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Influence of environmental and nutritional factors on succinic acid production and enzymes of reverse tricarboxylic acid cycle from Enterococcus flavescens
Enzyme and Microbial Technology 40 (2007) 629–636
Influence of environmental and nutritional factors on succinic acid production and enzymes of reverse tricarboxylic acid cycle from Enterococcus flavescens Lata Agarwal, Jasmine Isar, Gautam K. Meghwanshi, Rajendra Kumar Saxena ∗ Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India Received 30 April 2006; accepted 19 May 2006
Abstract Effect of different environmental and nutritional factors on succinic acid production by batch fermentation of Enterococcus flavescens and enzymes involved in the acid production through reverse tricarboxylic acid cycle (TCA) cycle was investigated. An overall seven-fold increase in succinic acid production (from 0.92 g l−1 in 72 h initially to 6.7 g l−1 in 48 h) was achieved in 300-ml of optimized medium having 3% sucrose, 1:0.5 ratio of tryptone and ammonium hydrogen phosphate, 15 mM MgCO3 at pH 6.5 when inoculated with 4% (v/v) of seed inoculum and incubated at 39 ◦ C for 48 h, as against initial un-optimized medium. Subsequent scale-up in a 10-l bioreactor using these optimized fermentation conditions under controlled pH and continuous CO2 supply resulted in 14.25 g l−1 of succinic acid in 30 h. Optimization of the environmental and nutritional parameters resulted in a maximum production of succinic acid by affecting the levels of the enzymes involved in its production via the reverse TCA cycle. A linear relationship was observed between succinic acid production and in the enzyme activities. The enzyme activity was found to be increase in order phosphoenol pyruvate carboxykinase < malate dehydrogenase < fumarase < fumarate reductase < phosphoenol pyruvate carboxylase. © 2006 Elsevier Inc. All rights reserved. Keywords: Succinic acid; Fermentation; Anaerobic production; Enterococcus flavescens; Scale-up; Reverse TCA cycle enzymes
1. Introduction Succinic acid, known as butanedioic acid, a member of C-4 dicarboxylic acid family, has been drawing much industrial interest because it can serve as a feedstock for the manufacture of many commodity chemicals such as 1,4 butanediol and tetrahydrofuran or new “green” solvents or polymers through esterification reactions [1–6]. Succinic acid and its derivatives are widely used as specialty chemicals for applications in food, pharmaceuticals and cosmetic industries [7,8]. Currently, the acid is produced commercially through chemical synthesis involving hydrolysis of petroleum products, which is associated with certain environmental hazards. Therefore, much attention has been focused in the past few years on fermentative production of succinic acid by anaerobic or facultative anaerobic microorganisms [9–12]. This is because it
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Corresponding author. Tel.: +91 11 24116559; fax: +91 11 24115270/24116427. E-mail address: [email protected] (R.K. Saxena). 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.05.019
is a common intermediate in the metabolic pathway of several anaerobic and facultative anaerobic microorganisms [13]. This fermentation process for the production of succinic acid can be regarded as a “green technology” not only because renewable substrates are used for its production but also because CO2 , a green house gas is fixed during microbial production of succinic acid [4]. Many anaerobic and facultative anaerobic microorganisms ferment carbohydrates to a mixture of acids, e.g. formate, acetate, lactate and succinate as end products. Phosphoenol pyruvate (PEP) is one of the central intermediates during the mixed acid fermentation. It is either converted into pyruvate resulting in the formation of the fermentation products acetate, formate, ethanol and lactate or it is converted into oxaloacetate resulting in the formation of end products succinate and propionate via the reversible arm of tricarboxylic acid (TCA) cycle (Fig. 1) [14,15]. Under anoxic conditions, the flux of PEP towards either oxaloacetate or puruvate is affected by environmental factors such as pH, temperature, hydrogen, carbon dioxide and nutritional factors as carbon, nitrogen sources and metal ions of the growth medium.
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L. Agarwal et al. / Enzyme and Microbial Technology 40 (2007) 629–636 with a column temperature of 50 ◦ C using 5 mM H2 SO4 as mobile phase with a flow rate of 0.6 ml min−1 [9,16,17].
2.3. Preparation of crude enzyme The fermentation broth was centrifuged at 8000 × g at 4 ◦ C for 15 min. Supernatant was removed and pellet was suspended appropriately in 0.1 M Tris–H2 SO4 buffer (pH 8.5) to prepare 5% of the cell slurry. One millimolar of EDTA (prepared in phosphate buffer, pH 7.0) was added to this slurry. The cell in the slurry was disrupted with ultra sonicator (Soniprep—150, USA) for 15 min (at a cycle of 10 s each). The mixture was allowed to stand for 10 min at 4 ◦ C. The cell lysate was centrifuged at 8000 × g for 30 min at 4 ◦ C. The pellet was discarded and the supernatant was used as crude enzyme (or cell free extracts) for the assays of different enzymes involved in succinic acid production via the reverse arm of the TCA cycle. Fig. 1. Pathway leading to production of succinic acid and pyruvate from phosphoenol pyruvate.
The anaerobic production of succinate follows a pathway involving phosphoenol pyruvate carboxylase (PPC)/phosphoenol pyruvate carboxykinase (PPCK), malate dehydrogenase, fumarase and fumarate reductase. Therefore, it was felt worthwhile to examine the effect of different environmental and nutritional parameters on succinic acid production and on the activities of these TCA cycle enzymes involved in the production pathway. In the present investigation, isolate B20, a potent succinic acid producer was isolated after an extensive screening programme carried out in our laboratory [16]. Further, process optimization was attempted for maximum succinic acid production by the organism (later identified as Enterococcus flavescens). Subsequently, scale-up of production was carried out in a 10-l bioreactor under the conditions optimized. The activities of each of the enzymes involved in succinic acid production were also determined during the process optimization. 2. Materials and methods 2.1. Organism and growth condition The isolate B20 was selected as a potent succinic acid producer after screening more than 300 anaerobic and facultative anaerobic bacteria and fungi. The isolate was identified as E. flavescens by 16s rRNA sequencing carried out at MIDILABS (Newark, DE, USA). The strain of E. flavescens was isolated from rumen of buffalo. For growth and maintenance, the strain was grown in 500-ml sealed anaerobic bottles containing 300-ml of the medium containing (g l−1 ): glucose 20; peptone 10; yeast extract 5.0; K2 HPO4 3.0; NaCl 1.0; (NH4 )2 SO4 1.0; CaCl2 ·2H2 O 0.2; MgCl2 ·6H2 O 0.2 and Na2 CO3 1.0. The medium was sterilized (15 min, at 121 ◦ C) in bottles sealed with butyl rubber bungs with N2 headspace. To the sterilized medium, a few drops of concentrated sulphuric acid were added aseptically to adjust the pH to 6.5. The N2 headspace was replaced by CO2 and Na2 S·9H2 O (0.02 g l−1 ) was added to remove traces of dissolved oxygen [4,9]. The reduced medium was inoculated with 2% (v/v) seed inoculum and incubated at 39 ± 1 ◦ C for 24 h under static conditions with intermittent gentle shaking.
2.2. Estimation of succinic acid Concentrations of succinic acid was analyzed on HPLC (Shimadzu RID 10A, LC-10AD pump, CTO-10AS column oven, Tokyo, Japan) equipped with an ion exchange column (Aminex HPX-87H, 300 mm × 7.8 mm, Hercules, CA)
2.4. Enzyme assays Unless stated otherwise, all enzyme activities were measured spectrophotometrically under strict anoxic conditions as described previously [9,18]. All chemicals for the reaction except for the cell extract and the substrate were added to an optical glass cuvette. The cuvette was sealed with a soft rubber stopper and was made anoxic by flushing with N2 for 5 min. The additions of cell extract and anoxic substrate solutions to the anoxic cuvettes were made with a microliter syringe to give a final liquid volume of 1 ml. All activities were measured by standard or specified assay methods. Enzyme activities were determined from a minimum of three separate measurements. All assays were performed at 37 ◦ C with the exception of phosphoenol pyruvate carboxylase. The reactions were started by the addition of the substrate. The wavelength and the millimolar extinction coefficient for NAD, NADH, NADP and NADPH were 340 nm and 6.22 cm−1 mM−1 . The wavelength for methyl viologen and benzyl viologen was 578 nm, and the millimolar extinction coefficients were 9.78 and 8.65 cm−1 mM−1 , respectively. Fumarate and PEP formation were recorded at 240 nm, where their extinction coefficients were 2.53 and 1.50 cm−1 mM−1 , respectively. One International Unit of the enzyme is defined as the amount of enzyme catalyzing the conversion of 1 mol of substrate per min into specific products. The activity of phosphoenol pyruvate carboxylase was determined at 25 ◦ C by measuring the oxidation of NADH at 340 nm in a coupled system [19]. The standard assay mixture contained, in a total volume of 1.0 ml, 5 mol of potassium PEP, 10 mol of MgSO4 , 10 mol of KHCO3 , 0.1 mol of NADH, 100 mol of Tris–H2 SO4 buffer (8.5), 1.5 IU of malate dehydrogenase and the enzyme sample. The mixture was placed in the spectrophotometer for 4–5 min to achieve temperature equilibration (25 ◦ C). The formation of oxaloacetate was monitored spectrophotometrically in a malate dehydrogenase coupled system. The reaction velocity was measured as a decrease in A340 resulting from the oxidation of NADH. One international unit (IU) is defined as the amount of enzyme that oxidizes 1 mol of NADH per minute at 25 ◦ C and pH 8.5 under specified conditions.
2.5. Process optimization Effect of different environmental and nutritional parameters on the production of succinic acid and on the activities of the enzymes involved in its production via the reductive arm of the TCA cycle was examined. The effect of different pH on succinic acid production was studied upto 72 h of incubation at 39 ◦ C by preparing medium in a set of buffers (0.2 M citrate phosphate buffer (pH 4.0, 4.5, 5.0, 5.5 and 6.0), 0.2 M sodium phosphate buffer (pH 6.5, 7.0 and 7.5), 0.2 M Tris–HCl (pH 8.0, 8.5 and 9.0)). Similarly, effect of different temperatures on production of acid was studied after 60 h in the range of 25–50 ◦ C (25, 30, 35, 37, 39, 45 and 50 ◦ C). With respect to optimization of carbon source, different carbohydrates (glucose, fructose, maltose, xylose, sucrose, lactose, galactose, whey, cane molasses, starch, glycerol, sorbitol, rhamnose, arabinose and mannitol) at 2% concentration were investigated by replacing glucose (2%) from the medium. The selected sugar was optimized for its concentration for maximum production of both succinic acid and enzyme activity. With respect to optimization of
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nitrogen sources, peptone (1%, w/v) was replaced by different inorganic and organic nitrogen sources in the production medium with the same percent equivalent. Different nitrogen sources examined were ammonium chloride (0.6%, w/v), ammonium phosphate (0.76%, w/v), ammonium nitrate (0.45%, w/v), ammonium carbonate (0.59%, w/v), ammonium sulphate (0.75%, w/v), potassium nitrate (1.16%, w/v), sodium nitrate (0.97%, w/v), urea (0.34%, w/v) (inorganic nitrogen sources) and yeast extract (1.88%, w/v), tryptone (1.1%, w/v), corn steep liquor (4%, v/v), beef extract (1.77%, w/v) (organic nitrogen sources). Different metal carbonates (CaCO3 , MgCO3 , Fe2 CO3 , Na2 CO3 and Mn2 CO3 at the concentration of 10 mM) were added in the optimized medium to assess their effect on production of succinic acid. With a view to evaluate whether it was the metal ion or carbonate ions which was influencing the succinic acid production, the effect of different salts of the metal ion of the best metal carbonate was studied in the form of its chlorides, sulphates and phosphates (10 mM) on the production of succinic acid. Subsequently, the concentration of the most suitable metal salt was optimized for maximum production. The optimized medium thus obtained was inoculated with 1, 2, 3, 4, 5 and 6% (v/v) of inoculum (with an absorbance of 0.6 at 660 nm). Finally, the incubation period was optimized by estimating the amount of succinic acid produced at different time intervals up to 72 h at a regular interval of 12 h. Scale-up was carried out in a 10-l bioreactor (Bioflow IV, New Brunswick Scientific Inc. Co., USA) with 7.5-l working volume of the optimized medium (pH 6.5). Prior to inoculation, strict anaerobic conditions were established with 0.025% Na2 S·9H2 O. The production medium was sterilized at 121 ◦ C for 15 min and was inoculated with 4% (v/v) of the inoculum with an absorbance of 0.6 at 660 nm. Fermentation was carried out at 39 ± 1 ◦ C with a carbon dioxide flow rate of 0.5 volume of air per unit volume of the medium (vvm) at an agitation rate of 200 rpm. Foaming was controlled by adding silicon antifoam agent (0.1 ml of 50%, v/v, prepared in distilled water). pH was controlled by addition of 1N NaOH. Samples were withdrawn periodically at an interval of 6 h till 60 h and analyzed for succinic acid production and enzyme activities. All the experiments were conducted in triplicates and results are presented along with standard deviation.
3. Results and discussion The pH is an important factor that affects both growth and growth-associated production of molecules. Succinic acid production is a CO2 fixing process [20] and the pH of the medium affects the solubility and the availability of the CO2 , it is therefore most critical factor affecting succinic acid production [9,21]. It was observed that E. flavescens was capable of producing succinic acid in the wide pH range of 4.0–9.0 with maximum production at pH 6.5 (0.92 g l−1 in 72 h) and there was a subsequent decrease in the amount of succinic acid produced with the increasing pH (Fig. 1a). Similarly it was observed that Mannhemia succiniciproducens MBEL 55E, a succinic acid producer grows well in the pH range of 6.0–7.5 [12]. In an anaerobic bacterium, Anaerobiospirillum succiniciproducens, maximum succinic acid was produced at pH 6.2 [9]. There are a few reports where it has been observed that on increasing the pH to 7.2, there was a decrease in succinic acid yield [20,22]. The activity of reverse TCA cycle enzymes was also high at pH 6.5 (Fig. 2a). Thus, indicating that in the cells grown at pH 6.5, both succinic acid production as well as the activities of the enzymes involved in its production are maximum. Similar has been reported by Samuelov et al. [9] who studied the influence of pH on the level of fermentative enzymes responsible for end-
Fig. 2. (a) Effect of different pH on succinic acid production and enzyme activities in E. flavescens. (b) Effect of different temperatures on succinic acid production and enzyme activities in E. flavescens.
product formation and found that in the cells grown at pH 6.2, both the PPCK activity and succinic acid production was high. In our study, PPC was the least active thereby indicating that this enzyme is not actively involved in succinic acid production from E. flavescens. Among different temperatures studied, maximum succinic acid production was achieved at 39 ◦ C (Fig. 2b). However, the organism failed to grow at 50 ◦ C. Similarly M. succiniciproducens KCTC 0769 grew best and produced succinic acid at 39 ◦ C in 72 h [23]. The effect of temperature on the activities of the enzymes involved in the production of succinic acid (via the reverse TCA cycle) was also determined. The results showed that the activities of these enzymes are maximum at 39 ◦ C.
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Table 1 Effect of different of types of carbon sources (2%) on succinic acid production and enzyme activities in E. flavescens Carbon concentration (%)
Succinic acid (g l−1 )
Glucose (control) Fructose Maltose Xylose Sucrose Lactose Galactose Whey Cane molasses Starch Glycerol Sorbitol Mannitol Rhamnose Arabinose
0.95 0.93 1.3 0.52 2.82 2.1 0.66 0.54 0.5 0.13 1.3 0.61 0.21 0.24 0.13
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.05 0.04 0.07 0.02 0.12 0.09 0.03 0.03 0.02 0.006 0.07 0.04 0.03 0.04 0.04
PPCK (IU ml−1 ) 100 99.0 134.0 66.0 241.0 198.0 70.0 64.2 61.0 13.0 135.0 65.0 23.2 25.2 14.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.17 5.15 6.43 2.49 12.77 9.70 3.29 2.83 2.80 0.69 6.48 2.99 1.2 1.5 0.54
PPC (IU ml−1 ) 7 55.0 75.0 32.2 98.0 95.0 38.0 35.2 32.0 2.0 74.0 31.0 4.0 5.3 2.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.62 2.53 3.98 1.5 4.80 4.94 1.71 1.52 1.47 0.09 0.21 1.46 0.12 0.17 0.1
Mal. dehyd. (IU ml−1 ) 108 105.0 145.0 67.2 256.0 210.0 74.0 67.0 65.0 16.0 143.0 70.0 27.2 30.15 17.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.95 4.83 7.68 3.14 14.34 9.66 3.85 3.2 3.12 0.77 7.72 3.71 0.81 1.2 0.83
Fumarase (IU ml−1 ) 128 125.0 163.0 78.3 267.0 220.0 82.0 81 77.0 25.0 162.0 79.0 37.3 41.5 23
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.0 6.12 8.47 3.6 13.88 10.12 3.77 3.6 3.54 1.30 7.94 3.79 1.72 1.92 1.1
Fumarate reductase (IU ml−1 ) 178 175.0 195.0 117.0 296.0 235.0 121.0 117.0 110.0 53.0 192.0 112.0 65.2 69.4 51
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
9.0 9.10 10.53 6.23 13.61 12.45 5.80 6.19 6.16 2.90 9.41 5.15 3.1 3.4 1.9
PPC: phosphoenol pyruvate carboxylase. PPCK: phosphoenol pyruvate carboxykinase. Mal. dehyd.: malate dehydrogenase.
On studying the effect of different carbon sources on the production of succinic acid, it was observed that sucrose supported maximum succinic acid production of 2.82 g l−1 , which is followed by lactose (2.1 g l−1 ). Maltose, glycerol, fructose and glucose were moderately utilized by E. flavescens, while rhamnose, arabinose and mannitol were not consumed by the organism (Table 1). Similarly, it has been reported that glucose, sucrose and fructose were efficiently utilized by A. succiniciproducens resulting in the production of 2.2, 1.9 and 1.6 g l−1 of succinic acid, while rhamnose, arabinose and mannitol were not consumed by the organism [22]. However, glycerol has been reported as the best carbon source for production of succinic acid in case of A. succiniciproducens [4]. The activities of each of the enzymes tested were also maximum in sucrose, followed by lactose as sole source of carbon (Table 1). This shows that sucrose affects succinic acid production by directly affecting the activities of the enzymes of the reverse TCA cycle. It has also been reported that succinic acid production and the level of enzymes involved in its acid production in E. coli was remarkably affected by the carbon source in the growth medium [24]. It was observed that in the cells grown on glucose and fructose, there was about two-fold higher enzyme (PPC) level than that on glycerol. However, here we report that succinic acid production as well as the level of enzymes was the highest in cells grown in the media containing sucrose as the sole source of carbon. Subsequently, upon optimizing the concentration of sucrose in the medium it was observed that at 3% concentration, sucrose resulted in the maximum of 3.4 g l−1 of succinic acid and the maximum enzyme levels (Fig. 3). However, beyond this concentration of sugar, the osmolarity of the medium changed so much that it affected the enzyme activity leading to lesser production of succinic acid. Among the various organic nitrogen sources tested, tryptone maximally enhanced the production of both succinic acid
(3.8 g l−1 ) as well enzyme activities (Table 2). Amongst the inorganic nitrogen sources, ammonium hydrogen phosphate [(NH4 )2 HPO4 ] resulted in the maximum of 2.43 g l−1 of succinic acid with maximum enzyme activities. However, when tryptone and ammonium hydrogen phosphate were used in the ratio of 1:0.5, a maximum of 4.4 g l−1 of succinic acid was observed (Fig. 4). A similar trend of enzyme activities was observed (i.e. being maximum at 1:0.5 ratio). The most probable reason for the maximum enzyme activities and succinic acid yield could be the availability of hydrogen along with the nitrogen source. The addition of H2 in the form of ammonium
Fig. 3. Effect of sucrose concentrations on succinic acid production and enzyme activities in E. flavescens.
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Table 2 Effect of different nitrogen sources on succinic acid production and enzyme activities in E. flavescens Nitrogen conc. (%)
Succinic acid (g l−1 )
PPCK (IU ml−1 )
PPC (IU ml−1 )
Mal. dehyd. (IU ml−1 )
Fumarase (IU ml−1 )
Fumarate reductase (IU ml−1 )
Peptone NH4 Cl (NH4 )2 HPO4 NH4 NO3 NH4 CO3 NH4 SO4 KNO3 NaNO3 Urea Yeast extract Tryptone Corn steep liquor Beef extract
3.41 ± 0.18 1.82 ± 0.09 2.43 ± 0.12 0.00 1.92 ± 0.09 2.10 ± 0.10 0.20 ± 0.09 0.0 1.72 ± 0.08 2.50 ± 1.15 3.80 ± 0.17 3.25 ± 0.14 3.58 ± 0.18
272 ± 12.24 185.0 ± 9.80 220 ± 12.32 0.0 194 ± 10.09 205 ± 11.48 32 ± 1.73 0.0 174 ± 8.00 224 ± 10.08 295 ± 12.68 283 ± 14.71 276 ± 14.07
100 ± 4.2 92 ± 4.87 90 ± 4.86 0.0 95 ± 4.37 98 ± 4.21 80 ± 3.6 0.0 87 ± 4.00 93 ± 3.99 110 ± 5.61 106 ± 4.88 100 ± 5.40
285 ± 15.10 194 ± 8.34 232 ± 10.67 0.0 217 ± 9.98 220 ± 9.46 34 ± 1.80 0.0 185 ± 9.62 237 ± 12.32 317 ± 15.22 295 ± 12.68 288 ± 12.28
297 ± 13.66 205 ± 9.43 245 ± 11.03 0.0 229 ± 10.53 236 ± 10.15 42 ± 2.26 0.0 196 ± 8.82 249 ± 11.21 325 ± 13.65 311 ± 18.97 299 ± 12.85
332 ± 17.59 236 ± 10.86 281 ± 15.17 0.0 265 ± 14.04 286 ± 15.16 95 ± 5.32 0.0 237 ± 10.19 288 ± 13.25 365 ± 18.98 331 ± 17.21 337 ± 14.49
PPC: phosphoenol pyruvate carboxylase. PPCK: phosphoenol pyruvate carboxykinase. Mal. dehyd.: malate dehydrogenase.
hydrogen phosphate accelerated the conversion of sucrose to succinic acid due to the incorporation of electrons derived from H2 . Similar had been reported by Van der Werf et al. [18], who observed that succinic acid was produced by Actinobacillus sp. 1302, when 100% H2 was present in the culture. Lee et al. [20] have also reported that the supply of H2 gas (5%) significantly enhanced succinic acid from A. succiniciproducens. Succinic acid is a highly reduced fermentation product, therefore, the external supply of H2 , a potential electron donor enhances its production. Metal ions are known to play an important role in maintaining cellular metabolism and enzyme activities [25]. An increase in the production of succinic acid (5.32 g l−1 ) was recorded on supplementing this optimized medium with MgCO3 . The activ-
Fig. 4. Effect of ratio of tryptone and ammonium hydrogen phosphate on succinic acid production and enzyme activities in E. flavescens.
ities of the enzymes of the reverse TCA cycle were also high in MgCO3 (Fig. 5a). The reason could be that Mg2+ is a cofactor for most of the enzymes involved in the anaerobic pathway. This is in accordance to the findings of Meyer et al. [26] who reported that in E. coli the rate of PPC activity for substituted cation Mg2+ ions was higher than for metal ions like Mn2+ , Co2+ , Zn2+ . Mg2+ has also been reported as the cofactors for the activity of PPC [27]. Subsequently, in the present investigation, different salts of magnesium ion were investigated and it was found that amongst the chloride, carbonate, sulphate and phosphate tested, carbonate was most effective for the production of succinic acid (data not shown). The most probable reason could be the supply of carbondioxide from the carbonate salt, which was fixed during the fermentation process for the production of succinic acid. Similarly, Van der Werf et al. and Samuelov et al. [18,28] suggested that the production of succinate requires CO2 fixation and also that the CO2 concentration regulates the level of key enzymes of the PEP carboxykinase pathway in A. succiniciproducens. They further confirmed that high levels of CO2 stimulated PEP carboxykinase levels, whereas alcohol dehydrogenase and lactate dehydrogenases were significantly decreased. It has been reported by Zeikus et al. [3] that CO2 functions as an electron acceptor and alters the flux of PEP, which metabolizes to pyruvate and lactate/ethanol at low CO2 levels but makes succinate at high CO2 concentration. The activities of the TCA cycle enzymes were also high when the cells were grown in the medium containing MgCO3 . On subsequent optimization of the concentration of MgCO3 , 5.8 g l−1 of succinic acid was produced in 60 h at 15 mM MgCO3 (Fig. 5b). Production of 6.7 g l−1 of succinic acid was obtained in 48 h when the medium was inoculated with 4% (v/v) of inoculum (Fig. 6a and b). There was no increase in amount of succinic acid produced on subsequent increase in inoculum density (Fig. 6a). This is because the inoculum density of 4% (v/v) shortened the lag period and increased the final cell concentra-
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Fig. 5. (a) Effect of different metal ions on succinic acid production and enzyme activities in E. flavescens. (b) Effect of different concentrations of MgCO3 on succinic acid production and enzyme activities in E. flavescens.
Fig. 6. (a) Effect of different inoculum densities on succinic acid production and enzyme activities in E. flavescens. (b) Effect of different incubation periods on succinic acid production and enzyme activities in E. flavescens.
tion resulting in reduction of total fermentation time. However, further increase in the inoculum density beyond this did not enhance the production of succinic acid, because there was competition of nutrients and certain essential nutrients became limiting much faster. It was also observed that beyond 48 h of incubation, there was no increase in production of succinic acid (Fig. 6b). Finally, scale-up studies in a 10-l bioreactor in the optimized medium showed that succinic acid production starts at 6 h and a maximum of 14.25 g l−1 was obtained in 36 h of incubation (Table 3). The activity of PPC, PPCK, malate dehydrogenase, fumarase and fumarate reductase reached to the maximum. Improvement in product yield is expected in the fermentor as compared to that in shake flask/bottle because of better control
of process parameters in the former. Similar has been reported by Humphrey [29]. In scale-up, i.e. in biorecator, better yield could be attributed to high availability of CO2 and maintainance of pH of the medium to 6.5 by addition of 1N NaOH, which otherwise decreases due to the formation of acids. This was not possible inside the sealed anaerobic bottles. Similar trend has been reported where 13.5 g l−1 of succinic acid was produced when 100% CO2 was supplied to the bacterium M. succiniciproducens and the pH was maintained between 6.5 and 7.0 [12]. This is also supported by the findings of other workers, who reported that under high CO2 levels, succinate is a major reduced product [3,9].
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Table 3 Fermentation profile of the succinic acid production and enzyme activities in E. flavescens Incubation period (h)
Succinic acid (g l−1 )
PPCK (IU ml−1 )
PPC (IU ml−1 )
Mal. dehyd. (IU ml−1 )
Fumarase (IU ml−1 )
Fumarate reductase (IU ml−1 )
0 6 12 18 24 30 36 42 48 54 60
0 0.83 ± 0.03 2.5 ± 0.58 4.7 ± 0.24 8.5 ± 0.45 11.3 ± 0.48 14.25 ± 0.69 14.25 ± 0.75 14.26 ± 0.65 14.27 ± 0.87 14.27 ± 0.65
0 79 ± 4.03 220 ± 10.12 345 ± 16.22 580 ± 30.16 751 ± 39.80 792 ± 48.31 793 ± 42.82 795 ± 38.16 796 ± 41.39 798 ± 41.49
0 44 ± 2.28 87 ± 3.74 130 ± 6.89 156 ± 9.51 185 ± 8.51 195 ± 8.97 195 ± 12.48 198 ± 9.10 198 ± 10.29 198 ± 9.10
0 84 ± 4.03 229 ± 10.99 354 ± 15.22 595 ± 26.78 762 ± 32.76 810 ± 36.73 811 ± 38.88 813 ± 39.83 813 ± 37.39 814 ± 38.26
0 96 ± 4.12 242 ± 11.61 362 ± 19.18 620 ± 27.90 775 ± 33.32 821 ± 42.69 821 ± 37.76 823 ± 38.68 825 ± 42.90 825 ± 37.99
0 141 ± 7.19 276 ± 11.86 887 ± 38.14 657 ± 33.50 792 ± 35.64 847 ± 38.96 848 ± 35.62 857 ± 39.14 852 ± 44.30 854 ± 39.28
PPC: phosphoenol pyruvate carboxylase. PPCK: phosphoenol pyruvate carboxykinase. Mal. dehyd.: malate dehydrogenase.
4. Conclusions Initial optimization in 300-ml medium contained in 500ml anaerobic bottles with uncontrolled CO2 and pH resulted in seven-fold increase in succinic acid production by affecting the levels of the enzymes involved in its production. Subsequent scale-up in this optimized medium under controlled CO2 and pH conditions in a 10-l bioreactor resulted in further twofold increase in succinic acid production with a final yield of 14.25 g l−1 in 36 h. This fermentation-derived succinic acid has the potential to become a large volume commodity chemical that would form the basis for supplying many important intermediate and specialty chemicals for the consumer product industries. Acknowledgements Authors acknowledge with thanks the financial support from Council of Scientific and Industrial Research (CSIR) under a New Millennium Indian Technology Leadership Initiative (NMITLI) project sanctioned to R.K.S. L.A. and J.I. acknowledge with thanks the fellowships of CSIR to carry out this work. References [1] Jain MK, Datta R, Zeikus JG. High-value organic acids fermentation: emerging processes and products. In: Bioprocess engineering: the first generation. Chichester, England: Ellis Harwood Ltd.; 1989. pp. 366–389. [2] Millard CS, Chao YP, Liao JC, Donnelly MI. Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli. Appl Microbiol Biotechnol 1996;62:1808–10. [3] Zeikus JG, Jain MK, Elankovan P. Biotechnology of succinic acid production and markets for derived industrial products. Appl Microbiol Biotechnol 1999;51:545–52. [4] Lee PC, Lee WG, Lee SY, Chang HN, Chang YK. Fermentative production of succinic acid from glucose and corn steep liquor by Anaerobiospirillum succiniciproducens. Biotechnol Bioprocess Eng 2000;5:379–81. [5] Lee SY, Hong SH, Lee SH, Park SJ. Fermentative production of chemicals that can be used for polymer synthesis. Macromol Biosci 2004;4:157–64. [6] Hong SH, Kim JS, Lee SY, In YH, Choi SS, Rih JK, et al. The genome sequence of the capnophilic rumen bacterium Manheimia succiniciproducens. Nat Biotechnol 2004;22:1275–81.
[7] Guettler MV, Jain MK. Method for making succinic acid, Anaerobiospirillum succiniciproducens variants for use in process and methods for obtaining variants. US Patent; 1996, 5,573,931. [8] Vemuri GN, Eiteman MA, Altman E. Effect of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli. Appl Environ Microbiol 2002;68:1715–27. [9] Samuelov NS, Lamed R, Lowe S, Zeikus JG. Influence of CO2 -HCO3 + levels and pH on growth, succinate production and enzyme activities of Anaerobiospirillum succiniciproducens. Appl Environ Microbiol 1991;57:3013–9. [10] Landucci R, Goodman B, Wyman C. Methodology for evaluating the economics of biologically producing chemicals and materials from alternative feedstocks. Appl Biochem Biotechnol 1994;45–46:678–96. [11] Lee PC, Lee WG, Lee SY, Chang HN. Succinic acid production with reduced by-product formation in the fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source. Biotechnol Bioeng 2001;72:41–8. [12] Lee PC, Lee SY, Hong SH, Chang HN. Isolation and characterization of new succinic acid producing bacterium, Mannheimia succiniciproducens MBEL 55E, from bovine rumen. Appl Microbiol Biotechnol 2002;58:663–8. [13] Chotani G, Dodge T, Hsu A, Kumar M, LaDuca R, Trimbur D, et al. The commercial production of chemicals using pathway engineering. Biochim Biophys Acta 2000;1543:434–55. [14] Macy JM, Ljungdahl EG, Gottschalkm G. Pathway of succinate and propionate formation in Bacteriodes fagilis. J Bacteriol 1978;134:81–91. [15] Clark DP. The fermentation pathways in Escherichia coli. FEMS Microbiol Rev 1989;63:223–34. [16] Agarwal L, Isar J, Saxena RK. Rapid screening procedures for identification of succinic acid producers. J Biochem Biophys Methods 2005;63:24–32. [17] Chatterjee R, Millard CS, Champion K, Clark DP, Donnelly MI. Mutation of the pts G gene results in increased production of succinate in fermentation of glucose by Escherichia coli. Appl Environ Microbiol 2001;67:148–54. [18] Van der Werf MJ, Guettler MV, Jain MK, Zeikus JG. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch Microbiol 1997;167:332–42. [19] Yoshinga T, Izui K, Katsuki H. Purification and molecular properties of allosteric phosphoenol pyruvate carboxylase from Escherichia coli. J Biochem 1970;68:747–51. [20] Lee PC, Lee WG, Kwon S, Lee SY, Chang HN. Succinic acid production by Anaerobiospirillum succiniciproducens: effects of the H2 /CO2 supply and glucose concentration. Enzyme Microb Technol 1998;24: 549–54. [21] Jones RP, Greenfield PF. Effect of carbon dioxide on yeast growth and fermentation. Enzyme Microb Technol 1982;4:210–23.
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[22] Lee PC, Lee WG, Lee SY, Chang HN. Effects of medium components on the growth of Anaerobiospirillum succiniciproducens and succinic acid production. Process Biochem 1999;35:49–55. [23] Huh YS, Hong YK, Hong WH, Chang HN. Selective extraction of acetic acid from the fermentation broth produced by Mannheimia succiniproducens. Biotechnol Lett 2004;26:1581–4. [24] Teraoka H, Nishikido T, Izui K, Katsuki H. Control of the synthesis of phoshoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in Escherichia coli. J Biochem 1970;67:567–75. [25] Podkovyrov SM, Zeikus JG. Purification and characterization of phosphoenol pyruvate carboxykinase, a catabolic CO2 fixing enzyme, from Anaerobiospirillum succiniciproducens. J Gen Microbiol 1993;139:223–8.
[26] Meyer CR, Rustin P, Wedding RT. A simple and accurate spectrophotometric assay for phosphoenol pyruvate carboxylase activity. Plant Physiol 1988;86:325–8. [27] Kai Y, Matsumura H, Izui K. Phosphoenolpyruvate carboxylase: threedimensional structure and molecular mechanisms. Arch Biochem Biophys 2003;414:170–9. [28] Samuelov NS, Datta R, Jain MK, Zeikus JG. Whey fermentation by Anaerobiospirillum succiniciproducens for production of succinate based animal feed additive. Appl Environ Microbiol 1999;65:2260–3. [29] Humphrey A. Shake flasks to fermentor: what have we learnt? Biotechnol Prog 1998;14:3–7.
Influence of environmental and nutritional factors on succinic acid production and enzymes of reverse tricarboxylic acid cycle from Enterococcus flavescens Lata Agarwal, Jasmine Isar, Gautam K. Meghwanshi, Rajendra Kumar Saxena ∗ Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India Received 30 April 2006; accepted 19 May 2006
Abstract Effect of different environmental and nutritional factors on succinic acid production by batch fermentation of Enterococcus flavescens and enzymes involved in the acid production through reverse tricarboxylic acid cycle (TCA) cycle was investigated. An overall seven-fold increase in succinic acid production (from 0.92 g l−1 in 72 h initially to 6.7 g l−1 in 48 h) was achieved in 300-ml of optimized medium having 3% sucrose, 1:0.5 ratio of tryptone and ammonium hydrogen phosphate, 15 mM MgCO3 at pH 6.5 when inoculated with 4% (v/v) of seed inoculum and incubated at 39 ◦ C for 48 h, as against initial un-optimized medium. Subsequent scale-up in a 10-l bioreactor using these optimized fermentation conditions under controlled pH and continuous CO2 supply resulted in 14.25 g l−1 of succinic acid in 30 h. Optimization of the environmental and nutritional parameters resulted in a maximum production of succinic acid by affecting the levels of the enzymes involved in its production via the reverse TCA cycle. A linear relationship was observed between succinic acid production and in the enzyme activities. The enzyme activity was found to be increase in order phosphoenol pyruvate carboxykinase < malate dehydrogenase < fumarase < fumarate reductase < phosphoenol pyruvate carboxylase. © 2006 Elsevier Inc. All rights reserved. Keywords: Succinic acid; Fermentation; Anaerobic production; Enterococcus flavescens; Scale-up; Reverse TCA cycle enzymes
1. Introduction Succinic acid, known as butanedioic acid, a member of C-4 dicarboxylic acid family, has been drawing much industrial interest because it can serve as a feedstock for the manufacture of many commodity chemicals such as 1,4 butanediol and tetrahydrofuran or new “green” solvents or polymers through esterification reactions [1–6]. Succinic acid and its derivatives are widely used as specialty chemicals for applications in food, pharmaceuticals and cosmetic industries [7,8]. Currently, the acid is produced commercially through chemical synthesis involving hydrolysis of petroleum products, which is associated with certain environmental hazards. Therefore, much attention has been focused in the past few years on fermentative production of succinic acid by anaerobic or facultative anaerobic microorganisms [9–12]. This is because it
∗
Corresponding author. Tel.: +91 11 24116559; fax: +91 11 24115270/24116427. E-mail address: [email protected] (R.K. Saxena). 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.05.019
is a common intermediate in the metabolic pathway of several anaerobic and facultative anaerobic microorganisms [13]. This fermentation process for the production of succinic acid can be regarded as a “green technology” not only because renewable substrates are used for its production but also because CO2 , a green house gas is fixed during microbial production of succinic acid [4]. Many anaerobic and facultative anaerobic microorganisms ferment carbohydrates to a mixture of acids, e.g. formate, acetate, lactate and succinate as end products. Phosphoenol pyruvate (PEP) is one of the central intermediates during the mixed acid fermentation. It is either converted into pyruvate resulting in the formation of the fermentation products acetate, formate, ethanol and lactate or it is converted into oxaloacetate resulting in the formation of end products succinate and propionate via the reversible arm of tricarboxylic acid (TCA) cycle (Fig. 1) [14,15]. Under anoxic conditions, the flux of PEP towards either oxaloacetate or puruvate is affected by environmental factors such as pH, temperature, hydrogen, carbon dioxide and nutritional factors as carbon, nitrogen sources and metal ions of the growth medium.
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L. Agarwal et al. / Enzyme and Microbial Technology 40 (2007) 629–636 with a column temperature of 50 ◦ C using 5 mM H2 SO4 as mobile phase with a flow rate of 0.6 ml min−1 [9,16,17].
2.3. Preparation of crude enzyme The fermentation broth was centrifuged at 8000 × g at 4 ◦ C for 15 min. Supernatant was removed and pellet was suspended appropriately in 0.1 M Tris–H2 SO4 buffer (pH 8.5) to prepare 5% of the cell slurry. One millimolar of EDTA (prepared in phosphate buffer, pH 7.0) was added to this slurry. The cell in the slurry was disrupted with ultra sonicator (Soniprep—150, USA) for 15 min (at a cycle of 10 s each). The mixture was allowed to stand for 10 min at 4 ◦ C. The cell lysate was centrifuged at 8000 × g for 30 min at 4 ◦ C. The pellet was discarded and the supernatant was used as crude enzyme (or cell free extracts) for the assays of different enzymes involved in succinic acid production via the reverse arm of the TCA cycle. Fig. 1. Pathway leading to production of succinic acid and pyruvate from phosphoenol pyruvate.
The anaerobic production of succinate follows a pathway involving phosphoenol pyruvate carboxylase (PPC)/phosphoenol pyruvate carboxykinase (PPCK), malate dehydrogenase, fumarase and fumarate reductase. Therefore, it was felt worthwhile to examine the effect of different environmental and nutritional parameters on succinic acid production and on the activities of these TCA cycle enzymes involved in the production pathway. In the present investigation, isolate B20, a potent succinic acid producer was isolated after an extensive screening programme carried out in our laboratory [16]. Further, process optimization was attempted for maximum succinic acid production by the organism (later identified as Enterococcus flavescens). Subsequently, scale-up of production was carried out in a 10-l bioreactor under the conditions optimized. The activities of each of the enzymes involved in succinic acid production were also determined during the process optimization. 2. Materials and methods 2.1. Organism and growth condition The isolate B20 was selected as a potent succinic acid producer after screening more than 300 anaerobic and facultative anaerobic bacteria and fungi. The isolate was identified as E. flavescens by 16s rRNA sequencing carried out at MIDILABS (Newark, DE, USA). The strain of E. flavescens was isolated from rumen of buffalo. For growth and maintenance, the strain was grown in 500-ml sealed anaerobic bottles containing 300-ml of the medium containing (g l−1 ): glucose 20; peptone 10; yeast extract 5.0; K2 HPO4 3.0; NaCl 1.0; (NH4 )2 SO4 1.0; CaCl2 ·2H2 O 0.2; MgCl2 ·6H2 O 0.2 and Na2 CO3 1.0. The medium was sterilized (15 min, at 121 ◦ C) in bottles sealed with butyl rubber bungs with N2 headspace. To the sterilized medium, a few drops of concentrated sulphuric acid were added aseptically to adjust the pH to 6.5. The N2 headspace was replaced by CO2 and Na2 S·9H2 O (0.02 g l−1 ) was added to remove traces of dissolved oxygen [4,9]. The reduced medium was inoculated with 2% (v/v) seed inoculum and incubated at 39 ± 1 ◦ C for 24 h under static conditions with intermittent gentle shaking.
2.2. Estimation of succinic acid Concentrations of succinic acid was analyzed on HPLC (Shimadzu RID 10A, LC-10AD pump, CTO-10AS column oven, Tokyo, Japan) equipped with an ion exchange column (Aminex HPX-87H, 300 mm × 7.8 mm, Hercules, CA)
2.4. Enzyme assays Unless stated otherwise, all enzyme activities were measured spectrophotometrically under strict anoxic conditions as described previously [9,18]. All chemicals for the reaction except for the cell extract and the substrate were added to an optical glass cuvette. The cuvette was sealed with a soft rubber stopper and was made anoxic by flushing with N2 for 5 min. The additions of cell extract and anoxic substrate solutions to the anoxic cuvettes were made with a microliter syringe to give a final liquid volume of 1 ml. All activities were measured by standard or specified assay methods. Enzyme activities were determined from a minimum of three separate measurements. All assays were performed at 37 ◦ C with the exception of phosphoenol pyruvate carboxylase. The reactions were started by the addition of the substrate. The wavelength and the millimolar extinction coefficient for NAD, NADH, NADP and NADPH were 340 nm and 6.22 cm−1 mM−1 . The wavelength for methyl viologen and benzyl viologen was 578 nm, and the millimolar extinction coefficients were 9.78 and 8.65 cm−1 mM−1 , respectively. Fumarate and PEP formation were recorded at 240 nm, where their extinction coefficients were 2.53 and 1.50 cm−1 mM−1 , respectively. One International Unit of the enzyme is defined as the amount of enzyme catalyzing the conversion of 1 mol of substrate per min into specific products. The activity of phosphoenol pyruvate carboxylase was determined at 25 ◦ C by measuring the oxidation of NADH at 340 nm in a coupled system [19]. The standard assay mixture contained, in a total volume of 1.0 ml, 5 mol of potassium PEP, 10 mol of MgSO4 , 10 mol of KHCO3 , 0.1 mol of NADH, 100 mol of Tris–H2 SO4 buffer (8.5), 1.5 IU of malate dehydrogenase and the enzyme sample. The mixture was placed in the spectrophotometer for 4–5 min to achieve temperature equilibration (25 ◦ C). The formation of oxaloacetate was monitored spectrophotometrically in a malate dehydrogenase coupled system. The reaction velocity was measured as a decrease in A340 resulting from the oxidation of NADH. One international unit (IU) is defined as the amount of enzyme that oxidizes 1 mol of NADH per minute at 25 ◦ C and pH 8.5 under specified conditions.
2.5. Process optimization Effect of different environmental and nutritional parameters on the production of succinic acid and on the activities of the enzymes involved in its production via the reductive arm of the TCA cycle was examined. The effect of different pH on succinic acid production was studied upto 72 h of incubation at 39 ◦ C by preparing medium in a set of buffers (0.2 M citrate phosphate buffer (pH 4.0, 4.5, 5.0, 5.5 and 6.0), 0.2 M sodium phosphate buffer (pH 6.5, 7.0 and 7.5), 0.2 M Tris–HCl (pH 8.0, 8.5 and 9.0)). Similarly, effect of different temperatures on production of acid was studied after 60 h in the range of 25–50 ◦ C (25, 30, 35, 37, 39, 45 and 50 ◦ C). With respect to optimization of carbon source, different carbohydrates (glucose, fructose, maltose, xylose, sucrose, lactose, galactose, whey, cane molasses, starch, glycerol, sorbitol, rhamnose, arabinose and mannitol) at 2% concentration were investigated by replacing glucose (2%) from the medium. The selected sugar was optimized for its concentration for maximum production of both succinic acid and enzyme activity. With respect to optimization of
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nitrogen sources, peptone (1%, w/v) was replaced by different inorganic and organic nitrogen sources in the production medium with the same percent equivalent. Different nitrogen sources examined were ammonium chloride (0.6%, w/v), ammonium phosphate (0.76%, w/v), ammonium nitrate (0.45%, w/v), ammonium carbonate (0.59%, w/v), ammonium sulphate (0.75%, w/v), potassium nitrate (1.16%, w/v), sodium nitrate (0.97%, w/v), urea (0.34%, w/v) (inorganic nitrogen sources) and yeast extract (1.88%, w/v), tryptone (1.1%, w/v), corn steep liquor (4%, v/v), beef extract (1.77%, w/v) (organic nitrogen sources). Different metal carbonates (CaCO3 , MgCO3 , Fe2 CO3 , Na2 CO3 and Mn2 CO3 at the concentration of 10 mM) were added in the optimized medium to assess their effect on production of succinic acid. With a view to evaluate whether it was the metal ion or carbonate ions which was influencing the succinic acid production, the effect of different salts of the metal ion of the best metal carbonate was studied in the form of its chlorides, sulphates and phosphates (10 mM) on the production of succinic acid. Subsequently, the concentration of the most suitable metal salt was optimized for maximum production. The optimized medium thus obtained was inoculated with 1, 2, 3, 4, 5 and 6% (v/v) of inoculum (with an absorbance of 0.6 at 660 nm). Finally, the incubation period was optimized by estimating the amount of succinic acid produced at different time intervals up to 72 h at a regular interval of 12 h. Scale-up was carried out in a 10-l bioreactor (Bioflow IV, New Brunswick Scientific Inc. Co., USA) with 7.5-l working volume of the optimized medium (pH 6.5). Prior to inoculation, strict anaerobic conditions were established with 0.025% Na2 S·9H2 O. The production medium was sterilized at 121 ◦ C for 15 min and was inoculated with 4% (v/v) of the inoculum with an absorbance of 0.6 at 660 nm. Fermentation was carried out at 39 ± 1 ◦ C with a carbon dioxide flow rate of 0.5 volume of air per unit volume of the medium (vvm) at an agitation rate of 200 rpm. Foaming was controlled by adding silicon antifoam agent (0.1 ml of 50%, v/v, prepared in distilled water). pH was controlled by addition of 1N NaOH. Samples were withdrawn periodically at an interval of 6 h till 60 h and analyzed for succinic acid production and enzyme activities. All the experiments were conducted in triplicates and results are presented along with standard deviation.
3. Results and discussion The pH is an important factor that affects both growth and growth-associated production of molecules. Succinic acid production is a CO2 fixing process [20] and the pH of the medium affects the solubility and the availability of the CO2 , it is therefore most critical factor affecting succinic acid production [9,21]. It was observed that E. flavescens was capable of producing succinic acid in the wide pH range of 4.0–9.0 with maximum production at pH 6.5 (0.92 g l−1 in 72 h) and there was a subsequent decrease in the amount of succinic acid produced with the increasing pH (Fig. 1a). Similarly it was observed that Mannhemia succiniciproducens MBEL 55E, a succinic acid producer grows well in the pH range of 6.0–7.5 [12]. In an anaerobic bacterium, Anaerobiospirillum succiniciproducens, maximum succinic acid was produced at pH 6.2 [9]. There are a few reports where it has been observed that on increasing the pH to 7.2, there was a decrease in succinic acid yield [20,22]. The activity of reverse TCA cycle enzymes was also high at pH 6.5 (Fig. 2a). Thus, indicating that in the cells grown at pH 6.5, both succinic acid production as well as the activities of the enzymes involved in its production are maximum. Similar has been reported by Samuelov et al. [9] who studied the influence of pH on the level of fermentative enzymes responsible for end-
Fig. 2. (a) Effect of different pH on succinic acid production and enzyme activities in E. flavescens. (b) Effect of different temperatures on succinic acid production and enzyme activities in E. flavescens.
product formation and found that in the cells grown at pH 6.2, both the PPCK activity and succinic acid production was high. In our study, PPC was the least active thereby indicating that this enzyme is not actively involved in succinic acid production from E. flavescens. Among different temperatures studied, maximum succinic acid production was achieved at 39 ◦ C (Fig. 2b). However, the organism failed to grow at 50 ◦ C. Similarly M. succiniciproducens KCTC 0769 grew best and produced succinic acid at 39 ◦ C in 72 h [23]. The effect of temperature on the activities of the enzymes involved in the production of succinic acid (via the reverse TCA cycle) was also determined. The results showed that the activities of these enzymes are maximum at 39 ◦ C.
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Table 1 Effect of different of types of carbon sources (2%) on succinic acid production and enzyme activities in E. flavescens Carbon concentration (%)
Succinic acid (g l−1 )
Glucose (control) Fructose Maltose Xylose Sucrose Lactose Galactose Whey Cane molasses Starch Glycerol Sorbitol Mannitol Rhamnose Arabinose
0.95 0.93 1.3 0.52 2.82 2.1 0.66 0.54 0.5 0.13 1.3 0.61 0.21 0.24 0.13
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.05 0.04 0.07 0.02 0.12 0.09 0.03 0.03 0.02 0.006 0.07 0.04 0.03 0.04 0.04
PPCK (IU ml−1 ) 100 99.0 134.0 66.0 241.0 198.0 70.0 64.2 61.0 13.0 135.0 65.0 23.2 25.2 14.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.17 5.15 6.43 2.49 12.77 9.70 3.29 2.83 2.80 0.69 6.48 2.99 1.2 1.5 0.54
PPC (IU ml−1 ) 7 55.0 75.0 32.2 98.0 95.0 38.0 35.2 32.0 2.0 74.0 31.0 4.0 5.3 2.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.62 2.53 3.98 1.5 4.80 4.94 1.71 1.52 1.47 0.09 0.21 1.46 0.12 0.17 0.1
Mal. dehyd. (IU ml−1 ) 108 105.0 145.0 67.2 256.0 210.0 74.0 67.0 65.0 16.0 143.0 70.0 27.2 30.15 17.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4.95 4.83 7.68 3.14 14.34 9.66 3.85 3.2 3.12 0.77 7.72 3.71 0.81 1.2 0.83
Fumarase (IU ml−1 ) 128 125.0 163.0 78.3 267.0 220.0 82.0 81 77.0 25.0 162.0 79.0 37.3 41.5 23
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.0 6.12 8.47 3.6 13.88 10.12 3.77 3.6 3.54 1.30 7.94 3.79 1.72 1.92 1.1
Fumarate reductase (IU ml−1 ) 178 175.0 195.0 117.0 296.0 235.0 121.0 117.0 110.0 53.0 192.0 112.0 65.2 69.4 51
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
9.0 9.10 10.53 6.23 13.61 12.45 5.80 6.19 6.16 2.90 9.41 5.15 3.1 3.4 1.9
PPC: phosphoenol pyruvate carboxylase. PPCK: phosphoenol pyruvate carboxykinase. Mal. dehyd.: malate dehydrogenase.
On studying the effect of different carbon sources on the production of succinic acid, it was observed that sucrose supported maximum succinic acid production of 2.82 g l−1 , which is followed by lactose (2.1 g l−1 ). Maltose, glycerol, fructose and glucose were moderately utilized by E. flavescens, while rhamnose, arabinose and mannitol were not consumed by the organism (Table 1). Similarly, it has been reported that glucose, sucrose and fructose were efficiently utilized by A. succiniciproducens resulting in the production of 2.2, 1.9 and 1.6 g l−1 of succinic acid, while rhamnose, arabinose and mannitol were not consumed by the organism [22]. However, glycerol has been reported as the best carbon source for production of succinic acid in case of A. succiniciproducens [4]. The activities of each of the enzymes tested were also maximum in sucrose, followed by lactose as sole source of carbon (Table 1). This shows that sucrose affects succinic acid production by directly affecting the activities of the enzymes of the reverse TCA cycle. It has also been reported that succinic acid production and the level of enzymes involved in its acid production in E. coli was remarkably affected by the carbon source in the growth medium [24]. It was observed that in the cells grown on glucose and fructose, there was about two-fold higher enzyme (PPC) level than that on glycerol. However, here we report that succinic acid production as well as the level of enzymes was the highest in cells grown in the media containing sucrose as the sole source of carbon. Subsequently, upon optimizing the concentration of sucrose in the medium it was observed that at 3% concentration, sucrose resulted in the maximum of 3.4 g l−1 of succinic acid and the maximum enzyme levels (Fig. 3). However, beyond this concentration of sugar, the osmolarity of the medium changed so much that it affected the enzyme activity leading to lesser production of succinic acid. Among the various organic nitrogen sources tested, tryptone maximally enhanced the production of both succinic acid
(3.8 g l−1 ) as well enzyme activities (Table 2). Amongst the inorganic nitrogen sources, ammonium hydrogen phosphate [(NH4 )2 HPO4 ] resulted in the maximum of 2.43 g l−1 of succinic acid with maximum enzyme activities. However, when tryptone and ammonium hydrogen phosphate were used in the ratio of 1:0.5, a maximum of 4.4 g l−1 of succinic acid was observed (Fig. 4). A similar trend of enzyme activities was observed (i.e. being maximum at 1:0.5 ratio). The most probable reason for the maximum enzyme activities and succinic acid yield could be the availability of hydrogen along with the nitrogen source. The addition of H2 in the form of ammonium
Fig. 3. Effect of sucrose concentrations on succinic acid production and enzyme activities in E. flavescens.
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Table 2 Effect of different nitrogen sources on succinic acid production and enzyme activities in E. flavescens Nitrogen conc. (%)
Succinic acid (g l−1 )
PPCK (IU ml−1 )
PPC (IU ml−1 )
Mal. dehyd. (IU ml−1 )
Fumarase (IU ml−1 )
Fumarate reductase (IU ml−1 )
Peptone NH4 Cl (NH4 )2 HPO4 NH4 NO3 NH4 CO3 NH4 SO4 KNO3 NaNO3 Urea Yeast extract Tryptone Corn steep liquor Beef extract
3.41 ± 0.18 1.82 ± 0.09 2.43 ± 0.12 0.00 1.92 ± 0.09 2.10 ± 0.10 0.20 ± 0.09 0.0 1.72 ± 0.08 2.50 ± 1.15 3.80 ± 0.17 3.25 ± 0.14 3.58 ± 0.18
272 ± 12.24 185.0 ± 9.80 220 ± 12.32 0.0 194 ± 10.09 205 ± 11.48 32 ± 1.73 0.0 174 ± 8.00 224 ± 10.08 295 ± 12.68 283 ± 14.71 276 ± 14.07
100 ± 4.2 92 ± 4.87 90 ± 4.86 0.0 95 ± 4.37 98 ± 4.21 80 ± 3.6 0.0 87 ± 4.00 93 ± 3.99 110 ± 5.61 106 ± 4.88 100 ± 5.40
285 ± 15.10 194 ± 8.34 232 ± 10.67 0.0 217 ± 9.98 220 ± 9.46 34 ± 1.80 0.0 185 ± 9.62 237 ± 12.32 317 ± 15.22 295 ± 12.68 288 ± 12.28
297 ± 13.66 205 ± 9.43 245 ± 11.03 0.0 229 ± 10.53 236 ± 10.15 42 ± 2.26 0.0 196 ± 8.82 249 ± 11.21 325 ± 13.65 311 ± 18.97 299 ± 12.85
332 ± 17.59 236 ± 10.86 281 ± 15.17 0.0 265 ± 14.04 286 ± 15.16 95 ± 5.32 0.0 237 ± 10.19 288 ± 13.25 365 ± 18.98 331 ± 17.21 337 ± 14.49
PPC: phosphoenol pyruvate carboxylase. PPCK: phosphoenol pyruvate carboxykinase. Mal. dehyd.: malate dehydrogenase.
hydrogen phosphate accelerated the conversion of sucrose to succinic acid due to the incorporation of electrons derived from H2 . Similar had been reported by Van der Werf et al. [18], who observed that succinic acid was produced by Actinobacillus sp. 1302, when 100% H2 was present in the culture. Lee et al. [20] have also reported that the supply of H2 gas (5%) significantly enhanced succinic acid from A. succiniciproducens. Succinic acid is a highly reduced fermentation product, therefore, the external supply of H2 , a potential electron donor enhances its production. Metal ions are known to play an important role in maintaining cellular metabolism and enzyme activities [25]. An increase in the production of succinic acid (5.32 g l−1 ) was recorded on supplementing this optimized medium with MgCO3 . The activ-
Fig. 4. Effect of ratio of tryptone and ammonium hydrogen phosphate on succinic acid production and enzyme activities in E. flavescens.
ities of the enzymes of the reverse TCA cycle were also high in MgCO3 (Fig. 5a). The reason could be that Mg2+ is a cofactor for most of the enzymes involved in the anaerobic pathway. This is in accordance to the findings of Meyer et al. [26] who reported that in E. coli the rate of PPC activity for substituted cation Mg2+ ions was higher than for metal ions like Mn2+ , Co2+ , Zn2+ . Mg2+ has also been reported as the cofactors for the activity of PPC [27]. Subsequently, in the present investigation, different salts of magnesium ion were investigated and it was found that amongst the chloride, carbonate, sulphate and phosphate tested, carbonate was most effective for the production of succinic acid (data not shown). The most probable reason could be the supply of carbondioxide from the carbonate salt, which was fixed during the fermentation process for the production of succinic acid. Similarly, Van der Werf et al. and Samuelov et al. [18,28] suggested that the production of succinate requires CO2 fixation and also that the CO2 concentration regulates the level of key enzymes of the PEP carboxykinase pathway in A. succiniciproducens. They further confirmed that high levels of CO2 stimulated PEP carboxykinase levels, whereas alcohol dehydrogenase and lactate dehydrogenases were significantly decreased. It has been reported by Zeikus et al. [3] that CO2 functions as an electron acceptor and alters the flux of PEP, which metabolizes to pyruvate and lactate/ethanol at low CO2 levels but makes succinate at high CO2 concentration. The activities of the TCA cycle enzymes were also high when the cells were grown in the medium containing MgCO3 . On subsequent optimization of the concentration of MgCO3 , 5.8 g l−1 of succinic acid was produced in 60 h at 15 mM MgCO3 (Fig. 5b). Production of 6.7 g l−1 of succinic acid was obtained in 48 h when the medium was inoculated with 4% (v/v) of inoculum (Fig. 6a and b). There was no increase in amount of succinic acid produced on subsequent increase in inoculum density (Fig. 6a). This is because the inoculum density of 4% (v/v) shortened the lag period and increased the final cell concentra-
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Fig. 5. (a) Effect of different metal ions on succinic acid production and enzyme activities in E. flavescens. (b) Effect of different concentrations of MgCO3 on succinic acid production and enzyme activities in E. flavescens.
Fig. 6. (a) Effect of different inoculum densities on succinic acid production and enzyme activities in E. flavescens. (b) Effect of different incubation periods on succinic acid production and enzyme activities in E. flavescens.
tion resulting in reduction of total fermentation time. However, further increase in the inoculum density beyond this did not enhance the production of succinic acid, because there was competition of nutrients and certain essential nutrients became limiting much faster. It was also observed that beyond 48 h of incubation, there was no increase in production of succinic acid (Fig. 6b). Finally, scale-up studies in a 10-l bioreactor in the optimized medium showed that succinic acid production starts at 6 h and a maximum of 14.25 g l−1 was obtained in 36 h of incubation (Table 3). The activity of PPC, PPCK, malate dehydrogenase, fumarase and fumarate reductase reached to the maximum. Improvement in product yield is expected in the fermentor as compared to that in shake flask/bottle because of better control
of process parameters in the former. Similar has been reported by Humphrey [29]. In scale-up, i.e. in biorecator, better yield could be attributed to high availability of CO2 and maintainance of pH of the medium to 6.5 by addition of 1N NaOH, which otherwise decreases due to the formation of acids. This was not possible inside the sealed anaerobic bottles. Similar trend has been reported where 13.5 g l−1 of succinic acid was produced when 100% CO2 was supplied to the bacterium M. succiniciproducens and the pH was maintained between 6.5 and 7.0 [12]. This is also supported by the findings of other workers, who reported that under high CO2 levels, succinate is a major reduced product [3,9].
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Table 3 Fermentation profile of the succinic acid production and enzyme activities in E. flavescens Incubation period (h)
Succinic acid (g l−1 )
PPCK (IU ml−1 )
PPC (IU ml−1 )
Mal. dehyd. (IU ml−1 )
Fumarase (IU ml−1 )
Fumarate reductase (IU ml−1 )
0 6 12 18 24 30 36 42 48 54 60
0 0.83 ± 0.03 2.5 ± 0.58 4.7 ± 0.24 8.5 ± 0.45 11.3 ± 0.48 14.25 ± 0.69 14.25 ± 0.75 14.26 ± 0.65 14.27 ± 0.87 14.27 ± 0.65
0 79 ± 4.03 220 ± 10.12 345 ± 16.22 580 ± 30.16 751 ± 39.80 792 ± 48.31 793 ± 42.82 795 ± 38.16 796 ± 41.39 798 ± 41.49
0 44 ± 2.28 87 ± 3.74 130 ± 6.89 156 ± 9.51 185 ± 8.51 195 ± 8.97 195 ± 12.48 198 ± 9.10 198 ± 10.29 198 ± 9.10
0 84 ± 4.03 229 ± 10.99 354 ± 15.22 595 ± 26.78 762 ± 32.76 810 ± 36.73 811 ± 38.88 813 ± 39.83 813 ± 37.39 814 ± 38.26
0 96 ± 4.12 242 ± 11.61 362 ± 19.18 620 ± 27.90 775 ± 33.32 821 ± 42.69 821 ± 37.76 823 ± 38.68 825 ± 42.90 825 ± 37.99
0 141 ± 7.19 276 ± 11.86 887 ± 38.14 657 ± 33.50 792 ± 35.64 847 ± 38.96 848 ± 35.62 857 ± 39.14 852 ± 44.30 854 ± 39.28
PPC: phosphoenol pyruvate carboxylase. PPCK: phosphoenol pyruvate carboxykinase. Mal. dehyd.: malate dehydrogenase.
4. Conclusions Initial optimization in 300-ml medium contained in 500ml anaerobic bottles with uncontrolled CO2 and pH resulted in seven-fold increase in succinic acid production by affecting the levels of the enzymes involved in its production. Subsequent scale-up in this optimized medium under controlled CO2 and pH conditions in a 10-l bioreactor resulted in further twofold increase in succinic acid production with a final yield of 14.25 g l−1 in 36 h. This fermentation-derived succinic acid has the potential to become a large volume commodity chemical that would form the basis for supplying many important intermediate and specialty chemicals for the consumer product industries. Acknowledgements Authors acknowledge with thanks the financial support from Council of Scientific and Industrial Research (CSIR) under a New Millennium Indian Technology Leadership Initiative (NMITLI) project sanctioned to R.K.S. L.A. and J.I. acknowledge with thanks the fellowships of CSIR to carry out this work. References [1] Jain MK, Datta R, Zeikus JG. High-value organic acids fermentation: emerging processes and products. In: Bioprocess engineering: the first generation. Chichester, England: Ellis Harwood Ltd.; 1989. pp. 366–389. [2] Millard CS, Chao YP, Liao JC, Donnelly MI. Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli. Appl Microbiol Biotechnol 1996;62:1808–10. [3] Zeikus JG, Jain MK, Elankovan P. Biotechnology of succinic acid production and markets for derived industrial products. Appl Microbiol Biotechnol 1999;51:545–52. [4] Lee PC, Lee WG, Lee SY, Chang HN, Chang YK. Fermentative production of succinic acid from glucose and corn steep liquor by Anaerobiospirillum succiniciproducens. Biotechnol Bioprocess Eng 2000;5:379–81. [5] Lee SY, Hong SH, Lee SH, Park SJ. Fermentative production of chemicals that can be used for polymer synthesis. Macromol Biosci 2004;4:157–64. [6] Hong SH, Kim JS, Lee SY, In YH, Choi SS, Rih JK, et al. The genome sequence of the capnophilic rumen bacterium Manheimia succiniciproducens. Nat Biotechnol 2004;22:1275–81.
[7] Guettler MV, Jain MK. Method for making succinic acid, Anaerobiospirillum succiniciproducens variants for use in process and methods for obtaining variants. US Patent; 1996, 5,573,931. [8] Vemuri GN, Eiteman MA, Altman E. Effect of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli. Appl Environ Microbiol 2002;68:1715–27. [9] Samuelov NS, Lamed R, Lowe S, Zeikus JG. Influence of CO2 -HCO3 + levels and pH on growth, succinate production and enzyme activities of Anaerobiospirillum succiniciproducens. Appl Environ Microbiol 1991;57:3013–9. [10] Landucci R, Goodman B, Wyman C. Methodology for evaluating the economics of biologically producing chemicals and materials from alternative feedstocks. Appl Biochem Biotechnol 1994;45–46:678–96. [11] Lee PC, Lee WG, Lee SY, Chang HN. Succinic acid production with reduced by-product formation in the fermentation of Anaerobiospirillum succiniciproducens using glycerol as a carbon source. Biotechnol Bioeng 2001;72:41–8. [12] Lee PC, Lee SY, Hong SH, Chang HN. Isolation and characterization of new succinic acid producing bacterium, Mannheimia succiniciproducens MBEL 55E, from bovine rumen. Appl Microbiol Biotechnol 2002;58:663–8. [13] Chotani G, Dodge T, Hsu A, Kumar M, LaDuca R, Trimbur D, et al. The commercial production of chemicals using pathway engineering. Biochim Biophys Acta 2000;1543:434–55. [14] Macy JM, Ljungdahl EG, Gottschalkm G. Pathway of succinate and propionate formation in Bacteriodes fagilis. J Bacteriol 1978;134:81–91. [15] Clark DP. The fermentation pathways in Escherichia coli. FEMS Microbiol Rev 1989;63:223–34. [16] Agarwal L, Isar J, Saxena RK. Rapid screening procedures for identification of succinic acid producers. J Biochem Biophys Methods 2005;63:24–32. [17] Chatterjee R, Millard CS, Champion K, Clark DP, Donnelly MI. Mutation of the pts G gene results in increased production of succinate in fermentation of glucose by Escherichia coli. Appl Environ Microbiol 2001;67:148–54. [18] Van der Werf MJ, Guettler MV, Jain MK, Zeikus JG. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch Microbiol 1997;167:332–42. [19] Yoshinga T, Izui K, Katsuki H. Purification and molecular properties of allosteric phosphoenol pyruvate carboxylase from Escherichia coli. J Biochem 1970;68:747–51. [20] Lee PC, Lee WG, Kwon S, Lee SY, Chang HN. Succinic acid production by Anaerobiospirillum succiniciproducens: effects of the H2 /CO2 supply and glucose concentration. Enzyme Microb Technol 1998;24: 549–54. [21] Jones RP, Greenfield PF. Effect of carbon dioxide on yeast growth and fermentation. Enzyme Microb Technol 1982;4:210–23.
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L. Agarwal et al. / Enzyme and Microbial Technology 40 (2007) 629–636
[22] Lee PC, Lee WG, Lee SY, Chang HN. Effects of medium components on the growth of Anaerobiospirillum succiniciproducens and succinic acid production. Process Biochem 1999;35:49–55. [23] Huh YS, Hong YK, Hong WH, Chang HN. Selective extraction of acetic acid from the fermentation broth produced by Mannheimia succiniproducens. Biotechnol Lett 2004;26:1581–4. [24] Teraoka H, Nishikido T, Izui K, Katsuki H. Control of the synthesis of phoshoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in Escherichia coli. J Biochem 1970;67:567–75. [25] Podkovyrov SM, Zeikus JG. Purification and characterization of phosphoenol pyruvate carboxykinase, a catabolic CO2 fixing enzyme, from Anaerobiospirillum succiniciproducens. J Gen Microbiol 1993;139:223–8.
[26] Meyer CR, Rustin P, Wedding RT. A simple and accurate spectrophotometric assay for phosphoenol pyruvate carboxylase activity. Plant Physiol 1988;86:325–8. [27] Kai Y, Matsumura H, Izui K. Phosphoenolpyruvate carboxylase: threedimensional structure and molecular mechanisms. Arch Biochem Biophys 2003;414:170–9. [28] Samuelov NS, Datta R, Jain MK, Zeikus JG. Whey fermentation by Anaerobiospirillum succiniciproducens for production of succinate based animal feed additive. Appl Environ Microbiol 1999;65:2260–3. [29] Humphrey A. Shake flasks to fermentor: what have we learnt? Biotechnol Prog 1998;14:3–7.