Journal of Biotechnology 193 (2015) 123–129
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Improved propionic acid and 5,6-dimethylbenzimidazole control strategy for vitamin B12 fermentation by Propionibacterium freudenreichii Peng Wang a,b,c,∗ , Zhiwei Zhang a , Youjing Jiao a , Shouxin Liu c , Yunshan Wang d,∗∗ a
College of Chemical & Pharmaceutical Engineering, Hebei University of Science & Technology, Shijiazhuang 050018, China Hebei Research Center of Pharmaceutical and Chemical Engineering, Hebei University of Science & Technology, Shijiazhuang 050018, China c State Key Laboratory Breeding Base-Hebei Province Key Laboratory of Molecular Chemistry for Drug, Hebei University of Science & Technology, Shijiazhuang 050018, China d National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100190, China b
a r t i c l e
i n f o
Article history: Received 11 August 2014 Received in revised form 22 October 2014 Accepted 21 November 2014 Available online 29 November 2014 Keywords: Vitamin B12 Propionic acid 5,6-Dimethylbenzimidazole Feed-back inhibition Expanded bed adsorption bioreactor
a b s t r a c t An efficient fermentation-strengthening approach was developed to improve the anaerobic production of vitamin B12 by cultivation process optimization with Propionibacterium freudenreichii. The effects of the byproduct propionic acid and the precursor 5,6-dimethylbenzimidazole (DMB) on vitamin B12 biosynthesis were investigated. Byproduct inhibition experiments showed that maintaining propionic acid concentration in broth below 10–20 g/L in the early stage and 20–30 g/L in the late stage can efficiently improve vitamin B12 biosynthesis. Batch fermentation indicated the occurrence of feed-back inhibition in intracellular intermediate biosynthesis. In addition, the incorporation of the precursor DMB depended on the fermentation level of the vitamin B12 intermediate. High vitamin B12 concentration (58.8 mg/L) and production (0.37 mg/g) were obtained with an expanded bed adsorption bioreactor by using the propionic acid and DMB control method. The optimum concentration and production of 59.5 and 0.59 mg/L h for vitamin B12 production were respectively achieved after five continuous batches. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Vitamin B12 has various applications in the food and medicine industries (Wang et al., 2010) and also it is an important coenzyme in the metabolism of human tissues. Vitamin B12 can be biosynthesized in two ways: aerobic and anaerobic fermentation. Anaerobic fermentation with Propionibacterium has attracted increasing research interest because of the low-cost production of food-grade vitamin B12 (adenosylcobalamin) (Martens et al., 2002; Murooka et al., 2005; Kosmider et al., 2012). Considerable efforts have been exerted to improve vitamin B12-producing strains and vitamin B12 biosynthesis. Some studies have implemented random mutagenesis and genetic engineering method (Bykhovskii et al., 1998; Piao et al., 2004), whereas the others have attempted to optimize vitamin B12 fermentation by using various substrates
∗ Corresponding author at: College of Chemical & Pharmaceutical Engineering, Hebei University of Science & Technology, Shijiazhuang 050018, China. Tel.: +86 311 81668397. ∗∗ Corresponding author. E-mail address: [email protected] (P. Wang). http://dx.doi.org/10.1016/j.jbiotec.2014.11.019 0168-1656/© 2014 Elsevier B.V. All rights reserved.
(e.g., whey, tomato pomace, and crude glycerol) (Marwaha et al., 1983; Haddadin et al., 2001; Kosmider et al., 2012). However, the yields and productivities in the current industrial fermentation continue to significantly fluctuate since the traditional approaches result in retarded cell growth and low efficiency for vitamin B12 synthesis. Therefore, in-depth investigations focusing on cultivation process optimization are important. Propionic acid is a byproduct of the cultivation process of food-grade vitamin B12 by Propionibacterium freudenreichii; this byproduct may accumulate to high concentrations of 30–40 g/L and cause feed-back inhibition in microbial cell growth. Among the methods employed to prevent propionic acid feed-back inhibition during fermentation, in situ product removal (ISPR) has shown advantages in improving production. This technique may also be used for large-scale production. Various ISPR processes have been reported using membrane-integrated bioreactors, plant fibrousbed bioreactors, and expanded bed adsorption bioreactors (EBABs) (Bovayal et al., 1994; Feng et al., 2011; Wang et al., 2012a). However, most studies focused only on the increased production and/or yield of the extracellular product (propionic acid). Little is known about the effects of ISPR systems on intracellular product (vitamin B12) biosynthesis.
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In addition, in the industrial anaerobic fermentation of vitamin B12, exogenous 5,6-dimethylbenzimidazole (DMB) is usually used as a precursor to promote vitamin B12 biosynthesis (Woodson and Escalante-Semerena, 2006; Chandra and Brown, 2008). However, the main obstacle in this process is the addition of DMB. Current studies have used microexamination to determine the microbial growth state. However, this method is influenced by human factors, leading to an unsteady fermentation concentration. With regard to the fermentation mechanism, a key intermediate (adenosylcobalamide, AdoCbi) of vitamin B12 has been isolated and characterized in our laboratory. This compound can be used to determine the condition of vitamin B12 biosynthesis corresponding to the molecular weight (Wang et al., 2012b). However, whether or not AdoCbi synthesis induces feed-back inhibition remains unknown, that is needed to be further clarified by the analysis of AdoCbi biosynthesis mechanism. This study aims to develop an economic and efficient approach to improve vitamin B12 production. Research efforts have focused on determining the reciprocal effects of propionic acid biosynthesis on cell growth and vitamin B12 biosynthesis. An optimal precursor DMB supply strategy was proposed on the basis of the results of AdoCbi biosynthesis analysis. EBAB was employed to produce vitamin B12 using the propionic acid and DMB control method. Repeated fermentative properties in terms of substrate consumption, cell growth, and product biosynthesis were also discussed.
2. Materials and methods 2.1. Microorganisms and medium P. freudenreichii CICC 10019 was obtained from the Chinese Industrial Microorganism Conservation Center. The stock culture was incubated in deep agar slants and then stored at 4 ◦ C. The culture was transferred to new agar monthly. The preculture medium was composed of the following (per liter of deionized water): glucose, 35 g; corn steep liquor (CSL), 21 g; ammonium sulfate, 5 g; potassium dihydrogen phosphate, 4 g; and cobalt chloride, 0.005 g. The pH before autoclaving was within 6.8–7.0 (adjusted by 12% ammonia solution). The stock sugar solution was autoclaved separately before mixing with the rest of the medium. The fermentation medium was composed of the following (per liter of deionized water): glucose, 60 g; CSL, 40 g; potassium dihydrogen phosphate, 4.6 g; and cobalt chloride, 0.0127 g. The pH before autoclaving was within 6.8–7.0. Glucose was autoclaved separately for medium preparation.
2.2. Propionic acid inhibition fermentation with the EBAB system Four growth conditions without DMB addition were used in this study to examine the effect of propionic acid biosynthesis on vitamin B12 biosynthesis. The fermentation medium was prepared with different propionic acid concentrations ranging from 0 to 10 g/L, 10 to 20 g/L, and 20 to 30 g/L, respectively, using the EBAB system. The medium with propionic acid but not regulated by the EBAB system was set as a control. The EBAB columns were made of three glass columns packed with Duolite A30 resin; each column was equipped with a stainless steel wire mesh at both ends. A schematic of the columns is shown in Fig. 1, and a detailed description of the EBAB system can be found in the study by Wang et al. (2012a). Sampling and analysis of cell growth and AdoCbi biosynthesis (for vitamin B12 concentration determination) were regularly analyzed throughout the fermentation process.
2.3. DMB addition fermentation in shake flasks Seed culture grown at 30 ◦ C for 24 h and was used to provide 10% (v/v) inocula for fermentation at 30 ◦ C. Five cultivations (50 mL) were employed in a fermentation broth supplemented with 0.9 mg/L DMB solution at 0, 24, 48, 72, and 96 h of fermentation to examine the effects of DMB on intermediate and vitamin B12 biosyntheses. The medium without DMB was set as a control. Accordingly, propionic acid control was dismissed after optimizing DMB fermentation. The samples were examined at specific time intervals to determine cell mass, residual sugars, and products, including AdoCbi and vitamin B12. 2.4. EBAB repeated-batch fermentation Repeated-batch fermentation was performed in the EBAB system on the basis of the optimal approach using the propionic acid and DMB control strategy. Through this process, the fermentation broth with free cells was circulated through the EBA column when propionic acid was separated in the expanded bed column by the semi-continuous mode at specific time intervals. The expanded bed was operated wherein the fermentation broth flowed with a rate of 5 L/h. The columns were alternated and switched to their corresponding valves to repeatedly operate the circulation system after the resin reached saturation. Vitamin B12 was obtained according to the intermediate biosynthesis level; DMB was added to the fermenter when necessary. Afterward, 90% volume of Propionibacterium cells in the fermenter was removed for vitamin B12 separation and purification. Consequently, fresh medium was shifted to the fermenter together with the remaining 10% broth for repeated fermentation. This cycle was repeated five times to obtain high production and yield in the reactor system. Fermentation kinetics was studied, and the total liquid volume in each batch was 1.5 L, including 200 mL in EBAB. 2.5. Analytical methods Cell growth was determined with a spectrophotometer (UV721, Shanghai Precision & Scientific Instrument, China) at a wavelength of 600 nm. Before measuring cell concentrations, 1 mL of broth was centrifuged at 10,000 × g for 10 min, the supernatant was removed, and the cell pellets were resuspended in 1 mL of phosphate buffer. The concentrations of the principal fermentation products (AdoCbi and vitamin B12) were determined through HPLC as previously described by Li et al. (2008a) with slight modifications. Biosynthetic intermediate AdoCbi and commercial vitamin B12 were used in this study as standards for quantitation. Adocbi was synthesized from dicyanocobinamide [(CN)2 Cbi] using homogeneous CobA and CobU enzymes as previously described by O’Toole and Escalante-Semerena (1995). The broth sample (25 mL) was added with 2.5 mL of 8% (w/v) NaNO2 and 2.5 mL of glacial acetic acid. The mixture was boiled for 30 min and then filtered. The upper aqueous phase was injected into a Waters (Milford, MA) 2695 system automated gradient controller, a Beckman C18 column (5 m, 4.6 m × 25 cm) with a flow rate of 1.0 mL/min, and a 2996 Diode Array Detector (Waters Corporation, Milford, MA) in full wavelength at 25 ◦ C. The mobile phase was 250 mmol phosphoric acid/acetonitrile (30/70, v/v). The fermentation byproduct propionic acid was analyzed by HPLC using the Beckman C18 column, with 0.005 M H2 SO4 as the mobile phase at a flow rate of 0.6 mL/min and a wavelength of 215 nm at 50 ◦ C. Commercially available propionic acid was used as external standard.
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Fig. 1. Schematic diagram of the EBAB system used for propionic acid inhibition studies and repeated batch fermentations.
3. Results and discussion 3.1. Effect of controlling propionic acid concentration on AdoCbi production With regard to multi-product biosynthesis by mono-strain fermentation, the effects of propionic acid concentration on AdoCbi production were concerned in terms of carbon flux distribution (from substrates to products). Experiments on different propionic acid concentrations (0–10, 10–20, and 20–30 g/L) were conducted using the EBAB system. Time course profiles of cell growth, product biosynthesis, and substrate consumption are described in Fig. 2. All fermentation experiments in this study successfully produced AdoCbi and propionic acid. During fermentation, propionic acid was more growth associated than AdoCbi because AdoCbi synthesis occurred later than propionic acid synthesis during cell growth. A 22 h lag phase was observed before glucose was metabolized during fermentation without propionic acid control (Fig. 2A). The substrate was rapidly consumed, and product synthesis steadily increased. After 84 h of fermentation, propionic acid concentration reached 30.9 g/L and both cell growth and AdoCbi biosynthesis decreased. This result may be attributed to feed-back inhibition. The maximum AdoCbi concentration of 40.4 mg/L was achieved after 160 h of fermentation, with a yield of 0.67 mg/g. During fermentation at 20–30 g/L propionic acid concentration, feed-back inhibition was slightly severed, and both cell accumulation and product biosynthesis increased (Fig. 2B). After 160 h of fermentation, the maximum AdoCbi concentration of 48.4 mg/L was obtained. Accordingly, the glucose consumption rate was aggravated, and the lag phase was shortened to 10 h. A similar tendency of fermentation profile was also observed, and feed-back inhibition was significantly mitigated by controlling propionic acid concentration at 10–20 g/L (Fig. 2C). Interestingly, AdoCbi biosynthesis remarkably increased from 36 to 72 h with 10–20 g/L and 72–160 h with 20–30 g/L (Fig. 2B and C). These data indicate that propionic acid concentration should be controlled at a relatively low range (e.g., 10–20 g/L) in the early stage and at a high range (e.g., 20–30 g/L) in the late stage of vitamin B12 fermentation. Excessively controlling propionic acid in broth (e.g., 0–10 g/L) accelerated
substrate consumption and cell growth but decreased vitamin B12 biosynthesis, with a maximum AdoCbi of 42.3 mg/L (Fig. 2D). Maintaining the propionic acid concentration in the broth at a low level (e.g., below 10.02 g/L) with the EBAB system yields a high propionic acid concentration since feed-back inhibition was remarkably relieved. However, this condition can decrease vitamin B12 yield (Wang et al., 2012a). In the present work, maintaining propionic acid concentration at a relatively a high range in the late fermentation stage yielded a high vitamin B12 concentration. This finding indicated that a certain degree of feed-back inhibition may be beneficial for vitamin B12 biosynthesis in the late fermentation stage. This result supports previous studies and could be considered as a supplement for vitamin B12 fermentation. 3.2. Effect of DMB addition on AdoCbi and vitamin B12 syntheses The effect of DMB addition on AdoCbi and vitamin B12 syntheses was investigated. Time course kinetics of sugar consumption and product production under different concentrations of DMB addition are described in Fig. 3. In the present work, all fermentation experiments showed that cell growth can be accomplished and that the maximum cell mass did not significantly vary (Table 1). However, product biosynthesis presented a different condition. In the fermentation experiment without DMB (Fig. 3A), AdoCbi production steadily increased and reached the maximum concentration of 40.4 mg/L in step 1 (0–84 h). In step 2, the biosynthesis underwent a stationary phase and gradually reduced. Accordingly, cell growth and glucose consumption also decreased. Feed-back inhibition occurred in the biosynthesis of AdoCbi when DMB was not added. Similar results were also reported by Wang et al. (2010) in vitamin B12 aerobic bioprocess. With regard to DMB addition at different times, these bioprocesses should be divided into two phases: AdoCbi biosynthesis phase and vitamin B12 biosynthesis phase. The addition of DMB decreased AdoCbi concentration in the broth and increased vitamin B12 concentration. In the fermentation experiment with DMB addition at 0 and 24 h (Fig. 3B and C), the obtained vitamin B12 concentrations were 6.5 and 5.2 mg/L, respectively. As the time of DMB incorporation was delayed, AdoCbi production significantly
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Fig. 2. Time course profiles for vitamin B12 production with different propionic acid concentrations in EBAB. (A) Without controlling propionic acid concentration; (B) propionic acid concentrations controlled to 20–30 g/L; (C) propionic acid concentrations controlled to 10–20 g/L and (D) propionic acid concentrations controlled to 0–10 g/L.
increased and vitamin B12 biosynthesis correspondingly increased (Fig. 3D and E). The vitamin B12 concentration peaked at 42.6 mg/L upon the addition of DMB at 84 h and then decreased. As illustrated in Fig. 3F, only 31.5 mg/L of vitamin B12 was obtained when DMB was added at 96 h. This finding may due to the phenomenon that AdoCbi synthetic capacity reached the maximum level and then decreased by feed-back inhibition. In each DMB addition strategy of fermentation, the yield of vitamin B12 from AdoCbi was maintained in the range of 0.97–1.04 mg/mg (Table 1). This result indicates that AdoCbi can be used as a marker for quantization in vitamin B12 fermentation before DMB incorporation. Feed-back inhibition on intracellular AdoCbi biosynthesis in vitamin B12 fermentation must also be considered. DMB addition should depend on the AdoCbi biosynthesis level. The addition time of DMB significantly influenced product biosynthesis since DMB might either induce low amounts of AdoCbi to vitamin B12 before the best time or convert to other pseudo-cobalamins (e.g., 2-methyladenine as the lower ligand of vitamin B12) in a delay, resulting in a low amount of vitamin B12 concentration (EscalanteSemerena, 2007). Therefore, the concentration of vitamin B12 increased upon the addition of DMB. Vitamin B12 synthesis was
more influenced by DMB than by propionic acid because of the fluctuating concentration (5.2–42.6 mg/L). However, both DMB and propionic acid should be considered to further optimize vitamin B12 fermentation. 3.3. Repeated fermentation by the propionic acid and DMB addition control strategy with the EBAB system Basing on the above results, we employed the propionic acid and DMB control strategy for repeated vitamin B12 fermentation by the EBAB system. The vitamin B12 concentration in the time course was identified on the basis of the AdoCbi concentration. The kinetic profile of the repeated batch fermentation is shown in Fig. 4 and Table 2. In the first batch, a 12 h lag phase was observed, and then cell accumulation rapidly increased upon controlling the propionic acid concentration at 10–20 g/L (relatively low level to get maximum cell growth) in the first stage. The maximum cell growth was attained after 84 h of fermentation. In the second stage, the propionic acid concentration increased to 20–30 g/L (sufficient high level to get maximum productivity of vitamin B12) by regulating the EBAB system, and the cell growth was slightly inhibited
Table 1 Effect of different supplement time points of DMB on the cell growth, AdoCbi biosynthesis production in the bioprocess by P. freudenreichii. DMB supplemented strategy at different time
Residue sugar (g/L)
0 h addition 24 h addition 60 h addition 84 h addition 96 h addition
12.3 8.5 4.6 0 0
a b
± ± ± ± ±
0.05 0.06 0.08 0.02 0.07
Maximum cell mass (OD600nm ) 22.5 23.7 23.9 24.7 24.4
± ± ± ± ±
0.04 0.05 0.03 0.08 0.02
AdoCbi titer (mg/L) when DMB added 0.0 5.1 16.6 42.5 32.5
± ± ± ± ±
0.06b 0.11 0.19 0.16 0.12
Maximum VB 12 titer (mg/L) 6.5 5.2 16.5 42.6 31.5
± ± ± ± ±
0.14 0.16 0.17 0.19 0.16
The yields were calculated based on the maximum vitamin B12 (mg/L)/AdoCbi (mg/L), which concentration at the time of DMB supplemented. AdoCbi titer with 0 h addition of DMB was approximately 0 mg/L since the data was quantitated before the addition of DMB.
Yield (mg/mg)a 0.97 1.04 0.99 1.00 0.97
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Fig. 3. Time course profiles for vitamin B12 production with a gradual DMB incorporation strategy in flask shakes. (A) Without addition of DMB; (B) 0 h addition of DMB; (C) 24 h addition of DMB; (D) 60 h addition of DMB; (E) 84 h addition of DMB and (F) 96 h addition of DMB.
for vitamin B12 biosynthesis. DMB incorporation was depended on AdoCbi production and added eligible. Consequently, a high vitamin B12 concentration of 58.8 mg/L was achieved with a production of 0.37 mg/L/h. Repeated batch cultures were performed using the method described in Section 2.4. The lag phase was eliminated in
batch 2 because the cells from batch 1 were reused to inoculate the next batch. The carbon resource was consumed immediately, and the fermentation time shortened from 160 h (batch 1) to 148 h (batch 2). No lag phase was observed in batch 3. The fermentation time was only 127 h. The fermentation efficiency was improved
Fig. 4. Repeated batch fermentations for vitamin B12 production with propionic acid and DMB control strategy in EBAB. Fermentation conditions: 30 ◦ C, pH 7.0 and standing cultivation. Initial glucose concentration was 60 g/L.
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Table 2 Expanded bed adsorption bioreactor (EBAB) repeated batch fermentation by propionic acid and DMB control strategy.a Batch number
Fermentation time (h)
Maximum cell mass (OD600nm )
1 2 3 4 5
160 148 127 101 94
28.5 30.5 33.3 36.8 34.2
a b
± ± ± ± ±
0.07 0.05 0.02 0.06 0.08
Maximum vitamin B12 titer (mg/L) 58.8 58.9 58.5 59.5 53.9
± ± ± ± ±
0.15 0.18 0.11 0.14 0.16
Productivity (mg/L/h) 0.37 0.40 0.46 0.59 0.57
± ± ± ± ±
0.15 0.18 0.11 0.13 0.19
Yield (mg/g)b 0.98 0.98 0.98 0.98 0.90
Initial substrate concentration was 60 g/L. The yields were calculated based on the vitamin B12 (mg/L)/consumed glucose (g/L).
in the following repeated culture cycles of batches 4 and 5. As described in Table 2, the fermentation time significantly shortened from 160 h (for batch 1) to 94 h (for batch 5) and the productivity of vitamin B12 was improved accordingly. The cell mass continuously increased in batches 1 to 4 and then slightly decreased in batch 5 as EBAB proceeded. This result may be attributed to the limitation of the inhibitors produced in the broth. This explanation was supported by a previous study (Zhang et al., 2014a). A similar tendency was obtained in vitamin B12 concentration and yield. Although the vitamin B12 concentration fluctuated from batches 4 to 5, the production remained relatively high. As a result, the maximum vitamin B12 concentration of 59.5 mg/L was achieved and the production increased from 0.37 mg/L/h (batch 1) to 0.59 mg/L/h (batch 4). This result indicates that the existing viable cells in batch 5 were enough to sustain fermentation. Similar results were reported in repeated lactic acid fermentation (Zhang et al., 2014b). The inhibitory effects of propionic acid and DMB on vitamin B12 biosynthesis can be reduced by the EBAB process through a precise byproduct and precursor control strategy. During this process, P. freudenreichii cells were reused. As a result, the lag phase was eliminated during the subsequent batch culture. Therefore, the productivity of vitamin B12 remarkably increased. In the present industrial fermentation, DMB as a medium composition is often added in the initial fermentation, and the broth containing DMB cannot be inoculated as seed liquid into the next batch. Therefore, DMB addition hinders continuous fermentation. The present study realized continuous vitamin B12 fermentation by repeated batch mode. In addition, from the engineering point of view, it is easy to accomplish for controlling byproduct concentration real-time online by using EBAB system and it could answer why use this system for B12 production in this study. Metabolic pathway for vitamin B12 biosynthesis in P. freudenreichii which includes the route involving the AdoCbi, propionic acid and DMB is shown in Supplementary Fig. S1. Propionyl-CoA and Succinyl-CoA are important nodes for propionic acid and vitamin B12 synthesis, respectively. In this study, partitioning carbon fluxes between propionic acid and vitamin B12 synthesis was occurred. When propionic acid concentration was controlled at a relatively low level, EBAB process might enabled more nutrients to the route of propionic acid synthesis instead of vitamin B12 production. While propionic acid concentration was controlled at a relatively high level, the route of vitamin B12 synthesis was strengthened. Based on these results, a combination of controlling the propionic acid concentration strategy was devised for improving vitamin B12 production. Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec.2014.11.019. Many microorganisms have been employed to produce vitamin B12 with various substrates under anaerobic conditions. These microorganisms include Butyribacterium methylotrophicum with methanol (1.7 mg/L), P. freudenreichii with lactate (3.6 mg/L), and Bacillus megaterium (B. megaterium) with Luria–Bertani (8.5 g/L)
(Bykhovskii et al., 1998; Piao et al., 2004; Biedendieck et al., 2010). A high production of approximately 60 mg/L with glucose and DMB by Propionibacterium subsp. Shermanii (Bykhovskii et al., 1998) was reported. In the present study, a comparable vitamin B12 concentration of 59.5 mg/L was obtained. Compared with previous studies in our laboratory, the present study improved vitamin B12 production from 0.33 mg/L/h to 0.59 mg/L/h (Wang et al., 2012a). Many studies have attempted to optimize culture medium composition and thus improve vitamin B12 production. Precursor addition (e.g., betaine) and statistical experimental design have been widely and deeply studied from different aspects with aerobic microbes (Kim et al., 2003; Li et al., 2008b; Kosmider et al., 2012). Vitamin B12 production has also been improved by optimizing cultivation parameters, such as dissolved oxygen content (Wang et al., 2010), pH (Li et al., 2008a), and medium components (Li et al., 2008c). ␦-Aminolevulinate (ALA) is an important precursor of vitamin B12; ALA can also be used as an indicator of vitamin B12 production in fermentation (Kang et al., 2012). However, the metabolic pathway from ALA to vitamin B12 is more distant from than that from AdoCbi to vitamin B12 (Scott and Roessner, 2002). In addition, a certain stream branch exists. Therefore, AdoCbi is a suitable marker to determine vitamin B12 biosynthesis during fermentation. Comparing with current research methods, the most valuable spot in this work emphasize multi-angular optimization combine byproduct control, principal product biosynthesis and fermentation reactor. Metabolic engineering has been used as a powerful tool to improve anaerobic vitamin B12 fermentation. Recently, Biedendieck et al. (2010) have reported an antisense RNA strategy for modifying the anaerobic vitamin B12 synthesis pathway in B. megaterium. This strategy can significantly increase the intracellular concentration of vitamin B12. Metabolic engineering methods, such as 13 C-labeled substrate, have also been used for carbon flux analysis in Pseudomonas denitrificans (Wang et al., 2012). The novel control strategy with EBAB developed in this study may be applicable to other biotechnological processes, such as methane and vitamin B12 by methanogens (Yang et al., 2004) or folate and vitamin B12 by P. freudenreichii (Wyk et al., 2011). 4. Conclusions An efficient and economic strategy for vitamin B12 fermentation was developed by cultivation process optimization. Controlling propionic acid concentration 10–20 g/L in the early stage and 20–30 g/L in the late stage can mitigate feed-back inhibition and improve vitamin B12 biosynthesis using the EBAB system. Intracellular intermediate feed-back inhibition occurred and can be severed by optimal DMB incorporation. In addition, optimizing the cultivation process with the propionic acid and DMB control strategy realized vitamin B12 repeated fermentation and increased the production from 0.37 mg/L/h (batch 1)–0.59 mg/L/h (batch 5). This strategy obtained in this study could be a promising option for industrial application of vitamin B12 fermentation.
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Acknowledgements We are grateful for financial assistance received from the National Basic Research Program of China (2013CB733604), the National Natural Science Foundation of China (21302039), the Natural Science Foundation of Hebei Province (B2014208136) and the Hebei Province Department of Education Fund (QN2014188). References Biedendieck, R., Malten, M., Barg, H., Bunk, B., Martens, J.H., Deery, E., Leech, H., Warren, M.J., John, D., 2010. Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium. Microb. Biotechnol. 3, 24–37. Bovayal, P., Corre, C., Madec, M.N., 1994. Propionic acid production in a membrane bioreactor. Enzyme Microb. Technol. 16, 883–888. Bykhovskii, V., Zaitseva, N.I., Eliseev, A.A., 1998. Tetrapyrroles: diversity, biosynthesis, biotechnology. Prikl. Biokhim. Mikrobiol. 34, 3–21. Chandra, T., Brown, K.L., 2008. Vitamin B12 and ␣-ribonucleosides. Tetrahedron 64, 9–38. Escalante-Semerena, J.C., 2007. Conversion of cobinamide into adenosylcobamide in bacteria and archaea. J. Bacteriol. 189, 4555–4560. Feng, X.H., Chen, F., Xu, H., Wu, B., Li, H., Li, S., Ouyang, P.K., 2011. Green and economical production of propionic acid by Propionibacterium freudenreichii CCTCC M207015 in plant fibrous-bed bioreactor. Bioresour. Technol. 102, 6141–6146. Haddadin, M.S.Y., Abu-Reesh, I.M., Haddadin, F.A.S., Robinson, R.K., 2001. Utilisation of tomato pomace as a substrate for the production of vitamin B12-a preliminary appraisal. Bioresour. Technol. 78, 225–230. Kang, Z., Zhang, J.L., Zhou, J.W., Qi, Q.S., Du, G.C., Chen, J., 2012. Recent advances in microbial production of ␦-aminolevulinic acid and vitamin B12. Biotechnol. Adv. 30, 1533–1542. Kim, S.K., Choi, K.H., Kim, Y.C., 2003. Effect of acute betaine administration on hepatic metabolism of S-amino acids in rats and mice. Biochem. Pharmacol. 65, 1565–1574. Kosmider, A., Bialas, W., Kubiak, P., Drozdzynska, A., Czaczyk, K., 2012. Vitamin B12 production from crude glycerol by Propionibacterium freudenreichii sp. shermanii: optimization of medium composition through statistical experimental designs. Bioresour. Technol. 105, 128–133. Li, K.T., Liu, D.H., Li, Y.L., Chu, J., Wang, Y.H., Zhuang, Y.P., Zhang, S.L., 2008a. An effective and simplified pH-stat control strategy for the industrial fermentation of vitamin B12 by Pseudomonas denitrificans. Bioprocess. Biosyst. Eng. 31, 605–610. Li, K.T., Liu, D.H., Li, Y.L., Chu, J., Wang, Y.H., Zhuang,.Y.P., Zhang, S.L., 2008b. Improved large-scale production of vitamin B12 by Pseudomonas denitrificans with betaine feeding. Bioresour. Technol. 99, 8516–8520. Li, K.T., Liu, D.H., Li, Y.L., Chu, J., Wang, Y.H., Zhuang, Y.P., Zhang, S.L., 2008c. Influence of Zn2+ , Co2+ and dimethylbeidazole on vitamin B12 biosynthesis by Pseudomonas denitrificans. World J. Microbiol. Biotechnol. 24, 2525–2530.
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Martens, J.H., Barg, H., Warren, M.J., Jahn, D., 2002. Microbial production of vitamin B12. Appl. Microbiol. Biotechnol. 58, 275–285. Marwaha, S.S., Sethi, R.P., Kennedy, J.F., Rakesh, K., 1983. Simulation of fermentation conditions for vitamin B12 biosynthesis from whey. Biotechnol. Adv. 11, 481–493. Murooka, Y., Piao, Y., Kiatpapan, P., Yamashita, M., 2005. Production of tetrapyrrole compounds and vitamin B12 using genetically engineering of Propionibacterium freudenreichii. An overview. Lait 85, 9–22. O’Toole, G.A., Escalante-Semerena, J.C., 1995. Purification and characterization of the bifunctional CobU enzyme of Salmonella typhimurium LT2, Evidence for a CobU-GMP intermediate. J. Biol. Chem. 270, 23560–23569. Piao, Y.Z., Yamashita, M., Kawaraichi, N., Asegawa, R., Ono, H., Murooka, Y., 2004. Production of Vitamin B12 in genetically engineered Propionibacterium freudenreichii. J. Biosci. Bioeng. 98, 167–173. Scott, A.I., Roessner, C.A., 2002. Biosynthesis of cobalamin (vitamin B12). Biochem. Soc. Trans. 30, 613–620. Wang, Z.G., Huang, H.Y., Li, Y.L., Chu, J., Huang, M.Z., Zhuang, Y.P., Zhang, S.L., 2010. Improved vitamin B12 production by step-wise reduction of oxygen uptake rate under dissolved oxygen limiting level during fermentation process. Bioresour. Technol. 101, 2845–2852. Wang, P., Wang, Y.S., Su, Z.G., 2012a. Novel in situ product removal technique for simultaneous production of propionic acid and vitamin B12 by expanded bed adsorption bioreactor. Bioresour. Technol. 104, 652–659. Wang, P., Wang, Y.S., Su, Z.G., 2012b. Improvement of adenosylcobalamin production by metabolic control strategy in Propionibacterium freudenreichii. Appl. Biochem. Biotechnol. 167, 62–72. Wang, Z.J., Wang, P., Liu, Y.W., Zhang, Y.M., Chu, J., Huang, M.Z., Zhuang, Y.P., Zhang, S.L., 2012. Metabolic flux analysis of the central carbon metabolism of the industrial vitamin B12 producing strain Pseudomonas denitrificans using 13 C-labeled glucose. J. Taiwan Inst. Chem. Eng. 43, 181–187. Woodson, J.D., Escalante-Semerena, A., 2006. The cbiS gene of the archaeon methanopyrus kandleri AV19 encodes a bifunctional enzyme with adenosylcobinamide amidohydrolase and ␣-ribazole-phosphate phosphatase activities. J. Bacteriol. 188, 4227–4235. Wyk, J.V., Witthuhn, R.C., Britz, T.J., 2011. Optimisation of vitamin B12 and folate production by Propionibacterium freudenreichii strains in kefi. J. Int. Dairy 21, 69–74. Yang, Y.N., Zhang, Z.Y., Lu, J., Maekawa, T., 2004. Continuous methane fermentation and the production of vitamin B12 in a fixed-bed reactor packed with loofah. Bioresour. Technol. 92, 285–290. Zhang, Y.M., Chen, X.R., Luo, J.Q., Qi, B.K., Wan, Y.H., 2014a. An efficient process for lactic acid production from wheat straw by a newly isolated Bacillus coagulans strain IPE22. Bioresour. Technol. 158, 396–399. Zhang, Y.M., Chen, X.R., Luo, J.Q., Qi, B.K., Shen, F., Su, Y., Khan, R., Wan, Y.H., 2014b. Improving lactic acid productivity from wheat straw hydrolysates by membrane integrated repeated batch fermentation under non-sterilized conditions. Bioresour. Technol. 163, 160–166.
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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Improved propionic acid and 5,6-dimethylbenzimidazole control strategy for vitamin B12 fermentation by Propionibacterium freudenreichii Peng Wang a,b,c,∗ , Zhiwei Zhang a , Youjing Jiao a , Shouxin Liu c , Yunshan Wang d,∗∗ a
College of Chemical & Pharmaceutical Engineering, Hebei University of Science & Technology, Shijiazhuang 050018, China Hebei Research Center of Pharmaceutical and Chemical Engineering, Hebei University of Science & Technology, Shijiazhuang 050018, China c State Key Laboratory Breeding Base-Hebei Province Key Laboratory of Molecular Chemistry for Drug, Hebei University of Science & Technology, Shijiazhuang 050018, China d National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100190, China b
a r t i c l e
i n f o
Article history: Received 11 August 2014 Received in revised form 22 October 2014 Accepted 21 November 2014 Available online 29 November 2014 Keywords: Vitamin B12 Propionic acid 5,6-Dimethylbenzimidazole Feed-back inhibition Expanded bed adsorption bioreactor
a b s t r a c t An efficient fermentation-strengthening approach was developed to improve the anaerobic production of vitamin B12 by cultivation process optimization with Propionibacterium freudenreichii. The effects of the byproduct propionic acid and the precursor 5,6-dimethylbenzimidazole (DMB) on vitamin B12 biosynthesis were investigated. Byproduct inhibition experiments showed that maintaining propionic acid concentration in broth below 10–20 g/L in the early stage and 20–30 g/L in the late stage can efficiently improve vitamin B12 biosynthesis. Batch fermentation indicated the occurrence of feed-back inhibition in intracellular intermediate biosynthesis. In addition, the incorporation of the precursor DMB depended on the fermentation level of the vitamin B12 intermediate. High vitamin B12 concentration (58.8 mg/L) and production (0.37 mg/g) were obtained with an expanded bed adsorption bioreactor by using the propionic acid and DMB control method. The optimum concentration and production of 59.5 and 0.59 mg/L h for vitamin B12 production were respectively achieved after five continuous batches. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Vitamin B12 has various applications in the food and medicine industries (Wang et al., 2010) and also it is an important coenzyme in the metabolism of human tissues. Vitamin B12 can be biosynthesized in two ways: aerobic and anaerobic fermentation. Anaerobic fermentation with Propionibacterium has attracted increasing research interest because of the low-cost production of food-grade vitamin B12 (adenosylcobalamin) (Martens et al., 2002; Murooka et al., 2005; Kosmider et al., 2012). Considerable efforts have been exerted to improve vitamin B12-producing strains and vitamin B12 biosynthesis. Some studies have implemented random mutagenesis and genetic engineering method (Bykhovskii et al., 1998; Piao et al., 2004), whereas the others have attempted to optimize vitamin B12 fermentation by using various substrates
∗ Corresponding author at: College of Chemical & Pharmaceutical Engineering, Hebei University of Science & Technology, Shijiazhuang 050018, China. Tel.: +86 311 81668397. ∗∗ Corresponding author. E-mail address: [email protected] (P. Wang). http://dx.doi.org/10.1016/j.jbiotec.2014.11.019 0168-1656/© 2014 Elsevier B.V. All rights reserved.
(e.g., whey, tomato pomace, and crude glycerol) (Marwaha et al., 1983; Haddadin et al., 2001; Kosmider et al., 2012). However, the yields and productivities in the current industrial fermentation continue to significantly fluctuate since the traditional approaches result in retarded cell growth and low efficiency for vitamin B12 synthesis. Therefore, in-depth investigations focusing on cultivation process optimization are important. Propionic acid is a byproduct of the cultivation process of food-grade vitamin B12 by Propionibacterium freudenreichii; this byproduct may accumulate to high concentrations of 30–40 g/L and cause feed-back inhibition in microbial cell growth. Among the methods employed to prevent propionic acid feed-back inhibition during fermentation, in situ product removal (ISPR) has shown advantages in improving production. This technique may also be used for large-scale production. Various ISPR processes have been reported using membrane-integrated bioreactors, plant fibrousbed bioreactors, and expanded bed adsorption bioreactors (EBABs) (Bovayal et al., 1994; Feng et al., 2011; Wang et al., 2012a). However, most studies focused only on the increased production and/or yield of the extracellular product (propionic acid). Little is known about the effects of ISPR systems on intracellular product (vitamin B12) biosynthesis.
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In addition, in the industrial anaerobic fermentation of vitamin B12, exogenous 5,6-dimethylbenzimidazole (DMB) is usually used as a precursor to promote vitamin B12 biosynthesis (Woodson and Escalante-Semerena, 2006; Chandra and Brown, 2008). However, the main obstacle in this process is the addition of DMB. Current studies have used microexamination to determine the microbial growth state. However, this method is influenced by human factors, leading to an unsteady fermentation concentration. With regard to the fermentation mechanism, a key intermediate (adenosylcobalamide, AdoCbi) of vitamin B12 has been isolated and characterized in our laboratory. This compound can be used to determine the condition of vitamin B12 biosynthesis corresponding to the molecular weight (Wang et al., 2012b). However, whether or not AdoCbi synthesis induces feed-back inhibition remains unknown, that is needed to be further clarified by the analysis of AdoCbi biosynthesis mechanism. This study aims to develop an economic and efficient approach to improve vitamin B12 production. Research efforts have focused on determining the reciprocal effects of propionic acid biosynthesis on cell growth and vitamin B12 biosynthesis. An optimal precursor DMB supply strategy was proposed on the basis of the results of AdoCbi biosynthesis analysis. EBAB was employed to produce vitamin B12 using the propionic acid and DMB control method. Repeated fermentative properties in terms of substrate consumption, cell growth, and product biosynthesis were also discussed.
2. Materials and methods 2.1. Microorganisms and medium P. freudenreichii CICC 10019 was obtained from the Chinese Industrial Microorganism Conservation Center. The stock culture was incubated in deep agar slants and then stored at 4 ◦ C. The culture was transferred to new agar monthly. The preculture medium was composed of the following (per liter of deionized water): glucose, 35 g; corn steep liquor (CSL), 21 g; ammonium sulfate, 5 g; potassium dihydrogen phosphate, 4 g; and cobalt chloride, 0.005 g. The pH before autoclaving was within 6.8–7.0 (adjusted by 12% ammonia solution). The stock sugar solution was autoclaved separately before mixing with the rest of the medium. The fermentation medium was composed of the following (per liter of deionized water): glucose, 60 g; CSL, 40 g; potassium dihydrogen phosphate, 4.6 g; and cobalt chloride, 0.0127 g. The pH before autoclaving was within 6.8–7.0. Glucose was autoclaved separately for medium preparation.
2.2. Propionic acid inhibition fermentation with the EBAB system Four growth conditions without DMB addition were used in this study to examine the effect of propionic acid biosynthesis on vitamin B12 biosynthesis. The fermentation medium was prepared with different propionic acid concentrations ranging from 0 to 10 g/L, 10 to 20 g/L, and 20 to 30 g/L, respectively, using the EBAB system. The medium with propionic acid but not regulated by the EBAB system was set as a control. The EBAB columns were made of three glass columns packed with Duolite A30 resin; each column was equipped with a stainless steel wire mesh at both ends. A schematic of the columns is shown in Fig. 1, and a detailed description of the EBAB system can be found in the study by Wang et al. (2012a). Sampling and analysis of cell growth and AdoCbi biosynthesis (for vitamin B12 concentration determination) were regularly analyzed throughout the fermentation process.
2.3. DMB addition fermentation in shake flasks Seed culture grown at 30 ◦ C for 24 h and was used to provide 10% (v/v) inocula for fermentation at 30 ◦ C. Five cultivations (50 mL) were employed in a fermentation broth supplemented with 0.9 mg/L DMB solution at 0, 24, 48, 72, and 96 h of fermentation to examine the effects of DMB on intermediate and vitamin B12 biosyntheses. The medium without DMB was set as a control. Accordingly, propionic acid control was dismissed after optimizing DMB fermentation. The samples were examined at specific time intervals to determine cell mass, residual sugars, and products, including AdoCbi and vitamin B12. 2.4. EBAB repeated-batch fermentation Repeated-batch fermentation was performed in the EBAB system on the basis of the optimal approach using the propionic acid and DMB control strategy. Through this process, the fermentation broth with free cells was circulated through the EBA column when propionic acid was separated in the expanded bed column by the semi-continuous mode at specific time intervals. The expanded bed was operated wherein the fermentation broth flowed with a rate of 5 L/h. The columns were alternated and switched to their corresponding valves to repeatedly operate the circulation system after the resin reached saturation. Vitamin B12 was obtained according to the intermediate biosynthesis level; DMB was added to the fermenter when necessary. Afterward, 90% volume of Propionibacterium cells in the fermenter was removed for vitamin B12 separation and purification. Consequently, fresh medium was shifted to the fermenter together with the remaining 10% broth for repeated fermentation. This cycle was repeated five times to obtain high production and yield in the reactor system. Fermentation kinetics was studied, and the total liquid volume in each batch was 1.5 L, including 200 mL in EBAB. 2.5. Analytical methods Cell growth was determined with a spectrophotometer (UV721, Shanghai Precision & Scientific Instrument, China) at a wavelength of 600 nm. Before measuring cell concentrations, 1 mL of broth was centrifuged at 10,000 × g for 10 min, the supernatant was removed, and the cell pellets were resuspended in 1 mL of phosphate buffer. The concentrations of the principal fermentation products (AdoCbi and vitamin B12) were determined through HPLC as previously described by Li et al. (2008a) with slight modifications. Biosynthetic intermediate AdoCbi and commercial vitamin B12 were used in this study as standards for quantitation. Adocbi was synthesized from dicyanocobinamide [(CN)2 Cbi] using homogeneous CobA and CobU enzymes as previously described by O’Toole and Escalante-Semerena (1995). The broth sample (25 mL) was added with 2.5 mL of 8% (w/v) NaNO2 and 2.5 mL of glacial acetic acid. The mixture was boiled for 30 min and then filtered. The upper aqueous phase was injected into a Waters (Milford, MA) 2695 system automated gradient controller, a Beckman C18 column (5 m, 4.6 m × 25 cm) with a flow rate of 1.0 mL/min, and a 2996 Diode Array Detector (Waters Corporation, Milford, MA) in full wavelength at 25 ◦ C. The mobile phase was 250 mmol phosphoric acid/acetonitrile (30/70, v/v). The fermentation byproduct propionic acid was analyzed by HPLC using the Beckman C18 column, with 0.005 M H2 SO4 as the mobile phase at a flow rate of 0.6 mL/min and a wavelength of 215 nm at 50 ◦ C. Commercially available propionic acid was used as external standard.
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Fig. 1. Schematic diagram of the EBAB system used for propionic acid inhibition studies and repeated batch fermentations.
3. Results and discussion 3.1. Effect of controlling propionic acid concentration on AdoCbi production With regard to multi-product biosynthesis by mono-strain fermentation, the effects of propionic acid concentration on AdoCbi production were concerned in terms of carbon flux distribution (from substrates to products). Experiments on different propionic acid concentrations (0–10, 10–20, and 20–30 g/L) were conducted using the EBAB system. Time course profiles of cell growth, product biosynthesis, and substrate consumption are described in Fig. 2. All fermentation experiments in this study successfully produced AdoCbi and propionic acid. During fermentation, propionic acid was more growth associated than AdoCbi because AdoCbi synthesis occurred later than propionic acid synthesis during cell growth. A 22 h lag phase was observed before glucose was metabolized during fermentation without propionic acid control (Fig. 2A). The substrate was rapidly consumed, and product synthesis steadily increased. After 84 h of fermentation, propionic acid concentration reached 30.9 g/L and both cell growth and AdoCbi biosynthesis decreased. This result may be attributed to feed-back inhibition. The maximum AdoCbi concentration of 40.4 mg/L was achieved after 160 h of fermentation, with a yield of 0.67 mg/g. During fermentation at 20–30 g/L propionic acid concentration, feed-back inhibition was slightly severed, and both cell accumulation and product biosynthesis increased (Fig. 2B). After 160 h of fermentation, the maximum AdoCbi concentration of 48.4 mg/L was obtained. Accordingly, the glucose consumption rate was aggravated, and the lag phase was shortened to 10 h. A similar tendency of fermentation profile was also observed, and feed-back inhibition was significantly mitigated by controlling propionic acid concentration at 10–20 g/L (Fig. 2C). Interestingly, AdoCbi biosynthesis remarkably increased from 36 to 72 h with 10–20 g/L and 72–160 h with 20–30 g/L (Fig. 2B and C). These data indicate that propionic acid concentration should be controlled at a relatively low range (e.g., 10–20 g/L) in the early stage and at a high range (e.g., 20–30 g/L) in the late stage of vitamin B12 fermentation. Excessively controlling propionic acid in broth (e.g., 0–10 g/L) accelerated
substrate consumption and cell growth but decreased vitamin B12 biosynthesis, with a maximum AdoCbi of 42.3 mg/L (Fig. 2D). Maintaining the propionic acid concentration in the broth at a low level (e.g., below 10.02 g/L) with the EBAB system yields a high propionic acid concentration since feed-back inhibition was remarkably relieved. However, this condition can decrease vitamin B12 yield (Wang et al., 2012a). In the present work, maintaining propionic acid concentration at a relatively a high range in the late fermentation stage yielded a high vitamin B12 concentration. This finding indicated that a certain degree of feed-back inhibition may be beneficial for vitamin B12 biosynthesis in the late fermentation stage. This result supports previous studies and could be considered as a supplement for vitamin B12 fermentation. 3.2. Effect of DMB addition on AdoCbi and vitamin B12 syntheses The effect of DMB addition on AdoCbi and vitamin B12 syntheses was investigated. Time course kinetics of sugar consumption and product production under different concentrations of DMB addition are described in Fig. 3. In the present work, all fermentation experiments showed that cell growth can be accomplished and that the maximum cell mass did not significantly vary (Table 1). However, product biosynthesis presented a different condition. In the fermentation experiment without DMB (Fig. 3A), AdoCbi production steadily increased and reached the maximum concentration of 40.4 mg/L in step 1 (0–84 h). In step 2, the biosynthesis underwent a stationary phase and gradually reduced. Accordingly, cell growth and glucose consumption also decreased. Feed-back inhibition occurred in the biosynthesis of AdoCbi when DMB was not added. Similar results were also reported by Wang et al. (2010) in vitamin B12 aerobic bioprocess. With regard to DMB addition at different times, these bioprocesses should be divided into two phases: AdoCbi biosynthesis phase and vitamin B12 biosynthesis phase. The addition of DMB decreased AdoCbi concentration in the broth and increased vitamin B12 concentration. In the fermentation experiment with DMB addition at 0 and 24 h (Fig. 3B and C), the obtained vitamin B12 concentrations were 6.5 and 5.2 mg/L, respectively. As the time of DMB incorporation was delayed, AdoCbi production significantly
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Fig. 2. Time course profiles for vitamin B12 production with different propionic acid concentrations in EBAB. (A) Without controlling propionic acid concentration; (B) propionic acid concentrations controlled to 20–30 g/L; (C) propionic acid concentrations controlled to 10–20 g/L and (D) propionic acid concentrations controlled to 0–10 g/L.
increased and vitamin B12 biosynthesis correspondingly increased (Fig. 3D and E). The vitamin B12 concentration peaked at 42.6 mg/L upon the addition of DMB at 84 h and then decreased. As illustrated in Fig. 3F, only 31.5 mg/L of vitamin B12 was obtained when DMB was added at 96 h. This finding may due to the phenomenon that AdoCbi synthetic capacity reached the maximum level and then decreased by feed-back inhibition. In each DMB addition strategy of fermentation, the yield of vitamin B12 from AdoCbi was maintained in the range of 0.97–1.04 mg/mg (Table 1). This result indicates that AdoCbi can be used as a marker for quantization in vitamin B12 fermentation before DMB incorporation. Feed-back inhibition on intracellular AdoCbi biosynthesis in vitamin B12 fermentation must also be considered. DMB addition should depend on the AdoCbi biosynthesis level. The addition time of DMB significantly influenced product biosynthesis since DMB might either induce low amounts of AdoCbi to vitamin B12 before the best time or convert to other pseudo-cobalamins (e.g., 2-methyladenine as the lower ligand of vitamin B12) in a delay, resulting in a low amount of vitamin B12 concentration (EscalanteSemerena, 2007). Therefore, the concentration of vitamin B12 increased upon the addition of DMB. Vitamin B12 synthesis was
more influenced by DMB than by propionic acid because of the fluctuating concentration (5.2–42.6 mg/L). However, both DMB and propionic acid should be considered to further optimize vitamin B12 fermentation. 3.3. Repeated fermentation by the propionic acid and DMB addition control strategy with the EBAB system Basing on the above results, we employed the propionic acid and DMB control strategy for repeated vitamin B12 fermentation by the EBAB system. The vitamin B12 concentration in the time course was identified on the basis of the AdoCbi concentration. The kinetic profile of the repeated batch fermentation is shown in Fig. 4 and Table 2. In the first batch, a 12 h lag phase was observed, and then cell accumulation rapidly increased upon controlling the propionic acid concentration at 10–20 g/L (relatively low level to get maximum cell growth) in the first stage. The maximum cell growth was attained after 84 h of fermentation. In the second stage, the propionic acid concentration increased to 20–30 g/L (sufficient high level to get maximum productivity of vitamin B12) by regulating the EBAB system, and the cell growth was slightly inhibited
Table 1 Effect of different supplement time points of DMB on the cell growth, AdoCbi biosynthesis production in the bioprocess by P. freudenreichii. DMB supplemented strategy at different time
Residue sugar (g/L)
0 h addition 24 h addition 60 h addition 84 h addition 96 h addition
12.3 8.5 4.6 0 0
a b
± ± ± ± ±
0.05 0.06 0.08 0.02 0.07
Maximum cell mass (OD600nm ) 22.5 23.7 23.9 24.7 24.4
± ± ± ± ±
0.04 0.05 0.03 0.08 0.02
AdoCbi titer (mg/L) when DMB added 0.0 5.1 16.6 42.5 32.5
± ± ± ± ±
0.06b 0.11 0.19 0.16 0.12
Maximum VB 12 titer (mg/L) 6.5 5.2 16.5 42.6 31.5
± ± ± ± ±
0.14 0.16 0.17 0.19 0.16
The yields were calculated based on the maximum vitamin B12 (mg/L)/AdoCbi (mg/L), which concentration at the time of DMB supplemented. AdoCbi titer with 0 h addition of DMB was approximately 0 mg/L since the data was quantitated before the addition of DMB.
Yield (mg/mg)a 0.97 1.04 0.99 1.00 0.97
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Fig. 3. Time course profiles for vitamin B12 production with a gradual DMB incorporation strategy in flask shakes. (A) Without addition of DMB; (B) 0 h addition of DMB; (C) 24 h addition of DMB; (D) 60 h addition of DMB; (E) 84 h addition of DMB and (F) 96 h addition of DMB.
for vitamin B12 biosynthesis. DMB incorporation was depended on AdoCbi production and added eligible. Consequently, a high vitamin B12 concentration of 58.8 mg/L was achieved with a production of 0.37 mg/L/h. Repeated batch cultures were performed using the method described in Section 2.4. The lag phase was eliminated in
batch 2 because the cells from batch 1 were reused to inoculate the next batch. The carbon resource was consumed immediately, and the fermentation time shortened from 160 h (batch 1) to 148 h (batch 2). No lag phase was observed in batch 3. The fermentation time was only 127 h. The fermentation efficiency was improved
Fig. 4. Repeated batch fermentations for vitamin B12 production with propionic acid and DMB control strategy in EBAB. Fermentation conditions: 30 ◦ C, pH 7.0 and standing cultivation. Initial glucose concentration was 60 g/L.
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Table 2 Expanded bed adsorption bioreactor (EBAB) repeated batch fermentation by propionic acid and DMB control strategy.a Batch number
Fermentation time (h)
Maximum cell mass (OD600nm )
1 2 3 4 5
160 148 127 101 94
28.5 30.5 33.3 36.8 34.2
a b
± ± ± ± ±
0.07 0.05 0.02 0.06 0.08
Maximum vitamin B12 titer (mg/L) 58.8 58.9 58.5 59.5 53.9
± ± ± ± ±
0.15 0.18 0.11 0.14 0.16
Productivity (mg/L/h) 0.37 0.40 0.46 0.59 0.57
± ± ± ± ±
0.15 0.18 0.11 0.13 0.19
Yield (mg/g)b 0.98 0.98 0.98 0.98 0.90
Initial substrate concentration was 60 g/L. The yields were calculated based on the vitamin B12 (mg/L)/consumed glucose (g/L).
in the following repeated culture cycles of batches 4 and 5. As described in Table 2, the fermentation time significantly shortened from 160 h (for batch 1) to 94 h (for batch 5) and the productivity of vitamin B12 was improved accordingly. The cell mass continuously increased in batches 1 to 4 and then slightly decreased in batch 5 as EBAB proceeded. This result may be attributed to the limitation of the inhibitors produced in the broth. This explanation was supported by a previous study (Zhang et al., 2014a). A similar tendency was obtained in vitamin B12 concentration and yield. Although the vitamin B12 concentration fluctuated from batches 4 to 5, the production remained relatively high. As a result, the maximum vitamin B12 concentration of 59.5 mg/L was achieved and the production increased from 0.37 mg/L/h (batch 1) to 0.59 mg/L/h (batch 4). This result indicates that the existing viable cells in batch 5 were enough to sustain fermentation. Similar results were reported in repeated lactic acid fermentation (Zhang et al., 2014b). The inhibitory effects of propionic acid and DMB on vitamin B12 biosynthesis can be reduced by the EBAB process through a precise byproduct and precursor control strategy. During this process, P. freudenreichii cells were reused. As a result, the lag phase was eliminated during the subsequent batch culture. Therefore, the productivity of vitamin B12 remarkably increased. In the present industrial fermentation, DMB as a medium composition is often added in the initial fermentation, and the broth containing DMB cannot be inoculated as seed liquid into the next batch. Therefore, DMB addition hinders continuous fermentation. The present study realized continuous vitamin B12 fermentation by repeated batch mode. In addition, from the engineering point of view, it is easy to accomplish for controlling byproduct concentration real-time online by using EBAB system and it could answer why use this system for B12 production in this study. Metabolic pathway for vitamin B12 biosynthesis in P. freudenreichii which includes the route involving the AdoCbi, propionic acid and DMB is shown in Supplementary Fig. S1. Propionyl-CoA and Succinyl-CoA are important nodes for propionic acid and vitamin B12 synthesis, respectively. In this study, partitioning carbon fluxes between propionic acid and vitamin B12 synthesis was occurred. When propionic acid concentration was controlled at a relatively low level, EBAB process might enabled more nutrients to the route of propionic acid synthesis instead of vitamin B12 production. While propionic acid concentration was controlled at a relatively high level, the route of vitamin B12 synthesis was strengthened. Based on these results, a combination of controlling the propionic acid concentration strategy was devised for improving vitamin B12 production. Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec.2014.11.019. Many microorganisms have been employed to produce vitamin B12 with various substrates under anaerobic conditions. These microorganisms include Butyribacterium methylotrophicum with methanol (1.7 mg/L), P. freudenreichii with lactate (3.6 mg/L), and Bacillus megaterium (B. megaterium) with Luria–Bertani (8.5 g/L)
(Bykhovskii et al., 1998; Piao et al., 2004; Biedendieck et al., 2010). A high production of approximately 60 mg/L with glucose and DMB by Propionibacterium subsp. Shermanii (Bykhovskii et al., 1998) was reported. In the present study, a comparable vitamin B12 concentration of 59.5 mg/L was obtained. Compared with previous studies in our laboratory, the present study improved vitamin B12 production from 0.33 mg/L/h to 0.59 mg/L/h (Wang et al., 2012a). Many studies have attempted to optimize culture medium composition and thus improve vitamin B12 production. Precursor addition (e.g., betaine) and statistical experimental design have been widely and deeply studied from different aspects with aerobic microbes (Kim et al., 2003; Li et al., 2008b; Kosmider et al., 2012). Vitamin B12 production has also been improved by optimizing cultivation parameters, such as dissolved oxygen content (Wang et al., 2010), pH (Li et al., 2008a), and medium components (Li et al., 2008c). ␦-Aminolevulinate (ALA) is an important precursor of vitamin B12; ALA can also be used as an indicator of vitamin B12 production in fermentation (Kang et al., 2012). However, the metabolic pathway from ALA to vitamin B12 is more distant from than that from AdoCbi to vitamin B12 (Scott and Roessner, 2002). In addition, a certain stream branch exists. Therefore, AdoCbi is a suitable marker to determine vitamin B12 biosynthesis during fermentation. Comparing with current research methods, the most valuable spot in this work emphasize multi-angular optimization combine byproduct control, principal product biosynthesis and fermentation reactor. Metabolic engineering has been used as a powerful tool to improve anaerobic vitamin B12 fermentation. Recently, Biedendieck et al. (2010) have reported an antisense RNA strategy for modifying the anaerobic vitamin B12 synthesis pathway in B. megaterium. This strategy can significantly increase the intracellular concentration of vitamin B12. Metabolic engineering methods, such as 13 C-labeled substrate, have also been used for carbon flux analysis in Pseudomonas denitrificans (Wang et al., 2012). The novel control strategy with EBAB developed in this study may be applicable to other biotechnological processes, such as methane and vitamin B12 by methanogens (Yang et al., 2004) or folate and vitamin B12 by P. freudenreichii (Wyk et al., 2011). 4. Conclusions An efficient and economic strategy for vitamin B12 fermentation was developed by cultivation process optimization. Controlling propionic acid concentration 10–20 g/L in the early stage and 20–30 g/L in the late stage can mitigate feed-back inhibition and improve vitamin B12 biosynthesis using the EBAB system. Intracellular intermediate feed-back inhibition occurred and can be severed by optimal DMB incorporation. In addition, optimizing the cultivation process with the propionic acid and DMB control strategy realized vitamin B12 repeated fermentation and increased the production from 0.37 mg/L/h (batch 1)–0.59 mg/L/h (batch 5). This strategy obtained in this study could be a promising option for industrial application of vitamin B12 fermentation.
P. Wang et al. / Journal of Biotechnology 193 (2015) 123–129
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