EFFECT OF ENVIRONMENTAL FACTORS ON VITAMIN

B12 PRODUCTION BY PROPIONIBACTERIUM FREUDENREICHII

AND BACILLUS MEGATERIUM
 INTRODUCTION
 Vitamin B12 (cobalamin) is an important cofactor for metabolism of
carbohydrates, lipids, amino acids and nucleic acids.
 Vitamine B12 is a crucial B vitamin .

 Cobalamin is another name of vitamin B12.

 Vitamin B12 is a water-soluble vitamin.

 Deficiency of vitamin B12 levels too low ,several neurological

symptoms are show.
 WHOLE BODY: anaemia, fatigue,or poor balance.
ALSO COMMON: memory loss, reduced sensation of touch, shortness of
breath .
SOURCES OF VITAMINB 12
 FUNCTIONS OF VITAMIN B12

Metabolism of every DNA synthesis and

Functioning of brain
cell of the human regulation
and nervous system
body

Production of healthy
red blood cells that Fatty acid synthesis
carry oxygen around and energy
the body production
MATERIALS AND METHODS-.
MICROORGANISM USED – Propionibacterium freudenreichii and
Bacillus megaterium.

MEDIA USED- Medium (1): Semi-solid lactate agar (SLA) recommended by Hettinga
et al. (1968) was used for the maintenance of Propionibacterium sp. It has the
following composition: Tryptone 10.0g, yeast extract 10.0g, sodium lactate 16.7ml,
MgSO4. 7H2O 0.5g, KH2PO4 0.25g in one litre distilled water and adjusted pH to 7.0
– 7.2.

Medium (2): Nutrient Agar (Difco Manual, 1977) was used for the maintenance of
Bacillus megaterium. Its composition is as follows: Bacto peptone 5.0g, Bacto beef
extract 3.0g and agar 15g in one litre Tap water and adjusted pH to 7.0.
Propionibaterium freudenrichii Bacillus megaterium
FERMENTATION PROCESS
 FERMENTATION PROCESS
 Fifty ml portions of the medium were dispensed into 250ml cotton
plugged conical flasks. Each flask was inoculated with a suitable
inoculum of Propionibacterium sp.(3.5 x 105 ml) and incubated at 30 ºC
for 4 days as static culture then 4 days as submerged culture, while flasks
of Bacillus megaterium inoculated with (5.5 x 105 ml) and incubated at
30 ºC on rotary shaker (150 rpm) for 4 days.
 5,6 Dimethylbenzimidazole (DMB) was added, as a vitamin B12
precursor, to the fermentation cultures 24 hr before the end of incubation
period as recommended by Pedziwilk, et al., (1979) and Marwaha, et al.,
(1983 a).
 FLOWCHART OF FERMENTATION PROCESS

Propionibacterium sp. 3.5 x 105 ml

Incubation at 30°C for 4 days in static culture

250 ml then 4 days in submerged culture

250 ml

Mixed with
5,6
Dimethyl
FERMENTATION
Bacillus megaterium 5.5 X 105 ml benzimidazole
COMPLETED
(DMB)

(VITAMIN B12)
Incubation at 30°C for 4 days in rotatory shaker
(150 rpm)

5.5 X 150 ml
 Effect of Initial pH: Twelve values of pH ranged between 6.0 and 9.0 were chosen for
studying their effects on vitamin B12 production to select the most suitable pH of the
production medium.
 Incubation temperature: To determine the optimum growth temperature for vitamin
B12 production, fermentation was carried out at various temperatures in the range of
5, 30, 32, 35 and 40 ºC for vitamin B12 production by bacterial strains.
 Aeration: An experiment was performed to study the effect of aeration conditions on
vitamin B12 production.
 Determination of Vitamin B12: The production of vitamin B12 was determined in
the culture and in the cells according to the method of Mazumder, et al., (1987).
RESULT AND DISCUSSION
EFFECT OF INITIAL PH

 Twelve values of pH ranged between 6.0 and 9.0 were chosen for
studying their effects on vitamin B12 production.
 The optimum pH for vitamin B12 production by strains of
Propionibacteria was 7.0 ± 2.
 vitamin B12 production by B. megaterium reached the highest level at
initial pH 6.8.
Effect of initial pH on the production of vitamin B12 by
Propionibacterium spp. and Bacillus megaterium.
EFFECT OF INCUBATION TEMPERATURE

 An experiment was conducted to find out the effect of

different degrees of incubation temperatures namely, 20,
25, 30, 32, 35 and 40 ºC on vitamin B12 production.
 The temperatures from 30 to 35ºC achieved the highest
vitamin B12 production in all organisms.
 The optimum incubation temperature for higher vitamin
B12 production by Propionibacteria and Bacillus
megaterium was 30ºC.
Effect of incubation temperature °C on the production of vitamin B12 by
Propionibacterium spp. and Bacillus megaterium.
 EFFECT OF AERATION
 Result show that the growth under static culture conditions (4 days) and
followed by submerged culture conditions (4 days) for Propionibacterium
freudenreichii subsp. freudenreichii CCM 1857 and Propionibacterium
freudenreichii subsp. shermanii P1NRC gave the highest vitamin B12 in
culture being 560.08 & 395.31 μg / 100 ml.
 The maximum production of vitamin B12 was recorded using shaking culture
condition, followed by static culture condition and again followed by shaking
culture condition being 233.5 & 215.4 μg / 100 ml in cultures for Bacillus
megaterium DSM 2894 and Bacillus megaterium 1066
Effect of aeration on the production of vitamin B12 by Propionibacterium
spp.
 CONCLUSION
 The result was reported that vitamin B12 reached a maximum
values when the culture was incubated anaerobically for the first
half of the fermentation, followed by aerobic conditions for latter
half of the fermentation by Propionibacterium shermanii.
 Results indicated that that pH 6.6 to 6.8 were the most
convenient for vitamin B12 by Bacillus megaterium, however the
pH 7.0 was the best level for Propionibacterium spp.
 The optimum incubation temperature for vitamin B12 production
ranged between 30 - 35ºC.
 The bacterium can also make vitamin B12 when grown
aerobically and anaerobically.
 REFERENCES
Abdel-Hafez, A. M.; W. A. Mashhoor; Rawia F. Gamal; A. A. Rafaat and
Azhar A. EL-Sayed (1981). Production of vitamin B12 by propioic acid
bacteria. II- Effect of nutritional and environmental factors on vitamin B12
production by Propionibacterium shermanii P-16. Bull.1686, Agric. Microiol.
Dept., Fac. Of Agric., Ain shams Univ., Cairo, Egypt.
Barisova, T. G.; L. I. Vorob and L. S. Kozlevnikova (1971). Synthesis of
vitamin B12 in Propionibacterium shermanii in relation to cultivation
condition. Biol. Nauki, 14 (7): 22 - 26.
Berry, E. C. and L. B. Bullerman (1966). Use of cheese whey for vitamin B12
production. Appl. Microbiol. Biotechnol., 14: 356 - 357.
Bielecka, M.; H. Czranocka-Roczniakowa and A. Gradzinska (1978).
Optimization of whey medium composition for biosynthesis of vitamin B12
by propionibacteria. Acta Aliment. Polon., 4: 133 - 140.