H6 'Wis.—lO INFLUENCE OF NITRATES ON NITROGEN- ASSIMILATING BACTERLV BY T. L. HILLS IT ^.V!* 'lO Reprinted from JOURNAL OF AGRICULTURAL RESEARCH Vol. XII, No. 4 : : : Washington, D. C., January 28, 1918 PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE. WITH THE COOPERATION OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS WASHINGTON : GOVERNMENT PRINTING OFFICE : 1918 INFLUENCE OF NITRATES ON NITROGEN-ASSIMILAT- ING BACTERLV By T. L. Hills, - Research Bacteriologist, Idaho Agricultural Experiment Station INTRODUCTION RElvATlON OF NITRATES TO VARIOUS FORMS OF PLANT LIFE The importance of nitrogen to plant life can not be overestimated. It is one of several elements essential to plant growth, one, moreover, which is apt to be deficient in arable soils. These facts are well brought out by the almost innumerable investigations which have been made concerning the source of nitrogen for plants. The influence of nitrate nitrogen on various plants has been the con- trolling idea in many of these experiments. Very little attention has been placed on the effect of nitrates on the lower plants, especially the bacteria. Because of the relation that exists between higher plants and bacteria it seems advisable to consider the effect of nitrates on the soil bacteria. Indeed, progress in the knowledge of nitrogenous fertilizers depends on a study of the effect of the fertilizer on the soil organisms as well as on the higher plants. The action of fertilizers on the different groups of soil organisms, the relation of these organisms to higher plants, and the separation of the important from the unimportant groups are some of the factors involved in the problem of soil fertility. REVIEW OF LITERATURE The relation of nitrates to the germination of seeds has been studied by De Chalmot (Ix)^ who found that corn germinated in solutions con- taining nitrate was more robust than corn germinated under similiar conditions without nitrate. He also noted that if too concentrated solutions of nitrate were used germination was retarded rather than hastened. The presence of nitrate also increased the amount of al- buminous material in the seed. The direct influence of nitrate nitrogen on the growing plant is too well known to justify any lengthy discussion here. Jost (26, p. 134) gives the results of experiments made by Boussingault, who grew the sunflower (Helianthus argophyilus') in sand with and without nitrate. 'Major portion ot a paper submitted in partial fulfillment of the requirements for the degree of doctor of philosophy in bacteriology in the Graduate School of the University of Wisconsin, December. 1916. 2 The writer wishes to acknowledge his appreciation oi the suEgestions and criticisms obtained through- out the progress ol this work from Prof. E. B. Fred and E. G. Hastmgs, of the University of Wisconsin. 3 Reference is made by number (italic) to "Literature cited," pp. 227-230. Journal of Agricultural Research, Vol, XII, No. 4 Washington, D. C. J*n. 28. 1918 Ir Key No. Wis. — (183) i84 Journal of Agricultural Research Vol. XII, No. 4 During the three months' growth of the plants 1.40 gm. of potassium nitrate were added. At the end of the period the dry weight of the plant suppHed with nitrate was nearly 60 times greater than that of the plant where no nitrate was added. The relation between the growth of nonleguminous plants and the amount of nitrate nitrogen supplied is shown in a very striking manner in the following table taken from Hell- riegel and Wilfarth {21, p. 53-54) : Nitrogen as Ca (N03)8 added to pots, gm Dry weight of oats (grain and straw) . gin. . None o. 3605 .4191 0.056 5- 9024 5-8510 5- 2867 10. 9814 10. 9413 0.168 21. 2732 21. 4409 0-336 But little work has been done on the direct influence of nitrates on the development of the Eumycetes. Some investigations have been made as to the ability of certain fungi to assimilate nitrate nitrogen directly. Ritter {42) studied many species and found that some forms would assimilate nitrate directly, while others reduced it to nitrite and am- monia. He found some forms which failed to grow on media containing nitrate. Kossowicz {28) found that various fungi utilized nitrates and that nitrite and ammonia were produced. Miinter {36) studied the influence of inorganic salts on the growth of various Actinomycetes. He found that potassium and sodium nitrates in quantities equivalent to 5 per cent permitted good growth of the organisms 'but retarded spore formation. Calcium, barium, and stron- tium nitrates in small quantities affected some species but not others. Small quantities of these nitrates did not affect growth to any extent, but larger quantities were detrimental to growth and spore formation. Silver nitrate in all amounts studied almost entirely prohibited growth. Nitrates appear to exert some influence on the yeasts. Drabble and Scott {13) studied the effect of sodium nitrate on these organisms. They found that the greatest reproduction took place in solutions containing 0.2 gram-molecule of the nitrate. Increasing amounts of the salt led to a decrease in reproductive activity until with 0.7 gram-molecule present no reproduction took place. From their results it is evident that small quantities of nitrate stimulated reproduction, whereas larger amounts proved detrimental. Kayser (27) studied the effect of man- ganese nitrate on yeasts. He found that the amount which produced the maximum increase in the alcoholic fermentation of sugar varied with the strain of yeast employed. He likewise found that manganese nitrate produced greater increase than did the same quantity of potassium ni- trate. Fernbach and Lanzenberg {14) concluded that nitrates hindered the rapidity of cell multiplication of yeasts but greatly accelerated the action of the zymase. More alcohol was formed in the presence than in the absence of nitrate. According to Kossowicz {28), nitrates are not a suitable source of nitrogen for yeasts. •k"^* Jan. 28, i9i8 NitYogen-Assimilating BacteHa 185 The direct influence of nitrates on bacteria has been studied to a limited extent. The influence of various nitrates on soil bacteria has been studied by Greaves (jp). He added sodium, potassium, calcium, mag- nesium, manganous and ferric nitrates to soil in varying quantities. The amount added to the soil was such that in each case equivalent quantities of the anion (NO3) in the various forms were added. The efiFect of these salts on the bacteria was determined by using ammonifi- cation as an index of the bacterial activity. He found that sodium- potassium, manganous and ferric nitrates in small amounts, approxi- mately 0.97 to 5.5 mgm. of nitrate in 100 gm. of soil, slightly stimulated ammonification. Greater concentrations of these salts proved toxic as evidenced by a decrease in the amount of ammonia formed. Sodium nitrate was much more beneficial to ammonification than potassium nitrate. From his results as a whole Greaves concludes that it is the electronegative ion which stimulates bacterial activity. Calcium and magnesium nitrates proved toxic in all concentrations studied. However, a majority of the investigations have been directed toward a determination of the effect of the bacteria on the nitrates. But little work appears to have been done on the direct action of nitrates on bacteria. Pfeffer {38, p. 351) cites some experiments showing the repellant action of potassium nitrate toward certain bacteria. Spir- illum undula was repelled by a solution of potassium nitrate having an osmotic concentration equivalent to 0.5 to i.o per cent. With Spirillum volutans a much higher concentration was necessary to bring about the same reaction. It was found that different organisms required different quantities of the same nitrate to repel them. It can be readily seen that by far the greatest amount of work on the relation of nitrates to plant growth has been done in the realm of the higher plants. Obviously further investigations should be made in respect to the effect of nitrates on the lower forms of plant life, especially the bacteria. In this paper an attempt is made to set forth the results secured in a study of the influence which nitrates exert on certain groups of soil bacteria, including not only their reproduction but also some of their physiological properties. EXPERIMENTAL WORK OUTLINE OP PROBLEM The results of much careful experimentation show that nitrate nitro- gen is most readily assimilated by higher plants. As a rule it seems to stimulate the plant to increased activity. In some cases this is un- doubtedly due to increased nutrition, while in others it is a result of nuclear stimulation with a consequent cell multiplication. No sharp line can be drawn between these two effects. Probably one overiaps the other, and the increased growth of the organism can be attributed to a combination of the two actions. 1 86 Journal of Agricultural Research voi. xii, no. 4 From a practical standpoint the relation of nitrates to the nitrogen- assimilating organisms of the soil is of importance. Hence, it was arranged to study the effect of nitrates on soil bacteria, especially those forms concerned with the fixation of atmospheric nitrogen. The work naturally falls into two rather distinct lines of investigation. First, the influence of nitrates on Azotobacter was determined. Here studies were made of the effect of nitrates on the growth of the organism in soil and also the effect of these salts on the nitrogen-fixing property of these bacteria. The action of Azotobacter on nitrates in solution, the relation of nitrates to pigment production and to the formation of volutin bodies were studied. Second, the influence of nitrates on the growth of Bacillus radicicola in soil was studied. The action of B. radicicola on nitrates in solution and the possible nitrogen-assimilating properties of the legume in the presence of nitrates were investigated. Also the influence of nitrates on gum production was determined. The latter part of the investigations included a study of the relation of nitrates to nodule formation on alfalfa. METHODS USED IN EXPERIMENTS Nitrates were determined by the reduction method with Devarda's alloy and also by the phenolsulphonic acid (colorimetric) method. The total nitrogen content of all samples was determined by the modified Kjeldahl method with sulphuric acid, salycilic acid, sodium thiosulphate, and copper sulphate. Where nitrate nitrogen was present, 50 c. c. of concentrated sulphuric-sal ycilic acid (25 c. c. of concentrated acid plus 25 c. c. of distilled water) were added to the cultures slowly and with constant stirring. This acid was allowed to react for a few days, after which the usual procedure was carried out. Digestion was continued for five to six hours subsequent to the clarification of the liquid. The amount of ammonia was determined by distillation with steam in the presence of magnesium oxid. Nitrites (qualitative test) were tested for with Trommsdorf's reagent. In all distillations NI14. acid and alkali were used. In the preparation of agar cultures of alfalfa seedlings the seeds were treated with a 0.25 per cent solution of mercuric chlorid and rinsed in sterile distilled water. Three bacteria-free seeds were transferred to the surface of soft mannit agar (0.7 per cent agar) in each tube. The nitrates were added in solution to all cultures. Gram-molecular quantities of potassium, sodium, calcium, and ammoniun nitrates (Merck's) were weighed into sterile distilled water. These solutions were prepared in such a manner that 5 c. c. contained 450 mgm. of nitrate. In all nitrate solutions the nitrate radical, or anion, was present in the same quantities, while the cation, or metal, was present in varying quantities, depending upon the particular salt. Jan. 28. i9i8 Nitrogen-Assimilating Bacteria 187 Plate counts of all soil cultures were made by weighing 20 gm. (dry weight) of the soil into a 200-c. c. water blank. From this suspension all subsequent dilutions were made. Mannit agar ^ was used for the plate counts in the cultures of Azotobacter and B. radicicola. Duplicate plates were made for each dilution poured. SOIL USED Only one type of soil was employed, Miami silt loam obtained from the Experiment Station farm. No chemical analyses of the soil were made other than an estimation of its organic matter content, which was approximately 2.75 per cent. The soil was neutral in reaction and its nitrate content was approximately 1.5 mgm. of nitrogen as nitrate in 100 gm. of the dry soil. ISOLATION OF AZOTOBACTER AND BACILLUS RADICICOLA Azotobacter. — (i) Strain A was isolated from a silt loam soil. This strain grew well on mannit agar, but produced no pigment after three weeks' growth. (2) Strain B was isolated from a sandy loam soil. This strain grew equally well on mannit agar and produced a brownish black pigment within one week's growth. Both strains assimilated practically the same amount of atmospheric nitrogen under laboratory conditions. Bacillus radicicola.— A stock laboratory culture of B. radicicola was replated twice before taking the final culture. The nodule produc- ing power of the organism was determined by inoculating bacteria-free alfalfa seedlings (in soft agar). After sufficient incubation nodules were produced in abundance. INFLUENCE OE NITRATES ON AZOTOBACTER INFLUENCE OF NITRATES ON THE GROWTH AND REPRODUCTION OF AZOTOBACTER IN STERILIZED SOIL What effect do nitrates have on pure cultures of Azotobacter in ster- ilized soil? Do these salts cause a decrease in the numbers of the organ- isms? Do they cause an increase in numbers? Or do they exert no particular influence one way or the other? It is difficult to believe that the latter could be true, inasmuch as nitrates have such a profound effect on higher forms of plant life. Such readily soluble and assimilable substances as nitrates could hardly remain without affecting either an increase or a decrease in the number of organisms existing in their presence. With the idea of determining what effect nitrates might have on Azotobacter when grown in sterilized soil, the following experiments were planned. In this work both strains of the Azotobacter (described on 1FRED.E.B. A I^BORATORYMANUAl, OF SOIL BACTERIOLOGY, p. io8. Philadelphia and London, 1916. i88 Journal of Agricultural Research Vol. XII, No. 4 p. 187) were employed and conditions governing the preparation and incubation of the cultures were similar in the case of each strain. The only variation was the periods used in incubating the cultures. Counts were made after one and two weeks' incubation with strain A and after one, two, and three weeks with strain B. TABiyE I. — Influence of potassium nitrate on the growth of Azotobacter (strain A) in sterilized soil Treatment (nitrate in 100 gm. of dry soil). Number of organisms in i gm. of drj- soil. Culture No. At begin- ning. After I week. Relative. After 2 weeks. Relative. I Mgm. 10 10 25 25 50 50 100 100 150 150 200 200 300 300 15, 600 15, 600 15, 600 15, 600 15, 600 15, 600 15,600 15, 600 15, 600 15, 600 15,000 15, 600 15, 600 15, 600 15, 600 15, 600 825, 000 935. 000 I, 500, 000 Per cent. > 100 } 170 } 523 } 2, 233 } 1.295 } 179 } 27 } " f 315,000 L 360, 000 f 1, 175, 000 Per cent. 2 > 100 ■7 } 348 t 4, 200, 000 5, 000, 000 20, 400, 000 18, 900, 000 11,000,000 II, 820,000 1' 12, 350, 000 t 10, 750, 000 r 27, 750, 000 6 } 3.418 7 8 1 8,210 Q f 9, 000, 000 t 9, 150, 000 f 25, 000 [ 55, 000 r I { c 10 } 2, 68s 12 I, 575. 000 225, 000 250, 000 1 ^^ I^ TA ( ° je 16 1 ° Table II. — Infltience of sodium nitrate on the growth of Azotobacter {strain A) in sterilized soil Treatment (nitrate in 100 gm. of dry soil). Number of organisms in i gm. of dry soil. Culture No. At begin- ning. After I week. Relative. After 2 weeks. Relative. I. Mgm. 10 10 25 25 SO SO 100 100 150 150 200 200 300 300 13,800 13, 800 13, 800 13, 800 13, 800 13, 800 13, 800 13, 800 13, 800 13, 800 13, 800 13, 800 13, 800 13, 800 13, 800 13, 800 310, 000 225, 000 575. 000 430, 000 2, 850, 000 5, 800, 000 15, 200,000 12, 750, 000 17.750,000 16, 200, 000 550, 000 400, 000 Per cent. \ 100 1 188 f I. 615 } 5.217 } 6,335 } 177 1 ° 1 ° r 425, 000 \ 490, 000 f 875, 000 Per cent. 2 t 100 } 191 } 492 } 3, 150 1 2, 800 2 4 e / 2, 250, 000 6 7 , f IS, soo, 000 I i3> 300, 000 f 9, 850, 000 I 15.750,000 f 690, 000 I 375. 000 r . I r \ 8 Q 10 II } - } ° } ° 12 . XX 14 IC 16 Jan. 38. 1918 Nitrogen-A ssimilating Bacteria 189 Table III. — Influence of calcium nitrate on the growth of Azotobacter {strain A) in sterilized soil Treatment (nitrate in 100 gm. of dry soil). Number of organisms in i gm. of dry soil. Culture No. A,t begin- ning. After I week. Relative. After 2 weeks. Relative. I Mgm. 10 10 25 25 50 50 100 100 150 150 200 200 300 300 p p p pppppppppppp p OOOOOOOOOOOOOOOO 260, 000 330, 000 5, 800, 000 Per cent. \ 100 1 1, 966 1 3.440 \ 3 10, 000 1 260, 000 1 975. 000 [ 1, 090, 000 / 9, 200, 000 \ 8, 600, 000 r 13, 200, 000 \ 12, 600, 000 / 8, 750, 000 \ 8, 000, 000 f 2,000,000 I 2,350,000 / ° r I ° Per cent. 2 \ 100 •2 I 362 e 10, 700, 000 9, 600, 000 i^, 2t;o, 000 6 \ 3. 122 7 1 } 4, 526 8 11,600,000 f 4,213 1 6, 600, 000 6, 050, 000 3, 500, 000 3, 900, 000 I 2, 144 } 1.254 1 ° } 2,938 10 I 763 12 1-2 14, 1 ° le ll 16 1 ° 1 One hundred and fifty gm. of soil (dry weight) were weighed into 500- c. c, Erlenmeyer flasks and the nitrates added in solution, as indicated in the following tables. At the same time i per cent of mannit was added in solution and the moisture content was raised to approximately 18 per cent. The flasks were allowed to remain at room temperature for one day, when the contents were thoroughly mixed. The flasks and contents were then sterilized at 15 pounds' pressure for three hours. Upon cooling they were inoculated with 5 c. c. of a suspension of the organisms in sterile distilled water. The cultures were incubated at 28° C. and counts made at the intervals already indicated, Mannit agar was used in pouring the plates. Each number in the following tables represents an average of duplicate plates. Tables I, II, and III show the results of the work with strain A and Tables V, VI, and VII the results with strain B. It will be seen at a glance that all three nitrates exerted an enormous influence on the growth of the Azotobacter. The smallest concentration did not appear to exert much influence either in increasing or decreasing the number of Azotobacter. There was a slight gain, but it was not so marked as that brought about by higher concentrations of nitrates. When 25, 50, and 100 mgm. of nitrate were present in 100 gm. of soil, very large increases were obtained in practically all instances. In one instance sodium nitrate caused the greatest relative gain, but the most consistent increase was produced by calcium nitrate. Beginning with 150 mgm, the number of Azotobacter began to decrease. This decrease was especially noticeable in the cultures containing potassium and sodium nitrates. At the end of the first week, Azotobacter organisms IQO Journal of Agricultural Research Vol. XII, No. 4 were still found in the potassium-nitrate cultures where 200 mgm. were present. However, at the end of the second week the organisms were dead. The same concentration of sodium and calcium nitrates proved even more toxic. No evidences were secured, indicating that these organisms can resist concentrations in excess of 300 mgm. of nitrate per 100 gm. of soil. The question may be raised in regard to the influence of sterilization on the nitrate present in the soil, Does the prolonged heating in the presence of soil organic matter reduce the nitrate? In order to study this point, a few cultures were prepared similar to those already described. They were subjected to sterilization under pressure of 15 pounds for two, three, and five hours. Nitrate determinations at the end of these periods failed to show any reduction. In the presence of i per cent of mannit the nitrate content remained unchanged during sterilization. From these results it is evident that small amounts of nitrate up to 150 mgm. of nitrate in 100 gm. of soil greatly increased the reproduction of x\zotobacter. In regard to the toxicity of higher concentrations, sodium nitrate appeared to exert the greatest influence in this direction, followed by calcium and potassium nitrates in the order named. The results of the experiment are recorded in Table IV. Table IV. — Influence of ammonium nitrate on the growth of Azotohacter {strain A) in sterilized soil Treatment (nitrate in 100 gm. 01 dry soil). Number of organisms in i gm. of dry soil. Culture No. At begin- ning. After I week. Relative. After 2 weeks. Relative. I Mgm, 25 25 100 100 200 200 18, 500 18, 500 18, 500 18, 500 18, 500 18, 500 18, 500 18, 500 I, 400, 000 1, 050, 000 5, 600, 000 4, 900, 000 2, 900, 000 2, 600, 000 1, 100, 000 950, 000 Per cent. > 100 ; 427 } 223 } 84 r 975, 000 \ 1, 100, 000 f 5, 000, 000 \ 3, 900, 000 / 3. 950, 000 \ 4, 100,000 f 875, 000 I 915) 000 Per cent. > 100 2. .. . . ■J 4 e. 6 } 388 1 86 7. .......... . 8.... That the nitrate radical and not the combined metal was the causal agent in the increase in the number of Azotobacter was indicated from the results of the next test. Here ammonium nitrate was used. It will be seen from the data of this experiment that ammonium nitrate caused an increase in the number of Azotobacter when present in small amounts. However, the increase in the presence of ammonium nitrate was less marked than when equal quantities of the other nitrates were used. Since the experiments with ammonium nitrate were not made at the same time as the preceding experiments (discussed on pp. 189-190), it is possible that conditions varied sufficiently to account for the less pro- nounced results. When 200 mgm. of nitrate were present in loo gm. of Jan. 38, X918 Nitrogen- A ssimilating Bacteria 191 soil the number of Azotobacter showed a decrease. Apparently ammo- nium nitrate is more toxic than potassium, sodium, and calcium nitrate. However, the main point at issue seems fairly well established — namely, that the increase in the number of Azotobacter is caused by the nitrate radical and not by the combined metal. Table V. — Influence of potassium nitrate on the growth of Azotobacter (strain B) in sterilized soil Treat- ment (nitrate in 100 gm. of dry soil). Number of organisms in i gm. of dry soil. Cultu No. At begin- ning. After I week. Relative. After 2 weeks. Relative. After 3 weeks. Rela- tive. I. . 2 . . Mgm. 10 10 25 25 50 50 100 100 150 150 200 200 300 300 12, 600 12, 600 12, 600 12, 600 12, 600 12, 600 12, 600 12, 600 12, 600 12, 600 12, 600 12, 600 12, 600 12, 600 12, 600 12, 600 235, 000 Per cent. \ 100 >I, 510 }2, 436 |i.340 |l,320 } 851 r 373 f ° / 112,500 \ no, 500 / 2, 100, 000 \ 2, 250, 000 / 1,575.000 \ 1,950,000 r 3,250,000 \ 4, 900, 000 { 4, 000, 000 \ 3, 300, 000 f 2, 000, 000 \ 2, 100, 000 { 800, 000 1 750, 000 { Per cent. > 100 }i,9So }i,58r J3, 65s ]i, 3(>l }i.838 } 695 1 ° f 116,000 \ 117,000 { 875, 000 \ I, 260, 000 / I, 700, 000 '1 1,325,000 f 3,525,000 \ 2, 960, 000 f 2, 500, 000 \ 2, 900, 000 f I, 500, 000 \ 2, 000, 000 f 630, 000 \ 700, 000 { I Per ct. \ 100 }i,300 }2, 783 }2,3i7 \i, 502 } 580 } ° 3- ■ 4- ■ 5- ■ 6. . !■■ 8.. 9.. 10. . 11 . . 12. . 13- • 14. . 15- • 16. . 3, 750, 000 3, 300, 000 5, 750, 000 5, 700, 000 3, 100, 000 3, 200, 000 3, 200, 000 3, 000, 000 2, 100, 000 I, 900, 000 875, 000 880, 000 Table VI. — Influence of sodium nitrate on the growth of Azotobacter {strain B) in sterilized soil Treat- ment (nitrate in 100 gm. of dry soU). Number of organisms in i gm. of dry soil. Cultu No. At begin- ning. After I week. Relative. After 2 weeks. Relative. After 3 weeks. Rela- tive. 1. . 2. . 3-- 4- • 5-- 6. . 7-- 8.. 9.. 10. . 11 . . 12. . 13- ■ 14.. . Mgm. 10 10 25 25 50 50 100 100 150 150 200 200 300 . 300 15, 600 15, 600 15, 600 15,600 15,600 15, 600 15,600 15,600 15, 600 15, 600 15, 600 15, 600 15,600 15,600 15,600 15,600 158, 000 149, 000 I, 250, 000 990, 000 I, 765, 000 1, 825, 000 1,875,000 2, 250, 000 2, 200, 000 I, 950, 000 165, 000 170, 000 Per cent. > 100 } 727 }l, 165 }i,338 }i,35o 1 108 } ° 1 ° f 110,500 \ 126, 000 f I, 750, 000 \ 1,350,000 f 6, 600, 000 \ 5, 300, 000 f 2, 025, 000 \ 3, 040, 000 f 2,775,000 \ 3, 200, 000 f 530, 000 L 785, 000 { I { I Per cent. > 100 [1,310 }5,o29 >2, 141 }2,525 } 556 / 112,500 I 115,000 f 5,000,000 \ 6, 600, 000 1 9, 150, 000 \ 7, 150, 000 ri5,95o,ooo \i4, 600, 000 f 5, 800, 000 1 5, 250, 000 1 3, 100, 000 \ 2, 750, 000 / ° 1 { Per cl. \ 100 } 5,097 \ 7, 161 }i3, 423 1 4,860 } 2, 573 I ° 1 ° 192 Journal of Agricultural Research Vol. XII, No. 4 Table VII. — Influence of calcium nitrate on the growth of Azotohacter {strain B) in sterilized soil Culture No. Treat- ment (nitrate in 100 gm of dry soil). Mgm. o o 10 10 25 25 50 50 100 100 150 150 200 200 300 300 Number of organisms in i gm. of dry soil. At begin- ning. 22, 000 22, 000 22, 000 22, 000 22, 000 22, 000 22, 000 22, 000 22, 000 22, 000 000 1, 000 000 000 000 000 22 After I week. 905> 860, 23, 200, 19, 600, 17, 200, 19, 600, II, 800, 14, 000, 7, 500. II, 000, 2, 55o» 3. 500. 107, 87, 000 000 000 000 000 000 000 000 000 000 000 000 500 500 Relative. Per cent. \ 100 y, 423 >2, 084 |l, 461 } 342 After 2 weeks. 475, 000 460, 000 28, 000, 000 36, 000, 000 52, 000, 000 43, 500, 000 22, 500, 000 20, 000, 000 12, 000, 000 5, 300, 000 6, 500, 000 2, 750, 000 3, 225, 000 Relative. Per cent. > 100 W 181 ]z, 25s jl, 448 I 818 402 203 After 3 weeks. 130, 000 157, 500 050, 000 600, 000 750, 000 250, 000 400, 000 850, 000 750, 000 950, 000 800, 000 130, 000 120, 000 o Rela- tive. Per ct. \ 100 |3, 002 }2-> 273 J2, 633 }i,7& o 420 II A glance at the figures of Tables V, VI, and VII shows that the small- est concentration of nitrate used produced a much more marked relative increase in numbers with strain B than it did with strain A. On the other hand, the greater resistance of this strain to the higher nitrate concentrations is clearly evident. In the potassium- and calcium- nitrate cultures the organisms were present in an active state where the nitrate was added in amounts equivalent to 200 mgm. of nitrate in 100 gm. of soil. However, this same concentration of sodium nitrate prevented the development of the Azotobacter. The first five concen- trations of all three nitrates caused a very large increase in the number of Azotobacter when compared with control cultures where no nitrate was added. In one instance an enormous increase was noted after three weeks' incubation in the presence of 50 mgm. of nitrate as sodium nitrate. This increase far excelled that noted with other concentrations of the same salt. The writer can ofifer no conjecture as to this occurrence. Similar results were obtained by the writer in 19 14 (23) with a strain of Azotobacter isolated -from a silt loam soil at the Pennsylvania Experi- ment Station. It was found that soil and liquid cultures containing small amounts of potassium, sodium, and calcium nitrates caused an increase in the number of Azotobacter in pure culture compared with control cultures containing no nitrate. An increasing concentration of the nitrates continued favorable to the growth of the organism up to a certain limit, but higher concentrations retarded its growth. Finally a nitrate concentration was attained at which Azotobacter growth altogether ceased. Jan. 28, igis NUrogen-Assimilating Bactcria 193 The results of the study of nitrates and their influence on Azotobacter in sterilized soil show very clearly that small amounts of nitrate cause a great increase in the number of Azotobacter cells. Higher concentra- tions are not so favorable to the growth of the organisms, and the highest concentrations studied prevented the development of the Azotobacter in sterilized soil. From a study of the results of these experiments, it seems that the increase in number of Azotobacter in the presence of small amounts of nitrate is a direct result of nuclear stimulation. Later studies to be cited {pp. 205-208) show that nitrates exerted considerable influence on the internal structure of the Azotobacter cell. It appears reasonable to expect that the nitrate affected the nuclear structure in such a manner that an increase in cell multiplication resulted. It seems probable that the action of nitrate as a simple nutrient would be shown by a slower increase in cell multiplication. INFLUENCE OF NITRATES ON THE FIXATION OF NITROGEN BY AZOTOBACTER It has been shown in the preceding paragraphs that the presence of small quantities of nitrate in sterilized soil bring about a large increase in the number of Azotobacter. This increase was noted in the case of both strains of Azotobacter. It would be of interest to know whether the increase in bacterial numbers was accompanied by a corresponding increase in the amount of nitrogen assimilated. The results secured by a few investigators indicate that in the presence of combined nitrogen as nitrates the nonsymbiotic nitrogen-fixing organisms will not fix atmospheric nitrogen. Stoklasa (44, p. 492-503) studied the influence of Azotobacter on sodium nitrate in aerobic and anaerobic liquid cultures. He found only a small gain in organic nitro- gen and from these results he concluded that in the presence of nitrates Azotobacter could not assimilate atmospheric nitrogen. It has been shown by Hanzawa {20) that in a liquid culture containing 12 mgm. of nitrate (from potassium nitrate) in 100 c. c. of medium, a mixed culture of Azotobacter fixed 5.25 mgm. of nitrogen. Under the same conditions with 60 mgm. of nitrate present in 100 c. c. of medium he found but 5.35 mgm. of nitrogen fixed. He concluded that nitrates remained, as far as small quantities were concerned, almost without influence on the amount of atmospheric nitrogen fixed by Azotobacter. Some studies have been carried on with respect to the influence of ni- trates on the nonsymbiotic anaerobic nitrogen-assimilating organism, Clostridium spp. Bredemann (9) showed that ammonium nitrate in solution caused a decrease in the amount of nitrogen fixed by species of Clostridium. Pringsheim (40) grew cultures of C. americanum in solutions containing potassium nitrate. He found that in the presence of available energy the organism fixed some nitrogen when nitrate was 194 Journal of Agricultural Research voi. xii, No. 4 present but to a less extent than did control cultures containing no nitrate. From these results it appears that nitrates do not stimulate the nitro- gen-assimilation of the nonsymbiotic nitrogen-fixing bacteria. Inasmuch as nitrates in small amounts caused such an increase in the number of Azotobacter in sterilized soil, it was thought advisable to determine just what influence these salts exert on nitrogen fixation by Azotobacter. Accordingly, experiments were carried out with Azotobacter on agar films, in soil cultures and in solution. Agar-film cultures. — In this work both strains of the Azotobacter were used. One hundred c. c. of mannit agar were placed in liter Erlenmeyer flasks and nitrates of potassium, sodium and calcium added in varying quantities. The flasks and contents were sterilized at 10 pounds' pressure for 25 minutes, cooled, and inoculated with 10 c. c. of a suspension of the organism in sterile distilled water. The flasks were incubated at 28° C. for three weeks. The weight of both inoculated and uninoculated flasks was maintained throughout the experiment by the addition of sterile distilled water. At the end of the incubation period total nitrogen analyses were made. Because of the high nitrate content dilute sulphuric-salycilic acid was added slowly and carefully to prevent loss of nitrogen by the evolution of gaseous oxids of nitrogen. The acid was allowed to react for a few days before continuing the total nitrogen determination. The results of the experi- ments are presented in Tables VIII and IX. Table VIII. — Influence of nitrates on the fixation of nitrogen by Azotobacter {strain A) on agar films Cul- ture No. Treatment (nitrate in looc. c. of medium). o 50 mgm. of NO3 potassium nitrate . . . . .do 100 mgm. of NO3 potassium nitrate . . . .do 50 mgm. of NO3 sodium nitrate. . . . . . .do 100 mgm. of NO3 sodium nitrate. 50 mgm. of NO3 calcium nitrate. . ....do 100 mgm. of NO3 calcittm nitrate . ....do Nitrogen contained in loo c. c. of medium. Inoculated. Found. Average, Mgm. 13.00 12. 70 12. 60 18.50 18. 40 27. 60 27-75 18.65 18.30 27. 00 27.65 13-75 13. 70 18.80 19-15 Mgm. 12.80 18.45 70 Uninoculated. Found. Average. Mq'^). 4- •■> 4. y 4. I 7. oy 7. 20 16.80 15-70 7- 50 7-30 15. 00 i_5. 20 8.00 8. 50 14. 50 14.30 Mgm.. 4-05 I 7- 10 } 16. 25 } 8-25 \ 14-40 Nitrogen fixed. Mgm. 8.75 "•35 11-45 II. 10 12.25 5-50 4-55 Jan. 28, 1918 Nitrogen- A ssimilating Bacteria 195 Table IX. — Influence of nitrates on the fixation of nitrogen by Azoiobacter {strain B) on agar films Cul- ture No. 3 4 5 6 7 8 9 10 II 12 13 14 IS Treatment (nitrate in 100 c.c. of medium). o o o 75 mgm, of NO3 as potassium ni- trate ...do 150 mgm. of NO3 as potassium ni- trate ....do 75 mgm. of NO3 as sodium nitrate . . . .do 1 50 mgm, of NO3 as sodium nitrate ....do 75 mgm. of NO3 as calcium nitrate ....do 1 50 mgm . of NO3 as calcium nitrate ....do Nitrogen contained in 100 c. c. of medium. Inoculated. Found. Average. Mgm. Mgm 15-50 15-70 15.60 25. 20 25.40 36.40 36.90 25. 60 25.70 37- 60 37- 20 20. 10 19. 60 32.80 33-30 15.60 25-30 } 36 } 37 \ 33 Uninoculated. Found. I Average. Mgm. 6. 50 6.30 6. 40 \ 14 {; 14-70 24. 00 23.20 12.80 13.20 26. 20 25.40 12. 00 2. 70 24-50 2^. 20 MgTti. 6. 40 Nitrogen fixed. Mgm: 9. 20 11-45 13-05 12.65 II. 60 7-50 8. 20 A glance at the results (Tables VIII and IX) shows that an increase in nitrogen fixation occurred where potassium and sodium nitrates were present, whereas a marked decrease in the total nitrogen content was observ'ed where calcium nitrate was used. Whether the calcium itself is detrimental to an increase in organic nitrogen or whether it is the com- bination of calcium with nitrate can not be stated. It is significant, however, that this decrease in fixation of nitrogen was noted throughout all the experiments where calcium nitrate was employed. It is very evident that calcium nitrate exerts some detrimental effect on the nitro- gen assimilating properties of the organism. There seems to be but a slight difference in the nitrogen-fixing ability of the two strains studied. In the absence of nitrates the amount fixed varies but little. Also in the presence of potassium and sodium nitrates the relative increase in amount of nitrogen fixed remains about the same. Calcium nitrate offers an exception where it is employed. The detrimental efifect seems to be more marked in the case of strain A than with strain B. Strain A under normal conditions fixed slightly less nitro- gen than strain B, so it may be possible that this strain is weaker. The formation of pigment by the Azotobacter in the presence of the nitrates is of interest. Strain A normally produced no pigment by the end of three weeks' incubation. But when grown on the agar films in the presence of nitrate a most marked pigment production appeared. This pigment was especially noticeable in the presence of the calcium 196 Journal of Agricultural Research voi. xii, No. 4 salt. Since strain B normally produces a good pigment, the influence of nitrate on this strain was not very marked. The relation of nitrates to pigment formation will be taken up later (pp. 203-205). From the results of the experiments with agar films containing various amounts of nitrate, it seems apparent that potassium and sodium nitrates in amounts of 50 and 100 mgm. of nitrate in 100 c. c. of medium cause a small increase in the amount of nitrogen fixed. However, this increase in fixation is not at all parallel with the increase in number of Azoto- bacter caused by nitrates in sterilized soil. It may be concluded that an increase in the number of Azotobacter in sterilized soil as a result of nitrate stimulation does not mean a corre- sponding increase in nitrogen fixation on agar films. Soiiv CULTURES. — The conditions obtaining in these experiments were strictly comparable with those heretofore cited dealing with the influence of nitrates on Azotobacter in sterilized soil (pp. 187-193). The fixation of nitrogen was studied in pure culture in sterilized soil and in unsterilized soil. One hundred and fifty gm. of soil (dry weight) were weighed into i -liter Erlenmeyer flasks, nitrates were added in vary- ing amounts from 10 to 200 mgm., and i per cent of mannit was also added. Triplicate flasks were prepared for each amount of nitrate studied. The moisture content was raised to approximately 18 per cent and the flasks allowed to remain at room temperature for one day. The con- tents were then thoroughly mixed and a fine crumb structure produced. The flask-s for the experiments with pure cultures in sterilized soil were immediately steriHzed at 15 pounds' pressure for three hours. After cooling, two of each set were inoculated with 5 c. c. of a suspension of Azotobacter (strain A) in sterile distilled water. The remaining flask of each set was not inoculated, but was incubated at 28° C. with the inocu- lated flasks. The moisture lost by evaporation was replaced from time to time by the addition of sterile distilled water. At the end of the incu- bation period the soil was removed and spread out in thin layers and allowed to dry. It was then thoroughly ground in a porcelain-ball mill for one hour. At the end of this time all of the soil passed through a loo-mesh sieve. Soil cultures used in the study of the effect of nitrates on nitrogen fixation in unsterilized soil were prepared in a similar manner, except that the flasks were not sterilized. Previous to incubation a small inoculum of Azotobacter (strain A) was added to insure the presence of the nitrogen-fixing organism in the soil cultures. The proper moisture content was maintained in the same manner as in the case of the pure cultures in sterilized soil and the incubation period was the same for both. The results are given in Tables X, XI, XII, and XIII. Jan. 28, 1918 Nitrogen- Assimilating Bacteria 197 Table X. -Influence of sodium nitrate on the fixation of nitrogen by Azotobacter in sterilized soil Treatment (nitrare in 100 gm. of dry soil). Total tiitrogen in 100 gm. of dry soil. Nitrogen Culture No. Inoculated. Uninoculated. fixed in 100 gm. of Found. Average. Found. Average. dry soil. I Mgm. 10 10 10 10 10 10 50 50 50 50 50 50 150 150 150 150 150 150 Mgm. 135-0 134. 132. 133-0 137-0 Mgm. ■ 133- 7 ■ 135-0 136.6 137. 149- 149. 2 162. 3 162. 5 Mgm. 132. 131- 5 131. Mgm. \ 131- 5 Mgm. I 2-7 I 2 2 3-5 2 ■J 137-0 136.0 137-0 136.5 137.5 137.0 149.0 149.0 149.0 148. 5 149-5 149-5 163.0 162. 162. 162. 5 163.0 162. 134. 133-5 134- ^33- 7 ■i 2.9 •J 4 4 [ 3-3 4 C 140. 137-0 138- 5 138.5 C 10.5 C 6 6 i [ 10.7 6 7 7 152. 150.0 152. 5 [ 151-5 10. 8 7 . , 8 1 8 > II. 8 J Table XI. — Influence of sodium nitrate on the fixation of nitrogen by Azotobacter in unsterilized soil Treat- ment (ni- trate in 100 gm. ol dry soil). Total nitrogen in 100 gm. of dry soil. Nitrogen Culture No. Inoculated. Uninoculated. fixed in 100 gm. of Found. Average. Found. Average. dry soil. I Mgm. 10 10 10 10 10 10 50 50 50 50 50 50 150 150 150 150 150 150 Mgm. 132.0 135-0 135-0 132.0 134.0 134.0 137-5 138.8 138.8 137-5 137-5 138.0 150. 151. 150. 149.0 149-5 150. 5 169. 167. 168.0 167.5 168.0 168. s Mgm. [ 134- [ ^33- 3 137- 8 1 137- 7 [ 150- 3 [ 149- 7 [ 168. i 168. Mgm. f 130. \ ^33- 5 I 132- Mgm. 131. 8 Mgm. I 2 2 I 2 2 I- 5 2 . . . •2 f 134- 1 ^33- I 133- 1 ^33- 3 •3 4-5 ■3 A 1 A 4-4 4 c [ 140. \ 140. 5 t 142. \ 140. 8 c 9-5 c 6 1 6 8.9 6 7 [ 148. { 154- I 153- 5 151. 8 7 16. 2 7 8 1 8 \ 16. 2 8 J 198 Journal of Agricultural Research Vol. XII, No. 4 Tabids XII. — Influence of calcium nitrate on the fixation of nitrogen by Azotobacter in sterilized soil Treat- ment (ni- trate in 100 gm. of dry soil). Total nitrogen in 100 gm. of dry soil. Nitrogen Culture No. Inoculated. Uninoculated. fixed in 100 gm. of Found. Average. Found. Average. dry soil. Mgm. 10 10 10 10 10 10 50 50 SO SO SO 50 200 200 200 200 200 200 Mgm.. 133-0 ^33- (> 133-3 133-0 134.2 133-4 137.0 137.0 136.5 136.5 137-0 137-5 148. 148.5 149.0 148.5 149-0 148. 173-0 173.0 174.0 173-5 173-0 174.0 Mgm. \ ^33- 3 [ ^33- 5 136. 8 I 137- [ 148. 5 148. 5 1 173- 7 173- 5 Mgm. \ 131- 1 131- [ 132- Mgm. \ 131- 3 Mgm. 2. ] \ 2. 2 J [ 135- 1 134- 135- [ 134- 7 2. I 1 2.3 J e [ 140. 5 141. I 140. 5 i 140. 7 e 7.8 c 6 1 6 7-8 6 ] ' 7 f 163. 164. I 164- 5 j 163. 8 7 9. 9 7 8 1 8 . .... 1 .. 8 Table XIIL — Influence of calcium nitrate on the fixation of nitrogen by Azotobacter in unsterilized soil Treat- Total nitrogen in 100 gm. of dry soil. Nitrogen Culture No. ment (ni- trate in 100 gm. of dry soil). Inoculated. Uninoculated. fixed in 100 gm. of Found. Average. Found. Average. dry soil. I Mgm.. 10 10 10 10 10 10 SO 50 50 so 50 50 200 200 200 200 200 200 Mgm. 134-5 136. 136.5 135.0 135.5 135-5 138-5 138.0 139.0 138.0 137-5 138.5 151- 5 152.0 151. 150.0 151-5 151.0 177.0 178.0 176. 176.5 177.0 178.0 Mgm. 135- 7 [ 135- 3 138. 5 138. 151-5 i 150. 8 [ 177-0 [ 177- 2 Mgm. 1 134. 1 ^33- 5 [ 132. f Mgm,. \ ^33- 2 Mgm. I 2- 5 1 2 2 I ^' '■ 2 [ ■^ [ 133- 5 133- [ ^33- [ ^33- 2 3 5. 3 2 4 1 4 1 \ 4-8 A J 5 [ 140. 5 141. I 141. 5 f \ 141.0 c 10.5 c 6 6 9-8 6 7 f 164. 165. [ 164. f \ 164- 3 7 12. 7 7 8 1 8 1 12.9 8 |.. ....... Jan. 28. 1918 Nitrogen-Assimilatifig Boctena 199 It will be seen at a glance that a greater relative increase in nitrogen fixation in the presence of nitrates occurred in the soil cultures than on the agar films. But in the latter instance the amount of nitrogen as- similated in the absence of mistakes is far in excess of that assimilated in the soil cultures under similar conditions. The amount of nitrogen fixed in the soil cultures is surprisingly low, but as relative increases or decreases are desired this does not materially influence the results. The influence of sodium nitrate on the fixation of nitrogen by pure cultures of Azotobacter in sterilized and unsterilized soil is brought out very clearly in the figures of Tables X and XI. In both cases, where no nitrate was added, an equal fixation of nitrogen occurred. Where 10 mgm. of nitrate were added to 100 gm. of soil, slightly more nitrogen was assimilated in the unsterilized soil than in st'Crilized. The reverse seemed to be true when 50 mgm. of nitrate were added. But in the presence of 150 mgm. of nitrate, the fixation by the pure culture in sterilized soil did not increase materially in comparison with that which occurred in the 50 mgm. of nitrate concentration. Evidently the maximum fixation under these conditions had been reached. The gain in the unsterilized soil at the highest concentration of nitrate studied almost doubled the amount fixed in the pure culture. It appears evident that the presence of sodium nitrate causes a greater fixation of nitrogen in unsterilized soil than it does under similar conditions in sterilized soil inoculated with Azotobacter. In the case of calcium nitrate, somewhat comparable results were ob- tained. The fixation where no nitrate was added was equivalent to that obtained in the controls for the sodium nitrate. Where nitrate was added in amounts equal to 10 mgm. of nitrate in 100 gm. of soil, an increased fixation w^as obtained in the unsterilized soil, but practically no increase occured in the pure culture in sterilized soil. Fifty mgm. of nitrate in 100 gm. of soil produced an increase in fixation. In the highest concentration of calcium nitrate the difference in nitrogen fixed between the pure culture in sterilized soil and unsteriUzed soil was not so great as in the case where sodium nitrate was used. In the sterilized soil where the two nitrates were present in equal amounts it can be seen that more fixation took place in the presence of sodium nitrate. The difference is not marked, but it exists neverthe- less. It will be remembered that calcium nitrate had a detrimental effect on nitrogen fixation by Azotobacter on agar films. However, in soil cultures this same nitrate stimulated Azotobacter to an increased assimilation of nitrogen. This difference is not suprising as it has been shown repeatedly that bacterial activities in soil and in artificial cultures are not always comparable. From the results of the experiments performed with reference to the influence of nitrates in soil on the fixation of nitrogen therein, it appears 200 Journal of Agricultural Research voi. xii. no. 4 evident that in pure cultures both sodium and calcium nitrates in the amounts studied produced an increase in the amount of nitrogen fixed. The sodium salt stimulated this process to a slightly greater extent than did the calcium salt. In unsterilized soil nitrates exerted the same action but to a more marked extent. The amount of nitrogen fixed under these conditions was generally in excess of that fixed under similar conditions in sterilized soil inoculated with a pure culture of Azotobacter. Such large relative increases in total nitrogen in the soil in the presence of nitrates would not normally take place under field conditions for here no accumulations of nitrate occur in quantities sufficiently large enough to influence this process. Summing up all the experiments performed in relation to the influence of nitrates on the fixation of atmospheric nitrogen by Azotobacter, it appears that the increase in total nitrogen in the presence of these salts is by no means comparable to the increase in the number of organisms in sterilized soil under the same conditions. An increase in the number of Azotobacter does not mean a parallel increase in the amount of nitrogen fixed. INFLUENCE OF AZOTOBACTER ON NITRATES IN SOLUTION Attention has been thus far directed toward the influence exerted by nitrates on the growth and nitrogen-assimilating power of Azotobacter. The following points are now to be considered: Do the nitrogen-fixing bacteria reduce nitrates to nitrites and ammonia? Is there an increase or decrease in the amount of organic nitrogen as a result of the presence of nitrate in the medium? Beijerinck and Van Delden (5) found that Azotobacter chroococcum reduced nitrate directly to ammonia. Stoklasa (44, p. 492-503) studied the changes in a nutrient solution containing 0.2 per cent of sodium nitrate inoculated with Azotobacter. He found under an- aerobic conditions that the nitrate was largely reduced to nitrite and ammonia and that a very small amount of organic nitrogen was formed. Under aerobic conditions there was more nitrite formed than under anaerobic conditions and very little ammonia or oganic nitrogen. He concluded, therefore, that Azotobacter did not fix atmospheric nitrogen in the presence of nitrates. The following experiments were performed in an endeavor to answer the questions raised in the initial paragraph of this section. To Erlen- meyer flasks of 500-c. c. capacity, containing loo-c. c. portions of mannit solution, sodium and ammonium nitrates were added in amounts equiva- lent to 150 mgm. of nitrate in loo c. c. of the solution. Nine flasks were prepared for each nitrate and the same number for the controls containing no nitrate. The flasks and contents were sterilized at 10 pounds pres- sure for 30 minutes. After cooling, six of each set were inoculated, three Jan. 28, 1918 Nitrogen- A ssimilating Bacteria 201 with strain A and three with strain B, and the remaining three were left uninoculated to serve as controls. The flasks were incubated at 28° C, for 21 days. The total weight was maintained throughout the experi- ment by the addition of sterile distilled water from time to time. At the end of three weeks the contents of each set of triplicate flasks were poured together and 50-c. c. samples drawn for analysis. Nitrate ammonia and total nitrogen were determined as given under "Methods." The results are shown in Tables XIV, XV, and XVI. Table XIV. — Influence of Azotobacter on nitrates in solution, giving the quantity of nitrate lost Treatment (ni- trate in 100 c. c. of me- dium). Nitrate in loo c. c. of medium. Cul- Strain A. strain B. ture No. Inoculated. Uninoculated. Nitrate lost. Inoculated. Uninoculated. Found. Aver- age. Found. Aver- age. Found. Aver- age. Found. Aver- age. lost. Mgm. 0.00 .00 80.9 80.6 100.3 102. 1 Mgm. 1 0.00 jso. 75 >I0I.2 Mgm. ( 0.00 \ .00 /iSO- 4 li5i-3 /i49- 6 \i50. Mgm. > 0. 00 }i50.8 }i49-8 Mgm. 0.00 0— 70.0s a— 48. 60 Mgm,. f 0.00 \ 00 /105. 6 1105. 2 /l3l- I \i30. 7 Mgm.. 1 0.00 }ios-4 1 130.9 Mgm. f o-oo \ .00 /iSO- 4 \iSi-3 /149- 6 Mgm. > 0. 00 |iS0.8 }i49.8 Mgm. 0.00 lo-iS isogm.of NO3 as sodium ni- trate . . do ..... 6—45-40 19-27 19-27 isomgm.of NO3 as ammonium nitrate do C-18.90 o Strong NO2 r,eaction. b Medium NO2 reaction. c Slight NO2 reaction. Table XV. — Influence of Azotobacter on nitrates in solution, giving the quantity of ammonia produced Treatment (ni- trate in 100 c. G. of me- dium). Nitrogen as ammonia in 100 c. c. of medium. Cul- Strain A. Strain B. ture No. Inoculated. Uninoculated. Am- monia pro- duced. Inoculated. Uninoculated. Am- Found. Aver- age. Foimd. Aver- age. Found. Aver- age. Found. Aver- age. pro- duced. 1-9 1-9 10-18 Mgm. 0.20 . 10 2.00 1.80 13-90 13- 9S Mgm. I 0.15 I I- 90 }i3-97 Mgm.. ( 0.00 \ .00 f-.IO \ . 20 / 13-90 I 13-90 Mgm. >• 0. 00 }i3-90 Algm. 0.15 1.85 .•07 Mgm. ( o^oo \ .20 f 2.20 I 2-40 / 13-80 1 13-80 Mgm. \ 0. 10 } s-30 }i3-8o Mgm. f 0.00 \ .00 f-.io \ .20 / 13-90 I 13-90 Mgm. } 0. 00 }i3-90 Mgm. isomgm.of NO3 as sodium ni- trate do 2- 25 19-27 19-27 isomgm.of NO3 as ammonium nitrate do . 10 202 Journal of Agricultural Research Vol. XII, No. 4 TablB XVI. — Influence of Azotobacter on nitrates in solution, giving the quantity of nitrogen fixed Treatment (ni- trate in 100 c. c. of me- dium). Total nitrogen in 100 c. c. of medium. Cul- Strain A. Strain B. ture No. Inoculated. Uninoculated. Nitrogen fixed. Inoculated. Uninoculated - Nitro- Found. Aver- age. Found. Aver- age. Found. Aver- age. Foimd. Aver- age. gen fixed. 1-9 1-9 lO-lS Mgm. 5.00 5.00 22. 50 22.60 47.00 46.90 Mgm- I S-oo I22.5S }46. 95 Mgm. f 2.60 I 2- 70 / 14.00 \ 14.20 / 43' 20 I 42.90 Mgm. }2.6s }i4- 10 |43-os Mgm. 2-35 8-45 3- 90 Mgm. f S-oo I 5- 10 / 28.00 \ 27- So f 48. TO \ 48. 20 Mgm.. I S-os J27.90 }48. IS Mgm. f 2.60 I 2.70 f 14.00 \ 14.20 / 43-20 I 42.90 Mgm.. } 2-6s |i4. 10 }43-0S Mgm. 2.40 iSomgm.ofNOs as sodium ni- trate do 13- 80 19-27 19-27 isomgm.of NO3 as ammonium nitrate do S-io Table XIV showing the effect on the^ total nitrate content will be discussed first. Strain A differed widely from strain B in its ability to reduce nitrates. It will be noted that strain A reduces nitrate more readily than strain B in the presence of both sodium and ammonium nitrate. In order to determine the nature of the reduction of the nitrates, qualitative and quantitative tests were made. The reduction of nitrates by Azotobacter takes place with the formation of nitrites as shown in Table XIV. Strain A effected a strong reduction of nitrate to nitrite with both sodium and ammonium nitrate. Strain B also reduced nitrate to nitrite, but to a lesser degree than did strain A. An inspection of the data in Table XV indicates that the reduction of nitrates ceased with the formation of nitrite, since no appreciable amounts of ammonia were produced by either strain of Azotobacter. In regard to the fixation of atmospheric nitrogen by these strains of Azotobacter, it was found that nitrogen was assimilated both in the presence and absence of nitrate. In the presence of nitrate there was a large increase in the total organic nitrogen. Sodium nitrate stimulated both strains, although strain B fixed the larger amount. Similar results were obtained when the fixation of nitrogen on agar films was studied. In the presence of ammonium nitrate the amount of nitrogen fixed was considerably decreased, but the amount fixed was in excess of the control cultures containing no nitrate. It seems evident that sodium and ammonium nitrate in the amounts studied did not prevent the fixation of atmospheric nitrogen. In fact, the presence of these salts seemed to stimulate the process. Under aerobic conditions both strains of Azotobacter studied caused a reduction in the total amount of nitrate present in the solution. This reduction may be accounted for in two ways: (i) The reduction of nitrate to nitrite and (2) the assimilation of nitrate by the organisms. Practically no ammonia was formed under the conditions of these experi- ments. These results agree with those of Stoklasa. However, in con- Jan. 28, i9i8 Nitrogen-Assimiloting Bacteria 203 trast to the work of Stoklasa, both strains of Azotobacter assimilated more atmospheric nitrogen in the presence of nitrates in solution than in the absence of these salts. INIfLUENCE OP NITRATES ON THE PRODUCTION OP PIGMENT BY AZOTOBACTER It has already been noted in the experiments dealing with the effect of nitrates on the fixation of atmospheric nitrogen on agar films that nitrates favor pigment production. This was true in the case of both strains of the Azotobacter. Moreover, it has been observed by other investigators that Azotobacter when grown in the presence of nitrate will produce a darker pigment than when grown in its absence. Beijerinck (4, p. 575) states that Azotobacter in pure culture will form a dark-bro\vn pigment in the presence of glucose and a small amount of nitrate. Sackett {43) found that nitrate caused an increase in pigment production by Azotobacter. In media without the nitrate the pigment formation was materially decreased and in some cases practically eliminated. He also noted that the amount of nitrate present has a direct influence on the intensity of the pigment formation. He found that when sodium nitrate was added to a suitable medium to give a content of 0.0, o.oi, 0.03, 0.05,0.08, o.i, 0.3, and 0.5 per cent, with glucose used as the source of energy, the organisms produced pigment. Streak inoculations were made, and after 14 days' incubation he found that the maximum of color was obtained at 0.05 to 0.08 per cent and that greater concentrations did not increase the intensity of the brown-black pigment. From his results it is evident that sodium nitrate caused an increase in pigment formation by azotobacter. In order to determine the possible effect of potassium, sodium, and cal- cium nitrate on pigment formation with strains A and B, the following experiment was performed. Under normal conditions on mannit agar free from nitrate strain A produced little or no pigment even after three weeks' growth. At the end of this time dirty-yellow streaks occurred throughout the growth, but no brown pigment was produced. However, with strain B at the end of two or three weeks a decided brown to brown-black pigment was produced in the absence of nitrate. Agar slope cultures containing increasing amounts of potassium, so- dium, and calcium nitrate, as indicated in Table XVII, were prepared. These were inoculated with both strains of Azotobacter and incubated at 28° C. for 10 days. Daily observ^ations were made for first evidences of pigment formation. In some of the cultures of strain A growing on media containing calcium nitrate this pigmentation was observed as early as 48 hours subsequent to inoculation. The following day pig- mentation developed in strain B. The cultures on the potassium and sodium-nitrate media began to show evidence of pigmentation in four to six days. The final results, obtained after 10 days' incubation, are found in Table XVII. 204 Journal of Agricultural Research Vol. XII, No. 4 <; ^ 8 a o 55^ ;-« SPS Qpq « a „ boooooo." tS o •=i S' o OtQ o O lU _ ^ o o 13 -i-"d .S 'O "d t3 ^ p bo'- .^^ _ ^ _ o o -*-" "d .^i Td "d "O .OOioOOOOO a Hcsvoomoo C H H N ro H N fO 'J' >oo r— 00 Jan. 28. i9i8 Nitrogefi-Assiniilating BocteHa 205 A general idea may be gained from Table XVII concerning the relative increase in pigment formation in the presence of the nitrates. A study of the table gives a fair idea of the relative differences in pigment pro- duction. Very interesting results were obtained with strain A. It will be seen from Table XVII that no pigment was produced in the control culture after 10 days, while in the presence of nitrates pigmentation was noted. The intensity of the pigment varied with the increase of nitrate up to 150 mgm. Beyond 150 mgm. there was no increase. Potassium and sodium nitrate did not exert such a decided influence on pigment production as calcium nitrate. The latter salt produced an intense dark-brown to brownish-black pigment. In the case of strain B the influence of nitrate was not so pronounced since this strain normally produced considerable pigment in the absence of nitrates. Potassium and sodium nitrate caused a slight increase in pigment formation. Here, again, the calcium salt brought about most pronounced increase. However, the relative increase in pigment forma- tion in strain B was not so pronounced as in strain A. Where the nitrate was present, a much more spreading growth was obtained. A heavy bacterial growth accumulated at the base of the slope except in the two cultures in which the highest concentrations were used. In the latter instances the accumulation was less than those in cultures growing on media containing no nitrate. Although the original inocula- tion could not be made absolutely uniform, so far as number of organ- isms was concerned; nevertheless it was evident that on those slopes containing 10, 25, 50, and 100 mgm. of nitrate in 100 c. c. of the medium a much more abundant growth was obtained than on those slopes free from nitrate. Here, again, it is seen, in a rough, comparative way, that the smaller amounts of nitrates caused an increase in the number of Azotobacter. The results of this work on pigment production are quite in accord with those of Sackett. Potassium, sodium, and especially calcium, nitrates in varying amounts increase pigment formation by Azotobacter with an increase in nitrate concentration. This effect is especially marked in strain A, which under normal conditions does not produce any pigment. INFLUENCE OK NITRATES ON THE FORMATION OF VOLUTIN BODIES IN AZOTOBACTER The presence of volutin bodies, or metachromatic granules, in Azoto- bacter has been shown by Bonazzi (7). These substances, according to Meyer (54, p. 238), are reserve food materials other than fat droplets, glycogen, and similar substances reacting with iodin stain which occur in the cytoplasm of the cells of various bacteria. With Millon's reagent they give no reaction. He believes that these bodies are composed of nucleic-acid compounds, but are not nuclear proteids. 2o6 Journal of Agricultural Research Vol. XII, No. 4 In connection with the foregoing investigations concerning the influ- ence of nitrates on pigment formation by Azotobacter, it was thought that some results of cytological interest might be obtained in regard to the effect of varying amounts of nitrates on the volutin bodies. Slope cultures of mannit agar were prepared containing the different nitrates as indicated in Table XVIII. These slopes were inoculated with both strains of Azotobacter and incubated at 28° C. for 10 days. At the end of this time each culture was stained and examined micro- scopically. The following method was used for demonstrating the presence of the volutin bodies. The organisms to be examined were air dried on a glass slide and then fixed in the flame of a Bunsen burner. The preparation was then flooded with a i to 10 aqueous solution of methylene blue (Merck's) prepared by adding 10 c. c. of a saturated aqueous solution of methylene blue to 90 c. c. of distilled water. The stain was washed off after five minutes with a i per cent solution of sulphuric acid and immediately rinsed in distilled water. The prepa- ration was dried and examined with the oil-immersion objective. The volutin bodies appeared within the cytoplasm as very dark blue dots, the outline of the cell wall was a lighter blue, while the cell net^work was stained a very light blue. Guignard's stain ^ was also used to demonstrate the presence of the volutin bodies. Fresh smears on a glass slide were fixed over 10 per cent osmic acid for three minutes. The preparation was then air-dried and fixed to the slide by rapidly passing the latter a few times through a Bunsen burner. The preparation was covered with the stain which was allowed to react for five minutes. The stain was then washed off with distilled water, dried, and examined with the oil-immersion objec- tive. The outline of the cell as well as the net work within-was stained light purple. The granules within the cytoplasm were a reddish purple. The results are given in Table XVIII. Tabi^e XVIII.- -Influence of nitrates on the formation of volutin bodies in Azotobacter in 10 days Treat- ment (nitrate in 100 c. c. of me- dium). Strain A. Strain B. Culture No. Potassium nitrate. Sodium nitrate. Calcium nitrate. Potassium nitrate. Sodium nitrate. Calcium nitrate. Mgm. 10 25 so 100 ISO 200 300 Present.o. . . . do.o do.o do.o do.o do.o Doubtful.... Present do.o do.ft do.ft ... do ft Doubtful. . . . Present do Doubtful.... Present o do.ft do.o do.ft Present. Do. 2 do.o Doubtful. . . . do.o do." do ft ... Do.o Do s 6 do do ft do ft Do ft 7 do. 6 do.ft . . do ft .. do.ft .... do.ft ... Do 8 Ao.b .... do.6 . do doi>. ... .. do ft Do ft o Representing an approximate average of two volutin bodies per cell. b Representing an approximate average of four volutin bodies per cell. ' Guignard'sstain. Fifty c. c. of 2 per cent fuchsin in i per cent acetic acid; 40 c. c. of 0.2 per cent methyl green in i per cent acetic acid; i c. c. of glacial-acetic acid. Distilled water was used in making the i per cent acetic-acid solution. Jan. 28, 1918 NitYogen-A ssimilating Bacteria 207 It will be seen that all three nitrates exerted considerable influence on the formation of volutin bodies. Not only was the number of bodies increased, but also the size. The relative increase in size of the granules was much more marked than was the numerical increase. In Azoto- bacter cells grown on mannit agar containing no nitrate the number of volutin bodies in each cell averaged about two ; in the presence of nitrate four to five volutin granules were found. The greatest increase in num- ber, as well as size, occurred where the nitrate concentration was highest. With both strains sodium nitrate apparently caused the greatest increase. This was true in the lower as well as in the higher concentrations. The volutin bodies in strain B seemed to respond to the presence of nitrates more noticeably than did those of strain A, especially in the presence of potassium nitrate. It is evident that nitrates of potassium, sodium, and calcium cause an increase in the number and size of volutin bodies in Azotobacter cells. Do these salts tend to hasten the appearance of these bodies, or do they at first retard their development? The following experiment was carried out in an endeavor to determine this point. Only sodium nitrate was used, since this particular salt proved most beneficial to the forma- tion of volutin bodies. Agar slopes were prepared containing the different amounts of nitrate as indicated in Table XIX. The cultures were incu- bated at 28° C. and examined daily for the presence of volutin bodies. The methylene blue — i per cent sulphuric acid — method of staining was employed. The iresults of the experiment are given in Table XIX. Table XIX. — Influence of sodium nitrate on the rate of formation of volutin bodies. in Azotobacter Time. Nitrate in 100 c. c. of medium. Strain A. Strain B. oMgm. 25 Mgm: 100 Mgm. 300 Mgm. Mgm. 25 Mgm. 100 Mgm. 300 Mgm. Day. Absent... Present". ...do.a ...do." Absent.. . Present «. ...do.a.... . . .do.o. . . . Doubtful. Present a. ...do.".... ...do. 6... Doubtful. ...do.6... ...do.6... ...do.b... Absent... ...do Present". . . .do.o Absent... Present". ...do.b... ...do.b... Doubtful. ...do.ft... ...do.b... ...do.b... Dbtful. Do.b. 3-- 4.. Dob. Do.b. o Representing an approximate average of two volutin bodies per cell. b Representing an approximate average of four volutin bodies per cell. A study of Table XIX shows that it is rather doubtful whether the nitrate present tended to hasten the appearance of the volutin bodies. No convincing evidence has been presented for or against this statement. No granules were seen^in the first day's growth of strain A, although the next day they were present in all four cultures. In strain B more convincing proof is furnished that the sodium nitrate hastened the appearance of these reserve food substances. The volutin bodies were not present in the control and lowest nitrate concentration cultures the first day, but they were very noticeable in the culture containing the highest concentration of nitrate and doubtful in the remaining one. On the second day volutin bodies were present in all cultures grown on 208 Journal of Agricultural Research Vol. XII, No. 4 nitrate media, v/hile the control culture was still free from them. The third day showed the presence of volutin bodies in all four cultures. Strain B offers the better proof that sodium nitrate tends to hasten the appearance of volutin bodies in the cells of Azotobacter. Further experiments were not made in an endeavor to determine what influence nitrates might have on the cytology of the Azotobacter cell. The brief studies reported here were made in connection with the pigment forma- tion experiments, but do not bear any particular relation to them. The increase in number and size of volutin bodies may bear some relation to the increased amount of nitrogen fixed or assimilated by Azotobacter in the presence of nitrates. INFLUENCE OF NITRATES ON BACILLUS RADICICOLA INFLUENCE OP NITRATES ON THE GROWTH AND REPRODUCTION OP BACILLUS RADICICOLA IN STERILIZED SOIL One hundred and fifty gm. (dry weight) of the soil were weighed into 500-c. c. Hrlenmeyer flasks and the nitrates added as indicated in Tables XX-XXII. Duplicate cultures for each amount of nitrate were prepared. One per cent of mannit (in 5 c. c. of distilled water) was also added. The flasks were kept at room temperature for one day and the contents then thoroughly mixed. The flasks were steri- lized at 1 5 pounds' pressure for three hours. Upon cooling they were inoculated with 5 c. c. of a suspension of Bacillus radicicola in sterile distilled water. The number of bacteria in the inoculum was deter- mined. The moisture content was then approximately 18 to 20 per cent. The flasks were incubated at 28° to 30° C. and mannit-agar plates poured at the end of one and two weeks. The results of these experiments are given in Tables XX, XXI, and XXII, in which each figure represents an average of duplicate plates. Table XX. — Influence of potassium nitrate on Bacillus radicicola in sterilized soil Culture No. Treat- inent (ni- trate in 100 gm. of dry soil). Number of organisms in i gm. of dry soil. At begin- ning. After I week. Relative. After 2 weeks. Relative. I 2 Mgm. 10 10 25 25 50 50 100 100 150 150 200 200 300 300 10, 670 10, 670 10, 670 10, 670 10, 670 10, 670 10, 670 10, 670 10, 670 10, 670 10, 670 10, 670 10, 670 10, 670 10, 670 10, 670 680, OOQ 825, 000 2, 195, 000 3, 410, 000 3, 900, 000 3, 885, 000 1,555,000 I, 585, 000 445, 000 320, 000 375, 000 330, 000 135, OOO 170,000 45, 000 50, 000 Per cent. 100 I 372 } 517 1 208 } ^^ } 46 > 2Q } 6.3 f 8, 015, 000 \ 7, 000, 000 f 14, 600, 000 \ 11,050,000 r IK. AOO. 000 Per cent. > 100 1 ■2 4 C 6 \ 14, 800, 000 1 1 7 f II, 500, 000 \ 14, 400, 000 f 2, 680, 000 \' 3, 290, 000 r 560, 000 \ 790, 000 f 90, 000 I («) J 25, 000 I 3°, 000 } 173 1 40 } 8.9 } - } -. 8 Q 10 II 12 JT. 14 IC 16 a Contamination. Jan. 98, 191S Nitrogen- A ssimilating Bacteria 209 Table XXI. — Influence of sodium nitrate on Bacillus radicicola in sterilized soil Treat- ment (ni- trate in 100 gm. of dry soil). Number of organisms in i gm. of dry soil. Culture No. At begin- ning. After I week. Relative. After 2 weeks. Relative. Mgm. 10 10 25 25 50 50 100 100 150 150 200 200 300 300 i5> 500 15, 500 i5> 500 i5,.5oo 15. 500 15. 500 15. 500 15, 500 15, 500 i5> 500 15, 500 15. 500 i5> 500 15. 500 i5> 500 15. 500 I, 500, 000 1, 250, 000 2, 560, 000 3, 000, 000 6, 150, 000 5. 375. 000 4, 850, 000 5, 570, 000 2, 000, 000 I, 850, 000 I, 060, 000 835, 000 760, 000 725, 000 250, 000 365, 000 Per cent. > 100 > 201 } 418 } 378 \ 140 } 69 } ^"^ 22 / 6, 750, 000 \ 5, 950, 000 f 10, 000, 000 \ 12, 500, 000 f 14, 650, 000 \ 15, 700, 000 \ 8, 500, 000 f I, 520, 000 \ I, 650, 000 / 850, 000 \ 940, 000 f 500, 000 \ 620, 000 f 150, 000 \ 210,000 Per cent. 2 > 100 1 177 e 6 > 240 7 8 I 134 } ^^ 1 ^'^ 1 8.8 IJ. TC 1 2.8 16 a Contamination. Table XXII. — Influence of calcium nitrate on Bacillus radicicola in sterilized soil Treat- ment (ni- trate in 100 gm.of dry soil). Number of organisms in i gm. of dry soil. Culture No. At begin- ning. After I week. Relative. After 2 weeks. Relative. Mgm. 10 10 25 25 50 50 100 100 150 150 200 200 300 300 oppppppppppppppP OOOOOOOOOOOOOOOO 960, 000 850, 000 3, 650, 000 3, 940, 000 5, 500, 000 6, 700, 000 4, 000, 000 3, 500, 000 Per cent. 100 1 419 } 674 } 414 1 Percent. j 4,675,000 :\ ^^ 2 i 4,590,000 J 1 / 6,000,000 1 A \ 5,450,000 J C / 10,650,000 1 6 I 14,700,000 J '^ 7 f 9,350,000 j1 1 8,670,000 1/ ^95 8 1, 200, 000 1 ^g^ 2, 050, 000 iJ S65.000JI ^^^ I, ot^o, 000 J 375.000:1 260, 000 ij "'-' 35.000 1 47,000 ,1 ^ ^ f 1,500,000 1 10 \ 1,750,000 j ^^ II f 765,000,1 12 \ 800,000 1 ^' 1-2 f 350,000 1 _ \ 300,000 / '■ 14 TC ( 25,0001 \ 40,000 J ' 16 1 An inspection of all three tables reveals two marked differences from the results obtained in similar work with Azotobacter. First, it will be noted that nitrates do not appear to exert such a marked stimulating effect with B. radicicola as wath Azotobacter. The numerical increase due to the presence of the nitrate is clearly shown in the percentage columns. Second, it will be noted that B. radicicola does not seem to be so sensitive to higher concentrations of nitrates as does Azotobacter. In all instances at concentrations equivalent to 300 mgm. of nitrate in 2IO Journal of Agricultural Research Vol. XII, No. 4 loo gm. of soil the legume organisms were still alive, although present in numbers far below those of the control cultures. In all Azotobacter cultures no organisms survived this concentration. No one nitrate produced an excessive stimulation in comparison with the others. The calcium salt present as 150 mgm. of nitrate in 100 gm. of soil at the end of the first week gave the greatest stimulation for con- centrations of that amount. However, at the end of the second week this concentration had caused a marked decrease in the number of or- ganisms. In the case of all three nitrates the concentration representing 25 mgm. of nitrate in 100 gm. of soil produced the greatest stimulation. This resulting stimulation also held true throughout the second week. The decrease in number below those of the control cultures, due to in- creasing concentrations of nitrate, began first in the presence of potassium nitrate at 100 mgm. of nitrate per 100 gm. of soil, then with sodium nitrate at 150 mgm., and lastly with calcium nitrate at 200 mgm. But the number of organisms present in the soil cultures containing sodium nitrate in amounts equivalent to 100 mgm. and calcium nitrate at 100 mgm. at the end of the second week was below those of the control cultures. It therefore appears from these results that small amounts of potassium, sodium, and calcium nitrate stimulate the reproductive activity of B. radicicola. Concentrations of nitrates greater than those amounts which produced maximum stimulation cause a decrease in the number of or- ganisms. The highest concentration of nitrate studied did not entirely prevent the growth of the bacteria, but it reduced the number of organ- isms far below those contained in control cultures where no nitrates were added. Ammonium nitrate was also employed. The soil cultures were pre- pared as already described and inoculated with B. radicicola. The cul- tures were incubated at 28° to 30° C. and counts were made at the end of one and two weeks' time. The results of the study with ammonium nitrate are dven in Table XXIII. Table XXIII. — Influence of ammonium nitrate on Bacillus radicicola in sterilized soil Treat- ment (ni- trate in 100 gm.of dry soil). Number of organisms in i gm. of dry soil. Culture No. At begin- ning. After I week. Relative. After 2 weeks. Relative. I Mgm. 25 25 100 100 200 200 P P P P P P P P OOOOOOOO 850, 000 765, 000 2, 500, 000 3, 050, 000 I, 350, 000 I, 050, 000 700, 000 655, 000 Per cent. \ 100 } 343 } 148 } 84 / , I, 365, 000 \ I, 400, 000 r 5, 060, 000 \ 4, 320, 000 / 1, 030, 000 \ 950, 000 1 635, 000 \ 605, coo Per cent. \ 2 > 100 7, 1 4 \ 338 C 1 6 / ^' 7 1 8 } ' 45 Jan. 28, i9i8 Nitrogen-Assimiloting BacteHa 211 From the results as a whole it appears that it is the nitrate radical and not the combined salt which causes the increase in the number of B. radicicola when small amounts of nitrates are present. A stimulation occurred, resulting in an increase in number which is quite comparable to that obtained with potassium, sodium, and calcium nitrates. The highest concentration of ammonium nitrate used did not appear to have such an inhibiting effect as did the corresponding concentrations of the three other salts. Throughout the work with B. radicicola in sterilized soil compara- tively low numbers of these organisms were found. Whether or not this depression was due to toxic substances formed as a result of steriliza- tion can not be stated. If this decrease in numbers as a result of the presence of toxic substances is true, it is very evident that the detrimental effect had not become materially lessened at the end of the incubation period. However, in any event the validity of the outcome is not im- paired, since comparative and not absolute data are of importance and since in all probability the same conditions obtained throughout the cultures. It seems certain from the results of these studies on the effect of potassium, sodium, calcium, and ammonium nitrates on the growth of B. radicicola in sterilized soil that small amounts of nitrate stimulate the growth of the organisms. It is also shown that B. radicicola is much more resistant than Azotobacter to higher concentrations of potassium, sodium, calcium, and ammonium nitrates. INFLUENCE OP BACILLUS RADICICOLA ON NITRATES IN SOLUTION The series of soil culture experiments just discussed served to give an idea concerning the effect of nitrates on the legume organism. It was found that in small amounts nitrates stimulated the bacteria to increased reproduction. But no study was made as to the effect of Bacillus radicicola on the nitrate. Does the organism break up the nitrate, reducing it to nitrite or ammonia ? Does it cause any loss in nitrate when grown in a solution containing that salt? Beijerinck (2, p. 762) as a result of physiological experiments with B. radicicola, states that the organism does not reduce nitrate. Prucha (41) also states that B. radicicola does not reduce nitrates. However, Zipfel (49) found that B. radicicola will reduce nitrates to nitrites but not to ammonia. The following experiments, somewhat similar to those already cited in relation to Azotobacter, were carried out in an endeavor to answer these questions. To twenty 500-c. c. Erlenmeyer flasks containing 200 c. c. of mannit solution, potassium, sodium, calcium, and ammonium nitrates were added as indicated in Tables XXIV, XXV, and XXVI. Quadruplicate flasks were prepared for each concentration of nitrate and for the control cultures without nitrate. The flasks and contents were sterilized at 212 Journal of Agricultural Research Vol. Xn, No. 4 lo pounds' pressure for 30 minutes. After cooling, two of each set of four flasks were inoculated with 5 c. c. of a suspension of B. radicicola in sterile distilled water. The remaining two flasks of each set (uninocu- lated) served as controls. The flasks were incubated at 28° C. for 2 1 days. The total weight of the flasks was maintained throughout the incuba- tion period by the addition of sterile distilled water from time to time. At the expiration of the period of incubation the nitrate, ammonia, and total nitrogen contents were determined as given under "Methods used in experiments." The contents of the duplicate inoculated flasks were poured together and 50 c. c. samples drawn for analysis. The same procedure was followed in the case of the uninoculated flasks. The results are given in Tables XXIV, XXV, and XXVI. Table XXIV. — Influence of Bacillus radicicola on nitrates in solution giving the quantity of nitrate lost Treatment (nitrate in loo c. c. of medium). Nitrate in loo c. c. of medium. Cul- ture No. Uninoculated. Inoculated. Nitrate lost. Found. Average. Foimd. Average. I None Mgm. 0. 00 . 00 151- 4 151. 148.8 148.8 154-8 155-6 151- 4 151. 6 Mgm. > 0. 00 }i5i- 2 [148. 8 }l55-2 }i5i-5 Mgm. ( 0. 00 \ . 00 /117-O I117.O flI4. 4 \ii4. / 76.6 I 76.8 fi42. 6 \i42. 6 Mgm. > 0. 00 [■117. >ii4. 2 } 76.7 V142. 6 Mgm. 2 do 0. 00 3 4 5 6 7 8 9 10 150 mgm. of NO3 as potassium nitrate . do 150 mgm.^of NO3 as sodium nitrate . . do 150 mgm. of NO3 as calcium nitibte. do 150 mgm . of NO3 as ammonium nitrate .... do -34-2 -34-6 -78.5 - 8.9 Table XXV. — Influence of Bacillus radicicola on nitrates in solution giving the quantity of nitrogen as ammonia formed Treatment (nitrate in loo c. c. of medium). Nitrogen as ammonia in loo c. c. of medium. Nitro- Cul- ture No. Uninoculated. Inoculated. gen as ammo- nia Found. Average. Found. Average. formed. I None Mgm. 0. 20 . 10 . 10 . 20 . 20 . 20 •40 -30 13.90 13-95 Mgm. } 0-15 } - > . 20 } 13-92 Mgm. f 0. 10 \ . 20 / . 00 I • 10 / .20 I -3° J -3° \ . 10 f 13. 80 I 13- 85 Mgm. } 0-15 } '°^ I -25 > . 20 } 13-82 Mgm. 2 do 0. 00 3 4 5 6 150 mgm. of NO3 as potassium nitrate . do — . 10 150 mgm. of NO3 as sodium nitrate . . do +-OS 7 8 150 mgm. of NO3 as calcium nitrate. do -- 15 9 10 150 mgm. of NO3 as ammonium nitrate do +.10 Jan. 38, 1918 Nitrogen- A ssimilating Bacteria 213 Table XXVI. — Influence of Bacillus radicicola on nitrates in solution giving the quantity of nitrogen fixed Cul- ture No. I 2 3 4 5 6 7 8 9 10 Treatment (nitrate in 100 c. c. of meditun). None ....do 150 mgm. of NO3 potassium nitrate. . . . . .do 150 mgm. of NO3 sodium nitrate ....do i5omgm. of NO3 calcium nitrate . . . . . . .do 150 mgm. of NO3 ammonium nitrate . do Total nitrogen in 100 c. c. of medium. Uninoculated. Inoculated. Found. Average. Found. Average. Mgm. Mgm. Mgm. Mgm. 2. 40 2.50 } 2.45 I 3-5° I 3- 40 18.00 17.90 } 17- 95 r 18. 70 \ 19. 00 } 18. 85 16.80 17. 00 ■ 16. 90 / 19- 30 \ 19. 20 I 19-25 14. 00 13.80 I 13-90 f 14. 60 I 14- 70 } 14-65 40. 50 41. 20 } 40. 8s / 41- 30 I 41- 70 } 41. 50 Nitro- gen fixed. Mgm.. 95 The data in Table XXIV show that a rather large reduction in the total nitrate content took place. This reduction varied rather markedly among the four dififerent nitrates studied. The greatest reduction oc- curred where calcium nitrate was used. Potassium and sodium were next in order; the loss was almost the same for both salts. Ammonium nitrate was last with but a very small comparative reduction in total nitrate. The question arises as to whether the nitrate is reduced to nitrite, ammonia, or elemental nitrogen or whether the reduction in amount is due to a natural assimilation of the nitrate by the organisms. The first possibility was precluded when qualitative tests for nitrites were made and none found. Table XXV reveals the fact that no ammonia was produced. Table XXVI shows no loss in total nitrogen. Therefore it seems obvious that reduction in total amount of nitrate present is brought about by the assimilation of those compounds by the organisms. An inspection of Table XXVI, which gives the results of the total nitrogen determinations, shows that a slight fixation of atmospheric nitrogen took place. This fixation is entirely possible, as will be shown later when the influence of nitrates on the fixation of nitrogen is taken up. In the presence of potassium, sodium, and ammonium nitrates the amount of nitrogen assimilated is somewhat decreased. But in the case of sodium nitrate a large increase in the amount of total nitrogen seems to have taken place. This is interesting in the light of results to be presented later. From the results of the work on the effect of B. radicicola on nitrates it may be concluded that the organisms do not reduce the nitrates to nitrite or ammonia or elemental nitrogen under aerobic conditions. 214 Journal of Agricultural Research voi. xii, no. 4 INFLUENCE OF NITRATES ON THE FIXATION OF ATMOSPHERIC NITROGEN BY BACILLUS RADICICOLA The ability of B. radicicola to fix atmospheric nitrogen in the absence of the host plant has been studied by numerous investigators. From the results of their work it seems fairly probable that the legume organ- ism can fix nitrogen to a slight extent when growing in a nonsymbiotic state. Beijerinck (5) was one of the earliest to make a study of the possible fixation of atmospheric nitrogen by B. radicicola under these conditions. He found that a small quantity, 0.91 to 1.82 mgm. of nitro- gen was fixed per 100 c. c. of the medium. Prasmowski (59, p. 55) and Berthelot (6) concluded as a result of their experiments that when the organism was grown outside the host plant the gain in nitrogen was small. The greatest gain in nitrogen was found by Maze {32) who re- ported an increase of 23.4 mgm. of nitrogen per 100 c. c. of the medium in 16 days. Lewis and Nicholson {30) found by incubating the cultures for a considerable length of time that a large increase in fixation occurred. Bottomley {8) found that a pure culture of B. radicicola fixed approxi- mately I mgm. of nitrogen in 15 days. Fred {17) made a study of the possible fixation of nitrogen by the legume organism and found that it fixed approximately 1.2 mgm. of nitrogen in 100 c. c. of the medium. He found that on agar films a greater fixation occurred than when the organisms were grown in a liquid medium. A few investigators, however, found that no increase in nitrogen occurred when B. radicicola was grown outside the host plant. Frank (j6) states that in a nitrogen-free medium the legume organisms did not fix enough nitrogen to be accurately measured. Immendorf (25) also found no increase in nitrogen when pure cultures of B. radicicola were grown in soil containing a nitrogen-free solution. It will be seen that the majority of investigators, especially the more recent ones, found that a slight amount of atmospheric nitrogen was fixed or assimilated by B. radicicola when grown outside the host plant and on a medium suitable for its development. It has already been shown that nitrates cause an increase in the num- ber of B. radicicola when grown in pure culture in sterilized soil. Does such an increase in the number of organisms necessarily mean an m- creased fixation of nitrogen? Three experiments using agar films were carried out in order to determine this point. Erlenmeyer flasks of I -liter capacity containing 100 c. c. of mannit agar were used. Before the medium solidified, the nitrates were added in the proportions indi- cated in Table XXVII. Six flasks for each different quantity of nitrate were prepared, except in one case, as shown in Experiment II. The flasks were plugged with nonabsorbent cotton and sterilized at 10 pounds' pressure for 30 minutes. After cooling, three of each set were inoculated with 5 c. c. of a suspension of B. radicicola in sterile distilled water. The organisms had been growing on mannit agar at 28° C. for six days. The flasks in Experiments I and III (Table XXVII) were incubated at Jan. 28, 1918 Nitrogen- Assimilating Bacteria 215 28° C. for three weeks and those in Experiment II for two weeks. The moisture lost by evaporation in both inoculated and uninoculated flasks was replaced from time to time by the addition of sterile distilled water. At the expiration of the incubation period the total nitrogen was deter- mined as given under "Methods used in experiments." The results of the experiments are given in Table XXVII. An inspection of the data reveals the fact that B. radicicola in pure culture fixecf a small amount of nitrogen when growing in a nonsymbiotic state with no nitrate present. In the presence of nitrates there was an increased fixation. Although the increase in total nitrogen is small, because of the number of determinations made, it may be considered as positive. The potassium and sodium salts seemed to be more effective than the calcium nitrate, with one exception (Table XXVII, Experiment I). It will be remembered that the latter salt appeared to depress nitro- gen fixation by Azotobacter and the two former somewhat to favor it (p. 194-195). TABtE XXVll— Influence of nitrates on the fixation of nitrogen by Bacillus radicicola, giving the increase in nitrogen EXPERIMENT I Culture No. Treatment (nitrate in 100 c. c. of medium.) Total nitrogen in 100 c. c. of medium. Uninoculated. Foimd. None ....do ....do 75 mgm. of NO3 as sodium nitrate . . do do 150 mgm. of NO3 as sodium nitrate. do do 75 mgm. of NO3 as calcium nitrate . do do 150 mgm. of NOjas calcium nitrate . do do Mgm. 4-5 4-4 4-4 -8.7 8.7 8.6 12.5 12. 7 Average. 8.9 9.0 13- I 13. 2 Mgm. 4-45 8. 70 12. 60 8.90 13. 20 Inoculated. Found. Mgm. Average. Mgm. 4. 60 11-75 14. 70 12. 40 14. 10 Nitrogen increase. Mgm. o. 15 3-05 3- SO o. 90 EXPERIMENT II None ....do 75 mgm. of NO3 as sodium nitrate . . do 150 mgm. of NO3 as sodium nitrate. . do 75 mgm. of NO3 as calcium nitrate . do 150 mgm. of NO3 as calcium nitrate. do 4.90 4.90 8. 70 8.50 13-30 13. 00 II. IS II. 10 14- 70 («) ¥■ 4.90 60 '13- IS ■II. 125 ^14. 70 }- }<,. 075 50 }i4- 35 11.65 15-25 o- 175 0. 90 1. 20 o. 525 0-550 a Lost by breakage during sterilization. 2l6 Journal of Agricultural Research vo1.xii.no. 4 Table XXVII. — Influence of nitrates on the fixation of nitrogen by Bacillus radicicola, giving the increase in nitrogen — Continued EXPERIMENT III Culture No. Treatment (nitrate in loo c. c. of medium): 9 10 II 12 13 14 15 16 17 18 19 20 None ...do ....do 75 mgm. of NO3 as potassium ni- trate ....do ....do _. 150 mgm. of NO3 as potassium ni- trate ...do ....do 75 mgm. of NO3 as sodium nitrate ....do ....do 150 mgm. of NOgas sodium nitrate ....do ....do 75 mgm. of NO3 as calcium nitrate ....do ....do 150 mgm. of NOjas calcium nitrate ....do, ....do Total nitrogen in ICX5 c. c. of medium. Uninoculated. Inoculated. Found. Average. Found. Average. Mgm. Mgm. Mgm. Mgm. 5.10 \ 5-50 1 5. 10 1 5-07 5- 40 [ 5-50 5. CO I 5-45 J 9-35 ) f 10.85 1 9-5" [ 9-37 { 10. 90 \ 10. 90 9-25 J I 10.95 J 14.50 [ 15-65 14. 20 [14. 28 \ 15-30 1 5-. 45 14-15 I 15-40 8. 50 [ 9-85 8.30 8.38 \ 9-90 9-83 8.35 I 9- 70 12.3s f 12.95 12. 40 [12.33 j 13-10 [ 13-03 12. 20 I 13-05 8-95 f 985 9. 10 r 9- °i \ 9- 90 [ 9-93 9. 00 I 10.05 13.90 f 14.40 13.80 13.80 14-50 [ 14- 42 13.70 \ 14-35 Nitrogen inca-ease. Mgm,. 0-43 1-53 I. 17 1-45 o. 70 o. 92 o. 62 It has been shown that, when nitrates are added in varying quantities to sterilized soil, the number of B. radicicola are increased. Provided the the organism can fix a small amount of nitrogen in the absence of nitrate nitrogen, is it not possible that this increase in nitrogen fixation may be due merely to the increase in the number of cells ? It seems that this is true according to the results in Table XXVII. It appears probable that the increase in nitrogen fixed in the presence of nitrates is very likely because of an increase in the number of bacterial cells and not to any physiological change brought about in the organism itself. There was a marked increase in bacterial growth on the media con- taining the nitrate compared with the same media free from nitrate. The growth on the latter medium exhibited a normal, whitish watery appearance, characteristic of this organism. On the cultures containing nitrates a much more profuse growth occurred. In, many instances a pinkish tint was observed. This pigment production was especially marked in the case of the culture containing the sodium salt. After the first experiment had been completed, it was thought that possibly this pigmentation was due to an impurity in the culture. Therefore the two remaining experiments were made, using a subculture from the original. Jan. 28. i9i8 Nitrogen-Assimilating Bacteria 217 This culture was plated three times, each plating being made from a well- isolated colony. The final subculture was taken from a similar well- isolated colony. However, pigment formation in the presence of nitrate persisted in the two final experiments, showing clearly that some reaction took place between the nitrate and the organism grown on the medium. It is of interest to note that the pigment formation in the presence of nitrate was observed in later work where the influence of nitrates on nodule formation was investigated. Prucha (41) found that on agar slopes of medium containing 0.5 per cent of potassium or calcium nitrate, the growth of B. radicicola became opaque and that an iridescent tint was produced. Although the results of these experiments may vary somewhat among themselves, taken as a whole it appears evident that B. radicicola may fix a small amount of atmospheric nitrogen when grown without the host plant and on a suitable medium. The addition of various amounts of nitrates as indicated increased somewhat the amount of nitrogen assimilated by B. radicicola. INFLUENCE OP NITRATES ON THE PRODUCTION OP GUM BY BACILLUS RADICICOLA , Since nitrates, especially in smaller amounts, cause an increase in the number of B. radicicola in pure culture, it was thought advisable to determine what influence these salts have on the production of gum. In culture media favorable to the growth of B. radicicola these bacteria will produce a gelatinous substance which is readily precipitated with 95 per cent alcohol or acetone. Upon the addition of either of these precipi- tants a fairly heavy, water-white, frothy gelatinous mass is formed which soon rises to the surface of the liquid. Upon standing, this mass contracts somewhat, and portions of it may fall to the bottom of the liquid from which it has been precipitated. Chemical analyses, according to Buchanan (10), have shown that this gum is a carbohydrate. Upon hydrolysis with 2 per cent sulphuric acid and 15 pounds' pressure for one hour, Fehling's solution is reduced, showing the presence of a sugar. The gum does not give proteid reac- tions with the Millon, biuret, or xanthoproteic tests. Hence, the gum is not protein in character; nor does it contain nitrogen in the combined form. Clearly it is a nonnitrogenous body. In the experiment undertaken to determine whether nitrates influence the formation of gum only relative difi"erences are noted. No attempt was made to obtain quantitative results. Erlenmeyer flasks of i -liter capacity containing 200 c. c. of mannit solution were used. The cultures contained various quantities of nitrate as indicated in Table XXVIII. Triplicate flasks for each amount of nitrate were prepared. In this table these three flasks are represented as "a," "b," and "c." After sterilization at 15 pounds' pressure for 25 minutes the flasks were cooled and inoculated with 5 c. c. of a suspension 2l8 Journal of Agricultural Research Vol. XII, No. 4 of B. radicicola in sterile distilled water. The cultures were then incubated at room temperature (approximately 25° C.) for eight weeks. At the expiration of the incubation period the contents of the flasks were poured into hydrometer cylinders of equal depth and diameter. One hundred and fifty c. c. of acetone were added to precipitate the gum. After careful shaking, the cylinders were covered with inverted petri dishes to prevent evaporation. At the end of 24 hours the amount ol gum precipitated was observed. The relative amounts are recorded in Table XXVIII. Table XXVIII. — Influence of nitrates on the production of gum by Bacillus radicicola Cul- Treatment (nitrate in loo c. c. of medium). Relative production of gimi— precipitated by acetone. No. Flask a. Flask b. Flask c. None Large Very large . Large Very large . Laree Large Very large . Large ...do ...do Large. 2 3 4 5 75 mgm. of NO3 as potassium nitrate . 450 mgm. of NO3 as potassium nitrate . 75 mgm. of NO3 as sodium nitrate .... 450 mgm. of NO3 as sodium nitrate. . . 7^ msrm of NO-> as calcium nitrate Very large. Large. Very large. Large. Do. . do ...do 7 4t;o mgm of NO3 as calcium nitrate. . ...do C nsider- able. Con sider- able. From the results it is certain that the nitrates, especially in the smaller of the two concentrates, caused a very considerable increase in the amount of gum produced by B. radicicola. The nitrates of potassium and sodium caused a production of more gum than did the calcium salt. It will be remembered that in the experiments where the influence of nitrates on the fixation of atmospheric nitrogen by B. radicicola was studied, less nitrogen was fixed in the presence of calcium nitrate than in the pres- ence of the other two salts. Here again the greater stimulative action of potassium and sodium nitrates is emphasized. Buchanan in his investigations on the formation of gum by B. radici- cola has found that varying amounts of potassium nitrate in a 2 per cent saccharose solution or in a 2 per cent saccharose-clover-extract solution caused a slight increase in growth and in gum production. It seems probable that the increased gum production in the nitrate cultures is caused not only by an increase in bacterial cells but also perhaps by an increased stimulation in the formation of gum by the cells themselves. The relative increase in the amount of gum produced in the presence of nitrates seems to be greater than the actual increase in number of organisms brought about by the stimulating effect of the nitrate. In the latter instance this stimulating effect has been deter- mined in soil cultures only and so a fair basis of comparison can not be Jan. 28, i9i8 Nttrogen-Assimilating Bocterta 219 found. Had the influence of nitrates on the growth and reproduction of B. radicicola been determined in liquid culture, as well as in soil cultures, then a comparison could have been made. Furthermore, the divergencies in the time element, eight weeks' incubation in the liquid cultures and three weeks in the soil cultures, are such as to render futile any attempt at correlation. It may be that the large formation of gum was due to the prolonged incubation. A shorter period of three weeks undoubtedly would show a relatively smaller amount of gum produced as a result of the presence of the nitrate. However, from the results of the experiment it is certain that potas- sium, sodium, and -calcium nitrate influence the formation of gum by B. radicicola. The three nitrates studied caused a large increase in the amount of gum obtained by precipitation with acetone. Calcium nitrate caused the least stimulation, but the difference was not large. INFLUENCE OF NITRATES ON NODULE FORMATION The results of numerous investigations have shown that nitrates retard and oftentimes entirely prevent the formation of nodules on leguminous plants when grown in soil or liquid cultures. Vines (45), working with the horse bean, found that the use of large amounts of nitrate iii the form of potassium nitrate retarded nodule formation. He concluded that a decrease in the amount of nitrates meant an increase in the number of nodules. Vv'oods {4S) found that leguminous plants assimilated more nitrogen when they were grown in the absence of potassium and calcium nitrate than* when thus supplied. His results seem to indicate that nodule development was retarded somewhat by these salts. Similar results were obtained by Frank (z6). Nobbe and Richter (57) in 1902 grew soybeans in a rich garden soil and found upon inoculation that a gain of 74.7 per cent of nitrogen occurred. However, upon the addition of nitrates this gain was considerably reduced, the extent of the reduction corresponding to the amount of nitrate added. About this same time, Wohltmann and Bergene (47) using many diflierent types of soils, found that nodules were not formed on the roots of peas when ammonium nitrate was added, Creydt {12) found that sodium nitrate retarded nodule formation on yellow lupines when these legumes were grown in soil, Fred and Graul {18) found that very small amounts of nitrates did not appreciably decrease nodule formation, but that larger amounts proved detrimental and finally prohibited entirely the development of nodules. The presence of nitrates in culture solutions has also been found to reduce and oftentimes to inhibit the formation of nodules on leguminous plants. Marchal {31) concluded that alkaline nitrates in concentrations of I to 10,000 in liquid cultures prevented the formation of nodules on peas. Flamand {15) grew vetch and beans in a nutrient solution and 220 Journal of Agricultural Research voi. xii, no. 4 found that nitrates in the following amounts prevented nodule forma- tions: potassium nitrate, i to 10,000, sodium nitrate i to 2,000, ammo- nium nitrate i to 2,000, and calcium nitrate i to 2,000 and i to 10.000. Hiltner's {24) experiments showed that 5 mgm. of nitrogen as potassium nitrate per liter prevented nodule formation on peas. In contrast to these experiments Bassler (j) claimed that results obtained from his work indicated that no effect was noticed by adding nitrates to lupines growing in quartz sand. The question naturally arises whether this condition is due to the weakening of the organism brought about by growth in a nitrated environment and to a consequent impairment or entire loss of its infect- ing power, or whether it is caused by some interreaction between the salt and the plant root, tending to increase the latter's resistance to the attack of this particular organism. INFLUENCE OF NITRATES ON THE INFECTING POWER OF BACILLUS RADICICOLA Some investigations have been carried out to determine what effect nitrates have on the legume organisms themselves. Wilson {46) showed that although nitrates inhibit the formation of nodules, the organisms capable of producing nodules did not lose their vitality or nodule-pro- ducing power when grown in the presence of nitrates. The results of Prucha {41) are in accord with those of Wilson. He found that B. radi- cicola does not seem to lose its infecting power when grown on media containing nitrate. During the course of his work he found that potas- sium and sodium nitrates inhibited the formation of nodules. Further evidence that the organisms appear to retain their vitality in the pres- ence of nitrates is produced by the results of Maze (jj, p. 15-iy), who showed that legume bacteria were able to fix a slight amount of nitrogen when grown in a soil extract solution containing i per cent sodium nitrate. Herke {22) states that potassium nitrate favors the growth of nodule bacteria. However, other investigators state that nitrates have a harmful effect on B. radicicola. Laurent (29, p. 134) found that legume organ- isms failed to grow in a pea or lupine decoction containing nitrate in the form of potassium and sodium salts in amounts equivalent to i to 500 and I to 1,000. Moore (55) in his experiments demonstrated that nitrates at I to 10,000 were sufficient to prevent nodule formation. He states that B. radicicola loses its power of infection when grown in a medium containing nitrates. From the results cited it can be seen that there is some disagreement as to the influence exerted by nitrates on B. rad'i/:icola. In some cases the organism seems to retain its vitality in the presence of nitrates, while in others it appears to have become weakened. It must be ad- Jan. 28, 1918 Nitrogen- Assimilating Bacteria 221 mitted, however, that the evidence seems to favor the former contention — namely, that nitrates do not cause the bacteria to lose their nodule- producing power. In order to determine whether or not nitrates weaken these organisms, the following experiments were made: Slopes of mannit agar (in test tubes) containing various amountsof sodium and calcium nitratesas indi- cated in Table XXIX were inoculated with B. radicicola. These cul- tures were incubated at 28° C. for one week, when transfers were made to fresh nitrate media and incubated at 28° C. for another week. At the expiration of this time, three 4-day-old seedlings of alfalfa were inoculated with three drops of a suspension of the organism in 5 c. c, of sterile distilled water. The same slope cultures were incubated at 28° C. and used for all subsequent inoculations in this experiment. The inoculated seedlings were placed in the greenhouse under cheese- cloth covering. The temperature here during the daytime averaged approximately 30° C. The seedlings were examined for the first appear- ance of nodules and in no case did they appear before 18 to 20 days. A total count of nodules on all plants was made at the end of 45 days. Three subsequent inoculations were made under the same conditions. In this way organisms in contact with nitrate for varying lengths of time could be used. The results of the inoculation experiments are given in Table XXIX. Table XX-IX. — Influence of nitrates on the infecting power of Bacillus radicicola Cul- Treatment (nitrate in loo c. c. of medium). Number of nodules after 43 days. ture No. Inoculated June 3. Inoculated June 15. Inoculated July II. Inoculated July 17. I None 5 5 7 4 5 6 2 7 4 9 5 7 5 6 4 4 6 5 8 5 4 8 4 4 5 5 7 8 7 5 15 3 4 5 6 7 9 8 II 9 4 6 3 5 5 II 8 7 3 8 2 3 4 5 6 7 8 15 mgm. of NO3 as sodium nitrate . . . 37 mgm. of NO3 as sodium nitrate . . . 75 mgm. of NO3 as sodium nitrate . . . 150 mgm. of NO3 as sodium nitrate . . 225 mgm. of NO3 as sodium nitrate . . 450 mgm. of NO3 as sodium nitrate . . None 9 10 II 12 13 14 15 i6 15 mgm. of NO3 as calcium nitrate . . 37 mgm. of NO3 as calcium nitrate. . . 75 mgm. of NO3 as calcium nitrate . . 150 mgm. of NO3 as calcium nitrate. . 225 mgm. of NO3 as calcium nitrate . 450 mgm. of NO3 as calcium nitrate . Uninoculated 4 6 8 9 3 3 do From the results given in Table XXIX it is very evident that under the conditions of the experiment the legume organisms did not lose their power of producing nodules when grown on a medium containing 222 Journal of Agricultural Research voi. xii, no. 4 varying amounts of sodium and calcium nitrates. The numbers of nodules produced on the alfalfa plants by organisms grown on media containing nitrate do not vary widely from those on the plants inocu- lated with organisms grown on media containing no nitrate. Not only did the organisms fail to lose their nodule-producing power, but from all appearances their infecting power did not seem to be materially weakened. It therefore seems apparent that an explanation for the failure of nodules to develop on leguminous plants in the presence of nitrates is not found in the theory that the organisms producing these nodules are weakened when grown in the presence of nitrates. INFLUENCE OF NITRATES ON ALFALFA ROOTS AND NODULE FORMATION The next step taken would naturally be in the direction of a study of the influence of the nitrates on the plant roots themselves in order to determine whether or not they thus are made more resistant to the attack of these organisms. A review of the literature shows that almost nothing has been done touching this phase of the question. Wilson {46) y studying the effect of certain salts on nodule production, states that possibly the salt has some effect on the root, making it resistant to the attack of the organism. Maze {33, p. 15-17), who also concluded that nitrates did not cause B. radicicola to lose its infecting power, says that nodules do not develop on roots of legumes when nitrates are present because the carbohydrate in the roots is changed into protein material by the absorption of the nitrate. Alfalfa seedlings (Medicago saliva) growing in soft agar containing potassium, sodium, and calcium nitrates, as indicated in Table XXX, were used in this study. Quadruplicate tubes were prepared for each amount of nitrate. The higher concentrations of the nitrate were not used, since it was found that germination and subsequent growth were considerably impaired in the presence of such large amounts. The tubes with the mannit agar and nitrate were sterilized at 1 5 pounds' pressure for 30 minutes. These were cooled and sterilized alfalfa seeds planted as given under "Methods used in experiments." The tubes were then placed in the greenhouse under cheesecloth covering and the seeds allowed to germinate. Germination took place in all instances, although it was retarded somewhat by the presence of the nitrate. At the end of five days the first tube of each set was inoculated with three drops of a suspension of B. radicicola in sterile distilled water. Subsequent inocu- lations were made as indicated in Table XXX. These were made at different intervals in order to allow the roots of the seedlings to remain for a longer time in contact with the media. It was hoped that in this way an idea might be obtained as to the time when the root first became resistant. The results are given in Table XXX. Jan. 28, 1918 Nitrogen- A ssimilating Bacteria 223 Table XXX.— Influence of nitrates on alfalfa roots and nodule formation Culture No. Total number of nodules in each tube of seedlings inoculated after — Treatment (nitrate in 100 c. c. of medium). None V 10 mgm. of NO3 as potassium nitrate . 25 mgm. of NO3 as potassium nitrate . 50 mgm. of NO3 as potassium nitrate . 100 mgm. of NO3 as potassium nitrate 150 mgm. of NO3 as potassium nitrate ID mgm. of NO3 as sodium nitrate . . . . 25 mgm. of NO3 as sodium nitrate . . . . 50 mgm. of NO3 as sodium nitrate . . . . 100 mgm. of NO3 as sodium nitrate . . . 150 mgm. of NO3 as sodium nitrate . . . 10 mgm. of NO3 as calcium nitrate . . . 25 mgm. of NO3 as calcium nitrate . . . 50 mgm. of NO3 as calcium nitrate . . . 100 mgm. of NO3 as calcium nitrate . 150 mgm. of NO3 as calcium nitrate . . 5 days' growth. 10 days' growth. l^) 18 days' growth. Q') (^) 22 days' growth. («) a Fungus contamination. & Plant died after few days' growth. It will be seen that in a few instances where a high concentration of nitrates occurred the development of the seedlings subsequent to ger- mination ceased. This condition may have been due to too high a con- centration of soluble salts or to inferior seed. However, losses were not sufficiently serious materially to affect the outcome of the experiment. In all cases the seedlings grown in agar without nitrate produced nodules when inoculated with B. radicicola. A few nodules appeared on seedhngs in cultures containing the lowest concentration of all three nitrates. The number of nodules in these cases was less than in the control cultures. No nodules whatever developed in any concentration above lo mgm. of nitrate in lOo c. c. of medium. Under normal condi- tions in test-tube cultures the nodules make their appearance at about 18 to 20 days after inoculation. The incubation of all cultures was extended 40 days after inoculation in order to make certain that no further nodule development would take place. The nonproduction of nodules was not due to the failure of the inoc- ulum. In all cases an excellent inoculum growth was obtained, espe- cially in the case where nitrate was present in the medium. Indeed, it was so luxuriant that in many cases the organism grew in considerable quantity far down into the root zone. In many cases where nitrates were present the same pink coloration was produced that was discussed under another caption, on page 216. As has been already stated, seedlings of varying ages were moculated for the reason that it was thought that a more or less prolonged contact of the roots with the nitrate in the medium might serve as an mdex to 224 Journal of Agricultural Research voi. xii. No. 4 the time in the growth of the seedling when permanent resistance to attack of the organisms was established. The results obtained do not seem to indicate that seedling roots 18 to 20 days' old are any more resistant to the attack of the organisms than are those that are younger. Evidently if any reaction takes place between the nitrate and the plant root it occurs during the very early stages in the development of the plant. These results seem to point to the conclusions that the nonformation of nodules in the presence of nitrates is due not to a weakening of the vitality of the organism, but to some reaction between the plant root and nitrate. One naturally queries whether the plant root cells are made more resistant to the bacteria seeking to gain entrance there or whether the naturally occurring carbohydrate food supply to be used by the organisms after gaining entrance is diminished by its conversion into protein material by the absorption of nitrate? Further studies were not made in an endeavor to solve this question. INFLUENCE OP NITRATES IN SOIL ON ALFALFA NODULES AND ON THE REFORMATION OF NODULES Additional studies were made with nitrates in relation to their influ- ence on nodules already formed and on the redevelopment of nodules once removed from alfalfa plants. The experiments were carried out in an endeavor to determine whether nitrates prevented an increase in the number of nodules on plants possessing nodules and whether they prevented the reformation of nodules when removed. Experiments revealed clearly that removed nodules were replaced by new ones pro- vided the plant was carefully replaced in the soil (soil with normal low nitrate content) and the proper amount of moisture maintained. In these experiments i -gallon earthenware jars were used. These were filled to within an inch of the top with 1,800 gm. of soil of a low nitrate content. Different amounts of the nitrates to be studied were added in the quantities indicated in Table XXXI. Concentrations of 100 and 300 mgm. of nitrate in 100 gm. of soil were also used, but the transplanted alfalfa seedlings were unable to withstand such excessive concentration, with the result that all died within a week or ten days after transplanting. Quadruplicate pots were prepared for each con- centration of nitrate. The nitrates in solution were mixed with the proper amount of distilled water which, when added to the pots, brought the moisture content to approximately 20 per cent. The pots were then allowed to remain undisturbed for one day at room temperature to allow the water containing the nitrate to become well diffused through- out the soil mass. Into two pots of each set were transplanted young alfalfa plants from which the nodules had been removed. The two remaining pots contained transplanted alfalfa plants with the nodules left on and their location noted. The plants used in this experiment Jan. 38, J918 Nitrogen- A ssimilating Bacteria 225 were removed from an alfalfa plot, the soil of which was a sandy loam. Previous to transplanting the roots of the young plants were carefully washed in running water and immediately transplanted. The pots were kept well watered, and after two or three days they were removed to the greenhouse. Here they were watered when necessary. Trans- plantations were made on the 27th of June and the experiment termi- nated on the 3d of August. The plants were removed from the pots, the roots carefully washed and examined for the presence of nodules. The results are presented in Table XXXI. Table XXXI. -Influence of nitrates in soil on alfalfa nodules and on the reformation of nodtdes Pot No. Al. A2. A3- A4. B I. B2. B3- B4. Ci. C2. C3- C4. D I. D2. D3- D4. Bi. E2. §3- E4. Fi. F2. F3. F4. Gi. G2. G3- G4. Nitrate ia loo gm. of dry soil. None ....do ....do ....do 25 mgm, of NO3 as potassium nitrate do ....do ...do 50 mgm. of NO3 as potassium nitrate. ... .do do do 25 mgm. of NO3 as sodium nitrate . . . do do do 50 mgm. of NO3 as sodium nitrated . . do do do 25 mgm. of NO3 as calcium nitrate . . do do do 50 mgm. of NO3 as calcium nitrate . . do do do Treatment of nodules. Removed ....do Not removed . do Removed do Not removed . do Removed . . . . do Not removed . do Removed.. .. do Not removed . do Removed.. .. do Not removed . do Removed.. .. do Not removed . do Removed.. .. do Not removed . do Number of nodules — At bedn- ning. At end. (a) («) " Plants died. It will be seen in the control pots, where no nitrate was present (ex- cept the small amount normally present in the soil at the beginning of the experiment), that if the nodules were removed, new ones formed. The location of the nodules before their removal was noted, and the new ones were found to occupy the same place. However, when nitrates were added to the soil no new nodules were formed. This statement holds true for both concentrations of all three salts in all experiments. 2 26 Journal of Agricultural Research voi. xii, N0.4 Some interesting results were obtained where the nodules were not removed. In the control pots an increase in nodule formation took place. It can not be stated definitely whether the new nodules appeared as a result of inoculation from the soil or whether the organisms had already gained entrance to the roots before the plants were removed from the field soil previous to transplanting. Nevertheless, it is shown that the number of nodules increased as compared with the number present at the time of transplanting. But where nitrates were added a reduction in number occurred rather regularly throughout all the pots. In two instances the number remained constant, in 10 it was reduced, and in none was it increased. The calcium salt appeared to effect the least reduction in number of nodules. Conclusions concerning the comparative in- fluence of the three salts in this regard cd,n not be drawn because of the small number of determinations made. It is sufficient to note that nitrates present in amounts equal to 25 and 50 mgm. of nitrate in 100 gm. of soil did not permit an increase in number of nodules, but rather caused a decrease. The conclusions drawn from the experiments relative to the influence of nitrates on nodule formation are: (a) the presence of nitrates is detrimental to the formation of nodules by alfalfa; (b) the nonformation of nodules is not due to a weakening of B. radicicola when grown in the presence of nitrates; (c) some reaction takes place between the nitrates and the plant root, thus preventing nodule formation; (d) nitrates in the soil prevent, the re-formation of nodules once removed and also cause a decrease in the number of those already present. SUMMARY (i) Small quantities of potassium, sodium, and calcium nitrates caused a great increase in the number of Azotobacter in sterilized soil.. Ammonium nitrate in the same quantities caused a less marked in- crease. Higher concentrations were not so favorable to the growth of the organisms. (2) Potassium and sodium nitrates in the concentrations studied caused an increase in the amount of nitrogen assimilated by Azoto- bacter on agar films. Calcium nitrate in the same amounts brought about a decrease in the amount of nitrogen fixed to a point even below that representing the amount assimilated in the absence of nitrates. In soil cultures nitrates of sodium and calcium caused an increase in total ni- trogen, which was more marked in the unsterilized cultures than in those cultures sterilized and inoculated with a pure culture of Azotobacter. However, the increase in total nitrogen is not commensurate Vv^ith the increase in the number of Azotobacter noted under the same conditions. (3) Under aerobic conditions Azotobacter in liquid cultures reduced nitrate to nitrite, but not to ammonia. More atmospheric nitrogen was assimilated in the presence of nitrate than in the absence of this salt. Jan. 28, i9i8 NUrogen-Asshnilating Bacteria 227 (4) Pigmentation occurred when potassium and sodium nitrates, and especially calcium nitrate, were used with Azotobacter, the colora- tion increasing with the concentration of the salt. This effect was more marked in Azotobacter strains which produce little or no pigment in the absence of nitrates. (5) All three nitrates studied caused an increase in the number and size of volutin bodies in Azotobacter cells. From all appearances these salts also tended to hasten the development of these bodies. (6) The number of Bacillus radicicola in sterilized soil was increased by the addition of small quantities of potassium, sodium, ammonium, and calcium nitrates. This increase was not so marked as in the Azoto- bacter cultures. B. radicicola appeared to be much more resistant to higher concentrations of nitrates than Azotobacter. (7) B. radicicola under aerobic conditions did not reduce nitrates in solution to nitrite, ammonia, or elemental nitrogen. The presence of nitrates did not materially influence the small amount of atmos- pheric nitrogen fixed under these conditions. (8) When grown on agar films, B. radicicola fixed a small amount of nitrogen, varying from 0.15 to 0.43 mgm. of nitrogen in 100 c. c. of the medium. The addition of various amounts of potassium, sodium, and calcium nitrates increased to a slight extent the amount of nitrogen assimilated. (9) In hquid cultures all three nitrates caused a large increase in the amount of gum obtained by precipitation with acetone. (10) The presence of large amounts of potassium, sodium, and cal- cium nitrates proved detrimental to the formation of nodules on alfalfa. B. radicicola did not appear to lose its infecting power when grown on media containing varying amounts of sodium and calcium nitrates. Alfalfa seedUngs grown in the presence of large amounts of nitrate did not produce nodules when inoculated with a viable culture of B. radicicola. Nitrates in soil cultures prevented the re-formation of nodules once removed and also caused a decrease in the number of nodules already present. LITERATURE CITED (i) Bassler, p. 1895. SANDKULTURVERSUCHE UBER DIE STICKSTOFPASSIMILATION DER GELBEN LUPINE IM STERILISIERTEN UND GEIMPFTEN BODE.N BEI DARGEBOT WECHSELNDER MENGEN VON SALPETERSAUREN SALZEN. In JahresbcT. Agr. Chem., n. F., Bd. 18 (Jahrg. 38), p. 131. (2) Beijerinck, M. W. 1888. DIE BACTERiEN DER P.VPILIONACEEN-KNGLLCHEM. In Bot. Ztg., Jahrg. 46, No. 48, p. 758-771. (Continued article.) (3) — 189 1. OVER OPHOOPING VAN ATMOSPHERISCHE STICKSTOP IN CULTUREN VAN BACILLUS RADICICOLA. In Vcfslag. en Meded. K. Akad. Wetensch. [Amsterdam], r. 3, deel 8, p. 460-475. 228 Journal of Agricultural Research voi. xii, No. 4 (4) Beijerinck, M. W. 1901. UEBER OLiGONiTROPHiLE MiKROBEN. In Centbl. 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