical EngineersPublished on Web 01/25/2007

a number of different E. coli mutants have been developed andtested for succinate production including pioneering work toproduce succinate under aerobic conditions (14-22). In orderto improve succinate yield, other studies used recombinantAFP111 overexpressing heterologous pyruvate carboxylase incomplex media with the main components tryptone (20 g L-1)and yeast extract (10 g L-1), which resulted in mass yields of1.10 and productivities of 1.3 g L-1 h-1 (23, 24). In the presentwork the aim was to investigate the fermentation characteristicsof AFP184 in a medium consisting of corn steep liquor,inorganic salts, and different sugars without supplementationof other additional nutrients. Corn steep liquor is a byproductfrom the corn wet-milling industry, but as opposed to yeastextract and peptone it is an inexpensive source of vitamins andtrace elements. The composition of corn steep liquor can varywidely depending mainly on the variables involved in starchprocessing and the type and condition of the corn, but averagescan be found in the literature (25, 26). For the purpose of thisstudy sucrose, glucose, fructose, xylose, and mixtures of glucoseand fructose and glucose and xylose were used as carbonsources. This work addresses the effect of different sugars onsuccinic acid kinetics and yields in an industrially relevantmedium.

Materials and MethodsStrain and Inoculum Preparation. The E. coli strain

AFP184, which lacks functional genes coding for pyruvateformate lyase, fermentative lactate dehydrogenase, and theglucose phosphotransferase system (9), was used in this study.Cultures were stored at -80 C as 30% glycerol stocks. Theinoculum was prepared by inoculating four 500-mL shake flaskscontaining 100 mL sterile medium (same medium as used inthe biorector; see below) with 200 L of the glycerol stockculture. The inoculated flasks were incubated for 16 h at 37 C(200 rpm).Fermentations. Batch fermentations were conducted in a 12

L bioreactor (BR12, Belach Bioteknik AB, Sweden) with a totalstarting volume of 8 L (including 0.4 L inoculum and 2 L sugarsolution). The medium used for strain AFP184 contained thefollowing components (g L-1): K2HPO4, 1.4; KH2 PO4, 0.6;(NH4)2SO4, 3.3; MgSO47H2O, 0.4; corn steep liquor (50%solids, Sigma-Aldrich), 15; and 3 mL antifoam agent (Antifoam204, Sigma-Aldrich). The medium was sterilized in the fer-menter at 121 C for 20 min; thereafter 2 L of a separatelysterilized sugar solution (400 g L-1) and 400 mL inoculum wereaseptically added to a final volume of 8 L and a total sugarconcentration of 100 g L-1. The fermentation temperature wascontrolled at 37 C, and the pH was maintained between 6.6and 6.7 by automatic addition of NH4OH (15% NH3 solution).The dissolved oxygen concentration (%DO), measured by a pO2electrode, was regulated by varying the agitation speed between500 and 1000 rpm. The total fermentation time was 32 h andconsisted of an aerobic growth phase and an anaerobic produc-tion phase. During the aerobic growth phase (7-9 h) the culturemedium was aerated with an air flow of 10 L min-1. When theoptical density at 550 nm had reached a value of 30-35, theanaerobic production phase was initiated by withdrawing theair supply and sparging the culture medium with CO2 at a flowrate of 3 L min-1. The agitation speed was set to 500 rpm forthe anaerobic phase. Succinic acid was produced during thisanaerobic production phase, which continued for the remainderof the fermentation. During the fermentations, samples wereaseptically withdrawn for analysis of optical density, viable cells,and sugars and organic acids concentrations. Sucrose was testedwith strain AFP184 in a 1 L bioreactor (Biobundle 1L, Applikon

Biotechnology, The Netherlands) to determine if the strain couldgrow and produce succinic acid when using sucrose as thecarbon source. The fermentation procedure was the same as inthe 12 L bioreactor. Three fermentations in the 1 L bioreactorswere also carried out to determine the specific growth rates ofAFP184 growing on glucose, fructose, and xylose, respectively.Samples were taken during the aerobic phase and analyzed foroptical density. No anaerobic phase was carried out.Analysis. Cell Growth. Cell growth was monitored by

measuring the optical density at 550 nm (OD550). Opticaldensities were then converted to dry cell weight (DCW, g L-1)using the following OD-DCW calibration curve developed inour laboratory:

To establish the number of viable cells, 10-fold serial dilutionsof the fermentation samples were plated on tryptone soy agarplates and incubated overnight at 37 C. The number of colonieswas calculated, and the number of viable cells was expressedas cells per milliliter fermentation broth.Organic Acid and Sugar Analysis. The organic acid con-

centrations were measured by HPLC (Series 200 QuaternaryLC pump and UV-vis detector, Perkin-Elmer) equipped witha C18 column (Ultra Aqueous, 5 m, 150 mm 4.6 mm,Restek) using a 50 mM KH2PO4 buffer with 2% acetonitrile(pH 2.5 adjusted by HCl) at a flow rate of 0.35 mL min-1 asthe mobile phase, with 20 L of sample injected manually foranalysis with UV detection at 210 nm. Samples for acid analysiswere centrifuged at 10 000 rpm for 10 min at 4 C. Thesupernatant was diluted with the mobile phase and 37%hydrochloric acid (volume sample:buffer:HCl) 0.1:0.8:0.1 mL)and filtered through a 0.2 m syringe filter. Sugar concentra-tions were determined using the same HPLC system as abovebut equipped with a Series 200 refractive index (RI) detector(Perkin-Elmer), guard column, and an ion exchange column(Aminex HPX87-P, BioRad). The column was kept at 85 C ina column oven for optimal performance. Prior to analysis, thesamples were centrifuged at 10 000 rpm for 10 min at 4 C.The supernatant was diluted with water and filtered through a0.2 m syringe filter, and 20 L of sample was injectedmanually. Water at a flow rate of 0.6 mL min-1 was used asthe mobile phase. All data was collected and processed usingPerkin-Elmers TotalChrom analytical software. Peak areasfrom the chromatograms were evaluated by comparison tostandard curves prepared from solutions with known concen-trations of organic acids (formic, pyruvic, malic, lactic, acetic,succinic, and fumaric acid), sucrose, glucose, fructose, andxylose.

ResultsFermentations with strain AFP184 using sucrose, glucose,

fructose, xylose, and equal mixtures of glucose/fructose andglucose/xylose were performed and analyzed.Single-Sugar Fermentations. AFP184 was able to use

glucose, fructose, and xylose, but not sucrose, as a carbon sourcefor biomass and succinic acid production (Figure 1a-c). Duringsucrose fermentation no sugar was metabolized, and sucrosewas therefore excluded from further analysis. Specific growthrates during the aerobic phase were calculated using datacollected from experiments carried out with glucose, fructose,and xylose in 1 L bioreactors (Table 1). It was observed that infermentations using glucose and fructose exponential growthwas initiated within 1 h after inoculation, but when xylose wasused exponential growth did not start until after 4 h. Once

DCW ) 0.324OD550 + 1.687

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growing exponentially xylose resulted in the highest specificgrowth rate and glucose in the lowest. It should be noted thatwhen glucose was used a slow increase in dry cell mass wasobserved during the first 7 h of the anaerobic phase.Viable cell counts were made for all of the different

fermentations (Figure 1a-c). During the first hours of theanaerobic phase the number of viable cells was constant or

increased slightly as an effect of continued growth. Afterapproximately 20 h the viable cell density started to decrease.Succinic, acetic, and pyruvic were the only acids produced

in concentrations over 1 g L-1. The highest succinic acidconcentration, 40 g L-1, was detected after 32 h when glucosewas used as the carbon source. After 32 h the succinateproduction did not increase significantly (data not shown), and

Figure 1. Sugar, product, and cell concentration profiles for (a) glucose, (b) fructose, (c) xylose, (d) glucose/fructose, and (e) glucose/xylosefermentations. Product and sugar concentrations are given in g L-1, dry cell weights in g L-1 and viable cells in (cells mL-1) 109. Symbols used:glucose (4), fructose ()), xylose (O), succinic acid (b), pyruvic acid (9), viable cells (/), and dry cell weight (). Staggered line indicates timeof switch to the anaerobic phase.Table 1. Summary of Fermentation Parameters for AFP184a

sugar (h-1) YP/S (g g-1) anaerobic QP (g L-1 h-1) overall QP (g L-1 h-1) anaerobic, T22d QP (g L-1 h-1) anaerobic, T32dglucose 0.42 0.83 1.27 2.89 1.76fructose 0.50 0.66 1.01 1.79 1.26xylose 0.57 0.50 0.78 1.49 1.08glu/frub 0.58 0.79 1.67 1.05glu/xylc 0.60 0.86 1.48 1.08a Specific growth rates were obtained from fermentations carried out in 1 L bioreactors. YP/S is the mass yield of succinic acid based on the glucose

consumed in the anaerobic phase, and QP is the volumetric productivity of succinic acid during the anaerobic phase. b glu/fru is an abbreviation for glucose/fructose. c glu/xyl is an abbreviation for glucose/xylose. d Anaerobic productivity at 22 and 32 h total fermentation time, respectively.

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hence 32 h was selected as the final fermentation time. Whenfructose and xylose were used, the final succinic acid concentra-tions reached values of 32 and 25 g L-1, respectively. Thesefermentations also resulted in lower acetate concentrations butproduced pyruvate. The highest yield, 0.83 g succinate per gramsugar consumed in the anaerobic phase, was obtained whenglucose was used as a carbon source. The lowest yield wasobtained from xylose (0.50 g g-1). The yield from fructose was0.66 g g-1.The overall volumetric productivities (QP) are presented in

Table 1. Productivities per viable cell were calculated asfunctions of time and succinic acid concentrations, respectively(Figure 2). Productivities per viable cell as function of time forfermentations using fructose and xylose were initially high butdecreased substantially during the first hours of anaerobicconditions (Figure 2a). During the remainder of the fermenta-tions productivities remained relatively constant. Glucosefermentations can be distinguished from fermentations on theother sugars as the productivity per viable cell showed only amarginal productivity decrease up until 16 h, after which itdecreased significantly. When investigating productivities as afunction of succinic acid concentration, the fructose and xylosefermentations resulted in, after a strong initial productivitydecrease, slightly decreasing productivities as the concentrationof succinic acid increased (Figure 2b). Glucose on the otherhand gave a constant productivity until a succinate concentrationof approximately 30 g L-1 was reached, after which theproductivity decreased in an almost linear manner.Mixed-Sugar Fermentations. Strain AFP184 grew well in

both glucose/fructose and glucose/xylose mixtures. No increasein dry cell weight was observed during the anaerobic phase(Figure 1d and e). An interesting observation is that the substrateconsumption rate was higher for both fructose and xylose thanfor glucose in the aerobic phase when AFP184 was grown onmixed sugars. In the anaerobic phase glucose and fructose weremetabolized at similar rates, but when glucose was mixed withxylose, xylose was metabolized faster during the first 16 h ofthe fermentation. After 16 h the consumption rates of glucoseand xylose were approximately equal. The succinic acid yieldsin the anaerobic phase were 0.58 and 0.60 g succinic acid pergram sugar consumed for glucose/fructose and glucose/xylosemixtures, respectively (Table 1). The final succinic acidconcentrations were in the range of 25-27 g L-1, and duringboth mixed-sugar fermentations between 8 and 12 g L-1 ofpyruvate was formed (Figure 1d and e). Most of the pyruvatewas excreted during the first 2-4 h of the anaerobic phase andat a rate similar to the succinic acid production rate. Thepyruvate concentration decreased in both cases to final con-

centrations of approximately 1-3 g L-1. Acetate levels increasedslowly during both fermentations. Less acetate was formed whenfructose was used, 2.7 g L-1 compared to 6.7 g L-1 when xylosewas used.The viable cell densities increased during the aerobic phases

and were approximately constant during the anaerobic phasesup until 20 h of the fermentations had passed, where after theydecreased (Figure 1d and e).Productivities during mixed-sugar fermentation are presented

in the same way as for single-sugar fermentations (Figure 3).When using gluose/fructose the productivity per viable cell asa function of time was initially high (Figure 3a). A majorproductivity decrease occurred at a succinate concentration of20 g L-1 (Figure 3b). For glucose and xylose the productivitywas constant throughout the anaerobic phase (Figure 3a andb).

DiscussionThe aim of the current work was to investigate the fermenta-

tion characteristics of E. coli strain AFP184 in a corn steepliquor based medium using different mono- and disaccharidesas carbon source. From the cell densities obtained after 8 h ofaerobic growth and under the conditions used, it could beconcluded that AFP184 grew on all sugars and sugar combina-tions except sucrose. However, specific growth rate on glucosewas slower than for growth on xylose or fructose. This result ismost likely an effect of the mutation in the phosphotransferasesystem (PTS) of AFP184. The PTS is a system of enzymesresponsible for uptake and phosphorylation of different sugarsin bacteria (27, 28). The PTS is sugar-specific, and the mutationin AFP184 is affecting EIICBglc, a glucose specific permeasein the PTS (12). There is also a phosphotransferase permeasefor fructose (27, 29), but in strain AFP184 this permease is notaffected by any mutations. Apart from the PTS, E. coli can alsotransport glucose by galactose permease and phosphorylate itby the enzyme glucokinase (30). Glucokinase is not subjectedto any mutations in AFP184. According to the literature glucoseuptake and phosphorylation by glucokinase is slower than bythe PTS (31), which explains why the growth rate on fructoseis higher than the growth rate on glucose. Unlike fructose, xyloseis not transported into the cells via the PTS. Instead xyloseuptake is facilitated by chemiosmotic effects (32, 33). Onceinside the cell it is isomerized by xylose isomerase to xyluloseand phosphorylated by xylulose kinase to xylulose 5-phosphate.Xylulose 5-phosphate then enters the pentose phosphate pathwayand can enter the glycolytic pathway after conversion to eitherfructose 6-phosphate or glyceraldehyde 3-phosphate. Interest-ingly, growth on xylose was initially very slow, whereas the

Figure 2. Succinic acid productivities for single-sugar fermentations in grams succinate per viable cell as a function of (a) time and (b) succinicacid concentration. Symbols used in the figure: glucose fermentation (4), fructose fermentation (]), and xylose fermentation (O).

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use of glucose and fructose rapidly initiated exponential growth.Because the inocula were grown in a glucose medium, glucoseand fructose fermentations may already have the enzymesrequired for utilization of the respective sugars, whereas in thecase of xylose expression of the enzymes first needs to beinduced before exponential growth can start.When glucose is mixed with other sugars, E. coli usually

consumes it first, and after all glucose has been metabolizedthe utilization of other sugars is initiated, a phenomenon calledcatabolite repression. From the sugar consumption (Figure 1)it was observed that in glucose/fructose mixtures, fructose wasmetabolized slightly faster during the aerobic phase. This effectwas expected as a result of the mutation strain AFP184 has inthe glucose PTS. More interesting is the observation that whenglucose and xylose were mixed, the sugars were fermentedsimultaneously but xylose was consumed at a substantiallyhigher rate. Other studies of glucose/xylose fermentations byE. coli PTS mutants show similar results where glucose andxylose are co-metabolized. The effect is attributed to a mutationin the PTS affecting EIICBglc, which in turn prevents glucosefrom repressing uptake of other sugars (30, 34-36).To estimate maximum yields and optimal metabolic flows

between intermediates, redox balances were carried out. Theanaerobic glucose, fructose, and xylose metabolism of AFP184for maximal succinate production is presented in simplified formin Figure 4. From a redox balance for the anaerobic phase themaximum succinate yield from 1 mol of glucose is 1.71 mol(1.12 g succinate per gram glucose), which is in accordancewith previous results (23).The redox balance assumes activity of the enzyme isocitrate

lysase, a key enzyme in the glyoxylate shunt. It has previouslybeen reported that AFP111 shows isocitrate lyase activity afteraerobic growth (23). The yield obtained in this study fromglucose was 1.27 mol succinate per mole glucose, which is 74%of the theoretical. If generation of new cells during the anaerobicphase is considered, a carbon balance can account for more than95% of the carbon.The theoretical yield from fructose is 1.20 mol succinate per

mole fructose, which corresponds to a mass yield of 0.79,although this requires another electron acceptor such as ethanol,which was not detected. A redox balance without consideringethanol yields 1 mol succinate and 1 mol pyruvate per molefructose. A molar yield of 1.00 was achieved (mass yield 0.66)for succinate from pure fructose. Redox balances under anaero-bic conditions indicate that 2.5 times greater flux through thereductive arm is required to match the flux through the oxidativeTCA plus the glyoxylate shunt for either glucose or fructose asa substrate. During fructose fermentation by AFP184, phos-

phoenolpyruvate (PEP) is converted to pyruvate by the PTS(29, 37), with 1.0 mol fructose yielding 1.0 mol PEP and 1.0mol pyruvate due to transport requirements. This metaboliteimbalance prevents a balanced redistribution of reducingequivalents, and as a result pyruvate will accumulate and beexcreted (38) as was observed in Figure 1b. Glucose mixed withfructose should provide an increased pool of PEP to balancethe pyruvate generated by fructose transport and result in higheryields than fructose alone. However, experimental results donot confirm this hypothesis (Table 1), although much morepyruvic acid was excreted initially with fructose/glucosemixtures relative to fermentation of pure fructose (12.4 vs 5.2g L-1). The accumulation of higher concentrations of pyruvicacid for the mixed substrate fermentation is suspected to be dueto the more rapid uptake of both sugars, resulting in a fasterintracellular accumulation of pyruvate with no available electronsink for further metabolism. For both fermentations, most ofthis excreted pyruvic acid was taken up again, requiring theexpenditure of reducing equivalents for active transport (39).This excretion and reutilization of pyruvate can explain boththe low yields and the lower rates found for fructose/glucosefermentation.A redox balance for xylose gives a maximum theoretical yield

of 1.43 mol succinate per mole xylose. In the case of xyloseonly about 45% of the theoretical yield was achieved (0.64 molmol-1). By carrying out a carbon balance, 60% of the carboncan be accounted for. Further work is required to identify theremaining carbon. When xylose was mixed with glucose, theyield increased to 0.83 mol succinate per mole sugar, whichwas expected because glucose gives higher yields.When glucose was used as the carbon source, the productivity

per viable cell decreased drastically after 16 h (Figure 2a).Succinic acid productivity as a function of succinic acidconcentration showed a sharp decrease in productivity atapproximately 30 g L-1 (Figure 2b). The succinic acid concen-tration at 16 h corresponds to almost 30 g L-1. From this resultit can be concluded that as long as the cells are viable, theyretain their productivity until the concentration of succinic acidis not too high. Fructose and xylose did not show any drasticdecreases in productivity (Figure 2). Neither of these fermenta-tions achieved succinate concentrations of more than 20-25 gL-1 during the first 20 h of the fermentations (Figure 1b andc). In the mixed-sugar fermentations glucose/xylose mixturesdid not result in very high succinate concentrations and nodecrease in productivity was observed (Figure 3c and d). Whenglucose and fructose were mixed and fermented, a sharpproductivity decrease was observed after 16 h (Figure 3a) whenthe concentration of succinate was approximately 20 g L-1

Figure 3. Succinic acid productivities for mixed-sugar fermentations in grams succinate per viable cells as a function of (a) time and (b) succinicacid concentration. Symbols used in the figure: Glucose/fructose fermentation (]), and glucose/xylose fermentation (O).

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(Figure 1d). When glucose was used as the carbon source,productivities did not decline until concentrations of 30 g L-1were reached. In the other fermentations the concentrations ofend products other than succinate were low, but in thisfermentation the concentration of pyruvic acid was 10 g L-1after 16 h and the total concentration of end products exceeded30 g L-1 (Figure 1d). From these results it seems likely thathigh final concentrations of end products inhibit productivity.Many investigations regarding the inhibition effects by organicacids on E. coli growth and productivity have been carried out(40-42). The effects of organic acid inhibition is greatest atlow pH when the acids are undissociated, but even at neutralpH high concentrations of the acids have shown inhibitoryeffects (42). The fermentations in this study were carried outwith ammonium hydroxide as base for neutralization of endproducts, and it has been reported that ammonium in concentra-tions above 3 g L-1 inhibits growth (43, 44). In the presentwork ammonium concentrations of approximately 10 g L-1 wereobtained at the end of the fermentations with high succinateproduction. In other studies changes in the growth characteristicsof E. coli under different ammonium sulfate concentrations wasinvestigated, and ammonium sulfate concentrations of 5% hadto be used before any severe inhibition was observed (45). Whenthe fermentations carried out in the present work showedproductivity decreases, they were in the anaerobic phase andonly very little growth would take place even without anyinhibition; it is not clear if productivity would be affected by

the achieved ammonium concentrations. Possible ammoniainhibition is being further investigated in studies where differentbases are compared to better understand the causes of theobserved productivity decrease.Finally, a comment should be made regarding the volumetric

productivities achieved. As mentioned in the Introduction,biological production of succinic acid should preferably reachvolumetric productivities of 2.5 g L-1 h-1 to be feasible. Inthis study volumetric productivities during anaerobic productionin the range of 1.5-2.9 g L-1 h-1 were demonstrated. Thereason the productivities reach almost 3 g L-1 h-1 is becausethe cells after the switch to the anaerobic phase are not sugar-limited. A high initial sugar load was used, but the same resultscould be achieved by feeding a high concentration sugar streamafter the end of the aerobic phase. The total volumetricproductivity is directly dependent on the cell density, and toachieve a high cell density requires the use of substantialamounts of the carbon source. This of course could compromisethe overall process economics, and the necessary cell densityfor feasible production of succinic acid must be considered ifthe technology is to be applied to a large scale productionfacility.

ConclusionsIn this paper we have demonstrated growth and succinate

production of E. coli strain AFP184 in a medium containingcorn steep liquor, salts, and different mono- and disaccharides.

Figure 4. Anaerobic glucose, fructose, and xylose metabolism of AFP184 for maximal succinate production. Note that some metabolites areexcluded. The reactions blocked by the mutations in AFP184 are indicated by crosses.

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The reduction of complex components in the medium has apositive effect on the final price of the produced succinate dueto lower cost of raw materials; however, final concentrationsare in the range of 30-40 g L-1 after 32 h and need to beimproved for the process to become more economical. Studieswill be directed to increase the final titer and to further improvethe process.

AcknowledgmentThis research was supported by Diversified Natural Products

Inc. and the Research Council of Norrbotten. Equipment wasprovided by the Kempe Foundation.

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Received October 2, 2006. Accepted December 21, 2006.BP060301Y

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