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Succinic acid production from sucrose by Actinobacillus succinogenes NJ113
Bioresource Technology 153 (2014) 327–332
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Succinic acid production from sucrose by Actinobacillus succinogenes NJ113 Min Jiang 1, Wenyu Dai 1, Yonglan Xi, Mingke Wu, Xiangping Kong, Jiangfeng Ma ⇑, Min Zhang, Kequan Chen, Ping Wei State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Puzhu South Road 30#, Nanjing 211816, PR China
h i g h l i g h t s A. succinogenes produced high concentration of succinic acid in high productivity. Sucrose is transported and utilized via a sucrose PTS by A. succinogenes NJ113. Succinate productivity increased by 35% via a fed-batch culture. A. succinogenes NJ113 could tolerate more formic acid in medium with sucrose. High succinic acid productivity made the process more economic.
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Article history: Received 25 September 2013 Received in revised form 20 November 2013 Accepted 23 November 2013 Available online 1 December 2013 Keywords: Succinic acid Sucrose Utilization pathways Fed-batch culture A. succinogenes NJ113
a b s t r a c t In this study, sucrose, a reproducible disaccharide extracted from plants, was used as the carbon source for the production of succinic acid by Actinobacillus succinogenes NJ113. During serum bottle fermentation, the succinic acid concentration reached 57.1 g/L with a yield of 71.5%. Further analysis of the sucrose utilization pathways revealed that sucrose was transported and utilized via a sucrose phosphotransferase system, sucrose-6-phosphate hydrolase, and a fructose PTS. Compared to glucose utilization in single pathway, more pathways of A. succinogenes NJ113 are dependent on sucrose utilization. By changing the control strategy in a fed-batch culture to alleviate sucrose inhibition, 60.5 g/L of succinic acid was accumulated with a yield of 82.9%, and the productivity increased by 35.2%, reaching 2.16 g/L/h. Thus utilization of sucrose has considerable potential economics and environmental meaning. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Succinic acid is an important precursor for many specialty chemicals in the food, agriculture, pharmaceutical, green solvent and biodegradable plastics industries (Song and Lee, 2006). The US Department of Energy has identified it as one of twelve organic acids that are key chemical building blocks (Werpy and Petersen, 2004). Succinic acid is mainly produced from butane through maleic anhydride by a chemical process. Because of petroleum shortages and the increasing demand for succinic acid, biological production of succinic acid, an environment-friendly method, is becoming a viable alternative. A great many strains have been investigated for producing succinic acid. Previous studies mainly examined Anaerobiospirillum ⇑ Corresponding author. Tel./fax: +86 2558139927. 1
E-mail address: [email protected] (J. Ma). These authors equally contributed to this study.
0960-8524/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.11.062
succiniciproducens (Davis et al., 1976; Lee et al., 2000a), Actinobacillus succinogenes (Guettler et al., 1999; McKinlay et al., 2010), Mannheimia succiniciproducens (Lee et al., 2002b; Oh et al., 2008), Corynebacterium glutamicum (Okino et al., 2008) and recombinant Escherichia coli strains (Andersson et al., 2007; Ma et al., 2010). Among the succinic acid-producing strains, A. succinogenes has been considered to be one of the most promising microorganisms for industrial succinic acid production because of its tolerance towards high concentrations of organic acids and ability to produce a relatively large quantity of succinic acid (Guettler et al., 1999). A. succinogenes is a capnophilic anaerobic rumen bacterium, and it can utilize abundant carbon sources, such as xylose, glucose, fructose, mannitol, sorbitol, cellobiose, and sucrose under anaerobic conditions (Adsul et al., 2011). Among all of the carbon sources A. succinogenes can utilize, sucrose is the most abundant disaccharide on earth and is mainly extracted from sugarcane and sugarbeet (Lee et al., 2010a). Sucrose and sucrose-rich sugarcane molasses are becoming an attractive
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raw material for cost-effective bio-based production (Van der Werf et al., 1997). It is the most important carbon source in the industrial production of yeast S. cerevisiae; more than half of ethanol production relies on sucrose (Reid and Abratt, 2005). More citric acid was produced from sucrose than from glucose on citric acid production by Aspergillus niger (Hossain et al., 1984). Sucrose could also serve as a potential raw material for the efficient production of lactic acid by R. oryzae GY18 (Guo et al., 2010). Sugarcane molasses is sugar industry residuals, and has been reported as an important sucrose resource of microorganism. Sugarcane molasses has been used to produce succinic acid by A. succinogenes CGMCC1593 (Liu et al., 2008). In addition, sucrose has beneficial effects in protecting cells. It has been shown to protect the native protein structure from degradation by heat (Kilimann et al., 2006), high pressure (Molina-Hoppne et al., 2004), and dehydration (Prestrelski et al., 1993). Therefore, sucrose is likely to be an appropriate carbon source for bio-product production. Liu et al. (2008) showed that A. succinogenes CGMCC1593 produced 40.3 g/L succinic acid with a productivity of 0.66 g/L/h using sucrose. Wang et al. (2011) also reported that one metabolically engineered E. coli strain utilized sucrose to produce 32.7 g/L succinic acid with a productivity of 0.34 g/L/h. Chan et al. (2012) reported metabolically engineered E. coli KJ122-pKJSUC-24T produced 47 g/L succinic acid using sucrose, at a productivity of 0.98 g/L/h. All of the above reported succinic acid productivities were less than satisfying. It is essential to enhance the productivity to make this approach suitable for industrial production. There are substantial differences between the various strains in their abilities to use sucrose, and the pathways by which sucrose is being utilized are also different. Some E. coli strains were constructed by cloning sucrose-utilizing genes to E. coli, and these E. coli strains could metabolize sucrose to glucose and fructose extracellularly (Lee et al., 2010a; Wang et al., 2011; Chan et al., 2012). Compared to metabolically engineered strains, some wild strains can use sucrose in different pathways. Lee et al. (2010b) studied the way M. succiniciproducens MBEL55E uses sucrose in detail via gene deletion, and demonstrated that it used a sucrose PTS, sucrose 6-phosphate hydrolase, and a fructose PTS to utilize sucrose. However, the pathways involving substrate utilization by A. succinogenes have not been studied intensively, and most studies related to metabolism pathways were only able to infer. Pathways dependent on sucrose utilization by A. succinogenes have never been reported. In the present article, the utilization of sucrose by A. succinogenes NJ113 was investigated. Furthermore, the sucrose utilization pathways of A. succinogenes NJ113 were analyzed to explain how succinic acid was produced from sucrose. Finally, a fed-batch culture strategy was adopted instead of batch culture to enhance the succinic acid productivity.
10.0 g NaHCO3, 8.5 g NaH2PO4H2O, and 15.5 g K2HPO4 and heat sterilized at 121 °C for 15 min. Anaerobic bottles were inoculated with 1 mL of a 70 °C glycerol stock culture and incubated at 37 °C. For anaerobic bottle cultivation, exponentially growing cells were inoculated into 100 mL sealed anaerobic bottles filled with 30 mL of fermentation medium containing the following (per liter): 3.0 g KH2PO4, 0.2 g MgCl26H2O, 0.2 g CaCl2, 1.0 g NaCl, and 10 g yeast extract. Carbon source (sucrose) was autoclaved separately. The pH of the medium was maintained by the addition equal quality carbon source of MgCO3. The anaerobic bottle cultivation was carried out in a rotary shaker at 37 °C and 180 rpm. Batch fermentation was conducted in a 3 L fermentor (Bioflo 110, USA) with an initial broth volume of 1.5 L, which contained (per liter): 3.0 g KH2PO4, 0.2 g MgCl26H2O, 0.2 g CaCl2, 1.0 g NaCl, 5.0 g corn steep liquor, and 10.0 g yeast extract. The carbon sources (sucrose) was separately sterilized at 121 °C for 15 min and added to the medium at a final concentration of 100 g/L. Carbon dioxide (CO2) was bubbled through the medium for 30 min to remove oxygen before inoculation. All fermentation processes were carried out at an agitation speed of 200 rpm and CO2 flow rate of 0.5 L/min. The pH was controlled at 6.8 by automatically adding 2.0 mol/L of Na2CO3. Fed-batch fermentation was carried out under the same conditions as the batch fermentation with an initial sucrose concentration of 50 g/L, as cell growth would not be inhibited when the sucrose concentration was lower than 50 g/L. When the sucrose in medium was lower than 20 g/L, a 600 g/L sucrose solution was fed to keep sucrose concentration between 20–50 g/L. Sucrose was added by a peristaltic pump at a flow rate of 540 ml/h. A total of 100 g/L sucrose was fed during the entire process. All experiments were carried out in triplicate. 2.3. Analytical methods The dry cell weight (DCW) was computed from a curve relating optical density at 660 nm (OD660) to dry weight. An OD660 of 1.0 represented 520 mg of dry weight per liter. Organic acid were analyzed by high-performance liquid chromatography (Chromeleon server monitor, UVD 170 U detector, P680 pump, Dionex, USA). To determine the fermentation products, an ion exchange chromatographic column (Prevail organic acid column, Grace, USA) was used, and 25 mM KH2PO4 (adjusted to pH 2.5 by H3PO4) was used as the mobile phase with a flow rate of 1 mL/min. Sucrose were determined by an Hypersil NH2 column (5 lm 4.6250 mm, Dalian Elite Analytical Instruments Co., Ltd., China) and a refractive index detector, RI101 (Shodex, USA). The yields of organic acid, including succinic acid and acetic acid, defined as the amount of final organic acid produced from 1 g glucose consumption, expressed as a percentage.
2. Methods 2.4. Kinase activity assays 2.1. Chemicals Yeast extracts were purchased from Oxoid Ltd. (Basingstoke, Hampshire, England). Other chemicals were of reagent grade and were from either Sinochem (Shanghai, P.R. China) or Fluka Chemical (Buchs, Switzerland) unless otherwise specified. CO2 was obtained from Nanjing Special Gases Factory (Nanjing, P.R. China). 2.2. Microorganism and growth conditions A. succinogenes NJ113 (China General Microbiological Culture Collection Center, CGMCC No. 1716) was used in all experiments. Cells were grown in 50 mL medium sealed in anaerobic bottles with the volume of 100 mL. The medium for inoculum cultures was composed of (per liter): 10.0 g glucose, 5.0 g yeast extract,
For preparation of cell extracts, cells were harvested by centrifugation (8000 g for 10 min at 4 °C) in the mid-exponential phase and washed twice with 100 mM Tris–HCl (pH 7.0) containing 20 mM KCl, 5 mM MnSO4, 2 mM DTT, and 0.1 mM EDTA. The washed cell pellet was resuspended in the same buffer, and was placed in an ice bath for ultrasonication. After ultrasonication, the suspension was centrifuged for 20 min at 10,000g and 4 °C to remove cell debris, and then the supernatant was stored at 80 °C until required. The fructokinase activity were measured by spectrometric method which couples the formation of F-6-P to the reduction of NADP+ via glucose phosphate isomerase (PGI; CAS: 9001-41-6, S. cerevisiae, Sigma) and glucose-6-phosphate dehydrogenase (Caescu et al., 2004).
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One unit (U) of enzyme activity was defined as the amount of enzyme necessary to catalyze the conversion of 1 nmol of substrate per minute into specific products. Specific activity was expressed as U/mg of protein. 2.5. Phosphoenolpyruvate phosphotransferase system (PEP-PTS) activity assay The PEP-PTS activity was estimated by a modification of the assay method (Jiang et al., 2013). The cells were harvested, washed twice as described above. The washed cell pellet was resuspended in 2 mM potassium phosphate buffer (pH 7.0) containing 150 mM KCl and 5 mM MgCl2. Toluene–ethanol (1:9, v/v) was added to the cell suspension at a final concentration of 1% by micro-pipette, and agitated vigorously for 60 s. After centrifugation at 1000g for 6 min at 4 °C, the supernate was removed and the cells were suspended in the same buffer. Equivalent to 30–100 lg dry weight of cells was added to the reaction mixture containing 5 mM phosphoenolpyruvate, 0.5 mM NADH, 10 U lactate dehydrogenase (EC1.1.1.27, rabbit muscle, Sigma), 60 mM MgCl2, 15 mM sucrose or fructose and sufficient buffer to give a final volume of 0.3 ml at 37 °C. The decrease of NADH was monitored by spectroscopy at 340 nm. Net PEP-PTS activity was expressed as nanomoles of pyruvate formed per milligram (dry weight) of cells per minute. 2.6. Protein assay Protein contents were determined by Bradford (Bradford, 1976), using bovine serum albumin (Sigma Chemical Co.) as a standard. 3. Results and discussion 3.1. Succinic acid fermentation of sucrose by A. succinogenes NJ113 Glucose utilization by A. succinogenes NJ113 has been studied in some detail, but no studies on sucrose utilization have been reported previously. A. succinogenes NJ113 was able to use sucrose as a carbon source in this study; therefore, it was necessary to investigate how it does so. Concentration gradient experiments were performed in serum bottles as described before. As shown in Table 1, the succinic acid concentration increased as the initial sucrose concentration increased. When the sucrose concentration was 100 g/L, the succinic acid concentration reached its highest value of 57.1 g/L with a yield of 71.5%. When the sucrose concentration was higher than 100 g/L, the succinic acid concentration decreased. It meant that higher concentrations of sucrose repressed the productive capacity of succinic acid. When 50 g/L of sucrose was used, a maximum cell OD of 13.41 was obtained, and then the OD decreased when the sucrose concentration was higher than 50 g/L. Osmotic pressure seems to be the main inhibitory factor for
succinic acid production and cell growth (Bretz and Kabasci, 2012). During the entire process, the acetic acid concentration was relatively stable, ranging from 5 to 7.5 g/L; however, the formic acid concentration increased as the initial sucrose increased and reached a maximum of 12.3 g/L. Table 1 shows the most suitable concentration of sucrose utilized by A. succinogenes NJ113 was 100 g/L. Subsequently, a batch culture was carried out in a 3-L fermentor to further investigate growth conditions utilizing sucrose. In Fig. 1, a final concentration of 57.5 g/L succinic acid accumulated within 36 h, with a yield of 77% and a productivity of 1.60 g/L/h. Both the production and productivity were higher than those reported by Xi et al. (2011), 51.6 and 1.52 g/L/h, respectively, using glucose as the carbon source with A. succinogenes NJ113. In addition, the succinic acid productivity and production were higher than that reported by Liu et al. (2008), Wang et al. (2011) and Chan et al. (2012) using sucrose as the carbon source. The maximum cell OD was 11.6 at 10 h, and it then decreased until the end of the fermentation. When sucrose was fed as the sole carbon source, A. succinogenes NJ113 grew rapidly, coupled with a fast formic acid and succinic acid productivities in the first 10 h. However, cell would be inhibited in high concentration of succinic acid and formic acid. In previous study with glucose as carbon source by our lab, A. succinogenes NJ113 showed that cells decreased when succinic acid was accumulated to 20 g/L. Cells produced succinic acid too fast with sucrose as carbon source, then cells biomass was inhibited in short time. During the process, formic acid was produced rapidly, increasing from 12.6 g/L at 6 h to 36.5 g/L at 8 h, and then decreasing. Formic acid was harmful for A. succinogenes cells (Li et al., 2010). However, it seemed that A. succinogenes could tolerate higher concentrations of formic acid in a medium containing sucrose according to the data got from serum culture and batch culture, as sucrose can protect cells compared to glucose. And when formic acid accumulated to a harmful concentration, it was metabolized to CO2 to supply NADH according to the A. succinogenes central metabolic network map (McKinlay et al., 2010). 3.2. Sucrose transport and metabolism pathways in A. succinogenes NJ113 Sucrose is composed of a glucose unit and a fructose unit connected by a glycoside linkage. Some bacteria can break down extracellular sucrose into glucose and fructose by cleaving the glycoside linkage using certain enzymes, such as the catabolic enzymes, the sucrose hydrolases and phosphorylases, and the biosynthetic glycosyl-nucleotide glycosyltransferases (Kitaoka and Hayashi, 2002). Intracellular sucrose metabolism can be categorized into three types: a PTS system and two non-PTS systems (Lee et al., 2010b). The PTS system includes the PEP-dependent sucrose-specific PTS, sucrose 6-phosphate hydrolase, and fructokinase. One non-PTS system includes sucrose permease and sucrose
Table 1 Sucrose utilization by A. succinogenes NJ113 in serum bottles.a Initial sucrose (g/L)
OD660
Succinic acid (g/L)
Formic acid (g/L)
Acetic acid (g/L)
Residual sucrose (g/L)
Succinic acid yield (%)
40 50 60 70 80 90 100 110 120
11.70 ± 0.57 13.41 ± 0.62 12.05 ± 0.51 12.46 ± 0.81 11.60 ± 0.31 10.97 ± 0.14 9.31 ± 0.59 7.53 ± 0.87 6.80 ± 0.95
26.68 ± 0.32 32.49 ± 0.59 37.53 ± 0.48 41.00 ± 0.88 46.30 ± 0.42 47.52 ± 1.05 57.13 ± 0.13 55.39 ± 0.79 44.49 ± 0.91
5.47 ± 0.11 5.94 ± 0.09 7.30 ± 0.13 6.52 ± 0.06 9.48 ± 0.61 9.14 ± 0.73 11.58 ± 1.31 11.03 ± 0.95 12.35 ± 0.87
6.49 ± 0.13 7.57 ± 0.22 7.17 ± 0.17 7.04 ± 0.24 7.40 ± 0.31 6.12 ± 0.58 5.98 ± 1.15 5.51 ± 1.06 6.40 ± 0.95
1.66 ± 0.15 3.15 ± 0.18 6.66 ± 0.05 9.04 ± 0.18 12.09 ± 0.61 18.39 ± 0.31 20.06 ± 0.7 28.69 ± 1.51 48.81 ± 1.71
69.6 ± 0.8 69.4 ± 0.2 70.4 ± 0.9 67.7 ± 0.3 68.2 ± 0.3 69.2 ± 0.8 71.3 ± 1.0 68.1 ± 0.9 62.5 ± 1.3
Each value is an average of three parallel replicates and is reported as mean ± standard deviation. a Cells were grown in anaerobic bottles for 48 h.
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100
OD660 Succinic Acid Formic Acid Acetic Acid Sucrose
90
70
12 10
60
8
50 6
40 30
OD660
Concentration g/L
80
14
4
20 2 10 0
0
0
4
8
12
16
20
24
28
32
36
40
44
Time(h) Fig. 1. Batch culture with 100 g/L of sucrose by A. succinogenes NJ113. The succinic acid concentration reached its highest value of 57.5 g/L in 36 h with a yield of 77% and a productivity of 1.60 g/L/h.
Table 2 PTS activities in A. succinogenes NJ113 grown on different carbon sources. Carbon sourcea
Sucrose PEP-PTS activityb
Glucose PEP-PTS activityb
Sucrose Glucose
60.3 ± 1.9 53.4 ± 0.8
9.3 ± 1.9 11.4 ± 0.8
Each value is an average of three parallel replicates and is reported as mean ± standard deviation. a Means cells grown in medium in the presence of the indicated carbon sources. b Activity unit defined as nmol/min per mg dry wt. of cells.
14
60
12 OD660 Succinic acid Formic acid Acetic acid Sucrose
40
30
10 8 6
OD660
Concentration g/L
50
20 4 10
2 0
0 0
4
8
12
16
20
24
28
32
36
40
44
48
to glucose and fructose extracellularly. We also found some genetic information, relating to Asuc 0914 and Asuc 1829, about sucrose PTS in A. succinogenes 130Z at the JGI Integrated Microbial Genomes (IMG) website (IMG Home). Asuc 0914 encodes the PTS system, sucrose-specific IIBC subunit, and Asuc 1829 encodes sucrose-6-phosphate hydrolase. Reports showed that a complete PTS consists of enzyme I, HPr, enzyme IIA, and enzyme IIBC. Enzyme I and HPr are common to all PTS carbohydrates (Deutscher et al., 2006). The EIIA received the phosphoryl group from PHPr and transferred phosphoryl group to EIIBC domain, however, the EIIA domain information was not found in A. succinogenes. In M. succiniciproducens MBEL55E, it seems that the EIIA domain for glucose PTS is used for sucrose as well (Lee et al., 2010b). A similar condition may exist in A. succinogenes, so that a complete sucrose PTS system may exist in A. succinogenes NJ113. This would mean that A. succinogenes NJ113 is able to utilize sucrose via sucrose PTS. To reveal the sucrose utilization pathways in A. succinogenes NJ113, some sucrose utilization associated enzymes were detected. The data listed in Table 2 were obtained from cells grown in medium in the presence of the indicated carbon sources. Data showed cells grown in sucrose got a sucrose PTS activity of 60.3 ± 1.9 nmol/min per mg dry wt. of cells. They proved that sucrose PTS activity exists in A. succinogenes NJ113. As mentioned above, the PTS system includes the sucrose-PTS, and sucrose 6-phosphate hydrolase, which means if sucrose is transported into cells via sucrose PTS pathway, it is phosphorylated to sucrose-6phosphate and then degraded to glucose-6-phosphate and fructose by sucrose 6-phosphate hydrolase. Glucose-6-phosphate would be metabolized via the EMP pathway immediately, while fructose would be utilized via the fructokinase pathway or fructose PTS pathway. Hence, fructose PTS activity and fructokinase activity were detected and compared. Both fructose PTS activity and fructokinase activity existed in A. succinogenes NJ113 cells grown on sucrose (Table 3), however, the fructokinase activity was negligible. This indicated that, after sucrose-6-phosphate was degraded to fructose, fructose PTS participated in fructose utilization. It seems clear that A. succinogenes NJ113 uses a sucrose PTS, sucrose-6-phosphate hydrolase and a fructose PTS to completely transport and metabolize sucrose (Fig. 3). According to the analysis above, sucrose metabolism contains two branches, EMP and fructose PTS pathways. And data in Table 2 showed sucrose PTS activity was much higher than glucose PTS activity. These two reasons might explain that why sucrose was more efficient in succinic acid production by A. succinogenes NJ113 comparing to glucose utilization.
Time (h) Fig. 2. Fed-batch culture with 100 g/L of sucrose by A. succinogenes NJ113. Cells were grown with an initial sucrose concentration of 50 g/l. After 28 h, the succinic acid production reached the maximum value of 60.44 g/L, with a yield and productivity of 83% and 2.16 g/L/h respectively.
phosphorylase, and the other non-PTS system involves sucrose permease and sucrase (Toth et al., 2000). A. succinogenes NJ113 was able to grow well in medium containing sucrose at a relatively high sucrose uptake rate (Fig. 1). It was worth mentioning that no glucose or fructose was detected during the entire process. This indicated that sucrose may not be cleaved
3.3. Fed-batch culture of succinic acid fermentation using sucrose The above results (Fig. 1) have shown that, in batch culture with 100 g/L of sucrose, succinic acid production was inhibited during the first 6 h by high concentration of the substrate for the low consumption of sucrose. Hence, a fed-batch culture was used to replace the batch culture to enhance the productivity of succinic acid. In fed-batch culture (Fig. 2), succinic acid production was not inhibited in the beginning of the fermentation compared to the batch culture. A maximum OD660 of 11.88 was achieved in 8 h,
Table 3 Fructose PTS and fructokinase activities in A. succinogenes NJ113 grown on different carbon sources. Carbon sourcea
Fructose PTS activity (nmol/min per mg dry wt. of cells)
Fructokinase activity (U/mg protein)
Sucrose Fructose
45.9 ± 1.3 39.7 ± 0.7
4.8 ± 0.9 5.7 ± 0.5
Each value is an average of three parallel replicates and is reported as mean ± standard deviation. a means cells grown in medium in the presence of the indicated carbon sources.
M. Jiang et al. / Bioresource Technology 153 (2014) 327–332 Sucrose
331
Extracellular Space
Suc -PTS
Fru-PTS
PEP PEP Pyc
suc-6-P
Pyc
Intracellular Space
Suc-6-P hydrolase
Fru Fru-1-P Glu-6-P Fructokinase
Fru-6-P Fru-1, 6-BP
PEP
OAA
Mal
Succinic Acid
Fig. 3. Sucrose metabolism pathway by A. succinogenes NJ113. A. succinogenes NJ113 uses a sucrose PTS, sucrose-6-phosphate hydrolase and a fructose PTS to completely transport and metabolize sucrose.
Table 4 Main fermentation parameters of the fed-batch culture and batch culture by A. succinogenes NJ113.
Fermentation timea (h) Succinic acid (g/L) Maximum yield (g/g) Productivity (g/L/h) Sucrose consumption Maximum cell concentration (g/L)
Batch
Fed-batch
Decreased or increased rateb (%)
36 57.5 0.77 1.60 74.4 11.6
28 60.4 0.83 2.16 72.6 11.9
22.2 5 7.8 35 – –
a Means the time is when A. succinogenes NJ113 accumulated the maximum succinic acid concentration. b Means data detected in fed-batch culture decreased ( ) or increased (+) value comparing with in batch culture divided by data in batch culture.
and after operating 28 h the succinic acid production reached the maximum value of 60.5 g/L, with a yield and productivity of 82.9% and 2.16 g/L/h, respectively. It’s the highest productivity reported about A. succinogenes NJ113 fermentation at present. The main fermentation parameters of the fed-batch culture and batch culture were listed in Table 4 for comparison. It can be seen that the succinic acid concentration, yield, sucrose consumption, and maximum cell concentration all increased a little; however the fermentation time decreased by 22.2%, which caused overall productivity to increase by 35.2%. It is an obvious enhancement. The decrease in fermentation time means a decrease in production cost; therefore, fed-batch culture is more advantageous for high sucrose concentration fermentation. In fed-batch fermentation, the maximum formic acid concentration was 19.9 g/L at 6 h; it decreased thereafter. The maximum formic acid concentration was lower than the value, 36.5 g/L, in batch culture. Data in serum fermentation, batch culture and fedbatch culture showed that formic acid concentration changed with sucrose concentration. This indicated that A. succinogenes NJ113 produced higher concentration of formic acid, and may tolerate a higher concentration of formic acid when more sucrose is present in the medium. 4. Conclusion This study has demonstrated the feasibility of utilizing sucrose as a carbon source to produce succinic acid by A. succinogenes NJ113. Through batch fermentation, 57.5 g/L of succinic acid were produced with a yield of 77% and a corresponding productivity of 1.60 g/L/h. Further analysis of the enzyme activity revealed that a sucrose PTS and a fructose PTS contribute to sucrose metabolism
in A. succinogenes NJ113. To alleviate substrate inhibition, fed-batch fermentation was used and succinic acid productivity increased 35.2%, reaching 2.16 g/L/h. It seems A. succinogenes NJ113 could tolerate higher concentration of formic acid when more sucrose is present. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21076105, 21106066), ‘‘973’’ Program of China (Grant No. 2013CB733901), ‘‘863’’ Program of China (No. 2011AA02A203), the State Key Laboratory of Materials-Oriented Chemical Engineering Foundation of Nanjing University of Technology (ZK201001), Key University Science Research Project of Jiangsu Province (11KJA530001), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, ‘‘Qinglan Project’’ of Jiangsu Province and ‘‘The six talent summit’’ of Jiangsu Province Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT 10662). References Adsul, M.G., Singhvi, M.S., Gaikaiwari, S.A., Gokhale, D.V., 2011. Development of biocatalysts for production of commodity chemicals from lignocellulosic biomass. Bioresour. Technol. 102, 4304–4312. Andersson, C., Hodge, D., Berglund, K.A., Rova, U., 2007. Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli. Biotechnol. Prog. 23, 381–388. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Bretz, K., Kabasci, S., 2012. Feed-control development for succinic acid production with Anaerobiospirillum succiniciproducens. Biotechnol. Bioeng. 109, 1187–1192. Caescu, C.I., Vidal, O., Krzewinski, F., Artenie, V., Bouquelet, S., 2004. Bifidobacterium longum requires a fructokinase (Frk; ATP: D-fructose 6-phosphotransferase, EC 2.7.1.4) for fructose catabolism. J. Bacteriol. 186, 6515–6525. Chan, S., Kanchanatawee, S., Jantama, K., 2012. Production of succinic acid from sucrose and sugarcane molasses by metabolically engineered Escherichia coli. Bioresour. Technol. 103, 329–336. Davis, C.P., Cleven, D., Brown, J., 1976. Anaerobiospirillum, a new genus of spiralshaped bacteria. Int. J. Syst. Bateriol. 26, 498–504. Deutscher, J., Francke, C., Postma, P.W., 2006. How phosphotransferase systemrelated protein phosphorylation regulates carbohydrate metabolism in bacteria. Microbiol. Mol. Rev. 70, 939–1031. Guettler, M.V., Rumler, D., Jain, M.K., 1999. Actinobacillus succinogenes sp. nov., a novel succinic-acid-producing strain from the bovine rumen. Int. J. Syst. Bacteriol. 49, 207–216. Guo, Y., Yan, Q.J., Jiang, Z.Q., Teng, C., Wang, X.L., 2010. Efficient production of lactic acid from sucrose and corncob hydrolysate by a newly isolated Rhizopus oryzae GY18. J. Ind. Microbiol. Biotechnol. 37, 1137–1143. Hossain, M., Brooks, J.D., Maddox, I.S., 1984. The effect of the sugar source on citric acid production by Aspergillus niger. Appl. Microbiol. Biotechnol. 19, 393–397. IMG Home. Available from: Jiang, M., Xu, R., Xi, Y.L., Zhang, J.H., Dai, W.Y., Wan, Y.J., Chen, K.Q., Wei, P., 2013. Succinic acid production from cellobiose by Actinobacillus succinogenes. Bioresour. Technol. 135, 469–474.
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Kilimann, K.V., Doster, W., Vogel, R.F., Hartmann, C., Ganzle, M.G., 2006. Protection by sucrose against heat-induced lethal and sublethal injury of Lactococcus lactis: an FT-IR study. Biochim. Biophys. Acta 1764, 1188–1197. Kitaoka, M., Hayashi, K., 2002. Carbohydrate-processing phosphorolytic enzymes. Trends Glycosci. Glyc. 14, 35–50. Lee, P.C., Lee, W.G., Lee, S.Y., Chang, H.N., Chang, Y.K., 2000a. Fermentative production of succinic acid from glucose and corn steep liquor by Anaerobiospirillum succiniproducens. Biotechnol. Bioprocess Eng. 5, 379–381. Lee, P.C., Lee, S.Y., Hong, S.H., Chang, H.N., 2002. Isolation and characterization of a new succinic acid-producing bacterium, Mannheimia succiniciproducens MBEL55E, from bovine rumen. Appl. Microbiol. Biotechnol. 58, 663–668. Lee, J.W., Choi, S., Park, J.H., Vickers, C.E., Nielsen, L.K., Lee, S.Y., 2010a. Development of sucrose-utilizing Escherichia coli K-12 strain by cloning b–fructofuranosidases and its application for L-threonine production. Appl. Microbiol. Biotechnol. 88, 905–913. Lee, J.W., Choi, S., Kim, J.M., Lee, S.Y., 2010b. Mannheimia succiniciproducens phosphotransferase system for sucrose utilization. Appl. Environ. Microbiol. 76, 1699–1703. Li, Q., Wang, D., Wu, Y., Yang, M.H., Li, W.L., Xing, J.M., Su, Z.G., 2010. Kinetic evaluation of products inhibition to succinic acid producers Escherichia coli NZN111, AFP111, BL21, and Actinobacillus succinogenes 130ZT. J. Microbiol. 48, 290–296. Liu, Y.P., Zheng, P., Sun, Z.H., Ni, Y., Dong, J.J., Zhu, L.L., 2008. Economical succinic acid production from cane molasses by Actinobacillus succinogenes. Bioresour. Technol. 99, 1736–1742. Ma, J.F., Jiang, M., Chen Ke, Q., Xu, B., Liu, S.W., Wei, P., Ying, H.J., 2010. Succinic acid production with metabolically engineered E. coli recovered from two-stage fermentation. Biotechnol. Lett. 32, 1413–1418. McKinlay, J.B., Laivenieks, M., Schindler, B.D., McKinlay, A.A., Siddaramappa, S., Challacombe, J.F., Lowry, S.R., Clum, A., Lapidus, A.L., Burkhart, K.B., Harkins, V., Vieille, C., 2010. A genomic perspective on the potential of Actinobacillus succinogenes for industrial succinate production. BMC Genomics 11, 680.
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Succinic acid production from sucrose by Actinobacillus succinogenes NJ113 Min Jiang 1, Wenyu Dai 1, Yonglan Xi, Mingke Wu, Xiangping Kong, Jiangfeng Ma ⇑, Min Zhang, Kequan Chen, Ping Wei State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Puzhu South Road 30#, Nanjing 211816, PR China
h i g h l i g h t s A. succinogenes produced high concentration of succinic acid in high productivity. Sucrose is transported and utilized via a sucrose PTS by A. succinogenes NJ113. Succinate productivity increased by 35% via a fed-batch culture. A. succinogenes NJ113 could tolerate more formic acid in medium with sucrose. High succinic acid productivity made the process more economic.
a r t i c l e
i n f o
Article history: Received 25 September 2013 Received in revised form 20 November 2013 Accepted 23 November 2013 Available online 1 December 2013 Keywords: Succinic acid Sucrose Utilization pathways Fed-batch culture A. succinogenes NJ113
a b s t r a c t In this study, sucrose, a reproducible disaccharide extracted from plants, was used as the carbon source for the production of succinic acid by Actinobacillus succinogenes NJ113. During serum bottle fermentation, the succinic acid concentration reached 57.1 g/L with a yield of 71.5%. Further analysis of the sucrose utilization pathways revealed that sucrose was transported and utilized via a sucrose phosphotransferase system, sucrose-6-phosphate hydrolase, and a fructose PTS. Compared to glucose utilization in single pathway, more pathways of A. succinogenes NJ113 are dependent on sucrose utilization. By changing the control strategy in a fed-batch culture to alleviate sucrose inhibition, 60.5 g/L of succinic acid was accumulated with a yield of 82.9%, and the productivity increased by 35.2%, reaching 2.16 g/L/h. Thus utilization of sucrose has considerable potential economics and environmental meaning. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Succinic acid is an important precursor for many specialty chemicals in the food, agriculture, pharmaceutical, green solvent and biodegradable plastics industries (Song and Lee, 2006). The US Department of Energy has identified it as one of twelve organic acids that are key chemical building blocks (Werpy and Petersen, 2004). Succinic acid is mainly produced from butane through maleic anhydride by a chemical process. Because of petroleum shortages and the increasing demand for succinic acid, biological production of succinic acid, an environment-friendly method, is becoming a viable alternative. A great many strains have been investigated for producing succinic acid. Previous studies mainly examined Anaerobiospirillum ⇑ Corresponding author. Tel./fax: +86 2558139927. 1
E-mail address: [email protected] (J. Ma). These authors equally contributed to this study.
0960-8524/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.11.062
succiniciproducens (Davis et al., 1976; Lee et al., 2000a), Actinobacillus succinogenes (Guettler et al., 1999; McKinlay et al., 2010), Mannheimia succiniciproducens (Lee et al., 2002b; Oh et al., 2008), Corynebacterium glutamicum (Okino et al., 2008) and recombinant Escherichia coli strains (Andersson et al., 2007; Ma et al., 2010). Among the succinic acid-producing strains, A. succinogenes has been considered to be one of the most promising microorganisms for industrial succinic acid production because of its tolerance towards high concentrations of organic acids and ability to produce a relatively large quantity of succinic acid (Guettler et al., 1999). A. succinogenes is a capnophilic anaerobic rumen bacterium, and it can utilize abundant carbon sources, such as xylose, glucose, fructose, mannitol, sorbitol, cellobiose, and sucrose under anaerobic conditions (Adsul et al., 2011). Among all of the carbon sources A. succinogenes can utilize, sucrose is the most abundant disaccharide on earth and is mainly extracted from sugarcane and sugarbeet (Lee et al., 2010a). Sucrose and sucrose-rich sugarcane molasses are becoming an attractive
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raw material for cost-effective bio-based production (Van der Werf et al., 1997). It is the most important carbon source in the industrial production of yeast S. cerevisiae; more than half of ethanol production relies on sucrose (Reid and Abratt, 2005). More citric acid was produced from sucrose than from glucose on citric acid production by Aspergillus niger (Hossain et al., 1984). Sucrose could also serve as a potential raw material for the efficient production of lactic acid by R. oryzae GY18 (Guo et al., 2010). Sugarcane molasses is sugar industry residuals, and has been reported as an important sucrose resource of microorganism. Sugarcane molasses has been used to produce succinic acid by A. succinogenes CGMCC1593 (Liu et al., 2008). In addition, sucrose has beneficial effects in protecting cells. It has been shown to protect the native protein structure from degradation by heat (Kilimann et al., 2006), high pressure (Molina-Hoppne et al., 2004), and dehydration (Prestrelski et al., 1993). Therefore, sucrose is likely to be an appropriate carbon source for bio-product production. Liu et al. (2008) showed that A. succinogenes CGMCC1593 produced 40.3 g/L succinic acid with a productivity of 0.66 g/L/h using sucrose. Wang et al. (2011) also reported that one metabolically engineered E. coli strain utilized sucrose to produce 32.7 g/L succinic acid with a productivity of 0.34 g/L/h. Chan et al. (2012) reported metabolically engineered E. coli KJ122-pKJSUC-24T produced 47 g/L succinic acid using sucrose, at a productivity of 0.98 g/L/h. All of the above reported succinic acid productivities were less than satisfying. It is essential to enhance the productivity to make this approach suitable for industrial production. There are substantial differences between the various strains in their abilities to use sucrose, and the pathways by which sucrose is being utilized are also different. Some E. coli strains were constructed by cloning sucrose-utilizing genes to E. coli, and these E. coli strains could metabolize sucrose to glucose and fructose extracellularly (Lee et al., 2010a; Wang et al., 2011; Chan et al., 2012). Compared to metabolically engineered strains, some wild strains can use sucrose in different pathways. Lee et al. (2010b) studied the way M. succiniciproducens MBEL55E uses sucrose in detail via gene deletion, and demonstrated that it used a sucrose PTS, sucrose 6-phosphate hydrolase, and a fructose PTS to utilize sucrose. However, the pathways involving substrate utilization by A. succinogenes have not been studied intensively, and most studies related to metabolism pathways were only able to infer. Pathways dependent on sucrose utilization by A. succinogenes have never been reported. In the present article, the utilization of sucrose by A. succinogenes NJ113 was investigated. Furthermore, the sucrose utilization pathways of A. succinogenes NJ113 were analyzed to explain how succinic acid was produced from sucrose. Finally, a fed-batch culture strategy was adopted instead of batch culture to enhance the succinic acid productivity.
10.0 g NaHCO3, 8.5 g NaH2PO4H2O, and 15.5 g K2HPO4 and heat sterilized at 121 °C for 15 min. Anaerobic bottles were inoculated with 1 mL of a 70 °C glycerol stock culture and incubated at 37 °C. For anaerobic bottle cultivation, exponentially growing cells were inoculated into 100 mL sealed anaerobic bottles filled with 30 mL of fermentation medium containing the following (per liter): 3.0 g KH2PO4, 0.2 g MgCl26H2O, 0.2 g CaCl2, 1.0 g NaCl, and 10 g yeast extract. Carbon source (sucrose) was autoclaved separately. The pH of the medium was maintained by the addition equal quality carbon source of MgCO3. The anaerobic bottle cultivation was carried out in a rotary shaker at 37 °C and 180 rpm. Batch fermentation was conducted in a 3 L fermentor (Bioflo 110, USA) with an initial broth volume of 1.5 L, which contained (per liter): 3.0 g KH2PO4, 0.2 g MgCl26H2O, 0.2 g CaCl2, 1.0 g NaCl, 5.0 g corn steep liquor, and 10.0 g yeast extract. The carbon sources (sucrose) was separately sterilized at 121 °C for 15 min and added to the medium at a final concentration of 100 g/L. Carbon dioxide (CO2) was bubbled through the medium for 30 min to remove oxygen before inoculation. All fermentation processes were carried out at an agitation speed of 200 rpm and CO2 flow rate of 0.5 L/min. The pH was controlled at 6.8 by automatically adding 2.0 mol/L of Na2CO3. Fed-batch fermentation was carried out under the same conditions as the batch fermentation with an initial sucrose concentration of 50 g/L, as cell growth would not be inhibited when the sucrose concentration was lower than 50 g/L. When the sucrose in medium was lower than 20 g/L, a 600 g/L sucrose solution was fed to keep sucrose concentration between 20–50 g/L. Sucrose was added by a peristaltic pump at a flow rate of 540 ml/h. A total of 100 g/L sucrose was fed during the entire process. All experiments were carried out in triplicate. 2.3. Analytical methods The dry cell weight (DCW) was computed from a curve relating optical density at 660 nm (OD660) to dry weight. An OD660 of 1.0 represented 520 mg of dry weight per liter. Organic acid were analyzed by high-performance liquid chromatography (Chromeleon server monitor, UVD 170 U detector, P680 pump, Dionex, USA). To determine the fermentation products, an ion exchange chromatographic column (Prevail organic acid column, Grace, USA) was used, and 25 mM KH2PO4 (adjusted to pH 2.5 by H3PO4) was used as the mobile phase with a flow rate of 1 mL/min. Sucrose were determined by an Hypersil NH2 column (5 lm 4.6250 mm, Dalian Elite Analytical Instruments Co., Ltd., China) and a refractive index detector, RI101 (Shodex, USA). The yields of organic acid, including succinic acid and acetic acid, defined as the amount of final organic acid produced from 1 g glucose consumption, expressed as a percentage.
2. Methods 2.4. Kinase activity assays 2.1. Chemicals Yeast extracts were purchased from Oxoid Ltd. (Basingstoke, Hampshire, England). Other chemicals were of reagent grade and were from either Sinochem (Shanghai, P.R. China) or Fluka Chemical (Buchs, Switzerland) unless otherwise specified. CO2 was obtained from Nanjing Special Gases Factory (Nanjing, P.R. China). 2.2. Microorganism and growth conditions A. succinogenes NJ113 (China General Microbiological Culture Collection Center, CGMCC No. 1716) was used in all experiments. Cells were grown in 50 mL medium sealed in anaerobic bottles with the volume of 100 mL. The medium for inoculum cultures was composed of (per liter): 10.0 g glucose, 5.0 g yeast extract,
For preparation of cell extracts, cells were harvested by centrifugation (8000 g for 10 min at 4 °C) in the mid-exponential phase and washed twice with 100 mM Tris–HCl (pH 7.0) containing 20 mM KCl, 5 mM MnSO4, 2 mM DTT, and 0.1 mM EDTA. The washed cell pellet was resuspended in the same buffer, and was placed in an ice bath for ultrasonication. After ultrasonication, the suspension was centrifuged for 20 min at 10,000g and 4 °C to remove cell debris, and then the supernatant was stored at 80 °C until required. The fructokinase activity were measured by spectrometric method which couples the formation of F-6-P to the reduction of NADP+ via glucose phosphate isomerase (PGI; CAS: 9001-41-6, S. cerevisiae, Sigma) and glucose-6-phosphate dehydrogenase (Caescu et al., 2004).
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One unit (U) of enzyme activity was defined as the amount of enzyme necessary to catalyze the conversion of 1 nmol of substrate per minute into specific products. Specific activity was expressed as U/mg of protein. 2.5. Phosphoenolpyruvate phosphotransferase system (PEP-PTS) activity assay The PEP-PTS activity was estimated by a modification of the assay method (Jiang et al., 2013). The cells were harvested, washed twice as described above. The washed cell pellet was resuspended in 2 mM potassium phosphate buffer (pH 7.0) containing 150 mM KCl and 5 mM MgCl2. Toluene–ethanol (1:9, v/v) was added to the cell suspension at a final concentration of 1% by micro-pipette, and agitated vigorously for 60 s. After centrifugation at 1000g for 6 min at 4 °C, the supernate was removed and the cells were suspended in the same buffer. Equivalent to 30–100 lg dry weight of cells was added to the reaction mixture containing 5 mM phosphoenolpyruvate, 0.5 mM NADH, 10 U lactate dehydrogenase (EC1.1.1.27, rabbit muscle, Sigma), 60 mM MgCl2, 15 mM sucrose or fructose and sufficient buffer to give a final volume of 0.3 ml at 37 °C. The decrease of NADH was monitored by spectroscopy at 340 nm. Net PEP-PTS activity was expressed as nanomoles of pyruvate formed per milligram (dry weight) of cells per minute. 2.6. Protein assay Protein contents were determined by Bradford (Bradford, 1976), using bovine serum albumin (Sigma Chemical Co.) as a standard. 3. Results and discussion 3.1. Succinic acid fermentation of sucrose by A. succinogenes NJ113 Glucose utilization by A. succinogenes NJ113 has been studied in some detail, but no studies on sucrose utilization have been reported previously. A. succinogenes NJ113 was able to use sucrose as a carbon source in this study; therefore, it was necessary to investigate how it does so. Concentration gradient experiments were performed in serum bottles as described before. As shown in Table 1, the succinic acid concentration increased as the initial sucrose concentration increased. When the sucrose concentration was 100 g/L, the succinic acid concentration reached its highest value of 57.1 g/L with a yield of 71.5%. When the sucrose concentration was higher than 100 g/L, the succinic acid concentration decreased. It meant that higher concentrations of sucrose repressed the productive capacity of succinic acid. When 50 g/L of sucrose was used, a maximum cell OD of 13.41 was obtained, and then the OD decreased when the sucrose concentration was higher than 50 g/L. Osmotic pressure seems to be the main inhibitory factor for
succinic acid production and cell growth (Bretz and Kabasci, 2012). During the entire process, the acetic acid concentration was relatively stable, ranging from 5 to 7.5 g/L; however, the formic acid concentration increased as the initial sucrose increased and reached a maximum of 12.3 g/L. Table 1 shows the most suitable concentration of sucrose utilized by A. succinogenes NJ113 was 100 g/L. Subsequently, a batch culture was carried out in a 3-L fermentor to further investigate growth conditions utilizing sucrose. In Fig. 1, a final concentration of 57.5 g/L succinic acid accumulated within 36 h, with a yield of 77% and a productivity of 1.60 g/L/h. Both the production and productivity were higher than those reported by Xi et al. (2011), 51.6 and 1.52 g/L/h, respectively, using glucose as the carbon source with A. succinogenes NJ113. In addition, the succinic acid productivity and production were higher than that reported by Liu et al. (2008), Wang et al. (2011) and Chan et al. (2012) using sucrose as the carbon source. The maximum cell OD was 11.6 at 10 h, and it then decreased until the end of the fermentation. When sucrose was fed as the sole carbon source, A. succinogenes NJ113 grew rapidly, coupled with a fast formic acid and succinic acid productivities in the first 10 h. However, cell would be inhibited in high concentration of succinic acid and formic acid. In previous study with glucose as carbon source by our lab, A. succinogenes NJ113 showed that cells decreased when succinic acid was accumulated to 20 g/L. Cells produced succinic acid too fast with sucrose as carbon source, then cells biomass was inhibited in short time. During the process, formic acid was produced rapidly, increasing from 12.6 g/L at 6 h to 36.5 g/L at 8 h, and then decreasing. Formic acid was harmful for A. succinogenes cells (Li et al., 2010). However, it seemed that A. succinogenes could tolerate higher concentrations of formic acid in a medium containing sucrose according to the data got from serum culture and batch culture, as sucrose can protect cells compared to glucose. And when formic acid accumulated to a harmful concentration, it was metabolized to CO2 to supply NADH according to the A. succinogenes central metabolic network map (McKinlay et al., 2010). 3.2. Sucrose transport and metabolism pathways in A. succinogenes NJ113 Sucrose is composed of a glucose unit and a fructose unit connected by a glycoside linkage. Some bacteria can break down extracellular sucrose into glucose and fructose by cleaving the glycoside linkage using certain enzymes, such as the catabolic enzymes, the sucrose hydrolases and phosphorylases, and the biosynthetic glycosyl-nucleotide glycosyltransferases (Kitaoka and Hayashi, 2002). Intracellular sucrose metabolism can be categorized into three types: a PTS system and two non-PTS systems (Lee et al., 2010b). The PTS system includes the PEP-dependent sucrose-specific PTS, sucrose 6-phosphate hydrolase, and fructokinase. One non-PTS system includes sucrose permease and sucrose
Table 1 Sucrose utilization by A. succinogenes NJ113 in serum bottles.a Initial sucrose (g/L)
OD660
Succinic acid (g/L)
Formic acid (g/L)
Acetic acid (g/L)
Residual sucrose (g/L)
Succinic acid yield (%)
40 50 60 70 80 90 100 110 120
11.70 ± 0.57 13.41 ± 0.62 12.05 ± 0.51 12.46 ± 0.81 11.60 ± 0.31 10.97 ± 0.14 9.31 ± 0.59 7.53 ± 0.87 6.80 ± 0.95
26.68 ± 0.32 32.49 ± 0.59 37.53 ± 0.48 41.00 ± 0.88 46.30 ± 0.42 47.52 ± 1.05 57.13 ± 0.13 55.39 ± 0.79 44.49 ± 0.91
5.47 ± 0.11 5.94 ± 0.09 7.30 ± 0.13 6.52 ± 0.06 9.48 ± 0.61 9.14 ± 0.73 11.58 ± 1.31 11.03 ± 0.95 12.35 ± 0.87
6.49 ± 0.13 7.57 ± 0.22 7.17 ± 0.17 7.04 ± 0.24 7.40 ± 0.31 6.12 ± 0.58 5.98 ± 1.15 5.51 ± 1.06 6.40 ± 0.95
1.66 ± 0.15 3.15 ± 0.18 6.66 ± 0.05 9.04 ± 0.18 12.09 ± 0.61 18.39 ± 0.31 20.06 ± 0.7 28.69 ± 1.51 48.81 ± 1.71
69.6 ± 0.8 69.4 ± 0.2 70.4 ± 0.9 67.7 ± 0.3 68.2 ± 0.3 69.2 ± 0.8 71.3 ± 1.0 68.1 ± 0.9 62.5 ± 1.3
Each value is an average of three parallel replicates and is reported as mean ± standard deviation. a Cells were grown in anaerobic bottles for 48 h.
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100
OD660 Succinic Acid Formic Acid Acetic Acid Sucrose
90
70
12 10
60
8
50 6
40 30
OD660
Concentration g/L
80
14
4
20 2 10 0
0
0
4
8
12
16
20
24
28
32
36
40
44
Time(h) Fig. 1. Batch culture with 100 g/L of sucrose by A. succinogenes NJ113. The succinic acid concentration reached its highest value of 57.5 g/L in 36 h with a yield of 77% and a productivity of 1.60 g/L/h.
Table 2 PTS activities in A. succinogenes NJ113 grown on different carbon sources. Carbon sourcea
Sucrose PEP-PTS activityb
Glucose PEP-PTS activityb
Sucrose Glucose
60.3 ± 1.9 53.4 ± 0.8
9.3 ± 1.9 11.4 ± 0.8
Each value is an average of three parallel replicates and is reported as mean ± standard deviation. a Means cells grown in medium in the presence of the indicated carbon sources. b Activity unit defined as nmol/min per mg dry wt. of cells.
14
60
12 OD660 Succinic acid Formic acid Acetic acid Sucrose
40
30
10 8 6
OD660
Concentration g/L
50
20 4 10
2 0
0 0
4
8
12
16
20
24
28
32
36
40
44
48
to glucose and fructose extracellularly. We also found some genetic information, relating to Asuc 0914 and Asuc 1829, about sucrose PTS in A. succinogenes 130Z at the JGI Integrated Microbial Genomes (IMG) website (IMG Home). Asuc 0914 encodes the PTS system, sucrose-specific IIBC subunit, and Asuc 1829 encodes sucrose-6-phosphate hydrolase. Reports showed that a complete PTS consists of enzyme I, HPr, enzyme IIA, and enzyme IIBC. Enzyme I and HPr are common to all PTS carbohydrates (Deutscher et al., 2006). The EIIA received the phosphoryl group from PHPr and transferred phosphoryl group to EIIBC domain, however, the EIIA domain information was not found in A. succinogenes. In M. succiniciproducens MBEL55E, it seems that the EIIA domain for glucose PTS is used for sucrose as well (Lee et al., 2010b). A similar condition may exist in A. succinogenes, so that a complete sucrose PTS system may exist in A. succinogenes NJ113. This would mean that A. succinogenes NJ113 is able to utilize sucrose via sucrose PTS. To reveal the sucrose utilization pathways in A. succinogenes NJ113, some sucrose utilization associated enzymes were detected. The data listed in Table 2 were obtained from cells grown in medium in the presence of the indicated carbon sources. Data showed cells grown in sucrose got a sucrose PTS activity of 60.3 ± 1.9 nmol/min per mg dry wt. of cells. They proved that sucrose PTS activity exists in A. succinogenes NJ113. As mentioned above, the PTS system includes the sucrose-PTS, and sucrose 6-phosphate hydrolase, which means if sucrose is transported into cells via sucrose PTS pathway, it is phosphorylated to sucrose-6phosphate and then degraded to glucose-6-phosphate and fructose by sucrose 6-phosphate hydrolase. Glucose-6-phosphate would be metabolized via the EMP pathway immediately, while fructose would be utilized via the fructokinase pathway or fructose PTS pathway. Hence, fructose PTS activity and fructokinase activity were detected and compared. Both fructose PTS activity and fructokinase activity existed in A. succinogenes NJ113 cells grown on sucrose (Table 3), however, the fructokinase activity was negligible. This indicated that, after sucrose-6-phosphate was degraded to fructose, fructose PTS participated in fructose utilization. It seems clear that A. succinogenes NJ113 uses a sucrose PTS, sucrose-6-phosphate hydrolase and a fructose PTS to completely transport and metabolize sucrose (Fig. 3). According to the analysis above, sucrose metabolism contains two branches, EMP and fructose PTS pathways. And data in Table 2 showed sucrose PTS activity was much higher than glucose PTS activity. These two reasons might explain that why sucrose was more efficient in succinic acid production by A. succinogenes NJ113 comparing to glucose utilization.
Time (h) Fig. 2. Fed-batch culture with 100 g/L of sucrose by A. succinogenes NJ113. Cells were grown with an initial sucrose concentration of 50 g/l. After 28 h, the succinic acid production reached the maximum value of 60.44 g/L, with a yield and productivity of 83% and 2.16 g/L/h respectively.
phosphorylase, and the other non-PTS system involves sucrose permease and sucrase (Toth et al., 2000). A. succinogenes NJ113 was able to grow well in medium containing sucrose at a relatively high sucrose uptake rate (Fig. 1). It was worth mentioning that no glucose or fructose was detected during the entire process. This indicated that sucrose may not be cleaved
3.3. Fed-batch culture of succinic acid fermentation using sucrose The above results (Fig. 1) have shown that, in batch culture with 100 g/L of sucrose, succinic acid production was inhibited during the first 6 h by high concentration of the substrate for the low consumption of sucrose. Hence, a fed-batch culture was used to replace the batch culture to enhance the productivity of succinic acid. In fed-batch culture (Fig. 2), succinic acid production was not inhibited in the beginning of the fermentation compared to the batch culture. A maximum OD660 of 11.88 was achieved in 8 h,
Table 3 Fructose PTS and fructokinase activities in A. succinogenes NJ113 grown on different carbon sources. Carbon sourcea
Fructose PTS activity (nmol/min per mg dry wt. of cells)
Fructokinase activity (U/mg protein)
Sucrose Fructose
45.9 ± 1.3 39.7 ± 0.7
4.8 ± 0.9 5.7 ± 0.5
Each value is an average of three parallel replicates and is reported as mean ± standard deviation. a means cells grown in medium in the presence of the indicated carbon sources.
M. Jiang et al. / Bioresource Technology 153 (2014) 327–332 Sucrose
331
Extracellular Space
Suc -PTS
Fru-PTS
PEP PEP Pyc
suc-6-P
Pyc
Intracellular Space
Suc-6-P hydrolase
Fru Fru-1-P Glu-6-P Fructokinase
Fru-6-P Fru-1, 6-BP
PEP
OAA
Mal
Succinic Acid
Fig. 3. Sucrose metabolism pathway by A. succinogenes NJ113. A. succinogenes NJ113 uses a sucrose PTS, sucrose-6-phosphate hydrolase and a fructose PTS to completely transport and metabolize sucrose.
Table 4 Main fermentation parameters of the fed-batch culture and batch culture by A. succinogenes NJ113.
Fermentation timea (h) Succinic acid (g/L) Maximum yield (g/g) Productivity (g/L/h) Sucrose consumption Maximum cell concentration (g/L)
Batch
Fed-batch
Decreased or increased rateb (%)
36 57.5 0.77 1.60 74.4 11.6
28 60.4 0.83 2.16 72.6 11.9
22.2 5 7.8 35 – –
a Means the time is when A. succinogenes NJ113 accumulated the maximum succinic acid concentration. b Means data detected in fed-batch culture decreased ( ) or increased (+) value comparing with in batch culture divided by data in batch culture.
and after operating 28 h the succinic acid production reached the maximum value of 60.5 g/L, with a yield and productivity of 82.9% and 2.16 g/L/h, respectively. It’s the highest productivity reported about A. succinogenes NJ113 fermentation at present. The main fermentation parameters of the fed-batch culture and batch culture were listed in Table 4 for comparison. It can be seen that the succinic acid concentration, yield, sucrose consumption, and maximum cell concentration all increased a little; however the fermentation time decreased by 22.2%, which caused overall productivity to increase by 35.2%. It is an obvious enhancement. The decrease in fermentation time means a decrease in production cost; therefore, fed-batch culture is more advantageous for high sucrose concentration fermentation. In fed-batch fermentation, the maximum formic acid concentration was 19.9 g/L at 6 h; it decreased thereafter. The maximum formic acid concentration was lower than the value, 36.5 g/L, in batch culture. Data in serum fermentation, batch culture and fedbatch culture showed that formic acid concentration changed with sucrose concentration. This indicated that A. succinogenes NJ113 produced higher concentration of formic acid, and may tolerate a higher concentration of formic acid when more sucrose is present in the medium. 4. Conclusion This study has demonstrated the feasibility of utilizing sucrose as a carbon source to produce succinic acid by A. succinogenes NJ113. Through batch fermentation, 57.5 g/L of succinic acid were produced with a yield of 77% and a corresponding productivity of 1.60 g/L/h. Further analysis of the enzyme activity revealed that a sucrose PTS and a fructose PTS contribute to sucrose metabolism
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