alexa Fermentation| Carbondioxide| Vitamin B12
ISSN: 2155-9821
Journal of Bioprocessing & Biotechniques
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Enhance Vitamin B12 Production by Online CO2 Concentration Control Optimization in 120 m3 Fermentation

Ze-Jian Wang1, Hui-Yuan Wang1, Ping Wang1, Yi-ming Zhang2, Ju Chu1, Ying-Ping Zhuang1* and Si-Liang Zhang1
1State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
2Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE-412 96 Göteborg, Sweden
Corresponding Author : Ying-Ping Zhuang
State Key Laboratory of Bioreactor
Engineering, East China University of Science & Technology
P. O. Box 329,130 Meilong Road
Shanghai 200237, People’s Republic of China
Tel: 86-21-64253702
E-mail: [email protected]
Received April 24, 2014; Accepted May 12, 2014; Published May 20, 2014
Citation: Wang ZJ, Wang HY, Wang P, Zhang Y, Chu J, et al. (2014) Enhance Vitamin B12 Production by Online CO2 Concentration Control Optimization in 120 m3 Fermentation. J Bioprocess Biotech 4:159 doi:10.4172/2155-9821.1000159
Copyright: © 2014 Wang ZJ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Great amounts of carbon dioxide generated by Pseudomonas denitrificans during high aerobic vitamin B12 fermentation, while the influence of CO2 concentration on vitamin B12 production remains unclear. In this paper, we present parallel experiments to investigate various levels of inlet CO2 fractions on the physiological metabolism of P. denitrificans in laboratory scale fermentation. The results demonstrated that the oxygen transfer rate, cell growth and glucose consumption were inhibited with CO2 fraction elevated from 0.03% to 8.86 ± 0.24%, while the most exciting results showed that the specific vitamin B12 production rate and the yield to glucose were greatly stimulated when dissolved CO2 increased to 8.86 ± 0.24%. Therefore, the optimal exhausted CO2 fraction control strategy in 120 m3 fermenter was established. With the exhaust CO2 concentration was well controlled at 7.5 ± 0.25% on-line, vitamin B12 production greatly improved to 223.7 ± 3.7 mg/L, which was 11.2% higher than that of control. This strategy was proved to be significant necessary and effective for successfully scale up optimization in industrial vitamin B12 fermentation.

Keywords
Dissolved carbon dioxide; Pseudomonas denitrificans; Vitamin B12; Fermentation; Scale up; Optimization
Introduction
Vitamin B12 is an important growth factor and has many applications in medicine and nutrition. Because of its increasing need in the world, vitamin B12 has received much more attention recently. Most of the vitamin B12 was produced by the culture of aerobic bacterium P. denitrificans in industrial production [1]. Due to the actively respiring characters of P. denitrificans, the poorly ventilated systems, high back pressure and hydrostatic pressure in large-scale fermenters, high dissolved CO2 concentration were always encountered in industrial process. As for the significant influence of dissolved CO2 on the growth and metabolism of many microorganisms [1], it must be taken into consideration in large-scale industrial fermentation.
The effects of CO2 on growth and product formation in submerged cultures have been investigated in various microorganisms, and the controversy results were always encountered [2,3]. Researches of Gill and Lacoursiere had revealed that cell growth and metabolism of Pseudomonas fluorescens and Escherichia coli could be stimulated at low dissolved CO2 level not more than 100 mm Hg pressure or 5% inlet gas phase respectively, while it could be greatly inhibited by the increased CO2 concentration [4,5]. In yeasts cultivation, the growth rates were seriously decreased under CO2-enriched condition [6,7]. For filamentous fungi, in which the secondary metabolites formation was always accompanied with morphology changes [1], it was proved that both the morphology and product formation were negatively affected by the elevated CO2 conditions [1,8,9]. However, many other reports demonstrated that CO2 could also have positive effect on the metabolism of some microorganisms [10], in which existed high CO2- fix pathway activity, the anaplerotic reactions were very important for replenishing the metabolites generated from tricarboxylic acid cycle’s pool, Researches had shown that enhanced CO2 could stimulate the activity of key enzymes for the anaplerotic reaction in Corynebacterium glutamicum [11,12] and succinate-producing strain [13] by accelerating the accumulation of glutamate and succinate. The former studies on industrial vitamin B12 fermentation have shown that P. denitrificans has high affinity to oxygen, and accompanied with higher CO2 evolution rate during fermentation [14]. However, at present, there was not clearly understood of the effect of CO2 on metabolism of vitamin B12 biosynthesis.
Therefore, the aim of present study was to evaluate the effect of CO2 on production and metabolism of vitamin B12 produced by P. denitrificans. Furthermore, an optimal industrial fermentation strategy based on online and CO2 controlling strategy was successfully applied to enhance industrial vitamin B12 production.
Materials and Methods
Microorganisms and medium
The industrial strain P. denitrificans used in this study was donated by Huarong pharmacy corporation (Shijiazhuang, China). The cultivations were carried out at a temperature of 32°C and agitation speed of 260 rpm. The seed medium was composed of (g/L): sucrose 40, corn steep liquor 20, betaine 5, (NH4)2SO4 1,(NH4)2HPO4 2, MnSO4⋅H2O 0.8, COCl2⋅6H2O 0.02, MgO 0.3, 6-dimethyl-benzimidazole (DMBI) 0.01, ZnSO4⋅7H2O 0.01, CaCO3 1.5, the pH was adjusted to 7.2-7.4 by NaOH.
The fermentation medium lab scale and industrial scale was composed of (g/L): glucose 80, corn steep liquor 45, betaine 14, (NH4)2SO41, KH2PO4 0.75, COCl2⋅6H2O 0.075, MgO 0.5, DMBI 0.05, ZnSO4⋅7H2O 0.08, CaCO3 1, pH 7.2-7.4. Feed media (FM) for the fedbatch fermentation were as follows (g/L), FM-1: Glucose 200, DMBI 0.15, COCl2⋅6H2O 0.15 and FM-2: betaine 30, COCl2⋅6H2O 0.3, DMBI 0.3.
Determination of dry cell weight (DCW) and residual sugar concentration
For determination of biomass concentration, 10 ml ferment broth was centrifuged at 4,000 g for 10 min. After washing twice with distilled water, the cell precipitate was dried to a constant weight at 105°C. The residual sugar concentration was measured by anthrone method [15].
Determination of Pyruvate Carboxylase and intracellular δ-aminolevulinic acid (δ-ALA)
Vitamin B12 is a highly complicated molecular structure [16,17] that is biosynthesized from eight molecules of δ-aminolevulinic acid (δ-ALA). This δ- ALA precursor can be generated by either of two pathways (Figure 1): In the C4 pathway, δ-ALA is made from glycine and succinyl CoA by the action of ALA synthase [1]. The δ-ALA was determined according to the literature [18]. For determination of Pyruvate Carboxylase, 10 ml of samples were taken quickly and centrifuged at 4°C, 12,000 g for 5 min, the obtained cells were frozen in -80°C nitrogen. After several times of repeated freezing and thawing, cell pellets were washed with 20 mM Tris–HCl (pH 7.6) for ultrasonication. The sonicated cell suspension was centrifuged and the cell extract was used for the enzyme assay. Pyruvate carboxylase activity was determined by the method of Uy [19].
Measurement of organic acids and amino acids
For the analysis of the extracellular organic acids and amino acids in metabolism, the HPLC systems (Agilent 1100, USA) were equipped with an Aqua Sep C18 (250 × 4.6 mm, 5 μm, ES, USA) and Zorbax Eclipse AAA column (150 × 4.6 mm, 5 μm, Agilent, USA) respectively, the determination conditions were the same to that of previously reported [14].
Quantification of vitamin B12 in the broth
The vitamin B12 concentration was determined by HPLC. Broth samples (25 ml), into which 2.5 ml of 8% NaNO2 and 2.5 ml of glacial acetic acid were added, were boiled for 30 min. Then the upper aqueous phase was measured using HPLC system equipped with C18 column (4.6 mm, 25 cm, 5 μm) and UV detector (361 nm). 5% of acetonitrile was used as the mobile phase with flow rate of 1.0 ml min-1 at 25°C combined with 95% of 0.25 M sodium acetate anhydrous, the pH was adjusted to 3.6 by acetic acid.
Fermentation process design
Firstly, fed-batch fermentation was carried out in a 50 L turbineagitated bioreactor (Shanghai Guoqiang Inc., China) with 30 L working volume. The cultivation conditions were same to that of previous report. When the cultivation turned into higher vitamin B12 biosynthesis phase, 2.5 L broth was incubated into four parallel 5 L bioreactors respectively (Shanghai Guoqiang Inc., China) for further investigation. Fermentations were performed at 32°C, stirrer speed was 450 rpm, and the pressure in the tar was all kept at 0.1 MP. The inlet gas stream was consisted of a defined mixture of air and pure carbon dioxide with the aeration rate at 1 vvm. The mixture was prepared by thermal mass flow controllers (Mass-Trak, SIERRA, Netherlands). In addition to the control process where air was the input gas, processes were also performed with 3.32 ± 0.12%, 8.86 ± 0.24%, and 13.84 ± 0.27% CO2 addition (v/v air) in the inlet gas mixture. Meanwhile, the dissolved CO2 concentration in fermentation was determined with using dissolved carbon dioxide concentration electrode (Mettler).
Fermentation in 120 m3 fermenter
Three-stage fermentation was performed in 150 L, 9 m3, and 120m3 fermenters gradually, involving two stages of seed growth and one stage of fed-batch vitamin B12 fermentation. The primary seed, secondary seed and large-scale fermentation were cultivated with the methods as previous reported [14]. The geometry and dimensions of 120 m3 fermenter tanks and agitators were given in Table 1.
The inlet and exhaust gas ingredients were analyzed by a mass spectrometer (MAX300-LG, Extrel). Oxygen Uptake Rate (OUR) and Carbon dioxide Evolution Rate (CER) was calculated and collected online Oxygen Uptake Rate (OUR), and were controlled by adjusting agitation and aeration conditions.
Carbon dioxide control strategy model in 120 m3 fermenter
The culture medium always enriched with CO2 due to the high evolution rate of growing cells during fermentation process [20], especially in industrial scale fermenters with high hydrostatic pressures, high cell density or high pressure [21,22]. Previous researches revealed that P. denitrificans has high affinity to oxygen, with the dissolved oxygen concentration maintained nearly zero, therefore the oxygen transfer rate (OTR) was equal to OUR during the steady fermentation process. The OTR was determined by the agitation, aeration and the pressure, so the equation was used to create their relationship. Meanwhile, the active respiration make the CO2 levels rise rapidly in large scale fermenters. In order to implement online control the oxygen supply levels and exhausted carbon dioxide concentration, we establish a response model about the OTR and exhausted CO2 concentration related to aeration and agitation
OUR ≈ OTR = m × Fα × Rβ × Pr (Equation.1)                                             (1)       
Where m=correction coefficient; α, β, γ=response coefficient of flow, agitation and pressure relate to oxygen transfer rate; OUR= oxygen uptake rate (mmol L/h); OTR=oxygen transfer rate (mmol L/h); F=air flow (L/min); P=pressure (MPa); R=agitator speed (rpm). Moreover, the relation between oxygen uptake rate and carbon dioxide evolution rate has presented by RQ which shown in Equation 2. In addition, equation 3 shows online determination formula of carbon dioxide evolution rate.
                                                                                                      (2)   
                                                                              (3)
Results and Discussion
Effect of CO2 on the dynamic of vitamin B12 fermentation
For investigating the effects of dissolved CO2 levels on vitamin B12 biosynthesis, the stationary cell growth broth in 50 L fermenter was transferred to the four 5 L reactors with 2.5 L working volume simultaneously. The fermentations were performed with operating conditions held constant to ensure that dissolved carbon dioxide was the only major variable. Samples were taken individually for analysis of biomass, glucose, inter-metabolites, and vitamin B12 concentrations; and the data points represent mean values from duplicate experiments. A gas-mixing unit was used to give input CO2 concentration in the influent gas of 0.03%, 3.32 ± 0.12%, 8.86 ± 0.24%, and 13.84 ± 0.27% respectively.
The time course of cell growth and pH under the four different CO2 addition were illustrated in Figure 2. During those four conditions, the dissolved oxygen concentration were all maintained at nearly zero levels, while the cell growth haven’t any effect when the CO2 addition elevated from 0.01% to 8.86 ± 0.24%; However, with influent CO2 concentration increased to 13.84 ± 0.27%, the cell growth rate was greatly inhibited and biomass concentration decreased to 30.7 g/L after 48h cultivation, which was 8.9% lower than that of control (33.68 g/L) (Figure 2a). These might be caused by the respiration inhibition of CO2 on biological membranes and cytoplasmic enzymes [23], and which may also interact with lipids of the cell membrane, the higher CO2 concentration would greatly inhibit cell growth [1,9]. It could be clear seen from Figure 2b that pH had hardly any different to the control when CO2 addition increased to 8.86 ± 0.24%, while the pH was remarkably decreased when the influent CO2 elevated to 13.84 ± 0.27%.
The time course of cell growth and pH under the four different CO2 addition were illustrated in Figure 2. During those four conditions, the dissolved oxygen concentration were all maintained at nearly zero levels, while the cell growth haven’t any effect when the CO2 addition elevated from 0.01% to 8.86 ± 0.24%; However, with influent CO2 concentration increased to 13.84 ± 0.27%, the cell growth rate was greatly inhibited and biomass concentration decreased to 30.7 g/L after 48h cultivation, which was 8.9% lower than that of control (33.68 g/L) (Figure 2a). These might be caused by the respiration inhibition of CO2 on biological membranes and cytoplasmic enzymes [23], and which may also interact with lipids of the cell membrane, the higher CO2 concentration would greatly inhibit cell growth [1,9]. It could be clear seen from Figure 2b that pH had hardly any different to the control when CO2 addition increased to 8.86 ± 0.24%, while the pH was remarkably decreased when the influent CO2 elevated to 13.84 ± 0.27%.
The effect of influent CO2 on vitamin B12 production rate, glucose consumption rate and specific carbon dioxide evolution rate were summarized in Table 2. Results revealed the inhibitory effects of CO2 concentration on the specific glucose consumption rate (SGCR). Little decrease of SGCR was appeared with CO2 addition increased to 3.32 ± 0.12%; However, the SGCR were dramatically decreased to 0.14 and 0.10 mmol/gDCW/h when the influent CO2 increased to 8.86 ± 0.24% and 13.84 ± 0.27% respectively, which were 19.2% and 46.7% lower than that of control (0.18 mmol/gDCW/h).
Just different from cell growth and SGCR, vitamin B12 productions were greatly stimulated when influent CO2 increased from 0.03% to 8.86 ± 0.24% (Figure 2c). The highest specific vitamin B12 production rate reached to 69.4 μg/gDCW/h under 8.86 ± 0.24% CO2 addition, 53.5% higher than that of control (45.2 μg/gDCW/h). While vitamin B12 productions and specific production rate were all significantly inhibited when the CO2 concentration raised to 13.84 ± 0.27%. Analysis of vitamin B12 yields to the glucose consumption (Yp/s) (Figure 2d) showed that the maximum yield of 480.5 μg/mmol (vitamin B12/ glucose consumption) were obtained when the influent CO2 reached 8.86 ± 0.24%, while 13.84 ± 0.27% CO2 addition led the yields dramatically decreased to lowest levels of 248.3 μg/mmol. Exhaust gas analysis showed that specific carbon dioxide evolution rate (SCER) of P. denitrificans decreased with elevated CO2 concentration in input gas. The statistics of the percent of glucose used for CO2 generation were decreased from 94 ± 2.1% to 85 ± 1.5% with influent CO2 increased from 0.03% to 8.86 ± 0.24%; while under the highest CO2 concentration of 13.84 ± 0.27%, nearly 93.3 ± 1.3% of the glucose consumption was seriously transformed to CO2. In conclusion, maintained an appropriate input CO2 concentration around 8.86 ± 0.24% was positive for the economical vitamin B12 production.
Previous researches on the effects of elevated CO2 on the industrial producing strains of bacteria, yeasts, and fungi [1,9] revealed that the inhibitory action of product biosynthesis was probably caused by the decreased substrate consumption with elevated CO2 concentration. In our research, however, glucose consumption decreased with the increased influent CO2 concentration, while the yields of vitamin B12 based on glucose consumption increased greatly with the elevated influent CO2 concentration. These results indicated that there must exist abnormal metabolism characters of P. denitrificans under the various influent CO2 concentrations. The reasons were further investigated by organic acid and amino acid analysis of the central metabolism.
Effect of CO2 on the inter-metabolites in fermentation
The researches on vitamin B12 biosynthesis had demonstrated that the concentrations of the precursor of δ-aminolevulinic acid (δ-ALA) played an important role on vitamin B12 biosynthesis [16]. δ-ALA could be synthesized from C4-pathway (from glycine and succiny CoA by δ-ALA synthesis) or C5-pathway (from glutamate by glutamyltRNA reductase) associated with central metabolism [24]. The dynamic processes of extracellular organic acids concentration under different CO2 levels were shown in Figures 3 and 4.
When influent CO2 increased from 0.03% to 8.86 ± 0.24%, α-ketoglutarate and succinate concentrations greatly increased, which were actually accelerated the biosynthesis of glutamate, and the other organic acids such as pyruvate, acetate, lactate and citrate hadn’t any different. However, the acetate and pyruvate were accumulated much higher when influent CO2 reached 13.84 ± 0.27%. These were probably caused by the inhibition of respiration under high CO2 concentration, and the resulted high NADH accumulation in the cell really repressed the activity of pyruvate dehydrogenase and TCA cycle, and then attributed to the accumulation of acetate and pyruvate.
The dynamic analysis on the concentrations of extracellular amino acids (Figure 3) showed that glutamate and glycine synthesis rate was significantly enhanced with elevated CO2 concentration. Under 8.86 ± 0.24% CO2 addition, glutamate and glycine concentration reached 1430.2 mgL-1 and 2716.4 mg/L, respectively, which were 55% and 122% higher than that of control. Threonine reached highest synthesis rate when inlet CO2 increased to 3.32 ± 0.12%. These metabolites accumulation could be accelerate the precursor’s biosynthesis for vitamin B12 synthesis.
δ-ALA is the critical precursors of vitamin B12 biosynthesis, the time course of δ-ALA concentration under different levels of inlet CO2 were shown in Figure 5a, experiment results demonstrated the positive effects of moderate CO2 concentration exerted to the δ-ALA synthesis. In bioprocess sparged with 8.86 ± 0.24% CO2 addition, the δ-ALA concentration reached the top point at 26.4 ± 1.2 mg/gDCW, which was 40.4% higher compared to that of control (18.6 ± 0.9 mg/gDCW). However, the δ-ALA biosynthesis was seriously inhibited with13.84 ± 0.27% CO2 addition, the final δ-ALA concentration was lowest as 13.02 ± 0.5 mg/gDCW, 30% lower than that of control.
Experiment results showed that the biosynthesis of glycine, succinic acid, and glutamate were elevated with the increased influent CO2 concentration from 0.03% to 8.86 ± 0.24%, which actually augmented δ-ALA generation for higher vitamin B12 biosynthesis.
Effect of CO2 on pyruvate carboxylase activity
Previous researches have illustrated that anaplerotic reaction catalysed by pyruvate carboxylase was mostly existed for replenishing the TCA cycle in Pseudomonas spp [25]. In this work, the dynamic analyses of specific pyruvate carboxylase activity in different input CO2 addition were shown in Figure 5b. In the process sparged with 8.86 ± 0.24% CO2, the maximal specific activity reached 37.2 U/gDCW, which was 35% higher than that of control (27.5 U/gDCW).
Tricarboxylic acid (TCA) cycle is one of the main central pathways in aerobic bacteria. It is responsible for the complete oxidation of acetyl-CoA and provision of precursors for many TCA-related metabolites biosynthesis. As precursors of the metabolites succinyl- CoA and glutamate for δ-ALA biosynthesis were all generated from TCA cycle. In the rapid VB12 synthesis phase, the relative high demand of succinyl-CoA and glutamate lead to an “overflow” of corresponding compounds of TCA cycle. Therefore, TCA cycle has to be replenished continuously through anaplerotic reaction for oxalacetate generation to maintain the cycle running.
The experiment results demonstrated that elevated levels of CO2 concentration had significant effect on vitamin B12 fermentation. When the influent CO2 concentration increased from 0.03% to 8.86 ± 0.24%, the specific vitamin B12 production rate and the yields of vitamin B12 to glucose were stimulated. However, cell growth, glucose utilization, specific vitamin B12 production rate, and the yields of vitamin B12 to glucose were all greatly inhibited when CO2 addition up to 13.84 ± 0.27%. Therefore, the influences of CO2 on industrial vitamin B12 production have to take into consideration.
Industrial fermentation strategy based on online exhaust CO2 controlling
Exhaust CO2 concentration controlling strategy: With the process parameters determined under various agitation speed, air flow, and pressure obtained from 120 m3 fermentation tank are shows in Table 3, we obtained relevant respond coefficient through regression analysis of these parameters between OTR and Process parameters determined (Equation.1). Then, oxygen transfer rate and oxygen uptake rate could be calculated according to these parameters (Equation.4).
OUR ≈ OTR = 1.0074 × F0.3729 × R0.4635 × P0.2472                                                                                 (4) 
The carbon dioxide fraction in exhausted gas was calculated from a mutual relation between RQ, CER, and OUR (Equation. 2,3,5)
                                         (5)
During the vitamin B12 fermentation process, glucose was used as the main carbon substrate, the respiration quotient (RQ) of this strain was always maintained at 1.03 ± 0.02 in microorganism growth period and RQ was calculated as 0.95 ± 0.04 in vitamin B12 synthesize period. Then OUR and exhausted CO2 concentration from 120 m3 fermentation tank could be obtained by adjusting agitator speed and air flow according to the created model above (Figure 6).
Scale-up fermentation based on exhaust CO2 concentration control: Based on the results obtained from 5 L vitamin B12 fermentation, the optimal controlling strategies based on exhausted CO2 concentration were implemented for vitamin B12 fermentation in 120 m3 fermenters. The exhausted CO2 concentration was controlled at 7.5 ± 0.25% by adjusting aeration and agitator speed, also OUR was maintained at needed level. This strategy greatly prevented the inhibition of high dissolved CO2 concentration on vitamin B12 synthesis.
Time course of OUR, dry cell weight, vitamin B12 production, and exhausted CO2 concentration were shown in Figure 7. By control the aeration and agitation throughout the fermentation process, the changes of OUR were maintained at similar levels in fermentation processes (Figure 7b), the optimal strategy by stepwise reduction of oxygen uptake rate under dissolved oxygen limiting level during fermentation process was used for oxygen supply levels control [11]. Meanwhile, the exhausted CO2 concentration changed greatly and maintained at the suitable levels with the adjustment of agitation and aeration conditions. In the contrast conditions, the exhaust CO2 concentration increased greatly to 9.1 ± 0.15%, cell growth rate was lower than that of optimal strategy, especially when it elevated to 12.0 ± 0.24% after 75 h cultivation, the vitamin B12 production rate was gravely inhibited (Figure 7c).
The results presented demonstrated that cell growth and vitamin B12 production were greatly stimulated by controlling a lower CO2 concentration of 6.0 ± 0.24% during the early fermentation phase. The highest vitamin B12 production rate was achieved when exhausted CO2 concentration were controlled at 7.5 ± 0.25%, The maximum vitamin B12 production reached 223.7 ± 3.7 mg/L at 190 h, which was 11.2% higher than that of without CO2 concentration controlled (201.4 ± 2.9 mg/L). It can be observed that higher production would be realized through simultaneously regulate the appropriate OUR and CO2 concentration levels are more beneficial to vitamin B12 synthesis.
Conclusion
CO2 was produced in aerobic and anaerobic fermentations through decarboxylation reactions in a number of metabolic pathways, and hence has an influence on the performance of microbial cultivations [1,9]. Especially in industrial large bioreactors with high total pressure, poorly ventilated system, and inefficiently aerated fermenters. In this work, we investigated the effects of dissolved CO2 concentration on vitamin B12 fermentation of P. denitrificans, established the correlation of exhausted CO2 concentration and the activity of the microbial metabolism by inter-scale observation and data association. The novel and optimal fermentation controlling strategy based on OUR and exhausted CO2 concentration was successfully applied in industrial scale up of vitamin B12 fermentation.
Acknowledgements
This work was financially supported by a Grant from the National Natural Science Foundation of China (Grant No. 31200024), Hebei Key Technology R&D Program (No. 13272803D), the Major State Basic Research Development Program of China (973 Program), No. 2012CB721000, and Doctoral fund No. 20110074110015. We also thank Huarong Pharmacy Corporation (Shijiazhuang, China) for donating the industrial strain P. denitrificans.
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  1. avnswamy
    Posted on Aug 06 2016 at 5:59 am
    what could be the vitamin b12 concentration in fermentation broth.? couldyou optimis this by controlling co2 concentration . Is it feed back control or feed forward control.
 

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