Chapter 97

Production of Vitamin B12 from Streptomyces Species

M. Camil Rex, B. Akshaya, Lokesh Ravi, and Kannabiran Krishnan

Abstract
Vitamins are organic compounds that are essential for the enzyme-catalyzed reactions and are also required
in trace amounts in the daily diet to carry out specific biological functions such as optimum growth and
maintenance of the health of organisms. Vitamin B12 is a fascinating molecule in the field of food and
medicine. It is otherwise known as cobalamin, a water-soluble vitamin with a major role in the function of
the nervous system, brain, and blood formation. Microbial biosynthesis of vitamin-B12 is restricted to few
species of bacteria and archaea. In the Phylum actinobacteria, Streptomyces is the largest genus and it has the
potential for the production of diverse secondary metabolites that have applications in biotechnological,
agricultural, and medical field. Based on several studies, it is apparent that Streptomyces are excellent vitamin-
B12 producers. Harvesting vitamin-B12 from Streptomyces species has gained immense commercial interest.
Vitamin-B12 is mainly employed in the production of mixed animal feed especially in processed foods,
poultry, vitamin pills, etc. This chapter provide the protocol for production of vitamin B12 using Streptomy-
ces species.

Key words Vitamin B12, Cobalamin, Streptomyces, Fermentation, Microbial production

1 Introduction

Vitamin B12(cobalamin) is an important member of water-soluble

vitamins. It supports the process of polymerase mediated replica-
tion of DNA and some important functions such as metabolism of
lipid particles, as a messenger in neurotransmission, detoxification,
etc. The daily requirement of vitamin B12 is recommended to be
1 μg for a healthy human. The rich sources of vitamin B12 are meat,
fish, milk, and eggs. Hence strict vegetarians often suffer from B12
deficiency, leading to serious conditions such as pernicious anemia
and megaloblastic anemia. During pregnancy, deficiency of vitamin
B12 may lead to birth defects such as Spina Bifida or neural tube
defects [1]. To overcome this, supplementation of vitamin B12 is
recommended daily, for which B12 is synthesized in large amounts
in pharma industries. Derivation of vitamin B12 from microorgan-
isms by fermentation is one of the popular methods of production.

Dhanasekaran Dharumadurai (ed.), Methods in Actinobacteriology,

Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-1728-1_97,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

661
662 M. Camil Rex et al.

Previously multiple sources of organisms were used for the produc-

tion of vitamin B12 such as Bacillus megaterium, Lactobacillus casei,
Propionibacterium freudenreichii, Streptomyces griseus, Streptomyces
aureofaciens, Flavobacterium devorans, Streptomyces fradiae, and
Ashbya gossypii [2]. Originally Streptomyces was found in Japanese
soil in 1949 as a producer of antibiotics; Later, Streptomyces was
identified as the best organism for the production of vitamin B12
because of its high yield potential [3]. It was reported that Strepto-
myces rochei produced the maximum amount of Vitamin B12
(45.3 μg/ ml) [1]. Streptomyces Olivaceus was the highest producer
of vitamin B12 among Streptomyces genera. Reports also show that
vitamin B12 is obtained as a by-product (non-specific -indirect
production) in the process of microbial synthesis of antibiotics (see
Note 1) such as chlortetracycline, streptomycin, and neomycin
[4]. The yield of vitamin B12 by various microorganisms (Table 1)
was already been reported [5]. Mixed culture of Bacillus firmus
AZ-78B and Streptomyces halstedii AZ-8A are reported to produce a
higher yield of vitamin B 12 (37.7 μg/ml) [6].

2 Materials
l Bennett media:
– Beef extract.
– Yeast extract.
– Casein digest.
– Agar.
– Glucose solution (25% w/v).
– Maltose solution (25% w/v).
– Nystatin solution (0.25%w/v).
– Seawater.
l Monopotassium phosphate.
l Ferrous sulfate heptahydrate.
l CoSO4∙7H2O.
l Calcium chloride dehydrate.
l Manganese sulfate monohydrate.
l Ammonium phosphate.
l Cobaltous sulfate heptahydrate.
l 0.2 M potassium phosphate buffer.
l 0.1% of KCN.
l Dextrose.
l Corn sleep liquor.
 Production of Vitamin B12 from Streptomyces Species 663

Table 1
The microorganisms and the processes recommended for the production of vitamin B12

Culture Fermentation Vitamin B12 production

Microorganism medium component (mg/L)
Propionibacterium Glucose Anaerobiosis, 206.0
freudenreichii 5,6-dimethyl
benzimidazole
Rhodopseudomonas protamicus Glucose 5,6-dimethyl 135.0
benzimidazole
Propionibacterium shermanii Glucose 5,6-dimethyl 60.0
benzimidazole
Pseudomonas denitrificans Sucrose Aerobiosis, betaine 60.0
Nocardiarugosa Glucose Aerobiosis 18.0
R. cobalaminogenum Sucrose Aerobiosis 16.5
Micromonospora sp. Glucose 5,6-dimethyl 11.5
benzimidazole
Streptomyces olivaceus Glucose 5,6-dimethyl 6.0
benzimidazole
Nocardia gardneri Hexadecane Aerobiosis 4.5
Butyribacterium Methanol Anaerobiosis 3.6
methylotrophicum
Pseudomonas sp. Methanol 5,6-dimethyl 3.2
benzimidazole
Arthrobacter hyalinus Isopropanol 5,6-dimethyl 1.1
benzimidazole

l Cobalt chloride(CoC12∙6H2O).
l Soyabean oil.
l Ammonium chloride.
l KH2PO4.
l MgSO4∙7H2O.
l KCl.
l Yeast extract.
l Calcium carbonate.
l NaCl.
l Glycerol.
l MnSO4∙7H2O.
l CoCL∙6H2O.
l FeSO4∙7H2O.
664 M. Camil Rex et al.

l ZnSO4∙7H2O.
l NaMO∙2H2O.

3 Methods

3.1 Sample 1. Collect soil samples (sediments) in a sterile glass bottle and
Collection and Serial store it in an icebox or thermal container and transferred to the
Dilution laboratory within 3 h for further processing.
2. Transfer the sample (20 g) into a 250 ml conical flask contain-
ing 100 ml of sterilized physiological saline and subject to serial
dilution(10
4) [1].

3.2 Isolation 1. Streptomyces is isolated using Bennett media (Beef extract—1 g,

of Streptomyces Yeast extract—1 g, Casein digest—2 g, Agar—17 g, Glucose
solution (25% w/v)—20 ml, Maltose solution (25% w/v)—
20 ml, Nystatin solution (0.25%w/v)—20 ml, Seawater—
470 ml, and Double distilled water—470 ml) followed by
pre-sterilization (autoclaving), pH of the medium should be
adjusted to 7.4 [1].
2. Heavy vegetative growth and sporulation of Streptomyces will
be observed within 3–5 days at 28–30  C [3].
3. Alternatively Streptomyces also cultured by using the
following [2].
(a) Dextrose % ¼ 0.5.
(b) Corn steep liquor% ¼ 0.5.
(c) Cobalt chloride (CoC12∙6H2O), ppm ¼ 2.0.
(d) Soyabean oil, % ¼ 0.1.
(e) Adjust the pH to 7.0.
(f) Sterilize at 250  F for 30–60 min.
4. Dextrose and cobalt chloride helps for efficient fermentation.
Yield of vitamin B12 is affected without cobalt and dextrose
(see Note 2).

3.3 Identification 1. Morphological methods include microscopic and macroscopic

of Streptomyces methods such as colony characteristics, coverslip culture
method, color, the structure of mycelium, spore chain, etc.,
are used for identification.
2. To carry out biochemical characterization, tests such as starch
hydrolysis, tween 20 hydrolysis, casein hydrolysis, urea hydro-
lysis, esculin hydrolysis, temperature tolerance, NaCl resis-
tance, etc., can be done. Bergey’s manual can be used as a
reference to identify the observed characteristics [1].
 Production of Vitamin B12 from Streptomyces Species 665

3.4 Inoculum 1. Streptomyces inoculum (CoSO4∙7H2O—0.005%; FeS-

Preparation O4∙7H2O—0.003%; KH2PO4—0.2%; MnSO4∙nH2O—
and Fermentation 0.002 g and CaCl2∙2H2O—0.001% [1, 3, 7]) is prepared by
adding 5 ml of broth culture suspension of the organism to
100 ml conical flask containing 45 ml of sterile inoculum
medium (Bennett’s agar), 50% double distilled water and an
equal volume of seawater and incubated in an electrical shaker
for 24 h at 100 rpm.
2. Inoculum (50 ml) is added to 9-L glass bottle containing 4-L
of sterile medium (Bennett’s agar). This should be aerated by
sterile air from a perforated tube at the base of the container
incubated at 80–85  F for 48 h.
3. The culture is transferred aseptically to 45 gallons of the sterile
medium (g/L) (CoSO4∙7H2O—0.005%; FeSO4∙7H2O—
0.003%; KH2PO4—0.2%; MnSO4∙nH2O—0.002 g;
CaCl2∙2H2O—0.001% and NH4PO4∙H2O—0.2% [1]) in the
deep tank and aerated at 83  F for 48 h [2].

3.5 Alternative 1. Streptomyces can also be actively grown in a submerged aerated

Fermentation Method culture medium (see Note 3) which contains assimilable carbon
sources, carbohydrates, mineral salts (cobalt), and organic
3.5.1 Submerged
nitrogen sources (soybean meal, distillers solubles, and myce-
Fermentation
lium), and other mineral nutrients and post inoculation, the
medium is fermented aerobically.

3.5.2 Solid State 1. The medium used for production of vitamin B12 from Strepto-
Fermentation (SSF) myces halstedii, AZ-8A under SSF consists of 3 g of agricultural
wastes supplemented with 30 ml mineral salts of following
basal medium. It contains (g/L) NH4Cl 2.0 g; KH2 PO4
1.0 g; MgSO4∙7H2O 0.5 g; KCl 0.5 g; Yeast extract 2.0 g;
CaCO3 2.0 g; NaCl 3.0 g; Glycerol 5.0 ml; MnSO4∙7H2O
0.2 g; CoCL∙6H2O 0.01 g; FeSO4∙7H2O 0.015 g;
ZnSO4∙7H2O 0.02 g; NaMO∙2H2O 0.005 g and distilled
water up to 1000 ml as reported earlier [6].

3.6 Sterilization 1. For batch fermentation adjust the pH of the medium to 7.0
Methods and it is necessary to hold the medium for 2 h at 250  F to
ensure sterility. Followed by adjusting the pH of the medium to
7.0 in copper, steel, or stainless steel fermenters [2].

3.7 Extraction 1. The fermentation broth is harvested and centrifuged at

of Vitamin B12 10,000 rpm (112,00  g) at
4  C for 10 min for extraction
of vitamin B12.
2. The pellets should be washed using 0.2 M potassium phos-
phate buffer (pH 5.5) and re-suspended in afresh buffer with
added 0.1% of KCN.
666 M. Camil Rex et al.

3. The suspension should be autoclaved for 15 min at 121  C,

filter by using a cellular acetate membrane filter with 0.2 μm
pore size and collect the supernatant which contains vitamin
B12 and store at
4  C [1].

3.8 Estimation 1. Vitamin B12 is estimated by using UV-Visible Spectrophotom-

of Vitamin B12 eter at 360 nm with potassium phosphate buffer (0.2 M) as a
reference standard.
2. The concentration of vitamin B12 is measured with the help of
standard graph and the formula is [1].
Conc:of Vitamin B12 ¼ ðTest O:D:=Standard O:D:Þ
 Concentration of standard solution:

4 Notes

1. Antibiotics production not affect the production and yield of

vitamin B12.
2. The concentration of dextrose, cobalt and soy meal content in
the culture media will increase vitamin B12 production.
3. Increased aeration and agitation will improve the vitamin B12
production.

References
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Microbial production of vitamin B12 and antimi-
crobial activity of glucose utilizing marine
derived Streptomyces species. Int J ChemTech
Res 4:976–982
2. Pfeifer VF, Vojnovich C, Heger EN (1954) Pilot
plant vitamin B12 by fermentation with Strepto-
myces olivaceus. Ind Eng Chem 46:843–849
3. Hall HH, Benedict RG, Wiesen CF et al (1953)
Studies on vitamin B12 production with Strepto-
myces olivaceus. Appl Microbiol 1:124–129
4. Musı́lek V (1959) Vitamin B12 production by a
strain of Streptomyces erythreus. Folia Microbiol
(Praha) 4:41–44
5. Martens JH, Barg H, Warren MJ, Jahn D (2002)
Microbial production of vitamin B12. Appl
Microbiol Biotechnol 58:275–285
6. Atta HM, Arafa RA, Salem MS, El-Meleigy MA
(2008) Microbiological studies on the produc-
tion of vitamin B12 from two mixed cultures
under solid state fermentation condition. J
Appl Sci Res 4(11):1463–1477
7. Nelson HA, Calhoun KM, Collingsworth DR
(1950) Neomycin and vitamin B12 activity in
Streptomyces 3553 fermentations. Abstr. In:
118th meeting, Am Chem Soc