SYNTHESIS OF VITAMIN B BY MICROORGANISMS - ASM
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SYNTHESIS OF VITAMIN B BY MICROORGANISMS
GERTRUDE SUNDERLIN AND C. H. WERKMAN
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Bacteriology Section, Iowa Agricultural Experiment Station, Ames, Iowa
Received for publication, January 7, 1928
Studies on the synthesis of vitamins have led to no general
agreement as to the ability of specific micro6rganisms to produce
vitamin B or as to the extent of this synthesis among the lower
plant forms.
This lack of agreement may be attributed in part to the failure
of investigators to keep clearly in mind the characteristics and
properties that define a vitamin and in part to the marked differ-
ences in methods which have been employed.
There is, on the one hand, the group which has adhered rigidly
to the conception of a vitamin as defined by Drummond (1924),
i.e., a substance of unknown constitution neither fat, protein,
carbohydrate, nor mineral salt, necessary for the life and well-
being of a suitable organism. On the other hand, there is a
group which has disregarded the requirements of Drummond and
has used as the criterion of the presence of vitamin B, the ability
of a substance to stimulate multiplication of a specific micro-
organism. The yeast growth test of Williams (1919) employs
the multiplication of Saccharomyces cerevisiae.
If we exclude from analysis results which are not in strict con-
formity with the definition of Drummond, we are still confronted
by the use of a multiplicity of methods all purporting to detect
the presence of vitamin B. The animal used may have been the
rat, mouse or pigeon. In certain cases a few milligrams have
constituted the entire quantity of microorganisms fed per rat per
day and the ability of an organism to synthesize vitamin B has
been decided upon the results obtained in such experiments.
Again, the period of feeding has varied from two to several
1718 GERTRUDE SUNDERLIN AND C. H. WERKMAN
weeks. Generally, no cognizance has been taken of the manner
of feeding, or the time required by the animals to become accus-
tomed to the taste of the micro6rganisms. In feeding certain
micro6rganisms, the animals may continue to lose weight for as
long as a week. In certain cases the organisms have been fed
apart from the basal ration, whereas, in other cases they have
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been intimately mixed with the constituents of the basal ration.
Again, too little attention has been given to the freedom of the
culture medium from vitamin. At the most, it should not con-
tain more than traces of vitamin. Vitamin-free synthetic media
are to be preferred.
For the purpose of this investigation Drummond's definition
of a vitamin is accepted; no other conception is justifiable in the
light of present knowledge and no adequate proof has been
offered that vitamin B, as defined by Drummond, has any
effect on the rate of multiplication of micro6rganisms. In fact,
Werkman (1927) has shown by an analysis of experimental data on
multiplication of Azotobacter chroococcum and Rhizobium legu-
minosarum that vitamin B does not stimulate multiplication of
these organisms.
The purpose of this investigation was (1) to determine whether
vitamin B is synthesized by certain bacteria, one torula and forms
intermediate between molds and bacteria, (2) to choose these
organisms from biologically separate groups of the lower plant
forms so as to give an indication of the general occurrence of
vitamin B synthesis among microorganisms, (3) to determine
rthe variation, if any, in the ability of different strains of the same
species to synthesize the B vitamin, (4) to make a quantitative
study of the production of the vitamin, (5) to determine the
effect of drying of the organisms on their vitamin content.
Feeding the microorganisms in testing for the vitamin has been
quantitative so far as practicable. As much as 15 grams of
organisms per rat per day have been fed. Care has been taken
to accustom the rats to the diet and experiments have been con-
tinued until definite results were secured. The culture media
have been synthetic or thoroughly extracted with 95 per cent
hot alcohol to insure suitability for use. The uninoculated
medium was always fed to control animals.SYNTHESIS OF VITAMIN B BY MICROORGANISMS 19
LITERATURE
Among the workers using rats in determining the presence of
vitamin B, were Wollman (1921) and Wollman and Vagliano
(1922) who tested the Bulgarian bacillus and Amylomucor B
for vitamins A and B. They concluded that neither vitamin A
nor B was present. Slanetz (1923) using mice found that Azoto-
bacter chroococcum, Bacterium lactis-acidi, Bacillus mycoides,
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Serratia marcescens, Rhizobium leguminosarum, Bacillus subtilis
Micrococcus agilis and three other soil forms produced neither
A nor B.
Cunningham (1924) fed young rats in order to test for both A
and B in three strains of the tubercle bacillus. She found neither.
Pacini and Russel (1918) in some work referred to by McCollum
and Simmonds (1925) as "not convincing" claim to have shown
stimulation of the growth of rats by extracts of typhoid bacilli.
Bierry and Portier (1918) injected under the skin and into the
peritoneal cavity living cultures of some normal intestinal bac-
teria. Their animals showed improvement within twenty-four
to forty-eight hours.
Damon (1921) has probably tested more organisms for vitamin
B synthesis than any other worker who has published. His
first results with para B, Bacterium coli and Bacillus subtilis were
negative. In a later publication (1923) he concluded that
Pfeiffer's bacillus and the timothy bacillus synthesized vitamin B
but that Bacillus adhaerens and Friedlander's pneumobacillus
were negative. Damon (1924) believed that a substance analo-
gous to vitamin B was produced by Mycobacterium smegmatis,
Mycobact. phlei and Mycobact. moelleri. Hunter (1923) con-
cluded that Azotobacter synthesized vitamin B. Kuroya and
Hosoya (1923) concluded that Bacterium coli was capable of
synthesizing B. Hoet, Leclef and Delarue (1924) using both rats
and pigeons found Torula rosea, and Mycoderma cerevisiae nega-
tive but Monilia candida positive.
Funk's pigeon test has been used extensively to determine the
presence of vitamin B. McCollum and Simmonds (1925) gave
evidence to support the view that "the pigeon test as ordinarily20 GERTRUDE SUNDERLIN AND C. H. WERKMAN
carried out is without value for the specific purpose for which
it has been so widely used."
Cooper (1914) fed Bacterium coli to pigeons on a polished rice
diet but found no evidence of vitamin B. Weill, Arloing and
Dufourt (1922) fed the colon bacillus and three spore formers
from the intestinal tract of pigeons to pigeons on a polished rice
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diet. The birds died in wbat was considered normal time for
polyneuritic pigeons.
Scheunert and Schieblich (1922, 1923) fed cultures of various
bacteria to pigeons. They concluded that there was some effect
due to vitamins in certain bacteria and no effect with other
orgams Eijkman, Van Hoogenhuije and Derks (1922) con-
cluded from their experiments that Bacterium coli contains no
antineuritic factor. They used pigeons in their work.
William's test for vitamin B depends upon the assumption
that the increased multiplication of yeast cells upon the addition
of alcoholic extracts of various substances is a measure of water-
soluble B and that only. Souza and McCollum (1920) and
Fulmer, Nelson and Sherwood (1921) have presented evidence to
the effect that the yeast growth test is unreliable.
Robertson (1924) using William's yeast growth test concluded
that Bacterium coli, Serratia marcescens, Bact. proteus, Pseudo-
monas pyocyanea, Bacillus subtilis and Sarcina lutea synthesized
a vitamn.
Thjotta (1921) obtained evidence of a growth promoting sub-
stance for the influenza bacillus from Bacillus proteus, Bacillus
ozaenae and Friedlander's pneuxnobacillus. Damon (1921), how-
ever, did not confirm these results.
METHODS AND MATERAL
The authors have felt that the only adequate test for the pre&-
ence of vtamin B is the effect of a substance upon the growth of
young 50-gram white rats. Weighings of the rats receiving the
substance to be tested, of control rats receiving the B deficient
ration, and of rats receiving the basal ration made complete by
the addition of 5 per cent wheat embryo or yeast, were made and
growth curves over a period of several weeks compared. MetalSYNTHESIS OF VITAMIN B BY MICROORGANISMS 21
cages with a small quantity of shavings were used. The cages
were cleaned weekly.
The basal ration consisted of alcohol extracted casein 18 parts,
corn starch or dextrin, 73.3 parts, salt mixture 3.7 parts, salt-
free butter fat 5 parts. The salt mixture was that of McCollum
and Davis (1915) modified by the addition of 0.002 gram of KI
and the substitution of ferric citrate for the lactate. The casein
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was continuously extracted with hot 95 per cent alcohol for
seventy-two hours.
Young 45- to 50-gram rats were kept on this ration until
definite symptoms of vitamin deficiency were observed before
feeding of the organisms was started.
The microorganisms were generally fed moist in a separate
container; at other times, they were mixed with the basal ration
as indicated in the individual experiment. The organisms were
generally grown on media in 18- by 40-inch pans. The media
used are indicated under the experimental results with each
organis. They were synthetic where practicable. Where pep-
tone or agar was used, each had been continuously extracted with
hot 95 per cent alcohol for never less than one week. The media
were always fed to control animals as a check.
EXPERIMENTAL RESULTS
Torula rosea
Torula rosea from the stock culture collection of the department
was grown on medium E of Nelson, Fulmer and Cessna (1921),
modified by the use of alcohol extracted agar and sucrose. Sur-
face growth from the pans was scraped off after three days and
fed moist. In the feeding of Torula rosea mixed with the basal
ration, it was found that the rats consistently refused to eat
appreciably of the mixture.
When the torula was fed separately, the rats are it readily.
This may account for the fact that Torula rosea is reported in the
literature as not synthesizing vitamin B. By actual determina-
tion it was found when the torula was mixed in the basal ration,22 GERTRUDE BUNDERLIN AND C. H. WER
the rats received approximately 2 grams per rat per week, an
amount too small to supply the vitamin need of the animal.
In figure 2 the animals received the mixed ration from the
third until the end of the fifth week. Beginning with the sixth
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20 2 4 6 8 /0 Wseere
Controls
accho'omyce ce'evsioe addedat t _- __
Wheat embryo ododed at t
Vitamin B. free raion.
FiG. 1. EFFJECT OF Saccharomyces cerer?8iae UPON THE GROWTH OF VITAMiN B
DEFICIENT RATS
Organism feeding started at
week the torula was fed separately at the rate of 8 grams per
rat per day. One animal was in a moribund condition and died.
The remaining two began to gain in weight immediately. A
gain of approximately a gram per rat per day body weight is
recorded for each day torula was fed apart from the basal ration.SYNTHESIS OF VITAMIN B BY MICRO6ORGANISMS 223
Oospora lactis
No record of the synthesis of vitamin B by O5spora lactis has
been found. We have found it a rich source of the vitamin.
The organism was grown on a synthetic medium:
grama
K2HPO4 ........................................................ 2.0
(NH4)2S04 .2.0
Succinic acid .................
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... 0.5
Ca12 .0.1
FeCl .0.1
MgSO .0.2
Glucose (extracted) .15.0
Distilled water ................................................1000.0
0a 2 4 6 we'
rQU/ oseco
FIG. 2. EFFECT OF Torula rosea UPON THE GROWTH OF VITAMIN B
DEFICIENT RATS
Organism feeding started at 'j
This medium gave a heavy pellicle growth within four days,
which could be removed en masse. Quantitative feeding
experiments were made with this organism to determine the
requirements of rats. One of several experiments is given in
figure 3. The organism was fed at the rate of 2.5 grams per rat
per day during the sixth week. It was then fed ad libitum during
the seventh week when the rats consumed approximately fifteen
grams per rat per day. During the eighth week it was again fed
inlamounts of 2.5 grams and ad libitum during the ninth and24 GERTRUDE BUNDERLIN AD C. H. WE
tenth weeks. When fed at the rate of 2.5 grams per rat per day
the animals made no gain and if larger quantities had not been
tried Oospora lactis would have been considered incapable of
B synthesis. Larger quantities fed to the same animals gave
excellent gains. Oospora lactis is a good source of vitamin B,
distinctly better than Torula rosea.
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'70
VW
z 4 6 8 /O wh:8
Oospor ladeis
FIG. 3. ErFcrT oF O6pora lacti8 UPON TE GROWTH Or VITAMN B
DEFICIENT RATS
Organism feeding started at I
Bacillus adhaeren
Bacillus adhaerens was chosen primarily because we were
desirous of employing in certain experimental work an organism
incapable of synthesizing vitamin B and this one had been so
reported in the literature.
The organism was grown on extracted peptone agar in large
petri dishes. The composition of the medium was:
Alcohol extracted peptone .......................... ............. 7.0
Alcohol extracted agar .......................................... 15.0
K,HPO4 ........................................ .......... 2.0
Distilled water ................................................. 1000.0SYNTHESIS OF VITAMIN B BY MICROORGANISMS 25
The growth was scraped from the medium after forty-eight
hours incubation and fed separately. Three grams per rat per
day were given to the rats in the experiment shown in figure 5.
In this particular experiment after feeding the organisms for two
weeks they were omitted from the ration. A drop from 16 to
2.6 grams occurred in the average gain per rat per week. Ba-
cillus adhaerens is an excellent synthesizer of vitamin B.
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/20
/00
60
4J
/0
/60
4 6
w_e__s
Actinornyces
Fla. 4. EFFECT OF Actinomyces UPON THE GROWTH OF VITMIN B DEFICIENT RATS
Organism feeding started at I
Bacterium coli
Three strains of Bacterium coli were tested in this experiment
to determine the relative potencies of the different strains.
Four grams per rat per day were fed separately. The results
demonstrated that Bacterium coli is capable of synthesizing
vitamin B and that no significant differences exist in the ability
of the different strains to perform the synthesis. A growth26 GERTRUDE StNDERLIN AND C. H. WER
curve showing the average increase in weight of three rats fed
strain no. 24 is shown in figure 5.
Bacillus subtilis
The organism was grown on extracted peptone agar, scraped
off and fed apart from the basal ration. In the experiment
graphed in figure 5 the rats received 6 grams per rat per day
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until the eighth week when they received 1 gram per rat per day.
90.~~~~~~~~~ A
Bacillus -subtilis
Bacterlum col; -
Bocillus odhaerens
THE.
Fio.5. EFFECToF BACTERIA UPON GROWTH o VITANaN B DEmcxzNTRATs
Organism feeding started at ~
In order to demonstrate that the marked loss of weight result-
ing was due to insufficient vitamin, the rats were again fed 6
grams per rat per day during the ninth week. Although Ba-
cillus subtilis serves as an excelent source of vitamin B, one gram
per rat per day of the micro6rganism is insufficient to maint
body weight.
Drying the bacterial growth at 37°C. for two days had no
detectable effect on the vitamin potency.SYNTHESIS OF VITAMIN B BY MICROORGANISMS 27
Bacillus mycoides
Quantitative feeding results with Bacillus mycoides are given
to demonstrate the effects of feeding various amounts of an
/60 1
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140
o /,~~~~
t~~~~~~
ooo
/:0 /'~~~
600 eo~~~~~~~
.4(/, /
4°% ~ X 4 6 8 weers
Azo6obbocer chroococcum
Rhl'zobium /eyum/nosorY//n
FxI. 6. EFFECT OF BACTERIA UPON THE GROWTH OF VITAMIN B DEFICIENT RATS
Organism feeding started at 4
organism. It is apparent that if 2 grams or less per rat per day
of mycoides were fed, the conclusion might be drawn that the
organism is unable to synthesize vitamin B, whereas, our results28 GERTRUDE SUNDERLIN AND C. H. WER1N
demonstrate that mycoides is a good source of vitamin B when
compared with other organisms. In the results of the experiment
given in figure 7, groups of rats were fed simultaneously 0.5,
1.0, 2.0, 4.0 and 8.0 grams of B. mycoides per rat per day. The
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C B.rnycol.es feedmn eladi
80 8
70
60
40 i 2 3 4 5r weeks
NeAoi9ve contro/--no oqonism ..........
0.5 9m". amycol'es pera rot perdo/_
g mn. .rnycos:des pernat per "y_
2 mns. 8.&nc0;dGs per rot peraby .
4 9mn. B.ivycoides per rot per coy
9ms. 5a myco'des per rot perdady
FIG. 7. GROWTH OF THE WHITE RAT ON VITAIN B DEFICIENT RATION AND
VARYING AMOUNTS OF BACILLUS MYCOIDES
group receiving 0.5 gram per rat per day lost weight just slightly
less rapidly than the rats receiving no organisms; while the group
receiving 1.0 gram per rat per day in two week's time averaged a
slight loss of weight. The groups receiving 2.0, 4.0 and 8.0
grams gained in order of increasing quantity fed.SYNTHESIS OF VITAMIN B BY MICROORGANISMS 29
During the third week of the feeding of the organism (fig. 8)
the rats that had been receiving 8.0 grams were given 2.0 grams
and vice versa. The rats that had received 2.0 grams per rat
per day had made an average gain of 2 grams per rat per week.
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qo
80A
l
o
60
40 , 2 3 4 5 6 Weems
Negoaive confrol- no olyoni3ms
Z9s. B.mycokdes per rot penday do
49oms. .fi rnycoiSes per rot per, do
8grms. amycoides per rot per ay _ ____
FIG. 8. GROWTH OF THE WHITE RAT ON VITAMIN B DEFICIENT RATION AND
2 GRAms B. MYCOIDES INTERCHANGED WITH 8 GRAMS
When fed 8.0 grams per rat per day they made an average gain
of 24 grams in one week. This marked increase can be accounted
for only as due to the increase in the amount of the organism fed.
The rats that had been given 8.0 grams per rat per day made30 GERTRUDE SUNDERLIN AND C. H. WERKMAN
an average gain for the two weeks of 12 grams per week. When
reduced to 2.0 grams the average gain per week was 7 grams.
Organisms dried at 370C. and at 10000. for forty-eight hours
were fed in order to determine the effect of desiccation on the
vitamin potency. The results indicated no effect of drying at
37°C. and only a slight effect at 1000C.
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Azotobacter chroococcum
This organism was grown on Ashby's medium modified by
the use of alcohol extracted sucrose. The animals ate readily
of the bacterial growth when mixed with the basal ration or
when fed separately. In figure 6 are shown the results of feeding
6 grams per rat per day separately. Two grams per rat per
day were found to be inadequate to provide sufficient vitamin B
for increase in body weight.
Rhizobium leguminosarum
Rhizobium was grown on Ashby's medium modified as for
Azotobacter. The strain happened to be one isolated from alfalfa
roots. Synthesis of vitamin B by this organism is indicated by
the growth curve in figure 6.
Actinomyces (species unknoyn)
An unknown soil actinomyces was grown on the following
medium:
grams
K2HPO4 .... 1.0
KNOs ........ ................................................. 2.0
Calcium succinate ............ 10.0
Extracted agar ............ 17.0
NH4Cl ............................. 1.S
Distilled water ......... 1000.0
Considerable difficulty was experienced in obtaining sufficient
growth to feed. Long periods of incubation were necessary so
that the growths scraped from large plate cultures were stored for
several days before the experiment began in order to have suffi-
cient growth to complete the feeding. Six grams per rat per daySYNTHESIS OF VITAMIN B BY MICROORGANISMS 31
to 2 rats constituted the only work done with actinomyces.
The growth curve is shown in figure 4. The results indicate a
vitamin B synthesis by the organism.
SUMMARY AND CONCLUSIONS
The synthesis of vitamin B by such biologically separated
genera of micro6rganisms as Torula, O6spora, Actinomyces and
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four genera of the order Eubacteriales reveals a general occur-
rence of vitamin B synthesis among widely separated groups of
the lower plant forms. Vitamin B, whatever its structure
chemically may be, is a constituent prevalent in microorganisms.
Specifically the following organisms were found to produce the
growth promoting vitamin for white rats: Torula rosea, Oospora
lactis, Bacillus adhaerens, Bacterium coli, Bacillus subtilis, Bacillus
mycoides, Azotobacter chroococcum, Rhizobium leguminosarum and
Actinomyces (species unknown).
The results showed no marked differences in the ability of
three strains of Bacterium coli to synthesize vitamin B.
Drying at 37°C. or at 100°C. for forty-eight hours does not
materially diminish the vitamin potency of the bacterial mass.
Several reasons are suggested to account for the discrepancies
present in the literature dealing with the synthesis of vitamin B
by micro6rganisms. Our conceptions of a vitamin are not in
accord and thus differently defined the term vitamin assumes an
ambiguous and obscured meaning until it is in direct conflict
with the original conception intended.
After limiting by definition the meaning of the term vitamin
to the legitimate conception expressed by Drummond we find a
multiplicity of methods employed to determine the synthesis of a
vitamin by micro6rganisms. Important among these are (1)
quantity of organism fed, (2) period of feeding and time allowed
for the rats to become accustomed to the bacterial diet, (3)
manner of feeding organisms to the rats, (4) species of animal used
as an indicator.32 GERTRUDE SUNDERLIN AND C. B. WERKMAN
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Soc. de Biol., 86, 832.You can also read
