Microbiological Synthesis of Vitamin B,, by Propionic Acid Bacteria ABRAHAM LEVITON AND ROBERT E. HARGROVE Bureau of Dairy Industry, Agricultural Research Administration, U . S . Department of Agriculture, Washington, D . C .
A
NUMBERofpapersconcernedchieflywithvitaminB11yields in actinomycetes cultures appeared between 1949and 1951.
I n 10-day shake bottle experiments, Saunders, Otto, and Sylvester (10)obtained yields of 0.5 mg. per liter or less with 70% of their cultures and yields greater than 1mg. per liter with 4 strains. Yields of up to 2 mg. per liter with a strain of Streptomyces olivaceus were reported by workers of the Northern Regional Research Laboratory (6). Garey and Downing (J), working with synthetic media unsupplemented and supplemented with natural materials, reported yields of from 0.7 to 1.2 mg. and 2.0 mg. per liter, respectively. I n a survey of vitamin Blz production by actinomycetes, Schull and Routien (11)obtained yields varying from 0.02 to 1.5 mg. per liter and an average yield of 0.455 mg. A strain of B. megalherium was found by Garibaldi, Kjichi, and Snell(4) to produce 0.8 mg. per liter, corresponding to a consumption of glucose of 10 grams. In a patent granted to Hall and Tsuchiya (7), the production of vitamin Blz by Flavobacterium devorans in a glucose, corn-steep solids, soy-meal medium was claimed in yields of 0.576 mg. per liter. Cobalt ions were shown by Hendlin and Ruger (8) to be a limiting factor in the synthesis of vitamin Biz. Working with 13 cultures, including a strain of S. griseus, unidentified rumen and soil isolates, a strain of Mycobacterium smegmatis, and a Pseudom m a s species, they reported that the addition of 1to 2 p.p.m. of cobalt ion gave rise to approximately a threefold increase in yield with S. griseus and t o significant increases with the other cultures. The discovery in this laboratory of vitamin B11 activity in cultures of propionic acid bacteria was followed by an extension of the work to include the cultivation, under a great variety of conditions, of strains belonging to all eight of the species of the genus. In this paper some of the conditions that have given rise t o excellent yields are described. The production of vitamin B12 active compounds is not species specific. All species of the genus when cultivated under the same conditions will produce active substances, yet in different quantities. Further, even with the same species, irregularities in yield from time to time have been observed. All of the factors that enter into the regularization of yield are not clearly understood, yet a number of factors have been investigated. The results of these investigations are reported in this paper. EXPERIMENTAL METHODS
Assays for vitamin Blz activity were conducted according to the
U.S.P. method (13) with slight modifications. A total volume of 5 ml. per tube was used instead of 10 ml. with satisfactory results. Carbon-treated tomato juice was dispensed with as superfluous and as a source of assay irregularities. Both turbidimetry after 17 hours and acidimetry after 72 hours gave satisfactory results. The use of a standardized powder medium, a Difco product as it became available, afforded the advantage of ease in preparation and reproducibility.
Volatile acids were determined by the method of Friedemann and Brook (8). Careful determination of the partition of the volatile acids between ethyl ether and water permitted the calculation of the ratio between the quantities of propionic and acetic acid. Lactic acid was determined in the ether extract of a sample which had been clarified with zinc hydroxide. The extraction was conducted discontinuously with 1-ml. samples by means of a specially designed tube which permitted easy decantation of the ether layer. Data pertaining t o this technique will be reported in another paper. p H measurements were made with a glass electrode; sugar determinations were done by the Hagedorn-Jensen (6) method on aliquots which had been clarified with zinc hydroxide. The dry weight of cells was determined in concentrated cell suspensions. From measurements of the weight of cell solids plus occluded solids, of occluded liquor, and of occluded liquor solids the weight of cell solids was calculated, Turbidimetry was accomplished by means of a colorimeter on 1-ml, samples diluted with 9 ml. of water. Fermentations were conducted in a variety of laboratory apparatus. I n some experiments, an all-glass fermentation apparatus was used, comprising essentially a long glass tube, 75 X 2.2 cm., expanded a t the top into a 500-ml. flask. A sealed-in sintered-glass disk was located near the bottom. A gas inlet tube joining the fermentation tube a t the bottom was connected by means of a stopcock with a cottop-packed filter. The tilting of the apparatus permitted the removal of samples through a pipet sealed onto the main tube. I n experiments on a larger scale, 4and 20-liter borosilicate glass bottles were used, equipped with the appurtenances required for agitation, fine aeration, and the aseptic introduction of materials and removal of samples. Shake bottle experiments were conducted in 8-ounce screw-capped bottles, either shaken in an International shaking machine a t 240 r.p.m. or rotated end over end a t 30 r.p.m. in a circle 10 inches in diameter. Experiments were conducted a t 30" C. unless otherwise indicated. Sterilization of media took place a t 120" C.; 15, 30, and BO minutes were allowed, res ectively, for batches ranging in volume from o to 1,1 to 3, anti 3 t o 20 liters. Propionibacterium freudenreichii (ATCC 6207) was used in all of the experiments. RESULTS AND DISCUSSION
The propionic acid bacteria are hardy and will grow and thrive under strictly anaerobic as well as aerobic conditions. They will not grow in strongly aerated media, yet they will grow in open tubes and in shallow layers, and this capacity is fostered by the use of large inocula. Corresponding to changes in these conditions of growth are changes in cellular and colonial morphology as Van Niel (14) has excellently shown. With the idea that such changes might be associated with anabolic changes by means of which vitamin B I ~is produced, experiments were designed in which vitamin BIZyields were determined under various conditions of anaerobiosis and aerobiosis. Table I contains results of two sets of experiments. I n one, two fermentation tubes were used (experiments 1 and la). Aeration
2651
INDUSTRIAL A N D ENGINEERING CHEMISTRY
2652 TABLE I.
so.
Time, Hr.
Bi2--Ac~1v~ SUBSTANCES AKD OTHER BIOCHB&iICAL DATA OBTAINED FERMENTATIOKS CNDER AEROBICAKD AXAEROBIC COSDITIONS
Y I E L D O F VITAMIN IN
Expt.
Turbidity log I o / I
PH
Lactic Acid G . per'L. 12
Volatile Acids, G. per L.
Ratio of Propionic a n d Acetic Acids
.. .. ..
0
24 48 96 144
... ...
6.8
..
... ...
..
, ,
...
, .
o:io
...
.. .. .. ..
20 0.0 , .
..
0
0 5.9 20 0 040 5.9 42 0.38 6.4 70 0.31 6.4 94 0.31 6.4 Acid added to bring pH from 7.1 to 6.5
:
0 02
12
20
3a
Vitamin B I ~ Activity, Mg. per I,. 0.02
..
la
Vol. 44, No. I1
20 20 0.0
..
..
through one tube and nitrogenation through the other a t the rate of 300 ml. per minute through 200 nil. of medium resulted in the passage of a fine turbulent stream of bubbles over the entire cross section of the tubes. During the first 24 hours, growth occurred in both tubes with an appreciable rise in pH, particularly in the aerated sample. The yield of vitamin Bin-active substances corresponding apparently to the same amount of growth was twice as great in the nitrogenated sample. After a pH adjustment had been made, the concentration of active substances continued to rise in the nitrogenated sample to 0.6 mg. per liter and to fall in the aerated medium from a maximum value a t the end of 24 hours of 0.1 to 0.02 mg. per liter concomitant with an apparent lysis of the cells. It would appear, therefore, that strong aeration is not only inhibitory but will lead also to the destruction of preformed vitamin active substances. I n the second series of experiments, the medium contained 1% N-Z-Amine (Type A ) , lyO peptone, 1.25% sodium lactate, 0.3%yeast extract, and 1 p.p.m. cobalt ion. Upon incubation for 192 hours at 30" C., the following results were obtained: Under anaerobic conditions in the tubular fermenter,%yieldof 1.2mg. of active substances per liter; in three shake bottles containing, respectively, 25, 25, and 175 ml. of medium, the first and third of which were shaken and the second of which was held in situ, the respective yields were 1.4, 0.36, and 0.54 mg. of active substances per liter. From this set of data, the conclusion may be reached that strict anaerobiosis is not essential for the achievement of high yields
0:22 0.25 0.34 0.56
.. ..
...
0.0 0.0
, . .
15.0 15.0 15.0
2.0 2 0 2 0
0:0 15.0 15.0 15.0
2.0
0.9
2.0 2.0
1.1 1.0
... ...
... ...
0:0 1.0 1.0
1.0
0:0
Comments Medium: 200 ml. containing 4 mi. iia lactate (0.6 g. lactic acid per ml,), 2 g. K-Z-amine (Type A ) , 0.6 g. yeast extract, 167 CoClz; air passed through fermentation tube Medium same as i n ( 1 ) ' nitrogen passed through( fermentation tube 3ledium: 2.5 liters containing per lit,er: 10 g. X-Z-Amine, 5 g. yeast extract, 3 6.(YHa)zSO1. 20 g. lactic acid. 9.1 g. N a O H , and 4 mg. CoCIz.6HzO; fermented anaerobically Medium same as i n ( 3 ) ; fermented aerobically
and that agitation conduces t o it. The results obtained with small shake bottles are not readily translatable in terms of what occurs in large scale experiments. Consequently, an experiment was designed in which vitamin 1312 synthesis in relation to other biochemical activity under anaerobic conditions in 4-liter bottles was compared with the corresponding synthesis under aerobic conditione. Nitrogen was passed through one bottle containing 2.5 liters of stirred medium a t the rate of 120 ml. per minute. The control sample was merely stirred a t 200 r.p.m. while exposed to the atmosphere through ports plugged with cotton. The results of this experiment a.re shown in Table I, experiments 3 and 3a. The rates showing the development of vitamin BIZ up to 1 mg. per liter of volatile acids coincide, as do the rates showing the consumption of lactic acid. The conclusion may be drawn, therefore, that under moderate conditions of agitation nothing is gained both with respect to rates and yields by the employment of anaerobic conditions. This observation is, of course, of practical importance because the conditions of anaerobiosis are easier to dispense with than to obtain. A point of further practical interest. is the rate a t which the fermentation proceeds. The impression has been creat>edin the literature (9) that the propionic acid fermentation is an abnormally slow one in comparison with other industrial fermentations. Van Niel ( 1 4 ) has shown that on a semicontinuous basis with a 100% inoculum the time required to ferment; a 2% sodium lactate broth may be reduced from 7 t o 2 days after 5 complete changes of medium. The data contained in Table I indicate,
A N D ASSAYDATAFOR ~ ~ - I , I TPOT E R EXPERIMENT TABLE 11. FERMENTATIOS
N-Z-Amine. 4 g. yeast extract, 12 g. lactic acid, 4.5 mg. CoCh. 6 1 1 ~ 0 ; sterilized 1 hour a t 120' C.) Assay Data Microbial assay ( L . leichmannii) .__ R a t assay -~ Fermentation D a t a Total Total Total Total Plate activity activity activity activity count, Vitamin cells, cells, cells, cells, Turbidity, cells Blr. Storage beer unsterilizedb Activity beer unbeer Activity Time, l o g I p / I x 10-1 Cell wt., per ml. activity, timea, sterilized, beer, beer, sterilized. sterilizedb, beer, hr. g. per 1. X 10-9 mg. per 1. days mg. per 1. mg. per 1. mg. per 1. mg. per 1. mg. per 1. mg. per 1. 0 0.018 0.08 .. 0.02 0 0.74 .. 0.40 .. .. . , .. .. 29 0.183 0.9 2.1 0.24 .. .. .. 1'.0 0 .' 67 0:33 59 0.210 1.1 2.6 0.60 0134 .. 76 0.234 1.2 2.9 0.70 .. , . .. 98 0.234 1.2 3.3 0.74 34' 0:68 0.'60 0:27 .. .. .. a At 5' C. and pH 4.5. autoclaved 15 minutes a t pH 7.0 and 120' C. resulted in 30% loss in activity; b Sterilized 3 minutes a t 72O C. three times with 5-hour holding interval; autoclaving 30 minutes a t p H 9.8 and 120° C. resulted in 98 to 99.5% loss; 0.890 gram of volatile acids produced p e r 100 inl. per I . 2 grams of lactic acid. (Medium consists per liter of 15
g.
November 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
.
TABLE111. YIELDS
Expt. No.
Time,
Hr.
O F IrITAMIN
Lactic Acid Consumed, G . per L.
BL2---Ac~1v~ SUBSTANCES AND OTHER BIOCHEMICAL DATAOBTAINED COMPARING GLUCOSE AND LACTICACID
Glucose Consumed, G. per L.
Cell Wt.,
G . per L
0.15 0.0 0 1.0 .... 18 9.5 1.7 .... 2 0 . 0 (10.0)G 40 2.5 .... 64 3 0 . 0 (10.0) 2.8 .... 88 40.0 2.5 112 2.4 ...* 136 0.0 0 0.0 0:4 24 .... 0.7 0 . 0 (2.5)b 486 1.1 2.5(3.8) 72 1.5 6 . 3 (7.4) 100 10.0 (7.2) 2.1 120 2.2 140 2.3 168 2.5 1 7 . 6 (6.5) 190 20.0 216 Values for lactic acid in parentheees indicate increment of lactic acid added. Values for glucose in parentheses indicate increment of glucose added. pH if under 6.0 adjusted t o 6.0 with NaOH.
....
..... ...
:E I;::]
a
b 0
pH 6.3 5.9 5.8 5.8 5.8 5.8 5.8 6.5 6.0 6.0 5.5c 5.5 5.5 5.3 5.3 5.2
..
..
however, that the fermentation of 201, sodium lactate broth may be achieved in a batch process in 40 hours or less, and this with only a 4y0inoculum. This rate certainly is comparable with the rates which obtain with other industrial fermentations. It may be worth while at this point to digress somewhat and to point out that from the results of numerous experiments, i t appears that the duration of the lag phase in the fermentation of lactates is dependent among other things on the size of the inoculum and on the lactate concentration. Related to the slow rates of fermentation reported in the literature is the observation that distillers solubles contain inhibitory substances. This observation points to the possibility that the quality of yeast extract (whether or not i t is associated to a large extent with occluded beer solids) may have been a ratelimiting factor in experiments in which large concentrations of yeast extract were used as a source of nitrogen. It is conventional in order to obtain results which may be taken to anticipate the results of plant scale fermentations to conduct laboratory investigations in pots which may range from 5 to 20 liters in capacity. Tables I1 and I11 contain the data obtained in such experiments. The inoculated medium in the first experiment stirred a t 200 r.p.m. was incubated a t room temperature, 28' to 30' C., for 4 days. At the end of 59 hours (see Table II), the fermentation was more than 90% complete, and the yield of vitamin BIZwas 0.6 mg. per liter. At the end of 76 hours the yield had risen to 0.7 mg. per liter and rose only insignificantly thereafter. At the end of 98 hours the run was discontinued, the medium was brought to pH 4.5, and a large sample was withdrawn and held in storage at 5' C. The bulk of the fermentation liquor was heated to 60" C. and held for 5 minutes, cooled, and then passed through a Sharples supercentrifuge a t 15,000 r.p.m. The cells were washed with 5 volumes of water and reserved for further analysis. Of the total activity approximately 40y0 was found to reside in the cells. This distribution, there is reason to believe, may have resulted from the heat treatment at pH 4.5 received by the liquor prior to the separation of the cells. Results which will be presented in another part of the paper indicate that all of the activity in untreated fermentation liquors is contained in the cells. The results of rat assays and microbiological assays given in Table I1 are in good agreement with respect to the activities averaged over a 34-day period in the sterilized fermentation liquor and its clear beer. However, the rat asshy value of 1.0 mg. per liter for the unsterilised liquor is higher than the corresponding average value 0.71 mg. per liter obtained microbiologically. This may signify that propionic acid bacteria have
Volatile Acids G. per
t.
i:0 15.0 22.7 30.0 30.0 30.0 3: 0 3.5
4.0
6.2
8.0
9.0 10.5 11.5 12.0
Ratio Pro; pionic Vitamin and Biz Acetic Activity Acids, % Mg.per i. 0.05 0.25 1.25 .. 1.70 1.60 2.0 .. 2.0 2.0
..
.. .. .. .. .. .. .. 1'.a
0:i5 ' 0.35
0.50 0.75 1.1
2653 IN POT
EXPERIMENTS
Remarks Medium: loliters containing 150 g. N-ZAmine, 50 g. yeast extract, 200 g. lactic acid, 91 g. NaOH, 20 g. glucose, and 40 mg. CoClz.6HzO; inoculum: 1 liter active culture containing 1.5 g. dry cells and 0.46 mg. vitamin Biz activity. held in situ 48 hr. prior to inoculation' Medium: 2.5 liters containing 37.5 g. N-Z-Amine, 12.5 g . yeast extract, 12.5 g. #ucose, 20 g. KHzPO4 3.75 Na H, and 10.5 mg. CdClz.6Hz8; inoculum: 125 ml., 24-hr. culture
.. 1.
1 :3
established themselves among the intestinal flora of the rat. However, at this time there is no intention to belabor this point which is obviously in need of further study. The heat liability of the active substance in the clarified beera 30% loss occurs on autoclaving for 15 minutes at p H 7.0pointed to the likelihood that a considerable part of the activity was due to vitamin B12b. Enrichment and purification of the active substances in the cells yielded a pinkish red solution, the absorption spectrum of which was more characteristic of vitamin B12b than of vitamin Biz. Comparison of the ratio, -3.4, between the extinction coefficients at 352 and 525 mp, with the ratio belonging to pure vitamin BIZb (2.8), indicated that a high degree of purity had been achieved with respect to light-absorbing impurities in this wave-length range. Calculation of the concentration of active substance from the absorption maximum a t 525 mp gave a value of 1.3 X lop4gram per ml. compared with the value of 1.4 X 10-4 gram per ml. or 3.5 mg. per gram of solids obtained microbiologically. That the activity is largely due to vitamin BIZbhas been confirmed b y means of partition chromatography ( l a )from which it appears that approximately 807, of the activity is due to vitamin BIZb and 20% to the cyanocobaltammine compound. The complete destruction of activity as a result of the autoclaving of the fermentation liquor a t pH 9.8 for one-half hour (see Table 11) is in corroboration of the observation that the microbiological activity of the liquor is associated almost entirely with its vitamin Blz and BIZb concentration. Because the low yield on a volume basis, 0.74 mg per liter, was most likely due to the low concentration of lactic acid, another experiment was designed in which the lactic acid concentration was increased in increments to much higher values. The results of this experiment are contained in Table 111, experiment 1. The small concentration of glucose contained in the medium served to prevent the pH from rising inordinately during the early stages of the fermentation. After an initial drop in 20 hours from 6.3 to 5.9, the p H remained approximately constant throughout the run of 136 hours. Table I11 contains a number of points of interest. The lag period is a comparatively short one and practically as much volatile acids and cellular substances are produced with a corresponding consumption of lactic acid in the first 20-hour period as in the second. This may be due to a short supply of lactic acid. The fermentation is a remarkably short one; only 40 hours are required for the fermentation of not less than 270 lactic acid. The development of vitamin Blz activity follows only roughly the development of protoplasm, and only during the period of most intensive growth does the increase appear to be linear. This may be due to a destruction of activity proportional to the activity.
INDUSTRIAL AND ENGINEERING CHEMISTRY
2654
Vol. 44, No. 11
TABLE IV. VITAMINBlzYIELDSOBTAINED UNDER A VARIETY OF COSDITIOKS AKD ITSDISTRIBUTION BETWEES CELLSA N D BEER Activity i n Beer, Mg. per L.
2.0
Activity in Cells, Mg. per L. 1.1
2.5
1.7
0.0
Expt. No.= 1
Time,
Cell Wt.
Hr.
G. per L:
120
2
65
0.0
Glucose Consumed, G. per L. 10.5
Lactic Acid Consumed, G. per L.
..
Volatile Acids, G. per L. 8.0
..
30
22.8
Remarks Glucose added i n inN-2crements. Amine-y&st medium extract
Lactic acid added in 20- a n d 10-g. portions per liter 3 90 2.5 3.0 0.0 53 40.2 Lactic acid added i n l o - , 20-, IO-, 13-g. portions per liter 4 90 2.6 2.2. 0.0 53 40.2 Lactic acid added in lo-, 20-, lo-, 13-g. portions per liter 5 90 2.4 2.1 0.0 53 40.2 Lactic acid added i n lo-, 20-, lo-, 13-g. portions per liter a Expt. 1: 2.5-liter p o t with stirring; expt. 2 : 10-liter p o t with stirring: expt. 3: shake bottle experiment, medium same as in ( 2 ) ; expt. 4 : shake bottle,experiment, medium same as in (2j, plus 0.3% distillerssolubles; a n d expt. 5 : shake bottle experiment, tryptic digest of casein substituted for N-%Amine.
..
..
hTotless than 1.25 mg. per liter of active substance is produced in a 40-hour incubation period, corresponding to the utilization of 20 grams per liter of lactic acid. An additional 0.45 mg. per liter is produced on the addition and utilization of another 10 grams of lactic acid. A further addition of lactic acid brings about only a proportionate increase in volatile acids but not in cell substance, and a slight insignificant drop in vitamin activity. Thirty grams of volatile acids are produced per 40 grams of lactic acid. The ratio, 2.0, between propionic and acetic acid found in these experiments agrees with the ratio reported by Van Niel ( 1 4 ) and other investigators. The determination of volatile and lactic acid taken together with microscopic examination of the fermentation liquor from time to time served as a convenient check on absence of contamination. CELLS 0 PER L.
5,
,5 1 5 0
I
L.CASEi PHASE --0---0-
CELLS
i
P. FREUDENRElCHii PHASE -0-0-
VITAMIN B \ 2
which are not clearly understood. S-Z-Amine (Type 4 )yields results equal or superior to those obtained with a number of other sources of nitrogen. I n Table IV, it is shown to be superior to a commercial tryptic digest of casein substituted for it on a weight basis. However, the yield obtained (2.1 mg. per liter) with the use of the tryptic digest of casein is high and satisfactory. Peptone and ammonium caseinate (data not recorded) are unsuited as sources of nitrogen in comparison with more highly degraded protein sources such as N-Z-Amine (Type A), tryptic digest of casein, tryptone, and tryptose, primarily because the fermentation rate with proteins and the more complex peptides is quite slow. This observation agrees with those of Van Niel ( 1 4 ) on the influence of the source of nitrogen on fermentation rate. The activity developed in the fermentations listed in Table I1 resides entirely in the cells. The harvested cells consequently furnish a highly concentrated source of vitamin B12 activity-1.2 mg. per gram in one instance. The active substances may easily be stripped from the cells to yield a concentrate containing 12 mg. per gram, with the color and absorption spectrum in the visible region belonging largely to vitamin &2b. This concentrate should furnish an excellent supplement for feeds and a starting point for the preparation of the crystalline vitamin.
>
eo
TIME (HOURS)
Figure 1. Yield of Vitamin BE i n Relation to Metabolic Processes When Whey-Hydrolyzed Casein-Yeast Extract Is Fermented with L. casei and P . freudenreichii Simultaneously with this large experiment, a number of smaller shake bottle experiments were started; in several of these the medium was varied. I n these experiments, 25 ml. of medium in &ounce medicine bottles were inoculated with a 1% inoculum, and these bottles were rotated end over end. Some of the results are contained in Table IV. Important is the finding that the maximum potential of the medium in the larger pot experiment was not fully realized. I n one of the shake bottles, a yield of vitamin Blz-active substances of 3.0 mg. per liter and 2.5 grams of dry cells was obtained. These values compared with corresponding ones of 1.7 mg. and 2.5 grams per liter obtained in the pot experiment indicate thatthereare limiting factors in the fermentation
FERMENTATION OF GLUCOSE
It is difficult to make a comparison of substrates as different as glucose and lactic acid under practical conditions, chiefly because glucose is a neutral substance and lactates are buffer salts. I n order to simulate as closely as possible buffer conditions in a lactate medium, it was decided to add glucose in increments to a highly buffered medium, a t such a rate that the pH drop within a 24-hour period would not bring the pH below 5.0. The fermentation proceeded slowly with stirring a t 30' C., without an appreciable lag period (Table 111, experiment 2). It was observed that the reducing power of the glucose added initially was lost on autoclaving. The metabolic activity during the first 48 hours is evidently associated with the dissimilation of nitrogen compounds. With the addition in increments of glucose after 48 hours, vitamin B12 activity rose from 0.35 mg. per liter a t the end of 48 hours to 1.1mg. per liter at the end of 120 hours, corresponding to a n over-all consumption of reducing substances of 10.5 grams per liter (calculated as glucose) and the production of 2.0 grams per liter of dry cells. Eight grams of volatile acids were produced, corresponding to a 76,2y0 yield. Cell growth was luxuriant and was more than twice as great in weight as the average growth corresponding to the dissimilation of the same quantity of lactic acid. On the basis of the quantity
November 1952
INDUSTRIAL AND ENGINEERING CHEMISTRY
of substrate dissimilated, glucose appears to be more efficient as a source of energy and of material in the activities of the cell associated with vitamin BIZ production. This result is consistent with the biochemistry of propionic acid bacteria. On the other hand the observation that more vitamin B12 activity per unit weight of cells is obtained in the lacticacid fermentation points to the possibility that in the two phases of glucose dissimilation, the first (leading to the formation of lactic acid) is not as productive of vitamin Blz as the second (leading to the dissimilation of lactic acid). AN EXPERIMENT IN SYMBIOSIS
Von Freudenreich and Jensen (1) first noted that in the dissimilation of lactose in milk the growth of lactic acid bacteria in symbiosis with propionic acid bacteria led to remarkable increases in the rate of fermentation. In experiments with P. freudenreichii and lactose, the need for symbiosis arises for another reason-lactose is not ordinarily dissimilated by P. freudenreichii. I n the present experiments with lactose-containing media, the symbiotic relationship has been found to be most useful. I n the following experiment 2.5 liters of medium were prepared to containper liter: 40grams of clarifiedwheypowder (80% lactose), 15 grams of N-%Amine (Type A), 20 grams of calcium carbonate, and 10 mg. of manganese sulfate. It was inoculated with 100 ml. of a light (0.2y0acid) culture of L.casei (ATCC 7469) in clarified whey, and the carbonate was added. Following incubation with stirring at 30” C. for 28 hours, 215 ml. of a 28-hour inoculum of P. freudenreichii were added as well as 12 mg. of CoC12.6HzO. The inoculum had been cultivated in a medium containing 1% lactic acid as sodium lactate, 3Oj, yeast extract, and 0,2y0N-%Amine (Type A) and at the time of inoculation contained 1 gram of dry cells per liter. The results of the experiment are contained in Figure 1. The fermentation consists of two phases, one in which only the lactobacilli, the other in which only the propionic acid bacteria, are active. Although available to the propionic acid bacteria, lactic acid was not dissimilated until all of the lactose had been consumed. Then, rapidly, for such high lactate levels, the greater part of the lactic acid was dissimilated within the next 24 hours. Both cell growth and dissimilation process came to a standstill within a second 24-hour period. Concomitant with the activity of the propionic acid bacteria, the vitamin Bl2 activity rose to 0.5 mg. per liter. Then the vitamin Bl2 activity continued to rise at a n increasing rate until at the end of 144 hours of active development the concentration of vitamin B,z-active substances had reached 2.5 mg. per liter, with indications that the rise had not reached its peak. Several increments of lactic acid were added thereafter and a final yield of 4.3 mg. of vitamin B12 activity was obtained, corresponding approximately to 3 grams per liter of dry cells. The time required to obtain this final high yield, 312 hours, is overlong. It should be emphasized that the first phase of the fermentation in which L. casei is active can easily be reduced from 72 t o 24 hours by the employment of heavier inocula and by the use of higher stirring rates in order to achieve a more intimate contact of calcium carbonate and lactic acid. It was found during the course of the fermentation that the p H had dropped
2655
to a very low suboptimal at the end of each of the first two 24-hour periods. It is hardly likely that the propionic acid phase of the fermentation can be shortened with respect to lactic acid dissimilation. Whether or not it can be shortened with respect to vitamin BIZsynthesis by the cells during what appears to be a resting stage is a question which can only be answered by further experimentation. What activities are associated with the development of vitamin BIZduring this stage is also a matter of interest which requires more study. CONCLUSIONS
Very high concentrations of vitamin BlP-active substances are synthesized by species of Propionibacterium. Contrary to the impression created in the literature of industrial microbiology, the fermentation of lactic acid to propionic, acetic, and carbonic acid may be carried out within a period of time comparable to that required for most industrial fermentations. It should be borne in mind that from an economic standpoint, not only the desired product but also by-products are of interest. This may prove to be particularly true with propionic acid bacteria. Although now considered of only academic interest, the production of propionic acid by propionic acid bacteria may merit re-evaluation in the light of the discovery of the new and useful product, vitamin Biz. ACKNOWLEDGMENT
The authors are deeply indebted to Arthur M. Hartman and Leslie P. Dryden for rat assay data, confirmatory data of which were very much needed. LITERATURE CITED
(1) Freudenreich, E. yon, and Jensen, O., Annuaire agr. Suisse, 7 (1906); Cent?. Bakt., 11, 529 (1906).
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