GUM PRODUCTION BY AZOTOBACTER CHROOCOC- CUM OF BEIJERINCK AND ITS COMPOSITION' W. BROOKS HAMILTON Department of Bacteriology, MacDonald College, Quebec, Canada Received for publication, March 5, 1931 INTRODUCTION In a paper in the JOURNAL OF BACTERIOLOGY (September, 1929), Sanborn and Hamilton described the effect of Azotobacter chroococcum upon the physiological activities of cellulose destroyers. The purpose of this article is to describe the production of gum by the Azotobacter and the availability of various nutrients for this purpose, also the chemical composition of the gum produced. The slime or gum produced by Azotobacter chroococcum has been discussed to some extent by Stapp (1924) and Heinze (1926). Buchanan (1909) in a discussion of gum production states: "It seems but fair to conclude from this array of evidence that there is a great possibility that bacterial slimes and gums of whatever kind are produced as a transformation or solution of the bacterial capsule." The best available description of the chemical properties of a bacterial gum is that of Buchanan (1909) who worked with Rhizobium radicicolum. The properties of the gum isolated by Buchanan are as follows: In a culture fluid of 2 per cent sucrose in tap water the gum was apparently partly in solution and partly as an insoluble modification, in the form of zoogloeal masses. The gum precipitated by alcohol was in the form of a soft rubbery mass when moist and hard and brittle when dry. I Parts I and III of a thesis presented to the Faculty of Graduate Studies and Research of McGill University in September, 1928, for the degree of Master of Science. 249
250 W. BROOKS HAMILTON The gum was precipitated by 65 per cent alcohol. The precipitate redissolved readily on the addition of water. It was also precipitated by ammonium sulphate and ammoniacal lead acetate. No precipitation occurred with sodium chloride, lead acetate, mercuric chloride, copper sulphate, acetic, hydrochloric, nitric, sulphuric, picric and tannic acids. Magnesium sulphate caused a precipitation only after the complete saturation of the solution with crystals and in the course of twentyfour hours. The gum reduced Fehling's solution only after autoclaving for an hour at one atmosphere pressure with 2 per cent sulphuric acid. Millon's xantho-proteic, and biuret tests for proteins were negative. A test for combined nitrogen, by converting the gum into a cyanid and obtaining the Prussian blue reaction, was also negative. ISOLATION OF THE AZOTOBACTER CHROOCOCCUM OF BEIJERINCK Ten grams of soil, which was classified as loam, were added to 150 cc. of sterile Ashby's solution in a large Erlenmeyer flask. The mixture was incubated at 30 C. for two days when loopfulls of the surface film were streaked on Ashby's agar. After two days' incubation on the agar these streaks showed the characteristic colonies of Azotobacter. Three species of the genus Azotobacter were fished from the above described plates. The species of Azotobacter isolated were identified with: (1) Azotobacter chroococcum of Beijerinck; (2) Azotobacter beijerinckii of Lipman; (3) Azotobacter vitreum of Lohnis and Westman. The strain of Azotobacter chroococcum isolated by the author was used throughout these experiments. GENERAL PROCEDURE The procedure for the production of gum was the same for the various nutrients used. The nutrient solution was prepared and sterilized in the usual manner. Each flask of 50 cc. of the media used in the various experiments was inoculated with 2 cc. of the Azotobacter chroococcum suspension. The suspension was standardized to a turbidity of ten according to the McFarland scale. The inoculated flasks were incubated at 30 C. for fourteen days. The gum was removed from the nutrient solutions after the
GUM PRODUCTION BY AZOTOBACTER CHROOCOCCUM 251 fourteen-day incubation period by a method suggested by Buchanan (1909). The media were filtered to remove coarse particles. The filtrates were then made alcoholic to the extent of 70 per cent of the total volume. The gum which precipitated in the alcoholic solutions was separated by filtration. The separated gum was dried to constant weight and the weights recorded for the amounts of gum produced per 100 cc. of the medium used. The gum was purified for further use by repeated dissolving and precipitating, the former with water and the latter with alcohol. The dissolved gum solution was filtered to remove any insoluble portions before each precipitation. To preserve the gum this method was devised: To every 100 cc. of gum solution 10 cc. of di-ethyl ether were added. The ether was allowed to evaporate when the gum was required for further experiments. The gum solution *2ontained approximately 5 per, cent gum. AMOUNTS OF GUM PRODUCED IN DIFFFIRENT MEDIA To determine the source of energy best suited to gum production by Azotobacter chroococcum various media were used. 1. The first medium tried was a modification of one used by Greig-Smith (1911). Potassium phosphate... Ammonium sulphate... 5.0 grams 0.6 grams Maltose...... 20.0 grams Distilled water... 1000.0 cc. The average yield of gum per 100 cc. of the above medium was 411.428 2. A medium of glycerinated beef broth was also used in pre-. liminary experiments. The medium contained: Difoo beef extract... Difco peptone... Glycerol C. P... 3.0 grams 5.0 grams 20.0 grams Distilled water... 1000.0 cc. The average amount of crude gum produced in this medium per 100 cc. of liquid was 2,226.50 3. Various sugars were used in 2 per cent solutions to deter-
252 W. BROOKS HAMILTON mine the ability of Azotobacter chroococcum to use them as sources of energy.for gum production. The only other nutrients available were the very minute quantities of mineral salts to be found in distilled water. The average amounts of gum produced by the Azotobacter chroococcum using the various sugars as sources of nutrient in concentrations of 2 per cent were: Xylose... 760.0 Glycerol... 760.0 Arabinose... 765.0 Mannitol... 745.0 Galactose... 765.0 Rhamnose... 765.0 Glucose... 735.0 Salacin... 765.0 Laevulose... 750.0 Raffinose... 800.0 Sucrose... 750.0 Dextrin... 1,080.0 Lactose... 755.0 Inulin... 1,055.0 Maltose... 765.0 TABLE 1 The effect of glucose concentration, (with a fixed nitrogen source), on the production of gum by Azotobacter chroococcum PERCENTAGE OF GLUCOSE WEIGT OF Gum PERCENTAGE OF GLUCOSE WEIGHT OF GUM 1 770.0 7 770.0 2 800.0 8 790.0 3 790.0 9 795.0 4 785.0 10 765.0 5 795.0 15 780.0 6 810.0 20 800.0 Figures are quoted for the amounts of gum produced in 100 cc. quantities of media. The above weights are the yields in milligrams per 100 cc. of liquid medium. 4. A nitrogen source was added to the sugars in the next series of experiments to increase the production of the gum. Peptone was used as the nitrogen source in concentrations of 0.5 per cent. The two common sugars, glucose and sucrose, were used in increasing concentrations as indicated in tables 1 and 2. 5. An attempt was made to produce gum from Azotobacter chroococcum in solutions of peptone alone. It was found that peptone would not furnish energy for the production of any gum.
GUM PRODUCTION BY AZOTOBACTER CHROOCOCCUM 253 ANALYSIS OF THE GUM PRODUCED BY AZOTOBACTER CHROOCOCCUM 1. Physical properties The gum is completely soluble in water, precipitated by alcohol (65 per cent), ether or acetone. The precipitated gum is translucent and grayish-white. When dry the gum has a smooth, glossy surface. When first precipitated, the gum is a translucent, amorphous, adhesive mass which darkens on hardening. The specific rotation of the gum is [a] D = -30.3, which would class it with the true gums. The effect of sucrose concentrations TABLE 2 (with a fixed nitrogen source), on the production of gum by Azotobacter chroococcum PERCENTAGE OF SUCROSE WEIGHT OF GUM PERCENTAGE OF SUCROBE WEIGHT OF GUM 1 890.0 7 1,235.0 2 950.0 8 1,895.0 3 1,230.0 9 960.0 4 1,285.0 10 1,305.0 5 1,165.0 15 975.0 6 1,195.0 20 920.0 Figures are quoted for the amounts of gum produced in 100 cc. quantities of media. 2. Chemical properties A solution of the gum (6.60 of solid gum per cubic centimeter of water) was submitted to the following tests: 1. Molisch's test: positive. 2. Fehling's test: solution not reduced after boiling with acid. 3. Biuret test: Negative. 4. Millon's test: Negative in both hot and cold solution. 5. The gum was submitted to the following precipitation tests and with these results: a. Gum precipitated by ethyl alcohol, 2N lead acetate, 2N ammonium sulphate, ammoniacal lead acetate. b. Gum not precipitated by tnagnesium chloride, sodium hydroxide, sodium chloride, hydrochloric acid, sulphuric acid, anmonium hydroxide, copper sulphate, acetic acid, nitric acid.
254 W. BROOKS HAMILTON Both concentrated and 2N acids were used. 6. Test for soluble starch-when iodine was added to the gum a yellow-green colour was produced which did not change on the addition of sulphuric acid. 7. When tested with phospho-tungstic acid for Vitamine B, no precipitate was formed Williams and Seidell (1916). 8. The Micro-Kjeldahl test for combined nitrogen showed a trace. (Koch and McMeekin, 1924). DISCUSSION AND CONCLUSIONS Azolobacter chroococcum of Beijerinck has been sbown to produce a gum using various carbohydrates as nutrients. The more complex carbohydrates are more readily available for the production of the gum than are the mono- and di-saccharides. The presence of a protein, such as peptone, enhances gum production but has not been found necessary for this function of the organism. A solution of peptone alone does not furnish material for the production of the gum. The gum produced by Azotobacter chroococcum is a carbohydrate of the higher series. Boiling with acid does not cause it to reduce Fehling's solution. It is levo-rotatory and therefore of the true gums and not the dextrins. The trace of combined nitrogen found is probably due to remains of bacterial cells not removed in the purifying process. The results of the analysis place the gum in "Class I" of Haas and Hill (1923) and under this classification it would be termed an arabin. REFERENCES BUCHANAN, R. E. 1909 Centralbl. f. Bakt., etc., II, 22, 371. WILLIAMS, ROBT. R., AND SEIDELL, ATHERTON. J. Biol. Chem., 1916, xxvi, pp. 436-437. GREIG-SMITH 1911 Centralbl. f. Bakt., etc., II, 30, 552. HAAS AND HILL 1923 An Introduction to the Chemistry of Plant Products. Longmans, Green and Company, London, England. HEINZE, B. 1926 Landw. Jahrb., 64, 127. KOCH AND MCMEEKIN 1924 Jour. Amer. Chem. Soc., 46, 2066. STAPP, C. 1924 Centralbl. f. Bakt. etc., II, 61, 276.