652 ;5 •py 1 SOIL INOCULATION WITH AZOTOBACTER BY PAUL EMERSON DISSERTATION SUBMITTED TO THE GRADUATE FACULTY OF THE IOWA STATE COLLEGE OF AGRICULTURE AND ME- CHANIC ARTS IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NO. 3 REPRINTED FROM RESEARCH BULLETIN No. 45 IOWA AGRICULTURAL EXPERIMENT STATION 1918 Digitized by the Internet Archive in 2010 with funding from The Library of Congress http://www.archive.org/details/soilinoculationwOOemer February, 1918 Research Bulletin No. 45 Soil Inoculation with Azotobacter <^ LSZ By PAUL EMERSON AGRICULTURAL EXPERIMENT STATION IOWA STATE COLLEGE OF AGRICULTURE AND MECHANIC ARTS AGRONOMY SECTION Soil Bacteriology AMESs IOWA IOWA AGRICULTURAL EXPERINENT STATION OFFICERS AND STAFF Raymond A. Pearson. M. S. A., LL. D.. President C. P. Curtiss. M S. A., D. S.. Director W. H. Stevenson. A. B.. B. S. A., Vice-Director AGRICULTURAL, ENGINEERING C. K. Shedd, B. S. A., B. S. in A. E., W. A. Foster, B. S. in Ed., B. Arch., Acting Chief Assistant AGRONOMY W. H. Stevenson. A. B., B. S. A., Cliief George E. Corson, B. S., M. S'., As- H. D. Hiiglies. B. S., M. S. A., Cliief sistant in Soil Survey in Farm Crops H. W. "Warner, B. S.. M. S., Soil Sur- P. E. Brown, B. S.. A. M.. Ph. D., Chief veyor (ab.=-ent on leave) in Soil Chemistry and Bacteriology -^ l, Rhodes, B. S., Soil Survey (ab- L. C. Burnett. M. S. A., M. S., Chief ^ent on leave) in Cereal Breeding .. , ^, . „ M. E. Olson, B. S., M. S'., Field Ex- L. W. Forman. B. S. A., M. S., Chief periments in Field Experiments . , ^ EI. Angell. Soil Surveyor John Buchanan, B. S. A Supermtend- j p gjg-g. g g pie\& Exnerimenta ent of Co-operative Experiment.^ q p j^^^^^,-, g g., m. S., Assistant R. S. Potter A, B.. M. b.. Pn. u.. . ^^ rmno Assista-t Ciiipf in Soil Chemistrv ^ ' Hnn=-on B q Eipli Exneri- R g S'lvdpr. B. S., Assistant in Soil H. P. Hanf:on. B. ^-^ -l^ield iixpen pviop^isfv ments (absent on leave) H. "^'■. .Tobn^nn B S.. M. S . Assist- ant in Soils (absent on le^-'^o'i ANIMAL HUSBANDRY W H Pew B S A Chief L. S. Gillette. B. S.. M. S., Assistant J. M. Evvard, M, S., Assistant Chief Chief in Dairy Husbandry _ in Animal Husbandry and Chief in A. C. McCandlisb, M. S. A., Assistant Swine Production in Dairv Husbandry R. Dunn. B. S., Assistant in Animal Rodney Miller. B. S. A., Assistant in Husbandry Poultry Husbandry H. A. Bittenbender, B. S. A., Chief in Poultry Husbandry BACTERIOLOGY R. E. Buchanan, B. S., iPh. D., Chief : Associate in Dairy and Soil Bacteriology BOTANY L. H. Pammel, B. Agr., M. S., Ph. D., I. E. Melhus, Ph. D., Chief in Plant Chief Patbolo.gy Charlotte M. Kin?, Assistant Chief in Botany CHEMISTRY A. W. Dox, B. S.. A. M., Ph. D., Chief S. B. Kuzirian, A. B., A. M., Ph. D., (absent on leave) Assistant W. G. Gaessler, B. S., Acting Chief G. W. Roark. Jr., B. S., Assistant A. R. Lamb. B. S., M. S'.. Assistant Lester Yoder, B. S., M. S'., Assistant DAIRYING M. Mortensen. B. S. A., Chief D. E. Bailey. B. S., Assistant Chief B. W. Hammer, B. S. A., Chief in in Dairying Dairv Bacteriology ENTOMOLOGY R. L. Webster, A. B., Acting Chief Wallace Park, B. S,, Assistant in Ag- riculture FARM MANAGEMENT H B Munger, B. S., Chief O. G. Lloyd. B. S., M. S'., Assist. Chief HORTICULTURE AND FORESTRY S. A. Beach. B. S.. M. S., Chief J. B. Kendrick, B. A., Research As- T. J. Maney, B. S., Chief in sistant in Pomology Pomology ' A. T. Erwin, M. S., Chief in Truck Harvey L. Lantz. B. S., Assistant in Crops Fruit Breeding Rudolph A. Rudnick, B. S., Assistant W. E. Whitehouse, B. S., Assistant in Truck Crops in Pomology G. B. McDonald, B. S. F., M. F., Chief Andrew Edward Murneek, B. A., Re- in Forestry search Fellow in Pomology Frank H. CuUey. B. S. A., M. L. A., Chief in Landscape Architecture RURAL SOCIOLOGY G. H. Von Tungeln. Ph. B., M. .\., Chief VETERINARY MEDICINE C. H. Stange, D. V. M., Chief GENERAL OFFICERS F. W. Beckman, Ph. B., Bulletin Editor F. B. Colburn, Photographer Gretta Smith. A. B., Assistant to Bulletin Editor C, E. Brashear,_B'._S. A., Assistant to Director """ JSaRYc QF CONGRESS j . .RECEIVED Soil Inoculation With Azotobacter* BY PAUL EMERSON. Following' tlie discovery of the nitrogen fixing' powers of the symbiotic bacteria in the soil, early investigators found that the power of utilizing the free atmospheric nitrogen was not confined to the symbiotic bacteria alone. They noted increases in soils which had borne no legumes and they found that fallow soils in particular increased appreciably in nitrogen content. These facts stimulated researches which led to the discovery of many forms of bacteria which are able, when growing alone, to fix nitrogen from the air. The chief of these is now known as the azotobacter group. It seems likely that the azotobacter will prove more effective in fixing nitrogen than the symbiotic bacteria, although the general requirements of the two classes of organisms are very similar. The azotobacter are active in practically all soils regardless of the kind of crop grown when conditions for their growth are satisfactory. These conditions are probably much the same as for the symbiotic bacteria except that these latter organisms require the presence of a specific legume for fixing the greatest amount of nitrogen. Azotobacter require a certain amount of carbonaceous material in the soils and are usually stimulated by . a small amount of nitrogen, but the exact optimum conditions for their growth are as yet unknown. These organisms are active in causing nitrogen increases in many soils, but the feasibility of introducing them into the soil or of attempting to increase their nitrogen-fixing powers by artificial means, and the effect of the presence of growing plants on their efficiency are ques- tions as yet unanswered, although Lipman has indicated that under proper conditions successful inoculation may be accom- plished in soils and Bottomley has successfully grown pure cul- tures of these organisms in the presence of growing plants with favorable results. HISTOBIGAL Beijerinck (2) isolated and described the first azotobacter (in 1901) . He found two species, one of which he named Azotohacter chroococcum and the other Azotohacter agilis. The former was isolated from the soil and the latter from a sample of water taken from one of the canals in the city of Delft. Two years later Lipman (36) added a third species, A. vinelandii, to the list and the following j^ear isolated and described two more, giv- ing them the names of A. deijerinckii and A. woodstownii. Of the five organisms of this species, A. cliroococcum., A. deijerincMi ♦Thesis submitted in partial fulfillment of the requirements for the Degree Df Doctor of Philosophy at the Iowa State College. '■h ■'>«<•■ ...' 28 and A. vinelandii are considered the most important in soil in- oculation studies. The frequency with which investigators in all parts of Europe and America have isolated azotobacter from various soils, indi- cates that they are widely distributed. Christensen (10) found that they were present throughout northwestern Europe, the activity of the organism apparently depending on the basicity of the soil. This view was later supported by the works of Fisher (14), Lohnis and Pillai (45) and others. Ashby (1) studied the soils of Mombasa, East Africa, Cairo, Egypt and Rothamsted, England and found azotobacter forms present in most eases. Lipman and Burgess (42) working with forty-six samples of soil from variotis parts of the world, found that over one-third of them contained azotobacter, the predominant form being A. chroococcum. Many of the soils examined were museum specimens and had been kept in tightly stoppered bottles for long periods of time. DESCRIPTION OF AZOTOBACTER. Bei.jerinck characterizes the azotobacter r-s stout bacteria, 4-6 microns or less in length, sometimes longer, occurring as large diplococci or short rods in young cultures, the hyaline cells often containing a vacuole and the entire organism enclosed in a mucilagenous wall of varying thiclaiess. They have a single polar flagellum or bundles of 4-10 polar flagella of about the same length as the organism itself. Beijerinck found no spores. Vagler (65) writes that the older colonies produce involution forms similar to those of yeasts while Heinz (22) and Fisher (15) showed that the organisms can resist drying for six to nine months. Later investigations by Mulvania (50) and Lohnis and Smith (47) demonstrated that the organism produces spores and completes a very complicated life cycle. Descriptions of azoto- bacter and detailed cultural characteristics of the organism were given by Lipman (35), Bei.jerinck (2), Prazmowski (54), "Warm- bold (70), Bonazzi (6), Lohnis and Westerman (48), Lohnis and Hanzawa (44), Jones (27) and others. ACTIVITIES OF AZOTOBACTER. Beijerinck first claimed that the isolated pure cultures of azotobacter were able to fix the atmospheric nitrogen in appre- ciable amounts; later, however, when working with Van Delden (4), he retracted this statement, claiming that pure cultures did not have this ability and that only in the presence of very small celled organisms called radiobacter could the free nitrogen of the air be fixed in the soil. Grcrlach and Vogel (18), Heinz (23), Lipman (37) and Freudenreich (17) proved conclusively that the earlier conclusions of Beijerinck were correct and that 29 the organism may fix considerable amounts of nitrogen in pure cultures. Lipman accounts for the fact that Beijerinck did not get a fixation of nitrogen in pure cultures by showing that the organism will not fix nitrogen unless the reaction of the medium is made neutral or slightly alkaline. When Beijerinck later accepted this suggestion he found that his pure cultures were able to fix atmospheric nitrogen. STUDIES OF AZOTOBACTER. Ver}' few investigators have attempted tO' inoculate soils with azotobacter or other non-symbiotic nitrogen fixing bacteria under conditions approximating those in the field. The influence of various kinds of sugars, cellulose, inorganic salts, and various organic compounds on the nitrogen-fixing power of the organ- isms have been studied extensively. Gerlach and Vogel (19), Pringsheim (55), Krainsky (33), Koch (30), Hoffman and Ham- mer (25) and Stranak (61) have found that various sugars and cellulose materially increase their nitrogen fixing powers while Fisher (16), Christensen (10), Lohnis and Pillai (46), Wilfarth and Wimmer (59) Kaserer (28), Rosing (59), Vogel (66), Greaves and Anderson (20) and Pringsheim (56) have shown that small amounts of lime, very small amounts of nitrogen, various inorganic salts and even a very small amount of arsenic will stimulate the nitrogen fixing power of the organisms in the presence of certain carbon compounds. Stoklasa (60) studied the products of the activities of the azotobacter organisms, con- fining his researches largely to the amounts and kinds of gases produced under different circumstances, under the influence of various substances supposed to be energy sources, and under varying temperature conditions. His results have been more or less confirmed by the works of Thiele (64), Hoffman (24), Keller- man and Smith (29) and Ehrenberg (13). The activity of the azotobacter in soils in general, and partic- ularly under laboratory conditions, was fully shown by the works of Lipman (39), Voorhees and Lipman (68), Lohnis (43), Kuhn (34), Freuclenreich (17), Dvorak (12), Remy (57), Remy and Rosing (58), Jacobitz (26), Stranak (62), Headden (21), Peter- son and Mohr (52), Koch and Seydel (31), Omeliansky and Ssewerowa (51), Warmbold (71) and others who demonstrated that under various conditions and in almost every type of known soil these organisms are able to fix appreciable amounts of the free atmospheric nitrogen. Only a few of these investigators, however, have made any attempt to secure an active flora of these organisms in the soil. Vogel (67) inoculated pure cultures of azotobacter into soils that had been treated with grape sugar, in some cases adding comparatively large amounts of nitrate of soda. In pot experiments with oats and mustard, increases were 30 noted for the inoculated series, altho the pots receiving nitrate of soda gave the greatest yields. When the experiment was re- peated in the field the inoculated plots gave smaller yields than the uninoculated, and the inoculation appeared to have an in- jurious effect upon the crop. A short time later Lipman and Brown (41) tried inoculation experiments with A. vinelandii and A. beijerinekn. They sunk four foot cylinders open at both ends into- the soil, filled the cylinders with soil and inoculated the soil with the organisms. The first summer the soils were left bare and then a rotation of crops was followed and oats, corn and rye grown in succession. While considerable variation Avas found in the nitrogen content of the crops and in the dry weight, the general conclusion reached was that the activities of the organisms did not increase the nitrogen content of the soil. The results do not preclude the pos- sibility that inoculation with the organisms in question may be made of practical value, provided proper conditions for the best growtli of the organisms are secured. Bottomley (7) and Bot- tomley and Hall (9) experimented with oats, barley and some root crops, and arrived at the same conclusions as did Lipman and Brown. Stranak (63) also inoculated soils with azotobacter and found a pronounced increase in the growth of potatoes, grain and beets. Altho the experiments dealing with the inoculation of soils with azotobacter have been inconclusive, it is believed that under proper conditions such inoculation may be extremely profitable. EXPERIMENTAL The wdde distribution of non-symbiotic nitrogen fixing bacteria in many types of soils is practically parallel with the distribution of the symbiotic organisms, and since it is practical and profitable to inoculate soil with the latter, even tho the particular organism may be present, the following cjuestions quite naturally arise: 1. If the azotobacter are not present in the soil, can inocula- tion be profitably accomplished? 2. What soil conditions are necessary for the greatest fixa- tion of nitrogen by these organisms ? These questions have an important bearing on the problem of the maintenance of permanent fertility in soils from the nitrogen standpoint and may govern the choice of the proper method of farming. Some commercial concerns have placed cultures on the market, claiming that they contain sufficient numbers of the non- symbiotic nitrogen fixing bacteria to enable the farmer to solve his nitrogen problem without growing legumes. However, results of experiments showing that such cultures are capable of inoculat- ing the soil were not found in the present investigation. 31 INCREASING THE NITROGEN FIXING POWER OF PURE CULTURES. Very little work has been done along the line of breeding pure cultures of bacteria to an increased efficiency in their specific actions, in fact, practically all the experiments have been carried out with the idea of finding a method whereby the organism could be kept alive for long periods without periodic transfers. The earliest investigation along this line was that of Czaplewski (11) who limited the amount of air in the tube by saturating the plug with paraffine. Later Lunt (49) found that certain cultures of water bacteria may be kept alive much longer in sterile water than in ordinary culture media. In some cases he kept certain organisms alive for two years by this method. BoUey (5) secured good growths of B. amylovorus and Bad. dianthi in agar and in bouillon by making transfers from cultures that had been hermetically sealed for nine years. It is not stated whether or not the organisms were tested for their pathogenicity and hence their virulence is left in doubt. This work supports that of Czaplewski in showing that cultures can be kept alive for long periods of time if the transpiration is reduced to a minimum. Some commercial concerns claim that they are able to increase the efficiency of their particular cultures of legume bacteria by alternate inoculations first on agar, then into sterile greenhouse soil, growing the specific legume to which the organism in ques- tion is adapted, and re-isolating the crsanism from the nodules produced on the roots of the legume. If this is possible for the symbiotic bacteria then it seems probable in the case of the non- symbiotic organisms. The following cpiestions naturally suggest themselves : 1. Can the nitrogen fixing power of azotobacter be increased by periodic transfers on nitrogen free media? 2. Can the nitrogen fixing powers of azotobacter be increased by growing the organism in the presence of growing plants ? In outlining work to answer the above questions it was realized that a large number of bacteria should be used. A number of large celled nitrogen fixing organisms that had all the staining reactions of the azotobacter type and closely resembled it in size and shape, were isolated in pure cultures from soil secured from the humus plots at the Iowa station and were designated with laboratory numbers. At the same time pure cultures were se- cured and their activities determined along with these of the unnamed cultures. The pure cultures were kindly furnished by Dr. J. G. Lipman of the New Jersey Agricultural Experiment Station and also by the American Museum of Natural History of New York, 32 MEDIA USED. The nitrogen free medium used thruout the experimental work was a modification of that proposed and used by Lipman (35), and its composition was as follows: Distilled water 1,000 cc Di-potassium phosphate 0.2 grams Magnesium sulphate 0.2 grams Calcium chloride 0.02 grams Dextrose 10.0 grams 10% Ferric chloride solution 2 drops The solution was brought to boiling and made neutral to phenolphthalein by the addition of N/10' NaOH. If a solid medium was desired 1 % powdered agar w^as added. Sterilization was accomplished by placing in the autoclave at ten pounds for 20 minutes. Inoculation was secured by scraping off a two days' growth from the agar slants with a sterile needle and transferring it to flasks containing 50 cc. of the above solution. In onler to de- termine w^hether the nitrogen fixing power of the organisms was stimulated by the addition of nitrogen, the above solution with the addition of 1 mg. of nitrogen as sodium nitrate was used. PRELIMINARY STUDIES. All of the organisms of the azotobaeter type including both the pure cultures and the unnamed cultures, were inoculated into 50 c. c. of both of the above solutions and tested for their nitrogen fixing powers. The inoculated solutions were incubated for tliree weeks at room temperature (22-25° C) and then Kjeldahlized. The amount of nitrogen fixed by each organism in the different solutions is shown in tal>le I. The same cultures were transferred 12 times at three to four day intervals on nitro- gen free media and their nitrogen fixing power tested in solu- tioiis with and without nitrogen. The results appear in table II. The laboratory organisms used in table I had been freshly isolated and purified from the soil, the named cultures had been kept on agar slants for varying periods of time. During the time that the inoculated culture solutions were incubating the transfers were being made in preparation for the inoculations for table II. Comparing the two tables we find that a ma.jority of the or- ganisms decreased in their ability to fix atmospheric nitrogen, altho a few showed a slight increase or at least retained their ef- ficiency. From these the following eight were selected for fur- ther study: No. 4, No. 22, No. 26, No 27, A. vinelandn, A. chroo- cocciim., A. heijerincMi and A. cliroococcum (HCM) . These eight organisms were studied under both laboratory and greenhouse conditions, 33 TABLE I^NITEIOGIEN-TTKATTON OBT PUBE ClUXiTUKES IN SQiLUTION WITH AJNID WITHODT MTKiOGEN. N. Fixed in Mgs. Organism c " ?, ■sjl Solution with Nitrogen Lab. No. 1 . - 2.24 2.38 0.28 0.98 i.9e 3.22! 0.42 0.42 0.42 lost 1.12 2.80 7.141 0.2S 1.12 0.56 0.50 0.42 0.70 0.70 0.2S 0.56 a. 12 lost 4.20 0.84 0.70 0.84 1.96 V.U Lab. No. 2- 1.54! Lab. No. 3 1.99 Lab. No. 6 Lab. No. 7 -__ 1.82 1.68 Lab. No. & 3.10 Lab. No. 10 0.84 Lab. No. 11 0.70 Lab. No. 12 - 1.40 Lab. No. 14 Lab. No. 15 lost 1.13 Lab. No. le - - lost Lab. No. IS Lab. No. 19 - __ U.43 1.54 Lab. No. 20- -. 1.90 Lab. No. 21 __ ... _ 2.10 Lab No. 22 2.53 Lab. No. 23- - . -- 2.52 Lab. No'. 24- 1.82 Lab. No. 25- - 1.82 Lab No. 26- 2.10 Lab. No. 27 A. vinelandii 5.60 2.66 A. chroo'coecum (IHCIM) A. chroocoecum A. chi'Oococeum (Oolo). A. beijerinekii 3.08 1.54 2.52 1.68 A. bsijerinekii No. 5 2.38 TAiBiLE n—NITBOOEN- FIXATION BT FORE! COLTURES IN SOLUTION WITH AND WITHOUT NITRIOGEN. After each org-anism has been transfer- red twelve times on nitrogen- free miedia at three to four day intervals. N. Fixed in Mgs, Organism 3 M .2 S, Lab. No. 1 -- 0.84 0.14 0.14 1.82 0.00 0.28 O.OO 0.98 0.00 0.00 0.42 0.00 O.OO 0.00 0.00 0.98 0.42 3.'53 2.10 0.00 0.00 1.12 1.40 0.00 1.12 2.52 O.OO 0.42 1.12 1.12 Lab No'. 2 O.OO Lab. No. S 0.99 Lab. No. 4 0.98 Lab. No. 6 _ 0.84 Lab No. 7 - 0.28 Lab No. 9- 1.26 Lab. No. 10 0.00 Lab No. 11 - - 1.6S 0.28 Lab. No. 14- 1.121 Lab No. 15 1.40 Lab No. 16- -— 0.14 Lab No 18 0.42 Lab. Noi. 19 --- 0.42 Lab No 20 1.54 Lab. No. 21 0.84 Lab. No. 22 Lab No. 23 1.12 1.54) Lab No 24 1.63 Lab No. 25 0.2S Lab No. 26. - - 1.82 Lab. No. 27- 2.52 0.00 A. chroocoecum A. chroocoecum (HICM) A. chroocoecum (Colo). 1.82 1.12 1.26 2.92 A. beijerinekii No. 0.— 0.00 LABORATORT STUDIES. The laboratory studies were arranged in a series of three ex- periments as follows : 1. To determine the effect of transfers made every other day on the nitrogen fixing- power of the organisms. 2. To determine the effect of transfers made once each week in sand cultures variously modified. 3. To determine the effect of growing four of the organisms on both agar and sand in large flasks with and without the presence of growing plants. 34 Series 1. To Determine the Effect of Transfers Made Every Other Day on the Nitrogen Fixing Power of the Orga!msms. Using the eight selected organisms transfers were made every other day on the nitrogen free medium for a period of three weeks. It was feared that such rapid transferring for so long a period on a medium practically free from nitrogen would re- duce the vitality of the organisms, accordingly each fifth trans- fer was made on a modification of the medium consisting in the addition of one milligram of nitrogen as sodium nitrate to each liter of the regular dextrose agar. At the end of the transfer period the organisms were inoculated into the nitrogen free and nitrogen containing solutions incubated for the same periods of time and the amount of nitrogen fixed determined by Kjeldah- lizing. The results of the determinations are shown in table III. TABIE nr— TIHiE EPFEiOT OF TIHuANiSIEEiHlS iMADE: EVElE;T OTHER DAT FOB FOUIB WEEOBCS ON THE' NTTRlOGEiN MXINIG POWEIB OF THE OCROAJSfTSMS . Organism Nitrogen Fixed in Mgs. Solution without Nitrogen Solution with Nitrogen Lab. No. 4 Lab. No. 25 Lab. No. 26 Lab. No. 27 A. vinelandii A. chrooeoecum A. ehrooeoccuni CHOM). A. beijerinckii (a) (b) (Av.) (a) (b) 0.14 0.42 0.28 0.70 0.42 0.00 O.OO 0.00 0.14 0.56 0.1+ 0.14 0.14 0.28 0.98 0.2S 3.. 50' 1.98 0.70 0.98 0.28 0.42; 0.35 0.98 0.98 r^r. 0.2S 0.42 0.98 1.12 2.66 lost 2.66 1.40 1.40 0.S4. 2.66 1.79 0.28 0.28 (Av) 0.56 0.35 0.63 0.84 0.98 l.Oo 1.40 0.28 That these transfers should have been made at longer intervals is evidenced by the fact that tables I and II showed that 12 of the cultures had increased in efficiency after they had been transferred every three days for 36 days. However, during the latter work the organisms did not show any indications of a loss of vitality and the growth at all times was vigorous and rapid. Tabic III shows a decrease in the nitrogen fixing powers of all the organisms except in the case of A. diroococcum (II CM) which appears to have retained its efficiency thruout the ex- periment. Series 2. To Determine the Effect on the Nitrogen Fixing Power of Transfers Made Each Week in Sand Cultures. In the following experiment sand was used instead of agar as the basis for the medium. Ground oats straw, ground red clover hay and either the regular dextrose solution, or the dextrosf; solution containing nitrogen were added. The tests were carried out in tubes arranged as follows : 35 6.25 gr. sand+2.5 cc N. free dextrose solution. 6.25 gr. sand-i-2.5 cc dextrose solution containing 0.2 gr. NaNOs per liter. 6.25 gr. sand + 3.5 cc N. free dextrose solution + 0.1 gr. clover nay. 6.25 gr. sand-|-3.5 cc N. free dextrose solution+0.1 gr. oats straw. The organisms were transferred directly from the slants into the tnbes and there allowed to incubate at room temperature for seven days. A small portion of the sand was then transferred to a fresh tube of the same medium as the original. As this par- ticular experiment did not directly follow the others the efficiency of the organisms was tested before they were inoculated into the sand. Table IV shows the amount of nitrogen fixed by the pure TAjB^LlE' IV-^TIBTE NITEiOOEN MXINO POWEIK OlF THE- PORIE CICPLiTUEElS IMMEIDIAT'ElLY BiEIPIOBIE: the SIANID' CIU'LITHJIRE' EXPEIRIIlMElN'TlS. Organi; [Solution witnout Nitrogen Solution with Nitrogen Lab. No. 4 Lab. No. 22 Lab. Nc 26 Lab. No. 27 A. vinelandii A. ehroococcum A. chroococcum (HJQM)- A. beijerinckii (a) 0.14' 0.00 O.OO 0.00 O.OO O.OO 0.00 0.98 (b) 0.70 0.00 0.14) 0.00 0.00 0.14 O.OO 0.84 (Av.) 0.42 O.OO 0.07 O.OO 0.00 0.07 0.00 0.91 (a) (b) 0.28 1.54 3.36 3.0s 2.914 2.66 2.38 2.52 1.40 1.40 2.80 2.66 2.52 2.38 2.94 lost (Av. 0.91 3.22 2.80 2.45 1.40 2.73 2.49 2.94 cultures at the beginning of this series of incubation, and the same methods as in the previous experiments. At the end of the fourth transfer period, i. e., four weeks after the start of the experimental series, the organisms were inoculated into dextrose solution and their nitrogen fixing powers determined. After four more weeks of transferring or in all eight weeks the final inoculation into dextrose solution was made. The influence of the oats and clover in the presence of sand on the nitrogen fixing power of the organisms used is shown in tables V and VI, by the fact that both the large celled organisms of the TAtBLE v.— NTTIBOiGEN FIXED BY THE PUBE COLTIPREIS APTE-B FOUR TBAiNSFElBS IN ISLAND' AT PElRilODS! 'OF' SEVEN D'AYS EACH. Nitrogen Fixed in Mgs. Organism dex. sol. dex. sol. + N dex. S0I.+ oats straw dex. S0I.+ clover hay Lab No. 4 0.28 0.07 0.77 0.42 0.14 0.21 O.07 0.14 0.35 0.2s 0.07 0.351 0.21 0.21 0.14 0.28 0.28 0.00 0.14 1.27 0.42: 0.07 1.19 0.35 0.98 Lab. No. '» 0.35 Lab. No. 26 Lab. No. 27 0.00 0.42 0.28 A. ehroococcum A. chroococcum) (HCIM) A. beijerinckii 0.21' 0.42 0.28 36 "HAiBLE' VI— NITRlOOEilSr iPIXED EiT THE FUBIE' OULTUElElS AFT'EIB EIGHT THAaSHSE'EIRlS AT PiElRODOIDlSi OP SEiyEN DAYS- EACH. Organism Lab. No. 4 Lab. No. 23 Lab. No. 20 Lab. No. 27 A. vinelandii A. chrooeoccum A. chrooeoccum! (HCiM) A. beijerinckii Nitrogen Fixed in Mgs. dex. sol. 0.30 0.20 0.70 0.20 0.20 0.30 0.10 0.00 dex. sol. dex. sol.+ + N oats straw 0.20 0.30 0.20 0.20 0.40 o.oo 0.10 0.40 0.40 0.40 o.ao 0.20 0.40 0.40 0.00 0.20 dex. S0I.4- clover hay 0.40 2.00 1.00 0.50 0.50 lost 1.40 O.b'O azotobaeter type and the azotobaeter themselves, made gains in their nitrogen fixing powers. There was no distinct gain due to any one kind of carbonaceous material. Of the six organisms showing gains A. cJiroococcum made the most notable, especially in the presence of the oats straw. The nitrogen fixing power of No. 4 appears to be rather constant thruout the series, with no appreciable gain or loss. A. heijerinckii showed a decided loss in its power to fix nitrogen in each of the four media, but gave a slight indication that in the presence of the clover hay it might be slowly regaining its power. Series 3. To Determine the Effect of Growing the Organisms on Both Agar and Sand With and WitJiont the Presence of Growing Plants. The main points considered in this experiment were: An increase in the surface area over which the organism could grow; an increase in the time between transfers and the grow- ing of the organisms in the presence of an undetermined species of alga3 and with growing oats and red clover plants. Two liter Erlenmeyer flasks were used and arranged in the following man- ner conforming to the outlines of the experiment: Flask No. 1. 1000 cc N. free dex. agar+1 gr. CaCOj planted to oats. Flask No. 2. 1000 cc N. free dex. agar-fl gr. CaCO 3 planted to red clover. Flask No. 3. 1000 cc N. free dex. agar-f 1 gr. CaCOa planted with an undetermined species of algse. Flask No. 4. 1000 gr. pure quartz sand + 180 cc N. free dex. solution neutralized with CaCO:!, planted with oats. Flask No. 5. 1000 gr. pure quartz sand + 180 cc N. free dex. solu- tion neutralized with CaCOs, planted with red clover. Flask No. 6. 1000 gr. pure| quartz sand+180 cc solution without dex. neutralized with CaCOs, planted with an undetermined species of algae. Check flasks of sand and dextrose agar. 37 The flasks of agar were sterilized in the autoclave at ten pounds for 30 minutes, but the flasks of sand were sterilized at 15 pounds pressure for four hours once a day for three consecutive days. Bacteriological tests on the sand at the end of that time showed it to be sterile. The culture of the algae used was so closely associated with a bacterial growth that a separation would have required a long time. For that reason it was not purified but was grown in sterile distilled water, for about three months before inoculation. The inoculation of the algae was made in the flasks about two weeks before the inoculation vrith the azotobacter cultures in order that the algae might make a sufficient gTowth to supply Vie bacterial cultures with the proper amount of carbonaceous material. To prevent contamination by the oat and red clover plants, the seeds were soaked three minutes in a 1-500 solution of mercuric chlor- ide, washed in sterile distilled water three times and then planted in sterile agar plates. By this means the seeds were sprouted and those which were contaminated were discovered and rejected. The sprouted seeds were transferred from the plates to the flasks by means of the platinum needle. A block of the agar containing the sprouted seed was cut out and placed in the proper position on the medium in the flask. The flasks were then carefully ob- served for five days tO' insure the absence of contamination. As all the flasks contained growing plants no attempt was made to exclude the light, but neither were they placed in the direct sunlight. They were kept on a table about eight feet from a large window facing the west. All the flasks were plugged with non-absorbent cotton and after inoculation a cap of paraffined paper was placed over the mouth and held in place with a rubber band. While the plants did not develop rapidly the oats grew much faster than the clover for about three weeks, after which time both began to lose chlorophyl and by the end of the five weeks' experimental period, the majority of the plants had died. The oats and clover in the flasks inoculated with A. chroococcum (HCM) and the clover in the flasks inoculated with B. radicicola showed a slight gain in growth and altho far from vigorous at the end of the experiment were still alive and growing slowly. ORGANISMS USET>. The organisms used were A. chroococcum (HCM), A. vine- landii, A. heijerinckii and for the purpose of comparison, B. radi- cicola isolated from the nodules of sweet clover. The latter were isolated and purifled especially for this series, and introduced to compare the effects of symbiotic and non-symbiotic organisms on the growth of the plant used. The results secured with it, how- ever, were of no great significance. 38 After the bacteria had remained midisturbed in the flasks for five weeks, transfers were made directly from the flasks into 50 CO. of the nitrogen-free dextrose solution, incubated for three weeks, and the nitrogen fixed determined in the usual manner. The total amount of nitrogen fixed by the bacteria themselves, as well as the amount fixed by the bacteria but due to the stimulative action of the plants on the bacterial activities, is shown in table VII. There was a stimulation of the nitrogen fixing power of the organisms due to the presence of a growing plant, especially no- ticeable in the case of A. vinelandii and A. chroococcum (HCM) and to some extent in the case of A. heijerinckii. A. vinelandii was stimulated thruout the entire series except when grown in sand in the presence of the algae. The oats and algae showed no TAB'LE Vn-THE EFFElOT OF' GEIOWKSTG' PLlAW^T'S; ON THIE NITROGEN FIXING FOWE'Ri OF PiU.RE' ICUXTUIRIES: Inoculum Medium Plant Used (a) (b) Nitrogen Fixed in Mgs. (Av) Z-5 Algae agar AJgae Vinelandii Vinelandii Vimlandii Vinelandii Vinelandii Vinelandii Vinelandii Vinelandii Olirooc'occmn (HCM) Chroococcum (HCM) Ohroococcum (HOM.) ._- Chroococcum (HOM) ..- Chroococcum (HCM) ..- Cbroococcum (HCM) .— Chroococcum (HOM) _.- Chroococcum (HCM) .._ Beijerinckii Beijerinckii Beijerinckii Beijerinckii Beijerinckii Beijennckii Beijerinckii Beijerinckii B. rad B. rad. B:. rad B. rad rad.. check sand check agar. sand- agar- agar- clo. clo. ClO'. ClO'. clo. rad., S. clo. agar sand and and agar sand agar agar igar sand sand sand agar sand agar agar Tgar sand sand sand agar agar agar sand sand Isand check check oats red clover- oats red clover- check check oatsi red clover. - algae oats red clover-- algae check check oats red clover- - oats red clovsr- oats' red clover- oats red clover. 0.84 0.84 0.^ 1.40 0.98 1.19 1.12 0.98 1.05 1.40 1.40 1.40 3.66 3. 53 3.59 2.10 2.38 2.24 4.20 lost 4.20 4.20 4.06 4.13 4.48 4.06 4.27 2.52 2.80 2.66 O.OO O.OO O.OO 0.28 0.14 0.21 0.28 0.44 0.30 1.82 3.22 3.52 S.92I 3.50 3.71 0.28 0.56 0.42 0.28 lost 0.28 3.22 5.18 4.20 0.00 0.00 O.OO 0.28 0.42 0.35 0.14 0.00 0.07 1.40 1.40 1.40 O.OO O.OO 0.00 1.39 1.39 1.39 1.39 1.39 1.39 1.39 1.40 1.40 0.14 0.14 .014 0.14 0.a4 .014 o.m 0.56 0.56 0.42 O.OO 0.21 0.14 0.00 0.07 0.42 0.50 0.4191 0.84 0.84 1.19 1.19 3.5t 1.05 2.54 2.24 1.05 1.19 3.36 1.09 2.31 4.13 1.40 2.73 4.27 1.40 2i.8'7 1.47 1.40 0.07 0.36 0.00 01.35 2.52 0.00 2.52 2.S7 0.00 2.87 0.42 0.21 0.21 0.28 0.21 0.07 3.01 0.21 2.80 0.07 0.00 o.or 1.40 0.00 1.40 1.39 0.35 1.04 1.39 0.3S 1.04 0.21 0.35 0.14 0.14 'o'.n 0.07 39 difference when gTown on the agar and in the sand medium the greatest stimulation was produced by the red clover. The activi- ties of A. chroococcum were stimulated to the greatest extent by the presence of algae in both sand and agar, the oats gave a poor stimulation in both cases, and red clover gave good results in the agar but not in the sand. The nitrogen fixing power of A. 'beijerinckii was retarded by the presence of algae, but was stimulated by red clover in both the agar and sand. The oats stimulated this organism only when grown on the agar. The nitrogen fixing power of B. radicicola was so low thruout the experiment that the results are not con- sidered. CONCLUSIONS FROM LABORATORY STUDIES. 1. Transfers made on a nitrogen free dextrose agar more often than once each week were detrimental to the nitrogen fixing power of azotobacter and other large celled nitrogen fixing organisms of the same type. 2. Transfers made once each week into a pure sand medium containing some carbonaceous material were beneficial to the nitrogen fixing power of the azotobacter in general, but the effect on A. heijerincMi was detrimental. 3. The nitrogen fixing power of A. vinelandii was stimulated to a marked extent when grown in large flasks for five weeks in the presence of red clover and oats on both agar and sand. It was stimulated by the presence of algae when grown on agar but not when grown on sand. The nitrogen fixing power of A. chroo'coccum was stimulated markedly when grown on agar for five weeks in the presence of growing oats and red clover, but to a less extent when grown with the same plants in sand. The greatest stimulation for this organ- ism was produced by growing it in the presence of algae in either sand or agar for the same period of time. 5. The nitrogen fixing power of A. beijerinckii was stimulated by the presence of red clover when grown on either sand or agar, and by oats when grown in sand. Algae in either agar or sand appeared to have a depressing effect on the nitrogen fixing power of this organism. GBEENHOUSE STUDIES. At the conclusion of the first experiment the eight organisms used in the laboratory series 1, 2 and 3 were also inoculated into soils in pots in the greenhouse. Ground oats straw or ground clover hay was added to these soils and the nitrogen fixing effi- ciency of the organisms both in fallow soils and in the presence of growing oats plants determined. Three experiments were car- ried out in this test, as soon as the soil in which one crop had been 40 grown was sampled, it was immediately reseeded and another crop grown. Strict account was kept of the amount of nitrogen added in the seed and in the organic matter. The dry weight of the crop and the N. content as well as the nitrogen content of the soil was determined at the end of each experiment. The soil used thruout the experiment was of the type classified by the United States Bureau of Soils as Miami silt loam, and according to tests in the laboratory did not contain azotobacter or any similar organisms. A large amount of this soil was thor- oly air dried^ sieved and mixed. Ten pounds were placed in each of eighty glazed pots, seventy-two of which were given the fol- lowing treatment : Half, or thirty-six pots received an applica- tion of 22.68 grams ground oats straw, and the other half received an equivalent amount of ground red clover hay. This application (22.68 grams) was equivalent to a five-ton application of this ma- terial per acre. The ground material was thoroly incorporated in the soil, which was packed firmly in the pots. The pots used were «lazed on the inside and made tight so there was no loss by leaching, neither was there any drainage provided. METHODS OP INOCULATION. The inoculum used was the dextrose solution described above. 1500 c e were placed in each of six 2 L. flasks, inoculated with the organism desired and incubated for seven days. Microscopic ex- aminations were made at the end of the incubation period to in- sure vigorous growth and the purity of the culture. 150 cc of the solution was used as the inoculum for each pot. This was poured over the surface of the dry soil and washed into it by the addition of sufficient water to bring the moisture content up to the optimum, in this case 25%. The pots were then weighed, covered with a cloth, and allowed to remain undisturbed for three days, in order to permit the moisture to become thoroly distrib'- uted thruout the soil. The pots were then arranged in the follow- ing manner and seeded to oats. Thirty grains of Early Champion oats were planted in each pot at each seeding. They were planted at five points. One in the center of the pot and the other four were arranged between the center and the edge at equal distances apart. Six seeds were planted at each place and when the plants appeared they were thinned out and but one plant left in each place. The discarded plants were allowed to remain and decay on the soil in the pot from which they were drawn. The length of the growing period was determined by the ap- pearance of the seed-bearing spike. This period varied slightly in each of the series, the first closed in sixty-three days, the second x'n sixty-nine days, and the third in seventy days after planting. 41 PLAN OP EXPERIMENT Pot. No. Treatment Inoculation 1— 3 2— i 5h- 7 6— 8 9—11 10—12 13—15 14—16 17—191 18—20 21—23 22—24 25—27 26—28-- 29—31 30— .32L 33-33 34—36 37—39 38-^0 41—43 42—44 45-4T 46—48 49-51 50—52 5S—5S 54—50 57—59 £8—60 61—63 62—61 65-67 66—68 69—71 70--72 73^74 75—76 77—78 79—80 Fallow iCropped Fallow Oropped Fallow Cl-opped Fallow lOlropped Fallow iCtopped Fallow Cropped Fallow (Oropped Fallow iCtropped Fallow 'Cropped Fallow Cropped FaUow Cropped Fallow iCtopped Fallow lOtopped Fallow Ctopped Fallow Chopped Fallow Cropped Fallow Chopped Fallow (Ctopped Fallow Cropped Fallow iCtoppeQ Oats straw Oats straw Olover hay Clover hay Oats straw Oats straw Clover hay 'Clover hay Oats straw Oats straw 'Clover hay Clover hay Oats straw Oats straw 'Clover hay Clover hay Oats straw Oats straw Clover hay Clover hay Oats straw Oats straw Clover hay Clover hay Oats straw Oats straw Clover hay Clover hay Oats straw Oats straw iClover hay Clover hay Oats straw Oats 'straw Clover hay Clover hay Oats straw Oats straw Clover hay Clover hay A. chroococcum (HCIM) A. chroorocciim (HOM) A. cliriKirdcciini (HOM) A. clii-c)<)c(.cciiiii eH'OM) A. chroococcum A. chroococcum A. chroococcum A. chroococcum A. beijerinckii A. beijerinckii A. beijerinckii A. beijerinckii A. vinelandii A. vinelandii A. vinelandii A. vinelandii 26 ID. 26 'D. 26 D. 26 r>. 27 D. 27 D. 27 D. 27 D. 23 D. 22 D. 22 D. 22 D. 4 D. 4 I>. 4 D. 4 D. Mixed culture Mixed culture Mixed culture Mixed culture Cheek Check Check Check The pots were watered with tap water ahout every other day and were weighed weekly. The loss in weight was replaced with water in order to keep tlie moisture content at the optimum. The growth of the plants was carefully noted and recorded by means of photographs at different periods. The harvested plants were dried, weighed, and tlie total nitrogen content determined by the Kjeldahl method. At the close of each series of experiments the soils were re- moved from the pots, placed on a sterile oil cloth, thoroly mixed, sampled and returned to the original pot. The sample taken at this time approximated 500 grams dry weight. The pots were seeded again as soon as possible and the experiment continued. During the short period between sampling and reseeding the moisture content was kept at the optimum. 42 The preliminary analyses, showing the nitrogen content of the original air dried soil, and of the same soil mixed with the ground oats or clover are as follows : 22.68 grs. ground oats straw contained 0.1416 grs. N. av. 6 dets. 22.68 grs. clover hay contained 0.4153 grs. N. av. 6 dets. 10 lbs. original soil contained 2.3494 grs. N. av. 6 dets. 10 lbs. original soil + oats straw contained. . 2.4910 grs. N. av. 6 dets. 10 lbs. original soil + clover hay contained. .2.7647 grs. N. av. 6 dets. ACTION OF DENITRIFYING BACTERIA. Some of the plants were very much stunted in their growth and an experiment was conducted to determine whether this was due to action by the denitrifying organisms. Samples weighing about six or eight grams were drawn from near the center of each pot by means of a sterile corkborer and placed immediately in sterile tubes. Sterile water was added and a soil suspension made from which inoculations were made into Giltay's denitrifying solution. The solution was incubated three weeks and the amount of nitrate nitrogen as well as the total nitrogen determined, the first by the aluminum reduction method of Potter and Snyder, and the second by the official method. The aluminum reduction was carried out by aeration, thus leaving the original solution available for analysis for total nitrogen. The results given in table VIII show that the denitrifying organisms were not the limiting factor in the growth of plants. Only the five soils in pots Nos. 29, 45, 62, 64 and 66 show any great loss in nitrogen and some of the pots show an actual gain in total nitrogen content. This gain is particularly noticeable in the soils inoc- ulated with A. heijerincMi, No. 26 and in the check pots. FABLE Vni— THE ACTIVITIES OF THE DENITRIFYING BACTERIA IN THE SOILS THREE WEEKS AFTER THE START OF THE EXPERIMENT Pot Nitrate N. mgs. N. mgs. Total N. mgs. Check Amt. denitri- fied 1 0.70 1.05 0.S8 0.91 0.90 0.56 0.70 0.791 0.70 0.42 0.70 0.84 0.86 0.65 0.56 lost 6.02 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 2 3 7.07 1.16 4 " 5 - _ __ 5.60 4.20 8.12 7.84 8.14 7.07 7.07 8.96 6.16 7.56 6.51 51.10 8.68 8.54 8.93 7.77 7.49 9.80 7.02 8.21 1.72: 3.13 6 »: 7 8 9 10 0.46 0.74 11 12 13 . '1.21 14 15 0.02 43 TABLE VIII— Continued. Pot N"itrate N. mgs. N. mgs. Total N. mgs. ^, . Amt c Check fj enitri- 16 „ . — 1.71 0..56 0.90 0.42' 0.84 0.S4 0.56 0.70 0.78 0.S6 0.06 0.58 0.87 0.56 "1.54, 0.431 0.70 0.56 0.S8 0.79 0.S6 0.90 1.54 0.63 0.63 0.59 0.70 0.98 0.77 1.05 0.77 0..5S O.W 0.91 0.81 O.EO 0.77 0.49 0.86 1.00 0.89 0.86 0.S6 1.33 0.91 0.78 1.26 1.31 i.v.e 0.9S 0.14 0.70 0.56 -0.84 0.49 0.67 0.87 1.03 0.65 0..51 6.10 7.50 7.70 8.61 8.40 7.91 9.38 7.87 8.12 8.66 9.08 9.24 8.751 9.94 8.23 8.23 8.23 8.23 8.23 8.2s 8.23 8 ''3 0.86 o.n 17 18 — 19 20 - 21 - .. 22 23 25 I"I"I~"III""I 26 -. - 7.21 6.44 7.42 8.04 7.28 5.04 6.80 5.32 9.10 7.98 7.30 7.48 8.62 8.15 5.60 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 0.25 0.93 0.75 27 28 0' 08 29 . . __ 2 63 30 31 .- 6. 86 9.53 1.37 32 33 34 . . 8.54 7.56 8.68 7.00 0.44 6.58 9.10 8. ,54 • 9.47 7.86 7.34 8.12' 8.23 8.23 8.23 8.23 8.23 8.23 8.23 35 36 . - 37 - 37 38 . 89 39 40 - 0.11 41 43 4.90 8.19 5.46 8.23 8.23 8.23 0.04 2.77 44 - 6.86 2.80 6.30 6.16 6.02 7.28 7.S4 3.57 7.3E 7.. 35 6.5-8 8.32 8.23 8.23 8.23 8.23 8.23 8.23 8.23 0.39 45 - - - 4.66 46 - -. . 0.88 47 48 . . 0.8» 1.65 49 50 51 7.77 7.70 8.12 7.56 6.44 6.09 5.40 7.70 5.60 S.'S 8.20 8.89' 8.05 7.30 7.09 6.32 S.56 6.46 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 . . 0.03 53 54 0.18 53 -. 0.83 56 l.U 57 58 — 59 60 1.81 1.77 61 8.23 63 .. .. . 1.96 5.60 4.76 7.28 1.54 7.28 6.16 6.72 4.6-2 8.40 7.98 6.02 7.70 9.80 8.12 2.74 6.86 6.07 8.. 54 2.52 7.42 6.86 7.28 5.46 8.89 8.65 6.89 8.75 10.43 8.63 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 8.23 5.49 63 1.37 64 — 2.10 65 . . 66 ._ .. . . ... 5.71 67 68 0.81 137 69 . - 0.95 70 n . . . - _ 2.77 72 - . 73 1.34 74 75 . . .. 76 *'So denitrification is shown by : 44 PRELIMINARY TESTS FOR NITROGEN FIXATION. To discover the action of the bacteria in the inoculated soils samples were taken from the fallow pots four weeks after t': e start of the experiment and their total nitrogen content determined. Table IX shows a gain in the nitrogen content over the original soil and the check soils but the actual gain due to the action of the bacteria introduced was very slight. Organisms 22, 4 and the mixed cultures showed no gain whatever, and the others showed only a slight gain in those soils to which clover had been TABLE IX — THE ACTIVITY OP THE BACTERIA IN THE INOCULATED FALLOW SOILS AFTER FOUR WEEKS. Grams of Nitrogen Per 10 Pounds of Soil. Pot Gi-ams N. 1 2 5 7 9 11 13 ]5 17 19 21 23 25 27 29 SI 33 35 3Y 3& 41 43 45 47 49 51 5S 55 57 59 &1 63 65 67 60 n Checks 73 74 77 78 M O'o o .Set. . C3 O 0643 0643 8942 8304 0643 2281 3411 9581 2281 2281 2134 0219 2281 ''239 7666 2772 2f281 0613 8942 8942 2281 1929 9581 7665 8409 9047 7027 5431 8409 8089 5431 .5750 9047 7451 2239 .5536 0043 93-66 0643 Average four checks- lost 3.2281 3.8942 3.9581 3.2281 3.2281 4.0S96 4.0S57 3.2281 3.06i!3 4.0219 3.9'561 3.2558 3.2239 4. 0857 4.0219 3.2281 3.1920 3.8304 3.8304 e.25S8 3.228.1 3.9561 3.8304 2.8409 2.7679 3.8942 3.6069 2.9047 2.7132 3.5752 3.6069 2.8408 a. 7451 3.1320 3.443a 2.8089' 3.0324 2. 8089 3.2239 3.0643 3.1462 3.8942 3.8942 3.1462 3.2281 4.2203 4.0219 3.2281 3.1462 4.1176 3.9900 3.2419 3.2239 3.9261 4.1490 3.228] 3.1281 3.8623 3.862f 3.2419 3.210:' 3.9581 3.7984 2.8409 2.8393 3.798! 3.5750 2.872? 2.7610 3.5590 3.5909 2.8727 2.745] 3.2079 3.5250 2.6S12 3.0483 2.8727 3.H4] 2.4910 0.5533 2.4910 0.5552 2.7647 1.1295 2.7647 1.1295 2.4910 0.6552 2.4910 0.7371 2.7647 1.4556 2.7647 1.2572 2.4910 0.7371 2.49110 0.6552 2.7647 1.3529 2.7647 1.2258 2.4910 0.7509 2.4910 0.7329 2.7647 1.1614 2.7647 1.3843 2.4910 0.7371 2.4910 0.6871 2.7647 1.0976 2.7647 1.0976 2.4910 0.7509 2.4910 0.7109 2.7647 1.1934 2.7647 1.0337 2.4910 0.8499 2.4910 0.3453 2.7647 1.0837 2.764.7 O.8103 2.4910 0.S818 2.4910 0.2700 2,7647 0.7943 2.7647 0.S262 2.4910 0.3817 2.4910 0.2541 2.7647 0.4432 2.7647 0.7603 2.3494 0.3318 2.3404 0.6989 2.3494 0.5233 2.3494 0.7947 0.7288 0.7288 1.0025 1.0025 0.7288 0.7288 1.0025 1.O025 0.7288 0.T288 1.0025 1.O025 0.7288 0.7288 0.0025 1.002s 0.7288 0.7288 0.0025 1.0025 0.7288 0.7288 1.0025 1.0025 0.7288 0.7288 1.0025 1.0025 0.7288 0.7288 1.0025 1.0025 0.7288 0.7288 1.0025 1.0025 §0. 5 cS ft _c g o o '" CS S Cj 2: 2; 1— 3— P oats C oats F clover — C clover F oats O oats F clover O clover .„ P oats C oats P clover .. O clover P oats C oats — _ P clover . O clover P oats O oats P clover O clover F oats O oats . P clover O clover P oats O oats P clover _- C clover F oats lO oats P clovsr O clover P oats C oats P clover C clover _— P. nothing _— - O. nothing P nothing C nothing A. chrooe. (HClM) — -4. chrooe. (HCM) — A. chrooe. (HCM) — A. chrooe. (HCM)— . A. chrooe. 3.3499 2.9S83 4.3058 4.1808 3.2210 3.5380 4.5726 4.4928 5.1701 5.2867 4.4S94 4.5801 3.9267 5.1120 2.4572 4.2388 3.4887 3.5170 4.4823 4.5722 3.6458 3.5720 4.5023 4.3114 3.4101 3.5823 4.4001 4.3114 3.6065 3.50C6 4.4630 4.30-23 3.3865 3.60S8 4.3787 4.4720 3.5942 3.5085 3.3002 3.5403 3.3833 2.9670 4.3488 4.-2684 3. -2582 3.5808 4.6183 4.5787 5.2218 5.28-24 4.4827 4.6613 3.95159 5.1794 2.4817 4.3457 3.5235 0.5133 4.5-271 4.6833 3.6822 3.6217 4.5473 ^4196 3.4442 3.5519 4.4441 4.3866 3.6423 3.. 5323 4.5076 4.3965 3.4203 3.60-21 4.4123 4.5794 3.6301 3.. 5689' 3.3332 3.6097 3.6232 3.7309 3.8963 4.0046 3.6232 3.7309 3.8963 4.O046 3.6232 3.7309 3.8963 4.0046 3.6232 3.7309 3.8063 4.0046 3.6232 3.7309 3.8963 4.0016 3.6232 3.7309 3.8963 4.0046 3.6232 3.7-209 3.8963 4.0046 3.62.32 3.7309 3.8963 4.0046 3.6232 3.7309 3.8963 4.0046 2— 4. 5— 7- 6— S-. &-11.- I(t— 1'^ 0.4525 0.-2638 13-15-- 14—16- 17—19 - A. chrooe. A. chrooe. A. beyer. .. -. - 0.7220 0.5741 1..5966 18—20— A. beyer. .. 1.5515 21—23— ')9 — Oi , A. teyer. A. beyer.^ .._ 0.5884 0.6567 25—27- A. vine. - - 0.34-27 26—28- A. vine. _. 1.4485 29^—31 A. vine. 30—32- A. vine. -, 0.3411 33—35 - No. 26 . 34—36 - No. 26 37— 3&- No. 26 - 0.6808 3S^}0.. 41—43- 42-44-- No. 26 No 27 No. 27 0.6787 O.O590 45-^7- 46—48 - No. 27 No. 27 0.6510 0.4149 49—51- No. 22 .- 50—52— 53—55- No. 22 No. 22 0.5478 .S4-56- 57— 59- 58—60 I.o. 22 No' 4-IZ-II-II-I." No. 4 0.3820 0.0193 61-63- 6113 62-61 No 4 - 3919 65—67- 66-68- Mixed culture Mixed culturs Mixed culture Mixed culturs Check - 6»-7l- 70—72— 73—74 0.5160 0.5748 7.5—76- Cheek 77— 7S- 79^80- Check Check 46 FIRST GROWING PERIOD. The determinations for this period are shown in appendix table I and in condensed table X. As indicated by table IX, there was a steady increase in the total amount of nitrogen fixed in all the soils. This increase is still more marked if the last columns of tables IX and X are compared. The bacteria were increasing-ly active in fixing- the free atmospheric nitrogen and in practically every case the total amount fixed due to the bacterial solution was more than doubled during- the latter five weeks of this series. These activities may be divided into two classes, as the bacteria were more markedly affected by the presence of clover hay or of oats straw. In the first class A. ckroococcmn, A. chroococcum {HCM), No. 26, No. 22 and the Mixed Culture stood out prom- inently. None of these four organisms showed any fixation due to the presence of the decaying oats straw, but they did show ap- preciable g'ains due to the presence of the clover hay. The pres- ence of the oats straw had apparently either inhibited the activi- ties of the organisms or increased the activities of the other forms that are incapable of fixing nitrogen for their own use and have utilized that fixed by the inoculating organisms. Organisms 4 and 27 showed a decided stimulation due to the clover hay and were able to utilize the oats straw as a source of energy, A heijerincki and A. vinelmidii were more markedly affected by the presence of oats straw. The stimulation of the activities of the former due to the presence of the decaying clover was parallel to that of the other organisms, and in addition the presence of the decaying oats straw stimulated its nitrogen fixing powers to over 250% of that of any other organism in the series with the single exception of A. vinelandii. On the other hand, A. vinelandii, while showing a marked stimulation due to the presence of the oats, also showed that the clover hay affected its activities much the same as the oats straw affected the other organisms, that is, the presence of the decaying clover hay in the cropped soils, de- creased its nitrogen-fixing power, and in the fallow soils, com- pletely inhibited it. SECOND GROWING PERIOD. The results for this period are shown in appendix table II, the more important parts of which are repeated in condensed table XI. In comparison with the first gTowing- period the results for the second period are decidedly lower thruout the second series. Not only are the total amounts of nitrogen found lower, but also the total amount of dry matter produced in the crop, indicating a possible direct relation between bacterial action and crop yields. These low results are explained by the fact that this series as 47 grown during' the hottest part of the summer, the pots being planted in the latter part of June and harvested during the earl- ier part of August. The results confirm those given in Table X except that in this series the only organism stimulated by the presence of the decaying oats straw was organism 27 in the cropped pots. Each of the inoculated organisms showed a direct stimulation due to the presence of the clover hay. The organisms may be divided into two classes according as their activities are stimulated or retarded by the presence of TIAJBCLE XI— THE NinHiOO'EJST MXEID BY BiAlCITIElRlLA— iSECIOiND' PERIOD. ('Oondensed from appendix Table 2.) Dupli- cate Pots Tieatment Bacterial inoculum used. Grams Nitrogen per 10 lbs. soil. g > 2 sou £ Z't tic 2 2g^ •^2 1— 3— 2— 4._ &— 7... &- 8... 9—11.. 10-412... 13—15.. 14^16... 17—19... IS— 20.. 21—23.. 22—24.. 25—27.- 2&— 28„ 291—31... iO— 32.. 33—35.. 34—36.. 87—39.. 38—40.. 41—13.. 42—44-. 45—47-. 46^48.. 49^51- 50^52.. 53—55.. 54—50- 57—59.. 5S— 60.- 61—68-. 62^64-. eo'— 67-. 66— 68-. 69—71- 70—72- 73—74-. 75-76-. 77—78-. 79—80- oats oats -. clover clover oats oats clover clover oats oats clover clover oats oats clover clover -.... oats oats clover clover oats oats clover clover oats oats clover clover oats oats clover clover oats oats clover clover nothing- ... nothing nothing A. chroocoecum (HCIM). A. chroocoecum (HiOM). A. chrooeoceum (HICM). .4.. chroocoecum (HIOM). A. chroocoecum A. chrooeoceum A. chroocoecum A. chroocoecum A. Beijerinckii A. Beijerinckii A. Beijerinckii .4. Beijerinckii A. vinelandii A. vinelandii A. vinelandii A. vinelandii No. 26 No. 26 No. 26 No. 26 No. 27 No. 27 No. 27 No. 27 No. 22 No. 22 No. 22 No. 22 No. 4 No. 4 No. 4 No. 4 Mixed culture Mixed culture Mixed culture Mixed culture Check Cheek Check nothing ... Oheck 2.7340 3.0128 2.8218 3.0916 3.3205 3.6592 3.3123 3.6402 3.6867 2.9607 e. 54791 2.7947 3.5242! 3.8836 3.1813 3.4901 2.7155 2.9924 2.6134 2.8626 3.3343 3.6743 3.21681 3.5857 2.8239 3.1115 2.6936 2.9763 3.3205 3.6591 3.4143 3.6822 2.7547 3.0350 2.8912 3.1730 3.4921 3.8492 3.0648 3.3747 2.7015 2.9770 8.O508 3.3429 3. 4487 3.8004 3.5089 3.8613 2.7083 2.9845 2.8804 3.170O 3.3977 3.7442 3.2667 3.5925' 2.6146 2.8812 2.8246 3.1262 3.3998 3.7465 3.1740 3.4913 2.5210 2.7781 2.6935 2.9S10 3.21396 3.6031 3.38521 3.7159 2.8835 3.1115 2.7518 3.0.537 2.9758 3.2798 2.7736 3.0364 3.3370 3.1866 3.6107 3.4603 3.3370 O.0485 0.1799 3.1866 3.6107 3.4603 3.3370 '"0^2729 0.0298 3.1866 3.6107 3.4603 3.3370 3.1866 3.6107 3.4603 3.3370 O.O630 0.1254 "o.0384 0.1719 3.1866 3.6107 3.4603 0.2385 3.3370 3.1860 3.6107 3.4603 3.3370 3.1866 0.1503 0.1897 0.4010 3.6107 3.4603 3.3370' 3 I860 0.1335 0.1322 3.6107 3.4003 3.3370 3.1866 0.1358 0.0310 3.6107 3.4603 0.2550 48 growing plants such as clover. In the first class are included A. chroococcum {RCM) , A. heijerinckii, A. vinelandii, No. 27 and the mixed cultures. The first three organisms have had their nitrogen-fixing powers stimulated by the presence of the plants in practically the same ratio and have fixed similar amounts in both the fallow and cropped soil. A. h&ijerincJdi showed the highest fixation of any of the eight for this series. The mixed cultures showed no fixation whatever in the fallow soils, but quite an appreciable amount in the cropped soils. No. 26, on the contrary, fixed an appreciable amount of nitrogen in the fallow soils but none at all in the presence of the growing oats plants. A. chroococcum. and No. 4 showed the same stimulation under practically the same conditions, namely, that they possess a greater nitrogen-fixing power in the presence of decaying clover if no crop is grown upon the soil, while No. 22 was apparently neither stimulated nor retarded by either fallow or cropped con- ditions, but was affected by the presence of the decaying oats straw. THIRD GROWING PERIOD. The results for this period are shown in appendix table III and condensed table XII. The total nitrogen content of the soil according to tables IX, X and XI, increased steadily through- TABLE XII— THE NITROGEN FIXATION BY BACTERIA— THIRD PERIOD (Condensed from Appendix Table III). Bacterial Inoculum Grams Nitrogen per 10 Pounds Soil + ,^ -Sll >> Treatment Used a l£-^ .^ O'S "S 53 «§ 3 o in s amt. mov crop .s-^l ■^ ^•^ fi; ^ 1 ^ ;z; ^ < 1 F oats A. chrooc. (HCM) 2.56-2:6 2.8618 2.8502 0.0146 R F oats A. chrooc. (HCM) 2.7342 3.3166 2.8502 0.4063 0.2405 'O oats O oats i chrooc. (HCM) 2. 3643 2,9647 3.3284 4 A. chrooc. (HCM) 2.1930 2.6754 3.32S4 5 F clover _ A. chrooc. (HCM) 2.0255 3.5486 3.1239 0.4247 7 F clover A. chrooc. (HCM) 2.8274 3.4296 3.1239 0.3057 0.3652 6 O clover . A. chrooc. (HCM) S.2852 4.0691 3.6021 0.4670 S O clover A. chrooc. (HCM) 2.S688 3.16142 3.6021 0.0121 0.2395 9 F oats — - A. Chroococcum.. 21.7489 3.3344 2.8502 0.4842 n F oats _ A. Chroococcum.. 2.3103 2.8024 2.8502 0.2421 10 oats A. Chroococcum.. 2.3652 2.9149 3.3284 12 O oats A. Chroococcum.. 2.S7S5 2.9537 3.3284 13 F clover A. Chroococcum... 2.8928 3.5089 3.1289 0.3850 13 F clover A. Chroococcum.. 3.1540 3.8265 3.1239 0.7026 0.5438 14 O clover A Chroococcum.. 2.8489 3.5649 3.6021 Ifi 'O Clover A Chroococcum. 2.7501 3.4326 3.6021 17 F oats A. beijerinckii 2.5789 3.1279 2.8502 0.2777 19 F oats A. beijerinckii 2.4S05 3.0088 2.8502 0.1586 0.218B 18 4 heijerinckii beijerinckii 2.2943 2.8469 3.3284 20 O oats A. 2.07317 2.5764 3.3284 21 F clover A. beijerinckii 3.5208 4.2705' 3.1239 1.1466 2i3' F clover .. A. beijerinckii S.0S68 3.6836 3.1239 0.5597 0.8532 49 TABLE XII— Continued Treatment Bacterial Inoculum Used Grams Nitrogen Per 10 Pounds Soil -0 -a 1 -° 5i ^"S-- w 3 ■" tj > a. ■-Ill .5 c c ^t O a. . Jl >-° 12 2i ^ z < 22 clover A. beijerinckii 2.7429' 3.4256 3.6021 24 clover A. beijerinckii 2.7694 3.4584 3.6021 Wi F oats A. vinelandii A. vinelandii A. vinchindii 2.5431 2.447S 2.3917 2 3491 3.0847 2.9601 2.9781 2.9494 2.8502 2.8502 3.3284 3.3284 0.2315 0.1189 97 F oata - 0.1767 26 oats 28 oats F clover 29 A. rinrliiniHi S.0172 3.6598 3.1289 0.5359 31 F clover A. r'nuUmiUi 3.0711 3.7252 3.1239 0.6013 0.5686 80 clover A. rhir'KiniHi 2.7429 3.4284 3.6021 32 clover A. rhicUindii 2.7363 3.3959 3.6021 33 F oats No. 26 2.4609 2.9850 2.80O2 0.1348 S3 F oats No. 26 2.3823 2.8887 2.8502 0.0385 0.0862 34 oats oats No. 26 ]So. 20 , ^*b. 25 2.4848 2.3321 2.9S95 3.0673 2.9746 3.6162 3.3284 3.3284 3.1239 .% ' 0^4923 m F clover , 39 F clover No. 26 2.0164 2.4459 3.1239 0.2463 38 lO clover No. 26 2.7749 S.4477 3.6021 40 'O clover No. 26 2.8877 3.5&S8 8.6021 41 F oats No. 27 No. 27 2.4478 2.4871 2.9691 3.0169 2.8502 2.8502 0.1189 0.1667 43 F Oatsi 0.1428 4f1 oats _, oats No. 27 .. 2.4583 2.4917 2.8143 3.0736 3.1595 S.4137 3.3284 3.3284 3.1289 44 No. 27 . No. 27 45 F clover 0.2898 47" F clover No. 27 3.0564 3.7074 3.1239 0.5835 0.4366 46 clover „„ No. 27 . 3.0181 3.7421 3.6021 0.1400 48 iO clover No. 27 2.8621 3.5807 3.6021 0.0700 49 F oats No. 22 2.4961 3.0278 2.8502 0.1776 51 F oats No. -221 2.2186 2.6911 2.8502 0.0888 50 oats oats . No 221 . 2.5580 2.4451 3.1751 3.0758 3.32S4 3.3284 5?; No. 22 53 F clover No. 22! 2.7230 3.3029 3.1239 0.1790 55 F clover No. 23 2.8667 3.4773 3.1239 0.3584 0.2662 54 clover clover _. F oats No. 23 No. 23 2.7893 2.6236 2.3430 3.5060 S.29S3 2.8321 3.6021 3.6021 2.8502 5fl 57- No. 4 59 F oats 'O oats No. 4 . 2.4094 2.3170 2.60S7 2.9126 2.8086 3.3108 2.8502 3.3284 3.3284 0.0624 0.0312 58 No. 4 No. 4 60 lO oats _. fil F clover F clover clover No. 4 3.0173 2.8208 2.8877 3.6599 3.4216 3.6172 3.1239 3.1239 3.6021 0.5860 0.2977 0.0151 m No. 4.^ 0.416S 62 No. i M a Clover F oats No. i_ 2.8224 2.3496 3.5098 2.8501 3.6021 0.0076 65 Mixed culture 2.8502' 67 F oats Mixed culture 2.3234 2.8183 2.8502 m oats - Mixed ciiltiire 2.4381 3.0435 3.3284 fiS lO oatsi - . Mixed culture 2.3586 2.89S7 3.3284 69 F clover Mixed culture 3.0761 3.7313 3.1239 0.6074 71 F clover Mixed culture 2.7831 3.3759 3.1239 0.2520 0.4297 70 2.8366 3.5867 3.6021 72 C clover . Mixed ci.lture 3.0306 3.7664 3.6021 0.1643 0.0822 73 F nothing- Check 2.3216 2.8161 * * * 74 F. nothing Check 2.0944 2.5405 * * * 77 F nothing Check 2.1074 2.5568 * * 78 F nothing Check 2.4085 2.9215 * 75 nothing Check 2.7760 3.3649 !t t t 7fi nothing Cheek 2.5375 3.1041 + + + 79 nothing Check 2.4911 3.1522 t t t 80 nothing Cfteek 2.4901 3.1362 t ' t t •Average four fallow checks 2.7086. ■fiAverage four cropped checks 3.1868. 50 out the first growing period, declined somewhat during the second, and according to table XII, there was a pronounced ten- dency to increase again during the third period. The crop re- sponse of this last period of growth confirms the results of the determinations, the amount of dry matter produced being prac- tically midway between the production of the first and second growing periods. Figs. 1-6, which show the growth of oats in rep- resentative pots for the three periods, show that the first crop when ready to harvest was in the majority of cases leafy and heavy and showed a decided tendency to lodge ; the second crop in the same stage of growth was somewhat dwarfed in appearance and with no indication of leafiness or weakness of stem ; the third crop, while not as heavy as the first, showed all of its characteris- tics except that as a whole the production was more uniform and did not show the variation in the total dry weight of the harvested crop. The bacterial activities, which are plotted in the tables shown in fig. 7, varied in the same proportion as the crop re- sponse of the treated soils, being practically parallel with the production of the dried weight cf the crop. The activities in- creased during the first growing period, declined thruout the second, but increased again during the third. The discus- sion of the third and last period of growth will be a combina- tion of the activities of the inoculated bacteria as discussed in the first and second growing periods. The last column in table XII indicates that each inoculated bacterial culture acted without exception in the same general manner instead of showing the expected variations. All of the inoculated bacteria fixed greater amounts of nitrogen in the soils to w^hich clover hay was added as organic matter than in soils that were treated with the same amount of oats straw, and the growing crop on these soils reduced the nitrogen-fixing power of each and every one of these organisms. The activities of any one of the eight organisms used during the third period of growth would be an accurate measure for the activities of any of the others, a fact not even indicated in the other periods of growth. Conclusion: Table XIII, recapitulating tables X, XI and XII, shows that inoculation, especially in fallow soils to which clover hay or oats straw was added, is not only possible but practical. The amounts of nitrogen shown in these tables are the actual amounts fixed by the organisms in ten pounds of soil and if these amounts are calculated on a 2,000,000 pound acre basis, the result is distinctly profitable. With proper soil condi- tions the greenhouse experiments can be duplicated in the field. All of the organisms have shown an appreciable fixation of nitrogen but A. beijerinckii and A. vinelandii have been de- cidedly the most active. This finding confirms the suggestion of 51 H^ 00 §> 8 IS CO r-i CO i> O 1-1 © d cot 00 ■^ 5s o S ^ S kn 00 o I* 00 (M CO lo as (M^OOrHc5!©(MQ0 f-1 t-- a; P 00 K3 -+ ■* O O i-l rH e> © O I O O O I o o CO © » -^ 00 rH in CO O S<6 ^ CO «i 1 ® -* CO rJ CC T1< cot-i>co!0©o©J IC0©©(M©l^f5-*-^"^i ! tC C^ So CO © O:; C C bo 4-. >> c o HfL, _ -^ --. ^-- - - - - t'«p©rHCOCOCO©rHC^LC5oSc-lC^OCO'T'COCO^o65^co-* gc^o03<5ei^p«2*coso{;-^c)5POTTHq;2jcoco(^rtScnLp(>icnoi^io-!fcocotLoJitic^ 000-*000»tP-*0®00-*OroeO-*0300l003i:^SSoOrHCO-ii<»©loSo3telftSfc{DKJ>t-l co5^cocococ^fioco^5c^lcoco6^c^lcOl^5c^oicoc*ic^co^ie^c^coco^5^^^cOcosi5^c^cofiOC^eO(^ co5ggiEjc-c-sgjjn'?ogt^>oco5Hcs^0 2at-pro3coioo«|-HtrlOQ3cf5J^-l.tIrrtCJOC01>^®oOc^lMicii>co©iM'--'l-ne-co OO^ab01r-COTt(OiOOCOlOrHgilc£)CC)C>rHOOCOOiCOOOOO^Jr-Oi©i>T-HOC-10 C0COCOCO(M^COC0C^ol9 5afer^eolo«®lScoi^co■!^^t-ilall^lt- C01>C0C0C0O(3SaDc^^C3r^kQ92rHrjQCQC0tTC0C5^»-H^^CvJC-lt-C0C>C^I^0iO00C0Q? COOO-ra510CO--Hir-C^OOCSCCimJ:>(X)TPe5i-IMOOCOK!-*rH^lOMt--©©V-t-©ot-l:-©©i--l^©©t~fc-©©I~b©©f-i>©©t-i~-^^-*Tf ■*^t~i>'*^^f^t--*-*j>J^-*-*j>l>^THj>J>^rti>-*-!)Jipicococo (M 0^1 ci c^ 7-1 c-i c^ c^ o-J c^ cvi c^i t^i cq c^ c^^ OQOO sa o o j^ H K S .i^ ^ -^ ,i«i •■ j CD a> CD cu•S■H.S.fclc^^(NCN(^lcN(^q(^^c^5Cl7;l5slsc^**Tf^-*T*, <{<|<|<]<]<;-3<O0rHCOQT-^(MLc:lOCb©io■*i>oo^-lc• i> J> 1^ J 52 Fio. 1. Oats at end of first growing period, immediately before harvest; in pots 2, 6, 10. 14, IS, 22 26, 30, 24, 38 \U Fig. 2. Oats at end of first growing period, mmediately before harvest; pots 44, 48, 52, 56, 60, 64, 68, 72, 76, 80 Fig 3. Oats at end of second growing period, immediately before harvest; pots 2, 6, 10, 14, 18, 22 , 26 30, 34, 38 Fig 4 Oats at end of second growing period, immediately before Harvest; pots 42, 46, 50, 54, 58, 62, 66, 70, 75, 79 \ -v. 1/7' \ \\\ r--^r IG. 5. Oats at end of third growing period, mmediatelv before harvest; pots 4, 8, 12, 16, 20 24. 28, 32. 36, 40 Fig. 6. Oats at end of third growing period, immediately before harvest; pots 44 48, 52, 56 60 64, 68, 72, 76, 80 54 -hr 'ro«, gPe wi.- ~Zni 3rQwr ng /* f/od- '-dd i™„„ 9 ^^ lOd:- f\7 Chr >oco TAm rrT^- rrT" ':rTi r=^ .:^::^ .... =^»^ 3^ HC m ^: -— — _^ '^ -._ i.fi ^ -^ ^ -^ R7 Cbr loca 'Clin .^ ^'' -^ -^ :^ ..^ '-^ "zzz. ^_ --, _ ----^ w 1.0 r ^ \ / y \ v. ^ ^ ftr hf.yf rim ;/f/ n.n ^ ^ s^^ ^ -^ .^ \ r:^ — ^ ,^ N ^N ^ f\7 vin 'Ian ii on ^ :._ ^' -^ ^^ -N. — .^^ ^ __^ \ / ^ ■■-^ -- -__ \ / / \ y ,^ ji^^ fir ?.fi n 0I\ --^ "^^ :::-- ^ — ~ """^ =^ ~~ — ^^"*" ^^^^ ^ ■-— ft^ ?1 n (in r^ ^- ^ - '*■' -- ^ ::^ ^ -.._ f^-- -~- ^^ "-' --- fl- ■?■> n nn -^r^ --' '^ ^^^ =,^ ' — -— - -^ rzL h— *- ___ __- ~^IZ^ ,-— e '^ ^ ftr 4 n M ^^ ^^- ^ --- — - -~^ ^ ' "^ — — -^ -"^ Z^^ — i--- —- LEGEND. Oata added as monure yjote HepffaHooJ OqH adaea as manure pais kept cmpped-- «- Oovei-Qddsd as manure oofs kept fallow C/over added os monure ;OoM HepfcmppecJ tr iip,( Cv Ifvn =s _-- ^.^^ '~~' " — ^ ;^ --■ --- ,^ ■rCT - ad — t=^ =::.: :::;;;;;;_ — - -^- ^ -^rl Fig. 7. This graph shows the variation in bacterial activity in the different growing periods 55 Lipman and Brown (41) by proving definitely that these organ- isms are capable of being profitably inoculated into field soils, provided that organic matter, carrying a sufficient amount of nitrogen as a stimulus, is supplied. Simimary. 1. When three crops of oats were grown continuously on this soil the nitrogen content of the soil increased during the first cropping period, decreased during the second, and increased slightly again during the third. 2. The nitrogen-fixing powers of the bacteria and the crop response were parallel with the total nitrogen content of the soil. 3. The nitrogen-fixing powers of some types of azotobacter and other large celled organisms of the same general character, were stimulated to a greater extent by the presence of decaying clover hay than of decaying oats straw. 4. The nitrogen-fixing powers of A. heijerinckii and A. vinelandii were stimulated to a greater extent by decaying oats straw than by clover hay, especially during the earlier stages of decomposition. , 5. The nitrogen-fixing powers of the azotobacter and other large celled organisms of the same general type eventually be- came greater in fallow than in cropped soils. 6. The non-symbiotic nitrogen-fixing organisms of the azoto- bacter group were all eventually influenced in their activities in the same manner and by the same materials. 7. Soils may be profitably inoculated by azotobacter and other large celled organisms of the same type, the best effects being secured in this work by an inoculation with A. heijerinckii or A. vinelandii. 8. The conditions necessary for the greatest fixation are : Good environmental factors such as tillage, drainage, etc. ; the presence of a rapidly decaying organic matter carrying a small nitrogen content, and freedom from growing plants. ACID EXTRACT, AMINO, NON-PROTEIN AND POLYPEP- TID NITROGEN CONTENT OF THE POT SOILS. Introduction : The nitrogen of the soil is found in many com- plex combinations, in the determination of which the Bureau of Soils has isolated a large number of nitrogenous compounds and many different forms have been discovered. In investigating methods for the determination of amino acids and nitrates in a limed and unlimed soil, both with and -without heavy applications of manures. Potter and Snyder (53) have found that they could accurately measure the 'amino nitrogen by a modification of the method devised by Kober and Sugiura (32). They discovered 56 no tendency for the amino acid to accumulate under the condi- tions of the experiment. Accordingly in the present investiga- tion determinations were made of the acid extract, non-protein, amino, and polypeptid nitrog^en of some of the soils inoculated with the azotobacter cultures used in the g:reenhouse experi- ments, in order to prove this point and also to discover if the bacterial action had any effect on the accumulation or disap- pearance of these nitrogenous forms. Soils used: Only the three soils inoculated with A. chroo- coccum, A. heijerinckii and A. vinelandii were analyzed. METHODS. Acid extract : Place 166 gr. of air dried soil on a wetted double filter paper in a Buchner funnel and extract with 600 e. c. of a 1% HCl solution using g-entle suction. Keep the soil barely covered with the solution and when extracted, wash with 200 to 300 c. c. of pure distilled water. Dry as quickly as possible, and determine the nitrogen content of the filtrate jjy the official salicylic acid method. Alkali extract: The non-protein, amino, and polypeptid ni- trogen determinations are based on the amounts extracted by a 1.5% NaOH solution. Shake 150 gr. of the air dried acid ex- tracted soil with 600 c. c. of the NaOH solution and centrifuge to a clear solution. At least 210 c. c. cf the clear solution must be obtained. Non-protein nitrogen : Pipette off 25 c. c. of tbe alkali extract, neutralize Avith a sulphuric acid solution and add sufficient tri- chloracetic acid to make a 2.5% solution. To do this use 4.3 c. e., of a 1 3/10 N. H2SO4 solution and 0.75 c. c. of a saturated tri- chloracetic solution. This method precipitates the proteins which are removed by filtering. Pipette 10 c. c. of the clear filtrate into large test tubes, add a couple glass beads, 2 drops of a 5% CuSO^ solution, 1 c. c. C. P. HoSO^, and approximately 1 gr. C P. potassium sulfate. Digest and distil as in the regular Kjeldahl method determining the ammonia colorimetrically. Amino acid nitrogen : Pipette 80 c. c. of the alkali extract into 100 €. c. measuring flasks, neutralize with strong HCl until neutral to litmus, add 7 c. c. saturated lead acetate solution, fill the flask to the mark with concentrated NH^OH and shake vig- orouly. Allow to settle for a few minutes then pass through double filter, using gentle suction and obtain at least 80 c. e. of the filtrate. Measure oft' 75 c. c. of this filtrate, add 25 c. c. saturated Ba(0H)2 and phenolphthalein as indicator and distill over steam bath under reduced pressure until there remains a volume of about 25 or 30 c. c. It is important that the reaction of the solution throughout this distillation should be at all times 57 alkaline. Discard the distillate, wash residue into 100 c. c. gradu- ate, cool, make up to 75 c. c, filter quickly to remove all car- bonates, pipette 50 c. c. into 100 c. c. measuring flasks, make ap- proximately neutral with N/10 HCl and add 40 c. c. of buffer solution, stopper tig'htly and keep in cool place, if possible, on ice. (The buffer is made by dissolving- 0.2 gr. molecules of boric acid in water, adding 100 c. c. of COo free N/10 NaOH solution and making up to 1000 c. c. with pure COo free water. Three volumes of this mixed with one volume of 0/1/N HCl makes the desired solution.) Use pure water as cold as possible to prepare fresh the fol- lowing solution: Place 10-20% copper chloride solution in 20-30 volumes cold Avater, add a few drops phenolphthalein and a sat- urated solution Ba(0H)2 until the purple color just forms. Centrifuge, decant oft' the clear liquid, wash with cold water and recentrifuge, repeating until there is no pink color formed by the addition of phenolphthalein in the wash water. Suspend the copper hydroxide in about 100 c. c. cold water and add ap- proximately 1 c. c. to the cool flasks, shake vigorously, make up to the mark, and allow to warm up to the room temperature. Fil- ter through No. 589 blue ribbon filter, pipette oft' 50 c. c. of the filtrate and determine the copper complex present as shown be- low as a measure of the amino nitrogen. Pipette off 40 c. c. of the filtrate for the determination of the polypeptid nitrogen. Polypeptid nitrogen: Hydrolize the polypeptids into amino acids by adding approximately 5 c. c. concentrated HaSO;^ to the 40 c. e. and placing under a steam pressure of 8-10 pounds for 10-12 hours. Remove the excess acid with a saturated solu- tion Ba(OH)o keeping the solution slightly alkaline to phenol- phthalein, filter and wash with carbonated water at least three times. Evaporate the filtrate to about 35 or 40 c. c, place in 100 c. c. measuring flasks, neutralize with N/10 HCl, add 40 c. c. buffer solution, 1 c. c. of the copper hydroxide solution in the cold water as for the amino determinations and determine the copper present in the same manner. Copper determination: Place the beakers containing the 50 c. c. on the hot plate, heat to boiling and neutralize with dilute HNO3. Boil down to about one-half and add bromine water until a decided bromine color appears, evaporate to about 10-15 c. c, add 20-30 c. c. pure water and a little more bromine water and evaporate down again to 10-15 c. c. Cool, add 2-3 c. c. glacial acetic acid, a few crystals potassium iodide, a few drops of starch solution and titrate immediately with .001/N sodium thio-sulfate until the blue color disappears. Each c. c. of the .001/N thio-sulfate solution is the equivalent of 0.000028 gr, amino acid nitrogen. 58 Preliminary determinations : In addition to the work on the soils, an unsuccessful attempt was made to determine tlie amount of non-protein and amino acid nitrogen fixed by the ba-ter:a in- oculated into the dextrose solution used in the other experiments. 250 e. c. of the dextrose solution was inoculated with the organ- isms indicated in Table 14 and incubated three weeks at room temperature. Enough c. p. sodium hydroxide w^as added di- rectly to the solution to make 1.5% and the determination car- ried out in the above manner. A slight trace was the greatest amount found. This table shows a decided increase in the soils under field conditions over the same soils in the dry state, the greatest in- crease taking place during the earlier periods of growth. The results of these determinations are grouped in three tables, each showing the amount of the different nitrogenous forms found at the end of each growing period. Discussion of results : A comparison of the results given in Tables XVI, XVII and XVIII, shows that there was a definite variation of the nitrogenous forms with the length of the time of cropping. In almost every case the amount extracted by the acid varied Avith the length of time that the soil had been cropped, growing smaller and smaller, and the amino and polypeptid nitrogen gave similar results. The amount of the^e nitrogenous 'MB/LlE XIV— lAlMINO' AlCID AND' NON-PiRlOT'EIN" NITKIO'GEN FIXED' BY THE PUlBE OULa'UIREiS' IN vSO'I/UTION. -id E Inoculum Non-protein N. Amino Acid N. 1 A. chroococcum CBCIMI- _-_ trace tracs s? A. chroococcum CHCIM) « A. chroococcuin (DfflCIM) - 4 No. 26 5 No. 26 fi No. 26 7 A. chroocoecum C.HCM) and A. ehroocoecum CHCIM) and A. ehroocoeeum (HCM) and No. 26 8 9 No. 20 No. 28— trace TAKLE' XV— THE' AMOHINT O'V iDIFFlEKENT NITKlOGENOTJiSi PORlMS IN THE SOIL AT THE' ■BElGiINNKNO OE' THE' 'EXIPEIRIIlM'ENTS, ALISO THE SAMIE SOIL PLtJiS the: EQUIVALEINT' OE' EH'K TOMS 01ROX.TND' OATS', STKAW OK GlROI]NI> OLOVEiR HAY ADIDEB' TO' 'J'll i: s.\.\I l"LE'. DEiTE'BMINATIONS BASED ON THE AiMOiUNT' IN 2ft GlR;. 'OiF' 'TH'i': S.\:iIJ''lvl<7 AND' iRIEIS'UlJT'Si ,KXPIRES!SED IN (MG. NTTROG'EflSr AND' IN PEK OENT' O'P THE TO'TALi NTTIBOGEN. Soil OZ8 c 1 '^ o c o.S ti Zi ii E < « E £ "o-O M ix-n E c Original . _ _ _ - 12.95 IS. 75 15.40 1.1243 1.1666 1.4424 8.7 8.5 9.3 2.22T5 2.339'0' 2.3475 17.2 17.0 15.2 O.CS40 0.1025 0.1050 0.7 0.7 0.7 0. 210O 0.2550 0.2625 1.6 Original + oatS— _ Original + red clover hay 1.8 1.7 59 TABLE SVT— (AIMOTHSTTS OF T!H'E iDIFFEIRENT FOKlMiSi OP NffTElOOEJST IN THE CRIOOPP'BID ANJy FALLOW Ii:N OiOULAT'EID SOILiS AT TCtlEi END OP THE PIBlST PEIBTOID OP GBOWTIH. (BEISUlLrPS EXPIRESBEID IN M'G. MT'KOiGEN FOUND AND' IN PEiB CENT OP T'HEI T'O'TIAIj NITfilOGlEIN' CIONTENIT, BASED ON 25 GR. SAMIPLE. Pots 5K. 2 2 M 1 2 ti C r- C 0.~ 3 i ° 6^3 c Ih c H S^ < t:^ c^ k, ii3 Ph < ^3 Oh £i.° IX 9— ll-_. 14.33 2.0242 14.1+ 3.1675 22.0 0.1260 0.8+ 0.4725 3.2+ 10—12— 15.60 1.9181 12.3— 2.6650 17.0+ 0.1505 0.9+ 0.2975 1.9 13—15— 20.39 2.2151 10.8 + 2.3690 11.6+ 0.1400 0.7— 0.4aS0 2.2 + 14—16— 19.81 1.9S18 10.0+ 3.0650 15.4+ 0.1085 0.5+ O.2O0O 1.0+ 17—19— 23.03 2.2939 9.91+ 3.5000 15.2+ 0.1015 0.4+ 0'.2625 1.1 + IS— 20l__ 23.09 1.1121 4.7 + 2.8500 a2.3i+ 0.1680 0.7 + 0.2100 0.9+ 21— 23-._ 19.77 2.3424 11.8+ 2.7175 13.8— 0.2810 1.1 + 0.4200 2.1 + 22—24— 20.19 0.S648 4.8+ 21.7325 13.5+ 0.1400 0.7— 0.3500 1.7+ 25—27 17.50 2.4030 13.7+ 2.6950 15.4 0.1820 1.0 + 0.4375 2.5 26—28-.. 22.54 1.9878 8.S+ 2.7425 12. a+ 0.2110 0.9+ 0.31.50 1.4— 29^31... 20.94 2. 3848 11.4— 2.5150 12.0+ 0.1750 0.8+ 0.2100 1.0 30^-S2... 118.69 0.7515 4.1— 2.5875 13.8+ 0.09SO 0.5+ 0.3200 1.7+ TABLE XVn— AM'O'UNTISi OP THE DIPPEBENT PIOIRMISI OP NTTEOGEN IN THE PALLO'W AND ORiOiPPED' IXOOULATE© SOIL® AT THE: END OP THE SECOND G'RiOiWING PERIOD. RESIULTS EXPRES'^'ED' IN M!G. N. POUND AND' IN PEK CENT OP THE TOTAIL N. CONTENT EASED O'N 25 -GEiAM SAiMP'LflB. Pots „ c . 2 g M 1 bi 'G re 3 c 2 M g.S§ a 0-T3 <^ *-' H SS < i:3 Oh 2^ 2^ o. < « S CU £-Z B 0- 9-11... 16.45 1.6515 10.0 1.8675 11.1+ O.O70O 0.4+ 0.2800 1.7+ 10—12... 15.44 lost 3.41S0 22.1 + 0.1110 0.T+ 0.3675 2.4^ 13-15... 21.48 2.1896 10.2 2.0700 9.0+ 0.1260 0.6— O.160O 0.7+ 14—16 19.32' 0.7257 3.V+ 3.99'2S 15.5— O.140O 0.7+ 0.1225 0.6+ 17—19 16.62 0.9212 5.5+ 2.6825 16.1 + 0.4750 2.8+ 0.1750 1.1— 18^20 15.84 0.7257 4.6— 3.4075 21.5+ O.02O1 0.1 + 0.3300 2.2+ 21—23 19.74 1.7S95 9.1— 3.5000 17.8— 0.1190 0.6— 0.2100 1.0+ 22—24 19.78 0.7500 . 3.8— 3.33OT 16.8+ O.063O 0.3 + 0.2800 1.4 + 25—27-.. 17.28 1.4727 8.5+ 2.6370 15.3— 0.2660 1.5+ 0.2100 1.2+ 26—28 16.32 0.6030 3.7— 2.6000 15.9+ 0.0630 0.4— 0.29'76 1.8+ 29—31... 20.31 2.1363 10.5+ 2.8330 13.9+ O.091O 0.4+ O.280O 1.3+ 30—32 20. 6® 1.1000 5.4 + 3.4900 16.9— 0.0'700 0.3+ 0.2275 1.1 + TABLE XVIIT— AMIO'UNTB OP THE DrPPEiBENT POIRMS OP' NITROGEN IN THE PALL'OW ANiD CiROiPPED INOCiULA,T'EID' iSiOTLlSi AT THEi EINlD OP THE THIBD AND LAST GR'O'WTNIG PEIRiTOD. iREISUODTS IBABED' ON 23 GIEAM S'AIMPLE, EXPRESSED IN IMG. N. POUND' u-m-D IN PEiR CENT O'P TOTAL N. CONTENT. Pots Total N. content 2Sgr. c u 1 TJ o c 2^ £ E c 'Ev. 'i < « E c a 0-T3 5 c^-s3 c £. 9^11... 16.93 1.2833 7.5+ 2.1475 12.7— 0.0420 0.2+ 0.2275 1.3+ 10—12... 15.65 0.5773 3.7 — 3.2850 20.9+ 0.0490 0.5+ O.210O 1.3 + 13—15... 20.23 0.7485 S.7— 2.3675 11.2+ O.042O 0.2+ 0.2450 1.2+ 14—16... 18.19 0.6306 3.4+ 3.3005 18.1 + O.091O 0.4t O.2100 1.1 + 17-^19... 16.90 1.1773 e.9+ 2.7025 15.9+ 0.0210 0.1 + 0.2625 1.6— 18—20..- 14.94 0.5560 3.7+ 3.6823 24.7— 0.06.30 0.4+ O.140O 0.9+ 21—23 22.03 1.7727 8.1— 2.8560 VZ.9+ 0.0420 0.2'— 0.2800 1.3— 2r2-24-.. 18.21 0.7000' 3.8+ 3. 03 GO 16.5+ 0.3240 1.8— 0.1750 0.9+ 25—27 a6.7l 1.506O 9.0+ 2.6925 16.1 + 0.0350' 0.2+ 0.5950 3.6— 26^28 15.66 0.7212 4.C + 3.6350 23.2+ 0.0420 0.31— 0.3150 2.0+ 29-31— 20.36 2.2060 10.8+ 2.740O 13.4+ O.056O 0.4 + 0.7075 3.4+ 3D--32 18.091 0.5939 3.3— 3.4500 11.3— 0.0770 0.4+ 0.2800 1.5 + 60 compounds became smaller, as decomposition of the oro:anic mat- ter proceeded, at a sligiitly faster rate than the total nitrogen content of the soil became depleted. The non-protein nitrog'eu also varied considerably, altho' not in the marked degree shown by the other forms. Neither the oats straw or the red clover hay, added as manures to the pots, showed any effect on the forms of nitrogen determined, further than the small amount shown in the preliminary determinations. If there was a difference in the soil under field conditions it evidently was too small to he measured by these methods. It is entirely possible that the amounts of these complex nitrogenous compounds are rapidly changing into other forms and that the per cent they bear to the total nitrogen content remains somewhat constant, varying only with the amount of organic matter present in the beginning, then as decomposition proceeds and the more complex combinations are broken up, this percentage relation becomes smaller and smaller until it reaches a constant. Once decomposition had begun in the soil there was absolutely no tendency for the more complex nitrogenous forms to accumu- late under conditions approximating those in the field. Instead of an accumulation there was a steady reduction. How closely this reduction is coupled with the decay of the organic matter and what would be the final equilibrium between the total nitrogen content and the nitrogenous compounds are questions for further study. Summary. 1. The acid extracted, non-protein, amino and polypeptid nitrogen changed into other forms with the advance of decompo- sition much faster than the total nitrogen contents of the soils in question decreased. 2. Oats straw and clover hay added as manures at the rate of five tons per acre had little effect in influencing this change. 3. The amounts of non-protein and amino acid nitrogen fixed by bacterial cultures in solution were negligible. 4. Bacterial inoculation had apparently no effect on the amounts of non-protein, amino or polypeptid nitrogen in the soil. 5. There was no tendency for the above forms of nitrogen to accumulate in the soil under conditions approximating those in the field. Acknowledgments: I wish to express my thanks to Dr. P. E. Brown for his help and suggestions thruout this work and to Dr. H. S. Potter for his suggestions in the determinations of the com- plex forms of nitrogen. APPENDIX TABLE 1. ===== == = ^ Found in Tot Soils, Cniculated 1 == —^ on B osia of 4491 vj. 'n Fallow and 4536 gr. 1 S = ° 1 1 liind of Crop Grown in cropred.— P..t Ji m izi 2 -"lias „ % c 0.^ n-c Inoculum Used and Pot No. Determinations 1 fi 2 [2 11 1 Q > 11.1 ill i,chrooc. (HCM) F P 3.0309 2.7668 4.2744 4.H171 3.3630 3.2079 4.3373 4.2547 3.3499 2.9SS3 4.305S 4.1809 "oroiio" 3.54SD 2.9747 4.3056 4.1073 U.835«" 7.1150" 6.0123" o.irai" 3.S499 2.9870 4.3058 4-2684 3.3833 2.9S70 4-3488 4.2684 3.6232 3.7309 3.8963 4.0046 Clover dover e'. {.'.'.'.'— — 0.2638 A, c/l">-toi.i""' F F ■'.SCOl 3.4450 4.6663 ■1.4770 3.6S20 3.0310 4.57S)S 4.50S7 3.2210 S.53S0 4.5726 4.492S ""o'oisis" 0.0136 3.2210 3.6244 4.5726 4.1702 "i.noo"' ""eSoo"" 1.0564" l^roS" 3.2210 3.5808 4.5726 4.5787 3.2532 3.5S0S 4.6183 4.57S7 3.6232 3.7309 3.8963 4.0046 11 ID-IZ 13-lS 14-lC 0at3 Clover Clover 0.7220 0.5741 4. iictjcwckii [7-19— IS-M- - - 21-2S F F C F P c F Oats _ Oats UKiVcr Clover Oats ,-- Oats CiOvcr -- Clover -.- Oats 5.1230 5.1392 4.5102 4.b8Sl S.OOOl 5.17.55 2. iOol 4.2S05 3.4tSV 5.2173 6.3343 4.3CS7 4.6722 3.8973 6.0-185 2.44V4 4.1912 3.4SS7 5.1701 5.28617 4.4394 4.E8C1 3.9267 5.1120 2.4572 4.23SS 3.4SSr "i'.mii' "o.oise" "aoiss" "oroise"' 5.1701 5.2731 4.4394 4-5666 3.0267 5.0984 2.45r2 4-2252 3.48S7 ""o^Sso"" "7?oi6o"" "i'.'dim" S.2050"' "6.6093" o.oSs" 0.0810 "6.1205 5.1701 5.2S24 4.4394 4.6613 3.9267 6.1791 2.4S72 4.3467 5.2218 6.as24 4.4S27 4.6613 3.9659 5.1794 2.4817 4.3457 3.6232 3.7309 3.8963 4.00-16 3.ia32 3.731-.' 3.'S«i 4-l«16 1.5086 1.5515 0.5SC4 25-27- 20-28 — - 20-31 — - 0.3427 1.44S5 Az. seD ,\3-»5 Oats 0.01-36 3.S034 0.4900 •n 19' 1' Glover 4.4823 SS-40 Olovcr 6.0136 4.55S6 ■t.VXili 6.1247 4-6833 4.0SS3 4.0046 0.6/87 F Oats --- Onts -. 3.5S30 3.556-2 3.70S7 3.5S79 3.6458 3.5/20 "oToiseT" 3.6458 .1.5584 i'.im' 3.6158 3.6^22 3.0232 F Clover _ - 4.5023 l6-(8 — Clover 4.3114 0.0136 4-2973 9.(H60 0.1217 4.4195 4.41i;5 4.0046 0.4149 F Oats 3.3«30 3.4573 3-41(1 3.4101 3.4442 Oats 3.6323 0.0136 3.5187 2. 1850 3.5519 3.5519 3.7309 F Clover - 4-4001 4.40.11 51-56 C aovcr - 4.272SI 4.S0OO 4.3114 0.0136 4.2978 6.7850 0.08S8 4.3866 4,3Mi6 4.0040 0.3t20 3.6M4 3.59S7 3.6065 3.5323 0.0:33 Oats 3.4'.i27 3.5085 3.5006 0.01S6 S.4S10 3.8650 3. 5323 3.730') F Clover ..- 4.^.310 4.4C44 4.4630 ._. 4.4630 4.4630 4..i076 3.8903 V.6113 62-(H Wis".d cultures 00-07 Clover 4.2230 4.3817 4.3023 O-0136 4.2fS7 7.1400 0.107S 4.3905 4.3905 4.O0W H.3919 Oats 3.5563 3.6514 3.603S 0.0136 3.5902 1-0450 3.1:021 3.6021 3.7109 F Clover 4.3087 4.3687 4.3ia7 4.4123 3.SJ63 0.516(1 C Clover .— 4.4135 4.5105 4.4720 0-0136 4.4684 9-530O i.l210 4.57114 4.5794 4.0046 0.5748 Check Average P Nothing _.- — 3.6369 5.5515 3.5912 3.594'. 3.5942 3.6301 3.4816 75-76 — Nothing 3.5562 3-4609 3.5CS5 0-0136 3.4949 5.5650 0.0740 3.5689 3.56S9 3.5S93 V Nothing 3.S002 3.3002 3.3002 3.3002 3.3002 3.3332 JS-90 - — Nothing 3.4609 3.6197 3.5103 0.0136 3.6267 C.7700 0.0830 3.6097 3.6097 APPENDIX TABLE II. Actual Grams N. Found in Pot Soils, Calculated on Basis 41J6 gr. in Fallow, 4101 gr. in Cropi ed. — Pots Determination , c S.5 SZ°^ Hi !|i ttoS.g g-ss oP°S6 •3.3-S o -^1 2.7340 3.0128 3.3370 2.8S64 3.0910 3.1866 3.3205 3.6592 3.6107 3.3397 3.6102 3.4603 2.6867 2.9607 3.3370 2.5610 2.7947 3.1866 3.5242 3.8S36 3.6107 3.2020 3.4901 3.4003 2.7165 2.9924 3.3370 2.6263 2.86-26 3.1866 3.3343 3.6743 3.6107 3.2897 3.5857 3.4003 2.8235 3.1115 3.3370 2.7306 2.9763 3.1866 3.3205 3.6591 3.6107 3.463(5 3.6322 3.4603 2.7517 3.0356 3.3370 2.9120 3.1730 3.1866 3.4921 3.8492 3.6107 3.0901 3.S747 3.4603 2.7015 2.9770 3.3370 3.0069 3.3J"9 3.1806 3.4487 3.S0O4 3.6107 3.5425 3-8613 3.4603 2.70S3 2.9845 3.3370 2.9083 3.17(0 3.1866 3.3977 3.7442 3.0107 3.2959 3.69-25 3.4603 2.6146 2.S812 .1.3370 2.8681 3.1-202 S.lwd 3.3998 3.7465 3.6107 3.2031 3.4913 3.4003 2.5210 2.7781 3.3370 2.7221 2.9310 3.186'j 3.2696 3.0031 3.01/7 3.4091 3.7159 3.4 .(3 Aver!i:--e 2.8235 3.1115 3.0537 2.9768 3.2793 t 2.7857 3.0364 II Onts ... C?lovt.r . Oats .. Oats ... Clover . Olovcr . Oats . . . Clover ... Clover . . Oats ... . Oats .... Clover .- Clover .. Oats ... Oats .... Clover .- Clover _. Oats .... Oats .... Clover .. Clover .. Oata .... Onts .... caover .. Clover .. Nothing . Nothing 2.7517 2.85.37 3.6443 2.7227 2.SCS3 3.3977 3.3051 2-8001 2.75IS 2.9696 2.fl»5 2.7900 3.2557 3.2177 2.7083 2.5910 3.4318 3.3(ftl 2.7577 2.!I-2S6 3.4400 3.04.<10 2.6790 3.2096 3.3183 2.8S79 2.7618 2.9S'20 2.7340 2.S21S 3.3-205 3.3123 2.6807 2.5479 3.5242 3.1813 2.6134 3.3343 3.2681 2.8235 2.0936 3.3-205 3.4143 2.7.547 2.S9I2 3.4921 8.0618 2.7015 3.0503 3.44S7 3.50S9 2.6146 2.8-246 3.3908 3.1740 2.8235 2.7518 2.9758 2.T7S6 0.0136 0.0130 0.0136 0.0136 O.OISO 6.0136 0.0136 '6.6l36 0.0136 6.0130 "o.6i36 "616136" "6.61S6 0.0130 6.0136 0.0136 3.2445 2.8235 2.7547 2.8776 8.4921 3.0512 2.8748 3.3977 3.2531 2.8235 2.7382 2.9758 2.7600 "'l?4995 " ' 016282'" "2?i746" "6"64i6"" "i.7S7"" "T6297" "ir6253" ""6!m43" 1.8895 0.0205 i?S346" "016452" "2"ol7s'" 6.6566" "s^nss"" ""6^629"" "2?2545" 6?6S4"' 2.2800 0.0149 "i"6s66'" "'6S362" "I'sffls" "6^6472" "'i'so-Is'" "6'6336"" 2.6103 0.0428 ""s^oior" li.mi ""i^iooo" 6^0127 2.2315 " 0.0422 1.6933 0.0376 'T.m&' ""6;6ie9" "ilm' '"o"657"" 0.1663 0.1897 0.4010 IIJ.O4B0 -t- clover N. -|- 3.4603 APPENDIX TABLE III. Az. 27D 41 42 43 64 -. 05 Ai-iual Grams N. Found in Pot Soils, Calculated on Basis of 3741 er. in Fallow and 3786 gr. in Cropped Pots Oats ... Oats -. Oats ... Clover . Clover - Clover . Clover . Onts Oats Oats Clover .. Olover _- Clover .. Olover — . Oats . , Oats Oats Oats Olov(!r ... Clover ... Clover ... Olover ... Onts Oats Oats Oats Clover ... Olover 11' Clover ... Oats Oats Oats Oats Clover ... Olover ... Olover ._ Clover .. Oats Oats Oati Oats Olover .. Clover .. Clover _- Olover .. Oats ._- Oats Oats Oats Clover .. Olover .- Cnover .. (/lover .- Oata Oats Oats .... Oats Clover .. Olover .- Clover .. (plover .- Oats Oats Oata -. Oats „-. (Jlovor .^. Clover .. Olover _. Olover .. Nothing Nothing Nothing Nothing Nothing Nothing Nothing Nothing 2.S30O 2.8131 2.7619 2.1864 2.3321 2.3038 2.3586 2.9059 2.S751 3.1808 2.5971 2.5018 2.81S9 3.0499 2.8773 2.6574 2.1074 2.4216 2.9583 3.2ni 2.8405 2.5656 2.32-27 2. 4478 3.0070 3.5203 2.7129 3.0761 2.7694 2.5731 2.3983 2.4478 2.3719 3.0-237 2.7429 2.4740 2.5044 2.3954 2.3050 2.9714 2.7969 2.0027 2.4478 2.4663 2.4871 2.4640 2.8405 2.9548 3.063O 2.4478 2.5713 2.1991 2.3321 2.4497 2.6971 3.0237 2.9152 2.^71 2.7959 2.8274 3. 0666 2.1074 2.3954 2.4616 3.-2852 2.8274 2.8688 2.7189 2.36-52 2.3103 2.3785 2.89-28 2.8489 3.1646 2.7501 2.57S7 2.2913 3.4805 2.5131 2.3917 2.4478 2.3491 3.0172 2.7429 s.oni 2.7363 2.4009 2.4848 2.3823 2.4478 2.4583 2.4871 2.4917 2.8143 3.0181 3.056-1 2.4451 2.7230 2.7893 2.S067 2.3160 2.4094 2.6037 3.0173 2.8877 2.8208 2.3496 2.4381 2.3234 2.3210 2.0944 2.7700 2.6375 2.1071 2.4085 2.4911 2.4901 0.0136 0.6136 0.0136 0.0136 "o'oiso 0.0130 "6?6i3«" "6?6i36' 0.0136 "o'orn" 0.0136 0.6136 "6^136" 0.0136 oToise" 0.0136 0.0136 6.6136 '6"6i3o' O.0136 "6?6ii6' "616136" 0.6136 "6^6136" "6'oi36" "6'6i36" 0.6136 2.3507 2.7S12 2.17t« 2.9-255 3.2710 2.8274 2.8562 2.57S7 2.2807 2.4S(B 2.0601 2.6431 2.3761 2.4478 2.3351 3-0172 2.7293 3.0711 2.7227 2.7113 2.0161 2.8741 2.4478 2.4447 2.4871 2.4781 2.S14S 3.0015 2.4961 2.5444 2.2180 2.4315 2.7230 2.7757 2.6607 2.6100 2.4085 2.4775 2.4765 3.0050 3. mo" 2.5500 2.7790 3.7675 3.1550 "3"iS6" 3.8030 1.1575 3.4015 2.2166' 3.9045 S.7480 "i'.imi 3.4770 2.9195 0.0715 0.0904 O.0S68 o.oiio 0.0975 '6ru68' 0.1002 0.0785 "6.6512' 0.1296' '6'i69i' 0.1103 "o'iisj' 0.1280 "6^940' 6'i263' o'issT 6.1293' 0.0420 '6ri6i6' '6?i3-27' 0.10S7 0.1055 "0.6664" 4.3356 j 0.15S5 2.9196 I 0.113.8 2.7489 2.4231 2.3103 2.4653 2.!,l)-28 2.y(;34 3.1540 2.863-1 2.6787 2.4178 2.4617 3.0172 2.4478 2.6650 2.4871 2.6261 2.S143 3.1106 3.0564 2.9705 2.4901 2.0393 2.21S0 2.6508 2.9047 3.3165 3.0817 2.9781 2.9091 2.9-191 3.0598 3.4381 3.7252 3.3V59 2.9091 3.0730 3.0109 3.1695 :'..4137 3.7431 3.027S 3.1751 2.0911 3.0758 2.8502 3.S-2&1 2.8502 1.3860 J. 7020 0.16S0 i.iioo" 2.8502 0.2345 3.3281 2.8502 0.11S9 3.3284 3.1239 0.6369 3.0021 3.1239 0.0013 3.00-21 2.8602 0.1348 3.3281 2.S0O2 0.0385 3.S-2S4 3.1239 0.40-23 3.3284 2.8602 3.3-28.1 3.1239 3.6U21 3.1239 3.C021 S.82S1 3.1239 3-6021 -'\.v. ck. fallow 2.70S6 -f N. c Av. ck. fa.llow 2.7086 + N. c jVv. cU. cropped 3.1.SfiS -t- N. Av. ck. cropped 3.1868 + N. ntent of oat.s ground 0.1410=2.8502 nlcnt ground clover 0.4163 = 3.1239. •ontent ground oats 0.1416=3.3284. ■ontent ground clover 0.4163=3.0021 *• 61 BIBLIOGRAPHY 1. ASHBY, S. F. 1907. Some observations on the assimilation of atmospheric nitro- gen by a free-living ovganisva-Azotobacter chroococcum of Bei- jerinck. Journ. Agr. Sci. 2:35. 2. BEIJERINCK, M. W. 1901. Nitrogen fixing bacteria. Centbl. f. Bakt. 2 Abt. 7:561. 3. BEI.JERINCK, M. W. 1908. Fixation of nitrogen by pure culture of azotobacter; dis- tribution of the organism. K. Akad. Wetensch. Amsterdam, Proc. Sect. Sci. 11:67. 1908. Abs. in Journ. Chem. Soc. 94:975. 4. BEIJERINCK, M. W. and VAN DELDEN, A. V. 1902. The assimilation of free nitrogen by bacteria. Centbl. f. Bakt. 2. Abt. 9:3. 5. BOLLEY, H. L. 1900. The duration of bacterial existence and trial environments. Centbl. f. Bakt. 2 Abt. 6:33. 6. BONAZZI, A. 1915. Cvtological studies of Azotobacter chroococcum. Journ. Agr. Rsch. 4:225. 7. BOTTOMLEY, W. B. 1907. Cereals and bacteria. Country Life, 22:466. 8. BOTTOMLEY, W. B. 1910. The fixation of nitrogen by free-living soil bacteria. Proc. Roy. Soc. Ser. B. 82:627. 1910. Abs. in Journ. Soc. Chem. Indus. 29:1171. 9. BOTTOMLEY, W. B. and HALL, A. D. 1909. Nitrogen-fixing bacteria and non-leguminous plants. Nature 82:218. 10. CHRISTENSEN, H. R. 1906. On the occurrence and distribution of Azotobacter chroococ- cum in different soils. Centbl. f. Bakt. 17:109, 161, 378. 11. CZAPLEWSKI, E. 1889. Bacteriological methods. Centbl. f. Bakt. Abt. 6:409. 12. DVORAK, J. 1912. Studies on nitrogen fixation in soil by micro-organisms. - Zeitschr. f. d. Landw. Versuchsvi^es in Oster. Jahrg. 13:1077. 1913. Abs. in Centbl. f. Bakt. 2 Abt. 37:106. 13. EHRENBERG, P. 1909. The bearing of carbon determinations upon nitrogen fixation in soils. Fiihling's Landw. 2tg. 58:663. 14. FISCHER, H. 1905. Contribution to the knowledge of the life conditions of nitrogen-collecting bacteria. Centbl. f. Bakt. 2 Abt. 14:33, 781. 15. FISCHER, H. 1905. Nitrogen fixing bacteria. Journ. Landw. 53:61. 16. FISCHER, H. 1905. Contribution to the knowledge of the life conditions of nitrogen-collecting bacteria. Journ. Landw. 53:289. 17. FRENDENREICH, E. DE 1903. Assimilation of atmospheric nitrogen by bacteria. Centbl. f. Bakt. 2 Abt. 10:514. 62 18. GBRLACH, M. and VOGEL, I. 1902. Nitrogen-gathering bacteria. Centbl. f. Bakt. 2 Abt. 8:669; 9:816; 9:881. 19. GERLACH, M., and VOGEL, I. 1903. Furtlier studies on nitrogen gathering bacteria. Centbl. f. Bakt. 2 Abt. 10:636. 20. GREAVES, J. E., and ANDERSON, H. P. 1914. The influence of arsenic upon the nitrogen fixing powers of the soil. Centbl. f. Bakt. 2 Abt. 42:244. 21. HEADDEN, W. P. 1910. The fixation of nitrogen in some Colorado soils. Bull. Colo. Agr. Exp. Sta. 155. 1911. Bull. Colo. Agr. Exp. Sta. 178. 1913. Bull. Colo. Agr. Exp. Sta. 186. 22. HBINZB, B. 1906. Some contributions to the microbiology of soils. Centbl. f. Bakt. 2 Abt. 16:640. 23. HEINZE, B. 1906. Concerning nitrogen fixation by lower organisms. Landw. Jahr. 35:889. 24. HOFFMAN, C. 1913. The protein and phosphorus content of azotobacter cells. Centbl. f. Bakt. 2 Abt. 36:474. 25. HOFFMAN, C, and HAMMER, B. W. 1911. Some factors concerned in the fixation of nitrogen by azoto- bacter. Res. Bull. Wise. Agr. Exp. Sta. 12. 26. JACOBITZ, E. 1901. The assimilation of free atmospheric nitrogen. Centb. f. Bakt. 2 Abt. 7:783, 833, 876. 27. JONES, D. H. 1913. A morphological and cultural study of some azotobacter. Centbl. f. Bakt. 2 Abt. 38:14. 28. KASERER, H. 1910. On the mineral needs of azotobacter. Ber. d. Deutsch. Bot. Gesell. 28:208. 29. KELLERMAN, K. F., and SMITH, N. R. 1914. The absence of nitrate formation in cultures of azotobacter. Centbl. f. Bakt. 2 Abt. 40:479. ?.0. KOCH, A. 1910. On nitrogen fixation in soils with cellulose as a source of energy. Centbl. f. Bakt. 2 Abt. 27:1. 1907. Mitt. Deut. Landw. Gesell. 22:117. 1909. Journ. Landw. 57:269. 31. KOCH, A., and SEYDEL, S. 1911. The utilization of cellubiose as a source of energy in nitro- gen fixation through bacteria. Centbl. f. Bakt. 31:567. 32. KOBER, P., and SUGIURA, S. 1913. A micro-chemical method for the determination of alpha and beta amino acids and certain derivatives in proteolysis, blood and urine. Journ. Amer. Chem. Soc. 35:1546. 33. KRAINSKY, A. 1908. Azotobacter chroococcum and its action in the soil. Centbl. f. Bakt. 2 Abt. 20:725. 1910. Centbl. f. Bakt. 2 Abt. 26:231. 34. KUHN, J. 1901. The assimilation of free nitrogen by soil bacteria without symbiosis with leguminous plants. Fiihling's Landw. Ztg. 50:2. 63 ?5. LIPMAN, J. G. 1904. Soil bacteriological studies. Rpt. N. J. Agr. Exper. Sta. 1904:237. 36. LIPMAN, J. G. 1903. Nitrogen-fixing bacteria. Doctor's thesis, Cornell University. 37. LIPMAN, J. G. 1903. Experiments on the transformation and fixation of nitrogen by bacteria. Rpt. N. J. Agr. Expt. Sta. 1903:247. S8 LIPMAN, J. G. 1904. Soil bacteriological studies. Rpt. N. J. Agr. Exp. Sta. 190-4:263. 39. LIPMAN, J. G. 1902. Nitrogen-fixing bacteria. Pop. Sc. Monthly. G2:97. 1903. Rpt. N. J. Agr. Exp. Sta. 1903:217. 1904. Bull. Bur. Chem. 81:146. 40. LIPMAN, J. G. and BROWN, P. E. 1911. Laboratory guide in soil bacteriology. 41. LIPMAN, J. G., and BROWN, P. E. 1907. Report of the soil chemist and bacteriologist. Rpt. N. J. Agr. Exp. Sta. 1907:141. 42. LIPMAN, C. B., and BURGESS, P. S. 1915. Studies on nitrogen fixation and azotobacter forms in soils in foreign countries. Centbl. f. Bakt. 2 Abt. 44:481. 43. LoHNIS, P. 1909. The importance of nitrogen fixation in cultivated soil. Fiihling's Landw. Ztg. 58:425. 44. LoHNIS, F., and HANZAWA, J. 1914. The classification of azotobacter. Centbl. f. Bakt. ^2:1. 45. LoHNIS, F., and PILLAI, N. K. 1908. Nitrogen-fixing bacteria. Centbl. f. Bakt. 2 Abt. 20:781. 46. LoHNIS, F., and PILLAI, N. K. 1907 Nitrogen-fixing bacteria. Centbl. f. Bakt. 2 Abt. 19:87. 47. LoHNIS. F., and SMITH, N. R. 1916. Life cycles of bacteria. Journ. Agr. Rsch. 6:675. 48. LoHNIS, F., and WESTERMANN. T. 1908. Nitrogen-fixing bacteria. Centbl. f. Bakt. 2 Abt. 22:234. ■49. LUTZ, L. 1905. The morphology and biology of nitrogen-assimilating organ- isms. Centbl, f. Bakt. 15:477. 50. MULVANIA, M. 1915. Observations on azotobacter. Science, N. S. 42:463. 51. OMELIANSKY, W. L. and SSEWEROWA, 0. P. 1911. Pigment formation in cultures of Azotobacter chroococcum Centbl. f. Bakt. 2 Abt. 29:643. 52. PETERSON, E. G., and MOHR. E. 1913. Non symbiotic nitrogen fixation by organisms in Utah soils. Centb. f. Bakt. 2 Abt. 38:494. 53. POTTER, R S., and SNYDER, R. S. 1915. Determination of amino acids and nitrates in so*ls. Res. Bull. la. Agr. Exp. Sta. 24. 54. PRAZMOWSKI, A. 1912. Natural history, morphology and cytology of Azotobacter chroococcum. Centbl. f. Bakt. 2 Abt. 33:292. 55. PRINGSHEIM, H. 1907. On the adaptability of different sources of energy to assimu- lation of atmospheric nitrogen and the distribution of nitro- gen-fixing bacteria in the soil. Centbl. f. Bakt. 2 Abt. 20:248, 1908. Centbl. f. Bakt, 2 Abt. 26:222, 64 56. PRINGSHEIM, H. 1914. Nitrogen assimilation in the presence of nitrates. Centbl. f. Bakt. 2 AM. 40:21. 57. REMY, T. 1909. Investigations on the process of nitrogen assimilation in its relation to the soil climate. Centbl. f. Bakt. 2 AM. 22:561. 58. REMY, T., and ROSING, G. 1911. The biological stimulation of natural humus. Centbl. f. Bakt. 2 Abt. 30:349. 59. ROSING, G. 1912. Results of the investigations on nitrogen fixation by Azoto- dacter cliroococcitm. Centbl. f. Bakt. 2 Abt. 33:292; 618. 60. STOKLASSA, J. 1908. Contribution to the knowledge of the chemical processes involved in the assimilation of free nitrogen by azotobacter and radiobacter. Centbl. f. Bakt. 2 Abt. 21:484. 61. STRANAK, J. 1909. The assimilation of atmospheric nitrogen by soil micro- organisms. Centbl. f. Bakt. 2 Abt. 25:320. 62. THIELE, R. 1906. The utilization of atmospheric nitrogen by micro-organisms. Landw. Vers. Sta. 63:161. 63. VAGLER, P. \ 1909. The fixation of atmospheric nitrogen. Centbl. f. Bakt. 2 Abt. 22:452. 64. VOGEL, J. 1912. Potash requirements by the nitrogen bacteria. Centbl. f. Bakt. 2 Abt. 32:411. 65. VOGEL, J. 1903. Some recent investigations in nitrogen assimilation by bac- teria without symbiosis. Piihling's Land\ik Ztg. 52:178. 1903. Reference Exp. Sta. Rec. 14:1048. 66. VORHEE§, E. B., and LIPMAN, J. G. 1905. Experiments on the accumulation and utilization of atmos- pheric nitrogen in the soil. Bull. N. J. Agr. Exp. Sta. 180. 67. WILFARTH, H., and WIMMER, G . 1907. The influence of mineral fertilization on the nitrogen fixa- ation by lower organisms in the soil. Landsw. Vers. Sta. 67:27, 68. WARMBOLD, H. 1906. Investigations on the biology of the nitrogen fixing bacteria. Landw. Jahs. 35, No. 1-2; 1., 35, No. 4-6; 63. LIBRARY OF CONGRESS 002 756 635 2