FERMENTATION PRODUCTS OF CERTAIN MANNITOL- FORMING BACTERIA.*

Br II. It. STILRS, \V. ft. l’II;Tb:RSOS. .\sn F;. 1%. l~‘KICD.

(From the De~~~~rlmenls of .lgricillturcll Chcnti,~ir~ and .Igricitlturul Bac- teriology, L’niversityJ o,j Wisconsin, Madison.)

(Recciwct for pul,lication, hI:~y T, 1925.~

The formation of mannitol in fermenting mixtures of plant, origin is now known to be due to a group of bacteria which act upon fructose and reduce this sugar to its corresponding alcohol. Mannitol-forming bacteria have been found in wine, sauerkraut, silage, canned goods, yeast, and cereal infusions. In some of t,hcse products such as sauerkraut and silage, their presence is not, objectionable, for it is probable t,hat t2hcy are among the chief microorganisms concerned in the natural souring and preservation of these materials. On the other hand, their presence and activi- ties in the manufacture of wine, canned goods, yeast,, and but,yl alcohol arc the cause of much trouble and financial loss.

It is in relation to the wine industry that this group of bacteria has received special study. The papers of Gayon and Dubourg (l), Miiller-Thurgau and Osterwalder (2), and others (3- 8) furnish abundant evidence to prove the injurious nature of these microorganisms. The sugars are fermented to mannitol, acetic and lactic acids, and other products which result, in a sour wine of lowered alcoholic content. Fruit juices are, however, not the only source of these bacteria. Their presence on cabbage and corn as indicated by the formation of mannitol in sauerkraut and silage has been shown by Fcder (9), Nelson and Beck (lo), Brunkow, Fred, and Pel;erson (ll), and Dox and Plaisance (12). From these same sources mannitol-producing bacteria have been isolated by Plaisancc and Hammer (13), and Fred, Peterson, and Anderson (14). Smit (15), Van Steenbcrgc (IG), and Fred, Pctcr-

* This work was supportctl in part by a grant from thr special rcsenrch fund of the University of \Visconsin.

c,43

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644 Mannitol-Forming Bacteria

son, and Davenport (17) have obtained them also from yeast, soil, and manure.

The products formed by these microorganisms from the prin- cipal sugars have been dctcrmined by Gayon and Dubourg (l), Smit (15), and more recently by Fred, Peterson, and their asso- ciates (17-19). Pentoses, if fermented, are converted into acetic and lactic acids. Glucose and the ot,her aldoses yield chiefly ethyl alcohol, lactic acid, and carbon dioxide. Resides mannitol, acetic acid, lactic acid, and carbon dioxide are formed from fruc- tose. Sucrose and rafhnose are destroyed by most of these bac- teria with t,he production of varying quantities of mannit,ol. In the fermentation of fructose, mannitol acc0unt.s for 50 per cent or more of t.hc sugar. IShy alcohol and carbon dioxide are each equivalent to about 25 per cent of the fermented glucose. The formation of mannitol, ethyl alcohol, and carbon dioxide is the most distinctive characteristic of this group of bacteria.

EXPERIMENTAL.

The mannitol cultures were secured by plating various samples of fermenting cereal infusions. A large number of cultures were secured in this way and t.heir fermentation characteristics studied.

From forty pure cultures thus obtained four were selected for detailed study. These four cultures wcrc replated three times on 9.25 per cent glucose-yeast water agar. Difference in the fer- mentation of the pentoses formed the basis of this sel&ion. Cul- ture 26 ferments both arabinose and xylose; Culture 19 attacks arahinose only; Culture 36 destroys xylose, but not arabinose; and Culture 23 ferments neither of these pentoses. Many of t.hese organisms are similar t.o those previously described in the litera- ture. Culture 26 presents a close analogy t,o Bacterium manni- topoeurrl described by Miiller-Thurgau and Osterwalder. Cul- ture 36 is very similar to Bacillus gayoni studied by t,he same investigators. Culture 23 resembles in many respects Lactobacihs fermentum described by Reijerinck (20). Except in the fermenta- tion of sucrose, Culture 19 agrees very well in fermentation characteristics with Culture 11 of the beta bacterium described by Orla-Jensen (21).

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Stiles, Peterson, and Fred

Fermentation Characteristics.

645

Test-tube fermen(,ations of various sugars and other carbon compounds are not only an aid in classifying organisms, but are also useful indexes as to t,he type of fcrmenta,tion. Many sources of carbon were tried in studying t.he fermentativo ability of these organisms. Very lit,tle or no acid product,ion was shown with man-

T.\DI,E I.

Permentation of Various Sugurs.

Computed for 100 cc. of culture; age, 120 hours.

Culture 26. Culture 36. I- Culture 19.

i $ 2,

,: -- -

cc.

2

35

3

54 45 2s 46

2

42 2

-

z. 2 z- m

Per cent

36

;’

69

91 72 93

73 36 73 s3

2

57 2

Per cent

0 25

5

84 91 8.1 33

94 83

Arabinose Xylose Rharnnosc

...... 36 1 22

...... “3 15 2: 3

Glucose . Fructose . Galactosc Mannose

Sucrose Maltose Lactose hlelibiose Trehalose

4.5 85

!

35 38 37

91 7.5 69

.i5

37 97 74 80 88

5

77 0

40 41 1::

J - 16

40 22 33 42

1

38 1

_- 9”;

3

39 45

1

78 52 1

Rafinose Mclczitose

nitol, dulcitol, dextrin, amygdalin, a-methylglucoside, esculin, and salicin. Table I presenk data for t,he fermentation of 100 cc.

portions of approximately 1 per cent sugar solutions. By com- paring the data given for Culture 19 on arabinose, glucose, and fructose, it is evident that. from the latter two sugars neutral products must be formed. Comparing glucose and fructose alone for the same organism it is apparent that more of a neutral

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646 Mannitol-Forming Bacteria

product is formed from fructose than from glucose. These indi- cations are confirmed in the quantitative dat,a which were later obtained.

Fermentation Products from Various Sugars.

The analytical methods followed were similar to those pub- lished in earlier work (17, 18). The amount of sugar at the begin- ning and end of the reaction was accurately determined. The major products were isolated and an attempt was made t.o account for the loss of sugar in a sum of the products.

Ferdentation of Pentoses.-Approximately 2 per cent solutions of t,he carbohydrate in yeast mater were used in this study. The data are given in Table II and arc comparable to those cited in

TABLE II.

’ ~~~ Cu!ture, I- __ Ilo.

Age. i - __- I ’ Kind. Amount Volatile fer-

mented. a.3 ncet1c

-. -

26

26 26 26 19 36 36

32 SylOSt?.

32 Arahinose. 39 I Xylosc. ;; 1 .\ral)j;lose.

i 32 Xylosc. 3% .\rahinose.

1.7G5 0.760 1.773 0.830 0.079 1 0.012

36 39 sylos~. 1 1.774 I 0.752 36 I 39 ’ .Irai)inose. , 1 0.105 1 0.008

-. -_

ym. per 100 cc.

0.999 0 686 0.952 0.910 0.852 0.960 0.0 0.854 0 006

Carbon dioxide.

0.039 0.014

--

Total ,roducts.

“% :c.

1.821 1.276 1.735 1.678 1 612 1.829 0.026 1.606 0 014

previous publications. If attacked at’ all, the pentose molecule is clcavcd into lactic and acetic acids. These are the major prod- ucts of the fermentation. Practically no alcohol is formed, and carbon dioxide is produced only in traces. Recovery of products ranges from 90 to 103 per cent. However, the ratio of acetic to lactic acid does not. corrcspond with the theory of 1: 1.5 for a simple cleavage. It is observed to vary from 1: 1.12 t,o 1:1.28, which indicates a side react.ion of some nature. As no appreciable quan- t.ities of carbon dioxide appear, there cannot be a secondary fer-

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Stiles, Peterson, and Fred 647

mentation of lactic acid. .\ possible explanation may lie in an aldol condensation of intermediat.e aldehydic product,s and t,heir subsequent conversion to acetic acid. Thus 2 molecules of an aldehyde with 3 carbon atoms in its molecule might unite to form an intermediate compound with a chain of 6 carbon atoms which could be fermented to acetic acid.

Scvcral of the cultures were set up with an excess of sterile CaCO, present. As the major fermentation products are acids, this method is to be preferred to that of successive alkali titrations. Although not adapted for a carbon dioxide determination, yet because of the constancy of its favorable hydrogen ion con- cent.ration, this method is conducive to a rapid and complete fermentation.

Some of these organisms arc limited in t.heir choice of pentose sugars. In the results of Table I Culture 36 is shown to attack xylose vigorously and to leave arahinose practically unt,ouched. This is equally true wit’11 large quantities of sugar. Even in the presence of CaC& the culture containing arabinose showed but very little destruction of sugar. The organism apparently uses just enough of the carbohydrate to keep itself alive with slow reproduct.ion. Culture 36 shows marked differences between the pent,oses which can be accounted for only by the different space arrangements of t.he sugar molecule. As will be shown later, xylose is as completely destroyed as glucose which is usually considered the most readily fermented sugar. The similarity of structure of these two sugars may be offered as an explanation for t,he practically compleie dest.ruct,ion of xylose. Culture 19 dis- tinguishes between t.hese two pentoses in an opposite manner. It shows a st,rong preference for arabinose and scarcely consumes xylose at all. &cause of the corresponding similarit,y in struc- ture between arabinose and galactose, this organism should be a poor fermenter of glucose and a strong fermenter of galactose and lactose. The figures of Table I in general bear out this relation- ship. Culture 26 destroys both of the pcntoscs, and Culture 23 attacks neither of these sugars. Rhamnose is not a favorable sugar for any of these organisms.

Ferment&ion of Fructose and Fructo Sugars.l---The initial quali-

1 Fructos-yielding sugars

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648 Mannitol-Forming Racteria

tative fermentation of fructose offered an interesting possibility. The sugar was rapidly and almost complctey deskoyed with only a moderate production of acid. In the larger fermentations all of the products were not, accounted for in the regular scheme of analysis. A major reaction product, which was found to be man- nitol, was st.ill in t.he r&due of the lactic acid extract,ion. The quantit.y of this hexahydric alcohol was determined by evaporat- ing the residue from the ether extraction to dryness in a porcelain

T.\BI.E III.

Fermentmtion Products of Fruc[ose, Sucrose, and Rafinose. --

---

26 19 36 43 26 19 36

12 9

16 15 15

23 I “0 41.-11 I 2%

26 j 16

-

r'rllctosc.

‘.

‘I

“

Sllcrosc

“

‘C

“

“

Itafinose. 36 1 16 1 ‘( L

1.82GI 0.2171 0.3001 0.0 1.580 0.201 0.3081 0.0 1.667 0.230 -I 0.202 0.0 1.578 0.215: 0.268 0.0 1.817 0.087 0.756, 0.401 1.842 O.OSl 0.717 0.37;

l.%il 0.074, 0.614 0.37- 1.857 0.091 0.707 0.36; 1.499 0.127 0.5661 0.m 1.630; 0.103 0 576’ 0 38: 1.435 0.085; 0.4931 0.x

‘. ‘i 4 j’ 1 ) 7 -

i a g a 2 8

0 .e e 3 5 x --

%i :iY “1% r 0.142 0.8‘23 0.150 0.780 0.102; 0.965 0.133 0.774 0,4031 Trace 0.3541 0.119 0.3d 0.m 0.394’ 0.074

0.0 0.237 +* 0.302 +*

vn. per 100 cc.

1.482 1.439 1.504 1.390 1.647 1.643 1.709 1.631 0.953 1.298 1.167

* Lkfinitc crystals, but sugar residue interfered with quantitative separation.

dish containing a layer of washed sand, and then extracting the mannitol with hot 80 per cent alcohol. The solution was evapo- rated and the mannitol crystallized in three fractions at a low tem- perature. However, all of the mannitol cannot be separated in this way, and for this reason the sum of the products in Table III is somewhat, low.

The nature of mannitol formation from fructose has been pre- viously described. The first fermentation products arc probably lactic and acetic acids, carbon dioxide, and hydrogen. This

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Stiles, Peterson, and Fred 649

reducing agent then converts half or more of the fructose to man- nitol, which is not readily suscept’ible to a secondary fermentation.

The fructo sugars, sucrose and rafinose, are readily fermented by these organisms. Sucrose, however, is quite readily hydrolyzed by heat, and precautions must bc taken in its sterilization. The sugar used for these fermentations was sterilized in carbon dioxide- free water in 12 per cent solution at. 15 pounds pressure for 20 minutes. The reducing sugar content after this treatment was found not to exceed 8 mg. per gm. of sucrose. The fact that Cul- ture 26 yielded but’ a mere t’race of mannitol is additional evidence that no apprcciablc quantities of invert sugar were present at the time of inoculation.

From the figures of Table III it is evident that the sucrose mole- cule is not first hydrolyzed to glucose and fructose and t’hese hex- oses then fermented. If, hydrolysis occurred, there would be produced, as compared with equivalent, quantities of glucose and fruct,osc, about half as much mannitol as is obtained from fructose, a decreased amount of alcohol, and an increased percentage of vola- tile acid and carbon dioxide. But t.his is not the case, and t.he sucrose molecule must bc either fermented directly or, if hydro- lyzed, the fructose component must be in some cnolic variation which is more susceptible to an alcohol than to a mannitol fermentation.

Lactobacillus pentoacclicus was included in the sucrose fermen- tations, but, did not attack the sugar until CaU& was added. Here again the more favorable hydrogen ion concentration of a medium containing CaC03 is evidenced. Even after the addition of this neutralizing agent t~he ferment,ation was not complete. Ko mannit’ol was found. The increased volatile acid content is probably due to a secondary fermentation of lactic acid, for, as previously reported, this organism ferments lactates.

Rafhnose is fermented fairly rapidly, but not all of the sugar is consumed. There arc t,wo possibilities in the hydrolysis of this trisaccharide. Either sucrose and galactose or melibiosc and fructose may be the first hydrolytic products. It it were hydro- lyzed in tbc lattm way, mannitol formation would likely follow; if hydrolyzed in the former may, mannitol could be formed onl\~ from the sucrose and would be formed only by those organisms which ferment sucrose wi1.h the production of mannitol. As

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650 Mannitol-Forming Bacteria

Culture 26 does not form mannitol from sucrose but does pro- duce it from raffinose, the hydrolysis of raffinose to melibiose and fructose is more probable. Culture 3G can produce mannitol from either sucrose or fructose, and either type of hydrolysis might take place.

Fermentation of Other Sugars.-As shown in Table IV the fer- mentation of glucose, galactose, and lactose is marked by the production of alcohol. Lactic acid and carbon dioxide are the other major products. Volatile acid is a minor product. Lac-

TABLE IV.

Perntentalion Proclucls 01 Altlwcs, C'alriwn Laclate, and Mnnnitol.

26 19 36 23

26 20 36 21 36 114 36 118

9

16 9

16 ‘73 26 --

36 1 21 I

days li

Glucose.

L‘

<<

“

Galactose. “

Carbon compound. / Acids

Lactose. ‘<

Calcium lactate. RIannitol.

* Control contained 1.200 gm. -

- -‘-

;yi ,PcT wo.cPc~ om. Pe 100 cc.

1.842 0.0610.942 1.7158 0.0870.729 l.889, 0.055iO. 9j8 1.813 0.0720.779 0.835 0.0090.437 1.54ii 0.0240.645 0.066 0.0 0.0 1.525 0.0330.721 0.100 0.0761.100

i 0.220,0.332

0.424 0.441’ 1.868 0.40s 0.435 1.659 0.442 0.439 1.894 0.431 0.429 1.711 0.182 0.182 0.810 0.410 0.404 1.483 0.010 0.010 0.452 0.466 1.672

0.076 0.0 / 0 552 _______-

tose is a different.iating carbohydrate for Cultures 26 and 36. The former does not attack it, whereas the latt.er destroys 90 per cent of t.hc sugar. Cult,ure 36 also shows a more vigorous destruc- tion of gslactosc than dots Culture 26. The type of cleavage is given by glucose, and the other two aldose sugars conform very closely to it. I,actose is probably hydrolyzed to its component hexoses which are then fermented.

Destruction of Products.-The possibility of secondary fermen- tations is also given by the figures of Table IV. Culture 36 very slowly destroys calcium lactate as shown by the production of

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Stiles, Peterson, and Fred 651

volatile acid. Also, it is the only organism of this group which ferments mannitol. A 2 per cent solution of the polyhydric alco- hol was used. Destruction of this fermentation product results in the production of la&c and acetic acids, and presumably of

Culture No.

26 Xylose. 36 “

26 Arabinose. 19 “

26 Glucose. 19 “

36 ‘i

23 ‘I

26 Ihctose. 19 CL

36 “

23 I‘

26 Galactosc. 36 “

36 Lactose. 20 Sucrose. 19 CL

36 “ 1

23 “ , 41..ll L‘

26 Ksffinose. 36 “

36 Rlannitol. Theory for Ba in

Ba(C&02)2.. .

Ba content of na content of Water of crystulli-

ention in tirst volatile acid. acid from nlcolml. crop of Zn

lactate.

53.1 53.1 52.9 53.2 50.1 51 4 50.1 52.3 53.S 53.7 53.0 53.0

54.1 51.1 50.X 51.2 50.6 50.9 52.0 50.9 53.s 32 8

53.8 Theory for Hz0 in Zn(C3HS03)2 + 3HzO

per cent

53.3 53.7 53.9 53.3

53 4 53.3 53.7 53.7 53.4 53.5 53.6 52.9 53.2 53.2

53.8 . . .

pm cent

1s. 12 18.13 18.11 18.16 17.98 IS. 14 18.16 18.16 18.00 1s. 17 lS.23 18.11 18.28 18.07 18.13 18.14 18.13 1s. 15 1s. 14 18.11 15.20 18.19 18.25

18.17

carbon dioxide. This fermentation proceeds with extreme slow- ness, for in 118 days only a little more than 25 per cent of the mannitol was dest,royed.

Identification of Volutile Acid and Alcohol.--Volatile acid is readily identified by making a clear solution of t’he barium salt,,

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652 1\IannitoLForming Bacteria

evaporat’ing a desired aliquot to dryness in a platinum dish, dry- ing to constant weight, at 13O”C., weighing, converting t.he barium present to t’he sulfate, and computing the barium content of the original salt.. Alcohol was; in t,he course of it,s determination, con- verted t,o the corresponding volatile acid which was t’hen iclenti- fied. The results are given in Table 1’. The majorit,y of the vola- tile acid samples arc low in barium content. This is no doubt due to the small amount, of lactic acid which passes over with t,he vola- tile acid, the barium content of which is 43.5 per cent. The amount. would bc influenced by the proportions of each acid prcs- cnt and the length of the tlistillation.

Fom oj Lactic 11 cd Produced.- pThe form of lactic acid produced is conveniently idcntificd by the wattar of crystallization of its zinc salt. The physical appearance of the crystallizing lactate generally indicates which form of salt is present. Inactive zinc lactate comes out of solution \-cry readily and is quite flaky. Xct,ive zinc lactate is much more soluble, crystallizes from small volumes of the mother liquor, and t,he particles, which are very fine, form a cake on the filter. Table V also gives t,he data for these salts. Most of the lactic acid produced from all of these organisms is inactive, and the results are in nearly every case in close agreement w&h the theoretical.

Description of Olga&?~as.-All of these organisms are Gram- positive rod forms, non-motile, non-sport formcrs, catalasc- negative, do not liquefy gelatin , grow best, in an atmosphere of lowered oxygen tension, and have a thermal death-point in a neu- tral medium between 60” and 70°C. Cultures 26, 23, and 36 arc small rods 0.4 to 0.6 by 1.2 to 2.8 microns, The cells of Cul- ture 19 are much larger, 0.6 t.o 0.75 by 2.0 to 9.0 microns, and occur singly or in chains. Carbolfuchsin is perhaps the best stain, but thionin is almost equally satisfactory. Culture 26 does not curdle milk; the others produce acid and form curd in from 10 to 20 days. Floccule format,ion in the fermented medium is evi- denced by Cultures 26 and 19 only. Methylene blue is reduced by all t,he cultures but more rapidly by Culture 36. The stronger reducing power of this organism is also shown by its being the only one which reduces nitrates, and again by the better yields of rnannit.01 obtained from both fructose and sucrose.

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Stiles, Peterson, and Fred 653

SUMMARY.

Four strains of m:Lnllitol-forming bacteria have been isolated from -cereals and their fermentation characteristics and products determined.

Culture 26 ferments both xylose and arabinosc; Culture 19 destroys onIy arabinose; Culture 36 attacks xylose onIy; and Cul- t,ure 23 fcrrrwnts neither of these sugars. The pentose molecule is cleaved wit,h the product.ion of lactic and acetic acids.

Fructose is completcl)- fermented with the format,ion of lactic and acetic acids, carbon dioxide, and mannitol. A11 of the cul- t,urcs ferment sucrose and three of them produce small quantities of mannitol from it. Rafinose is partially destroyed by (lulturcs 26 and 36 with the production of lact,ic and acetic acids, alcohol, carbon tlioxidc, and mannitol.

(~lucosc is fermented with the production of ethyl alcohol, carbon dioxide, and lactic acid. Galact.osc and lactose are aHacked to a lesser degree, but if fermented, result, in the forma- t,ion of similar products.

Irrespective of the sugar fermented, the lactic acid produced was found to bc mainly inactive. Calcium lactate is slowly fcrmenkd by Culture 36 with the production of volatile acid. The same organism slowly destroys mannitol with the formation of lact,ic and acetic acids? and probably of carbon dioxide.

BIJ3LIOGRAPHY.

2. IIiiller-Thurgau, II., andOsterwalder, A., Centr. Bakt., 2. AM., 1912-13, xsxvi, 11’9; 191Tp18, slviii, 1.

3. Kaywr, E., Ann. Insl. Pastezw, 169-l, viii, 737. 4. Maze, P., and Perrier, A., .4nn. Inst. Pasteur, 1903, xvii, 5X7. 5. RIazG, I’., and Pacottet, I’., Ann. Inst. Pusteur, 1904, xviii, 245, 6. Lsborde? ,J., Compl. rend. ~lcad., 1904, cssxviii, 2%. i. hIiillcr-Thurgau, II., Lands. Jahrb. Schwiz, 1907, xxi, 230. 8. Ihbourg, E., Ann. fnsl. PU.S~CUT, 1912, xxvi, 923. 9. Fcclcr, E., Z. Unlersuch. Kuhrur~gs- u. Genussmittel., 1911, xxii, 295.

10. Nelson. XT. IX., and Ueck, -1. J., .T. Am. Chem. Xoc., 1918, xl, 1001. 11. Brunkow, 0. R., Peterson, \V. II., and Fred, E. B., J. Am. Chem. Sot.,

1921, xliii, 22-M.

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Mannitol-Forming Bacteria

12. 1)0x, A., and Plaisance, CT. P., Iowa Agric. E.zp. Station, Research Bull. . 42, 1917.

13. Plaisance, G. P., and IIammer, B. R., J. Bact., 1921, vi, 431. 14. Fred, E. B., Peterson, W. H., and Anderson, J. A., J. Biol. Chem., 1921,

xlviii, 385. 15. Smit,, J., Z. G‘iiru?qsphgsiol, 1915, v, 273. 16. Van Steenberge, P., Snn. Inst. Pusteur, 1920, xxxiv, 503. 17. Fred, IX. IS., Pctcrson, W. If., and L)avenport, A., J. Biol. Chem., 1919,

xxxix, 347. IS. Peterson, N’. II., and Fred, E. B., J. Lfiol. (‘hem., 1920, xli, 431. 19. Peterson, \\-. H., and Fred, E. B., J. Biol. Chem., 1920, xlii, 273. 20. Ueijerinck, M. W., Arch. N6erbad. SC., Serie II, 1901, vi, 212. 21. Orla-Jensen, S., Mcm. acad. TOY. SC. et letfres Dunemwl;, 1919, v, 176.

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H. R. Stiles, W. H. Peterson and E. B. FredBACTERIA

CERTAIN MANNITOL-FORMING FERMENTATION PRODUCTS OF

1925, 64:643-654.J. Biol. Chem. 

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