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Production of VitaminB12 by improved strains of


Propionibacterium freudenreichii
M. Mazharuddin Khan, Naiman Ali Mir, M. Munawer Khan
PG Department of Microbiology and Biotechnology, Mumtaz College, Malakpet, Hyderabad,
Andhra Pradesh, 500036, India; E-mail: dr_mazhar_khan@yahoo.co.in

ABSTRACT
VitaminB12 was produced by physically and chemically induced mutant strains. The technique of anaerobic
and aerobic fermentation was adapted. Highest production of vitaminB12 was recorded by longer time UV
irradiated physically induced mutants. In a similar way the organisms treated with high concentration of
chemical mutagen also produced high level of VitaminB12. The highest yield of vitaminB12 was recorded at
30oC. The findings show that the longer the duration for irradiation, the higher quantity of chemical mutagen.
30o C temperature was favorable in improving the tolerance in the organism towards the propionic acid,
leading to maximum yield of vitaminB12.
Keywords: VitaminB12, Propionibacterium freudenreichii, UV irradiation, nitrosoguanidine, Lactobacillus
delbrueckii, anaerobic fermentation, aerobic fermentation

INTRODUCTION
Vitamin B12 is an important biological compound active as haematopoietic factor in mammals and
growth for many microbial and animals species. Vitamin B12 helps in the formation and regeneration
of red blood cells thus preventing anemia and is also important as a dietary supplement for animals
and human beings. Since the chemical synthesis of vitaminB12 requires more than 70 steps [1,2] the
industrial production by chemical method and subsequent purification steps made it industrial
production by chemical method and subsequent purification steps made it technically too difficult,
expensive, unsafe to the operators and the process is not eco-friendly. Therefore, VitaminB12 has
been produced on an industrial scale using the batch or fed-batch process of microbial fermentation
[3]. Various microorganisms including those of the genera Streptomyces, Bacillus,
Methanobacterium, Pseudomonas, Klebsiella and Propionibacterium have been used to produced
vitaminB12 on an industrial scale [4]. Among the above microorganisms in general
Propionibacterium species are preferred for the production of vitaminB12 particularly P.
freudenreichii is mostly preferred as this species has obtained the GRAS (Generally Regarded As
Safe) status from the US FDA (Food and Drug Administration) and produce neither endotoxin nor
exotoxins [5] and produce highest yield of 20 mg/lit. The major problem in vitaminB12 production
using Propionibacterium is the growth inhibition of the cell due to the accumulation of inhibitory
metabolites such as propionic acid and acetic acid in the process.
To overcome this situation and for enhancement of the production of vitaminB 12, in present
study random mutagenesis has been used in anticipation to generate mutant strains which can
positively produce high yield of product. In general a high yield of vitamin B12 has been achieved
by treating the microorganisms with mutagenic agents such as UV light or chemicals and selecting
the strains with practical advantages, such as productivity, genetic stability, reasonable growth rates
and resistance to high concentrations of intermediates such as propionic acid [6]. In the present
study an effort was made in anticipation to get regular and more quantity of vitaminB 12. Hence, the
Research Article, Biotechnol. Bioinf. Bioeng. 2011, 1(1):19-24
2011 Society for Applied Biotechnology. Printed in India.

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obtained P. freudenreichii was subjected to mutagenesis (physical and chemical) by which we are
able to develop desirable resistant characters in this organism against the propionic acid leading to
continuous production of vitaminB12 in fermentation process.

MATERIALS AND METHODS


Microorganisms Propinibacterium freudenreichii MTCC* 1950 and Lactobacillus delbrueckii
Subsp. Lactis type MTCC 911 were obtained from Microbial Type Culture Collection Centre,
Chandigarh, India, for production of vitamin B12 and for bioassay respectively. For anaerobic
fermentation (Mc. Intosh Mark III) anaerobic jar and for aerobic fermentation orbital shaking
incubator were used. The production of vitamin B12 was measured by the growth of Lactobacillus
delbrueckii subsp. lactis in response to vitamin B12 produced through spectrophotometric method.

Method of culture
Preparation of nutrient medium and Inoculation
For the initial culture of P. freudenreichii, nutrient agar and yeast-extract lactate agar medium was
used. All the ingredients were mixed and sterilized by autoclaving at 121oC for a period of 15 min.
After sterilization the culture from lyophilized vial was inoculated in the medium plates under
anaerobic conditions and kept in Hi Media Mc Intosh Jars Anaerobic system Mark-III with gaspak.

Preparation of inoculum
The inoculum was prepared from inoculating pure culture from above plates into a 200ml of
inoculum media in 500ml Erlenmeyer flask and incubated at 30oC for 24 hours in Hi-Media Mc
Intosh Jars Anaerobic-system. (a) Production medium: The production medium for vitaminB12
consists of sucrose, (as carbon source) glutamic acid, (as nitrogen source) betaine, cobalt and delta
aminolevulinic acid hydrochloride. Betaine acts as a source of methyl groups as it aids in the
synthesis of methionine by acting as a methyl donor. The medium was prepared by combining all of
the ingredients except the Delta-aminolevulinic acid hydro chloride and sterilized at 15 p.s.i. steam
pressure (121oC) for 15 minutes. Delta-aminolevulinic acid is activated by heat and hence it was
sterilized by a membrane filtration technique. The concentration of cobalt in the production medium
is critical as excess of cobalt suppresses the formation of cobinamide. Delta aminolevulinic acid act
as a precursor in the synthesis of vitaminB12 and the production is facilitated by the addition of it in
the production medium. (b) Mutagenesis: 20 (Twenty) sets of culture tubes were prepared for
physical and chemical mutagenesis. Induced mutation was carried out, the first 10 culture tubes
were treated with UV light (300 erg/mm2) and second 10 culture tubes were treated with
Nitrosoguanidine. The survivors of the physical and chemical mutagenesis were again subjected to a
second dose of mutagenesis. Finally the survivors of second dose were used for the production of
vitaminB12.
About 5% of the above mutant inoculum of P. freudenreichii (physical and chemical treated
mutants) was added in the 20 sets of production medium and 10 sets (100ml flask) were incubated
at 30oC and 10 sets were incubated at 32oC for a period of 7 days. The vitaminB12 fermentation is
divided into two fermentation phases: (1) Anaerobic fermentation phase: the anaerobic fermentation
phase lasts for 84hours, during which formation of cobinamide occurred. During the anaerobic
phase pH fall was recorded from 7.2-6.5. The production media was supplemented with 5,6Dimethyl benziminazole as. P freudenreichii cannot readily synthesize this base. (ii) Aerobic

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fermentation phase: After 84 hours the flask with production media were shifted from Mc. Intosh
jars to orbital shaking incubator for aerobic phase. The aerobic phase of fermentation also lasted for
another 84 hours during which the nucleotide was formed .This nucleotide under aerobic conditions
links with previously formed cobinamide to give cobalamin.

Assay of vitaminB12
The amount of vitaminB12 produced was estimated in broth samples by a microbiological bioassay
method using Lactobacillus delbrueckii subsp. lactis MTCC 911(formerly L. leichmanii). It is an
auxotrohpic strain requiring vitaminB12 for the growth. It is a reference method according to the
official Method of Analysis of AOAC international for assay [7]. The lyophilized culture of L.
delbrueckii subsp. lactis was maintained in tomato juice yeast-extract milk medium and transferred
biweekly. The culture was incubated at 37oC for 18-24 hours and then refrigerated until transferred.
A standard stock solution of vitaminB12 (Hi Media) was prepared by diluting stock solution to a
series of tubes to give 0.025, 0.05, 0.075, 0.10, 0.25, 0.5 and 0.75 mg (milli microgram) of
Vitamin B 12 per tube. Distilled water and double strength medium were added to bring the final
volume to 10 ml. The tubes were covered with metal cover lined with cotton and were autoclaved
for 5 min at 121oC. The tubes were cooled and 1 drop of inoculum was added to each tube except
the blank. The assay was read turbidometerically at 600nm in an spectrophotometer after 15-18 hrs
of incubation in a constant temperature water bath at 37oC.

RESULTS AND DISCUSSION


VitaminB12 has been produced in the laboratory at 30oC and 32oC temperature by the mutant strains
of P. freudenreichii through fermentation technique. During the production when the vitaminB12
reaches certain limit i.e., 18 mg/liter, the further production of vitaminB12 is stopped as
simultaneous gradual production of propionic acid also takes place in the process which inhibits the
activity of vitaminB12 production, as the growth of the organism is inhibited by propionic acid.
Treating conditions for the UV irradiation, such as the dose and time for each set was properly
selected. All sets (set A-5 tubes and set B-5 tubes) were irradiated with a dose of 300erg/mm2 for 30
sec, 60sec, 90 sec, 120sec and 150sec respectively (Table 1).
In the similar way for the chemical mutagenesis, the another culture sets (Set A-5 tubes, Set-B5
tubes) were treated with inducing agent Nitrosoguanidine with different concentrations (60 mg, 70
mg, 80 mg, 90 mg and 100 mg/lit) for 30 min, then centrifugation method was adopted for
separating the treated cells (Table 2). The parental type and above 20 sets of mutant strains (10
physical mutants and 10 chemical mutants) were cultivated in a liquid production medium anaerobic
and aerobic conditions at 30oC and 32oC respectively as discussed in material and methods. After 24
hours the growth was observed in all sets (flask) in the form of turbidity which indicates growth of
the organism and production of vitaminB12.
Investigations have shown that during the production of vitaminB 12 propionic acid also formed
in the production media and gradually increases in amount, when the amount of propionic acid in
the production system exceeds certain limits (i.e., 18 mg/lit), the growth of the organism was
inhibited and stops further production of vitaminB12. So parental strain and No-1 and No-2 flasks of
the physically mutant strains of set A and B could not produce vitaminB12 after reaching 18 and 19
mg/lit. Such toxicity of propionic acid towards the propionic bacteria in the fermentation process for
the vitaminB12 production was also shown by [8] for P. shermanii. The propionic acid effect is also
demonstrated by [9] for P. acidi-propionici, where the growth rate is drastically reduced.
In present investigation also the production of vitaminB12 and growth of parental strain P.
freudenreichii was arrested and the same type of performance of certain mutant strains was also

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Table 1. Details of dose and time of UV exposure for physical mutagenesis.
Number of test tube
1
2
3
4
5
Total

For the fermentation at 30C


Set - A (Sec)
30
60
90
120
150
5 Tubes

For the fermentation at 32C


Set - B (Sec)
30
60
90
120
150
5 Tubes

Table 2. Details of dose of Nitrosoguanidine for chemical mutagenesis.


Number of test tube
1
2
3
4
5

For the fermentation at 30C


Set - A (mg)
60
70
80
90
100

For the fermentation at 32C


Set- B (mg)
60
70
80
90
100

Total

5 Tubes

5 Tubes

noted, particularly in strains which were exposed for less duration for irradiation. This situation
indicates that the mutant strains, which were irradiated for 30 sec, 60 sec did not developed
tolerance towards propionic acid, so they ceased their activity after reaching 18 mg/l and 19mg/l,
perhaps the less irradiation time was not helping in the development of resistant character in the
organism towards propionic acid (Table 3, 4). In contrast to this the sets which were exposed for
longer duration, i.e., 90, 120 and 150 sec were showing their ability to produced more quantity of
vitaminB12 as higher production of vitaminB12 was recorded in their flasks. This condition indicates
that these organisms have developed tolerating ability towards propionic acid. It suggests that when
the organisms were exposed to UV radiation for longer time, i.e., 120 sec and 150 sec, they acquired
the resistance to propionic acid and as a result they produce more vitaminB12 (Table 3, 4).
Table 3. Comparison of the amount of Vitamin B 12 produced by parental and different physically
mutant strains at 30C.
No.
1
2
3
4
5
6

Strains
Parental
Set A mutant
Set B mutant
Set C mutant
Set D mutant
Set E mutant

Dose of Physical
Mutagen (UV
light) (erg/mm2)
300
300
300
300
300

Time of exposure
(Sec)

Yield of vitaminB12
(mg/liter) at 300C

30
60
90
120
150

19
19
20
21
22
22

Regarding the chemical mutant strains and their vitaminB12 producing ability the results show
the following findings: The lower concentrations of chemical mutagen Nitrosoguanidine has not
shown any positive effect in developing the tolerance in the organism towards the propionic acid,

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but higher concentrations (i.e., 90 mg and 100 mg of nitrosoguanidine/lit) have shown positive
effect in developing tolerance in the organism. Hence, more quantity of vitamin B12 was produced
by the mutants which were treated with high concentration of mutagen when compared with the
parental strain and also mutants which were treated with lower concentrations (Table 5, 6). This
condition also suggests that higher concentrations of nitrosoguanidine is developing tolerating
ability towards propionic acid in the organism. Further the influence of temperature is also noted in
the fermentation process on the vitaminB12 production ability of parental, physical and chemically
treated mutant strains. High activity of the organism i.e. production of vitaminB12 was recorded at
30oC temperature and low yield was recorded at 32oC temperature (Table 5, 6).
Table 4. Comparison of the amount of VitaminB12 produced by parental and different physically
mutant strains at 32C.
No.

Strains

1
2
3
4
5
6

Parental
Set A mutant
Set B mutant
Set C mutant
Set D mutant
Set E Mutant

Dose of physical
mutagen (UV
Light) (erg/mm2)
300
300
300
300
300

Time of Exposure
(Sec)

Yield of vitaminB12
(mg/liter) at 30C

30
60
90
120
150

18
18
18
18
20
21

Table 5. Comparison of the amount of VitaminB12 produced by parental and chemically


induced mutant strains at 30C.
No.
1
2
3
4
5
6

Strains
Parental
Set A1
Set B1
Set C1
Set D1
Set E1

Chemical mutagen
used in
mutagenesis
Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine

Conc. of chemical
mutagen (mg/liter)

Time
(min)

60
70
80
90
100

30
30
30
30
30

Yield of vitB12
(mg/liter)
at 300C
19
19
20
21
22
22

Table 6. Comparison of the amount of VitaminB12 produced by parental and chemically induced
mutant strains at 32C.
No.

1
2
3
4
5
6

Strains

Chemical
mutagen used in
mutagenesis

Parental
Set A 1
Set B 1
Set C 1
Set D 1
Set E 1

Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine
Nitrosoguanidine

Conc. of
chemical
mutagen
(mg/liter)
60
70
80
90
100

Time
(min)

Yield of vitB12 (mg/liter)


at 32C

30
30
30
30
30

18
18
18
18
20
21

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From tables it became clear that the growth inhibition which is caused by higher concentration
of propionic acid under anaerobic conditions can be minimized when temperature was decreased.
The above results infer the ability if the mutant culture strains in the production of vitaminB12. It
was observed from studies that the selected UV radiation (dose and duration), selected quantity of
chemical mutagen nitrosoguanidine were improving the resistance in the organisms against the
propionic acid. Further the fermentation parameter temperature also influenced the yield, as high
yield was observed at 30oC. Based on this it can be concluded that the conditional mutagenesis can
improve the vitaminB12 production capacity in the organism, modification of temperature in
fermentation process may also be beneficial. Hence this technique can be used in strain
improvement program and also in the production of vitaminB12 at industrial level.

REFERENCES
[1] Eschenmoser A. CIBA Found Symp. 1994, 180:309-329.
[2] Woodward RB. Pure Appl. Chem. 1973, 33:145-177.
[3] Youngsmith B, Sonomoto K, Tanaka A, et al. Eur. J. Appl. Microbiol. Biotechnol. 1982, 16:70-74.
[4] Piao Y, Yamashita M, Kawaraichi N, et al. J. Biosci. Bioeng. 2004, 98:167-173.
[5] Salminen S, Von Wright A, Morelli T, et al. Int. J. Food Microbiol. 1998, 44:93-106.
[6] Martens JH, Barg H, Warren MJ, et al. Appl. Microbial. Biotechnol. 2002, 58:275-285.
[7] Taranto MP, Vera JL, Hugenholtz J, et al. J. Bacteriol. 2003, 185:5643-5647.
[8] Nanba A, Nukada R, Nagai S, et al. J. Ferment. Technol. 1983, 61:551-556.
[9] Blanc P, Goma G. Bioproc. Eng. 1987, 2:175-179.

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