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Enhanced production of succinic acid by Actinobacillus succinogenes with reductive carbon source
Process Biochemistry 45 (2010) 980–985
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Enhanced production of succinic acid by Actinobacillus succinogenes with reductive carbon source Jian Li a , Min Jiang a,∗ , Kequan Chen a , Longan Shang b , Ping Wei a , Hanjie Ying a , Qi Ye a , Pingkai Ouyang a , Honam Chang c a
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, No. 5 Xinmofan Road, Nanjing 210009, Jiangsu, PR China Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, PR China c Department of Chemical Engineering and Bioprocess Engineering Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 18 October 2009 Received in revised form 15 January 2010 Accepted 1 March 2010 Keywords: NADH/NAD+ Succinic acid Actinobacillus succinogenes NJ113 Redox balance Metabolite distribution
a b s t r a c t Carbon sources with different oxidation states were used to investigate the possibility increasing the availability of NADH and the NADH/NAD+ ratio and to determine the effect of this manipulation on the distribution of metabolites in Actinobacillus succinogenes NJ113. The sugars glucose, sorbitol and gluconate were each used at an initial concentration of 40 g/L. The yield of succinic acid (0.75) and the ratio of succinic acid to acetic acid (5.06) were both higher for sorbitol than the values obtained with glucose (0.66 and 2.68, respectively). In contrast, with gluconate as the carbon source the yield of succinic acid was 0.54 and the ratio of succinic acid to acetic acid was only 1.70. This work showed that different levels of NADH availability and the NADH/NAD+ ratio can be achieved by using carbon sources that have different oxidation states. Highly reduced sorbitol was examined as a possible carbon substrate for maximizing the redox potential during the production of succinic acid. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Cofactors have a major role in the production of different fermentation products. The cofactor pair NADH and NAD+ have a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ and producing reducing equivalents in the form of NADH. It is crucially important for cell growth that NADH is oxidized to NAD+ and a redox balance is achieved [1–4]. Berrios-Rivera et al. investigated the effect of different carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in Escherichia coli. They found that the 1,2-propanediol reduction system is not limited by NADH, but rather by the pathways following the formation of methylglyoxal [3]. The cofactor pair NADH and NAD+ , which can be transformed reversibly between the reduced and the oxidized forms, serves this purpose very effectively in a living cell. Therefore, cofactor manipulation offers an additional tool to achieve certain goals in metabolic engineering. Succinic acid is valued as one of the key platform chemicals in the preparation of biodegradable polymers such as polybuty-
∗ Corresponding author. Tel.: +86 25 83172062. E-mail address: [email protected] (M. Jiang). 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.03.001
lene succinate and polyamides or as a raw material for producing bulk chemicals of the C4 family, which include 1,4-butanediol, tetrahydrofuran, N-methyl pyrrolidinone, 2-pyrrolidinone, and ␥butyrolactone [5–7]. In a report from the U.S. Department of Energy (USDOE), succinic acid was considered as one of the top 12 chemical building blocks manufactured from biomass [8]. The fermentative production of succinic acid has been investigated for a wide variety of bacteria, including Bacteroides ruminicola, Bacteroides amylophilus, Corynebacterium glutamicum, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens and E. coli [9–11]. A. succinogenes is a facultatively anaerobic bacterium and has ability to produce a relatively large amount of succinic acid under anaerobic condition from a broad range of carbon sources, including arabinose, cellobiose, fructose, galactose, glucose, lactose, maltose, mannitol, mannose, sorbitol, sucrose, xylose and salicin. A. succinogenes metabolizes glucose to phosphoenolpyruvate (PEP) via glycolysis with involvement of the oxidative pentose phosphate pathway (OPPP). PEP was thought to serve as the branchpoint to the formate-, acetate-, and ethanol-producing C3 pathway and the succinate-producing C4 pathway [12] (Fig. 1). The pathway to succinic acid requires NADH for its synthesis. There are reports describing the investigation of cofactor manipulation strategies, including the choice of carbon sources with different oxidation
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Fig. 1. Central anaerobic metabolic pathway of A. succinogenes using glucose, sorbitol and gluconate as carbon sources [7]. Black bold arrows: succinate-producing C4 pathway. Open arrows: formate-, acetate-, and ethanol-producing C3 pathway.
states [4], increasing intracellular availability of NADH by an NADH regeneration strategy [2,13], and increasing the total NAD levels by enhancing the NAD salvage pathway [14]. Andersson et al. have used sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose as carbon sources to study the succinic acid production with E. coli AFP184 in a low cost medium from a variety of sugars with only small amounts of by-products formed [9]. Lin et al. have investigated the effect of carbon sources differing in oxidation state and transport route on succinate production in metabolically engineered E. coli and shown the importance of NADH in the production of succinate [15], but the NADH/NAD+ ratio or the availability of NADH was not determined in the fermentation process. In this work, the strategy of using carbon sources with different oxidation states was used to manipulate the intracellular availability of NADH. The effect on the distribution of metabolites in A. succinogenes, in particular, on the production of chemicals that require NADH for their synthesis, such as succinate, ethanol, and lactate, was investigated. 2. Materials and methods 2.1. Microorganism A. succinogenes NJ113 [16] was used for succinic acid fermentation. 2.2. Media composition The medium used for preparation of the inoculum contained (in g/L): 10.0 glucose, 5.0 yeast extract, 10.0 NaHCO3 , 8.5 NaH2 PO4 ·H2 O and 15.5 K2 HPO4 . The culture medium contained (in g/L): 3.0 KH2 PO4 , 0.2 MgCl2 ·6H2 O, 0.2 CaCl2 , 1.0 NaCl, 5.0 corn liquor steep, and 10.0 yeast extract [16]. Glucose, sorbitol and gluconate were autoclaved separately and added aseptically.
fermentation and was sparged with nitrogen gas for 30 min to remove oxygen before the inoculation. All fermentation was done at an agitation speed of 200 rpm and CO2 flow-rate of 0.5 L/min. The pH was maintained at 6.8 by addition of 10 mol/L Na2 CO3 . All experiments were repeated three times. 2.4. Analytical methods Fermentation samples were centrifuged 9400 × g and 4 ◦ C for 10 min in a microcentrifuge. The supernatant was filtered through a 0.22-m syringe filter and stored chilled for HPLC analysis. The concentrations of organics were measured by HPLC (Chromeleon server monitor, UVD 170U detector, P680 pump, Dionex, USA) equipped with an ion-exchange column (PrevailTM organic acid column, Grace, USA). The column was maintained at 48 ◦ C, and KH2 PO4 (25 mmol/L; adjusted to pH 2.5 with H3 PO4 ) was used as the mobile phase at a flow-rate of 1 mL/min. Sugar concentrations were determined with the HPLC system described above but using a Series 3000 refractive index (RI) detector (Perkin-Elmer), a guard column, and an ion-exchange column (Aminex HPX87-P, BioRad) as described previously [9]. Dry cell weight (DCW) was obtained as described [16]. An optical density at 660 nm (OD660 ) of 1.0 represented a dry weight of 567 mg/L. 2.5. Enzyme assays For measurement of intracellular enzyme activity, cells were harvested by centrifugation at 9400 × g for 10 min at 4 ◦ C and washed twice with cold Tris–HCl (100 mmol/L; pH 7.5). The washed cells were suspended in the same buffer containing EDTA (0.1 mmol/L) and sonicated on ice for 90 cycles (a working period of 3 s in a 10 s interval for each cycle) at a power output of 200 W with an ultrasonic disruptor (GA92-IID, ShangJia Biotechnology Co., WuXi, China). The cell debris was removed by centrifugation at 13,400 × g for 20 min at 4 ◦ C, and the supernatant was used for the measurement of enzyme activity. Enzyme activity was measured in a temperature-controlled spectrophotometer (UV-2100, Unico, USA). The activity of phosphoenolpyruvate carboxykinase (PCK), malate dehydrogenase (MDH), fumarate dehydrogenase (FRD) and alcohol dehydrogenase (ADH) was measured as described [17,18]. Enzyme activity is expressed in units (U) and 1 U is defined as the amount of enzyme necessary to catalyse the conversion of 1 mol of substrate/min into specific products. Specific activity is expressed as U/mg of protein.
2.3. Cultivation conditions 2.6. Protein assays Fermentation was done in anaerobic bottles as described [16]. Batch fermentation was done at 37 ◦ C with an initial broth volume of 1.5 L in a 3 L fermentor (Bioflo 110, New Brunswick Scientific, Edison, NJ, USA). The culture medium was used for
Protein contents were determined by the Bradford method [19], using bovine serum albumin (Sigma Chemical Co.) as the standard.
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2.7. NADH/NAD+ assays The intracellular concentrations of NADH and NAD+ were measured as described [20]. 2.8. Measurement of redox potential Redox potential was measured with a METTLER TOLEDO pH 2100e (Mettler Toledo, Switzerland) and a redox potential electrode (P+ 4805-SC-DPAS-K8S/225 Redox, Mettler Toledo, Switzerland).
3. Results and discussion 3.1. Carbon source experiments Experiments were done with A. succinogenes NJ113 under anaerobic conditions in a 3 L fermentor to investigate the effect of
different carbon sources (sorbitol, glucose and gluconate) on cell growth and the distribution of metabolites. The results of these experiments, including the final DCW, the amount of substrate consumed, and the concentration of metabolites produced after 14 h are given in Table 1 and illustrated in Fig. 2. The identity of the carbon source had a significant effect on the production of succinic acid. The maximum DCW using sorbitol as the carbon source was much higher relative to glucose, but cells given glucose as the carbon source grew faster in the exponential phase than those given sorbitol as the carbon source. The inhibition of organic acid may be the reason why cell growth using glucose as the carbon source was suddenly stopped at 6 h [21,22]. The DCW was the lowest for gluconate as the carbon source, and was constant at around 1.7. At a low bacterial density, gluconate was the most slowly consumed carbon source, and the amount of metabolites produced was the smallest. The produc-
Fig. 2. Effect of different carbon sources on cell growth and the distribution of metabolites. The time-course is shown for: (A) carbon source, (B) DCW, (C) succinic acid, (D) acetic acid, (E) ethanol, (F) formic acid. () Sorbitol; () glucose; () gluconate.
J. Li et al. / Process Biochemistry 45 (2010) 980–985 Table 1 Metabolite concentrations using different carbon sources with A. succinogenes NJ113. Sorbitol (−1)a Substrate consumed (g/L) Maximum DCW (g/L) Succinic acid (g/L) Acetic acid (g/L) Formic acid (g/L) Ethanol (g/L) SAc /ACd (concentration) SAc yielde (g/g) SAc productivity (g/(L h))
39.5 5.06 29.8 5.89 2.83 1.03 5.06 0.75 2.13
± ± ± ± ± ± ± ± ±
0.48b 0.55 0.86 0.22 0.23 0.18 0.24 0.02 0.06
Glucose (0) 39.9 4.48 26.4 9.85 5.13 0.85 2.68 0.66 1.89
± ± ± ± ± ± ± ± ±
0.72 0.47 0.67 0.35 0.13 0.16 0.12 0.02 0.05
Gluconate (+1) 15.5 1.81 8.3 4.89 1.63 0.38 1.70 0.54 0.59
± ± ± ± ± ± ± ± ±
0.29 0.14 0.41 0.29 0.15 0.13 0.13 0.03 0.03
a
The value in parentheses represents the oxidation state. Each value is an average of three parallel replicates and is reported as mean ± standard deviation. c Succinic acid. d Acetic acid. e SA yield: g of SA formed/g of glucose utilized. b
tion of succinic acid (29.8 g/L) and ethanol (1.03 g/L) was greatest with sorbitol as the carbon source. In addition, the level of acetic acid and formic acid were decreased significantly (40% and 45% respectively) in sorbitol cultures relative to glucose, increasing the succinic acid to acetic acid ratio (SA/AC) to 5.06. Some reports suggest that acetic acid accumulation inhibits cell growth [23–25]; therefore, the sharp decrease in the level of acetic acid was of more benefit to cell growth in the late stage for the sorbitol cultures relative to the glucose cultures. The yield (0.75 g/g) and productivity (2.13 g/(L h)) of succinic acid in the sorbitol cultures were by far the greatest. 3.2. Influence of carbon source on redox potential A carbon source is the raw material of cell composition, and the energy necessary for cell growth. Biosynthetic transformations involving redox reactions offer potential advantages for the production of bulk chemicals compared to conventional chemical processes [2]. Different carbon sources have different oxidation states. The internal redox state, reflected by the NADH/NAD+ ratio, is influenced by environmental factors such as the oxidation state of the substrate [26]. Fig. 3 shows the changes of redox potential of three different carbon sources during fermentation. As shown in Fig. 3, the redox potential of the fermentation system decreased steeply in the first 2 h and then gradually as the fermentation proceeded. Although this is an anaerobe fermenta-
Fig. 3. Redox potential of three different carbon sources during fermentation. () Sorbitol; () glucose; () gluconate.
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tion process, at the beginning there is still trace oxygen in the medium. After inoculation, the trace oxygen was gradually consumed by A. succinogenes NJ113 which is a facultative anaerobe, that may be the reason why the redox potential of the fermentation system decreased steeply during the first 2 h. The redox potential of the glucose culture was much higher relative to that of the sorbitol culture, but lower relative to that of gluconate culture. In sharp contrast, sorbitol as a carbon source induced a fermentation behaviour by achieving a more reduced intracellular environment, and using gluconate as a carbon source induced a fermentation behaviour by achieving a more oxidized intracellular environment. In general, more reduced substrates yield a higher proportion of reduced metabolites, while more oxidized substrates yield a higher proportion of more oxidized metabolites compared to those produced with glucose as the carbon source [27]. Therefore, when sugars similar to glucose, but differing in their oxidation states, were used as substrates, the cell can redistribute its fermentation pattern to achieve a redox balance. The use of a more reduced carbon source (sorbitol) led to a significant increase in the yield of succinic acid, a reduced product, accompanied by a significant decrease in the yield of acetic acid, a more oxidized product that does not require NADH for its synthesis. In contrast, the use of gluconate as the carbon source resulted in a significant increase in the yield of acetic acid and a decrease in the yield of succinic acid. In summary, using a more reduced carbon source (sorbitol) provides a more reduced environment, as evidenced by the higher SA/AC ratio (Table 1). 3.3. Influence of carbon source on the NADH/NAD+ ratio Succinate production from glucose involves a single NADHgenerating step, from glucose-6-phosphate to PEP, whereby 1 mole of glucose generates 2 moles of NADH. In the next pathway, however, there are several NADH consumption steps. In the succinate-producing C4 pathway, a total of 4 moles of NADH is required for the steps from oxaloacetate to malate and from fumarate to succinate. In the formate-, acetate- and ethanolproducing C3 pathways, the producing of ethanol and of lactate require 2 moles of NADH each (Fig. 1). In the whole pathway in A. succinogenes, the requirement for NADH is greater than its generation, so the reducing power is insufficient. Fig. 1 shows the glycolysis metabolic pathway of A. succinogenes NJ113 with glucose, sorbitol or gluconate as the carbon source. As the figure shown, more reducing equivalents in the form of NADH are produced from sorbitol compared with glucose. Gluconate produces less NADH than that from glucose because every molecule goes directly to phosphoenolpyruvate (PEP), which skips the NADH-producing step in glycolysis. This provides a simple way of testing the effect of manipulating the NADH/NAD+ ratio on the metabolic patterns of A. succinogenes NJ113 under anaerobic conditions and on the production of succinic acid, which requires NADH. Table 2 presents the concentrations of NADH and NAD+ using different carbon sources with A. succinogenes NJ113. Over the time-course of the experiment, 0–4 h represents the early exponential phase, 4–8 h represents the late exponential phase and 8–12 h represents the stationary phase of cell growth. After 4 h the NADH/NAD+ ratio using sorbitol or gluconate as the carbon source was lower relative to glucose. After 8 h DCW was higher for the sorbitol cultures relative to glucose, and the NADH/NAD+ ratio increased from 0.94 for glucose to 2.71 for sorbitol, which is more reduced and can therefore produce more reducing equivalents in the form of NADH. In a similar way, the NADH/NAD+ ratio decreased to 0.31 for gluconate. These changes in NADH/NAD+ ratio affected the distribution of metabolic fluxes in A. succinogenes NJ113, which is reflected in the SA/AC ratio. This ratio was increased from 2.68 with glucose to 5.06 with sorbitol and was decreased to 1.70 with
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Table 2 Concentration of NADH and NAD+ , and the NADH/NAD+ ratio with A. succinogenes NJ113 using different carbon sources. Substrate
Time (h)
NADH (mmol/g DCW)
NAD+ (mmol/g DCW)
NADH/NAD+
Sorbitol
4 8 12
0.40 ± 0.05 0.38 ± 0.03 0.21 ± 0.02
0.23 ± 0.04 0.14 ± 0.01 0.62 ± 0.01
1.74 ± 0.37 2.71 ± 0.29 0.34 ± 0.03
Glucose
4 8 12
0.52 ± 0.05 0.31 ± 0.03 0.27 ± 0.02
0.28 ± 0.04 0.33 ± 0.01 0.55 ± 0.01
1.86 ± 0.32 0.94 ± 0.10 0.49 ± 0.04
Gluconate
4 8 12
0.29 ± 0.05 0.20 ± 0.03 0.32 ± 0.02
0.33 ± 0.04 0.64 ± 0.01 0.71 ± 0.01
0.88 ± 0.19 0.31 ± 0.05 0.45 ± 0.03
Each value is an average of three parallel replicates and is reported as mean ± standard deviation.
gluconate, suggesting that the cell adjusts its partitioning at the PEP step by changing the ratio of succinic acid (consumes 4 NADH) to acetic acid (consumes no NADH) ratio to achieve a redox balance. Therefore, a change in the SA/AC ratio can be used as an indirect indicator of a change in the NADH/NAD+ ratio. These results demonstrate that it is possible to manipulate the availability of intracellular NADH through the use of carbon sources with different oxidation states. The use of gluconate, a more oxidized carbon source, decreased intracellular NADH availability and resulted in a more oxidized environment. In contrast, the use of sorbitol, a more reduced carbon source, increased intracellular NADH availability and resulted in a more reduced environment. The cells utilize this extra NADH to reduce metabolic intermediates leading to the formation of fermentation products in order to achieve a redox balance. 3.4. Influence of carbon source on enzyme activity PCK is a key enzyme in the path to succinic acid formation in A. succinogenes NJ113. The activity of PCK determines the fraction of the carbon from PEP that goes to oxaloacetate. Malate dehydrogenase (MDH), fumarate dehydrogenase (FRD), and alcohol dehydrogenase (ADH), enzymes that involve reduction, were also investigated (Table 3). The results demonstrated that enzyme activity is influenced by carbon sources with different oxidation states. The use of sorbitol resulted in a more reducing intracellular environment, as reflected by a significant increase in both the NADH/NAD+ and the SA/AC ratios, which was beneficial for cell growth. The level of activity among the four key enzymes was the highest with sorbitol as the carbon source. The use of gluconate resulted in a more oxidized intracellular environment, as reflected by a significant decrease in both the NADH/NAD+ and the SA/AC ratios. The level of activity among four key enzymes was lowest with gluconate as the carbon source. It was found that a highly reducing substrate, such as sorbitol, may result in an increased content of ethanol content, because NADH was also consumed during the metabolic pathway from PEP to ethanol, and the increased of availability of NADH could increase the metabolic flux of ethanol synthesis. Therefore, the reduced the
Table 3 Effect of carbon source on key enzyme activity. Substrate
Sorbitol Glucose Gluconate
Specific activity (U/mg protein) PCK
MDH
FRD
ADH
2.87 ± 0.06 2.35 ± 0.08 1.25 ± 0.05
1.35 ± 0.04 0.86 ± 0.05 0.41 ± 0.04
0.63 ± 0.05 0.34 ± 0.02 0.18 ± 0.01
0.23 ± 0.02 0.15 ± 0.01 0.13 ± 0.01
Each value is an average of three parallel replicates and is reported as mean ± standard deviation.
synthesis of ethanol and the increased availability of NADH at the same time needs further study. 4. Conclusions The results of this work demonstrated that the production of succinic acid by anaerobic fermentation by A. succinogenes NJ113 could be enhanced by the choice of reductive carbon sources. We showed here that carbon sources with different oxidation states can be used to manipulate the NADH/NAD+ ratio. Further, we have shown that this manipulation has a direct effect on the distribution of metabolic fluxes in A. succinogenes NJ113, especially in those requiring NADH, such as succinic acid and ethanol. So, the method described here can be used as a simple way of determining whether a particular pathway that requires NADH is limited by the availability of this cofactor. This strategy of using carbon sources with different oxidation states can regulate the NADH/NAD+ ratio or the availability of NADH in the fermentation process, thereby change the key enzymes’ activity in the metabolic pathway, and redistribute metabolites to increase succinic acid production and reduce by-products generation. Therefore, there is a significant perspective of substituting glucose with further reduced form in economic sense. Acknowledgements This study was supported by grant 2009CB724701 from “973” Program of China, grant no. 20606017 from National Natural Science Foundation of China, State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of Technology, China, Science and Technology Achievement Transformation Project of Jiangsu province, and Qing Lan Project of Jiangsu province. References [1] Berrios-Rivera SJ, Sanchez AM, Bennett GN, San KY. Effect of different levels of NADH availability on metabolite distribution in Escherichia coli fermentation in minimal and complex media. Appl Microbiol Biotechnol 2004;65:426–32. [2] Berrios-Rivera SJ, Bennett GN, San KY. The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures. Metab Eng 2002;4:230–7. [3] Berrios-Rivera SJ, San KY, Bennett GN. The effect of carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in Escherichia coli. J Ind Microbiol Biotechnol 2003;30:34–40. [4] San KY, Bennett GN, Berrios-Rivera SJ, Vadali RV, Yang Y-T, Horton E, et al. Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli. Metab Eng 2002;4:182–92. [5] Lu SY, Eiteman MA, Altman E. Effect of flue gas components on succinate production and CO2 fixation by metabolically engineered Escherichia coli. World J Microbiol Biotechnol 2009; doi:10.1007/s11274-009r-r0185-1. [6] Song H, Lee SY. Production of succinic acid by bacterial fermentation. Enzyme Microb Technol 2006;39:352–61. [7] McKinlay JB, Vieille C, Zeikus JG. Prospects for a bio-based succinate industry. Appl Microbiol Biotechnol 2007;76:727–40.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Enhanced production of succinic acid by Actinobacillus succinogenes with reductive carbon source Jian Li a , Min Jiang a,∗ , Kequan Chen a , Longan Shang b , Ping Wei a , Hanjie Ying a , Qi Ye a , Pingkai Ouyang a , Honam Chang c a
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, No. 5 Xinmofan Road, Nanjing 210009, Jiangsu, PR China Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, PR China c Department of Chemical Engineering and Bioprocess Engineering Research Center, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-gu, Taejon 305-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 18 October 2009 Received in revised form 15 January 2010 Accepted 1 March 2010 Keywords: NADH/NAD+ Succinic acid Actinobacillus succinogenes NJ113 Redox balance Metabolite distribution
a b s t r a c t Carbon sources with different oxidation states were used to investigate the possibility increasing the availability of NADH and the NADH/NAD+ ratio and to determine the effect of this manipulation on the distribution of metabolites in Actinobacillus succinogenes NJ113. The sugars glucose, sorbitol and gluconate were each used at an initial concentration of 40 g/L. The yield of succinic acid (0.75) and the ratio of succinic acid to acetic acid (5.06) were both higher for sorbitol than the values obtained with glucose (0.66 and 2.68, respectively). In contrast, with gluconate as the carbon source the yield of succinic acid was 0.54 and the ratio of succinic acid to acetic acid was only 1.70. This work showed that different levels of NADH availability and the NADH/NAD+ ratio can be achieved by using carbon sources that have different oxidation states. Highly reduced sorbitol was examined as a possible carbon substrate for maximizing the redox potential during the production of succinic acid. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Cofactors have a major role in the production of different fermentation products. The cofactor pair NADH and NAD+ have a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ and producing reducing equivalents in the form of NADH. It is crucially important for cell growth that NADH is oxidized to NAD+ and a redox balance is achieved [1–4]. Berrios-Rivera et al. investigated the effect of different carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in Escherichia coli. They found that the 1,2-propanediol reduction system is not limited by NADH, but rather by the pathways following the formation of methylglyoxal [3]. The cofactor pair NADH and NAD+ , which can be transformed reversibly between the reduced and the oxidized forms, serves this purpose very effectively in a living cell. Therefore, cofactor manipulation offers an additional tool to achieve certain goals in metabolic engineering. Succinic acid is valued as one of the key platform chemicals in the preparation of biodegradable polymers such as polybuty-
∗ Corresponding author. Tel.: +86 25 83172062. E-mail address: [email protected] (M. Jiang). 1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.03.001
lene succinate and polyamides or as a raw material for producing bulk chemicals of the C4 family, which include 1,4-butanediol, tetrahydrofuran, N-methyl pyrrolidinone, 2-pyrrolidinone, and ␥butyrolactone [5–7]. In a report from the U.S. Department of Energy (USDOE), succinic acid was considered as one of the top 12 chemical building blocks manufactured from biomass [8]. The fermentative production of succinic acid has been investigated for a wide variety of bacteria, including Bacteroides ruminicola, Bacteroides amylophilus, Corynebacterium glutamicum, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens and E. coli [9–11]. A. succinogenes is a facultatively anaerobic bacterium and has ability to produce a relatively large amount of succinic acid under anaerobic condition from a broad range of carbon sources, including arabinose, cellobiose, fructose, galactose, glucose, lactose, maltose, mannitol, mannose, sorbitol, sucrose, xylose and salicin. A. succinogenes metabolizes glucose to phosphoenolpyruvate (PEP) via glycolysis with involvement of the oxidative pentose phosphate pathway (OPPP). PEP was thought to serve as the branchpoint to the formate-, acetate-, and ethanol-producing C3 pathway and the succinate-producing C4 pathway [12] (Fig. 1). The pathway to succinic acid requires NADH for its synthesis. There are reports describing the investigation of cofactor manipulation strategies, including the choice of carbon sources with different oxidation
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Fig. 1. Central anaerobic metabolic pathway of A. succinogenes using glucose, sorbitol and gluconate as carbon sources [7]. Black bold arrows: succinate-producing C4 pathway. Open arrows: formate-, acetate-, and ethanol-producing C3 pathway.
states [4], increasing intracellular availability of NADH by an NADH regeneration strategy [2,13], and increasing the total NAD levels by enhancing the NAD salvage pathway [14]. Andersson et al. have used sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose as carbon sources to study the succinic acid production with E. coli AFP184 in a low cost medium from a variety of sugars with only small amounts of by-products formed [9]. Lin et al. have investigated the effect of carbon sources differing in oxidation state and transport route on succinate production in metabolically engineered E. coli and shown the importance of NADH in the production of succinate [15], but the NADH/NAD+ ratio or the availability of NADH was not determined in the fermentation process. In this work, the strategy of using carbon sources with different oxidation states was used to manipulate the intracellular availability of NADH. The effect on the distribution of metabolites in A. succinogenes, in particular, on the production of chemicals that require NADH for their synthesis, such as succinate, ethanol, and lactate, was investigated. 2. Materials and methods 2.1. Microorganism A. succinogenes NJ113 [16] was used for succinic acid fermentation. 2.2. Media composition The medium used for preparation of the inoculum contained (in g/L): 10.0 glucose, 5.0 yeast extract, 10.0 NaHCO3 , 8.5 NaH2 PO4 ·H2 O and 15.5 K2 HPO4 . The culture medium contained (in g/L): 3.0 KH2 PO4 , 0.2 MgCl2 ·6H2 O, 0.2 CaCl2 , 1.0 NaCl, 5.0 corn liquor steep, and 10.0 yeast extract [16]. Glucose, sorbitol and gluconate were autoclaved separately and added aseptically.
fermentation and was sparged with nitrogen gas for 30 min to remove oxygen before the inoculation. All fermentation was done at an agitation speed of 200 rpm and CO2 flow-rate of 0.5 L/min. The pH was maintained at 6.8 by addition of 10 mol/L Na2 CO3 . All experiments were repeated three times. 2.4. Analytical methods Fermentation samples were centrifuged 9400 × g and 4 ◦ C for 10 min in a microcentrifuge. The supernatant was filtered through a 0.22-m syringe filter and stored chilled for HPLC analysis. The concentrations of organics were measured by HPLC (Chromeleon server monitor, UVD 170U detector, P680 pump, Dionex, USA) equipped with an ion-exchange column (PrevailTM organic acid column, Grace, USA). The column was maintained at 48 ◦ C, and KH2 PO4 (25 mmol/L; adjusted to pH 2.5 with H3 PO4 ) was used as the mobile phase at a flow-rate of 1 mL/min. Sugar concentrations were determined with the HPLC system described above but using a Series 3000 refractive index (RI) detector (Perkin-Elmer), a guard column, and an ion-exchange column (Aminex HPX87-P, BioRad) as described previously [9]. Dry cell weight (DCW) was obtained as described [16]. An optical density at 660 nm (OD660 ) of 1.0 represented a dry weight of 567 mg/L. 2.5. Enzyme assays For measurement of intracellular enzyme activity, cells were harvested by centrifugation at 9400 × g for 10 min at 4 ◦ C and washed twice with cold Tris–HCl (100 mmol/L; pH 7.5). The washed cells were suspended in the same buffer containing EDTA (0.1 mmol/L) and sonicated on ice for 90 cycles (a working period of 3 s in a 10 s interval for each cycle) at a power output of 200 W with an ultrasonic disruptor (GA92-IID, ShangJia Biotechnology Co., WuXi, China). The cell debris was removed by centrifugation at 13,400 × g for 20 min at 4 ◦ C, and the supernatant was used for the measurement of enzyme activity. Enzyme activity was measured in a temperature-controlled spectrophotometer (UV-2100, Unico, USA). The activity of phosphoenolpyruvate carboxykinase (PCK), malate dehydrogenase (MDH), fumarate dehydrogenase (FRD) and alcohol dehydrogenase (ADH) was measured as described [17,18]. Enzyme activity is expressed in units (U) and 1 U is defined as the amount of enzyme necessary to catalyse the conversion of 1 mol of substrate/min into specific products. Specific activity is expressed as U/mg of protein.
2.3. Cultivation conditions 2.6. Protein assays Fermentation was done in anaerobic bottles as described [16]. Batch fermentation was done at 37 ◦ C with an initial broth volume of 1.5 L in a 3 L fermentor (Bioflo 110, New Brunswick Scientific, Edison, NJ, USA). The culture medium was used for
Protein contents were determined by the Bradford method [19], using bovine serum albumin (Sigma Chemical Co.) as the standard.
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2.7. NADH/NAD+ assays The intracellular concentrations of NADH and NAD+ were measured as described [20]. 2.8. Measurement of redox potential Redox potential was measured with a METTLER TOLEDO pH 2100e (Mettler Toledo, Switzerland) and a redox potential electrode (P+ 4805-SC-DPAS-K8S/225 Redox, Mettler Toledo, Switzerland).
3. Results and discussion 3.1. Carbon source experiments Experiments were done with A. succinogenes NJ113 under anaerobic conditions in a 3 L fermentor to investigate the effect of
different carbon sources (sorbitol, glucose and gluconate) on cell growth and the distribution of metabolites. The results of these experiments, including the final DCW, the amount of substrate consumed, and the concentration of metabolites produced after 14 h are given in Table 1 and illustrated in Fig. 2. The identity of the carbon source had a significant effect on the production of succinic acid. The maximum DCW using sorbitol as the carbon source was much higher relative to glucose, but cells given glucose as the carbon source grew faster in the exponential phase than those given sorbitol as the carbon source. The inhibition of organic acid may be the reason why cell growth using glucose as the carbon source was suddenly stopped at 6 h [21,22]. The DCW was the lowest for gluconate as the carbon source, and was constant at around 1.7. At a low bacterial density, gluconate was the most slowly consumed carbon source, and the amount of metabolites produced was the smallest. The produc-
Fig. 2. Effect of different carbon sources on cell growth and the distribution of metabolites. The time-course is shown for: (A) carbon source, (B) DCW, (C) succinic acid, (D) acetic acid, (E) ethanol, (F) formic acid. () Sorbitol; () glucose; () gluconate.
J. Li et al. / Process Biochemistry 45 (2010) 980–985 Table 1 Metabolite concentrations using different carbon sources with A. succinogenes NJ113. Sorbitol (−1)a Substrate consumed (g/L) Maximum DCW (g/L) Succinic acid (g/L) Acetic acid (g/L) Formic acid (g/L) Ethanol (g/L) SAc /ACd (concentration) SAc yielde (g/g) SAc productivity (g/(L h))
39.5 5.06 29.8 5.89 2.83 1.03 5.06 0.75 2.13
± ± ± ± ± ± ± ± ±
0.48b 0.55 0.86 0.22 0.23 0.18 0.24 0.02 0.06
Glucose (0) 39.9 4.48 26.4 9.85 5.13 0.85 2.68 0.66 1.89
± ± ± ± ± ± ± ± ±
0.72 0.47 0.67 0.35 0.13 0.16 0.12 0.02 0.05
Gluconate (+1) 15.5 1.81 8.3 4.89 1.63 0.38 1.70 0.54 0.59
± ± ± ± ± ± ± ± ±
0.29 0.14 0.41 0.29 0.15 0.13 0.13 0.03 0.03
a
The value in parentheses represents the oxidation state. Each value is an average of three parallel replicates and is reported as mean ± standard deviation. c Succinic acid. d Acetic acid. e SA yield: g of SA formed/g of glucose utilized. b
tion of succinic acid (29.8 g/L) and ethanol (1.03 g/L) was greatest with sorbitol as the carbon source. In addition, the level of acetic acid and formic acid were decreased significantly (40% and 45% respectively) in sorbitol cultures relative to glucose, increasing the succinic acid to acetic acid ratio (SA/AC) to 5.06. Some reports suggest that acetic acid accumulation inhibits cell growth [23–25]; therefore, the sharp decrease in the level of acetic acid was of more benefit to cell growth in the late stage for the sorbitol cultures relative to the glucose cultures. The yield (0.75 g/g) and productivity (2.13 g/(L h)) of succinic acid in the sorbitol cultures were by far the greatest. 3.2. Influence of carbon source on redox potential A carbon source is the raw material of cell composition, and the energy necessary for cell growth. Biosynthetic transformations involving redox reactions offer potential advantages for the production of bulk chemicals compared to conventional chemical processes [2]. Different carbon sources have different oxidation states. The internal redox state, reflected by the NADH/NAD+ ratio, is influenced by environmental factors such as the oxidation state of the substrate [26]. Fig. 3 shows the changes of redox potential of three different carbon sources during fermentation. As shown in Fig. 3, the redox potential of the fermentation system decreased steeply in the first 2 h and then gradually as the fermentation proceeded. Although this is an anaerobe fermenta-
Fig. 3. Redox potential of three different carbon sources during fermentation. () Sorbitol; () glucose; () gluconate.
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tion process, at the beginning there is still trace oxygen in the medium. After inoculation, the trace oxygen was gradually consumed by A. succinogenes NJ113 which is a facultative anaerobe, that may be the reason why the redox potential of the fermentation system decreased steeply during the first 2 h. The redox potential of the glucose culture was much higher relative to that of the sorbitol culture, but lower relative to that of gluconate culture. In sharp contrast, sorbitol as a carbon source induced a fermentation behaviour by achieving a more reduced intracellular environment, and using gluconate as a carbon source induced a fermentation behaviour by achieving a more oxidized intracellular environment. In general, more reduced substrates yield a higher proportion of reduced metabolites, while more oxidized substrates yield a higher proportion of more oxidized metabolites compared to those produced with glucose as the carbon source [27]. Therefore, when sugars similar to glucose, but differing in their oxidation states, were used as substrates, the cell can redistribute its fermentation pattern to achieve a redox balance. The use of a more reduced carbon source (sorbitol) led to a significant increase in the yield of succinic acid, a reduced product, accompanied by a significant decrease in the yield of acetic acid, a more oxidized product that does not require NADH for its synthesis. In contrast, the use of gluconate as the carbon source resulted in a significant increase in the yield of acetic acid and a decrease in the yield of succinic acid. In summary, using a more reduced carbon source (sorbitol) provides a more reduced environment, as evidenced by the higher SA/AC ratio (Table 1). 3.3. Influence of carbon source on the NADH/NAD+ ratio Succinate production from glucose involves a single NADHgenerating step, from glucose-6-phosphate to PEP, whereby 1 mole of glucose generates 2 moles of NADH. In the next pathway, however, there are several NADH consumption steps. In the succinate-producing C4 pathway, a total of 4 moles of NADH is required for the steps from oxaloacetate to malate and from fumarate to succinate. In the formate-, acetate- and ethanolproducing C3 pathways, the producing of ethanol and of lactate require 2 moles of NADH each (Fig. 1). In the whole pathway in A. succinogenes, the requirement for NADH is greater than its generation, so the reducing power is insufficient. Fig. 1 shows the glycolysis metabolic pathway of A. succinogenes NJ113 with glucose, sorbitol or gluconate as the carbon source. As the figure shown, more reducing equivalents in the form of NADH are produced from sorbitol compared with glucose. Gluconate produces less NADH than that from glucose because every molecule goes directly to phosphoenolpyruvate (PEP), which skips the NADH-producing step in glycolysis. This provides a simple way of testing the effect of manipulating the NADH/NAD+ ratio on the metabolic patterns of A. succinogenes NJ113 under anaerobic conditions and on the production of succinic acid, which requires NADH. Table 2 presents the concentrations of NADH and NAD+ using different carbon sources with A. succinogenes NJ113. Over the time-course of the experiment, 0–4 h represents the early exponential phase, 4–8 h represents the late exponential phase and 8–12 h represents the stationary phase of cell growth. After 4 h the NADH/NAD+ ratio using sorbitol or gluconate as the carbon source was lower relative to glucose. After 8 h DCW was higher for the sorbitol cultures relative to glucose, and the NADH/NAD+ ratio increased from 0.94 for glucose to 2.71 for sorbitol, which is more reduced and can therefore produce more reducing equivalents in the form of NADH. In a similar way, the NADH/NAD+ ratio decreased to 0.31 for gluconate. These changes in NADH/NAD+ ratio affected the distribution of metabolic fluxes in A. succinogenes NJ113, which is reflected in the SA/AC ratio. This ratio was increased from 2.68 with glucose to 5.06 with sorbitol and was decreased to 1.70 with
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Table 2 Concentration of NADH and NAD+ , and the NADH/NAD+ ratio with A. succinogenes NJ113 using different carbon sources. Substrate
Time (h)
NADH (mmol/g DCW)
NAD+ (mmol/g DCW)
NADH/NAD+
Sorbitol
4 8 12
0.40 ± 0.05 0.38 ± 0.03 0.21 ± 0.02
0.23 ± 0.04 0.14 ± 0.01 0.62 ± 0.01
1.74 ± 0.37 2.71 ± 0.29 0.34 ± 0.03
Glucose
4 8 12
0.52 ± 0.05 0.31 ± 0.03 0.27 ± 0.02
0.28 ± 0.04 0.33 ± 0.01 0.55 ± 0.01
1.86 ± 0.32 0.94 ± 0.10 0.49 ± 0.04
Gluconate
4 8 12
0.29 ± 0.05 0.20 ± 0.03 0.32 ± 0.02
0.33 ± 0.04 0.64 ± 0.01 0.71 ± 0.01
0.88 ± 0.19 0.31 ± 0.05 0.45 ± 0.03
Each value is an average of three parallel replicates and is reported as mean ± standard deviation.
gluconate, suggesting that the cell adjusts its partitioning at the PEP step by changing the ratio of succinic acid (consumes 4 NADH) to acetic acid (consumes no NADH) ratio to achieve a redox balance. Therefore, a change in the SA/AC ratio can be used as an indirect indicator of a change in the NADH/NAD+ ratio. These results demonstrate that it is possible to manipulate the availability of intracellular NADH through the use of carbon sources with different oxidation states. The use of gluconate, a more oxidized carbon source, decreased intracellular NADH availability and resulted in a more oxidized environment. In contrast, the use of sorbitol, a more reduced carbon source, increased intracellular NADH availability and resulted in a more reduced environment. The cells utilize this extra NADH to reduce metabolic intermediates leading to the formation of fermentation products in order to achieve a redox balance. 3.4. Influence of carbon source on enzyme activity PCK is a key enzyme in the path to succinic acid formation in A. succinogenes NJ113. The activity of PCK determines the fraction of the carbon from PEP that goes to oxaloacetate. Malate dehydrogenase (MDH), fumarate dehydrogenase (FRD), and alcohol dehydrogenase (ADH), enzymes that involve reduction, were also investigated (Table 3). The results demonstrated that enzyme activity is influenced by carbon sources with different oxidation states. The use of sorbitol resulted in a more reducing intracellular environment, as reflected by a significant increase in both the NADH/NAD+ and the SA/AC ratios, which was beneficial for cell growth. The level of activity among the four key enzymes was the highest with sorbitol as the carbon source. The use of gluconate resulted in a more oxidized intracellular environment, as reflected by a significant decrease in both the NADH/NAD+ and the SA/AC ratios. The level of activity among four key enzymes was lowest with gluconate as the carbon source. It was found that a highly reducing substrate, such as sorbitol, may result in an increased content of ethanol content, because NADH was also consumed during the metabolic pathway from PEP to ethanol, and the increased of availability of NADH could increase the metabolic flux of ethanol synthesis. Therefore, the reduced the
Table 3 Effect of carbon source on key enzyme activity. Substrate
Sorbitol Glucose Gluconate
Specific activity (U/mg protein) PCK
MDH
FRD
ADH
2.87 ± 0.06 2.35 ± 0.08 1.25 ± 0.05
1.35 ± 0.04 0.86 ± 0.05 0.41 ± 0.04
0.63 ± 0.05 0.34 ± 0.02 0.18 ± 0.01
0.23 ± 0.02 0.15 ± 0.01 0.13 ± 0.01
Each value is an average of three parallel replicates and is reported as mean ± standard deviation.
synthesis of ethanol and the increased availability of NADH at the same time needs further study. 4. Conclusions The results of this work demonstrated that the production of succinic acid by anaerobic fermentation by A. succinogenes NJ113 could be enhanced by the choice of reductive carbon sources. We showed here that carbon sources with different oxidation states can be used to manipulate the NADH/NAD+ ratio. Further, we have shown that this manipulation has a direct effect on the distribution of metabolic fluxes in A. succinogenes NJ113, especially in those requiring NADH, such as succinic acid and ethanol. So, the method described here can be used as a simple way of determining whether a particular pathway that requires NADH is limited by the availability of this cofactor. This strategy of using carbon sources with different oxidation states can regulate the NADH/NAD+ ratio or the availability of NADH in the fermentation process, thereby change the key enzymes’ activity in the metabolic pathway, and redistribute metabolites to increase succinic acid production and reduce by-products generation. Therefore, there is a significant perspective of substituting glucose with further reduced form in economic sense. Acknowledgements This study was supported by grant 2009CB724701 from “973” Program of China, grant no. 20606017 from National Natural Science Foundation of China, State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of Technology, China, Science and Technology Achievement Transformation Project of Jiangsu province, and Qing Lan Project of Jiangsu province. References [1] Berrios-Rivera SJ, Sanchez AM, Bennett GN, San KY. Effect of different levels of NADH availability on metabolite distribution in Escherichia coli fermentation in minimal and complex media. Appl Microbiol Biotechnol 2004;65:426–32. [2] Berrios-Rivera SJ, Bennett GN, San KY. The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures. Metab Eng 2002;4:230–7. [3] Berrios-Rivera SJ, San KY, Bennett GN. The effect of carbon sources and lactate dehydrogenase deletion on 1,2-propanediol production in Escherichia coli. J Ind Microbiol Biotechnol 2003;30:34–40. [4] San KY, Bennett GN, Berrios-Rivera SJ, Vadali RV, Yang Y-T, Horton E, et al. Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli. Metab Eng 2002;4:182–92. [5] Lu SY, Eiteman MA, Altman E. Effect of flue gas components on succinate production and CO2 fixation by metabolically engineered Escherichia coli. World J Microbiol Biotechnol 2009; doi:10.1007/s11274-009r-r0185-1. [6] Song H, Lee SY. Production of succinic acid by bacterial fermentation. Enzyme Microb Technol 2006;39:352–61. [7] McKinlay JB, Vieille C, Zeikus JG. Prospects for a bio-based succinate industry. Appl Microbiol Biotechnol 2007;76:727–40.
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