MICROBIOLOGY TERMPAPER

BTC-013

VITAMIN B12 PRODUCTION

SUBMITTED TO:
Ms. SUJATA DASS
SUBMITTED BY:
PARUL MAHAJAN
B-TECH M-TECH
BIOTECHNOLOGY
SECTION: A
ROLL No: 4
REGISTRATION No.: 3040070085
 VITAMIN B12 PRODUCTION
AIM & OBJECTIVE:

Vitamin B12 Production

Vitamin B-12 is normally involved in the metabolism of every cell of the body,
especially affecting the DNA synthesis, normal functioning of brain and nervous system,
and formation of blood and regulation but also fatty acid synthesis and energy
production. However, many (though not all) of the effects of functions of B-12 can be
replaced by sufficient quantities of folic acid, since B-12 is used to regenerate folate in
the body. Most "B-12 deficient symptoms" are actually folate deficient symptoms, since
they include all the effects of pernicious anemia and megaloblastosis, which are due to
poor synthesis of DNA when the body does not have a proper supply of folic acid for the
production of thymine. A transdermal vitamin B12 delivery patch is applied to the skin
of a user for the delivery of vitamin B12 to the bloodstream of the user. The patch
includes a fabric backing and a skin-adhesive polymer matrix that is attached to one side
of the fabric backing. The matrix contains a vitamin B12 compound. The vitamin B12
compound diffuses from the matrix through the stratum corneum layer of the user's skin,
through the dermis layer of the skin, and into the user's bloodstream. On the application
of serum vitamin B12 radio-assay is used to the diagnosis of cobalt deficiency in
sheep.

Vitamin B-12 cannot be made by plants or animals as only bacteria have the enzymes
required for its synthesis. Vitamin B-12 deficiency can potentially cause severe and
irreversible damage, especially to the brain and nervous system. At levels only slightly
lower than normal, a range of symptoms such as fatigue, depression, and poor memory
may be experienced. So vitamin b12 is produced by the process of fermentation in
bacteria as well as industrially. it is produced by methanol utilizing bacteria, use of
cheese whey , production by citrobacter ferenduii by tempeh fermentation, lactobacillus
reuteri, efficient production by propionic acid bacteria under periodic variation of
dissolved oxygen concentration, production in fermented milk products continuous
methane fermentation and production of vitamin b12 in fixed bed reactors packed with
loopah. All these processes of fermentations in bacteria are used for the production of
vitamin b12.

INTRODUCTION
Vitamin B-12 is one of eight B vitamins which are important for the normal functioning
of the brain and nervous system, and for the formation of blood. It is normally involved
in the metabolism of every cell of the body, especially affecting DNA synthesis and
regulation, but also fatty acid synthesis and energy production.

Vitamin B-12 is the name for a class of chemically-related compounds, all of which
have vitamin activity. It is structurally the most complicated vitamin. Biosynthesis of
 the basic structure of the vitamin can only be accomplished by bacteria, but
conversion between different forms of the vitamin can be accomplished in the human
body. A common synthetic form of the vitamin, cyanocobalamin, does not occur in
nature, but is used in many pharmaceuticals, supplements and as food additive, due to
its stability and lower cost. In the body it is converted to the physiological forms,
methylcobalamin and adenosylcobalamin, leaving behind the cyanide, albeit in
minimal concentration. More recently, hydroxocobalamin, methylcobalamin and
adenosylcobalamin can also be found in more expensive pharmacological products
and food supplements. The utility of these is presently debated.

Vitamin B-12 is a collection of cobalt and corrin ring molecules which are defined by
their particular vitamin function in the body

The term B-12 may be properly used to refer to cyanocobalamin, the principal B-12 form
used for foods and in nutritional supplements. This ordinarily creates no problem, except
perhaps in rare cases of eye nerve damage.

Below: A microscopic picture of vitamin B12

Synthesis: Vitamin B-12 cannot be made by plants or animals as only bacteria

have the enzymes required for its synthesis. The total synthesis of B-12 was reported by
Robert Burns Woodward and Albert Eschenmoser, and remains one of the classic
feats of organic synthesis.

Species from the following genera are known to synthesize B-12: Aerobacter,
Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium,
Flavobacterium, Micromonospora, Mycobacterium, Nocardia, Propionibacterium,
Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia,
Streptomyces, Streptococcus and Xanthomonas. Industrial production of B-12 is through
fermentation of selected microorganisms. The species most often used, Pseudomonas
denitrificans and Propionibacterium shermanii, are frequently genetically engineered and
grown under special conditions to enhance yield.

Functions
Vitamin B-12 is normally involved in the metabolism of every cell of the body, especially
affecting the DNA synthesis and regulation but also fatty acid synthesis and energy
production. However, many (though not all) of the effects of functions of B-12 can be
replaced by sufficient quantities of folic acid, since B-12 is used to regenerate folate in
the body. Most "B-12 deficient symptoms" are actually folate deficient symptoms, since
they include all the effects of pernicious anemia and megaloblastosis, which are due to
poor synthesis of DNA when the body does not have a proper supply of folic acid for the
production of thymine.

Foods

Vitamin B-12 is naturally found in meat (especially liver and shellfish), milk and eggs.
Animals, in turn, must obtain it directly or indirectly from bacteria, and these bacteria
may inhabit a section of the gut which is posterior to the section where B-12 is absorbed.
Thus, herbivorous animals must either obtain B-12 from bacteria in their rumens. Eggs
are often mentioned as a good B-12 source, but they also contain a factor that blocks
absorption. Certain insects such as termites contain B-12 produced by their gut bacteria,
in a manner analogous to ruminant animals.

Plants do not supply B-12 to humans. Vegan humans who eat only plant based foods must
ordinarily take special care to supplement their diets accordingly. According to the U.K.
Vegan Society, the only reliable vegan sources of B-12 are foods fortified with B-12
(including some soy products and some breakfast cereals), and B-12 supplements.

Supplements

The Vegan Society, the Vegetarian Resource Group, and the Physicians Committee for
Responsible Medicine, among others, recommend that vegans either consistently eat
foods fortified with B-12 or take a daily or weekly B-12 supplement. Fortified breakfast
cereals are a particularly valuable source of vitamin B-12 for vegetarians and vegans.
Cyanocobalamin is converted to its active forms, first hydroxocobalamin and then
methylcobalamin and adenosylcobalamin in the liver. Vitamin B-12 can be supplemented
in healthy subjects also by liquid, strip, nasal spray, or injection and is available singly or
in combination with other supplements.

Symptoms and damage from deficiency

Vitamin B-12 deficiency can potentially cause severe and irreversible damage, especially
to the brain and nervous system. At levels only slightly lower than normal, a range of
symptoms such as fatigue, depression, and poor memory may be experienced.

REVIEW OF LITERATURE:
The molecular structure of vitamin B12 vitamin. B12 (cobalamin), the most complex of
all known vitamins, was announced in 1955 by several scientists, including British
biochemists A. R. Todd and Dorothy Hodgkin. In 1973 the vitamin was reported to have
been synthesized by organic chemists. Vitamin B12 and closely related cobalamins are
necessary for folic acid to fulfill its role; both are involved in the synthesis of proteins.
American physicians G. R. Minot and W. P. Murphy in 1926 fed large amounts of liver to
patients with pernicious anemia and cured them; the curative substance in this case was
probably vitamin B12. Vitamin B12 was first isolated in 1948 in a crystalline form. This
triggered an intense investigation of its structure by X-ray crystallographers, including
Dorothy Hodgkin, a British scientist who finally elucidated the complete structure of
this red, cobalt-containing substance in 1955. As well as this structural study, there was
also an increasing effort to obtain a large amount of vitamin B12 from natural sources for
therapeutic and experimental uses. In the early 1950s, it was found that the sludge
remaining in the fermentation tanks of sewage disposal plants had unusually high
amounts of vitamin B12 compounds, and that the final fermentation process was carried
out almost exclusively by methane-producing, anaerobic bacteria.

In the late 1950s, by studying the formation of methane by Methanosarcina barkeri and
other anaerobic bacteria, Thressa demonstrated that vitamin B12 is involved in the
methane-producing process. Furthermore, she explained that the free form of vitamin B 12
can function as a methyl group carrier, and that its coenzyme forms serve as hydrogen
carriers. This knowledge provided the basis for much of the current understanding of
methane biosynthesis. In 1958, another breakthrough in the study of vitamin B12 was
made by Horace A. Barker, Thressa's mentor, who discovered the biologically active
forms (coenzyme forms) of B12 vitamins while working on the anaerobic metabolism of
glutamate. Coenzymes are non-protein molecules that help the catalytic function of
enzymes. In the end, Thressa and her co-workers discovered 5 of the 12 known vitamin
B12 -dependent enzymes, some of which, as coenzyme forms, function in lysine
fermentation

The study began in 1821 with a mysterious disease now known as pernicious anemia,
caused by B12 deficiency. It took almost one hundred years to gain knowledge into the
signs of the disease and it still remained uncurable. Following a lead from W. Whipple in
California (1925), physians W. P. Murphy and G. R. Minot in Boston (1926) reported a
remarkable improvement in patients fed on a diet of raw liver. All three were awarded
noble prize in physiology and medicine in 1934 for their discovery concerning liver
therapy against anemia.

The first phase of research was to isolate the compound (liver therapy factor) that was
present in liver in a concentration of only 1 ppm. Glaxo and Merck scientists isolated this
red crystalline material which they named it as vitamin B12. It was extremely active
against pernicious anemia.

The second phase of research was to elucidate the structure of B12 by chemical and
physical methods and to evolve methods for large scale production. Dorothy Hodgkin of
Oxford was awarded the noble prize in 1964 for elucidating the structure of this
molecule. The work was an exciting episode in the history of X-ray crystallography
because B12 was the largest structure studied successfully upto that time.

The third phase of research activity was aimed at the synthesis, properties, reactions and
their precise role in enzymatic reactions. The total synthesis of this molecule with the
correct number of substituents and stereochemistry was achieved in 1976. It took 11
years and a dedicated contribution of 100 chemists in a collaborative effort directed by R.
B. Woodward, the noble laureate (Harvard) and A. Eichenmoser (Zurich).

So, coenzyme B 12 has long fascinated chemists due to its complex structure and because
it offers the only biological instance of a stable organometallic bond. Its unique property
arises from the different catalytic activity of two different coenzymes.

Sally Fallon is founding president of the Weston A Price Foundation, a non-profit nutrition
education foundation with over 400 local chapters and 9000 members. She is also the
founder of A Campaign for Real Milk, which has as its goal universal access to clean raw
milk from pasture-fed animals. Author of the best-selling cookbook Nourishing Traditions
and also of Eat Fat Lose Fat (Penguin), both with Mary G. Enig, PhD, Sally has a
encyclopedic knowledge of modern nutritional science as well as ancient food ways. Her
grasp on the work of Weston Price is breath taking and her passion for health freedom,
inspiring

David Hunter and colleagues carried out a genome-wide association study in search of
common genetic variation that might influence plasma levels of B12. They found three
highly correlated single-nucleotide polymorphisms (SNPs) in the gene FUT2 to be
associated with B12 levels.

FUT2 encodes an enzyme that links certain types of sugars and that, among other things,
regulates the secretion of the factors that constitute an individual's blood type. One of the
B12-associated SNPs renders FUT2 nonfunctional, and has been referred to as the
'nonsecretor' variant because it prevents secretion of the blood-type factors. The 'secretor'
version of FUT2 has been associated with infection by the bacterium Helicobacter pylori,
which can cause gastritis. The authors suggest that genetic variation in FUT2 may
influence B12 levels via H. pylori, which others have shown to be associated with B12
malabsorption.
 Vitamin B12 Production by a Methanol-Utilizing Bacterium
Tetsuo Toraya, Busaba Yongsmith, Atsuo Tanaka, and Saburo Fukui
Department of Industrial Chemistry, Faculty of Engineering, Kyoto University, Yoshida,
Sakyo-ku, Kyoto, Japan
Vitamin B12 production by a newly isolated strain of a methanol-utilizing bacterium was
studied. The maximal yield of the vitamin, 2.6 mg/liter of medium was attained by
optimization.

Douglas Rees, Ph.D.
The Dorothy Crowfoot Hodgkin Award, sponsored by Genentech, is granted in
recognition of exceptional contributions in protein science, which profoundly influence
our understanding of biology.

The 2008 Award was presented to Dr. Douglas Rees (California Institute of Technology)
on Saturday, July 19, 2008 for his fundamental contributions to the understanding of the
structural biology of metalloproteins and membrane proteins, most notably by his
analyses of the nitrogenase molybdenum-iron protein that established the unprecedented
structure of the FeMo-cofactor providing the active site for biological nitrogen fixation.
Dr. Rees’ work also resulted in: the first structure determination of a physiologically
gated ion channel, the mechanosensitive channel of large conductance (MscL) from
Mycobacterium tuberculosis; and the first structure determination of an intact and fully
ordered member of the widespread family of ABC transporters, the E. coli importer
BtuCD for vitamin B12.

Daniel J. Repeta
. Composition and cycling of dissolved organic matter (DOM). In particular I am interested
in the metabolic pathways used to remineralize complex forms of dissolved organic C, N,
and P by microbial consortia, and in the impact of DOM composition (including vitamins,
allelopathic compounds, and trace metal organic ligands) on microbial diversity.
Keywords: dissolved organic carbon, metabolic pathways, microbial consortia, vitamins,
ligands, allelopathic compounds
Craig Taylor
. Taylor is interested in the development and application of automated instrumentation for
long-term in situ time-series analysis of phytoplankton production and other microbial
activities; time series acquisition and preservation of microbial samples for assessment of
phylogenetic and functional responses to environmental change.
Keywords: microbial activity, primary production, biogeochemistry, time series,
instrumentation development, in-water sensors
Mak Saito
Trace element biogeochemistry and marine bioinorganic chemistry. In particular, my
group studies the interactions between trace elements such as cobalt (including cobalt
containing vitamin B12), zinc, cadmium, and iron and their nutritional and toxicity
effects on cyanobacteria and other microbes. We utilize analytical, physiological, and
molecular techniques to examine these processes from the global biogeochemical scale to
the metalloenzyme and genome level.
Keywords: trace elements, marine bioinorganic chemistry, cobalt, vitamin B12, zinc,
cadmium, iron, cyanobacteria, metalloenzymes, proteomics
. Vitamin B12 Deficiency Leads to Shrinking Brain, Bone Loss
Jonny Bowden, PhD, C.N.S. is a nationally known expert on weight loss and nutrition
and natural healing.

Production: In the 1920s and 1930s, it was found that liver extracts and other
concentrates of animal origin stimulated the growth of farm animals and could be used as
a treatment for human pernicious anemia. For many years the active principle was known
as the animal protein factor, and its chemical nature eluded scientists. Then, in 1948, a
crystalline material with the same characteristics was isolated from liver and called
vitamin B12. Its chemical structure was clarified in 1955, but it was not synthesized until
1973. Meanwhile, supplies of vitamin B12 were prepared by extraction from
fermentation products, a method that is still the most economic method of commercial
production. The chemical structure of vitamin B12 is the most complex of all the
vitamins.

Product forms: DSM vitamin B12 (cyanocobalamin) is available as a pure crystalline

powder and in various dilutions in powder form.

PRODUCTION OF VITAMIN B12 BY GRAM VARIABLE

METHANOL-UTILIZING BACTERIA
Vitamin b12 production by two gram variable methanol utilizing bacteria was reported.
Effects of growth conditions on vitamin b12 productivity were examined in shake flasks.
The maximum vitamin B12 production was 227ug per liter culture broth.

INTRODUCTION

VITAMIN B12 is produced industrially by fermentation of carbohydrate media using

microorganisms such as streptomyces species

The accumulation of vitamin b12 by strain M-02323 isolated from soil sample was
demonstrated by Tanaka (1974). Among methanol utilizing bacteria isolated from soil
and air Nishio one of the best vitamin b12 producers is Klebsiella sp. And also that in
pseudomonas and microcyclus interesting production rates were obtained.

Use of Cheese Whey for Vitamin B12 Production

The patterns of growth and vitamin formation by Propionibacterium shermanii in whey
were similar to the patterns established in other substrates. The vitamin formation was
observed during the latter part of the fermentation after the organism approached
maximal growth. Lactose utilization by the organism corresponded to the logarithmic-
growth phase of the organism. Analyses of the dried culture showed a large increase of
vitamin B12 in the fermentation solids compared with unfermented dried whey. A feed
analysis showed a notable increase of protein and a large decrease in nitrogen-free extract
of the dried fermentation solid compared with dried whey.

Abstract:
The present invention relates to a process for producing vitamin B12, wherein said
process comprises the steps of (a) culturing a strain of the genus Propionibacterium in a
first fermenter under anaerobic conditions to obtain a culture of Propionibacterium, (b)
transferring at least part of the culture obtained in (a) to a second fermenter and
subjecting this culture to oxygen, (c) replacing in the first fermenter part of the volume
transferred in (b) with fresh culture medium

FIELD OF THE INVENTION

The present invention relates to a fermentation process for the production of vitamin B12.

BACKGROUND OF THE INVENTION

Vitamin B12 is an important vitamin for humans and animals. It is used to treat
pernicious anemia and peripheral neuritis, and is used as a dietary supplement. Vitamin
B12 is also an important animal feed supplement as growth enhancer.

Vitamin B12 is produced industrially by microbial fermentation, using almost exclusively

Pseudomonas denitrificans and Propionibacterium species. Contrary to Pseudomonas,
Propionibacteria are food-grade. Processes using Propionibacterium species thus have the
advantage that they allow to formulate natural vitamin B12 together with the biomass in
which it is produced. Such processes avoid the conversion of natural vitamin B12 into the
cyanocobalamin form by chemical processes including cyanidisation followed by
extraction and purification steps using organic solvents.

Propionibacteria are Gram-positive bacteria capable of producing valuable compounds in

a variety of industrial processes. Propionibacteria are, for instance, important in the
production of cheese, propionic acid, flavours and vitamin B12. This maximum amount
of propionic acid produced in such fermentations results in a maximum of 25-35 g/l
biomass that can be reached. Propionic acid concentration is thus one limiting factor for
biomass growth and thereby for high vitamin B12 yield.

Several Propionibacterium species are capable to produce vitamin B12 in large scale
fermentation processes. The process is described as two-stage fermentation with a 72-88
hours anaerobic fermentation followed by a 72-88 hours aerobic phase. The vitamin B12
concentration in the cells rapidly increases in the aerobic phase; with typical values of 25-
40 mg vitamin B12/l. Anaerobic growth followed by an aerobic phase with limited
growth is important for economic production of vitamin B12 using Propionibacterium
species. This requirement, however, limits the amount of biomass to 25-35 g/l as
described above. Several attempts have been made to overcome the barrier of propionic
acid toxicity in order to increase biomass and thereby the yield of vitamin B12.

Alternated anaerobic-aerobic phases are e.g. suggested to reduce the amount of acids. In
the aerobic phase the propionic acid is converted to less toxic acetic acid, with
simultaneous formation of vitamin B12. The relative yield of vitamin B12 has been
increased, but the final titre is rather low. This is probably due to inhibition early in the
synthesis of vitamin B12 and/or other oxygen related products limiting the synthesis of
vitamin B12. The final vitamin B12 produced with this method is 9 mg/l compared to 4.5
mg/l with the fully separated anaerobic and aerobic phases. Both values are rather low for
vitamin B12 production with Propionibacteria.

The suggestion to use immobilized cells is mainly focused on the production of propionic
acid. The propionic acid production is greatly enhanced. Use of this option for vitamin
B12 production will imply harvesting of the vitamin B12-containing cells with the
immobilization material. Repeated fed-batch fermentation with an anaerobic phase
followed by an aerobic phase and withdrawal of broth at the end of the aerobic phase is
not possible.

There is thus still a need for Propionibacterium-based fermentation processes for the
production of vitamin B12 with further improvements in efficiency and/or vitamin B12
yield.

DESCRIPTION OF THE INVENTION

The present invention provides a process for producing vitamin B12 (and precursors
thereof having detectable vitamin B12 activity) which is not a continuous process and
which comprises the steps of:

(a) Culturing a strain of the genus Propionibacterium in a first fermenter under anaerobic
conditions to obtain a culture of Propionibacterium,

(b) Transferring at least part of the culture obtained in (a) to a second fermenter and
subjecting this culture to oxygen,

(c) Replacing in the first fermenter part of the volume transferred in (b) with fresh culture
medium, and

(d) Repeating steps (a), (b), and (c) at least once.

One aspect of the process of the invention concerns the relation between the fraction of
the anaerobic culture which is transferred in (b) and replenished with fresh medium in (c)
and, on the one hand, the growth rate of the anaerobic culture (in (a)) and, on the other
hand, the time interval between the subsequent withdrawals. One embodiment of the
process of the present invention comprises the steps of:
(a) Culturing a strain of the genus Propionibacterium in a first fermenter under anaerobic
conditions,

(b) transferring at least 30 to 90%, preferably at least 40 to 90%, more preferably at least
50 to 90%,more preferably at least 60 to 90% and most preferably at least 70 to 90% of
the culture volume obtained in (a) to a second fermenter and subjecting this culture to
oxygen,

(c) Replacing in the first fermenter the same volume as the one withdrawn in step (b)
with fresh culture medium; and

(d) Repeating steps (a), (b), and (c) at least once.

Knowing the withdrawal volume and the growth rate of the microorganism used, the
skilled person can easily deduce from the formula the time interval between each draw
and thus the duration of the whole process.

In another preferred embodiment of the process of the invention, the dissolved Oxygen
concentration at inoculation of the anaerobic phase is less than 5% of air saturation,
preferably less than 2.5%, and more preferably less than 1% of air saturation. According
to one embodiment of the invention there is provided a process wherein the aerobic
second phase in (b) is performed in at least two serially connected aerobic fermenters.
Most preferably the aerobic second phase in (b) is performed in plug flow mode, wherein
e.g. the “second” aerobic fermenter comprises a series of aerobic fermenters.

Suitable culture media for the production of vitamin B12 with Propionibacteria are well-
known in the art (cf. Biochemical engineering and biotechnology handbook, 1991, B.
Atkinson ed., Macmillan Publishers Ltd, pp: 1213-1220). In a preferred embodiment of
the invention the culture medium is supplemented with 1-50 mM of one or more
compounds selected from the group consisting of betaine, methionine and glutamine.
Another preferred supplement for the culture medium is 5, 6-dimethylbenzimidazole
(DBI). DBI is preferably supplemented at 1-40 mg DBI per litre. Preferably, DBI is
added to the culture medium at the start of the aerobic phase or during that phase.

In a preferred embodiment of the process of the invention the concentration of

undissociated propionic acid is controlled such that it does not exceed 5 mM. This
conveniently may be done by increasing the pH of the culture medium according to
methods well known to the skilled person.

According to another embodiment of the invention, the temperature under anaerobic

conditions is different from the temperature under aerobic conditions. Preferably, the
temperature under anaerobic condition is higher than the temperature under aerobic
conditions. In the process of the invention, preferably a strain of a Propionibacterium
species is used which is selected from the group consisting of the classical or Dairy
Propionibacteria as described in Bergey's manual of systematic bacteriology, 1986, J. B.
Butler, Williams & Wilkins, p 1346-1353. This group comprises inter alia the species P.
freundenreichii with subspecies freudenreichii and shermanii, P. thoenii, P. jensenii and
P. acidipropionici. More preferably the strain P. freundenreichii CBS 929.97 is used. P.
freundenreichii CBS 929.97 was deposited Jul. 10, 1997 at the Central Bureau voor
Schimmelcultures, Baarn, The Netherlands.

In one embodiment of the invention, the process of the invention is performed using
Propionibacteria strains that are genetically modified by means of classical mutagenesis
and/or recombinant DNA technology.

One of the advantages of the process of the invention is the throughput increase of the
fermentation. The anaerobic phase is considerably reduced. For a growth rate of 0.06 h −1
the output of the anaerobic phase is three fermenter volumes in 72 hours, compared to
one fermenter volume for the classical process. In addition, the invention reduces the
turnaround time of the fermentation, with ten consecutive fills and draws, to 20% of the
classical process.

Another advantage of the process of the invention is that the size of the fill and draw
volumes may be relatively large. Consequently, the growth inhibition caused by high
concentration of propionic acid under anaerobic conditions does not occur, leading to an
increase in the biomass and thereby an increase in vitamin B12 yields.

• Methylcobalamin and the New Story of

Vitamin B12
There's a buzz over B12 these days for two reasons, one scientific and the other
economic. First, the science: Over the last decade or so, researchers have strongly
implicated the toxic amino acid homocysteine in a variety of disease states.
Homocysteine tends to accumulate in the body whenever B12 gets deficient, and this
accumulation has been linked with increased risk of Alzheimer's disease, cardiovascular
disease, chronic fatigue syndrome/fibromyalgia and multiple sclerosis among other
conditions.

Folic acid deficiency can also lead to increased homocysteine levels-that's because folate
and B12, in their active 'coenzyme' forms, are both necessary cofactors for the enzymatic
conversion of homocysteine to methionine. Until recently it's been thought that the
availability of folate was the most important determinant of the body's ability to
remethylate homocysteine.

The coenzyme form of vitamin B12 is known as methylcobalamin or methyl B12. It's the
only form of vitamin B12 which can directly participate in homocysteine metabolism. In
addition, converting homocysteine to methionine via methyl B12 generates an increased
supply of sAMe (S-adenosyl methionine), the body's most important methyl donor.
Around 1998, methylcobalamin first became widely available in this country at an
affordable price, thus offering new options for treating B12 deficiencies and lowering
elevated homocysteine. Before then, methyl B12 had been enormously expensive and
widely available only in Japan, where it still remains a prescription medication. Today
any health-conscious American consumer can easily access the most powerful known
form of vitamin B12.

Methylcobalamin and Cyanocobalamin

When most of us think of vitamin B12, the molecule we really have in mind is
cyanocobalamin or cyano B12. As its name suggests, cyano B12 has a cyanide group
(CN) attached, whereas methyl B12 carries a methyl group (CH3) instead. Very little of
the body's natural B12 is in the cyano form under normal circumstances; exceptions are
in cases of cyanide poisoning or chronic smoking, both of which can raise
cyanocobalamin levels.

Whenever we swallow a conventional vitamin pill, any cyano B12 present gets carried
along and absorbed by an intricate "bucket brigade" of B12-binding proteins. Operating
in the stomach and small intestine, this transport system provides a very efficient
mechanism for absorbing a few micrograms of B12, yet is quickly swamped by anything
larger. As a result, only about 1% of a large oral dose of any form of B12 usually makes it
into the bloodstream. Fortunately, we can bypass intestinal absorption entirely by giving
B12 by injection or sublingually. In particular, sublingual administration is a simple and
effective way of substantially raising blood levels by absorbing B12 through the oral
mucosa. It's also unquestionably the most convenient way to take B12, especially for
people taking supplements on a daily basis.

It should be obvious that there are certain advantages inherent in taking methyl B12 as a
supplement, versus 'ordinary' B12. For one thing, methyl B12 doesn't have to engage the
body's resources to convert it into coenzyme form, it's already there. Even more
important is the fact that methylcobalamin is the most highly reduced form of vitamin
B12 possible; this makes methyl B12 a very potent reducing agent (antioxidant) indeed.
In a body undergoing oxidative stress -- for example from a disease process or from a diet
deficient in antioxidants -- it's possible that methyl B12 production can become impaired.

Fermentation Plant for the production of Vitamin B12

This production plant has never been in operation and is therefore practically new.
Most of its components are already available and only few components have
to be added. Through fermentation an amount of 360 - 500 kg crude Vitamin B12 100%
can be produced per year.

The world market today faces rapidly growing demand for Vitamin B12. This production
plant can contribute to meeting those demands by producing Vitamin B12 through
efficient fermentation with high outputs.
The basis for the fermentation process are highly efficient bacterial strains. Molasses can
be used as nutrient solution which is derived as waste product from sugar production.

The fermentation process

the main process components are 3 fermenters of 25 m3 each. The required amount of
molasses, as nutrient solution, is at 200 - 250 tons per year.
Depending on the efficiency of the used bacteria "propionibacterium spec.",
the annual output can vary from 360 - 500 kg of crude Vitamin B12.It is assumed that an
average of 10 fermentations per month can be realized, considering one day each for
preparations beforehand and cleaning afterwards.

Vitamin B12 production by Citrobacter freundii or Klebsiella

pneumoniae during tempeh fermentation and proof of
enterotoxin absence by PCR.
The influence of some fermentation parameters on vitamin B12 formation by strains of
Citrobacter freundii and Klebsiella pneumoniae isolated from Indonesian tempeh samples
during tempeh fermentation was investigated. A decrease in fermentation temperature
from 32 to 24 degrees C led to a decrease in vitamin B12 formation. Inoculation of
soybeans with different numbers of cells of C. freundii at the beginning of solid-substrate
fermentation showed that only the velocity of vitamin formation and not the final amount
of vitamin formed depended on the number of cells. The addition of cobalt and 5, 6-
dimethylbenzimidazole increased the vitamin B12 content of tempeh. Nevertheless,
levels of incorporation of the two precursors into the vitamin B12 molecule were very
low. Neither C. freundii nor K. pneumoniae possessed the genes encoding the
enterotoxins Shiga-like toxin SLT IIA, heat-labile enterotoxin LT Ih, and heat-stable
enterotoxin ST Ih, as indicated by PCR. This result supports the suggested use of these
two strains to form vitamin B12 during tempeh fermentation in Indonesia.

Process for the production of fermentation broth with

increased vitamin B12 content by synchronizing the bacterium
population
The invention relates to a process for the production of a fermentation broth with
increased vitamin B12 content by a fermentation process carried out with a methane-
producing mixed bacterium population under anaerobic septic conditions in the presence
of known nutrient components and precursors. According to the invention, methanol,
preferably in an amount of 0.5 v/v %, is added for some days, preferably for 3 days to the
fermentation broth containing vitamin B12 used as the starting substance. Thereafter a
minor part, preferably 10% of the fermentation broth, is removed and an equal volume of
a nutrient broth containing the usual components in tenfold concentrations is added
periodically in every 5th to 12th day, then the removal of fermentation broth is
interrupted for 0 to 2 days, preferably for 1 day, and only 0.4 to 1.5 v/v% of methanol are
added to the fermentation broth, and subsequently a minor part, preferably 10% of the
fermentation broth, is removed daily and an equal volume of a nutrient broth containing
the usual components in the usual concentrations is added together with 0.4 to 1.5 v/v %
of methanol, depending on the biogas production of the fermentation broth. This series of
operations is repeated periodically during the complete fermentation procedure taking
into consideration the pH of the fermentation broth.

Description:

This invention relates to a process for the production of fermentation broth with increased
vitamin B 12 content, by anaerobic fermentation carried out with a methane-producing
mixed bacterium population under septic conditions. As is known, vitamin B 12 is
generally produced either by monoculture sterile fermentation or with a mixed bacterium
population of sewage sludge origin, adapted to a special culture medium.

It is also known that when using a mixed bacterium population, by contrast with the
sterile monoculture fermentation process, the yield of vitamin B 12 cannot be increased in
practice by increasing the individual productivities of the bacterium strains. This can be
attributed to the fact that in mixed cultures the metabolic processes of the individual
bacterium strains are not independent of each other. Their interaction may be positive,
leading sometimes to a very marked increase in the production of the desired metabolite
(B. Johan, A. Szabo, E. Keresztessy: Combined fermentation Acta Microbial. Acad. Sci.
Hung. VI/4 (1959), but it may also be negative, for instance in septic fermentations where
the common pathogenic bacteria are unable to proliferate in the fermentation broth.

Several methods are known for increasing the vitamin B 12 yields of fermentations carried
out with methane-producing mixed bacterium populations, these methods can usually be
applied, however, to the production of fermentation broths containing only about 10,000
to 11,500 μg./l. of vitamin B 12 (e.g. Hungarian patent specification No. 159.356).

The aim of the present invention is to provide an economical process for increasing the
yield of vitamin B 12 in the fermentation processes utilizing known, semicontinuously
maintained bacterium populations of sewage origin. The new process was developed by
taking into account the following facts and experimental results:

1. Investigations of the vitamin B 12 -producing sewage-originated methanobacterial

fermentation have revealed that 93 to 97% of the resulting vitamin accumulates in the
microorganism cells, and enters the broth (supernatant) only after the necrosis of the
bacteria. Thus, in order to increase the vitamin B 12 yield of the fermentation, the number
of vitamin-producing bacteria should be increased.
2. The bacteria of the methane-producing bacterium population of sewage sludge origin,
particularly the coccus-diplococcus bacteria, have a relatively prolonged duplication
period, and thus they have no real "logarithmic phase" characteristic of the reproduction
of most bacteria..

3. Upon measuring daily the bacterium content and vitamin B 12 content of a fermentation
broth produced in a semicontinuous fermentation (10% of the fermentation broth are
removed daily and a same amount of fresh nutrient broth is supplied), these values are
found to be approximately invariable, that is, within 24 hours both the number of the
bacteria and the amount of vitamin B 12 return to the levels observed before removal. Thus
the invention is based on achieving the most perfect synchronization of the reproduction
cycle of the bacterium mass, both at the start of a new fermentation and during the
semicontinuous maintenance of the fermentation broth with an already high vitamin B 12
content.

According to the invention one proceeds as follows: a semicontinuously maintained

fermentation broth with a vitamin B 12 content of about 10.000 μg. /l. is used as the
starting material, and only a small amount of methanol is added to the broth for three
days, to bring the population by starvation into a state where the partition of the bacteria
in the multiplication phase ceases and new partition cannot start owing to the lack of
nutrient. Thus the cells are induced to undergo a "stationary" or lag phase. On the fourth
day, a nutrient broth, which contains the usual components in tenfold concentration, is
added at once to the fermentation broth. This nutrient broth is designated in the following
as "concentrated broth". This procedure is also continued in the semi-continuous phase of
fermentation. In this phase 10% of the fermentation broth is removed daily, and an equal
volume of a nutrient broth containing the usual components in the usual (normal)
concentrations (i.e. in 10 times lower concentrations than in the concentrated broth) is
added to the remainder of the fermentation broth. This latter nutrient broth is termed in
the following as "usual broth or normal broth". The addition of the concentrated broth
necessary to the multiplication of the increased number of bacteria is carried out
periodically, in every 5th to 12th day in average. When the concentrated broth is added on
the average every 10th day, 10% of the fermentation broth is removed and replaced with
an equal amount of usual broth in the intercurrent days, and an appropriate amount of
methanol is added, 30,000 to 40,000 μg./l. concentrations of vitamin B 12 can be reached
and steadily maintained.

The invention thus comprises a process for the anaerobic septic fermentation of a broth to
produce vitamin B 12 in the presence of bacterial nutrients, methanol and a methane-
producing bacteria capable of fermenting methanol to produce vitamin B 12 wherein
portions of the fermenting broth are periodically removed and replaced by a nutrient
broth containing nutrient components in a normal concentration to sustain fermentation.
The main advantages of the process according to the invention can be summarized
as follows:

1. The process provides, under certain conditions on a laboratory scale, high vitamin B 12
values that could not be attained so far with septic fermentation processes and can hardly
be attained even with artificially activated monoculture fermentation processes. The high
vitamin B 12 level, about 3.5 to 4 times higher than that of the average industrial scale
processes (about 10,000 μg./l.) can be accomplished in laboratory scale with the
synchronized periodical procedure of the invention with a 2 to 2.5-fold nutrient
requirement and 3-fold methanol requirement compared to the average industrial values.

2. In the laboratory process, reproduced several times, vitamin B 12 levels of 30,000 to

35,000 μg. /l. could be maintained for about a one-month semicontinuous fermentation
process even in an iron fermenter of 50 m 3 .The process of the invention is the first one
to utilize a synchronization technique in a fermentation procedure, and also the first to
utilize this method for septic cultures consisting of several bacterium types. In our
process starvation is utilized for synchronization, thereby bringing the different bacteria
into an approximately uniform repose (inactive) stage. The addition of the nutrient and
energy sources to the thus-synchronized different bacteria in experimentally pre-
determined intervals and amounts results in an optimum production of vitamin B 12 , i.e.
leads to the overproduction of the coccus-diplococcus bacteria with the best specific
productivities as compared to the less productive rod bacteria.

Continuous methane fermentation and the production of

vitamin B12 in a fixed-bed reactor packed with loofah
YINGNAN YANG ZHENYA ZHANG; JUN LU; MAEKAWA Takaaki;

A fixed-bed reactor with acclimated methanogens immobilized on a loofah support was

studied on a laboratory scale to evaluate the system producing methane from the mixture
of CO2 and H2 gas, with the production of vitamin B12 as a by-product. Fermentation
using CO2/H2 acclimated methanogens was conducted in a jar fermentor with hydraulic
retention times (HRTs) of three and six days. The performance of the reactor was mainly
dependent on the HRT. With an HRT of three days, the methane production rate and the
vitamin B12 concentration in the culture broth The loofah carrier immobilized almost 95%
of the methanogens, which led to a more effective bio-reaction. It was also observed that
the fermentation system had a better ability to buffer pH, especially for an HRT of six
days.

Process for the production of fermentation broth with increased vitamin

B12 content by synchronizing the bacterium population
The invention relates to a process for the production of a fermentation broth with
increased vitamin B12 content by a fermentation process carried out with a methane-
producing mixed bacterium population under anaerobic septic conditions in the presence
of known nutrient components and precursors. According to the invention, methanol,
preferably in an amount of 0.5 v/v %, is added for some days, preferably for 3 days to the
fermentation broth containing vitamin B12 used as the starting substance. Thereafter a
minor part, preferably 10% of the fermentation broth, is removed and an equal volume of
a nutrient broth containing the usual components in tenfold concentrations is added
periodically in every 5th to 12th day, then the removal of fermentation broth is
interrupted for 0 to 2 days, preferably for 1 day, and only 0.4 to 1.5 v/v% of methanol are
added to the fermentation broth, and subsequently a minor part, preferably 10% of the
fermentation broth, is removed daily and an equal volume of a nutrient broth containing
the usual components in the usual concentrations is added together with 0.4 to 1.5 v/v %
of methanol, depending on the biogas production of the fermentation broth. This series of
operations is repeated periodically during the complete fermentation procedure taking
into consideration the pH of the fermentation broth.

Vitamin B12 production in fermented milk products

Abstract:
Disclosed is a fermented milk product having an increased content of in situ produced
vitamin B12, wherein at least bacteria selected from the group consisting of bacteria of
the strain Propionibacterium freudenreichi spp shermani B369, bacteria of the subspecies
Propionibacterium freudenreichi spp freudenreichi and bacteria of the species
Lactobacillus reuteri are present, which result in a content of in situ produced vitamin
B12 of preferably at least 120 % of the content of in situ produced vitamin B12 achieved
in absence of at least bacteria selected from the group consisting of bacteria of the strain
Propionibacterium freudenreichi spp shermani B369, bacteria of the subspecies
Propionibacterium freudenreichi spp freudenreichi and bacteria of the species
Lactobacillus reuteri. The invention also relates to a method of preparing such a
fermented milk product having an increased content of in situ produced vitamin B12,
wherein milk is fermented making use of a common starter culture, and to the use of the
aforementioned bacteria for increasing the content of in situ produced vitamin B12 in a
fermented milk product.

Efficient production of vitamin B12 from propionic acid bacteria under

periodic variation of dissolved oxygen concentration

Performance of Propionibacterium freudenreichii in anaerobic, aerobic and periodic

fermentations was studied. It was found that oxygen is a key parameter to the metabolic
regulation. Cells could grow faster during a short period (6 h) after a DO-shift. However,
long time aerobic fermentation (over 6 h) is disadvantageous in cell growth due to the
inhibition of oxygen on the synthesis of cytochromes. The propionate was decomposed
immediately and pyruvate was accumulated after DO-shift. Low DO concentration was
found to be advantageous in cell growth, in decomposition of propionate, and in lowering
the production of acetate. A novel cyclic operation where anaerobic and aerobic
conditions were alternately implemented was developed to take full advantages of
anaerobic and aerobic fermentations in order to enhance the production of vitamin B12.
Propionate was significantly produced and no pyruvate was accumulated in the anaerobic
cycle, whilst propionate was decomposed and pyruvate was accumulated in the aerobic
cycle. Vitamin B12 was synthesized in the anaerobic cycle and ceased to be produced in
the aerobic cycle. With this operation mode, a low concentration of propionate could be
maintained and high cell concentration could be attained. The volumetric production of
vitamin B12 using this method could be increased by about 2-fold as compared with that
attained in the anaerobic fermentation.

High-Level Folate Production in Fermented Foods by the B12

Producer Lactobacillus reuteri JCM1112
ABSTRACT
we observed that Lactobacillus reuteri JCM1112 produces B12 and folate. However, the
folate/B12 mass ratio found was far below that desired for human consumption ( 170:1).
We used metabolic engineering applying genetic and physiological approaches to improve
this ratio and developed a generic and natural process that significantly increases folate
production.

INTRODUCTION
Humans have an auxotrophic requirement for vitamin B12 and folate, and the
recommended intakes of these nutrients for healthy adults are 2.4 and 400 µg/day,
respectively. Suboptimal intake of either of these compounds has been linked to
cardiovascular disease, neuropathy, birth defects, cancer, and different types of anemia,
among other pathologies. Remarkably, the onset of vitamin B12 deficiency symptoms is
often delayed by an increased intake of folate. This masking of B12 deficiency has resulted
in the restriction of folate intake levels and prevented folate fortification in many
countries. Strict vegetarian dietary regimens tend to be poor in vitamin B12 and rich in
folic acid, increasing the risk of vitamin B12 deficiency masking. This has boosted the
popularity of fortifying vegetarian foodstuffs with B12..

Coenzyme B12 is synthesized by a few members of the bacterial and archaeal groups. In
situ microbial B12 production is a convenient strategy to achieve natural enrichment of
fermented foods, notably from vegetable sources. Lactobacillus reuteri is a gram-positive,
heterofermentative lactic acid bacterium with a long history of safe use by the food
industry. This microorganism ferments several sugars, and this flexibility leads to its
capacity to thrive on several substrates of vegetable origin. Strain CRL1098 has been
reported to produce different forms of B12, and the draft genome sequence of strain
JCM1112 suggests that it is able to produce folate, as well as B12.

SUMMARY:
Vitamin B-12 cannot be made by plants or animals as only bacteria have the enzymes
required for its synthesis. Species from the following genera are known to synthesize B-
12: Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium,
Corynebacterium, Flavobacterium, Micromonospora, Mycobacterium, Nocardia,
Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella,
Serratia, Streptomyces, Streptococcus and Xanthomonas. Industrial production of B-12 is
through fermentation of selected microorganisms. The species most often used,
Pseudomonas denitrificans and Propionibacterium shermanii, are frequently genetically
engineered and grown under special conditions to enhance yield. It is formed by
fermentation process in bacteria as well as industrially. it is produced by methanol
utilizing bacteria, use of cheese whey , production by citrobacter ferenduii by tempeh
fermentation, lactobacillus reuteri, efficient production by propionic acid bacteria under
periodic variation of dissolved oxygen concentration, production in fermented milk
products continuous methane fermentation and production of vitamin b12 in fixed bed
reactors packed with loopah. All these processes of fermentations in bacteria are used for
the production of vitamin b12.

REFERENCES:
• http://en.wikipedia.org/wiki/vitaminB12
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• http://www.vitamins.com/health
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• www.pubmedcentral.nih.gov/articlerender.fcgi?artid=242746
• www.freepatentsonline.com/6492141.htmlhttp://aem.org/cgi/content/full/74/10/32
91
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• plantpro.doae.go.th/worldfermentedfood/P7_Warawut.pdf
• www.biomedexperts.com/.../Influence_of_cobalt_concentration_on_vitamin_B12
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• www.howweheal.com/vitaminb12.htm