Optimization of Cyanocobalamin Production by Propionibacterium Freudenreichii in Different Concentrations of Folic Acid and Methionine

Introduction

Vitamin B₁₂ or cobalamin, is a water-soluble vitamin that belongs to the corrinoid compound group (1) which has various exclusive biochemical functions in the brain, nervous system, and cellular metabolisms. It especially plays an important role in fatty acids synthesis and energy production in the human body (2). Vitamin B₁₂ is essential for the function of the nervous system (3), and its deficiency may have a variety of symptoms such as neuropathy, nervous system disease, and megaloblastic anemia (4). The chemical production of vitamin B₁₂ is not profitable due to its complicated process which involves 70 steps (1). Therefore, vitamin B₁₂ production is limited to the industrial fermentation process of bacteria such as Propionibacterium freudenreichii and Pseudomonas denitrificans, which is a rapid and low-cost procedure (5). Two different pathways are applied for this purpose, including the aerobic pathway in Pseudomonas denitrificans and the anaerobic pathways in Salmonella typhimurium, Propionibacterium freudenreichii subspecies shermanii, and Bacillus megaterium (6). Propionibacterium freudenreichii belongs to the Propionibacteriaceae family and uses various sources of carbon to produce vitamin B₁₂, such as glucose (7), molasses (8), and crude glycerol (9). Propionibacterium ssp.are capable of producing a wide range of biological compounds that enhance human health such as folic acid, proline, conjugated linoleic acid, and vitamin B(10). Folate is the generic name for a large number of chemical derivatives of pteroylglutamic acid (PGA), is also known as folic acid. It has interference with the basic activities of cellular metabolisms, such as DNA repair, methylation, and the biosynthesis of nucleotides, as well as vitamins and amino acids (11). Folate deficiency is associated with a large number of disorders, including Alzheimer’s disease, cardiovascular diseases, osteoporosis, the increased risk of breast and colorectal cancer (12-14). Folic acid is commonly applied to improve the nutritive value of the foods and is used as a nutritional supplement (15). Various compounds are necessary in the synthesis of vitamin B₁₂ including 5, 6-dimethylbenzimidazole (DMBI). The presence of oxygen in the growth medium of Propionibacterium plays an essential role in the production of DMBI (16, 17). Cobalt and DMBI are often added to the industrial production of vitamin B₁₂ because they are required for the formation of the corrin ring and the lower ligand molecule respectively and are considered limiting factors in vitamin B₁₂ (18, 19). The role of amino acids on the growth and production of vitamin B₁₂ has been studied. For example; the presence of methionine increases the levels of vitamin B₁₂ because methionine is needed for various methylation processes in the ovarian environment (20). Although many studies have been proposed on the anaerobic biosynthesis of Vitamin B₁₂ in Propionibacterium in terms of its genetic and biochemical characterization, the effect of folic acid on this bacterium has not been addressed yet. In this study, the authors have tried to develop new strategies for Propionibacterium freudenreichii to enhance vitamin B₁₂ production. In particular, this study aimed to determine the effect of methionine and different concentrations of folic acid on the optimal production of vitamin _B12 and dry cell weight in aerobic and anaerobic conditions.

Materials and Methods

Microorganisms and Mediums: Based on a preliminary investigation regarding the effect of folic acid on the cell growth and vitamin B₁₂ biosynthesis in Propionibacterium freudenreichii, four concentrations of folic acid (control, 250, 750, and 1000 mg/L) were added to the medium culture. Table 1 shows various methods for optimizing vitamin B₁₂ production used in this study, including the effect of anaerobic and aerobic conditions and the presence of methionine on bacterial culture medium at different concentrations of folic acid.

Table 1. Different Methods for Optimizing the Production of Vitamin B12

Propionibacterium freudenreichii (PTCC: 1674) was purchased from the Iranian Research Organization for Science and Technology (IROST, Karaj, Iran) (ptcc.irost.org). Each culture medium was composed of the following components (per liter of deionized water): lactate sodium 1%, extract yeast 1% (as nitrogen source) (Merck, Germany), trypticase soy broth 1% (Sigma-Aldrich), 10 g of tryptone (Biolife), 90 g of glucose (as carbon source), and 20 g of Corn Steep Liquor (CSL). The fermentation medium supplements were composed of the following (per liter of deionized water): control, 250, 750 and 1000 mg/L concentrations of folic acids (single carbon donor or a recipient) )Merck, Germany(, 100 mg of DMBI (Sigma-Aldrich), 100 mg of cobalt chloride (2), 0.05% (v/w) of methionine as methyl donor (20Marwaha et al. 1983), KCN: 1% (w/v) (21), 2.5 mL of glacial acetic acid, and NaNO₂ 8 %( w/v) (22).

Production of Vitamin B₁₂ in the Anaerobic Conditions and Different Concentrations of Folic Acid: The culture medium was incubated for 80 h at 30 ºC in the anaerobic jar systems and the fermentation medium (50 mL) was approximately normalized at the pH of 7. Folic acid was added to the different fermentation media at the concentration of control, 250, 750, and 1000 mg/L and subsequently, the media were kept for 4 days at 30 ºC.

Production of Vitamin B₁₂ in the Aerobic Conditions and Different Concentrations of Folic Acid: Propionibacterium freudenreichii were cultured under the same conditions as done in the anaerobic ones, but the aerobic conditions were created for them. The culture medium was extracted from jars anaerobic system; subsequently, the cap was removed and placed on a shaker (Lap companion) for a period of 72 h at 134 rpm (23, 24).

The Effect of Folic Acid in the Anaerobic Conditions and the Presence of Methionine: The bacteria were cultured similar to the anaerobic conditions, and methionine 0.05 % (w/v) was simultaneously added to the different media with folic acid (at the concentration of control, 250, 750, and 1000 mg/L) and subsequently, the media were kept for 4 days at 30 ºC.

The Effect of Folic Acid in the Aerobic Conditions and the Presence of Methionine: The bacteria were cultured in anaerobic conditions the same as previous conditions, and folic acid (at concentrations of control, 250, 750 and 1000 mg/L) along with methionine 0.05% (w/v) were added to the different media. Subsequently, the aerobic conditions were created for them, as done before.

Quantification of the Dry Cell Weight: The cultures medium of Propionibacterium freudenreichii were sterilized by autoclaving at 121 ºC for a period of 15 min followed by centrifugation at 14000 rpm for 10 minutes at 4 ºC. The cell precipitation was oven-dried at 70 ºC for 48 h and then weighted.

Determination of the Produced Vitamin B₁₂: The concentrations of the fermentation products were determined through HPLC (model: SY-8100). Biosynthetic vitamin B₁₂ was used in this study as the standard for quantification (Merck Company). The broth sample (25mL) was added with 2.5 mL of NaNO₂ 8 % (w/v) and 2.5 mL of glacial acetic acid. The mixture was boiled for 30 min, followed by adding potassium cyanide 1% (w /v) and filtering. The mixture was injected by a C18 column (5μm, 4.6μm× 25cm) with a flow rate of 1.0 mL/min and a wavelength of 254 nm in 25 ºC. The mobile phases consisted of KH₂PO₄ (.02 M) methanol (70/30 v/v).

Statistical Analysis: The mean values were obtained based on the measurements of three replicates and all quantifications were done based on Duncan’s multiple range tests for the discrimination of significance (defined as P˂0.05). Data were represented as the mean ± standard errors of the mean (SEM). Analyses were carried out using the statistical analysis system software (SPSS 19) and graphs were drawn by Excel 2007.

Results

Vitamin B₁₂ Production and Cell Growth under Aerobic and Anaerobic Conditions: Production levels of vitamin B₁₂ with different concentrations of folic acid increased in the aerobic conditions, compared to the non-aerobic ones. In the anaerobic conditions, there was no significant difference in vitamin B₁₂ production between the various concentrations of folic acid. At 750 mg/L concentration of folic acid, the amount of vitamin B₁₂ production in the aerobic conditions was about 5-times (P˂0.05) more than that obtained in similar conditions in the anaerobic medium. However, at the 1000 mg/L and 250 mg/L concentrations of folic acid vitamin B₁₂  level increased 3 and 2.1 times in comparison to the anaerobic conditions, respectively, but the differences were not significant (P>0.05). Under the anaerobic conditions, no significant difference was observed between different concentrations of folic acid (Fig. 1).

In the aerobic conditions, the highest cell dry weight was recorded in the concentration of 750 mg/L of folic acid which was significantly different from other concentrations (P˂0.05), while there was no significant difference between other concentrations (P˃0.05). In the anaerobic conditions, there was no significant difference between each concentration (P>0.05). The dry cell weight increased in the aerobic conditions in comparison to the anaerobic conditions with a 1.8-time increase at the concentration of 750 mg/L (Fig. 2).

The Effect of Folic Acid and Methionine in the Anaerobic Conditions on the Production of Vitamin B₁₂ and the Cell Growth: The results of HPLC indicated a significant difference in the production of vitamin B₁₂ in the presence of methionine and at the 750 and 1000 mg/L concentrations of folic acid, in which the highest level was observed at 750 mg/L of folic acid. In the presence of methionine, the level of vitamin B₁₂ increased significantly, as compared to its absence. Also, at 750 mg/L concentration of folic acid together with methionine, the production of vitamin B₁₂ was approximately 6-folds more and in 1000 mg/L concentration of folic acid, it was 4 times more than the recorded level in the absence of methionine. However, no significant difference was observed in the control group (Fig. 3).

Fig. 1- The effect of different concentrations of folic acid (control, 250, 750, and 1000 mg/L) on the amount of vitamin B12 production by P.freudenreichii in the aerobic and anaerobic. The data are expressed as the mean of three replicates ± standard error of the mean. Non-similar letters express the meaning of the data according to the Duncan's (P˂0.05) assay: A indicates the highest value, B is the highest value after A, C is the lowest value, and BC is the intermediate value between B and C.

Fig. 2- The effect of different concentrations of folic acid (control, 250, 750, and 1000 mg/L) on the bacterial growth in aerobic and anaerobic conditions. The data are expressed as the mean of three replicates ± standard error of the mean. Non-similar letters express the meaning of the data according to the Duncan's (P˂0.05) assay: A indicates the highest value, B is the highest value after A, C is the value after B, and BC is the intermediate value between B and C, D indicates the lowest value, BCD is the average value between B, C and D, and CD is the intermediate value between C and D.

Fig. 3- The effect of different concentrations of folic acid (0, 250, 750, 1000mg/L) on the production of B12 in the presence (open bar) and absence (close bar) of methionine (Met) under the anaerobic conditions by P.freudenreichii. The data are expressed as the mean of three replicates ± standard error of the mean. Non-similar letters express the meaning of the data according to the Duncan's (P˂0.05) assay: A indicates the highest value, B is the highest value after A, and C is the lowest value (Met=Methionine).

In anaerobic conditions, there was no significant difference in the dry cell weight between the groups whether methionine was available in the medium or not (P>0.05). At 750 mg/L concentration of folic acid in combination with methionine, the dry weight decreased significantly in relation to the control group. Besides, adding methionine to the fermentation medium increased the dry weight of the control group approximately 2.4 times more than the recorded data in the absence of it. The dry weight of the bacteria decreased as folic acid concentration increased the concomitant with methionine in the anaerobic conditions, which showed the negative effect of the high concentration of folic acid on the bacterial growth (Fig. 4).

Fig. 4- The effect of folic acid (control, 250, 750, and 1000 mg/L) on the bacterial growth and dry cell weight in the presence and absence of methionine under the anaerobic conditions. The data are expressed as the mean of three replicates ± standard error of the mean. Non-similar letters express the meaning of the data according to the Duncan's (P˂0.05) assay (Met=Methionine). A indicates the highest value, B is the highest value after A, AB is the intermediate value between A and B, C is the value after B, and BC is the intermediate value between B and C, D indicates the lowest value, CD is the intermediate value between C and D.

.The Effect of Folic Acid and Methionine in the Aerobic Conditions on the Production. of Vitamin B₁₂ and the Cell Growth:In the aerobic and anaerobic conditions and presence of methionine, the highest level of vitamin B₁₂ production was at the 750 mg/L of folic acid and subsequently at 1000 mg/L. The production of vitamin B₁₂ with control and 250 mg/L of folic acid was approximately the same. However, there was a slight difference between the production of vitamin B₁₂ in the presence of methionine under aerobic and anaerobic conditions. Therefore, it is likely to believe that methionine is more effective in vitamin B₁₂ production than the aerobic conditions. The highest level of vitamin B₁₂ was observed in the 750 mg/L concentration of folic acid; as compared to the control group, in which there was a 7-time increase. On the other hand, in 1000 mg/L concentration of folic acid, there was a 5-time increase in comparison to the control group (Fig. 5).

When methionine was available in anaerobic conditions along with an increasing concentration of the folic acid, the cell dry weight decreased, and there was no significant difference between different groups (250, 750, and 1000 mg/L). In the aerobic conditions, however, there was a significant difference between the concentrations of 750 and 1000 mg/L of folic acid compared to the similar concentrations in anaerobic conditions in term of dry weight with 1.5 and 1.3 times increase in 750 and 1000 mg/L concentration of folic acid respectively as compared to the same treatments in anaerobic conditions (Fig. 6).

Fig. 5- The effect of different concentrations of folic acid (0, 250, 750, 1000mg/L) on the production of B12 in the presence of methionine under the aerobic and anaerobic conditions by P.freudenreichii. The data are expressed as the mean of three replicates ± standard error of the mean. Non-similar letters express the meaning of the data according to the Duncan's (P˂0.05) assay: A indicates the highest value, B is the highest value after A, and C is the lowest value (Met=Methionine).

Fig. 6- The effect of different concentrations of folic acid (0, 250, 750, 1000mg/L) on dry cell weight (mg/l) in the presence of methionine under the aerobic and anaerobic conditions by P.freudenreichii. The data are expressed as the mean of three replicates ± standard error of the mean. Non-similar letters express the meaning of the data according to the Duncan’s (P˂0.05) assay: A indicates the highest value, B is the highest value after A, and C is the value after B, and BC is the intermediate value between B and C, ABC is the average value between A, B and C, D indicates the lowest value, BCD is the average value between B, C, D, and CD is the intermediate value between C and D (Met=Methionine).