Sugar-Tolerant Yeasts in Chocolate-Coated Creams1 - Industrial

Sugar-Tolerant Yeasts in Chocolate-Coated Creams1. Margaret B. Church, H. S. Paine, and John Hamilton. Ind. Eng. Chem. , 1927, 19 (3), pp 353–357...
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March, 1927

INDUSTRIAL A N D ENGINEERING GI-IEMISTRY

353

possible by decantation during the process of washing. One form of the precipitate was very fine with a creamy color, the other was white and consisted of fairly large granules. Table I11 gives the analyses for PzOj in each.

per cent Ca3PzOs and 90.6 per cent CaHP04.2H20. The amount of pure tricalcium phosphate obtained was about 19.5 per cent of the total, which, when added to that in the fines, amounted to about 27 per cent of the total precipitate. A predetermined amount of ground limestone was added Table 111-Composition of Two Forms of Precipitate Obtained to a similar solution and the reaction allowed to take place b y Precipitating Phosphates with Calcium Carbonate a t room temperature over a period of about 10 days. A p20r CITRATE-SOLUBLE CITRATE-INSOLUBLE SAMPLE determination of the citrate-insoluble PzO5 in the precipitate PzOs €’PO6 so obtained showed 8 per cent present in this form out of a Per cent . Per cent Per cent Fine 41.7 37.4 4.3 total of about 40 per cent. Similar results were obtained Coarse 45.8 None 43.8 by allowing the solution, either hot or cold, to trickle down through a tower filled with lumps of marble. PRECIPITATION WITH MILKOF LIME-Precipitates obtained The theoretical percentage of PzOj in CaHPOr.2H20 is 41.26 and in Ca3P208 is 45.79. It is evident, therefore, that by neutralizing with milk of lime contained, as has already the coarse fraction was pure tricalcium phosphate and the been indicated, only traces of citrate-insoluble PZOSand were fines a mixture of the tri- and dibasic forms, containing 9.4 presumed to be almost pure dicalcium phosphate.

Sugar-Tolerant Yeasts in Chocolate-Coated Creams‘ By Margaret B. Church, H. S. Paine, and John Hamilton BUREAUO F CHEMISTRY, U. S. D E P ~ R T M EOF N TAGRICULTURE, WASHINGTON, D. C.

T

H E yeasts as a class of microorganisms are well known sirup, and invert sirup. The invert sugar sirup is the sirup because baking, brewing, wine- and vinegw-making, resulting (a) from the inversion of part of the sucrose during and other fermentation processes in which they are boiling, (b) from intentional inversion of a certain quantity commonly utilized have been established for several thousand of the sucrose by such agents as cream of tartar, citric acid, It is not always realized, however, that the groups and invertase (an enzymic yeast product), and (c) from the of yeasts associated with these industries represent only a addition of invert sugar sirup. Cream also contains a few of the thousands of forms known to exist. Yeasts are marshmallow whip (frapp6, etc.), commonly composed of generally distributed in nature. Certain yeasts are un- dried egg white and sometimes gelatin, as well as sugar and doubtedly distributed in significant numbers only about those confectioner’s glucose. areas where the p r o d u c t s Fondant or cream is thus with which their growth and a composite habitat or subreproduction are associated Thousands of dollars are lost to candy manufacturers stratum in which the bacare found. Highly sugaryearly through the bursting of chocolate-coated t e r i a a n d yeasts present tolerant yeasts which are cream-center candies. The Bureau of Chemistry may eventually die, remain unable to exist in products has found that yeasts of high sugar tolerance are dormant, or flourish. The of negligible sugar content capable of active growth in the cream centers and cause cream represents a unique are, therefore, naturally bursting through the formation of gas. Proper sanip h y s i c a l c o n d i t i o n of a associated with sugar prodtary conditions at the factory, the use of sound inliquid or sirup enveloping ucts about sugarhouses3 and gredients, and the use of a cream of high sirup density, closely packed particles in refineries, and soft-drink and when permitted by the character of the cream, will t h e f o r m of microscopic candy factories. reduce this trouble. sucrose crystals and of fragAt the request of the Re1 ments of partially coagus e a r c h Committee of the l a t e d e g g w h i t e . There National Confectioners’ Association of the United States, the are included also minute air pockets, unavoidable in the proc- . Bureau of Chemistry has investigated the “bursting” of choco- ess of manufacture. The sucrose crystals and colloidal late-coated cream-cent er candy. “Leaks” in home-made egg n-hite fragments maintain their individuality in a cream, hand-rolled creams occurring within 24 hours and due to faulty no matter how smooth in texture i t may look, and they dipping were excluded from the investigation. Samples also continue their natural relationship to one another and of exploded chocolates were obtained through the iffiliations to the mother sirup in which they are supported. Each of the National Confectioners’ Association and the Carbo- sugar crystal retains about it, by means of molecular forces, hydrate Laboratory of the Bureau of Chemistry with the a very thin film of sirup, just as sand grains in soil have a industry and through relations established with factories tenacious hold on the water and solutes in the s0il.j The in Baltimore by the Microbiological Laboratory of the Bu- faces of sugar crystals, however, are more readily dissolved reau of Chemistry. The assistance from commercial con- and broken down by forces a t work in adjacent sirup films. fectioners was of value and sincerely appreciated. Microscopic scraps of partially or entirely coagulated egg An explanation of what constitutes a commercial cream white in the cream present an animal protein attractive to the or fondant in modern candy practice is best given in order bacteria found in dried egg products. The conspicuous that the conditions which it offers to microbial activity may bacteria in dried egg white would be those which utilize be u n d e r s t o ~ d . The ~ ~ ~term “cream” is applied to a type of proteins and resist a drying process through spore-formation, confection which in its final composition includes sugar in not those which develop slowly in products concentrated by the form of sucrose crystals, confectioner’s glucose, or corn the inclusion of carbohydrate. The spores of yeasts are not comparable to those forms 1 Received October 11, 1926. termed spores in bacteria of, for example, the Bacillus * Numbers in text refer to bibliography at the end of articlv.

I

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I S D C S T R I A L A N D ENGINEERISG CHE-VISTRY

zcefchii group. -Ilthough spores of yeasts are claimed t o be somewhat resistant to heat and drought,ll they also are comparable to the slightly resistant spores or conidia of molds.20 All sugar products would most naturally contain sugar-tolerant organisms in addition to other occasional resistant forms. Even granulated white cane sugar, a product of unique purity for one obtained directly from the vegetative parts of a plant by an extensive process of manufacture, is said to contain sugar-tolerant yeasts and spore-forming bacteria.lOJ122 Although the number of such yeasts in a highgrade granulated sugar is not large, the presence of even less than 50 such microorganisms per gram is of significance as a primary, though doubtless unavoidable, source of infection. As has been outlined previously,4 subsequent careless handling in the manufacture of candy is more properly identified with fermentation spoilage than such slight seeding of the raw products with yeast cells. The trouble known as bursting or “explosion” occurs in chocolate-coated creams of both the hand-rolled and cast type. The appearance of cracks in the coating, of extruded sirup a t such cracks, and of fondant in long curls, columns. or even layers a t cracks, and extensive breaks is evidence of such spoilage. Previous Investigations

Xeinzirl,21 in 1922, continuing the work of Hill,7 claimed that “explosion” in chocolates was due to B. sporogenes. As the controls in his experiments did not “explode,” it is possible to come to one of two conclusions: (a) That the strain of B. sporogenes used was actually sugar-tolerant; or ( b ) that the controls were not treated to the technic of inoculation and, therefore, were not open to contamination incident to this technic. We agree with his statement that “apparently true explosions in chocolates are always due to the accumulation of gases formed by fermentative microorganisms.” He notes that exploded chocolates contained yeasts, but disregards yeasts as a cause of bursting: that Saccharomyces cerevisiae inoculated into chocolates caused “explosion” in 10 days; and that two lots of exploded chocolates did not develop gas-forming bacteria on culturing into glucose Smith tubes (bacteriological medium with 1 to 1.5 per cent glucose). Hill,8in experiments reported in 1925 and working with an organism which she believed to be Clostridium multifermentans, obtained gas production in inoculated and uninoculated tubes of imitation cream and in 9 out of 15 samples of inoculated chocolate creams. The organism did not grow when studied in sugar-free media, but its sugar tolerance was only ’ tested in 1 to 1.5 per cent of the usual fermentation sugars. Philbrick,” in 1926, notes that molding starch contains anaerobic spore-formers and suggests that explosions in chocolates are due to the inclusion of these organisms. His investigations do not present evidence of experimental work controlled in such a manner as to eliminate activities of sugar-tolerant yeasts. The presence of the forms ordinarily developing on standard bacteriological media is not of primary significance in this problem unless their tolerance for heavy sugar solutions is also proved. While not denying the possibility of sugar tolerance being proved for bacteria and microorganisms other than yeasts, we feel that Philbrick’s investigations do not prove this point to the satisfaction of either the biologist or the candy maker. Shuttlg describes an instance in one factory where losses due to the bursting of chocolate-coated creams were controlled by eliminating vessels which could not be sterilized between batches. He shows that the yeasts which were developing in an uncleaned wooden barrel were capable of bursting paraffin seals in test tubes of nougat cream. Pink yeasts obtained from exploded creams did not cause bursting.

1-01. 19, Nu. 3

Preliminary Experiments

-4 fondant base, composed of 4 parts of sugar and 1 part

of glucose brought to a temperature of 117” C., and a “bob,” corrrposed of 4 parts of sugar and 1 part of glucose brought to a temperature of 115’ C., were made. il “bob” is a batch of sirup, which, when carefully cooked and mixed with fondant, produces a candy almost identical with one made entirely from fondant. A “bob” saves the fondant, the production of which takes much time and labor. The creams were composed of equal batches of the fondant and “bob” or sirup and 10 per cent (10 parts to 100) of frappe, heated together a t 64” C. A commercial frapp6, obtained from a confectioners’ supply house, was used. The cream was inoculated before casting with B. welchii (pathogenic strain (No.HL) from the U. S. Public Health Service and a straing from salt rising bread) grown in the usual liquid culture media for anaerobes. Sterile water was added to the controls before casting. No bursting or fermentation occurred in the candies inoculated with B. welchii or in the controls (Table I). I n the same connection Colsonf used B. welchii (No. HL), B. sporogenes, an unidentified diplococcus, and another anaerobe from commercial cream centers. Five creams were made in these experiments. S o . 1, 5 parts of sugar and 1 part of glucose were brought t o a temperature of 114’ C. for hand-rolled creams. KO. 2 , the same proportions of ingredients were cooked to 117” C. for hand-rolled creams. S o . 3, 4 parts of sugar and 1 part of glucose were brought to a temperature of 114’ C., the batch being cast at 60” C. KO.4, 4 parts of sugar and 1 part of glucose were brought to a temperature of 117” C., and the batch was cast a t 68” C. KO.5, 5 parts of sugar and 1 part of glucose were brought to a temperature of 115’ C., and a “bob” was composed of 5 parts of sugar and 1 part of glucose brought to a temperature of 110” C. The cream in S o . 5 was composed of 5 parts of the fondant base t o which mere added 4.6 parts of “bob.” The two batches were remelted together and cast a t 62” C. Ten per cent of marshmallow whip was included in each of these creams. The four organisms were grown in suitable media for 48 hours a t 37” C. The diplococcus was grown on dextrose agar slants and the three anaerobes in anaerobic beef ball medium. The growth on the agar slants was removed with a platinum needle and inoculated into the center of each piece of candy as described later. The growths from the anaerobic broth cultures were combined separately for each organism and centrifuged a t high speed. The clear liquor was decanted and the sediment used to inoculate the creams. I n one series of experiments two platinum loopfuls of this sediment were used to inoculate each piece. I n another series the entire sediment from 10 tubes was thoroughly stirred and incorporated into the mass of cream before casting the individual pieces. Uninoculated checks were carried on together with the inoculated pieces. No “exploded” candy was obtained from these experiments, even after a very long period of storage (Table I). T a b l e I-Results

of I n o c u l a t i n g Bacteria i n t o Five Creams

ORGANISM B. welchiia No.

HL

B. sbovogenesa Diplococcus’ Unknown anaerobea Controla B. welchii (two strains)b Controlb ~~

BCRST

SOUND

Per cenl

Per cenl

58

0

:8 08

0 0

100 100

PIECES

58

5s 80 40

0

0 0 0

100 100 100 100 100

~

a

b

Colqon’s experiments. Church’s experiments.

The possibility of sugar tolerance being proved for the bestknown spore-forming anaerobes in these constituents will t H. C. Colson, Jt , formerly with the Bureau of Chemistry

up ;tiid that uo controlr, in the present experiiiients burst, in spit.e of the spure-forntiug anaerobes included wit11 tlie marshinellom ildiip and inoliling starch.

foro m y ohser\-nt,ioii i- IM~IC O i i ilii: tilc prrscnt lr:ti.e published evidence oi such tolcrnncc

khrlior st,ndics of niicronrganisms in sugaihouse products US I,tiiiisiann bail lcd to the adoption of a wort extract mediittnt' tcl which soSt sugar, a variable product, even when obixitiwl under trlie label, was added for the detectioii of siigirto!rr:tnt forms. :\Itbough wort broth nial soft sugar ilre too ccmiglicatcd Cor rcady use where chemical control of fen~ierit i i i i e j n activity i y carried on with microbiological siudics, ilwir \-cry complexity is valuable in the isolation oS organisms, i l i c c:?rhohydrrites being supplied as sucrose, invert sirup, guns, etr. Tlie soit sugar was added in the proportio :15, atid 50 parts to 100 parts of wort agar or broth. sort, i,rotli or ngar was adjusted to a pH of 5.5 to 6.0: the ;tvcrage Sor sriniplcs oi confections received aid also for the creernii lnttde in the laboratory. I n the preliminary expcriinenis iarious d l u r e media were used. In the course of the experiments a somewhat routine prneedure of a simplifiecl ii;stnre became essential, and, therefore, the wort extract trwliunr fortifiedwith soft sugar was relied upon entirely. Cultural Examination

Cultural examination of each cornniercial sample OS ,'lriirst" ciimolates received revealed the presence of largc iiimihers of biglily sugar-tolerant yemts. The method of riiiarosciqjic examination used revealed the presence of yeasts i n significant rinmbers in all samples of "exploded" chocolates. 'L'iiese were either spore-iorming yeast.s or toruia-like yeasts, or mixture of both types. The yeasts obtained by cultural inet,liods isere purified as far as possible without resorting to siiigle spore culture. Isolation froin the edge of a colony u r Srom varying edges in a mixed colony yields pure cultures, especially where purification has been praciically accomplished by the previous use of seIective media. The sugartolerant yeasts in question can grow in comparatively high sugar concentrations, which suppress the activity of most other organisms. Microscopic Examination

A satisfactory microscopic method of detecting yeasts in cream consisted in dissolving a portion of the cream in distilled irater and centrifuging the solution. On decanting the soluiion from the material thrown down in centrifuging, a microscopic water mount was made directly from the material, using rose bengal as a stain. Bacteriological smears are not feasible where organisms are suspended in a heavy sugar solution, nor are they satisfactory even in routine examinations for tlie presence of yeasts. This is true because yeasts like fungi are large, delicate protoplasmic cells as compared vith bacteria. The making of a bacteriological smear involves extremes of heating and staining, which dist,ort Laid ruin the nat.ura1 structure of yeast cells. Tlie direct examination of a drop of fluid in which yeast cells are suspended gives a truer picture of the cells, for which the rose bengal provides a contrast stain, or, at least, a background. In irionriting yeast cells from agar slants of a concentrated inediuni a mounting fluid approximating the concentrat,ioii of the agar medium is necessary. Ckiierwiie, the yeast cells are alniust instantly affected by tlie o.iniot,ic teiidencies oi tlie timinting fluid and their cell sap. Xi tiie fluid used i n mounting is ivaier or a solution much lower in concentrat.ion than the culture niediuni, the yeast cells bnrat before om's eye.;. or. if nrounting bas proceeded slowly. the? diiintegrnt.e be-

.I Sodnnt b:ae of 4 parts o? sugar and 1 part of glucose. h r w g l ~to t ll5'C; and a "hob" of 4 parts of sugar a i d 1 part of Klucose, bronght to 114.5' C., were made. The c r e a ~ ~ i s were compwed of two equal parts of these batches xiid 10 per rant of marshmallow whip licated together at 8 range of tenipertitures (WJ', io", and 80" C.) recognized in confeeanufitcture as resulting in distinctive creams. For tis reasonable to expect fewer crystals to dissolve i n hetted (remelted) and cast at. 62" C . than a t 80" C. Is are not: Iiowever, dissolved a t a remelt tenipcr:iturc of 80" C., Tor if this were so the cream as such would I x destroyed. ?%e sirup which surrounds the crystals of the cream Ims a correspondingly lower density with a 62" ('. remelt. tbnn wit,Ii ail 80" C. remelt. On cooliiig, tlie excess of sugar in Llie supersaturated sirup aradually crystallizes. Controls

Controls were made for each batch of fondant. No controls crmked or burst. Therefore, any discussion of the presence of sugar-tolermt bacteria is iinnecessary liere. Inoculation

'l'lie method of i n o c u l a t i o n employed excludes any contnminations in inoculated samples to n,hich controls were not also subjected. Exposure to the air, occurring when the small holes were made, was negligible meure i---soft sugar inin comparison to that occurring during the casting of the ~ $ ~ ~ ,~ $ ~ ~ ~ cream. From the results of preliminary experimentation, it seemed best to inoculate each cream after casting with yeast cells from fresh agar slants. A very small hole, reaching to tlie center of each piece, w~lsmade with a sterile small panknife blade or scalpel. A loop of culture from an agar slant was introduced into the hole and the bole was closed compactly with fresh cream from the same batch. The pieces were then covered with chocolate in the usual manner. The following yeasts were used for inoculation: Zygoaacckaromyces mayor, a strain of the salt-tolerant group of yeasts utilized in cxxlerimenting - with culture control of the moromi fermentation of Japanese soy sauce; A (large), a large yeast obtained froni a refinery soft sugar, whicll had not p r c ~ v d sugar-tolerant in another group of experiments; 4708, a strain of torula-like (inclination to yoke forins in 80" C. remelt cream on drying out. of the inoculated sriniples) ycast nsed by Owen in experiments on t,lre cultural eont.rol of inversion in raw sugar^;'^ 4766, obtained from a comnierciai sample of burst walnut-maple chocolate creams; 4717a. a to&-like yeast causing tho spotting of refinery soft sngar (Figure 1); 4770, a yeast obtained from "exploded" chor~late creams (five flavors) pnt iip by one manufaeburer; 4740c and 4740p, obtained ?rmi cherry and piiirapple pieces i n :I burst £ East,er egg

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INDUSTRIAL AND ENGINEERING CHEMISTRY

March, 1927

sugar crystals were observed supporting layer on layer of yeast cells. These layers of yeast cells become displaced and float away in mounts made with diluted glycerol. Those yeasts most closely related to the crystal face are the least readily detached. If eosin is added to the mounting fluid, on the detachment of the yeast cells from the sugar crystal, there are revealed pits filled with the eosin-colored mounting fluid. These pits represent points where evident, inroads by the yeasts have occurred. Reduced sharpness in the faces of sugar crystals indicates partial solution of the crystals. Yeasts were seen on the faces of sugar crystals in coconut, “golden cream,” peppermint, and vanilla butter-cream chocolate-coated candies. They have been observed on faces of crystals in spotted soft sugar from a refinery (4717a), in soft sugar from a refinery (4791), in Cuban raw sugar (4796), in Louisiana raw sugar (4790), and in a soft sugar sample (No. 8) obtained from C. A. Browne. I n order to reproduce fermentation of chocolate-coated creams with highly sugar-tolerant yeasts as agents, 40 lots of chocolate-coated creams, including controls, were made. Xost of the yeasts used were from commercial T a b l e 111-contamination MATERIALS

Sterile cherry Infected cherry Sterile pineapple Infected pineapple Infected fondant

1 I

Pieces hoc.

19 16 54

Burst

Per cent 0 100 0 100 100

capable of causing bursting in chocolate-coated creams, and that the bursting is primarily due to the forming of minute quantities of gas in the cream through microbial activity. The yeasts obtained from burst commercially manufactured confections and utilized in these conclusive experiments were of various types. While certain ones would be termed “torulae” when grown on the usual culture media, their growth in sugar media and in fondant resulted in spore formation or, a t least, i n a tendency to yoke formation in all but one yeast (4717a) from spotted soft sugar. The presence in chocolate-coated creams of yeasts active in fermenting sugar solutions indicates that some means must be devised in candy factories to develop a cream of high sirup d e n ~ i t y . ~ ~ JSuch ~ , l ~a cream may be made by the use of a high proportion of glucose and invert sirup, or by the addition of invertase, wherever such an adjustment does not materially alter the qualities desired. The development of specific means to remedy the loss to confectioners through bursting will be presented in a later article (page 358, this issue). The sanitary conditions of the factory2J2J9and the soundness of ingredients, such as fruit pieces, should also be re-

t h r o u g h Y e a s t - I n f e c t e d F r u i t Pieces a n d F o n d a n t

I

62‘ C. REMELT

-

357

Sound

Per cent 100 0

1 yii:s I...

0 0

samples of fermented creams (Figure 3), as already described. Cultural and microscopic examinations checked exactly with the occurrence of fermentation and with the known abilities of the yeasts. The sugar-tolerant yeasts introduced were recovering in living condition from the inoculated candy after many weeks. The results of inoculating creams with bacteria are summarized in Table I. Six creams in the form of pieces of chocolate-coated centers were inoculated with two strains of B. welchii, B. sporogenes, an unknown anaerobe, and a species of diplococcus. The inoculated pieces and the controls remained unchanged. Tables I1 and I11 give the results obtained with yeast strains varying conspicuously in sugar tolerance and with control experiments. Inociilations with strains of yeasts other than those listed were made and corresponding results obtained. The composition of‘ the creams was the same, but variation was obtained by albering the remelt temperature, 62”, 70”, and 80” C. being selected. The order of the sample numbers (4766 through 2.major) follows the order of the sugar tolerance of the strains of yeasts used under the conditions of these experiments, 4766 being the most active strain in the bursting of confections and in testtube cultures. These numbers are followed by the controls and the list of uncontaminated substances used as inoculum. An increase in the number of sound pieces occur> with ( a ) the use of the yeast strains of lower sugar tol6rancc: and (b) the increase in the remelt temperature. The comparative results of making fruit centers with sterile and with yeastcontaminated fruit pieces are shown in Table 111. Experience in the laboratory and factory has plaiiily indicated that the manufacturer may expect as high as 100 per cent of bursting in fruit centers or cordials where contamination with sugar-tolerant yeasts exists. Summary

A11 the evidence presented here points to the conclusion that yeasts tolerant of high percentages of sugar sirup are

...

...

I

7‘0’ C. REMELT

Burst

Sound

Per cent

Per cent

...

...

0 100

...

...

...

80’ C. REMELT

1

Pieces hoc.

I

...

1

...

18

33“.

Burst

Sound

Per cent

Per cent

... ...

0 94

...

... ...

100 6

...

garded by the manufacturer. Otherwise, the bursting of chocolates now causing a large annual loss to the confectionery industry, both retail and wholesale, will continue as it is today an always present problem. Bibliography l-.4nonymous, J . Inst. Brewing, 32, 1925 (1926). 2--Bryant, Proc. 38 Ann. Convention Natl. Confectioners Assoc., p. 136 (1921). 3-Church, Sugar, 23, 413, 491 (1921). 4-Church and Hamilton, Manuf. Confectioner, 6 , 28 (1926). 5-Church and Thom, Science, 64, 470 (1921). 6-Guilliermond-Tanner, “The Yeasts.” New York, 1920. 7-Hill, Thesis, University of Washington (unpublished). 8-Hill, J. Bad., 10, 413 (1925). 9-Koser, J . Infectious Diseases,32, 208 (1923). 10-Levine, Buchanan, and McKelvey, Fruit P r o d f c l s J . , 4 (a), 5 (1925). 11-McKelvey, A n . Food J., 21, 39 (1926). 12-McKelvey, J . Bact., 11, 98 (1926). Facts About Sugar, 20 (1925). la-Owen, 16, 513 (1924). 14-Paine, THISJOURNAL, 15-Paine, Proc. 42 Ann. Convention Natl. Confectioners Assoc., p. 67 (1925). 16-Paine and Hamilton, U. S. Patent 1,502,207 (1924). 17-Philbrick, J . Bact., 11. 99 (1926). 18-Reddish. Abstracfs Bact., 3, 6 (1919). Ig-Shutt, Sci. Agr., 6, 118 (1925). 20-Thorn and Ayers, J . A g r . Research, 6, 153 (1916). 21-Weinzirl, J . Bact., 7, 599 (1922). 22-Weinzir1, Ibid., 11, 100 (1926).

Anzin-Kuhlmann Producing Synthetic Ammonia-The synthetic ammonia plant of the Anzin-Kuhlmann Chemical Products Co., at Anzin, France, began operation on December 3 . The initial daily returns show a n output slightly in excess of 7.5 tons of anhydrous ammonia for 24 hours. This installation will be supplemented by a second of the same capacity. Hydrogen is obtained from the coke ovens of the S. A. 5lines d’Anzin. The ammonia probably will be oxidized t o nitric acid a t the Madeleine-les-Lille works of the Kuhlmann concern.