Steps taken to eliminate the spontaneous fermentation of soap

Transcription

1 BACTERIA PRODUCING TRIMETHYLENE GLYCOL' C. H. WERKMAN AND G. F. GILLEN Department of Bacteriology, Iowa State College, Ames, Iowa Received for publication, July 3, 1931 Trimethylene glycol was first observed as a product of fermentation in 1881 by Freund. It was shown, later, to be a product of the spontaneous fermentation of soap lyes and assumed considerable importance in this respect because its presence impaired the quality of the glycerol prepared from these lyes, giving rise to a product of low specific gravity known as "light stuff." Moreover, the fermentation resulted in a considerable loss of glycerol. Steps taken to eliminate the spontaneous fermentation of soap lyes have so reduced the supply of trimethylene glycol on the market as to create a demand by manufacturers of certain pharmaceuticals. Cheaper production would undoubtedly lead to the development of new and more extensive uses. Comparatively little study of the chemism of the fermentative production of trimethylene glycol has been made, and no systematic investigation of the organisms responsible has come to the attention of the authors. Such a study constitutes the purpose of the present paper. HISTORICAL SURVEY The occurrence of trinmethylene glycol, as a product of the fermentation of glycerol, was observed by A. Freund (1881), while attempting to prepare n-butyl alcohol by the method of Fitz (1877). Freund observed that the syrupy residue remaining I Supported by an appropriation from Industrial Research funds of Iowa State College, as a part of the program for the study of the utilization of wastes by fermentation. The assistance of Mr. Charles Davis in checking cultural characteristics of a number of organisms is acknowledged. 167

2 168 C. H. WERKMAN AND G. F. GILLEN after distillation of the fermented mixture could not be brought again into fermentation, even after the butyl alcohol had been completely removed by distillation. This material proved to be neither glycerol nor an isomer but a new product of fermentation, distilling between 2100 to 220 C. Repeated distillations gave a liquid boiling between 2160 and C. at 736 mm. pressure, which, together with its specific gravity of (180/4 ) (boiling point by Reboul 2160 and specific gravity at 190), showed it to be probably trimethylene glycol. The dichloride, bromide and iodide of the material were prepared. Freund believed that the trimethylene glycol was formed by the abstraction of oxygen from the middle hydroxyl group, but did not determine the exact mechanism. The yield of trimethylene glycol was usually between 10 and 20 per cent, and sometimes as high as 27 per cent. Freund's orgaisms were probably spore formers. He made no bacteriological study but undoubtedly his organisms are not related to the non-sporulating forms to be described. A. A. Noyes and W. H. Watkins (1895) investigated the presence of trimethylene glycol in glycerol and succeeded in obtaining a liquid from "light stuff" which boiled between 2140 and , and had a specific gravity of (200/150). They identified this material as trimethylene glycol, and suggested that its presence was due to the saponification of the fat, and subsequent spontaneous fermentation of the glycerol, thus causing an appreciable loss of the latter. The method of analysis developed by Cocks and Salway (1922) proved to be of value in the determination of the quality of glycerol, under the assumption that only glycerol (g), trimethylene glycol (x), and water axe present. They found the apparent glycerol content (acetin value), and the specific gravity of the material, setting up the following equations: Specific gravity of mixture = x g Apparent glycerol = z g The method was not applicable to materials containing less than 1 per cent of trimethylene glycol. Cocks and Salway be-

3 BACTERIA PRODUCING TRIMETHYLENE GLYCOL lieved that the occurrence of trimethylene glycol as an impurity in the "dynamite" glycerol was a source of danger. They sought to improve their approximation of yields in their previous work, which was in error due to the contraction of volume on mixing of the three constituents, glycerol, trimethylene glycol, and water. They, therefore, used mixtures of known content, and constructed tables by which the quantity of trimethylene glycol can be determined from the specific gravity of the mixture. This procedure was worked out in detail, and its accuracy checked by using known mixtures, to within 0.2 per cent of the true percentage of trimethylene glycol. The boiling point of trimethylene glycol was found to be 2100 to 2110C., and the specific gravity C. A. Rojahn (1919) suggested that, if the water present in glycerol is known, the amount of trimethylene glycol may be calculated from the specific gravity of the mixture. He determined the water content by drying 2 grams on asbestos, under reduced pressure, over P205, with care not to prolong the drying longer than forty-eight hours, as the glycol was somewhat volatile. He then constructed graphs and tables giving the percentage of glycol for the varying specific gravity of the mixture and its water content. In reviewing the work of Noyes and Watkins (1896), Archibald Rayner (1926), concluded that the trimethylene glycol must be formed subsequently to the liberation of the glycerol in the case of that obtained from rancid fats. He found that fermentation took place more readily in a slightly alkaline medium. From the progress and products of the fermentation, it was concluded that there were three types of organisms fermenting glycerol: (a) acid producers, (b) gas producers, (c) trimethylene glycol producers. The gas producers were thought to cause a loss as high as 60 per cent of the glycerol. Following his previous experience, Rayner thought it advisable, in order to prevent the formation of trimethylene glycol, to keep the lyes as strong as possible, and to work them up as quickly as practicable. Rayner found that crude glycerol contained, usually, less than 1 per cent trimethylene glycol, occasionally 2 or 3 per cent. He also found an isomer, a-propanediol present. Rayner gave the properties of trimethyl- 169

4 170 C. H. REKMAN AND G. F. GILLEN ene glycol as a pale yellow, odorless liquid, with density /200 according to Cocks and Salway's method and boiling at 2100 to 2110C. The material was described as being less viscous than glycerol, and its solutions were almost identical in freezing point with equivalents of glycerol solutions. Contrary to previous belief, he found the nitration product of trimethylene glycol to be quite stable and that the trouble experienced with dynamite containing it was caused probably by another impurity. He likewise suggested that trimethylene glycol might be manufactured on a large scale, using a Van Ruymbeke still for the distillation of the fermented liquors. Braak (1928) in his excellent monograph on the fermentation of glycerol, has discussed in some detail the chemism of glycerol fermentation by organisms of the coli-aerogenes group. He succeeded in isolating from canal water, an organism which he named Bacterium Freundii which produced considerable quantities of trimethylene glycol from glycerol. Braak placed his organitsm among the coli-aerogenes "intermediates." METHODS For purposes of the present study, the experimental work was limited to the organis broadly known as the "coli-aerogenes" intermediate forms. Following the work of Braak which indicated the close relationship of Bacterium Freundii to species of Escherichia and Aerobacter, we used horse, sheep, mouse and cow manures and various soils as sources for the isolation of organisms producing trimethylene glycol. Glycerol media were employed for enrichment because the glycol has been found only among the products of the fermentation of glycerol. It has never been reported among the dissimilation products of the carbohydrates. A glycerol enrichment medium of the following composition was employed: 20 grams glycerol 1 gram di-potassium phosphate 3 grams peptone or ammonium chloride 1 liter water

5 BACTERIA PRODUCING TRIMETHYLENE GLYCOL The medium was adjusted to ph 7.0 and sterilized for fifteen minutes at 20 pounds pressure. The enrichment culture was smeared on a medium of the same composition with 1.5 per cent of agar added. From the glycerol medium colonies were transferred to sodium citrate agar slants: grams sodium citrate 3 grams ammonium chloride 1 gram di-potassium phosphate 1 liter of water 15 grams agar The citrate medium was used to eliminate typical Escherichia coli forms after they had not been shown to produce trimethylene glycol. Braak (1928) had previously shown Esch. coli to be unable to produce the glycol and we have as yet to obtain an organism producing trimethylene glycol which fails to utilize the citrate radical. Experience led us to believe that the true acetoin-producing bacteria do not form trimethylene glycol and, later, typically V-P positive forms were discarded. The production of trimethylene glycol was determined in a medium containing: glycerol 20 grams, dipotassium phosphate 1 gram, ammonium chloride 3 grams, and water 1000 cc. The culture was adjusted to neutrality daily by the addition of sodium hydroxide, or calcium carbonate was added to the medium in excess before sterilization. The fermentations were carried out in 6-liter flasks equipped with two-hole stoppers. Into one hole was inserted a small sterile cotton-plugged separatory funnel extending to the bottom of the flask; through the other was placed a cotton-plugged delivery tube for the discharge of gases. The flask and medium were sterilized for thirty minutes at 20 pounds pressure. Hydrogen was bubbled through until the flask had cooled to incubating temperature, thus displacing oxygen and providing anaerobic conditions. The flasks were inoculated with 30 cc. of a forty-eighthour culture and incubated at 30 C. For purposes of analysis, fermentation was considered complete after a period of ten to twelve days. Determinations were made on 4.5 liters of medium.

6 172 C. H. WERKMAN AND G. F. GILLEN Cultures found to produce trimethylene glycol were subjected to a detailed cultural and systematic examination. Uric acid medium was made according to Koser (1918). The utilization of the formate ion was determined in only one instance, and, then, by replacing sodium citrate in Koser's (1923) citrate medium with 0.5 per cent sodium formate. Indol was determined in tryptophane broth after two, five and seven days using the Gore test. The reduction of nitrate to nitrite was determined by the dimethyl-alpha-naphthylamine test and also by the standard alpha-naphthylamine test. The former is more satisfactory, in that the color of the positive test does not fade. Determinations were made daily for seven days. The Voges-Proskauer test for the production of acetoin was conducted according to standard methods as well as by that proposed by Werkman (1930) employing ferric chloride. Motility was determined by the soft agar method and by means of the hanging drop. Flagellation was observed by the aid of Muir's modified Pitfield stain. Trimethylene glycol was determined by removing insoluble salts by filtration and concentrating the liquor to one-third its volume by distillation. This concentrate was further reduced to one-fourth of its volume by vacuum distillation and soluble calcium salts were precipitated by oxalic acid. The calcium oxalate was filtered off and the liquor evaporated to a syrup on the water bath. The syrup was dissolved in alcohol (96 per cent) and the salts removed by filtration. The filtrate was then distired under a 29-inch vacuum. Everything coming over up to 100 C. was discarded. The fraction between 110 to 1300C. was a clear liquid which was refractionated under vacuum and the portion coming over between and 120 C. was collected separately. This fraction was further purified by distilling at a constant temperature of 2100C. at atmospheric pressure. This distillate was weighed and identified by the preparation of the dibenzoate. The glycerol used in the fermentation was subjected to the same procedure. No trimethylene glycol was ever found. The dibenzoate prepared by the use of Eastman trimethylene glycol was compared with the fermentation product.

7 BACTERIA PRODUCING TRIMETHYLENE GLYCOL Appreciation is expressed for transplants of cultures received as follows: V33B, M8B... Dr. S. A. Koser, Chicago University Br... Dr. A. J. Kluyver, Delft, Holland 21R, 23R, 24R, 26R, 28R... Dr. C. C. Ruchhoft, Sanitary District of Chicago 17, Dr. Roger Patrick of this laboratory EXPERIMENTAL RESULTS During the initial stages of the work, the scope was limited to a study of the "coli-aerogenes" group; it was subsequently restricted to the Gram-negative, non-sporulating rods fermenting lactose and glycerol and utilizing the citrate radical as a sole source of carbon. Only in the latter and more limited group were organisms found which produce trimethylene glycol. It was not found to be produced by typical Escherichia coli or Aerobacter aerogenes. All of the twelve cultures which were found to produce the glycol belong to a group generally known as the coli-aerogenes "intermediates" from the fact that they are methyl-red positive, Voges-Proskauer negative and fail to utilize uric acid as a sole source of nitrogen, but utilize citrate as a source of carbon. It becomes necessary to determine whether such "intermediate" forms are in fact to be allocated to Escherichia or to Aerobacter or should be grouped to form a new genus. These forms may well form a natural group deserving generic recognition. It would be of distinct value in continuing the necessary studies to confirm the systematic position of these organisms, to have a simple, rapid qualitative test to detect the presence of trimethylene glycol in a culture medium. The close relationship of glycerol to trimethylene glycol makes the task of developing a test difficult. On the other hand, it may prove true that the production of trimethylene glycol will show a perfect correlation with cultural characteristics more easily determined. 173 CHARACTERIZATION OF CITROBACTER GEN. NOV. All of the cultures examined for the production of trimethylene glycol belong to the coli-aerogenes group. Those found to pro-

8 174 C. H. WERKMAN AND G. F. GILLEN duce the glycol are "intermediates" in this group, and show a homogeneity as a subgroup within the larger coli-aerogenes division. The trimethylene glycol-producing organisms will be characterized as a group as follows: Morphological. Gram-negative, non-sporulating, short rods with rounded ends. Physiological. The V-P test is negative with glycerol. In the case of two cultures (nos. 17 and 18) a faintly positive reaction is detectable with glucose after incubation at 30 C. for five days. The citrate ion is used as the sole source of carbon. The urate ion is not used as the sole source of nitrogen or serves poorly. In the case of culture 8 the urate medium shows some growth. Nitrates are reduced to nitrites. Milk is not peptonized. Glycerol, mannitol, sorbitol, glucose, levulose, mannose, maltose, lactose, trehalose, xylose, rhamnose, and starch are dissimilated with the production of acid and gas. Erythritol, inulin and amygdalin are not dissimilated with the production of either acid or gas. Biochemical. Trimethylene glycol, lactic acid, acetic acid, formic acid, C02, H2, ethyl alcohol and succinic acid produced from glycerol; 2,3-butylene-glycol, acetoin, and acetone not produced from glycerol. The methyl-red test does not prove satisfactory for the differentiation of the genus because of the intermediate nature of the organisms. Their behavior is generally to produce a positive test but occasionally the results are not definite. The genus will be diagnosed with the purpose in mind of including the coli-aerogenes intermediates utilizing the citrate radical, methyl-red positive or weakly so and not producing acetoin from glycerol or glucose or in traces only from the latter. The diagnosis will not be limited, for the present, to orgamsms producing trimethylene glycol although production of the glycol occurs with all the cultures so far examined. Diagnosis. Gram-negative, non-sporulating short rods with rounded ends. Methyl-red positive or intermediate. Acetoin not produced from glycerol and rarely from glucose and then only in traces. Citrate radical utilized. Nitrates reduced to nitrites.

9 BACTERIA PRODUCING TRIMETHYLENE GLYCOL Many carbohydrates, alcohols and glucosides attacked with the production of acid and gas. Ammonium salts utilized as a source of nitrogen. Type species: The type species is Citrobacter Freundii (Braak) comb. nov. ALLOCATION OF CITROBACTER GEN. NOV. It is necessary to allocate the citrate-positive, coli-aerogenes intermediates for purposes of systematic reference. The group as diagnosed is accorded generic ranking along with Escherichia and Aerobacter in the family Bacteriaceae. The generic name Citrobacter is proposed. The group shows considerable homogeneity as a whole, whereas the individual organisms show dissimilative differences in their fermentative reactions which permit well-defined species differentiations. In the differential diagnosis of the genus, it proves impracticable to use the characters employed in the separation of Escherichia and Aerobacter. Citrobacter is intermediate to these two genera in certain characters which serve to distinguish them, i.e., methyl-red test, and uric acid utilization. Even the Voges-Proskauer test must be used under standard conditions. The organisms show luxuriant growth on citrate media which serves as a characterizing cultural reaction of the group. Three allocations of the group may be considered: (1) to the genus Escherichia, (2) to a new genus (3) to the genus Bacterium as a subgenus. This assumes recognition of the genus Bacterium. Allocation to Escherichia is probably unwise from the point of view of the systematist as well as the sanitary worker. The marked use of the citrate radical as a sole source of carbon is opposed to allocation to Escherichia. Guided by the present tendency to recognize the splitting of the genus Bacterium into several genera, it is logical to accord generic ranking to this group of Escherichia-Aerobacter intermediate microorganisms. They are definitely intermediate in characters which are of decisive value in characterizing these genera. Furthermore, we know from unpublished results, supported by the work of Braak (1928) that not only do the metabolic, products of the trimethylene glycol forms produced from 175

10 176 C. H. WERKMAN AND G. F. GILLEN glycerol differ from those of other organisms, but that marked quantitative differences also exist when the substrate is glucose. If future study should show the advisability of recognizing the genus Bacterium, Citrobacter would become a subgeneric name for the group. Systematic study of the intermediate forms is being continued. DESCRIPTIONS OF SPECIES A key to the species of organisms producing trimethylene glycol has been prepared. Individual descriptions of the species are given. Key to the 8pecie8 of Citrobacter A. Gelatin liquefied. b. Acid and gas from sucrose (18a) (18). Citrobacter decolorans. bb. No acid and gas from sucrose (48) (48a). Citrobacter album. AA. Gelatin not liquefied. b. Acid and gas from sucrose. c. Acid and gas from aesculin and salicin. d. Acid and gas from galactose (Br) (17). Citrobacter Freundii. dd. No acid and gas from galactose (8) (8a). Citrobacter diversum. cc. No acid and gas from aesculin and salicin (55) (51). Citrobacter anindblicum. bb. No acid and gas from sucrose. c. Acid and gas from dulcitol. No acid or gas from raffinose (28R) (23R) (24R) (M8BK). Citrobacter intermedium n. sp. cc. No acid and gas from dulcitol. Acid and gas from raffinose (V33B). Citrobacter glycologenes n. sp. Citroba Freundii (Braak) comb. nov. Source. Canal water. (Br); Soil (17). Cultures: Br. received from Dr. A. J. Kluyver. Isolated By Dr. H. R. Braak in Kluyver's laboratory. No. 17 isolated by Mr. Roger Patrick in this laboratory. Morphology. Medium: Sodium citrate agar. Age: Twenty-four hours. Temperature: 300C. Form: Short rods. Arrangement: Single, in pairs, or short chains. Ends: Rounded. Motility: Motile with peritrichous flagella. Staining reactions. Gram-negative. Cultural characteristic8. Agar streak: Smooth, grey, shining, ffliform and butyrous. Litmus milk: Acid in two days. Reduced and subsequently coagulated in twenty days. Gelatin stab: Growth apparent; no liquefaction.

11 BACTERIA PRODUCING TRIMETHYLENE GLYCOL Biochemical characters. Weak production of indol. Acetoin not produced from glucose. Utilizes ammonium chloride as a source of nitrogen. Methyl red positive. Produces H2S in lead acetate agar. Gelatin not liquefied. Catalase produced. Citric acid utilized. Uric acid not assimilated. Reduces nitrates to nitrites. Fermentation reactions. Acid and gas from aesculin, arabinose, glucose, galactose, glycerol, inositol, lactose, levulose, maltose, mannitol, mannose, raffinose, rhamnose, salicin, sorbitol, starch, sucrose, trehalose, and xylose. Neither acid nor gas from amygdalin, dextrin, dulcitol (?), erythritol, glycogen, inulin, or melezitose. Braak was aware that his organism was an intermediate and gave it the name Escherichia Freundii because it behaved more like Esch. coli than Aerobacter aerogenes. Braak's organism becomes Citrobacter Freundii and type species of the genus. Culture 17 was isolated from soil by Mr. Roger Patrick (1929) working in this laboratory. The description of no. 17 shows good agreement with Braak's description of his organism. Braak considered that the ability of his culture to attack dulcitol was doubtful, Patrick reported it positive. We have found the results with dulcitol indecisive and unreliable. Culture 17 will show a slight production of acetoin after at least five days' incubation in glucose medium at 37 C. Citrobacter allum (Schive, comb. nov.) Sources. Feces. Cultures. 48, 48a Morphology. Medium: Sodium citrate agar. Age: Twenty-four hours. Temperature: 30 C. Form: Short rods. Arrangement: Single, pairs, or chains. Ends: Rounded. Motility. Motile, peritrichous flagellation. Staining reaction. Gram-negative. Cultural characteristics. Agar streak: Growth moderate, filiform, glossy, raised. Plain broth: No surface growth, cloudy. Litmus milk: Acid and coagulated. Gelatin stab: Gelatin liquefied. Biochemical characters. Indol production doubtful. Acetoin not produced from glycerol or glucose. Nitrates reduced to nitrites. Ammonium chloride utilized as source of nitrogen. Methyl red reaction positive. Hydrogen sulfide produced. Catalase produced. Uric acid not utilized. Citric acid utilized as a source of carbon. 177

12 178 C. H. WERKMAN AND G. F. GILLEN Fermentation reactions. Acid and gas from arabinose, glucose, dextrin, dulcitol, galactose, glycerol, lactose, levulose, maltose, mannitol, mannose, raffinose, rhamnose, sorbitol, starch, trehalose, xylose. No acid or gas from aesculin, amygdalin, erythritol, inulin, melezitose, salicin, starch and sucrose. Citrobacter glycologenes n. sp. Source. Feces. Culture. V33B. Morphology. Mediuim: Sodium citrate agar. Age: Twenty-four hours. Temperature: 300C. Form: Shortrods. Arrangement: Single, pairs, or chains. Ends: Rounded. Motility. Motile with peritrichous flagella. Staining reactions. Gram-negative. Cultural characteristics. Agar streak: Growth moderate, filiform, raised, glossy. No change in medium. Plain broth: No surface growth, slight ring, cloudy, no odor. Medium unchanged after twelve days at 300C. Litmus milk: Acid in two days, no other change after twenty days. Gelatin stab: Growth evident along line of puncture but no liquefaction after thirty days at 200C. Biochemical characters. Indol not formed. Acetoin not produced from glucose. Nitrates reduced. Uses ammonium chloride as a source of nitrogen. Methyl red positive. No production of H2S. Gelatin not liquefied. Catalase produced. Uric acid not assimilated. Citric acid utilized. Formic acid not utilized. Fermentation reactions. Acid and gas from glucose, galactose, glycerol, lactose, levulose, maltose, mannitol, mannose, raffinose, rhamnose, sorbitol, starch, trehalose, and xylose. No acid or gas from aesculin, amygdalin, arabinose, dextrin, dulcitol, erythritol, inulin, melezitose, salicin, sucrose. Citrobacter intermedium n. sp. Source. Feces (M8B). Cultures. M8B, 23R, 24R and 28R. Morphology. Medium: Sodium citrate agar. Age: Twenty-four hours. Temperature: 300C. Form: Short rods. Arrangement: Single, pairs, or chains. Ends: Rounded. Motility. Motile with peritrichous flagella. Usually two flagella at one pole. Staining reactions. Gram-negative.

13 BACTERIA PRODUCING TRIMETHYLENE GLYCOL Cultural characteristics. Agar streak: Growth moderate, flifform, raised, glossy. No change in medium. Plain broth: no surface pellicle, slight ring, cloudy, no odor, medium unchanged in appearance after twelve days at 200C. Litmus milk: Acid in two days, followed by reduction. No coagulation. Gelatin stab: Growth evident along line of puncture but no liquefaction after thirty days at 20'C. Biochemical characters: Indol not formed. Acetoin not produced from glucose. Nitrates reduced. Uses ammoniulm chloride as a source of nitrogen. Methyl red positive. No production of H2S. Gelatin not liquefied. Catalase produced. Uric acid not assimilated. Citric acid utilized. Formic acid not utilized. Fermentation reactions. Acid and gas from arabinose, glucose, dulcitol, galactose, glycerol, lactose, levulose, maltose, mannitol, mannose, rhamnose, sorbitol, startch, trehalose and xylose. No acid or gas from aesculin, amygdalin, dextrin, erythritol, inositol, inulin, melezitose, raffinose, salicin and sucrose. Citrobacter decolorans (Burkey) comb. nov. Source. Soil. Culture. 18, 18a. Morphology. Medium, glucose phosphate peptone agar. Age: 24 hours. Temperature: 300C. Form: Short rods (0.9A by 1.61A). Arrangement: Single or in pairs. Ends: Rounded. Spores: Absent. Capsules: Absent in milk. Motility. Non-motile. Staining reactions. Gram-negative. No granular appearance with iodine. Cultural characteristics. Agar slant, growth moderate, filiform spreading, glistening, smooth. Plain broth: No surface pellicle, turbidity. Litmus milk: Acid after twenty-four hours, reduction after four days. Coagulation on tenth day. No peptonization. Gelatin stab: Line of puncture filiform growth. Liquefaction. Potato slant: Yellow-white, lustrous growth. Biochemical characteristics. Indol formed. Nitrates reduced to nitrites. Ammonium salts utilized. Methyl red reaction intermediate to negative. H2S produced. Gelatin liquefied. Diastase production slight. Weak test for acetoin from glucose after five days. Fermentation reactions. Acid and gas from adonitol, aesculin, arabinose, glucose, galactose, glycerol, inositol, lactose, levulose, maltose, mannitol, mannose, pectin, raffinose, rhamnose, salicin, sorbitol, starch, 179

14 180 C. H. WERKMAN AND G. F. GILLEN sucrose, trehalose, and sylose. No acid or gas from amygdalin, arabitol, dextrin, dulcitol, erythritol, inulin and melezitose. Culture (18) was isolated by Mr. Roger Patrick (1929) during studies on the fermentation of xylan. It agrees with one minor exception, in characteristics with the organism which Burkey (1928) isolated and named Aerobacter decolorans. Burkey (1928) in his description of the species states that acid and not acid and gas is formed in rhamnose medium. In his description of the individual cultures of Aerobacter decolorans he shows nos. 3 and 4 as producing acid but no gas but cultures 24, 30 and 31 are shown as producing both acid and gas from rhamnose. It gives a weak Voges-Proskauer positive reaction after five days' incubation in glucose medium. Its name therefore should be Citrobacter decolorans comb. nov. Citrobacter diversum (Burkey) comb. nov. Source. Soil (8); feces (8a). Cultures. 8, 8a. Morphology. Medium: Glucose-phosphate-peptone broth. Age: Twenty-four hours. Temperature: 37PC. Form: Short rods. Arrangement: Single or pairs. Ends: Rounded. Capsules: Capsulated in twenty-four-hour culture of litmus milk. Endospores. Absent. Motility. Non-motile. Staining reactions. Gram-negative. Cultural characters. Colony: Size 5 to 8 mm. in diameter, round semiopaque, finely granular. Medium not discolored. Agar streak: Growth abundant, ffliform, shiny and butyrous. Plain broth: Cloudy, with slight ring. Litmus milk: Acid and gas. No reduction or coagulation. Biochemical reactions. Indol produced. Acetoin not produced from glucose or glycerol. Nitrates reduced. Uses uric acid as source of nitrogen and citric acid as sole source of carbon. Methyl red reaction indefinite. H2S produced. Gelatin not liquefied. Diastase produced. Fermentation reactions. Acid and gas from adonitol, arabinose, aesculin, glucose, glycerol, glycogen, inositol, lactose, levulose, maltose, mannitol, mannose, melezitose, pectin, raffinose, rhamnose, salicin, sorbitol, starch, sucrose, trehalose and xylose. Acid and gas not produced from amygdalin, dextrin, dulcitol, erythritol, galactose and inulin.

15 BACTERIA PRODUCING TRIMETHYLENE GLYCOL No. 8 is the original strain isolated and described by Burkey (1928) and called by him Aerobacter diversum. It becomes Citrobacter diversum comb. nov. Citrobacter anindolicum (Lembke) comb. nov. Source. Soil. Cultures. 51 and 55. Morphology. Medium, sodium citrate agar. Age: 24 hours. Temperature: 30 C. Arrangement: Single, pairs or chains. Ends: Rounded. Motility. Motile with peritrichous flagella. Staining reaction. Gram-negative. Cultural characteristics. Agar streak: Growth fair, ffliform, convex, glossy. Plain broth: No surface pellicle, slight ring, turbid. Litmus milk: Acid in two days. Gelatin stab: Growth along line of puncture but no liquefaction after thirty days at 20 C. Biochemical characterisics. Indol not formed. Acetoin not produced from glycerol or glucose. Nitrates reduced to nitrites. Uses ammonium salts as a sole source of nitrogen. Methyl red positive. H2S produced. Gelatin not liquefied. Catalase produced. Uric acid not assimilated. Citric acid utilized as sole source of carbon. Fermentation reactions. Acid and gas from arabinose, glucose, dulcitol, galactose, glycerol, lactose, levulose, maltose, mannitol, mannose, raffinose, rhamnose, sorbitol, starch, sucrose trehalose and xylose. No acid or gas from aesculin, amygdalin, dextrin, erythritol, inulin, melezitose and salicin. Bergey (1930) described Escherichia anindolica as coagulating milk. Cultures 51 and 55 agree with Bergey's description in all other characters. CONCLUSIONS Fifteen cultures of bacteria have been found to produce trimethylene glycol from glycerol. All of the cultures belong to the citrate-positive, coli-aerogenes intermediates. It is proposed to recognize the group as diagnosed as a new genus in the family Bacteriaceae. The name Citrobacter is proposed for the genus. Seven species are recognized in the genus; Citrobacter Freundii (Braak), comb. nov. is the type species. Should the genus Bacterium be retained Citrobacter becomes the name of a subgenus. 181

16 182 C. H. WERKMAN AND G. F. GILLEN REFERENCES BRAAK, H. R Onderzoekingen over Vergisting van Glycerine. Thesis, Delft. CocKs, L., AND SALWAY, A. H A method for the determination of trimethylene glycol in crude glycerol. Jour. Soc. Chem. Indus., 41, 17T- 20T. FITZ, ALBERT Ueber Schizomyceten-Gahrungen. II. Ber. Deut. Chem. Gesell., 10, FREUND, A Ueber die Bildung und Darstel1ung von Trimethylene-alkohol aus Glycerin. Monatsh. Chem., 2, ; Sitzber. K. Akad. Wiss. 84, KOSER, S. A The employment of uric acid synthetic medium for the differentiation of B. coli and B. aerogenes. Jour. Infect. Dis., 2, Kosum, S. A Utilization of the salts of organic acids by the colon aerogenes group. Jour. Bact., 8, NOYES, A. A., AND WATKINS, W. H The occurrence of trimethyleneglycol as a by-product of the glycerol industry. Jour. Amer. Chem. Soc. 17, PATRICK, ROGER Bacteria fermenting xylan. Unpublished thesis. Iowa State College. RAYNER, ARCHIBALD The occurrence, properties, and use of trimethylene glycol, and the fermentation of glycerol lyes. Jour. Soc. Chem. Ind., 45, 265-T, 187-8T. ROJAHN, C. A Ueber ein Methode zur quantitativen Bestimanung von Trimethyleneglycol im Destillierten-Glyzerin (Dynamitglyzerin) und dessen VorlAufer. Ztechr. Analyt. Chem., 58, WEBKMAN, C. H An improved technic for the Voges-Proskauer test. Jour. Bact. 20,